12/15-lipoxygenase pathway is required for alveolar macrophages homeostasis
Erwan Pernet
A Novel Approach to Predicting Adverse Cardiovascular Events in COPD Using Respiratory Biomarkers
Suurya Krishnan
Acceptance, safety and effectiveness of exercise interventions in solid organ transplant candidates: a systematic review
Fernanda Pesce de Souza
Agreement between measured and predicted oxygen cost of constant-rate stair stepping and shuttle walking exercise in people with chronic obstructive pulmonary disease
Emily Koch
An Individual Patient Data Meta-Analysis to Estimate the Diagnostic Accuracy of a Deep Learning-based Software for Analyzing Chest X-Rays of Persons With Symptoms of Pulmonary Tuberculosis: preliminary findings
Syed Kumail Abidi
Aryl hydrocarbon receptor orchestrates macrophage polarisation
Hussein Traboulsi
Associations between Fat Free Mass and Exercise Physiological and Perceptual Outcomes in People with Chronic Obstructive Pulmonary Disease: A Research Project Proposal
Kaveh Gaynor-Sodeifi
Asthma and fixed airflow obstruction: Long-term trajectories suggest distinct endotypes
Nan Zhao
Bone Marrow Macrophage Precursors of Duchenne Muscular Dystrophy (mdx) Mice Are Differentially “Trained” Based on Muscle Disease Stage
Salyan Bhattarai
Can BCG-mediated protection be vertically transmitted?
Eva Kaufmann
Characterizing the role of PGE2 in an Irritant-induced Murine Model of Asthma
Emily Nakada
Combined pulmonary and rheumatology clinics improve the care of patients with interstitial lung diseases
Ketaki Rawal
Constant-Rate Shuttle Walking and Stair Stepping Tests to Assess Exertional Breathlessness in COPD: Responsiveness to Pulmonary Rehabilitation and Estimation of the Minimal Important Difference
Lauren Tracey
Control of Nonlinear, Viscoelastic Tissue
Amanda Bianco
Cross-protection of BCG vaccination in influenza infection: Trained adaptive immunity?
Kim Tran
Dissecting mechanisms by which intestinal macrophages maintain homeostasis during helminth infection
Yue Li
EFFECTIVENESS OF A HOME-BASED PRE-HABILITATION PROGRAM FOR LUNG TRANSPLANT CANDIDATES: A RETROSPECTIVE CHART REVIEW
Daniela Massierer
Effects of Amino Acid Restriction on Macrophage Function
Mikaela Eng
Efficacy and objective adherence to mandibular advancement splints for treatment of sleep-disordered breathing during pregnancy and the postpartum period.
Nelly Huynh
Energy Expenditure and Physical Activity in Pregnant Women with Gestational Diabetes: Is there a relationship with Sleep-Disordered Breathing?
Julia Leonard
Fgf10-deficiency alters airway development and response to chronic cigarette smoke injury increasing susceptibility to COPD-like phenotype.
Motahareh Vameghestahbanati
From the clinic to the laboratory: azithromycin for prevention of COPD exacerbations
Nafiseh Naderi
Gender partially explains the variation in the airflow obstruction in dysanapsis
Motahareh Vameghestahbanati
Heart rate variability in extremely preterm infants receiving NCPAP, NIPPV and NIV-NAVA after extubation: a randomized crossover trial.
Samantha Latremouille
Identification of Pseudomonas aeruginosa genetic variants in chronic lung infections of cystic fibrosis patients and their effect on lung disease severity
Jannik Donner
Immunity to Influenza A virus infection during pregnancy
Julia Chronopoulos
Implications of HuR in Idiopathic Pulmonary Fibrosis
Joshua Trivlidis
Inhibitory activity of STAT6-IP on IL-33-mediated IL-13 production and eosinophil activation in a Type 2 innate murine inflammatory model
Vanessa Moarbes
Late diagnosis and undertreatment of COPD – A Canadian perspective from the CanCOLD observationnal study
Sébastien Gagnon
Leukotriene C4 synthase deficiency causes spontaneous emphysematous changes in mice
Yumiko Ishii
M. tuberculosis reprograms hematopoietic stem cells in type I IFN dependent manner to impair trained immunity against TB
Nargis Khan
Maternal Sleep-Disordered Breathing in Pregnancy Increases Nocturnal Glucose Levels in Women with Gestational Diabetes
Raphieal Newbold
Mechanisms Underlying Corticosteroid Insensitivity Induction by Interleukin-17A in Bronchial Epithelial Cells
Wided Akik
Modelling the Epidemiology of Tuberculosis in Nunavut from 1948-2018
Aashna Uppal
Modulation of CD4+ T cell Fate by Airway Smooth Muscle Cell Mediated Inflammation
Joyce H. Jang
New insights into macrophage dynamics in the mouse diaphragm
Qian Li
Obstructive sleep apnea treatment of multiple sclerosis patients with severe fatigue using positive airway pressure (SAMS-PAP trial): initial study results.
S. Khadadah
Oscillometry to Assess Respiratory Function in Adult Cystic Fibrosis
Arash Rad
Potentiation of antibiotic lethality by sulfonamides against stationary phase Pseudomonas aeruginosa
Tianxiao Yang
Protective roles of autophagy in sepsis-induced skeletal muscle dysfunction, metabolic derangements and mortality
Jean-Philippe Leduc-Gaudet
PSEUDOMONAS AERUGINOSA EVASION OF NEUTROPHIL ANTIBACTERIAL FUNCTIONS IN EARLY CYSTIC FIBROSIS LUNG INFECTION
K. Kwong
Regulatory mechanisms of eicosanoids in hematopoietic stem cell-mediated trained immunity
Orsolya Lapohos
Smooth Muscle Molecular Mechanics
Megan Hammell
Systemic inflammation is key to understanding the link between concomitant Chronic Obstructive Pulmonary Disease and cardiovascular disease
Mira Abou Rjeili
The Cost of Multidrug-Resistant Tuberculosis in Canada
Jonathon R. Campbell
The effect of nasal dilator strips on obstructive sleep apnea-hypopnea in pregnant women
Noa Goldscher
The Impact of Enteric Helminth Infection on Pulmonary Innate Immune Modulation
Danielle Karo-Atar
The Impact of Parkin Overexpression on Muscle Atrophy and Mitochondrial Morphology in Sepsis-Induced Skeletal Muscle Dysfunction
Tomer Jordi Chaffer
The impacts of airway smooth muscle cells on CD4+ T cell trafficking and subset composition
Rui Sun
The role of CD4 T cell derived HB-EGF in allergen driven airway remodeling
Niusha Khazaei
The role of innate lymphoid cells (ILCs) in mice model of irritant-induced asthma
Utako Fujii
The Role of las Quorum Sensing in the Host-Pathogen Interactions of Intracellular Pseudomonas aeruginosa in Airway Epithelial Cells
Cedric Hupperetz
Understanding how Pseudomonas aeruginosa quorum sensing modulates lung inflammation
Lisa Hennemann

12/15-lipoxygenase pathway is required for alveolar macrophages homeostasis


Erwan Pernet1,2, Dnyandeo Amberkar1,2, Maziar Divangahi1,2
1RI-MUHC 2McGill University

Rationale. Alveolar macrophages (AM) are the lung tissue-resident macrophages that reside in the airways. AM are seeded during embryogenesis and maintain their pool independent of blood circulating monocytes throughout adulthood. AM-deficiency leads to surfactant accumulation, respiratory failure and increased susceptibility to pulmonary infection. Therefore, understanding the mechanisms involved in regulation of AM functions is critical for maintaining pulmonary homeostasis and immunity to infections. Although the contribution of cytokines to AM functions have been extensively studied, little is known about the role of eicosanoids, the host bioactive lipid mediators, which play important roles in macrophages function and pulmonary homeostasis. Eicosanoids are produced by the cyclooxygenase (e.g. prostaglandins), 5-lipoxygenase (e.g. leukotrienes and lipoxins) or the 12/15-lipoxygenase pathways (e.g. lipoxins and 12-/15-HETE). Herein, we aimed to investigate the potential contribution of the 12/15-lipoxygenase pathway in regulation of AM functions.

Methods. In age and gender-matched wild type (WT) and 12/15-lipoxygenase knockout (Alox15-/-) mice, the frequency and numbers of AM as well as other lung innate immune cells in both broncho-alveolar lavage (BAL) and lung tissue were determined by flow cytometry at various ages (3 days, 3 weeks or 6-8 weeks). AM proliferative capacities were assessed in vivo and ex vivo using Ki67 staining. The levels of key AM maturation cytokines GM-CSF and TGF-β1 were determined in BAL and lung tissue by ELISA.

Results. The number of AM were significantly reduced in both BAL and lung tissue of Alox15-/- mice compared to WT mice (6-8 weeks old). Additionally, no difference in other innate immune cells or other tissue-resident macrophages (e.g. brain, spleen and liver) were observed. Despite reduced AM numbers in Alox15-/-, there was no increased BAL turbidity or protein content (total or SP-A), indicating that AM are functional in Alox15-/- mice. Interestingly, the reduction of AM in adult Alox15-/- mice was due to proliferation and not maturation as there was no difference in AM populations at the early development stage (PND3 and 3 weeks). In line with this finding, GM-CSF and TGF-β1 levels in BAL and lung tissue were similar between genotype. However, the frequency of Ki67+ proliferating AM was lower in Alox15-/- compared to WT mice both in vivo and in vitro, indicating an impairment in self-renewal capacity in Alox15-/- AM.

Conclusions. Our findings indicate that the proliferative capacity of AM is impaired after birth in Alox15-/- mice and that the 12/15-lipoxygenase pathway plays a critical role in AM homeostasis.

 

A Novel Approach to Predicting Adverse Cardiovascular Events in COPD Using Respiratory Biomarkers


Suurya Krishnan1,2, Raquel Farias1,2, Jean Bourbeau1,2
1Université McGill 2McGill University Health Centre

Rationale

Chronic obstructive pulmonary disease (COPD) patients are at higher risk for cardiovascular disease (CVD), yet cardiovascular (CV) risk is not routinely assessed in COPD care. Identifying biomarkers of COPD severity and progression that are associated with new CV events is the first step in development of COPD specific CV risk model.

Methods
We collected data from CanCOLD, a longitudinal population-based study of adults over 40 years. Subjects were assessed at baseline with imaging, clinical questionnaires, pulmonary function and cardiopulmonary exercise tests. A total of 1112 subjects (580 COPD and 532 non-COPD) completed follow-up after 3 years. CV events were defined as a previous history of myocardial infarction, angina, stroke, transient ischemic attack or heart failure and collected form healthcare utilization questionnaires. COPD status (FEV1/FVC<0.07) and severity (FEV1>80% GOLD1, FEV1<80% GOLD2+) were measured on post-bronchodilator (bd) spirometry.  Respiratory exacerbations were defined as worsening of respiratory symptoms and use of antibiotics/corticosteroids/ER or hospital visit and was collected via telephone interviews every 3 months during follow-up. Multivariable regression analysis was done to determine association of CVD with spirometry and history of exacerbations after adjusting for age, sex, BMI and smoking.  

Results
Participants with COPD were more likely to have existing CVD such as ischemic heart disease (IHD) or heart failure (HF) OR of 1.56 (1.04-2.34) compared to those without COPD. After stratifying the COPD group by severity according to GOLD status, we found that subjects with moderate COPD (GOLD2+) were even more likely to have cardiac disease (IHD or HF) OR 2.00 (1.34-2.34) and CVD (IHD, HF, TIA or stroke) OR 1.60(1.05-2.44) compared to the non-COPD group. COPD subjects that had a recent exacerbation in the past year were also more likely to have cardiac disease OR 2.88 (1.43-4.80) and CVD OR of 2.06 (1.08-3.95) compared to those without COPD. Furthermore, COPD subjects with multiple exacerbations in the past year had the greatest likelihood of coexisting cardiac disease OR 4.25 (1.25-15.96) and CVD OR 5.14 (1.62-16.28) compared to those without COPD.

Conclusions
COPD increases the odds of having ischemic heart disease and heart failure by over 50%. Moderate airflow obstruction, recent exacerbations and multiple exacerbations in the previous year all increase the likelihood of CVD. Airflow obstruction and exacerbation history should be considered when developing cardiovascular risk prediction and prevention strategies for COPD patients.    

Acceptance, safety and effectiveness of exercise interventions in solid organ transplant candidates: a systematic review


Fernanda Pesce de Souza1, Uma Raje1, Daniela Massierer1,2,4, Catherine Tansey1, Jill Boruff1, Tania Janaudis-Ferreira1,2,3,4
1McGill University 2Centre for Health Outcomes Research, McGill University Health Centre Research Institute 3Respiratory Epidemiology and Clinical Research Unit, McGill University Health Center 4Canadian Donation and Transplantation Research Program

Background: Exercise training may be recommended to solid organ transplant (SOT) candidates to prevent further deconditioning related to the end-stage diseases of the lung, heart, kidney, pancreas and liver. It may improve fitness as well as tolerance for the upcoming physiologic stressor (surgery). However, the acceptance, safety and effectiveness of exercise training in SOT candidates remain unclear. Our objective was to systematically review the literature to 1) determine the acceptance and safety of exercises interventions in SOT and 2) examine the effect of exercises interventions on exercise capacity and quality of life (QoL) in this population. 

Methods: Medline, EMBASE and Cochrane were searched up to 2019. Studies of any design were included. Acceptance was defined by the proportion of patients that agreed to participate in the intervention. Safety was evaluated from the number of reported adverse events during exercise training and effectiveness as the change in maximal and functional exercise capacity and QoL.The Cochrane risk of bias tool and the modified Downs and Black checklist were used to appraise methodological quality of randomized control trials (RCTs) or non-RCTs respectively. 

Results: Out of 1940 possible articles, 58 were assessed in full for eligibility and 20 (8 RCTs, 3 non-RCTs and 9 pre-post) were included. All organs were represented, with exception of pancreas. The majority of the studies included lung transplant candidates (n=9/20). The exercise training ranged from 4 to 25 weeks of duration, and the type of exercise was diverse, with the majority of the studies offering aerobic and/or strength exercises. An average of 80% of patients approached agreed to participate in the interventions. Thirteen studies assessed adverse events but just one reported four minor medical complication.Three out of five studies reported an increase in maximal exercise capacity in the intervention group after the exercise training (the highest mean change observed in the VO2was 2.5mL/Kg and the lowest was 0.69mL/kg). Seven out of twelve studies reported an increase in functional exercise capacity (highest mean change was 100 meters and the lowest of 40 meters). SF-36 was the most common questionnaire used to assess QoL. Five out of nine articles showed an improvement in the mental component summary in the exercise group after the intervention (highest mean change of 18.4 points and the lowest was 2 points). Five out of eight articles showed an improvement in the physical component summary (mean change ranged from 1.9 to 15 points). 

Conclusion: The majority of approached patients in the included studies accepted to participate in an exercise intervention pre-transplantOur findings show that there is some evidence that exercise training is safe and that can improveexercise capacity and quality of life in to SOT candidates. 

Agreement between measured and predicted oxygen cost of constant-rate stair stepping and shuttle walking exercise in people with chronic obstructive pulmonary disease


Emily Koch1, Hayley Lewthwaite1, Jean Bourbeau2, Francois Maltais3, Magnus Ekstrom4, Dennis Jensen1,2
1McGill University 2McGill University Health Centre 3Université Laval 4Lund University

Background: The 3-minute constant-rate stair stepping (3MST) and shuttle walking tests (3MWT) quantify the breathlessness response to a standardized exercise stimulus in people with chronic obstructive pulmonary disease (COPD). Unlike traditional constant-load bicycle and treadmill exercise tests, the 3MST and 3MWT are short, simple, inexpensive and require little space or expertise to perform. To be able to estimate the oxygen cost (V’O2) of these tests would allow health care providers to equate stepping and walking rates to other activities of daily life that commonly induce breathlessness in their patients with COPD. We sought to assess the level of agreement between the V’O2 of these tests predicted by the American College of Sports Medicine (ACSM) metabolic equations and the V’O2 directly measured during the 3MST and 3MWT in people with COPD.  

Methods: This study was a retrospective analysis of V’O2 collected breath-by-breath using a metabolic cart from people with COPD who completed all 3-minutes of a 3MST (n=294; % male=81; FEV1%pred 52 ± 17; 66 ± 6 years) ranging from 14-32 steps/min and/or all 3-minutes of a 3MWT (n=161; % male=74; FEV1%pred 49 ± 19; 65 ± 6 years) ranging from 1.5-6 km/hr. We performed (i) Pearson correlations between predicted V’O2 and measured V’O2 averaged over the last 30-sec of each test and (ii) Bland-Altman analyses to evaluate the level of agreement between measured and predicted V’O2 during each test.

Results: There was a strong correlation between predicted and measured V’O2 during both the 3MST (R=0.80) and 3MWT (R=0.86). The mean of the difference between measured and predicted V’O2 was -0.10±0.44 L/min (95%CI -0.53, 0.34) for the 3MST and 0.26±0.38 L/min (95%CI -0.13, 0.64) for the 3MWT.

Conclusion: Despite strong correlations, on average, the ACSM prediction equations over- and under-estimated the actual V’O2 measured during the 3MST and 3MWT in COPD, respectively. ACSM prediction equations could be used to estimate the oxygen cost of different stepping and walking rates during the 3MST and 3MWT; however, development of COPD-specific equations is warranted and the focus of ongoing work in our laboratory.

An Individual Patient Data Meta-Analysis to Estimate the Diagnostic Accuracy of a Deep Learning-based Software for Analyzing Chest X-Rays of Persons With Symptoms of Pulmonary Tuberculosis: preliminary findings


Syed Kumail Abidi1, Miriam Harris1,2, Monde Muyoyeta3,4, Keertan Dheda5, Aliasgar Esmail5, Klaus Reither6,7,8, Marianne Breuninger6,8,9, Amy Qi1, Claudia Denkinger10,11, Alexei Korobitsyn12, Madhukar Pai1,2,11, Andrea Benedetti1,2,11, Faiz Ahmad Khan1,2,11
1Respiratory Epidemiology and Clinical Research Unit, Montreal Chest Institute & Research Institute of the McGill University Health Centre, Montreal, Canada. 2Department of Epidemiology, Biostatistics, and Occupational Health, McGill University, Montreal, Canada. 3ZAMBART, School of Medicine, University of Zambia. 4Centre for Infectious Disease Research in Zambia (CIDRZ), Lusaka, Zambia. 5Centre for Lung Infection and Immunity, Department of Medicine, University of Cape Town, Cape Town, South Africa. 6Swiss Tropical and Public Health Institute. 7University of Basel, Basel, Switzerland. 8Ifakara Health Institute, Bagamoyo, United Republic of Tanzania. 9Center for Infectious Diseases and Travel Medicine, University Hospital Freiburg, Freiburg, Germany. 10FIND, Geneva, Switzerland. 11McGill International TB Centre, Montreal, Canada. 12Global TB Programme, World Health Organization, Geneva Switzerland.

BACKGOUND: In some resource-constrained settings, a lack of personnel skilled at radiologic interpretation is an obstacle to using chest X-rays (CXRs) for the medical evaluation of persons presenting with symptoms of pulmonary tuberculosis (PTB). Recently, software that can replace human readers for analyzing CXRs to detect PTB have been developed using artificial-intelligence methods. Deep-learning is an artificial-intelligence method that is particularly promising for image analysis, but the diagnostic accuracy of deep-learning trained programs for analyzing CXRs to detect microbiologically-confirmed PTB has not been assessed.

 

METHODS: We performed an individual patient data (IPD) meta-analysis to assess the diagnostic accuracy of a deep-learning-trained software (qXR, QURE.AI, Mumbai, India) for analyzing digital CXRs of adults presenting with symptoms of PTB, as compared to a reference standard of sputum tested with TB culture or the GeneXpert MTB/RIF assay. Eligible studies were identified through a systematic review. Each CXR was analyzed with qXR, which output an abnormality score ranging from 0 to 1. ROC curves were generated for each study overall and by HIV status. Pooled sensitivity and specificity were calculated, overall and within strata of HIV, using bivariate random-effects two-step IPD meta-analysis, and pooled ROCs were generated. Accuracy was assessed using an abnormality score of 0.25 as the minimum (i.e. threshold) for classifying a CXR as compatible with PTB, and also using 0.6 as the threshold. We also identified the score at which a pooled sensitivity of 0.90 was obtained and estimated its pooled specificity.

 

RESULTS: CXRs and clinical data were obtained for 1581 participants from 3 published studies, all conducted in sub-Saharan Africa. Median age was 43 (IQR: 32-75); HIV prevalence was 44.3%, and prevalence of microbiologically-confirmed PTB was 21.0%. The deep-learning program had a pooled AUC of 0.836. The AUC was lower in people living with HIV (PLWH) as compared to individuals not infected with HIV, both within individual studies and when data were pooled together, where the pooled AUC was 0.910 for No HIV and 0.774 for PLWH. Using 0.25 as the threshold, pooled sensitivity and specificity (95%CI) were 0.84 (0.78-0.89) and 0.64 (0.57-0.71), respectively, and between-study heterogeneity was moderate for sensitivity and substantial for specificity. With 0.6 as the threshold, sensitivity and specificity were 0.62 (0.57-0.67) and 0.89 (0.86-0.91), respectively, and heterogeneity was not substantial. A score of 0.18 achieved a pooled sensitivity of 0.90 (0.79-0.96) with a pooled specificity of 0.54 (0.46-0.62), between-study heterogeneity was substantial for sensitivity and for specificity. The program was less sensitive for detection of PTB in PLWH at each of the evaluated thresholds, with a pooled sensitivity of 0.75 (0.61-0.85) at the 0.25 threshold, 0.46 (0.37-0.55) at the 0.60 threshold, and 0.84 (0.66-0.93) at the 0.18 threshold.       

 

CONCLUSION: In high HIV prevalence populations with a high pre-test probability of PTB, a deep-learning based CXR-analysis software could detect microbiologically-confirmed PTB with high sensitivity and moderate specificity, but with important between-study heterogeneity. Research is needed to identify sources of heterogeneity, to assess accuracy in low-HIV prevalence populations, and when CXRs are used for PTB screening.

Aryl hydrocarbon receptor orchestrates macrophage polarisation


Hussein Traboulsi1,2, Carolyn Baglole1,2
1McGill University Health Centre Research Institute 2Meakins Christies

Introduction: Chronic obstructive pulmonary disease (COPD) is a progressive respiratory disorder characterized by insufficient airflow in the lungs and episodes of worsening respiratory function known as exacerbations. COPD affects over 200 million worldwide and is currently the third leading cause of death. In Canada, approximately 4% of the population had COPD in 2014. Although the primary cause of COPD is tobacco smoke, outdoor (e.g. traffic-related air pollution) and indoor air pollution (biomass smoke) as well as genetic factors are also associated with the development of COPD. Cigarette smoke (CS) induces inflammation in the lung (including recruitment of immune cells such as neutrophils). Uncontrolled, this inflammation leads to obstruction of the small airways and destruction of lung parenchyma. Currently, there are no effective therapies for COPD. Therefore, there is a major unmet need to find new approaches to treat this disease. One unexpected regulator of cigarette smoke-induced inflammation is the aryl hydrocarbon receptor (AhR), a ubiquitously-expressed transcription factor historically associated with the metabolism of xenobiotic and aromatic hydrocarbons (e.g. dioxin). Previous work from our laboratory has established that AhR attenuates the number of neutrophils in the lung of mice exposed to cigarette smoke. However, this effect of AhR was not related to chemokines expression that are required for neutrophil infiltration. How AhR attenuates pulmonary neutrophilia remains to be fully understood, but we suggested that it is due to effective resolution of inflammation. This process ensures that the recruitment of neutrophils is terminated to prevent organ damage. During the resolution of inflammation, recruited neutrophils undergo apoptosis and are engulfed by macrophages in a process called efferocytosis. In support of this, previous work has shown that AhR affects macrophage phenotypes, specifically by regulating macrophage polarization to M1 or M2. M2 macrophages are characterised by their anti-inflammatory properties and their efficacy to phagocyte apoptotic neutrophils.  Hypothesis: Given that there is defective efferocytosis in COPD, we hypothesize that AhR promotes M2 polarization during cigarette smoke exposure, leading to an enhanced efferocytosis of neutrophils, thereby accounting for the ability of AhR to suppress CS-induced neutrophilia.

 

Research plan: First, macrophages isolated from AhRWT and AhRko mice were exposed to cigarette smoke extract (CSE) followed by determination of macrophages polarization by measuring the expression of M1 markers (e.g. TNF-α and iNOs) and M2 markers (e.g. FIZZ1, YM-1, inteurlekine-10 and arginase 1). Second, to evaluate the phagocytic activity, AhRWT and AhRko macrophages were incubated with labeled-neutrophils. Phagocytosis of neutrophils was then evaluated using flow cytometry. Third, to investigate the mechanism by wich AhR regulates macrophages phenotypes, we looked by western blot at the phosphorylation of STAT3 (key regulator of macrophages polarisation) after stimulation by IL-10 or CSE. Finally, we looked at the interaction between AhR and STAT3 by co-immunoprecipitation assay followed by western blot.

 

Results: We observed that macrophages that does not express AhR polarize with less efficiency to M2 in response to CS. The phagocytosis of apoptotic neutrophils by these macrophages was also less effective. Moreover, we did find that IL-10 induces AhR expression and interestingly the absence of AhR decreases STAT3 phosphorylation upon stimulation with IL-10 or CSE. We also found that in STAT3 interact physically with AhR.

 

Significance: These expected results can help us to understand the mechanism by which AhR regulates inflammation and neutrophilia during CS exposure, a feature believed to contribute substantially to the development of COPD. Overall, this proposed project will offer an understanding of a novel mechanism that may prevent the progression and facilitate the development of new treatments (e.g AhR ligands) for COPD, and other chronic inflammatory respiratory diseases.

Associations between Fat Free Mass and Exercise Physiological and Perceptual Outcomes in People with Chronic Obstructive Pulmonary Disease: A Research Project Proposal


Kaveh Gaynor-Sodeifi 1, Hayley Lewthwaite1, Lauren Tracey1, Jade Fraser1, Emily Koch1, Dennis Jensen1
1McGill University

BackgroundBody Composition, specifically fat-free mass (FFM), has been a parameter of growing interest to researchers within the chronic obstructive pulmonary disease (COPD) community, with loss of FFM commonly observed in people with COPD. The loss of FFM has been established as an independent predictor of adverse health outcomes in COPD, including premature death. However, there is limited research on the association between COPD-related alterations in FFM and physiological and/or perceptual responses that occur during a cardiopulmonary exercise test (CPET) in this patient population. Therefore, while there’s an established link between FFM and adverse health outcomes in COPD, little is known about the underlying physiological and/or perceptual mechanisms that lead to poor health outcomes and how this is related to FFM.  

 

Objective: This study aims to answer four research questions in people with COPD: 

  1. What evidence exists on associations between FFM and patient-reported and clinical outcomes?
  2. What is the association between FFM measured by dual-energy X-ray absorptiometry (DEXA) on physiological and perceptual responses to cardiopulmonary exercise testing (CPET)?
  3. What is the effect of rehabilitative exercise training (RET) on FFM?
  4. Are RET-induced changes in FFM associated with changes in CPET responses, in both physiological and perceptual domains? 

 

Methods: To address the first research question, a systematic literature review will be conducted. Studies will be included in the review if they measured FFM and report associations with patient-reported and/or clinical outcomes in people with COPD. Findings will be summarised descriptively and quantitatively, where data permits. The second question will be addressed with a retrospective analysis of cross-sectional data from 60 COPD patients with paired DEXA and incremental CPET data. The third and fourth questions will be addressed by retrospectively analyzing data from a longitudinal intervention-based study in 45 COPD patients. Specifically, this analysis will examine the effect of an 8-12 week RET program on DEXA-derived FFM and scrutinize the association of these changes in FFM with corresponding changes in physiological and perceptual responses to constant-load CPET. 

 

Anticipated results: We anticipate that the systematic review will support associations between FFM and patient-reported and clinical outcomes, with few studies identified that specifically explore associations between FFM and CPET responses. We hypothesize that the retrospective analyses will identify positive associations between DEXA-derived FFM and CPET responses, which are amendable to a RET intervention.

 

Asthma and fixed airflow obstruction: Long-term trajectories suggest distinct endotypes


Benjamin Smith1,3, Nan Zhao1, Ron Olivenstein1,3, Catherine Lemiere2, Qutayba Hamid1,3, James Martin1,3
1McGill University Health Centre Research Institute 2Hôpital du Sacré-Coeur de Montréal 3Meakins-Christie Laboratories of McGill University

Background: Long-term trajectories of asthma with fixed airflow obstruction (FAO) may reveal underlying disease endotypes. We investigated whether measures of asthma control and airways inflammation and remodeling differed by long-term FAO status in moderate-to-severe asthma.

 

Methods: Adults enrolled in the Difficult Asthma Study assessed initially using serial asthma control questionnaire (ACQ), exacerbation history, spirometry, and sputum cytology over 12-months, as well as endoscopic bronchial biopsy with airway smooth muscle (ASM) quantification, were revaluated three or more years later with questionnaires and spirometry. FAO was defined as a persistent post-bronchodilator forced expired volume in one-second (FEV1)-to-forced vital capacity ratio below 0.70.

 

Results: Sixty-two participants (48±11 years old; 50% female; 75% atopic; asthma duration 24±14 years) returned for follow-up assessment (median interval 7.9 years; IQR: 5.4-8.8 years). Compared to participants without FAO (n=28), those with FAO at baseline and long-term follow-up (n=18) had higher baseline sputum neutrophil content and ASM, and a higher exacerbation frequency that persisted long-term. Sputum eosinophils, ACQ and long-term FEV1 decline did not differ. Participants with incident FAO at long-term follow-up (n=16) had higher baseline exacerbation frequency, sputum eosinophil content, lower ACQ scores and accelerated FEV1 decline, whereas baseline ASM was similar to those without FAO.

 

Conclusion: In moderate-to-severe asthma, long-term FAO is characterized by neutrophilic sputum inflammation and airway remodeling, but FEV1 decline is similar to those without FAO. Long-term incident FAO is preceded by higher exacerbation frequency, higher sputum eosinophil content, and an accelerated FEV1 decline.

Bone Marrow Macrophage Precursors of Duchenne Muscular Dystrophy (mdx) Mice Are Differentially “Trained” Based on Muscle Disease Stage


Salyan Bhattarai1, Qian Li1, Feng Liang1, Ekaterina Gusev1, Eva Kaufman1, Maziar Divangahi1, Basil J Petrof1
1Meakins-Christie Laboratories, Research Institute of the McGill University Health Center, Montreal, Quebec, Canada

RATIONALE: Duchenne muscular dystrophy (DMD) is a genetic disease characterized by destruction of skeletal muscles, a process which involves the diaphragm and leads to respiratory failure. In dystrophic (mdx) mice, the disease manifests sequentially as pre-necrotic, necrotic (peak inflammation), and fibrotic phases. Macrophages exhibit pro- and anti-inflammatory phenotypes which are thought to drive the necrotic and fibrotic phases, respectively.

“Trained” innate immunity is the property that allows innate immune cells to show enhanced responsiveness upon re-exposure to the same or diverse pathogens. Epigenetic changes mainly drive trained immunity, which is also associated with enhanced glycolytic metabolism. Here, we hypothesized that factors released from chronically damaged DMD muscles might induce changes consistent with trained immunity in macrophage precursor cells of the bone marrow even prior to their recruitment into dystrophic muscle.

METHODS: Gene expression (at mRNA level) of prototypical macrophage phenotype markers were determined in bone marrow-derived macrophages (BMDM) from wild-type (WT) and mdx mice under basal and stimulated conditions at different stages of disease using qPCR. Potential epigenetic alterations were evaluated by immunoblotting and ChIP assays. Macrophage metabolism was studied by determining lactate production and oxygen consumption rate ("Seahorse" assay). Functional differences between WT and mdx BMDM in vivo were assessed by intravenous adoptive transfer of donor WT versus mdx bone marrow cells (necrotic phase) into host WT mice with experimentally-induced muscle injury; the recruitment and inflammatory potential of these donor cells were then determined by flow cytometry. Finally, to assess whether endogenous ligands for a receptor of innate immunity (Toll-like receptor [TLR]-4) could be involved in dystrophic muscle signaling to the bone marrow, we also analyzed the phenotype of BMDM from mdx mice lacking TLR4 (mdx-TLR4-/-).

RESULTS: No differences in basal expression of either pro- (iNOS, IL6, IL12a) or anti-inflammatory (CD206, YM1 and Arg1) genes were found between WT and mdx BMDM during the pre-necrotic phase. In contrast, during the necrotic phase both pro- and anti-inflammatory gene transcripts were higher in mdx BMDM, which subsided during the fibrotic phase but still remained elevated compared to WT. This activation of gene expression in mdx BMDM was associated with reduced global and local (at the promoter regions of iNOS, IL12a, CD206 and Arg1) levels of the trimethylation of histone 3 lysine 27 (H3K27me3). In contrast, no global alteration of H3K27me3 was observed during the fibrotic phase in mdx BMDM. Metabolic studies revealed that mdx BMDM switched from enhanced glycolysis at the necrotic phase toward greater oxidative metabolism at the later fibrotic phase. Moreover, mdx BMDM at the necrotic phase showed greater responsiveness to classical polarization stimuli (M1=IFNγ + LPS, M2=IL4) as well as the endogenous TLR4 ligand fibrinogen, which is consistent with the characteristics of “trained” innate immunity. In vivo, adoptive transfer of necrotic phase mdx bone marrow led to an increase of pro-inflammatory macrophages in acutely injured wild-type muscle. BMDM from mdx-TLR4-/- mice showed a general downregulation of pro- and anti-inflammatory genes compared to mdx mice during the necrotic phase along with restoration of metabolism towards a more oxidative state. Finally, adoptive transfer of mdx TLR4-/- bone marrow also led to a decrease of pro-inflammatory macrophages in acutely injured wild-type muscle

CONCLUSIONS: Our findings suggest that in mdx mice,  future macrophages are altered at the bone marrow precursor level by signals released from the dystrophic muscles , and that these changes are mediated in part through the TLR4 pathway. Thus, pharmacological modulation of this trained macrophage response, either by targeting the epigenetic machinery, cellular metabolism, or signals emanating from the damaged muscles, could be useful approaches to mitigate muscle pathology in DMD.

Supported by: CIHR, JT Costello Memorial Fund

Can BCG-mediated protection be vertically transmitted?


Eva Kaufmann1,2,5,6, Jeffrey Downey1,4,5,6, Marija Landekic3,6, Donald Vinh2,3,6, Maziar Divangahi1,2,3,4,5,6
1Meakins-Christie Laboratories, McGill University, Montreal, QC, H3A 0G4 2Department of Medicine, McGill University, Montreal, QC, H3A 0G4 3Department of Microbiology and Immunology, McGill University, Montreal, QC, H3A 0G4 4Department of Pathology, McGill University, Montreal, QC, H3A 0G4 5McGill International TB Centre, McGill University, Montreal, QC, H4A 3J1 6McGill University Health Centre, Montreal, QC, H4A 3J1, Canada

Humans are the only host for Mycobacterium tuberculosis (Mtb), which is the causative agent of tuberculosis (TB) and kills around 1.6 million people every year. BCG is the only licensed vaccine against TB and it is given to 80% of the newborn babies in high-endemic countries. While intradermal BCG vaccination protects against the disseminated form of TB in children, it does not provide protection in adults. Importantly, it has been well documented that BCG protection in children is not limited to TB, as BCG-vaccinated children are also protected against a range of pulmonary infectious diseases and sepsis. Additionally, recent epidemiological studies suggest that the protective signature of vaccinated individuals can be vertically transmitted to their progenies. Thus, we aim to test this possibility using a mouse model of BCG vaccination.

 

We have recently demonstrated that systemic administration of BCG leads to epigenetic reprogramming of hematopoietic stem cells to generate protective monocytes/macrophages against Mtb, which represents a novel vaccination approach against pulmonary TB. Based on these observations, we hypothesize that systemic BCG vaccination will epigenetically alter the germ cells in hosts that can then vertically transmit these protective signatures to the offspring.

 

To investigate vertically transmitted effects of BCG vaccination, we vaccinate 6-week old male and female mice with BCG intravenously (1x106CFU) or PBS (control). After 4 weeks, BCG- vaccinated or PBS-control mice were paired for breeding. The offspring of the BCG-vaccinated or PBS-control mice was then subjected to Mtb (aerosolized H37Rv; ~50 CFU) or Influenza A virus (IAV; intranasal 90 PFU) infections. Offspring of the parental BCG-vaccinated mice did not show any protection against subsequent Mtb as assessed by lung bacterial burden or IAV infection as evaluated by survival. These results collectively suggest that the protection of parental systemic BCG vaccination was not vertically transmitted to the offspring and further investigation is required to experimentally test this possibility.

Characterizing the role of PGE2 in an Irritant-induced Murine Model of Asthma


Emily Nakada1, Toby McGovern1, Utako Fujii1, Yumiko Ishii1, William S Powell1, James G Martin1
1Meakins-Christie Laboratories, The Research Institute of McGill University Health Centre and the Department of Medicine, McGill University, Montreal, QC, Canada

Background: A subtype of asthma called irritant-induced asthma can result from an exposure to low concentrations of chlorine (Cl2), a disease characterized by the influx of neutrophils and airway hyperresponsiveness (AHR). To circumvent the damaging effects of repeated Cl2-induced oxidative damage, the development of an adaptive response to Cl2 is necessary. Following multiple Cl2 exposures, mice have been shown to develop an adaptive response associated with the recruitment of macrophages expressing high levels of prostaglandin (PG)E2 and TGF-β. The role of PGE2, its mechanism of adaptation and whether PGE2 could be used to treat individuals following acute exposure to chlorine have not yet been explored. We hypothesize that PGE2 regulates the adaptive response to Cl2 and attenuates Cl2-induced airway injury.

Methods: BALB/c mice were nose-only exposed to Cl2 in air at 100ppm for 5 minutes on one occasion (1xCl2 group), on three occasions separated by 3.5 days (3xCl2 group), or exposed to air as a control (Air group). AHR was assessed 24 hours after the last Cl2 or air exposure and inflammation was quantified from bronchoalveolar lavages performed both 6 and 24 hours after exposure. The role of PGE2 was assessed in 1xCl2 mice injected intraperitoneally with indomethacin, an inhibitor of prostaglandin synthesis, at 4mg/kg, 1 hour before and 5 hours after a single Cl2 exposure. A separate group of mice was treated with 0.5µg of exogenous PGE2 30 minutes before and 5 hours after a single Cl2 exposure. Mice were assessed 24 hours later.

Results: Increases in airway neutrophil and total cell numbers induced following a single chlorine exposure (1xCl2) were significantly reduced in 3xCl2 mice at both 6 and 24 hours after the last Cl2 exposure. This adaptive response was accompanied by a decrease in AHR and less weight loss in 3xCl2 compared to 1xCl2-exposed mice. Following indomethacin-treatment, 1xCl2 mice had greater total inflammation, neutrophilia and epithelial cells in the bronchoalveolar lavage fluid at 6 hours and significantly more macrophages and greater weight loss at 24 hours. Interestingly, exogenous PGE2 further increased Cl2-induced AHR in mice.

Conclusions: Mice develop an adaptive response to Cl2 but contrary to our hypothesis PGE2 accentuated airway injury and therefore may be a potential target to prevent Cl2-induced airway injury.

Combined pulmonary and rheumatology clinics improve the care of patients with interstitial lung diseases


Ketaki Rawal1, Ilan Azuelos1, Christian Pineau1, Deborah Assayag1
1Department of Medicine, McGill University, Montreal, Canada

Rationale:  Interstitial lung disease (ILD) is due to an autoimmune connective tissue disease (CTD) in about 30% of cases. It is a significant cause of morbidity and mortality. An accurate diagnosis of CTD-ILD is important to determine management of associated lung disease and extra-pulmonary manifestations. Small studies have shown that rheumatologic evaluation often leads to a change in initial diagnosis, however its longitudinal impact on diagnosis and management remains unknown. This study assessed the longitudinal impact of a combined rheumatology and pulmonary ILD clinic on diagnosis and management.

 

Methods: This retrospective chart review study included all patients seen in a multidisciplinary clinic at a single tertiary care institution from February 2016 until March 2017 inclusively. During clinic visits, a pulmonologist and a rheumatologist reviewed the clinical, laboratory and radiological data in order to reach a diagnostic impression and determine management by consensus. Data collected for this study includes: demographics, pulmonary function, referring diagnosis, multidisciplinary diagnosis, suggested investigations and therapeutic management.

 

Results: There were 194 patient-visits: 114 (59%) were initial consultations, and 80 (41%) were follow-up visits. Mean patient age was 62.9 years (SD 13.5), and 71 (62%) were female. Mean forced vital capacity was 87% predicted (SD 25%) and mean diffusion capacity was 56% predicted (SD 20.4%). Most referrals were from respirologists (82, 72%), and rheumatologists (12, 11%). There were 8 (7%) patients with a prior lung biopsy. Diagnosis was changed in 57 (48%) initial visits and 20 (25%) cases at follow-up. Some of the them were deemed not to have an underlying CTD, while others were found to have a new CTD instead of an idiopathic ILD. There were 4 new diagnosis of hypersensitivity pneumonitis.12 were found not to have ILD.

Conclusions: A longitudinal multidisciplinary clinic has a substantial diagnostic and therapeutic impact on the management of patients with ILD at both initial consultation and during follow-up visits. Further longer-term studies are needed to determine if these changes in management affect prognosis and survival. 

 

 

Constant-Rate Shuttle Walking and Stair Stepping Tests to Assess Exertional Breathlessness in COPD: Responsiveness to Pulmonary Rehabilitation and Estimation of the Minimal Important Difference


Lauren Tracey1, Steven Murray1, Emily Koch1, Kaveh Gaynor-Sodeifi 1, Kim-Ly Bui2, Nathalie Saad3, Jean Bourbeau4, Benjamin M. Smith4, Francois Maltais2, Didier Saey2, Dennis Jensen1,4
1Clinical Exercise and Respiratory Physiology Laboratory, Department of Kinesiology and Physical Education, McGill University, Montreal, QC 2Centre de recherche, Institut universitaire de cardiologie et de pneumologie de Québec, Université Laval, Québec 3Jewish General Hospital and Mount Sinai Hospital, Montreal, QC 4Respiratory Epidemiology and Clinical Research Unit, Montreal Chest Institute, McGill University Health Centre, Montreal, QC

Breathlessness on exertion is the most common and distressing symptom experienced by patients with chronic obstructive pulmonary disease (COPD). Relief of exertional breathlessness is a primary goal of COPD management and there is a need for standardized tests to evaluate exertional breathlessness and its response to therapy in clinical care and research settings (Ekström et al., Eur. Respir. J. 51: 1800820, 2018; Mahler & Sethi, Chest. 154:235, 2018). Originally developed and validated by Perrault et al. (COPD. 6:330, 2009), the 3-min constant-rate shuttle walking (3MWT) and stair stepping tests (3MST) are responsive to changes in breathlessness following bronchodilation (Sava et al., COPD. 9:395, 2012; Borel et al. Int. J. COPD. 11:2991, 2016; Maltais et al., Eur. Respir. J. 53: 1802049, 2019); however, the responsiveness of these tests to pulmonary rehabilitation (PR) has not been studied nor has the minimal important difference (MID) for either test been estimated. Thus, we assessed the responsiveness of the 3MST and 3MWT to detect relief of exertional breathlessness in 52 adults with COPD ((23F; mean±SD FEV1%pred, 48±28% and age, 68±8 yrs)) following an 8-12 wk outpatient PR program. We also estimated the MID in Borg 0-10 category ratio scale (CR10) intensity ratings of breathlessness at the end of the 3MST and 3MWT. Stepping rates and walking speeds varied from 14-24 steps/min (18.6±2.4 steps/min) and 1.5-6 km/hr (3.9±1.0 km/hr), respectively. On average, the PR-induced change in breathlessness was -0.3±1.6 (p=0.323) and -0.6±1.3 Borg CR10 units (p=0.045) for the 3MST and 3MWT, respectively. When the analyses were restricted to people whose breathlessness ratings at baseline were >3 Borg CR10 units, the PR-induced change in breathlessness was -0.9±1.6 (p=0.024) and -1.6±1.4 Borg CR10 units (p<0.001) for the 3MST (n=21) and 3MWT (n=16), respectively.  Using a distribution-based approach (0.5*SD of the PR-induced change in breathlessness intensity ratings), a change in breathlessness of 0.8 and 0.7 Borg CR10 units was calculated as the MID for the 3MST and 3MWT, respectively.  The results of this study suggest that: (1) the 3MWT is responsive to detect relief of exertional breathlessness after PR in COPD, as is the 3MST, provided breathlessness ratings are >3 Borg CR10 units at baseline; and (2) a change in breathlessness of 0.7-0.8 Borg CR10 units during the 3MST and 3MWT is likely clinically meaningful in COPD.

Control of Nonlinear, Viscoelastic Tissue


Amanda Bianco1,4, Gijs Ijpma4, Raphael Zonis2,4, Anne-Marie Lauzon1,3,4, James Richard Forbes2
1Department of Biomedical Engineering, McGill University, Montréal, QC 2Department of Mechanical Engineering, McGill University, Montréal, QC 3Department of Medicine, McGill University, Montréal, QC 4Meakins-Christie Laboratories, McGill University, Montréal, QC

The Lauzon lab is developing a system that can test mechanical and contractile functionality of human or animal muscle tissues while exposed to environmental factors, biological mediators and/or drugs at relatively high-throughput level over a prolonged period of time, effectively bridging the gap between long-term cell culture studies and short-term whole tissue studies. Essential to the autonomous operation of this device is the development of a force control scheme for the measurement of a key parameter of muscle function, namely the muscle rate of shortening. However, the nonlinear viscoelastic mechanics of biological tissues combined with the muscle’s ability to change its properties by contracting provides a challenge for force control development. The main objective of this Master’s Thesis project is to develop a robust force control scheme for this device, whereby an isometrically contracted muscle will briefly be held at a set force with a prescribed rise time and percent overshoot before being returned to its initial length. The change in length occurring during this force hold will be used to calculate the muscle rate of shortening. To realize this objective, a reliable tissue model is needed to inform controller design. As such, input-output data and grey-box system identification will be used to identify a control-oriented model of the muscle. This project will subsequently explore whether model-informed PI or PID control with or without a feedforward component can provide accurate and robust force control or whether an adaptive or iterative control scheme is required.  The control scheme will be tested initially on airway smooth muscle and robustness of the control will be confirmed with other muscle types. Successful implementation will give researchers unprecedented insight into time dependent changes in the contractile properties, in particular the rate of shortening, of muscle tissues incubated with environmental factors, biological mediators and/or drugs. Moreover, the development of this system will further our understanding of the nonlinear, viscoelastic properties of muscle tissues.

 

Cross-protection of BCG vaccination in influenza infection: Trained adaptive immunity?


Kim Tran1,3,4, Jeff Downey1,3,4, Orsolya Lapohos2,3,4, Maziar Divangahi1,2,3,4
1Department of Pathology, McGill University 2Department of Microbiology and Immunology, McGill University 3Meakins-Christie Laboratories, Research Institute of the McGill University Health Centre 4Department of Medicine, McGill University

Seasonal vaccination programs have significantly reduced mortality rates of Influenza A virus (IAV) infections, but fail to protect against emergent highly pathogenic avian influenzas (HPAI), which bear considerable pandemic potential. Thus, novel strategies that enhance host immunity to IAV are needed. The Bacillus Calmette-Guerin (BCG) vaccine is administered intradermally worldwide to prevent a disseminated form of tuberculosis in children. Interestingly, BCG vaccination has been shown to provide heterologous protection against a broad range of infections. Recent literature suggests that the non-specific protection of BCG is attributed to a memory-like capacity of innate immunity, termed trained immunity. We have recently shown that the efficacy of BCG is also influenced by the route of administration as intravenous (iv) administration has increased protection against Mycobacterium tuberculosis (Mtb) infection due to access of BCG to the bone marrow which leads to reprograming of hematopoietic stem cells. Importantly, BCG vaccination also induces robust T cell responses, a critical component in antiviral immunity. Thus, we hypothesize that access of BCG to the bone marrow will generate a unique subset of T cells that provides protection against subsequent heterologous IAV infections. Here we show that BCG-iv vaccination in mice provides protection against IAV infection. Interestingly, BCG-iv vaccination induces the enrichment of CX3CR1+ CD4+ and CD8+ αβ T cell populations that are not generated following subcutaneous (sc)-immunization. Further characterization of these T cells revealed expression of effector memory markers as well as localization in the bone marrow, blood, spleen, lungs and airways. Given the unique presence of this population in BCG-iv vaccinated mice as well as the protection results, we envision that CX3CR1+ T cells contribute to protective immunity against IAV infection. Considering the safe track record of BCG vaccination in humans, which has been given to three billion newborns, this study may provide a rational of BCG vaccination for protection against IAV and potentially other pulmonary viral infections.

Dissecting mechanisms by which intestinal macrophages maintain homeostasis during helminth infection


Yue Li1,2, Marilena Gentile1,2, Ghislaine Fontes1,2, Irah King1,2
1McGill University 2Meakins-Christie Laboratories

Intestinal helminths are among the most pervasive pathogens of the animal kingdom. The chronicity of infection and inability to resist repeated infection suggest that our host defense system must balance resistance with pathogen tolerance. Macrophages are a sentinel immune cell that play a central role in host defense against parasitic helminths. Importantly, it has become clear that macrophages are a highly heterogeneous cell population with distinct functions related to tissue physiology and immunity. However, the macrophage subsets that regulate resistance vs. tolerance to helminth infection remain unclear. By applying multicolor flow cytometry and 3D whole-mount imaging analysis during infection of mice with the parasitic nematode Heligmosomoides polygyrus bakeri (Hpb), we observed two distinct subsets of CD64+ macrophages in small intestine: Ly6c-MHCII+CX3CR1intTim4- macrophages, which are monocyte-dependent, reside in proximity to parasite granulomas and rapidly upregulate a tissue repair phenotype including induction of PD-L2. Elimination of this latter subset of macrophages during Hpb infection results in a reduced Th2 response, but does not compromise host fitness. By contrast, specific depletion of MHCII+CX3CR1hiTim4+ macrophages which are monocyte-independent, leads to patent intestinal bleeding. We are currently performing transcriptional profiling and live imaging approaches to interrogate the distinct function of these two subsets of intestinal macrophages in host defense to helminth infection.

EFFECTIVENESS OF A HOME-BASED PRE-HABILITATION PROGRAM FOR LUNG TRANSPLANT CANDIDATES: A RETROSPECTIVE CHART REVIEW


Daniela Massierer1,2, Nicholas Bourgeois3, Agnès Räkel3, Karina Prevost3, Larry Lands2,3, Charles Poirier3, Tania Janaudis-Ferreira1,2
1School of Physical and Occupational Therapy, McGill University, Montreal, Quebec, Canada 2Translational Research in Respiratory Diseases program, Research Institute of the McGill University Health Centre, Montreal, Canada 3Lung transplant program, Centre hospitalier de l'Université de Montréal (CHUM)

Rationale:In Canada, most lung transplant programs offer mandatory hospital-based pre-transplant exercise programs to optimize fitness and prevent deconditioning in lung transplant candidates. Home-based programs offer an alternative for exercise pre-transplant, giving access to rehabilitation to a larger number of patients and lower healthcare cost. 

Objectives: to describe the changes in functional exercise capacity (6-minute walk distance (6MWD)) in lung transplant candidates who participated in a home-based exercise program and determine the relationship between changes in functional exercise capacity pre-transplant and post-transplant variables. 

Methods: a Retrospective cohort study of 178 individuals who received a lung transplant between 2011-2015, participated in a home-based exercise program while waiting for transplantation and had available pre and post-transplant data. 6MWD was assessed at 3-time points: time of assessment for transplant, last test prior to transplant and one-month post-transplant. Other variables included: age, sex, primary diagnosis, date of transplantation, body mass index (BMI), total hospital length of stay (LOS), intensive care unit (ICU) LOS and time on mechanical ventilation. 

Results: The median LOS on the waiting list was 1.8 yrs. There was a mean decrease of -27±119m between the 6MWD at the time of assessment and the last 6MWD prior to transplantation (p<0.001; whole sample). Forty-one patients (26%) increased their 6MWD (mean change 85±113m); 72 patients (46%) decreased their 6MWD (mean -110±112m); and 46 patients (29%) had no change in 6MWD (-1.5±108m). Those with a decrease in 6MWD were more likely to be diagnosed with idiopathic pulmonary fibrosis. There was a weak negative correlation between change in 6MWD prior to transplant and time on mechanical ventilation (r= -0.185; p<0.05). When adjusted for age, gender and BMI. Change in 6MWD prior to transplant was not associated with the time on mechanical ventilation, total hospital LOS or ICU LOS.   

Conclusions: Most patients (55%) were able to either increase or maintain their 6MWD by participating in a home exercise program while on the waiting list. These results are similar to what has been found for hospital-based programs. Home-based semi-supervised exercise program may be an effective rehabilitation approach prior to transplantation. 

Effects of Amino Acid Restriction on Macrophage Function


Mikaela Eng1,2, Maziar Divangahi1,2, Arnold Kristof1,2
1McGill University 2Meakins-Christie Laboratories

Acute lung injury (ALI) is a deadly clinical condition characterized by the breakdown of the lung alveolar-capillary membrane and respiratory failure that can occur after a direct (e.g., pneumonia) or indirect (e.g., sepsis) inflammatory insult. Macrophages play an important role in ALI, and their activity is regulated by changes in metabolism and gene transcription required for stress responses and cytokine production. These are tightly regulated by the protein kinase ‘mammalian target of rapamycin’ (mTOR), which nucleates two highly conserved macromolecular complexes – mTOR complex-1 (mTORC1) and mTORC2. Clinical studies demonstrate an association between protein availability and clinical outcomes in the critically ill, and mTORC1 is a cellular sensor of essential amino acid (EAA) availability. We therefore proposed that the availability of EAAs, particularly leucine and/or arginine, is critical for metabolic, stress, and inflammatory responses in macrophages that are related to ALI. Bone marrow-derived macrophages (BMDMs) were exposed to control media or that lacking leucine, arginine, and lysine (EAA restriction), in the presence or absence of saline or E. coli lipopolysaccharide and interferon-gamma (LPS/IFN-γ) for 6 h. The induction of interleukin-6 (IL-6) mRNA and protein by LPS/IFN-γ was inhibited by EAA restriction; IL-6 induction was restored by the addition of leucine alone. Moreover, IL-6 induction was inhibited by exposure to media lacking leucine alone. EAA restriction enhanced the induction of ER stress response genes (i.e., CHOP, GADD34) by LPS/IFN-γ. Our results point to mTOR and its leucine sensing mechanism as an important regulator of macrophage stress and inflammatory responses and form the basis for future studies identifying novel clinical biomarkers or therapeutic targets in ALI.

Efficacy and objective adherence to mandibular advancement splints for treatment of sleep-disordered breathing during pregnancy and the postpartum period.


Nelly Huynh1,2, Léa Drouin-Gagné2, Camille Gilbert2, Jean-Patrick Arcache2, Anne-Maude Morency3, Robert Gagnon3, John Kimoff3, Sushmita Pamidi3
1CHU Sainte-Justine 2Faculté de médecine dentaire, Université de Montréal 3McGill University Health Centre

Rationale: The prevalence and severity of sleep-disordered breathing (SDB) increases over the course of pregnancy. Even mild SDB has been associated with increased risks of gestational hypertension, gestational diabetes and delivery of small-for-gestational age infants. Although CPAP is the first-line treatment, pregnancy-specific changes (i.e., difficulties laying supine and increased nasal congestion) may be barriers to its use. The use of mandibular advancement splints (MAS) has not yet been evaluated in pregnancy and may be an effective treatment option.

Methods: This is a pilot uncontrolled experimental cohort study, which included 17 pregnant women with SDB (apnea-hypopnea index (AHI) ≥ 10 events/h). Patients were treated with nightly use of a MAS embedded with an adherence sensor, during the 2nd to 3rd trimester of pregnancy until 6 months postpartum. Ambulatory level 2 polysomnography scored with AASM Chicago respiratory criteria was performed at three time points: baseline prior to MAS treatment, with titrated MAS ante-partum, and 6 months post-partum (without MAS), to evaluate post-partum persistence of SDB. Health and sleep questionnaires were also completed at each time point.

Results: At baseline, the maternal age was 35.6±5.6 y (mean±SD), the BMI was 27.2±6.7 kg/m2 and gestational age was 28.5±7.0 wks. The use of treatment (≥ 4h/night over treatment period) was 68% [1.5-100%] (median [min-max]) during pregnancy, with treatment period = 73.5d [3-148] and days used = 29d [1-144].  Snoring time was significantly reduced from 15.4% [0.0-77.8] at baseline to 3.4% [0.0-24.9] with MAS (p≤0.001, figure). The baseline AHI was 20.3/h [9.5-23.0] and micro-arousal index was 31.9/h [20.7-52.7]. With MAS treatment, the AHI was reduced to 12.5/h [4.1-30.0] (p=0.02) and micro-arousal index to 27.7/h [14.7-38.7] (p=0.07, figure). Overall, 64% of patients were responders to MAS, defined as AHI reduction ≥ 30% and titrated AHI ≤ 15/h. High treatment satisfaction (88% patients) and few side effects were reported (23.5% at least once a week), consisting mostly of finding the MAS uncomfortable/cumbersome. At the postpartum assessment, 88% of patients had an AHI 10 (without MAS). The objective postpartum adherence data showed that 13% were regular users (≥ 4 h/night and ≥ 5d/week), while 63% were irregular users and 25% were non users.

Conclusion: MAS may be an effective treatment option in some women with SDB during pregnancy. Further research is warranted to better identify patients most likely to respond to MAS treatment, develop strategies to optimize adherence, and determine the impact on maternal and fetal pregnancy outcomes.

Research Funding Source: Funding sources were start-up funds of N.Huynh, research support funds from the Research committee of the Faculty of Dental Medicine of the Université de Montréal, and start-up funds from S.Pamidi, from Department of Medicine of McGill University.

Energy Expenditure and Physical Activity in Pregnant Women with Gestational Diabetes: Is there a relationship with Sleep-Disordered Breathing?


Julia Leonard1, Raphieal Newbold1, John Kimoff1,7, Sara Meltzer2, Andrea Benedetti1,3, Natasha Garfield2, Lorraine Lavigne1, Allen Olha1, Evenlyne Ray4, Kaberi Dasgupta1,5, Robert Gagnon6, Sushmita Pamidi1,7
1Centre for Outcomes, Research and Evaluation (CORE), Research Institute (RI) – McGill University Health Centre 2Division of Endocrinology, Department of Medicine, McGill University 3Departments of Epidemiology, Biostatistics and Occupational Health, McGill University 4Centre de recherche, Université de Montréal 5Divisions of Internal Medicine, Clinical Epidemiology and Endocrinology and Metabolism, Department of Medicine, McGill University 6Department of Obstetrics and Gynecology, McGill University 7Division of Respiratory Medicine, Department of Medicine, McGill University

Rational

Regular physical activity during pregnancy is beneficial for mother and child and is associated with improved pregnancy outcomes. Most studies have focused on the effect of physical activity interventions on the risk of pregnancy complications, such as excessive weight gain or gestational diabetes (GDM). Pregnancy complications, in particular GDM, are also associated with sleep-disordered-breathing (SBD), which affects 17-45% of pregnant women by the third trimester. SDB is associated with increased respiratory effort from breathing against an obstructed upper airway, and is often associated with daytime sleepiness. However, it is unknown whether untreated SDB in pregnancy is related to reduced physical activity and energy expenditure. In this study, we therefore sought to characterize patterns of physical activity and 24-hour energy expenditure in pregnant women with GDM and examine the relationship with SDB. 

 

Methodology

Women with GDM (WHO 2013 diagnostic criteria) underwent ambulatory level 2 sleep recordings and 24-hour continuous actigraphy. Participants wore the the Sensewear® Armband (BodyMedia), which uses an actigraphy analysis to record movements to estimate energy expenditure, physical activity intensity and sleep-wake activity, for one week. Descriptive statistics were performed to characterize daily total energy expenditure, duration of physical activity above 3.0 METS, daily step count and average daily sleep duration. We used linear regression models to estimate the association between apnea-hypopnea index (AHI) as a measure of SDB severity and daily total energy expenditure and physical activity patterns. 

 

Preliminary results

A total of 60 pregnant women with GDM underwent sleep recordings and continuous actigraphy with the following demographics (mean±SD): 35.6±4.2 years of age, BMI of 32.7±7.14 kg/m2, 29±3.5 weeks gestational age at enrolment. The mean AHI was 16.4±10.5. Daily physical activity of women with GDM and SBD was characterized (mean±SD): 4023±1675 steps/day, total energy expenditure of 9065.6±1473.4 kJ/day, active energy expenditure (defined as intensity >3.0 METS) of 340.5±357.0 kJ/day, and physical activity duration of 19±20.7 minutes/day. No relationship between physical activity or active energy expenditure and severity of SDB was observed. A preliminary analysis with a multiple linear regression, adjusting for BMI, demonstrated a positive relationship between AHI and daily energy expenditure (beta coefficient 42, p=0.01). 

 

Conclusion

This preliminary study quantifies the physical activity patterns of women with GDM. Overall, women with GDM had low physical activity levels and are not achieving physical activity recommendations for pregnant women described in current guidelines. While the presence of SDB did not alter physical activity levels, SDB was related to greater 24-h energy expenditure, likely via increased sleep-related energy expenditure. An ongoing interventional study (NCT02245659) by our group will examine whether treatment of SDB in this population has any impact on physical activity and energy expenditure. Such findings may have important clinical implications regarding weight management and prevention of adverse outcomes in the GDM population.

Fgf10-deficiency alters airway development and response to chronic cigarette smoke injury increasing susceptibility to COPD-like phenotype.


Motahareh Vameghestahbanati1,2, Hussein Traboulsi1, David Eidelman1,2, Qutayba Hamid1,2, Carolyn Baglole1,2, Benjamin Smith1,2
1Meakins-Christie Laboratories, Research Institute of McGill Health Center 2College of Medicine, McGill University

Introduction: Chronic obstructive pulmonary disease (COPD) is a major public health problem globally, and in Canada. Smoking is a major risk factor, but recent studies suggest that altered lung development may also contribute to COPD susceptibility. 

The airway tree, which forms early in life, is a major determinant of airflow and a filter of noxious particulates. We recently demonstrated that airway tree anatomy variation is common in the general population. Absence of a segmental airway (6.1% prevalence) is associated with narrower airway lumens in all lobes, higher odds of COPD, particularly among smokers, and genetic polymorphisms in fibroblast growth factor 10 (FGF10). Furthermore, computational fluid dynamic modelling of this FGF10-associated airway variant demonstrated higher particulate deposition compared with standard anatomy. 

FGF10 is a critical regulator of airway morphogenesis in utero, and airway epithelial maintenance post-natally.The production and maintenance of Basal stem cells (BSCs)during development depends on Fgf10 secreted by the stromal tissue located in between the cartilage rings. Postnatally, during homeostasis, basal stem/progenitor cells are only found in the trachea, where they maintain themselves by inducing Fgf10 expression in the tracheal stromal niche. Upon airway epithelial injury, Airway smooth muscles that envelop the non-cartilaginous airways, are activated to release Fgf10. Fgf10 then binds Fgfr2b on Basal stem cells(BSCs) to mobilize and expand this stem/progenitor pool by inhibiting their premature differentiation, thus facilitating efficient lung regeneration .The Fgf10-expressing basal stem cell niche extends into the non-cartilaginous airways allowing for BSC mobilization into the lower conducting airways.

 We recently demonstrated that Fgf10-haploinsufficient mice exhibit a phenotype that is highly consistent with human airway variant: fewer airway buds, narrower airway lumens, and higher airflow resistance. 

 

Objective: To investigate whether Fgf10-deficient mice  exhibit increased susceptibility to chronic cigarette smoke-induced airway narrowing and altered lung mechanics.

 

Methods: Heterozygous and  wild type 6-8-week-old mice were exposed to air or cigarette smoke (CS) for 8 weeks . After completion of exposure period, lung mechanics were measured by Flexivent system(n=10 in each group), followed by post-mortem in situmicro-computed tomography of the lung at fixed inflation pressure (25 cmH2O). Using OsiriX software to analyse CT image data(n=7-8 in each group), the airways were labeled along six prespecified paths in all lobes up to the 6th generation. Cross-sectional airway lumen diameters were measured within an image plane perpendicular to the local airway segment long axis. Total lung volume was measured using a standardized density-based seed-growing technique. The reproducibility of quantitative measures assessed by replicate scans was excellent. Data analysis was performed using SPSS 25 and SAS 9.3.

 

Results:  Fgf10+/- mice exhibited higher resistance and narrower airway lumens at every generation when compared with Fgf10+/+, whereas lung volumes were similar (i.e., dysanapsis). In both groups, CS exposure increased resistance (p=0.0409 in Fgf10+/- and p=0.0392 in Fgf10+/+), but there were no differences in central airway lumen diameters. Both the absolute and relative CS-induced increases in resistance were larger for Fgf10+/- (0.0618 [+15%]) when compared with Fgf10+/+ (0.0107 [+3%]), but this did not achieve statistical significance (p=0.0957). In addition,there was no difference in elastance between Fgf+/- and Fgf+/+ mice in the air or smoke conditions, nor in the smoke effect on elastance.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Conclusion: Fgf10 haploinsufficient mice exhibit dysanapsis and higher resistance when compared with wild-type mice, and may also be more susceptible to chronic CS induced resistance. These findings suggest that combined mechanisms of altered airway tree development, and airway response to chronic particulate injury may contribute to FGF10-associated COPD.

From the clinic to the laboratory: azithromycin for prevention of COPD exacerbations


Nafiseh Naderi1, Raquel Farias1, Mira Abou Rjeili1, Yousof Mostafavi1, Carolyn Baglole1, Jean Bourbeau1
1Research Institute of McGill University Health Center, McGill University, Montreal, Canada

Rationale: We recently demonstrated that long-term treatment with azithromycin reduces the number of exacerbations in severe COPD patients on optimal inhaled therapy. Beneficial effects were observed in both, ex-smokers and current smokersThese results led us to develop a translational project to study the mechanisms by which macrolides reduce COPD exacerbations. Beyond its antibiotic properties, azithromycin is known to act as an immune modulator in the host, decreasing the inflammatory mediators IL-6 and IL-8, two cytokines widely characterized in COPD exacerbations. However, the exact mechanism mediating the effects of azithromycin in COPD exacerbations is not completely understood. We hypothesized that treatment with azithromycin decreases exacerbation frequency by modulating inflammation in human airway epithelial cells exposed to cigarette smoke.

Objectives: 1)To characterize the expression of IL-6 and IL-8 in airway epithelial cells exposed to cigarette smoke;2)To evaluate the effectsof azithromycin on the expression and release of the inflammatory mediators IL-6 and IL-8 in airway epithelial cells exposed to cigarette smoke extract (CSE).

Methods: Beas 2B bronchial epithelial cells were incubated with 5% CSE for 3h, 6h and24h. Expression and release of IL-6 and IL-8 were analyzed by qRT-PCR and ELISA, respectively. Then, airway epithelial cells were pretreated with azithromycin and exposed to 5% CSE. Expression and release of IL-8 and IL-6 were measured by qRT-PCR and ELISA. 

Results: We observed a significant increase of IL-6 and IL-8 mRNA following 3h, 6h and 24h exposure to 5% CSE. Similarly, IL8 secretion was significantly increased after exposure to 5% CSE for 24h. When cells were pre-treated with azithromycin and exposed to 5% CSE for 3H, we observed a significant dose-dependent decrease in the expression of IL-6 mRNA. Finally, when cells were pre-treated with 9 ug/ml of azithromycinand exposed to 5% CSE for 3h, we observed a significant decrease in the expression of IL-6 and IL-8 mRNA. Furthermore, extracellular IL-8 protein levels were significantly decreased in 5% CSE-exposed-BEAS-2B cells when the cells were pretreated with azithromycin.

 

Conclusion:Incubation with azithromycin decreases the expression and release of the inflammatory mediators IL-6 and IL-8 in BEAS-2B airway epithelial cells exposed to CSE. These results suggest that azithromycin acts as an immunomodulator in human airway epithelium

Gender partially explains the variation in the airflow obstruction in dysanapsis


Motahareh Vameghestahbanati 1,2, Qutayba Hamid1,2, Benjamin Smith1,2
1Meakins-Christie Laboratories, Research Institute of McGill Health Centre 2Department of Experimental Medicine, McGill University

Introduction: Chronic obstructive pulmonary disease (COPD) is a leading cause of death globally, and in Canada. Tobacco smoke is a major risk factor, but recent studies demonstrate that half of older adults with COPD exhibited low lung function early in life. The airway tree, which forms early in life, is a major determinant of airflow and a filter of noxious particulates. Dysanapsis is a developmental concept introduced by Green et al in 1974 that refers to mismatch between airway tree and lung size. Since the airway tree is a major determinant of airflow resistance, a mismatch of airway tree to lung size (dysanapsis) may contribute to airflow obstruction. We recently demonstrated that dysanapsis quantified by computed tomography (CT) is common in a multi-ethnic population-based sample and explains more of the variation in FEV1/FVC , predicts COPD risk better than tobacco smoke and other major COPD risk factors and dysanapsis increases susceptibility to smoke-induced development of COPD.The role of gender in dysanapsis-associated airflow obstruction is unknown. In many developed countries, COPD has now become more common in women than men, they seem to have more severe COPD phenotype with early-onset disease and a higher predisposition to COPD with lower tobacco exposure suggesting that there might be a gender differences in dysanapsis. 

 

Objective: To investigate the contribution of gender to dysanapsis-associated airflow obstruction (FEV1/FVC). 

 

Methods: We selected men and women from a population-based cohortwho were free of traditional COPD risk factors (i.e, current and former cigarette, pipe and cigar smoking, Second-hand smoke exposure and occupational exposure to vapor-gas, dust or fumes) Full-lung computed tomography acquired at maximum inspiration was used to measure cross-sectional airway lumen diameters at 19 standard anatomic locations (trachea to subsegments), and the total lung volume. Spirometry was performed according to current guidelines. To elucidate the contribution of gender to dysanapsis-associated airflow obstruction we first computed an airway tree reference equation that accounts for age, height, lung volume, and gender. This equation was used to calculate a percent-predicted airway tree size, a measure of dysnapasis, where 100% represents the predicted “normal” value. Next, we computed the percent-predicted airway tree size using a reference equation that excluded gender. Finally, we calculated the proportion of FEV1/FVCexplained by dysanapsis using the reference equation with and without genderWe also dissected the contribution of gender to dysanapsis-explained FEV1/FVC along the central-to-peripheral airway tree axis. Data analyses were performed using SPSS 25 and SAS 9.3.

 

Results: Among 204 participants unexposed to traditional COPD risk factors, the mean age was 67±11, 41.2% were female, and mean FEV1/FVC was 0.73±0.08. Dysanapsis accounted for 23% of the variation in FEV1/FVC. Of all the variation in FEV1/FVC explained by dysanapsis, gender accounted for 14%, while 86% remained unaccounted for. When the airway tree was partitioned along the central-to-peripheral axis, peripheral airway dysanapsis explained more of the variation in FEV1/FVC (segments: 12%; subsegments: 11%) compared with central airway dysanapsis (trachea: 2%; mainstems: 4%). The contribution of gender to dysanapsis-explained FEV1/FVC was highest for the central airways (trachea [65%], and mainstems [48%]), and lowest for the peripheral airways (segments [9%], subsegments [16%]). 

 

Conclusion:Gender accounts for a relatively small proportion of the total variation of dysanapsis explained FEV1/FVC. The contribution of gender decreased along the central-to-peripheral airway tree axis, suggesting that our conclusion would not differ if more peripheral airways were quantified. The large proportion of FEV1/FVC explained by dysanapsis that is not accounted for by gender warrants further investigation.  

Heart rate variability in extremely preterm infants receiving NCPAP, NIPPV and NIV-NAVA after extubation: a randomized crossover trial.


Samantha Latremouille1, Monica Bhuller1, Smita Rao1, Wissam Shalish1, Guilherme Sant’Anna1
1Montreal Children's Hospital (MUHC), Montreal, Canada

Background: Nasal continuous positive airway pressure (NCPAP) and nasal intermittent positive pressure ventilation (NIPPV) are common post-extubation support modes but non-invasive neurally adjusted ventilatory assist (NIV-NAVA) is gaining popularity given the potential benefit of patient-ventilator synchronization. Heart rate variability (HRV) is a marker of well-being in neonates and differences in HRV were described in preterm infants receiving post-extubation support.

Objective:To investigate HRV during NCPAP, NIPPV and NIV-NAVA after extubation, and for differences in HRV between infants with extubation success or failure.

Methods: Randomized crossover trial in infants with birth weight (BW) ≤ 1250g undergoing their first extubation. Electrocardiogram recordings were performed during 30min (10min washout period between modes) while on each mode. Time domain, non-linear, and frequency domain parameters were calculated and compared using Friedman’s (Tukey-Kramer’s post-hoc) and Wilcoxon Rank Sum tests. The detrended fluctuation analysis short-term exponent (alpha1) is a measurement of complexity of the NN intervals (normal range: 0.5-1.5): 1.0 is a more balanced, complex system while 1.5 reflects a system with lower complexity and highly self-similar NN intervals. Extubation failure was defined as reintubation within 7d.

Results: Twenty-seven of 30 infants studied had full data recordings and were included for analysis, with median [IQR] gestational age of 26.0 wks [25.4 - 26.4], BW of 770g [610 - 915], and studied at day 7 [4 - 19] of life. A significant difference was noted for the Triangular Index parameter (HRVTi; p=0.035); post-hoc analysis showed that HRVTi on NCPAP > NIV-NAVA (p=0.013) and > NIPPV (p=0.062). Alpha1 was also significantly different between the 3 modes (p=0.032), with NIV-NAVA (close to 1.0) < NIPPV (p=0.041) and < NCPAP (p=0.014). In relation to extubation outcomes, significant differences were noted between success and failure infants in the high frequency power (HF) and sample entropy (SampEn) parameters during NCPAP (HF p=0.050, SampEn p=0.047) and NIPPV (HF p=0.042, SampEn p=0.013) but not NIV-NAVA.

Conclusion: During the period after extubation an increase in complexity of the NN intervals (alpha1) was observed only during NIV-NAVA suggesting a more adaptable system during this mode. Indeed, differences found between success and failure infants while on NCPAP and NIPPV were not observed during NIV-NAVA.

Identification of Pseudomonas aeruginosa genetic variants in chronic lung infections of cystic fibrosis patients and their effect on lung disease severity


Jannik Donner1, Emmanuel Faure1, Lin Liu1, Geoffrey McKay1, Manon Ruffin2, Brian Boyle3, Roger Levesque3, Emmanuelle Brochiero2, Dao Nguyen1
1Meakins-Christie Laboratories, Research Institute of the McGill University Health Centre (RI-MUHC), Montréal, QC, Canada 2Centre de recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM), and Département de médecine, Université de Montréal, Montréal, QC, Canada 3Institut de biologie intégrative et des systèmes (IBIS), Pavillon Charles-Eugène-Marchand, Université Laval, Quebec City, QC, Canada

Nearly 60% of adult CF patients are chronically infected by Pseudomonas aeruginosa (Pae) in their lifetime. Lung disease upon chronic infection is mainly caused by direct virulence of the infecting bacterial pathogens, and the presence of an excessive and injurious inflammatory response. Over time, progressive lung damage impairs its function to the point where lung transplantation becomes necessary. Interestingly, the rate of progression in CF lung disease is highly heterogeneous between patients. Even within the same patient, individual lungs can show significant regional variation in tissue damage and disease severity, which cannot be attributed to host genetics or environmental factors alone. Recent work indicates that regional disease heterogeneity could be due to the microevolution of Pae in the CF lung, as region-specific clonal Pae populations can differ markedly in phenotype, virulence, antibiotic resistance and immunogenicity.

Until now, the genetic variation of Pae in chronic lung infections has mostly been studied by whole-genome sequencing of single Pae isolates isolated from sputum samples. However, such approaches are limited to the analysis of only few Pae clones per patient and most likely overlook the extensive genetic heterogeneity present. Moreover, single genetic variants identified in Pae CF sputum cannot be directly linked to lung disease severity at the histopathological level because it is unknown where in the lung the Pae clones originate from.

We hypothesize that highly virulent Pae variants are present in severely damaged tissues, but not in mildly diseased ones, and that these cause more severe lung pathology due to increased cytotoxicity or inflammation.

In order to study the genetic diversity of Pae in chronic CF lung infection and to assess the effect of Pae variant populations on lung damage severity, we have collected tissue and mucus samples from whole lung explants obtained from CF patients with end-stage lung disease undergoing lung transplantation. The regional severity of lung disease was assessed based on clinical radiographic chest imaging and gross anatomical appearance of the lung explants. We performed tissue sampling in regions containing areas of mild, moderate or severe disease. To identify genetic variants in regional Pae populations, we designed two Ion AmpliSeq probe panels for massively parallel PCR amplification of 209 Pae target genes known to evolve during chronic CF lung infection or to be involved in the pathogenesis of Pae. To date, we have validated the IonAmpliSeq panels by analyzing total genomic DNA extracted from Pae-infected CF sputum and of clinical Pae isolates. Moreover, spike-in samples containing different absolute quantities of Pae genomic DNA (equivalent to 0.2 or 2% Pae DNA to human DNA) and at different proportion of two Pae strains (PAO1, PACS2) were analyzed with the Ion AmpliSeq technology. Known gene variants of the PACS2 strain (absent in PAO1) were accurately detected the expected frequency in the spike-in samples.

We are currently analyzing lung tissues and mucus DNA samples collected from CF lung explants to identify Pae genetic variants associated with regions of severe disease. The most promising candidate variants will be validated in cell culture-based pathogenicity assays.

The Ion AmpliSeq technique provides a novel, quick and cost-efficient approach to analyze genetic variants potentially associated with Pae pathogenicity and survival in the CF lung. The identification of Pae gene products contributing to host tissue damage will be a first step on the way to develop novel treatment strategies for chronic Pae infections in CF and to increase the patients’ well-being.

Immunity to Influenza A virus infection during pregnancy


Julia Chronopoulos1,2,3, Erwan Pernet2,3, Mikaela Eng1,2,3, Yumiko Ishii2,3, Mishaal Hukamdad1, Maziar Divangahi1,2,3, James G. Martin1,2,3
1McGill University 2Meakins-Christie Laboratories 3Research Institute of the McGill University Health Centre

Background: Higher mortality and morbidity rates from influenza A virus (IAV) infection have been reported in women. Pregnant women were more prone to severe IAV infection in the recent H1N1 2009 pandemic. It has been postulated that the maternal immune system changes over the course of gestation. This downregulation of immune mechanisms that adversely affect the clearance of viruses, such as IAV, are beneficial to the reproductive fitness of the fetus. In addition, epidemiological data on human pregnancy outcomes show IAV induces preterm labour, intrauterine growth restriction, and congenital malformations despite no evidence of vertical transmission to the fetus.

Hypothesis: Severe maternal morbidity in influenza A virus infection is caused by immunopathology rather than inadequate host resistance.

Methods and results: To assess whether pregnant mice are more susceptible to IAV infection, non-pregnant and pregnant C57BL/6 mice (gestation day 10 or 11), were infected with a lethal dose of IAV (PR8) intranasally and body weight was monitored. Non-pregnant and pregnant mice were equally susceptible to IAV infection. The kinetics of the viral load in the lungs were subsequently examined using an MDCK assay, RT-PCR to assess viral RNA, and flow cytometry to examine infected epithelial cells positive for IAV nucleoprotein (NP) in whole lung homogenates. Pregnant mice exhibited fewer infected epithelial cells and less viral replication in those infected epithelial cells shown by the mean fluorescence intensity of NP at day 3 post infection. The antiviral cytokines, type I (IFNα and IFNβ) and III (IFNλ) interferons were measured using a reporter cell line and ELISA assay. In early infection, higher levels of IFNλ were detected, but lower levels of type I IFNs were detected later in the course of the infection in the lungs and broncheoalveolar lavage (BAL) fluid of pregnant infected mice. There were lower levels of TNF-α and IL-6 (ELISA) in early infection in the BAL fluid of pregnant infected mice. Pregnant infected mice also did not develop airway hyperresponsiveness to inhaled methacholine and had significantly lower levels of IL-13 in the lungs. Despite pregnant mice controlling viral propagation and replication well as shown by the lower number of infected epithelial cells, pups born to mothers infected with either a sublethal or lethal dose of IAV were underweight, underdeveloped, and not viable. This observation was independent of vertical transmission of IAV to the placenta or the fetus by assessment of viral RNA.

Conclusions: The data suggest that the control of viral replication seen in the pregnant mice may be attributed to the early production of IFNλ, which prevents viral dissemination and an exuberant inflammatory response by negatively regulating type I IFNs and reducing the production of inflammatory cytokines in early infection. However, pups born to infected mothers are not viable. Thus, during infection, the maintenance of maternal fitness and survival might be an evolutionary strategy, which negatively impacts fetal viability.

Implications of HuR in Idiopathic Pulmonary Fibrosis


Joshua Trivlidis1, David Eidelman1, Ilan Azuelos1, Carolyn Baglole1
1McGill University

Background:Idiopathic Pulmonary Fibrosis (IPF) is a chronic and progressive fibrotic lung disease affecting an increasing number of people worldwide. Although the precise factors that cause IPF are still unknown, the disease pathogenesis is proposed to be driven by recurrent alveolar epithelial injury which leads to the differentiation of fibroblasts into α-smooth muscle actin (α-SMA)-expressing myofibroblasts (FMD, fibroblast to myofibroblast differentiation) and a subsequent excessive and relentless deposition of a collagen-rich extracellular matrix (ECM). Transforming growth factor beta-1 (TGFβ1) plays a key role in the process of FMD and is thought to be the most important pro-fibrotic mediator involved in the pathogenesis of IPF. Although the exact mechanisms that regulate FMD have not yet been fully elucidated, our recent data suggest that upon TGFβ1 stimulation, the RNA-binding protein Human Antigen R (HuR) translocates to the cytoplasm where it has been reported to stabilize various profibrotic mRNAs. Interestingly, cytoplasmic HuR has also been shown to stabilize the Hypoxia Inducible Factor (HIF-1α) mRNA which encodes a transcription factor critical for inducing a metabolic shift from oxidative phosphorylation towards glycolysis under hypoxic conditions. This metabolic shift has recently been shown to be necessary for TGFβ1-induced FMD and to occur even under normoxic conditions where the HIF1 protein is normally targeted for degradation. As one of the hallmark features of tissue fibrosis is the presence of a hypoxic microenvironment, we hypothesize that under hypoxic conditions, HuR plays a role in promoting fibroblast to myofibroblast differentiation by stabilizing HIF-1α mRNA and by inducing a metabolic shift towards glycolysis.

 

Aims:This hypothesis will be assessed through the following 3 aims:

1)Assess the effect of hypoxia on HuR expression and subcellular localization.

2)Investigate the effect of hypoxia on metabolic reprogramming and myofibroblast differentiation.

3)Investigate the role of HuR in hypoxia-induced metabolic reprogramming and myofibroblast differentiation.

 

Methods:To evaluate the effect of hypoxia on HuR expression and localization, metabolic reprogramming and myofibroblast differentiation, primary normal human lung fibroblasts (HLFs) will be incubated in either a standard incubator (normoxia) or in a hypoxia chamber (1% O2) for 0-48 hours. For aim 1) HuR mRNA and protein levels will be measured by qPCR and Western Blot respectively. The subcellular localization of HuR will be assessed by Immunofluorescence using confocal microscopy as well as by subcellular fractionation by Western Blot. For aim 2) The incubation of HLFs under hypoxic conditions will first be validated by measuring the expression of HIF-1α mRNA and protein levels by qPCR and Western Blot respectively. The process of FMD will be assessed by measuring the mRNA and protein levels of α-SMA and of the ECM markers collagen 1, collagen 3, and fibronectin by qPCR and Western Blot respectively. Metabolic shift will be examined by measuring the mRNA levels of the key glycolytic enzymes lactate dehydrogenase A (LDHA) and Hexokinase II (HKII) by qPCR. Moreover, lactate secretion as a surrogate for glycolytic activity will also be examined using a commercially-available calorimetric lactate assay. For aim 3) The role of HuR in hypoxia-induced metabolic reprogramming and myofibroblast differentiation will be determined by evaluating the effect of HuR siRNA on the expression of the aforementioned markers under normoxic and hypoxic conditions.

 

Preliminary Results: We observed a 6 ± 2.4-fold translocation of HuR to the cytoplasm after 4 hours of hypoxia when compared to normoxia. Next, we evaluated the expression of HIF-1α over the course of 48 hours and found that hypoxia increased HIF-1α protein levels at 6 hours by 13.6-fold when compared to normoxia . The mRNA and protein levels of the markers of α-SMA, collagen 1, and fibronectin were unaffected by hypoxia over 48 hours. However, when HLFs were stimulated with TGFβ1, hypoxia had a powerful effect on the mRNA levels of a-SMA and Collagen 1 which showed a 12.1-fold increase and 18.4-fold increase respectively when compared to normoxia at 48 hours. The addition of TGFβ1 only resulted in a 6.8-fold increase (α-SMA) and a 4.5-fold increase (Collagen 1) in mRNA levels at 48 hours compared with normoxia. mRNA levels of LDHA, when compared to normoxia, were increased by 2.7-fold under hypoxic conditions, by 5.2-fold with TGFβ1 stimulation and by 7.4-fold under hypoxia and TGFβ1 stimulation.

 

Inhibitory activity of STAT6-IP on IL-33-mediated IL-13 production and eosinophil activation in a Type 2 innate murine inflammatory model


Vanessa Moarbes1, Hedi Zhao1, Véronique Gaudreault1, Lydia Labrie1, Jichuan Shan1, Brian J. Ward1, Elizabeth D. Fixman1
1Research Institute of McGill University Health Center

Asthma and allergy rank among the costliest of all chronic diseases and are steadily increasing in occurrence. Data from asthmatic patients and murine models demonstrate that the STAT6 transcription factor promotes many responses in the allergic lung. Recent interest has focused on IL-33 as a promoter of Type 2 immunity. We have characterized mechanisms by which STAT6-IP, a cell penetrating peptide designed to inhibit STAT6, diminishes innate Type 2 inflammation induced upon IL-33 delivery to the lung. We compared responses in wild type and STAT6 knockout (KO) Balb/c mice treated daily for three days with saline or IL-33, either alone or with STAT6-IP or a negative control derivative, STAT6-CP. Mice were sacrificed 72h later.
Our data indicate that IL-33 increased bronchoalveolar lavage fluid (BALF) and lung eosinophil (Eos) numbers. Similarly, activated Eos and group 2 innate lymphoid cells (ILC2) were increased, as were BALF levels of Type 2 cytokines & chemokines in IL-33-treated mice. IL-13 production from minced lung explants, re-stimulated with saline or IL-33, was augmented by in vivo IL-33 administration. Each of these responses was reduced by STAT6-IP (but not STAT6-CP). Aside from IL-33-induced increases in lung Eos & ILC2, each of these responses was reduced in STAT6-KO mice treated with IL-33.
These data demonstrate that the ability of IL-33 to induce acute Type 2 airway inflammatory responses is largely dependent upon STAT6 and suggest that IL-33-induced IL-13 production and STAT6 activation participate in a positive feedback loop to promote IL-33-dependent airway inflammatory responses. STAT6-IP interrupts these maladaptive inflammatory responses, effectively reducing Type 2 inflammation in the lung.

Late diagnosis and undertreatment of COPD – A Canadian perspective from the CanCOLD observationnal study


Sébastien Gagnon1, Jean Bourbeau1, D.Marciniuk 8, F.Maltais 5, W.Tan 2, S.Aaron 3, P.Hernandez 7, B.Walker 4, D.O'Donnell 6, KR Chapman9
1McGill University 2University of British Columbia 3University of Ottawa 4Calgary University 5Université Laval 6Queens University 7Dalhousie University 8University of Saskatchewan 9Kremblin Research Institute

Introduction: COPD is an important disease, with a prevalence of 16,2 % in the COLD study. This burden has a great human and economic impact.  COPD is a progressive disease, however diagnosis and treatment often occur late in the disease process once patients experience significant symptoms.

 

The objective of this study is to evaluate physician diagnosis and treatment of individuals with COPD in the CanCOLD prospective cohort.

 

Methods: This research is part of the Canadian Cohort Obstructive Lung Disease (CanCOLD), which is a prospective multi-center cohort (9 sites), built on the prevalence study “COLD”. COPD patients were diagnosed based on a fixed post-bronchodilator FEV1/FVC < 0,7. Each COPD patient was compared with 2 groups of aged-matched non-COPD subjects (never smokers and ever smokers).  The results for this study are from the patient first visit for the cohort, which takes place between 2010 and 2015.  Among questionnaires, patients were asked to self-report the physician diagnosis that they received. 

 

Results: A total of 740 COPD patients were included, compared with 821 non-COPD (475 at-risk, 346 healthy).  In COPD patients, 53.9 % (399 patients) didn’t receive a physician diagnosis. Patient without a diagnosis were more likely to have a mild obstruction and a lowe burden of symptoms. Globally, only 28,3 % (265 patients) of COPD patients received a respiratory treatment. In COPD GOLD A patients, only 25 % of patients were receiving treatment. In COPD GOLD D there was 22 % of patients without respiratory treatment and an additional 19 % who were receiving only inhaled corticosteroid.  

 

Conclusion:  In this cohort, a majority of patients with COPD remained undiagnosed. The greater part of undiagnosed patients has low burden symptoms and mild obstruction. However, there is also a significant proportion of symptomatic or at-risk patients who remained undiagnosed or possibly  misdiagnosed. Finally, despite a high burden of symptoms or frequent exacerbations, a significant number of COPD patients don’t receive appropriate treatment, and this despite consideration of physician diagnosis.

Leukotriene C4 synthase deficiency causes spontaneous emphysematous changes in mice


Yumiko Ishii1, Toby McGovern1, Utako Fujii1, Niusha Khazaei1, Emily Nakada1, Soroor Farahnak1, James Martin1
1Meakins Christie laboratories

Introduction: Emphysema is an important component of chronic obstructive pulmonary disease (COPD) for which effective therapies are lacking. Emphysema is proposed to be caused by chronic inflammation induced by noxious particles but is also associated with lung development. Cysteinyl-leukotrienes (LTC4, LTD4, and LTE4, cysLTs) are inflammatory lipid mediators derived from arachidonic acid and the cysLT1 receptor expression is reportedly increased in COPD patients with severe exacerbations. However, whether cysLT synthesis influence the development emphysema is unknown.

Aim: To investigate the influence of the constitutive synthesis of cysLTs on lung structure. Methods: Lung structure and function were assessed in leukotriene C4 synthase knockout (LTC4S-KO) mice, ranging in age from 1-40 weeks, by the measurement of pulmonary mechanics using FlexiVent and the mean linear intercept (MLI), respectively. To examine the involvement of immune cells, differential cell counts in bronchoalveolar lavage (BAL) and immunohistochemistry of lung were performed. Emphysema-related molecules including protease/antiprotease expression were assessed by qPCR and ELISA. Results: Histological examination revealed premature emphysematous changes in the lungs, in evidence as early as 5 weeks of age. Static compliance and the K value (Salazar-Knowles equation) were higher and tissue elastance was lower in LTC4S-KO mice than in age matched wild type (WT) mice. Additionally, MLI was higher in LTC4S-KO mice. In BAL fluid from LTC4S-KO mice, hemosiderin-laden macrophages were increased. Conclusions: LTC4S deficiency in vivo leads to emphysematous changes spontaneously, and it may be caused by abnormal maturation of lung.

M. tuberculosis reprograms hematopoietic stem cells in type I IFN dependent manner to impair trained immunity against TB


Nargis Khan1, Jeffrey Downey1, Joaquín Sanz2, Eva Kaufmann1, Alain Pacis2, Erwan Pernet1, Miguel P Soares3, Luis Barreiro2, Maziar Divangahi1
1McGill University 22Department of Medicine, Genetic Section, University of Chicago, Chicago, IL, USA 3Instituto Gulbenkian de Ciencia, Oeiras, Portugal

 

Infections are associated with extensive reprogramming of hematopoietic stem cells (HSCs) residing in the bone marrow (BM) for producing immune cells to protect hosts. We have recently shown (Kaufmann et al, Cell 2018) that BCG reprograms HSCs in type II IFN dependent manner to promote myelopoisis, which leads to protective trained immunity against Mtb. Here we show that Mtb gains access to the BM at day 10 after low dose aerosol infection (H37Rv; ~50 CFU). Interestingly, the number of bacteria significantly increased by day 120 post-infection indicating that the growth of bacteria has not been controlled by the immune system within BM. This result was in sharp contrast to our published work of mice vaccinated intravenously (iv) with BCG (BCG-iv), in which the number of bacteria in the BM dramatically decreased over time. Additionally, in both models of infection, the number of BM-HSCs (as determined by a lineage-c-Kit+Sca-1+ (LKS) phenotype) directly correlated with the number of bacteria. Interestingly, the comparison of BCG-iv with Mtb-iv with the identical dose (1x106 CFU) indicate that while all the mice infected with Mtb died within 100 days, BCG infected mice survived. After Mtb-iv infection, the LKS population rapidly expanded and reached its max within 7 days and persisted up to day 90, when 80% of mice died. These results contrast with BCG-iv vaccination in which the LKS population gradually expanded and by day 90 returned to almost basal numbers. Finally, BM-derived macrophages from Mtb-infected mice were impaired to provide protection to subsequent Mtb infection, which was mediated via type I IFN. We furthermore show that distinct to BCG-iv vaccination, virulent Mtb reprograms HSCs in type I IFN dependent manner to suppress myelopoisis and promote lymphopoisis that impairs trained immunity again Mtb. Collectively, our results demonstrate that virulent Mtb hijacks the HSC transcriptional program to generate an immune response beneficial to the pathogen rather than host.

Maternal Sleep-Disordered Breathing in Pregnancy Increases Nocturnal Glucose Levels in Women with Gestational Diabetes


Raphieal Newbold1, Sara Meltzer1, Andrea Benedetti1, Natasha Garfield1, Evelyne Rey2, Lorraine Lavigne1, Allen Olha1, Kaberi Dasgupta1, Robert Gagnon1, R. John Kimoff1, Sushmita Pamidi1
1McGill University Health Centre 2Université de Montréal

Rationale: Sleep-disordered breathing (SDB) increases in prevalence over the course of pregnancy, occurring in 17-45% of women by the third trimester. Maternal SDB is associated with an increased risk of pregnancy complications, including gestational diabetes (GDM). It is unknown if maternal SDB is associated with worse glucose levels, particularly during sleep. Continuous glucose monitoring (CGM) is an emerging technique enabling continuous glucose data collection over several days. Our objective was to analyze glucose levels using CGM based on time of day and its relation to maternal SDB.

 

Methods: In this cross-sectional study, women with GDM (WHO 2013 diagnostic criteria) underwent ambulatory level 2 sleep recordings (Chicago Scoring Criteria) and continuous 72-hour glucose level measurements using CGM (iPro2, MedTronic). After subcutaneous insertion, the device measured interstitial glucose every five minutes across 72-hours consecutively. Glucose results were blinded from participants. We used linear regression to estimate the association between the apnea-hypopnea index (AHI) as a measure of SDB severity on glucose levels. We adjusted for BMI and use of insulin/metformin in the final model.

 

Results: Our sample of 65 participants had the following demographics (mean±SD): 35±4.7 years of age and BMI of 32±7.8 kg/m2 at enrolment. The mean AHI was 16±10.6 events/hour. Increasing AHI was significantly associated with elevated nocturnal glucose levels between 11pm-3am (p=0.01) and 3am-6am (p=0.02). AHI remained significantly associated with increases in nocturnal glucose in the adjusted model (11pm-3am, p=0.04; and 3am-6am, p=0.02). AHI was not associated with 24-hour glucose levels or daytime glucose.


Conclusion: Increasing AHI is associated with nocturnal glucose levels, even after adjustment for confounders. Without treatment, SDB may be a risk factor for the development of GDM in pregnancy. Interventional studies aimed at treating maternal SDB are needed to further determine the causal relationship between maternal SDB and glucose control in GDM.

 

Mechanisms Underlying Corticosteroid Insensitivity Induction by Interleukin-17A in Bronchial Epithelial Cells


Wided Akik1, Nurlan Dauletbaev2, Larry C. Lands2,3
1Division of Experimental Medicine, Faculty of Medicine, McGill University 2Department of Pediatrics, Faculty of Medicine, McGill University 3Meakins-Christie Laboratories, Research Institute of the McGill University Health Centre

Asthma is a heterogeneous chronic lung disease affecting both children and adults. Inhaled corticosteroids are typically used for the treatment of asthma. However, up to 10% of patients can be insensitive to high dose corticosteroids, rendering treatment options ineffective. These severe corticosteroid-insensitive patients account for up to 50% of asthma costs. A form of severe asthma is neutrophilic asthma, characterized by a type 2 T helper cells (Th2)-low and Th17 type of asthma unresponsive to treatments such as anti-IgE or anti-IL-5 antibodies. The pro-inflammatory cytokine Interleukin (IL)-17A enhances recruitment of neutrophils by inducing the production of IL-8, the primary chemoattractant for neutrophils into the airway, by bronchial epithelial cells. 

 

We hypothesize that it is through transcriptional mechanisms that IL-17A reduces the ability of corticosteroids to inhibit the production of IL-8 in airway epithelial cells. This work examines the effects of IL-17A as well as corticosteroids on the transcriptional regulation of IL-8 in Tumor Necrosis Factor (TNF)-alpha-stimulated bronchial epithelial cells. 

 

The human lung bronchial epithelial cell line (BEAS-2B) was studied. Cells were pretreated with IL-17A (10 ng/mL) for 2 hours, stimulated with TNF-alpha (10 ng/mL) for six hours. Eighteen hours post-treatment with budesonide (10-10M – 10-6M), supernatants were collected and assessed for secreted IL-8 levels (ELISA) to create dose-response curves to stimulated cells in the presence or absence of IL-17A. Alternatively, two hours post-treatment with budesonide (10-8M and 10-7M), transcriptional activity of IL-17A was assessed by quantifying the expression of the corticosteroid sensitive genes FKPB5, GILZ1 and DUSP1 (droplet digital PCR) in stimulated cells in the presence or absence of IL-17A. Fold change of gene expression was determined by normalizing to the reference gene, TBP. Thirty minutes post-treatment with budesonide (10-7M), nuclear translocation of the glucocorticosteroid receptor (GR) was analyzed (Western blot).

 

The results indicate that the observed corticosteroid insensitivity is not absolute. IL-17A induces a relative corticosteroid insensitivity by potentiating IL-8 production in TNF-alpha-stimulated BEAS-2B cells. Moreover, IL-17A 1) reduces the ability of corticosteroids to inhibit TNF-alpha-stimulated production of IL-8, 2) does not interfere with corticosteroid transcriptional ability, and 3) does not decrease glucocorticoid receptor (GR) nuclear translocation. The results will be further confirmed by studying protein-DNA interactions (ChIP-seq). Future work will explore post-transcriptional and epigenetic mechanisms as required to fully understand the mechanisms underlying IL-17A’s induction of corticosteroid-insensitivity. Confirmatory studies in primary airway epithelial cells from healthy donors and patients with severe neutrophilic asthma will follow.

 

Once the primary mechanisms for IL-17A’s role in the reduction of corticosteroid sensitivity are established and potential therapeutic targets identified, therapeutic genetic therapy approaches will be explored. These approaches will employ novel pH-sensitive liposomes capable of aerosol delivery of genetic material (mRNA, siRNA or oligonucleotides) to respiratory epithelial cells.

Modelling the Epidemiology of Tuberculosis in Nunavut from 1948-2018


Aashna Uppal1,2,3, Ntwali Placide Nsengiyumva1,2,3, Dieynaba S. N’Daiye1,2,3, Olivia Oxlade1,2,3, Kevin Schwartzman1,2,3
1Montreal Chest Institute, Montreal, Quebec, Canada 2Respiratory Epidemiology and Clinical Research Unit, Centre for Outcomes Research and Evaluation, Research Institute of the McGill University Health Centre, Montreal, Quebec, Canada 3McGill International Tuberculosis Centre, Montreal, Quebec, Canada

Background: Tuberculosis (TB) is a prominent public health problem in many Inuit communities in Canada. While it is a medical problem, TB pathogenesis is strongly influenced by social risk factors, such as tobacco use, heavy drinking, food insecurity and overcrowded housing. The objective of this study was to mathematically describe the historical epidemiology of TB in Nunavut while taking into account these social risk factors.

 

Methods: A dynamic transmission model was created using Berkeley Madonna (Version 8.3.23.0, University of California, 2015) to represent several TB health states. A hypothetical cohort of 1,000,000 individuals were stratified by social risk factor and transitioned between these health states over time according to a series of difference equations dependent on infection-related and risk factor-related parameters. Risk factors were not considered mutually exclusive and were informed by regional prevalence data. Key model parameters were calibrated against observed historical annual risk of infection (ARI) and TB incidence data.

 

Results: Using our model, in 2018, an estimated 0.15% of the cohort had prevalent active TB. Of the stratum characterized by a combination of all four social risk factors, 41% were in the susceptible uninfected state, 41% had latent TB, 1% had infectious active TB and 17% had recovered from previous active TB. The model estimated an overall TB incidence rate of 209 per 100,000 person-years and an ARI of 0.85% in 2018. These predicted values correspond well to the observed incidence rate of active TB, which, in year 2016 (the most recent year available) was 143 per 100,000 person-years.  

 

Conclusion: The model is able to predict trends that are representative of current TB epidemiology in Nunavut. Data from this model will be integrated into a decision-analysis model that investigates the cost-effectiveness of interventions targeting the social risk factors in question.

Modulation of CD4+ T cell Fate by Airway Smooth Muscle Cell Mediated Inflammation


Joyce H. Jang1, Michelle Chen1, Soroor Farahnak1, James G. Marti1
1Meakins Christie Laboratories, Research Institute of the McGill University Health Center and the Department of Medicine, McGill University, Montreal, Quebec, Canada

Background

The physiologic role of airway smooth muscle (ASM) is currently unknown. It is most commonly theorized as a vestigial remnant but ASM can play a damaging role in airway pathologies. Their “activation” in disease such as asthma has been linked to with the interaction of inflammatory mediators and inflammatory cells. CD4+ T cells were shown to mediate the induction of ASM remodeling as well as modulate contractile properties. However, evidence has also been presented demonstrating the ability for ASM to modulate T cell activity. Co-culture with T cells induced VLA-4, ICAM and CD44 in ASM cells (ASMC). ASMC were also shown to act as an antigen presenting cell for T cells after incubation with super-antigen. Mitochondria and anti-apoptotic factors can be passed to CD4+ T cells through membrane nanotubes. In this study, we investigate the possible roles ASMC play in modulating the immune system through CD4+ T cells. We hypothesis that ASMC will promote inflammatory activity of CD4+ T cells and thus contributing to the progression of airway diseases such as asthma.

Methods

Human ASM cells were isolated from airway tissues from donors with no history of airway diseases. Human peripheral blood mononuclear cells (PBMCs) were isolated from the blood collected from healthy volunteers after informed consent was given. CD4+ T-cells were purified by magnetic activated cell sorting (MACS) and activated with anti-CD3/28 Dynabeads™ for 72h before co-culture. ASM cells were seeded a day before co-culture. A 10:1 ratio of CD4+ T-cells:ASM cells was co-cultured for 24h before analysis. mRNA was harvested and expression of cytokines were measured by qPCR. Proliferation and apoptosis were assessed by measuring BrdU incorporation and Annexin V staining, respectively, by flow cytometry. Expression of cytokines and various surface markers was also assessed by flow cytometry. CD4+ T cells were isolated by collecting supernatant and washing ASMC. These cells were re-stimulated with anti-CD3/28 Dynabeads™ for 24h before flow cytometric analysis described above.

Results

ASMC reduced the proliferation and apoptosis of CD4+ T cells. There was a trending reduction in cytokine expression at the mRNA level in CD4+T cells after co-culture, however, this was not reproduced by flow cytometric analysis. IL-5 expression at the protein level was significantly increased in CD4+ T cells after co-culture. ASMC reduced the expression of the activation marker CD69 but increased the expression of CD44. CD4+ T cells were more responsive to CD3/28 stimulation after co-culture with ASMC.

Conclusion

ASMC seems to ‘prime’ CD4+ T cells to be more responsive to antigen stimulation.

New insights into macrophage dynamics in the mouse diaphragm


Qian Li1, Salyan Bhattarai1, Feng Liang1, Eva Kaufmann1, Junying Ding1,2, Basil J Petrof1
1Meakins-Christie Laboratories and Respiratory Division, McGill University, Montreal, Quebec, Canada 2Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing, China

Background: Diaphragm muscle injury occurs in several disease conditions and contributes to respiratory insufficiency. Successful muscle regeneration after injury depends upon the function of myogenic progenitor cells (called satellite cells), which in turn rely upon the appropriate regulation of macrophages including recruited bone marrow (monocyte)-derived macrophages (BMDM) and  tissue resident macrophages (TRM). TRM are macrophages residing in the tissue at steady state. They are highly heterogenous and tissue specific. Before birth, TRM can have different sources such as embryonic yolk sac, fetal liver and bone marrow. After birth, the maintenance of TRM is dynamic. TRM are able to maintain the population by local proliferation or by replenishment from BMDM . In skeletal muscle and particularly the diaphragm, the responses of BMDM vs TRM to acute injury are poorly understood.

Our Objectives were to: 1. Develop a model to study BMDM and TRM dynamics in the diaphragm; 2. Determine the contribution of BMDM to TRM maintenance at steady state; 3. Determine the responses of BMDM versus TRM at different phases after acute injury.

Methods: To distinguish between BMDM and TRM, congenic allelic variant mouse chimeras were generated by transplantation of donor bone marrow (CD45.2) into total body irradiated host mice (CD45.1). Lead shielding of the diaphragm (DIA) was employed to protect both satellite cells and TRM from radiation effects. Mice were sacrificed at 8 weeks and 16 weeks after bone marrow transplantation at steady state or 4 days and 60 days after acute muscle injury induced by cardiotoxin (CTX). Macrophage proliferation was examined by intracellular Ki67 and BrdU staining. A second approach of the parabiosis model was also used to address the same question.

Results: Satellite cells isolated from unshielded DIA had impaired proliferation and differentiation capacity, while DIA shielding restored these capabilities. In shielded DIA at steady state, approximately 55% of TRM were maintained by donor BMDM at both 8 weeks and 16 weeks post-bone marrow transplantation. At day 4 post injury, there is an increase of BMDM cell number and approximately 90% of intramuscular macrophages are of bone marrow origin. Ki67 and BrdU staining results suggest that local proliferation is not the major contributor of this increase. At day 60 post injury, the macrophage cell number decreased to pre-injury values. Importantly, the percentage of donor macrophages returned  to similar values (~60%) as those observed under steady state basal conditions .

Conclusion: At steady state, BMDM are the predominant contributor to TRM maintenance in the muscle. However, there also appears to be a substantial BMDM-independent population of TRM in the shielded diaphragm. After muscle injury, the acute increase in intramuscular macrophages is almost exclusively due to BMDM. Differences in local proliferation of macrophages do not appear to be a major contributor to this large dominance of BMDM after acute injury. After long- term recovery from diaphragm damage, the large predominance of BMDM observed in the acute phase of injury is partially reversed such that the pre-injury steady state levels and ratio of BMDM to TRM are re-established.

Funding support: CIHR and Chinese Scholarship Council  

Obstructive sleep apnea treatment of multiple sclerosis patients with severe fatigue using positive airway pressure (SAMS-PAP trial): initial study results.


S. Khadadah1, D. Trojan2, P. Duquette3, V. Jobin3, Y. Lapierre2, A. Benedetti4, F. Johara4, A. Robinson2, E. Roger3, A. Bar-Or2, M. Kaminska5, R.J. Kimoff1
1Respiratory Division and Sleep Laboratory, McGill University Health Centre 2Department of Neurology and Neurosurgery, Montreal Neurological Institute and Hospital, McGill University Health Centre 3Centre Hospitalier de l'Université de Montréal 4Department of Epidemiology, Biostatistics and Occupational Health, McGill University 5Respiratory Division and Respiratory Epidemiology and Clinical Research Unit, McGill University Health Centre, Montreal, QC, Canada

Introduction: We have described a significant relationship between severe fatigue and moderate-severe obstructive sleep apnea (OSA) in multiple sclerosis (MS) (MSJ 2012; 18:1159–69). The objective of the SAMSPAP trial (NCT01746342) was to evaluate the impact of CPAP treatment of OSA on fatigue and other clinical symptoms in MS patients using a randomized, double-blind, sham-CPAP controlled design. We present here the initial results of the study.

 

Methods: We recruited MS patients on stable immunomodulating medications with the following inclusion criteria: Expanded Disability Status Score (EDSS) ≤ 7, severe fatigue (Fatigue Severity Score, FSS ≥ 4), poor subjective sleep quality (Pittsburgh Sleep Quality Index, PSQI > 5), no more than mild cognitive dysfunction (Montreal Cognitive Assessment score ≥ 26), and OSA defined as an apnea-hypopnea index (AHI) ≥ 15 events/h on in-laboratory polysomnography using AASM research (Chicago) criteria. Patients with severe OSA (AHI > 30 and 4% O2 desaturation index > 15/h or severe somnolence (Epworth Sleepiness Scale, ESS ≥ 15)) were excluded. Participants were randomized to 6 months of active vs sham CPAP treatment. Change from baseline in outcome measures was assessed at 3 and 6 months, including FSS (primary outcome), Fatigue Scale of Motor and Cognitive Functions (FSMC), PSQI, ESS, pain visual analog scale, and EDSS.

 

Results: 103 patients were screened of whom 49 met inclusion criteria and were randomized. 34 patients (17 per group) completed the study protocol. The groups had similar baseline characteristics (CPAP vs Sham, Mean ±SD: AHI 31 ±11.5 vs 36 ±24.9/h; Arousal index 47.5 ±17.4 vs 55.4 ±26.9/h; FSS 6.7 ±0.7 vs 5.7 ±0.8). 71% of CPAP patients and 35% of Sham achieved adequate compliance (≥ 4h/night, ≥ 70% of nights). In a modified intention to treat analysis (n=34), there was a trend towards improvement of fatigue in the CPAP vs Sham group ( FSS 0.9 ±1.0 vs 0.3 ±0.9, p=0.09) and a significant improvement in sleepiness ( ESS 3.4 ±3.0 vs 0.8 ±3.4, p=0.03) at 3 months. These changes were not sustained at 6 months although there was a significant reduction in reported morning fatigue (41% vs 0%, p=0.03) with CPAP but not Sham at 6 months. We found no other significant differences in changes between groups in FSMC, PSQI, pain visual analog scale, and EDSS at 3 and 6 months.

 

Conclusion : This study showed a trend towards improved fatigue and a significant reduction in somnolence with CPAP compared to sham at 3 but not 6 months of treatment, although morning fatigue was significantly less with active CPAP at 6 months. Further analyses are in progress to evaluate the effect of compliance and MS disease-related variables on study results.

 

The authors gratefully acknowledge funding from the Multiple Sclerosis Society of Canada.

Oscillometry to Assess Respiratory Function in Adult Cystic Fibrosis


Arash Rad1, James Gruber2, Jennifer Landry2, Larry Dong3, Zoltan Hantos4, Andrea Benedetti3, Elie Matouk2, Stewart Gottfried5
1Department of Experimental Surgery, Faculty of Medicine, McGill University 2Respiratory Division, Department of Medicine, McGill University 3Departments of Medicine and of Epidemiology, Biostatistics & Occupational Health, McGill University 4Dept of Anesthesiology and Intensive Care, Semmelweis University, Budapest, Hungary 5Respiratory Division, Department of Medicine, Meakins-Christie Labs, McGill University

Background and Objective: Cystic fibrosis (CF) is a life-limiting multisystem disorder in which respiratory dysfunction predominates. Although spirometry (SPIRO) is the most commonly used test to assess lung function, it has recognized limitations. Oscillometry (OSC) is an alternative test of respiratory function which provides additional, complementary information. Surprisingly, little data exists in adults with CF. Accordingly, the aim of this study was to characterize OSC findings in a large adult CF cohort.

Methods: SPIRO and OSC were performed during a routine follow-up visit to the Montreal Chest Institute Clinic in 64 adults with established CF in stable state (mean age = 33.4 ± SD 12.9 yrs; 37 male). Parameters were expressed as Z-scores to facilitate comparison.

Results: Respiratory function was significantly reduced for the group. There was good correlation between various SPIRO and OSC parameters (r = 0.43-0.83), including indices of airway obstruction (FEV1/FVC ratio vs R5) and small airway dysfunction (FEF25-75 vs R5-20).  Z5, total impedance at 5Hz, was the most abnormal OSC parameter (Z = 5.6 ± 2.9). Tidal flow limitation was identified by OSC in 6 patients.

Conclusion: OSC appears to be a reliable measure of impaired respiratory function in stable adult CF. Further work is needed to assess its ability to reliably identify changes in function occurring either spontaneously or with therapeutic interventions, particularly in comparison with SPIRO, patient reported outcomes, and other measures.

 

Support: Department of Medicine, McGill University Health Centre.

Potentiation of antibiotic lethality by sulfonamides against stationary phase Pseudomonas aeruginosa


Tianxiao Yang1,3, Dorival Martins2,3, Dao Nguyen2,3,4
1Department of Parasitology, McGill University 2Department of Microbiology and Immunology, McGill University 3Meakins-Christie Laboratories, Research Institute of the McGill University Health Centre 4Department of Medicine, McGill University

Pseudomonas aeruginosa causes chronic lung infections in cystic fibrosis patients, which are difficult to treat due to the bacteria’s high antibiotic tolerance. Thus, there is an urgent need to enhance antipseudomonal drugs efficacy via bypassing tolerance. Previous work in our group showed that cyclopropane fatty acids (CFAs) present in P. aeruginosa cell envelope promotes antibiotic tolerance by decreasing the membrane permeability and thus the internalization of a wide range of antibiotics. This led us to hypothesize that CFA synthesis may be an interesting target to bypass tolerance. In Proteus mirabilis, there are reports that the sulfonamide drug sulfadiazine lowers bacterial CFA content and enhances polymyxin B activity, implying that sulfonamides act as CFA synthesis inhibitors. Herein, we probed whether various sulfonamides can potentiate bactericidal antibiotics in P. aeruginosa in a CFA-dependent manner. Using killing assays, we determined that sulfadiazine has no anti-bacterial activity in wild-type P. aeruginosa but enhances killing by colistin, a polymyxin antibiotic, by 1,000-fold. Such synergy was not observed in a CFA-depleted mutant (cfa), suggesting that sulfadiazine-driven potentiation of colistin is CFA-dependent.  In a checkerboard assay, we also saw synergy in the wild-type strain but not in a cfa mutant. To probe whether this effect was common to other sulfonamides, we tested colistin killing in combination with other sulfonamides, namely sulfamethazine, sulfadimethoxine, sulfamethoxazole, sulfameter and sulfacetamide. These drugs mostly antagonized colistin by up to 100-fold, suggesting that the synergy is specific to sulfadiazine. Interestingly, although CFAs depletion lowers tolerance to drugs other than colistin (e.g., ofloxacin and meropenem), sulfadiazine synergized only with colistin. We thus speculate that colistin, as a membrane-destabilizing antibiotic, may enhance the initial internalization of sulfadiazine, which in turn inhibit CFA synthesis, which in turns sensitizes P. aeruginosa to colistin. We conclude that sulfadiazine may be a strategy to bypass CFA-mediated colistin tolerance in P. aeruginosa but its mechanism remains to be fully understood. Furthermore, this antibiotic potentiation effect is not observed with other antibiotics nor other sulfonamides.

Protective roles of autophagy in sepsis-induced skeletal muscle dysfunction, metabolic derangements and mortality


Jean-Philippe Leduc-Gaudet1, Kayla Miguez1, Julie Faitg2, Felipe Eduardo Broering1, Jordi Chaffer1, Olivier Reynaud2, Dominique Mayaki1, Laurent Huck1, Gilles Gouspillou2, Sabah NA Hussain1
1Meakins-Christie Laboratories, Department of Medicine and Divison of Experimental Medicine, McGill University, Québec, Canada 2Département de Sciences de l’activité physique, Faculté des Sciences, UQAM, Québec, Canada

Rationale: Sepsis-induced skeletal muscle dysfunction contributes to physical disability, and increased mortality in intensive care unit patients. Autophagy is a catabolic process by which cells degrade their own components. Recent studies indicate that sepsis triggers sustained induction of autophagy in skeletal muscles; however, the impact of autophagy on sepsis-induced contractile and metabolic dysfunctions remains unclear. In this study, we evaluated the functional importance of autophagy in sepsis-induced skeletal muscle dysfunction.

 

Methods: Selective inhibition of autophagy in skeletal muscles was accomplished by cross breeding floxed Atg7 mice (Atg7f/f) with those expressing HSA-Cre-ERT2. Cre induction was achieved by feeding Tamoxifen. Control mice were Atg7f/f-HSA-Cre-ERT2 without Tamoxifen feeding. Sepsis was induced by the cecal ligation and perforation (CLP) procedure and animals were examined after 48 and 144 hours of sepsis. Animals undergoing sham procedure served as control.

 

Results: Deletion of Atg7 in skeletal muscle had a major impact on sepsis-induced loss of body weight and muscle mass as indicated by worsening of body weight loss, more severe sepsis-induced skeletal muscle atrophy and further decline in muscle contractility in mice with Atg7 deletions vs. those with intact Atg7 undergoing sepsis for 48 hours. In additions, body weight loss, hypoglycaemia and mortality were more severe over a 144 hours period in mice with Atg7 deletion as compared to those with intact Atg7. These results suggest that induction of autophagy in septic skeletal muscles play a protective role against sepsis-induced mortality. Furthermore, sepsis had no major effect on limb muscle mitochondrial respiration in animals with intact muscle Atg7 while those with deleted muscle Atg7 showed significant impairments of limb muscle mitochondrial respiration both in the sham and septic conditions. Finally, significant upregulation of ubiquitin E3 ligases (Atrogin-1 and MuRF1) was observed in septic muscles. Deletion of muscle Atg7 had no effect on this response when measured after 48 hours of sepsis. However, this response was more severe over 144 hours period in mice with Atg7 deletion as compared to those with intact Atg7.

 

Conclusion: We conclude that autophagy plays a protective role by preserving muscle mitochondrial quality and function and that autophagy inhibition in skeletal muscles worsens sepsis-induced loss of body weight and muscle atrophy possibly due to exaggerated responses to catabolic stimuli triggered by sepsis. Our results also indicate that autophagy in septic skeletal muscles play a protective role against sepsis-induced metabolic derangements and mortality.

 

 

Keywords: Sepsis; Skeletal Muscle Atrophy; Mitochondria, Autophagy 

 

Funding: This study is funded by grants from the Canadian Institutes of Health Research. Gilles Gouspillou salary support is provided by a FQRS Chercheur Boursier Junior 1 Award. Jean-Philippe Leduc-Gaudet is supported by CIHR Vanier Doctoral Scholarship. TJC is supported by an NSERC Undergraduate Summer Research Award.

 

PSEUDOMONAS AERUGINOSA EVASION OF NEUTROPHIL ANTIBACTERIAL FUNCTIONS IN EARLY CYSTIC FIBROSIS LUNG INFECTION


K. Kwong1,3, T. Beaudoin2, V. Waters2, D. Nguyen1,3
1Department of Microbiology and Immunology, McGill University, Montreal, Canada 2Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada 32Meakins Christie Laboratories, Research Institute of MUHC, Montreal, CA

Introduction

Pseudomonas aeruginosa(PA) is the predominant pathogen that causes chronic lung infection in patients with cystic fibrosis (CF). In order to delay chronic infections, inhaled tobramycin has demonstrated efficacy on improving PA eradication in CF children with initial PA infections. However, up to 40% patients still failed tobramycin eradication therapy and factors associated with failure of eradication therapy are unclear. Interestingly, microbiological analysis from clinical trials of PA eradication therapy suggested that certain PA bacterial phenotypes (loss pilus-mediated motility, mucoidy and wrinkly colony morphology)were associated with PA eradication failure, but results vary between studies and the mechanism remains to be determined. The adequate recruitment and neutrophils (PMNs)-mediated opsonophagocytic killing (OPK) of PA upon initial PA infection are likely required to eradicate PA even when antibiotic therapies are used.Thus, we hypothesize that PA isolates that persisted following inhaled tobramycin in CF patients with initial PA infection are resistant to PMN-mediated OPK compared to PA isolates from eradicated patients. 

Methods

We tested a collection of PA strains isolated from initial PA infection from CF children followed at SickKids as part of the “Early PA eradication” study. We comparedin vitrophagocytosis and intracellular bacterial killing by PMN-like cells (differentiated HL-60) in response to persistent clinical PA isolates (N = 10 persistent patients with 18 isolates) vs eradicated clinical PA isolates (N = 32 eradicated patients with 53 isolates). We characterized various bacterial phenotypes such as Type IV pilus mediated twitching and flagellum mediated swimming motility, mucoidy, pyocyanin and protease secretionproduction by plate assays; biofilm formation by crystal violet staining assay; total exopolysaccharide production by Congo red binding; and Psl exopolysaccharide production by binding of fluorescently labelled anti-Psl monoclonal antibody.

Results

We observed significantly lower PMN phagocytosis (n = 71, p < 0.01) and intracellular bacterial killing (n = 42, p < 0.05) of persistent PA compared to eradicated PA isolates. We compared various bacterial phenotypes usingunivariate and multivariable regression, PMN phagocytosis was significantly positively associated with twitching motility (r = 0.26, < 0.01) and negatively with mucoidy (r = -0.28, p< 0.01). Furthermore, preliminary data from a subset of PA isolates, persistent PA (n = 7) produce a remarkably higher levels of Psl (p < 0.01) compared to eradicated PA (n = 7), suggests that Psl among other PA bacterial factors likely reduces PMN antibacterial functions in addition to mucoidy and lack of twitching. The role of Psl in these clinical PA isolates and whether it is associated with impaired PMN antibacterial functions remains to be determined.

Conclusion

Our data thus far suggests that persistent PA isolates from CF patients with initial PA infection are resistant to PMN antibacterial functions compared to PA isolates from eradicated patients. This maybe an important mechanism that is significantly associated with failure of tobramycin eradication therapy. Moreover, multiple PA bacterial factors could contribute to impaired PMN antibacterial functions. Hence, identifying and targeting bacterial factors such as PA exopolysaccharides and potentiate PMN mediated bacterial clearance may improve current PA eradication therapy.

 

Regulatory mechanisms of eicosanoids in hematopoietic stem cell-mediated trained immunity


Orsolya Lapohos1,4, Kim Tran2,4, Eva Kaufmann3,4, Maziar Divangahi1,2,3,4
1Department of Microbiology and Immunology, McGill University 2Department of Pathology, McGill University 3Meakins-Christie Laboratories, RI-MUHC 4McGill International TB Centre

Despite the world-wide application of Bacillus Calmette-Guérin (BCG) vaccination and other anti–Mycobacterium tuberculosis (Mtb) interventions, Mtb remains one of the most successful human pathogens. Approximately 1.6 million people die of tuberculosis annually and eight to ten million new cases of active tuberculosis occur each year. While the BCG vaccination prevents the disseminated form of tuberculosis in childhood, its efficacy in adults is variable. Recently, our group has demonstrated that the access of BCG to the bone marrow initiates a unique program in hematopoietic stem cells (HSCs) to generate memory-like innate immunity (trained immunity) with enhanced protective capacity against Mtb infection. However, our understanding of regulatory mechanisms involved in reprogramming of HSCs and trained immunity is limited. Eicosanoids are host bioactive lipid mediators which play a critical role in the regulation of the immune response to infectious diseases, including Mtb infection. Importantly, they are also key mediators in HSC function. Taken together, these observations lead us to hypothesize that eicosanoid pathways are required for the generation of trained immunity through BCG-induced reprogramming of HSCs. We aim to determine the contributions of key eicosanoid pathways, the prostaglandin pathway (e.g. PGE2) and the lipoxygenase pathway (e.g. LTB4), in HSC reprogramming by BCG. Due to the commercial availability of drugs that target eicosanoid pathways, our study has tremendous potential to develop targeted therapy for tuberculosis.

Smooth Muscle Molecular Mechanics


Megan Hammell1,2, Linda Kachmar2, Gijs Ijpma2, Anne-Marie Lauzon2,3
1Department of Biomedical Engineering, McGill University 2Meakins-Christie Laboratories, McGill University 3Department of Medicine, McGill University

INTRODUCTION: Smooth muscle is found in all hollow organs of the body. In particular, tonic smooth muscle is responsible for tone generation. It has the ability to maintain force for long periods of time at low energy (ATP) cost. This characteristic is called the latch-state and was first described as a property of the myosin molecular motor solely. Myosin is composed of two heavy chains, two regulatory light chains (LC20) and two essential light chains. Smooth muscle myosin is activated through phosphorylation of LC20 by a Ca2+-calmodulin dependent myosin light chain kinase (MLCK). Once phosphorylated, smooth muscle myosin hydrolyzes ATP into ADP and Pi, attaches to actin and then generates force and motion through the release of Pi and ADP. It is believed that the latch-state occurs when the LC20 gets dephosphorylated by myosin light chain phosphatase (MLCP) while myosin is still attached to actin.

 

OBJECTIVES: To obtain in vitro force measurements generated by a population of myosin molecules pulling on an actin filament during LC20 dephosphorylation.

 

METHODS: Actin will be attached biochemically to a bead trapped by an optical laser trap. Actin will then be brought in contact with myosin on a pedestal. In the presence of ATP, myosin will pull on the actin-bead system until isometric force is reached. Force will be calculated as the product of the distance between the trap centre and bead (measured using a quadrant detector) and the trap stiffness (measured using Stoke’s drag force method). MLCP will be injected via a microporous membrane to dephosphorylate myosin during force measurement without creating any bulk flow.

 

PROGRESS: Biocompatibility of the assay chamber was verified and protocols were tested (protein concentrations, illumination level, amount of oxygen scavengers, etc.) to optimize the molecular mechanics measurements. A motile fraction as high as 80% was measured on the motility surface. Currently, the diffusion time is assessed by injecting ATP into chambers containing myosin bound to actin in rigor. A diffusion time ≤30s is expected and may require testing of other membrane porosities. Next, the dephosphorylation studies will begin.

 

SIGNIFICANCE: Smooth muscle force maintenance at low ATP has been recognised since the 1930s, but the latch-state mechanism was not proposed until 1981. Although studied thoroughly at the whole muscle level, this theory has never been verified at the molecular level and is essential for understanding the fundamental properties of smooth muscle.

Systemic inflammation is key to understanding the link between concomitant Chronic Obstructive Pulmonary Disease and cardiovascular disease


Mira Abou Rjeili1,2,3,4, Raquel Farias4, Sabah Hussain1,3,4, Carolyn Baglole1,3,4, Jean Bourbeau1,2,4
1Department of Medicine, Division of Experimental Medicine 2Respiratory epidemiology and clinical research unit 3Meakins- Christies laboratories 4Research Institute of McGill University Health Centre

Rationale: Chronic obstructive pulmonary disease (COPD) and cardiovascular disease (CVD) are often concomitant. Both diseases share similar risk factors, in particular cigarette smoking. Low-grade systemic inflammation is thought to be one of the mechanisms linking COPD and CVD. In response to cigarette smoke, airway epithelial cells (AECs) release the pro-inflammatory mediators interleukin (IL)-6 and IL-8 into the systemic circulation. Sustained tissue injury and cell death lead to the release of the Damage Associated Molecular Patters (DAMPS) S100A8, S100A9, High mobility group box 1 (HMGB1) and IL-1α.  This chronic inflammatory state could increase the expression of adhesion molecules including vascular cell adhesion protein 1(VCAM-1), intercellular adhesion molecule 1 (ICAM-1) and E-selectin in the vascular endothelium. This could negatively impact the cardiovascular system by promoting a pro-thrombotic state. The project is built on the hypothesis that sustained inflammation secondary to airway epithelial cell injury contributes to the development of CVD in COPD. The main objective of this project is to characterize the release of inflammatory biomarkers from AECs and endothelial cells (ECs) in an in vitro model of cigarette smoke exposure.

Methods: Human bronchial epithelial cells line (BEAS-2B), normal human primary bronchial epithelial cells (NHBE) and human microvascular endothelial cells (HMEC-1) were exposed to 2% and 5% cigarette smoke extract (CSE). Conditioned media from BEAS-2B cells exposed to CSE was collected and added to HMEC-1 cells to compare direct exposure and indirect exposure of ECs to CSE. Following exposure to CSE or to conditioned media, we assessed the expression of inflammatory biomarkers IL-6, IL-8,S100A8, S100A9, HMGB1 and IL-1α from AECs and the expression of adhesion molecules VCAM-1, ICAM-1 and E-selectin in ECs by qRT-PCR.

Results:  Following treatment of BEAS-2B cells with 5% CSE, we observed a significant increase in the expression of IL-6, IL-8 and IL-1a after 3, 6 and 24 hours. Similarly, the expression of IL-6, IL-8 IL-1α, S100A8 and S100A9 was significantly increased in NHBE cells exposed to 2% and 5% CSE. We did not observe an increase in HMGB1 expression in either cell type. When HMEC-1 cells were treated with 5% CSE, we observed an increased expression of VCAM-1 and E-selectin but not ICAM-1 expression. When HMEC-1 cells were treated with conditioned media, we observed a similar but attenuated response.

Conclusion:  Cigarette smoke promotes inflammation in both epithelial and endothelial cells. This suggests that sustained inflammation in patients could be a key mechanism linking COPD and CVD.

The Cost of Multidrug-Resistant Tuberculosis in Canada


Jonathon R. Campbell1, Placide Nsengiyumva1, Leslie Chang2, Hadeel Khadawardi3, Olivia Oxlade1, Hayden Rasberry1, Elizabeth Rea4,5, Kamila Romanowski2, Natasha Sabur3, Beate Sander4,6, Aashna Uppal1, Sarah K. Brode3,4, James C. Johnston2,7, Kevin Schwartzman1,8
1McGill International TB Centre 2British Columbia Centre for Disease Control 3West Park Healthcare Centre 4University of Toronto 5Toronto Public Health 6University Health Network 7University of British Columbia 8Montreal Chest Institute

Background

Multidrug-resistant tuberculosis (MDR-TB) requires prolonged treatment, expensive drugs, and close monitoring for treatment side effects. The costs of providing this care; however, are largely unknown. We estimated the health system costs for patients who completed MDR-TB treatment in British Columbia, Ontario, and Quebec, Canada.

Methods

We chart-reviewed all MDR-TB patients who initiated treatment at the British Columbia Centre for Disease Control (Vancouver), West Park Healthcare Centre (Toronto), and Montreal Chest Institute (MCI) between January 2010 and June 2016 and subsequently completed treatment. Information regarding consumables (e.g. drugs, supplies), services, and personnel time used during diagnosis, treatment, and follow-up was extracted. Quebec costs for each item were used to estimate the total cost per MDR-TB episode in all settings. For comparison with MDR-TB, we also calculated overall costs for 30 patients treated at the MCI for drug susceptible TB (DS-TB) and latent TB infection (LTBI).

Results

Forty-six MDR-TB patients were included. Mean age was 35 (SD 13.4) years, 25 patients (54%) were female, and 4 (9%) had extensively drug-resistant TB. Table 1 shows TB-related costs by phase of clinical care. Across all sites, the median (Q1, Q3) cost of MDR-TB was $123 183 ($94 895, $160 033) with treatment-related consumables ($60 654) and hospitalizations ($42 147) correspondingly accounting for 49% and 34% of costs. The median (Q1, Q3) costs for DS-TB and LTBI were $3371 ($2718, $9397) and $861 ($818, $956), respectively.

Conclusion

MDR-TB is very costly to treat and manage in Canada. The high costs of MDR-TB appear to be driven by hospitalizations and treatment-related consumables.

 

The effect of nasal dilator strips on obstructive sleep apnea-hypopnea in pregnant women


Noa Goldscher1, Robert Gagnon2, Sara Meltzer3, Natasha Garfield3, Evelyne Rey2, Kaberi Dasgupta4,5, Stella Daskalopoulou4,5, Natalie Dayan4,5, Lorraine Lavigne1, Kathleen Raiche1, Genevieve Tremblay1, Allen Olha1, Andrea Benedetti4,5, Sushmita Pamidi1,5, R John Kimoff1,5
1Division of Respiratory medicine, McGill University Health Center 2Division of Obstertrics and Gynecology, McGill University Health Center 3Division of Endocrinology, McGill University Health Center 4Department of Medicine, McGill University Health Center 5Center for Outcomes Research Evaluation

Objective: Nasal dilator strips (NDS) have been proposed as a placebo intervention for non-pregnant adults with obstructive sleep apnea-hypopnea (OSAH). However, their effect on OSAH in pregnant women is unknown. We aimed to prospectively determine the effects of NDS treatment on daytime sleepiness, subjective sleep quality and polysomnographically (PSG)-determined sleep quality and OSAH severity during pregnancy.

Study design: Participants were recruited from a tertiary obstetrical clinic  as part of two randomized controlled trials comparing the effects of CPAP to NDS (“Breathe Right”) in women diagnosed with OSAH (AHI > 10 events/h; Chicago scoring criteria) during a singleton pregnancy together with either a hypertensive disorder of pregnancy (HDP) and/or gestational diabetes mellitus (GDM). Level II in-home PSG was performed at baseline to determine eligibility. In patients randomized to NDS, PSG was repeated after initiation of treatment.  Epworth sleepiness scale (ESS) and Pittsburgh Sleep Quality Index (PSQI) scores were ascertained close to the time of each PSG. NDS compliance was assessed by strip counting at 2-4-weekly study visits.

Results: 17 women randomized to NDS completed both PSGs (6 with HDP, 11 with GDM). Patients were of mean age 37.2 ± 5.3 y and BMI 32.7 ± 9.6; baseline PSG was performed at 28.7 ± 3.7 weeks’ gestation and NDS PSG at 33.7 ± 3.3 weeks (mean time between PSGs = 38.3 ± 17.7 weeks). Mean compliance with NDS over the course of pregnancy was 83 ± 33.9 %, with 11 (65%) patients applying NDS 100% of nights, and 13 (76%) patients >70% of nights. There were no statistically significant differences between baseline and NDS treatment values (Table) for ESS or PSQI, or objective measures of sleep quality or OSAH severity, with the exception of an increase in mean snoring time and in supine AHI on NDS PSG.

Conclusions: Pregnant women with OSAH were generally compliant with NDS treatment.  NDS did not improve daytime sleepiness, subjective or objective sleep quality or OSAH severity. While there was no evident short-term “placebo” effect on symptoms, the lack of improvement in objective OSAH severity suggests that NDS represents an acceptable control intervention for randomized trials evaluating the effects of CPAP or other active OSAH treatments during pregnancy.

The Impact of Enteric Helminth Infection on Pulmonary Innate Immune Modulation


Danielle Karo-Atar1, Alexander P. Meli1, Makan Golizeh2, Yue Lee1, Gabriel Russell1, Ghislaine Fontes1, Momar Ndao2,3, Irah King1
1Meakins-Christie Laboratories, McGill University Health Center Research Institute, Montreal, Canada 2Infectious Diseases and Immunity in Global Health Program, McGill University Health Center Research Institute, Montreal, Canada 3Department of Medicine, Division of Infectious Diseases, McGill University, Montreal, Canada

In recent years, new hypotheses addressing the possible mechanisms underlying the epidemiological link between allergic disorders, autoimmune diseases and the modern lifestyle have risen. These hypotheses stipulate that the reason for the increased incidence in allergic disorders, in particular, is reduced exposure to beneficial symbiotic bacteria (commensals) and/or parasitic worms. Indeed, co-evolution between intestinal helminths, commensal bacteria and their hosts has resulted in mammals that tolerate the presence of helminths and microbes that modulate the host immune response.

Mucosal tissues such as the intestine and lung face the complex task of maintaining homeostasis while directly interacting with the outside world. These two tissues do not function in isolation, but reciprocally regulate one another, particularly in the context of infection and inflammation, to form a “gut-lung immune axis”. Infection with the strictly enteric nematode, Heligmosomoides polygyrus bakeri (Hpb) exemplifies this gut-lung cross talk.  Hpb chronically infects the small intestine and has been reported to inhibit allergic airway inflammation (AAI). Importantly, while the adaptive immune regulation behind this gut-lung crosstalk had been addressed before, the role of innate immune cells in regulating the gut-lung axis has not been examined. In this work, we show that Hpb infection causes a systemic, time dependent, increase of alveolar macrophages, eosinophils, T cells and dendritic cells in the lung. Importantly, Hpb infection induces a classic Th2 inflammation in the lung including elevated RNA levels of Il4, Il13, Il5, Eotaxin2 and Retnla. Using numerous genetic approaches to deplete type 2 cytokine-producing cells, we establish that the influx of eosinophils is Th2 dependent while the increase in alvMacs is independent of Th2 cells, ILC2 and monocytes. Interestingly, the systemic response induced by Hpb is also independent of the microbiota, as antibiotic treated mice exhibit similar inflammation as flora replete mice. Taken together, these results establish a significant modulation of the innate immune compartment in the lung following Hpb infection and sets the foundation for a further understanding of helminth-mediated inhibition of AAI.

The Impact of Parkin Overexpression on Muscle Atrophy and Mitochondrial Morphology in Sepsis-Induced Skeletal Muscle Dysfunction


Jean-Philippe Leduc-Gaudet1,2, Tomer Jordi Chaffer1, Felipe Eduardo Broering1, Olivier Reynaud2, Dominique Mayaki1, Gilles Gouspillou2, Sabah NA Hussain1
1Meakins-Christie Laboratories, Department of Medicine and Division of Experimental Medicine, McGill University, Québec, Canada 2Département de Sciences de l’activité physique, Faculté des Sciences, UQAM, Québec, Canada

Rationale: The accumulation of mitochondrial dysfunction is believed to contribute to skeletal muscle weakness and loss in severe sepsis. Changes in mitochondrial mitophagy, dynamics and morphology can greatly impact mitochondrial function, and vice versa. However, whether mitochondrial mitophagy, dynamics and morphology in skeletal muscle are altered in sepsis remain largely unexplored. Here, we aimed to (1) investigate the effects of sepsis on mitochondrial dynamics and morphology in skeletal muscle; and (2) evaluate the impact of parkin overexpression, a protein in charge of the removal of dysfunctional mitochondria, on muscle fiber size and mitochondrial morphology using a quantitative 2-dimensional transmission electron microscopy approach.

 

Methods:  Parkin was overexpressed for 4 weeks in the gastrocnemius (GAS) muscles of 4 week-old mice using intramuscular injections of Adeno-Associated Viruses (AAV). A control AAV, containing a sequence coding for the green fluorescent protein (GFP), was injected in the contralateral leg. Sepsis was induced by cecal ligation and perforation (CLP). Control (Sham) mice were subjected to the same surgery, but the cecum was neither ligated nor punctured. The impact of sepsis and Parkin overexpression on muscle myofiber size, mitochondrial morphology and gene expression were investigated.

 

Results: As expected, CLP resulted in a significant decrease in body weight 48 hours after surgery. Sepsis increased the expression of MuRF1 and Atrogen-1, suggesting increased protein degradation. In line with these findings, CLP mice displayed an increased abundance of small fibers, a clear sign of atrophy. Parkin overexpression attenuated myofiber atrophy in CLP mice and resulted in myofiber  hypertrophy in Sham mice. Septic muscle displayed enlarged and more complex InterMyoFribriallar (IMF) mitochondria. Interestingly, Parkin overexpression reduced the morphological complexity of IMF mitochondria in both Sham and CLP mice. Sepsis decreased the expression of TFAM, complex II and complex IV, while Parkin-overexpressing muscles were protected. Sepsis increased the expression and content of LC3-II, SQSTM1 and BNIP3. Parkin overexpression did not affect the expression and content of these proteins.

 

Conclusions: The present study shows that sepsis alters mitochondrial morphology in skeletal muscles, an effect attenuated by Parkin overexpression. Our results also indicate that Parkin overexpression attenuates sepsis-induced myofiber atrophy. Although further studies are required, our findings place Parkin as a potential therapeutic target to counter sepsis-induced muscle dysfunction. 

 

 

Keywords: Sepsis; Mitophagy, Muscle Atrophy; Mitochondria, Autophagy  

 

Funding: This study is funded by CIHR grants. GG salary support is provided by a FQRS Chercheur Boursier Junior 1 AwardJPLG is supported by a CIHR Vanier Doctoral Scholarship. TJC is supported by an NSERC Undergraduate Summer Research Award.

 

The impacts of airway smooth muscle cells on CD4+ T cell trafficking and subset composition


Rui Sun1, Joyce Jang1, James G. Martin1,2
1Meakins-Christie Laboratories, Research Institute of the McGill University Health Centre 2Department of Medicine, McGill University

Background

CD4+ T cells infiltrate the airway smooth muscle (ASM) bundles and may play a role in directly inducing ASM hyperplasia in asthma. In vitro studies have shown that CD4+ T cells are capable of establishing contact-dependent intercellular communications with the ASM cells (ASMCs), which promote T cell survival and ASMC proliferation.

Study aims

In the current study, we explored the potential chemotactic signals that may mediate CD4+ T cell migration to the ASMC in human, and the potential selectivity of the chemoattraction for certain CD4+ T cell subsets. In addition, the influence of ASMC on CD4+ T cell phenotypes following the interactions with ASMCs, especially the subset composition, was investigated.

Methods

Migration was studied using the modified Boyden Chamber Assay, in which activated CD4+ T cells derived from peripheral blood, and ASMCs are seeded in the upper and lower wells of the chambers, respectively, and incubated for 24 hours. The impact of ASMCs on T cell phenotype was studied by comparing the T cells cultured alone and co-cultured with ASMCs. In both experiments, the proportions of the common CD4+ T cell subsets, T helper 1 (T1), TH2, T17 and regulatory T cells (Treg) were evaluated using flow cytometry and a panel of antibodies targeting relevant surface markers, mainly C-X-C motif chemokine receptor 3 (CXCR3), C-C motif chemokine receptor 4 (CCR4) and CCR6. The mRNA levels of the relevant chemokines in the ASMCs were assessed using quantitative real time-PCR.

Results

Compared to the controls with blank lower wells, the presence of ASMCs in the lower wells induced an approximately two-fold increase in the number of migrated CD4+ T cells. However, none of the subsets among the migrated T cells showed an increased abundance in the ASMC-present condition compared to the control, indicating that the pro-migratory effect of ASMC on T cells was not selective for any of the subsets. The TH1 population, termed by surface phenotype CD4+CXCR3+CCR4-CCR6-, was significantly reduced among the T cells co-cultured with ASMCs. Furthermore, among all conditions involving the exposure to ASMCs in both experiments, the level of the surface marker CXCR3 in the total T cells showed marked downregulation. The transcripts of the CXCR3 ligands, CXCL9, 10 and 11 were detected in the ASMCs, and for CXCL9 and 10, the transcript levels were elevated upon the exposure to T cells.

Conclusions

ASMCs synthesize molecules with pro-migratory effects on CD4+ T cells and are consistent with the involvement of CXCR3 in the recruitment of the T cells to the ASM.

The role of CD4 T cell derived HB-EGF in allergen driven airway remodeling


Niusha Khazaei1, Soroor Farahnak1, Toby K. McGovern1, Yumiko Ishii1, Michael Chen1, James G. Martin1
1Meakins-Christie Laboratories, Translational Research in Respiratory Diseases Program, The Research Institute of the McGill University Health Centre, and Department of Medicine, McGill University, Montreal, Quebec, Canada

Background: Type2 helper T (Th2) cells participate in the development of allergy-triggered asthma. One of the most important features in this type-2 mediated asthma is airway remodeling. Structural changes of the airway arise from hypertrophy/hyperplasia of airway smooth muscle (ASM) cells and metaplasia of goblet cells. Heparin binding epidermal growth factor (HB-EGF), a potent mitogen and chemotactic factor, could promote migration and proliferation of cells, including smooth muscle cells and goblet cells and is expressed by CD4+ T cells.

Hypothesis: We hypothesized that CD4 T cell derived HB-EGF participates in airway remodeling.

Methods and Results: We crossed HB-EGF (flox) mice with CD4-Cre-ERT2 mice to deplete HB-EGF in CD4 T cells.  We established a house dust mite (HDM)-induced allergic model using prolonged allergen exposure. Tamoxifen administration specifically deletes HB-EGF in CD4 T cells. Inflammation, airway hyper-responsiveness (AHR), and histological features of the airways were measured to explore the effects of HB-EGF on airway remodeling. The results confirmed the efficiency of allergic model and showed inflammatory cells, AHR, goblet cells metaplasia, and increased ASM mass in the chronic HDM-induced model compared to the control group. Administration of the same allergen to CD4-HB-EGF knock out mice for evaluation of airway remodeling progression parameters indicated a significantly decreased level of inflammation and AHR.  Moreover, we showed that goblet cells metaplasia and ASM mass were diminished about 2.5-fold in compare to control group mice. In addition, regardless of the airway size there was a positive correlation between ASM mass and the degree of inflammation in any given airway.

Conclusions:  CD4 T cell derived HB-EGF plays a significant role in airway remodeling caused by repeated HDM exposure in the mouse. Whether the effect is mediated directly by HB-EGF or through modulation of inflammation requires further study. 

The role of innate lymphoid cells (ILCs) in mice model of irritant-induced asthma


Utako Fujii1, Yumiko Ishii1, Emily Nakada1, Niusha Khazaei1,2, Joyce Jang1, Julia Chronopoulos1,2, Rui Sun1,2, Soroor Farahnak1, Toby McGovern1, James G Martin1
1Meakins–Christie Laboratories, McGill University Health Centre Research Institute and the Department of Medicine, McGill University 2McGill University

RATIONALE

Innate lymphoid cells (ILCs) are a recently described part of the innate immune system. ILCs have been classified based on surface markers, cytokine production, and transcription factors. Type 1 ILCs (ILC1s) produce IFN-γ (Transcription factor is T-bet); type 2 ILCs (ILC2s) produce IL-5 and IL-13 (Transcriptional factor is GATA3), and type 3 ILCs (ILC3s) which produce IL-17 and IL-22 (Transcription factor is RORγt). ILC2s have 2 subsets, natural ILC2s (nILC2s) and induced ILC2s (iILC2s). ILCs are reported to have an important role in airway inflammation including allergic asthma. However, the role of ILCs in irritant-induced asthma remains unclear. The purpose of the current study was to examine the involvement of ILCs in irritant-induced airway inflammation and dysfunction.

Hypothesis

We hypothesized that ILCs exercise a protective function in irritant induced asthma.

METHODS

BALB/c mice were nose-only exposed to 100 ppm chlorine. One day later we examined ILCs in chlorine (Cl2)-induced airway inflammation by performing flow cytometry (ILC; Lineage-, CD45+, CD90+, CD127+) on lung digests. We assessed inflammation and responses to inhaled aerosols of methacholine 3, 12, and 24hours after exposure. Then, we expanded the ILC population in the lung by administering IL-33 or IL-1β intranasally. We attenuated the ILC population with anti-CD90 antibody intraperitoneally.

RESULT

ILC2s and ILC3s were significantly increased 24 hours after Cl2 exposure.

IL-33 administration significantly increased the population of ILC2s and ILC3s whereas IL-1β caused an expansion of ILC3s. IL-33 administration to naive mice caused airway hyperresponsiveness (AHR) to methacholine and airway eosinophilic inflammation but reduced the airway neutrophilia and abolished AHR in mice exposed to Cl2. IL-1β did not alter airway response To Cl2.

Reduction of ILCs did not affect Cl2-induced AHR and BAL neutrophilia.

CONCLUSIONS

These data suggest that while constitutive levels of ILCs do not influence airway responses to inhaled Cl2, an expanded ILC2 population has a significant modulating role in the development of airway inflammation and AHR following Cl2 exposure.

Supported by a grant from the Canadian Institutes of Health Research

The Role of las Quorum Sensing in the Host-Pathogen Interactions of Intracellular Pseudomonas aeruginosa in Airway Epithelial Cells


Cedric Hupperetz1, Lisa Hennemann1, Dao Nguyen1,2
1Department of Microbiology and Immunology, McGill University, Montreal 2McGill University Health Centre

Pseudomonas aeruginosa is an opportunistic gram-negative pathogen that causes severe infections in compromised hosts. It can control the expression of many of its genes, including virulence factors, through a cell-density-dependent mechanism known as quorum sensing. The quorum sensing transcriptional regulator LasR is a master activator that controls nearly 10% of the P. aeruginosa genes and other quorum sensing mechanisms. Despite its role, loss-of-function LasR mutants can be isolated in up to two-thirds of cystic fibrosis patients with chronic P. aeruginosa lung infections. Surprisingly, infections with lasR mutants are associated with worse lung function decline. How loss of LasR function alters host-pathogen interactions remains incompletely understood. Preliminary data from our lab suggests that P. aeruginosa, while classically considered an extracellular pathogen, can be internalized and persist within epithelial cells. This intracellular lifestyle may provide a mechanism for antibiotic and immune evasion. We hypothesized that loss-of-function lasR mutants are less cytotoxic and are internalized to a greater extent in bronchial epithelial cells. We tested the effects of a lasR-knockout mutation on the internalization and cytotoxicity of P. aeruginosa in Beas-2B bronchial epithelial cells using a tobramycin protection assay. We compared seven different lasR mutant and isogenic parental strains from laboratory or clinical genetic backgrounds. We show that there is no clear correlation between the loss of LasR function and intracellular cytotoxicity. However, the intracellular bacterial burden after 24 hours of infectionis greater in lasR compared to wild-type infections. These results suggest that loss of LasR function may promote an intracellular lifestyle, a potential mechanism for long term persistence in the host.

Understanding how Pseudomonas aeruginosa quorum sensing modulates lung inflammation


Lisa Hennemann1,2, Perrine Bortolotti1, Simon Rousseau1,2, Dao Nguyen1,3
1McGill University Health Centre 2McGill University 3McGill

Chronic Pseudomonas aeruginosa (PA) infections occur in 60-80% of all adult Cystic Fibrosis (CF) patients and are associated with an accelerated lung function decline and increased mortality. Due to host and bacterial factors that allow PA to rapidly adapt to the host environment and cause impaired bacterial clearance, PA is nearly impossible to eradicate once it has established a chronic infection. PA infections cause progressive lung disease by inducing an exuberant and non-resolving, primarily neutrophilic lung inflammation. Interestingly, CF-adapted PA strains isolated from late disease stages (“late isolates), when compared to their clonally related ancestral strain isolated from early disease stage (“early” isolates) in the same patients, share a number of genotypic and phenotypic adaptive changes. Loss of function lasR mutations are observed in up to 63% of late clinical PA isolates and are associated with accelerated lung function decline in CF patients. Recent work in our lab showed that lasR lung infections were associated with an exaggerated neutrophilic inflammatory response and immunopathology compared to wild-type PA infections. LasR is the major quorum sensing transcriptional regulator LasR, which controls the transcription of nearly 10% of all genes, including many acute virulence factors. Loss of LasR-regulated factors can thus alter host-PA interactions through multiple different mechanisms.

In this project, we hypothesize that LasR-regulated factors might modulate inflammation in the host lung by altering the expression of ICAM-1, an important pro-inflammatory mediator expressed by airway epithelial cells (AEC).

Our first aim is to analyze the impact of LasR loss of function on expression of membrane-bound ICAM-1 (mICAM-1) on AEC in response to PA extracellular diffusible bacterial products or live bacteria. Preliminary results show that expression of mICAM-1 is upregulated in response to lasR mutant filtrates compared to filtrates from wild-type PA. This mICAM-1 upregulation enhances neutrophil binding to AECs in vitro, as measured by a neutrophil adhesion assay, suggesting that it might promote neutrophilic lung inflammation in the CF airway. We further observed that mice infected with lasR mutant strains express significantly higher levels of mICAM-1 on the bronchial epithelium than mice infected with the wild-type.

Our second aim is to investigate which LasR-regulated factor(s) may be responsible for the increased mICAM-1 expression and neutrophil binding. Heat-treatment of PA filtrates abrogates the differences in mICAM-1 expression between wild-type and ΔlasR filtrates, indicating that heat-labile LasR-dependent compound(s) mediate the difference. PA expresses LasR-regulated secreted proteases potentially involved in modulating host cell surface receptor expression and cytokine production. We therefore show that LasR is protease deficient and complementation of LasR-deficient strains with the elastase LasB restores AEC expression of mICAM-1 to wild-type levels.

In conclusion, our results suggest that loss of LasR might increase lung inflammation due to loss of LasB activity. Our work highlights a possible new pathway through which a commonly observed adaptation may contribute to lung inflammation and thus exacerbate lung function decline.