Cannabis smoke suppresses antiviral immune responses to influenza A in mice.

Rationale: Despite its increasingly widespread use, little is known about the impact of cannabis smoking on the response to viral infections like influenza A virus (IAV). Many assume that cannabis smoking will disrupt antiviral responses in a manner similar to cigarette smoking; however, since cannabinoids exhibit anti-inflammatory effects, cannabis smoke exposure may impact viral infection in distinct ways. Methods: Male and female BALB/c mice were exposed daily to cannabis smoke and concurrently intranasally instilled with IAV. Viral burden, inflammatory mediator levels (multiplex ELISA), lung immune cells populations (flow cytometry) and gene expression patterns (RNA sequencing) were assessed in the lungs. Plasma IAV-specific antibodies were measured via ELISA. Results: We found that cannabis smoke exposure increased pulmonary viral burden while decreasing total leukocytes, including macrophages, monocytes and dendritic cell populations in the lungs. Furthermore, infection-induced upregulation of certain inflammatory mediators (interferon-γ and C-C motif chemokine ligand 5) was blunted by cannabis smoke exposure, which in females was linked to the transcriptional downregulation of pathways involved in innate and adaptive immune responses. Finally, plasma levels of IAV-specific IgM and IgG1 were significantly decreased in cannabis smoke-exposed, infected mice compared to infected controls, only in female mice. Conclusions: Overall, cannabis smoke exposure disrupted host-defence processes, leading to increased viral burden and dampened inflammatory signalling. These results suggest that cannabis smoking is detrimental to the maintenance of pulmonary homeostasis during viral infection and highlight the need for data regarding the impact on immune competency in humans.

Myeloid-specific deletion of activating transcription factor 6 alpha increases CD11b+ macrophage subpopulations and aggravates lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrotic interstitial lung disease of unknown etiology. The accumulation of macrophages is associated with disease pathogenesis. The unfolded protein response (UPR) has been linked to macrophage activation in pulmonary fibrosis. To date, the impact of activating transcription factor 6 alpha (ATF6α), one of the UPR mediators, on the composition and function of pulmonary macrophage subpopulations during lung injury and fibrogenesis is not fully understood. We began by examining the expression of Atf6α in IPF patients' lung single-cell RNA sequencing dataset, archived surgical lung specimens, and CD14+ circulating monocytes. To assess the impact of ATF6α on pulmonary macrophage composition and pro-fibrotic function during tissue remodeling, we conducted an in vivo myeloid-specific deletion of Atf6α. Flow cytometric assessments of pulmonary macrophages were carried out in C57BL/6 and myeloid specific ATF6α-deficient mice in the context of bleomycin-induced lung injury. Our results demonstrated that Atf6α mRNA was expressed in pro-fibrotic macrophages found in the lung of a patient with IPF and in CD14+ circulating monocytes obtained from blood of a patient with IPF. After bleomycin administration, the myeloid-specific deletion of Atf6α altered the pulmonary macrophage composition, expanding CD11b+ subpopulations with dual polarized CD38+ CD206+ expressing macrophages. Compositional changes were associated with an aggravation of fibrogenesis including increased myofibroblast and collagen deposition. A further mechanistic ex vivo investigation revealed that ATF6α was required for CHOP induction and the death of bone marrow-derived macrophages. Overall, our findings suggest a detrimental role for the ATF6α-deficient CD11b+ macrophages which had altered function during lung injury and fibrosis.

Multi-omics analyses of airway host-microbe interactions in chronic obstructive pulmonary disease identify potential therapeutic interventions.

The mechanistic role of the airway microbiome in chronic obstructive pulmonary disease (COPD) remains largely unexplored. We present a landscape of airway microbe-host interactions in COPD through an in-depth profiling of the sputum metagenome, metabolome, host transcriptome and proteome from 99 patients with COPD and 36 healthy individuals in China. Multi-omics data were integrated using sequential mediation analysis, to assess in silico associations of the microbiome with two primary COPD inflammatory endotypes, neutrophilic or eosinophilic inflammation, mediated through microbial metabolic interaction with host gene expression. Hypotheses of microbiome-metabolite-host interaction were identified by leveraging microbial genetic information and established metabolite-human gene pairs. A prominent hypothesis for neutrophil-predominant COPD was altered tryptophan metabolism in airway lactobacilli associated with reduced indole-3-acetic acid (IAA), which was in turn linked to perturbed host interleukin-22 signalling and epithelial cell apoptosis pathways. In vivo and in vitro studies showed that airway microbiome-derived IAA mitigates neutrophilic inflammation, apoptosis, emphysema and lung function decline, via macrophage-epithelial cell cross-talk mediated by interleukin-22. Intranasal inoculation of two airway lactobacilli restored IAA and recapitulated its protective effects in mice. These findings provide the rationale for therapeutically targeting microbe-host interaction in COPD.

Inflammatory Endotype-Associated Airway Resistome in Chronic Obstructive Pulmonary Disease.

Antimicrobial resistance is a global concern in chronic respiratory diseases, including chronic obstructive pulmonary disease (COPD). The collection of antibiotic resistance genes or resistome in human airways may underlie the resistance. COPD is heterogeneous, and understanding the airway resistome in relation to patient phenotype and endotype may inform precision antibiotic therapy. Here, we characterized the airway resistome for 94 COPD participants at stable disease. Among all demographic and clinical factors, patient inflammatory endotype was associated with the airway resistome. There were distinct resistome profiles between patients with neutrophilic or eosinophilic inflammation, two primary inflammatory endotypes in COPD. For neutrophil-predominant COPD, the resistome was dominated by multidrug resistance genes. For eosinophil-predominant COPD, the resistome was diverse, with an increased portion of patients showing a macrolide-high resistome. The differential antimicrobial resistance pattern was validated by sputum culture and in vitro antimicrobial susceptibility testing. Ralstonia and Pseudomonas were the top contributors to the neutrophil-associated resistome, whereas Campylobacter and Aggregatibacter contributed most to the eosinophil-associated resistome. Multiomic analyses revealed specific host pathways and inflammatory mediators associated with the resistome. The arachidonic acid metabolic pathway and matrix metallopeptidase 8 (MMP-8) exhibited the strongest associations with the neutrophil-associated resistome, whereas the eosinophil chemotaxis pathway and interleukin-13 (IL-13) showed the greatest associations with the eosinophil-associated resistome. These results highlight a previously unrecognized link between inflammation and the airway resistome and suggest the need for considering patient inflammatory subtype in decision-making about antibiotic use in COPD and broader chronic respiratory diseases. IMPORTANCE Antibiotics are commonly prescribed for both acute and long-term prophylactic treatment in chronic airway disorders, such as chronic obstructive pulmonary disease (COPD), and the rapid growth of antibiotic resistance is alarming globally. The airway harbors a diverse collection of microorganisms known as microbiota, which serve as a reservoir for antibiotic resistance genes or the resistome. A comprehensive understanding of the airway resistome in relation to patient clinical and biological factors may help inform decisions to select appropriate antibiotics for clinical therapies. By deep multiomic profiling and in vitro phenotypic testing, we showed that inflammatory endotype, the underlying pattern of airway inflammation, was most strongly associated with the airway resistome in COPD patients. There were distinct resistome profiles between neutrophil-predominant and eosinophil-predominant COPD that were associated with different bacterial species, host pathways, and inflammatory markers, highlighting the need of considering patient inflammatory status in COPD antibiotic management.

Cigarette smoke augments CSF3 expression in neutrophils to compromise alveolar-capillary barrier function during influenza infection.

BACKGROUND: Cigarette smokers are at increased risk of acquiring influenza, developing severe disease and requiring hospitalisation/intensive care unit admission following infection. However, immune mechanisms underlying this predisposition are incompletely understood, and therapeutic strategies for influenza are limited. METHODS: We used a mouse model of concurrent cigarette smoke exposure and H1N1 influenza infection, colony-stimulating factor (CSF)3 supplementation/receptor (CSF3R) blockade and single-cell RNA sequencing (scRNAseq) to investigate this relationship. RESULTS: Cigarette smoke exposure exacerbated features of viral pneumonia such as oedema, hypoxaemia and pulmonary neutrophilia. Smoke-exposed infected mice demonstrated an increase in viral (v)RNA, but not replication-competent viral particles, relative to infection-only controls. Interstitial rather than airspace neutrophilia positively predicted morbidity in smoke-exposed infected mice. Screening of pulmonary cytokines using a novel dysregulation score identified an exacerbated expression of CSF3 and interleukin-6 in the context of smoke exposure and influenza. Recombinant (r)CSF3 supplementation during influenza aggravated morbidity, hypothermia and oedema, while anti-CSF3R treatment of smoke-exposed infected mice improved alveolar-capillary barrier function. scRNAseq delineated a shift in the distribution of Csf3 + cells towards neutrophils in the context of cigarette smoke and influenza. However, although smoke-exposed lungs were enriched for infected, highly activated neutrophils, gene signatures of these cells largely reflected an exacerbated form of typical influenza with select unique regulatory features. CONCLUSION: This work provides novel insight into the mechanisms by which cigarette smoke exacerbates influenza infection, unveiling potential therapeutic targets (e.g. excess vRNA accumulation, oedematous CSF3R signalling) for use in this context, and potential limitations for clinical rCSF3 therapy during viral infectious disease.

Increased Monocyte-Derived CD11b+ Macrophage Subpopulations Following Cigarette Smoke Exposure Are Associated With Impaired Bleomycin-Induced Tissue Remodelling.

Rationale: The accumulation of macrophages in the airways and the pulmonary interstitium is a hallmark of cigarette smoke-associated inflammation. Notably, pulmonary macrophages are not a homogenous population but consist of several subpopulations. To date, the manner in which cigarette smoke exposure affects the relative composition and functional capacity of macrophage subpopulations has not been elucidated. Methods: Using a whole-body cigarette smoke exposure system, we investigated the impact of cigarette smoke on macrophage subpopulations in C57BL/6 mice using flow cytometry-based approaches. Moreover, we used bromodeoxyuridine labelling plus Il1a-/- and Il1r1-/- mice to assess the relative contribution of local proliferation and monocyte recruitment to macrophage accumulation. To assess the functional consequences of altered macrophage subpopulations, we used a model of concurrent bleomycin-induced lung injury and cigarette smoke exposure to examine tissue remodelling processes. Main Results: Cigarette smoke exposure altered the composition of pulmonary macrophages increasing CD11b+ subpopulations including monocyte-derived alveolar macrophages (Mo-AM) as well as interstitial macrophages (IM)1, -2 and -3. The increase in CD11b+ subpopulations was observed at multiple cigarette smoke exposure timepoints. Bromodeoxyuridine labelling and studies in Il1a-/- mice demonstrated that increased Mo-AM and IM3 turnover in the lungs of cigarette smoke-exposed mice was IL-1α dependent. Compositional changes in macrophage subpopulations were associated with impaired induction of fibrogenesis including decreased α-smooth muscle actin positive cells following intratracheal bleomycin treatment. Mechanistically, in vivo and ex vivo assays demonstrated predominant macrophage M1 polarisation and reduced matrix metallopeptidase 9 activity in cigarette smoke-exposed mice. Conclusion: Cigarette smoke exposure modified the composition of pulmonary macrophage by expanding CD11b+ subpopulations. These compositional changes were associated with attenuated fibrogenesis, as well as predominant M1 polarisation and decreased fibrotic activity. Overall, these data suggest that cigarette smoke exposure altered the composition of pulmonary macrophage subpopulations contributing to impaired tissue remodelling.

Development and validation of a mouse model of contemporary cannabis smoke exposure.

Cannabis is widely used for both recreational and medicinal purposes. Inhalation of combusted cannabis smoke is the most common mode of drug consumption, exposing the lungs to the pharmacologically active ingredients, including tetrahydrocannabinol (THC) and cannabidiol (CBD). While the relationship between cannabis smoke exposure and compromised respiratory health has yet to be sufficiently defined, previous investigations suggest that cannabis smoke may dysregulate pulmonary immunity. Presently, there exist few preclinical animal models that have been extensively validated for contemporary cannabis smoke exposure. To address this need, we developed a mouse model with readouts of total particulate matter, serum cannabinoid and carboxyhaemoglobin levels, lung cellular responses, and immune-mediator production. Using a commercially available smoke exposure system and a cannabis source material of documented THC/CBD composition, we exposed mice to a mean±sd total particulate matter of 698.89±66.09 µg·L-1 and demonstrate increases in serum cannabinoids and carboxyhaemoglobin. We demonstrate that cannabis smoke modulates immune cell populations and mediators in both male and female BALB/c mice. This modulation is highlighted by increases in airway and lung tissue macrophage populations, including tissue-resident alveolar macrophages, monocyte-derived alveolar macrophages, and interstitial macrophage subpopulations. No changes in airway or lung tissue infiltration of neutrophils were observed. Immune-mediator analysis indicated significant upregulation of macrophage-derived chemokine, thymus and activation-regulated chemokine, and vascular endothelial growth factor within the lung tissue of cannabis smoke-exposed mice. This accessible and reproducible smoke-exposure model provides a foundation to explore the impact of chronic cannabis exposures and/or co-exposures with pathogens of clinical relevance, such as influenza.

Cigarette smoke exposure attenuates the induction of antigen-specific IgA in the murine upper respiratory tract.

The upper respiratory tract is highly exposed to airborne pathogens and serves as an important inductive site for protective antibody responses, including mucosal IgA and systemic IgG. However, it is currently unknown to what extent inhaled environmental toxins, such as a cigarette smoke, affect the ability to induce antibody-mediated immunity at this site. Using a murine model of intranasal lipopolysaccharide and ovalbumin (LPS/OVA) immunization, we show that cigarette smoke exposure compromises the induction of antigen-specific IgA in the upper airways and systemic circulation. Deficits in OVA-IgA were observed in conjunction with a reduced accumulation of OVA-specific IgA antibody-secreting cells (ASCs) in the nasal mucosa, inductive tissues (NALT, cervical lymph nodes, spleen) and the blood. Nasal OVA-IgA from smoke-exposed mice also demonstrated reduced avidity during the acute post-immunization period in association with an enhanced mutational burden in the cognate nasal Igha repertoire. Mechanistically, smoke exposure attenuated the ability of the nasal mucosa to upregulate VCAM-1 and pIgR, suggesting that cigarette smoke may inhibit both nasal ASC homing and IgA transepithelial transport. Overall, these findings demonstrate the immunosuppressive nature of tobacco smoke and illustrate the diversity of mechanisms through which this noxious stimulus can interfere with IgA-mediated immunity in the upper airways.

Differential expression of sputum and serum autoantibodies in patients with chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a complex and progressive respiratory disease. Autoimmune processes have been hypothesized to contribute to disease progression; however, the presence of autoantibodies in the serum has been variable. Given that COPD is a lung disease, we sought to investigate whether autoantibodies in sputum supernatant would better define pulmonary autoimmune processes. Matched sputum and serum samples were obtained from the Airways Disease Endotyping for Personalized Therapeutics (ADEPT) study and at the Guangzhou Institute of Respiratory Health (GIRH). Samples were collected from patients with varying severity of COPD, asymptomatic smokers, and healthy control subjects. IgG and IgM autoantibodies were detected in sputum and serum of all subjects in both cohorts using a broad-spectrum autoantigen array. No differences were observed in sputum autoantibodies between COPD and asymptomatic smokers in either cohort. In contrast, 16% of detectable sputum IgG autoantibodies were decreased in subjects with COPD compared to healthy controls in the ADEPT cohort. Compared to asymptomatic smokers, approximately 13% of detectable serum IgG and 40% of detectable serum IgM autoantibodies were differentially expressed in GIRH COPD subjects. Of the differentially expressed specificities, anti-nuclear autoantibodies were predominately decreased. A weak correlation between increased serum IgM anti-tissue autoantibodies and a measure of airspace enlargement was observed. The differential expression of specificities varied between the cohorts. In closing, using a comprehensive autoantibody array, we demonstrate that autoantibodies are present in subjects with COPD, asymptomatic smokers, and healthy controls. Cohorts displayed high levels of heterogeneity, precluding the utilization of autoantibodies for diagnostic purposes.

Nasal Tissue Extraction Is Essential for Characterization of the Murine Upper Respiratory Tract Microbiota.

Respiratory infections are a leading cause of morbidity and mortality worldwide. Bacterial pathogens often colonize the upper respiratory tract (nose or mouth) prior to causing lower respiratory infections or invasive disease. Interactions within the upper respiratory tract between colonizing bacteria and the resident microbiota could contribute to colonization success and subsequent transmission. Human carriage studies have identified associations between pathogens such as Streptococcus pneumoniae and members of the resident microbiota, although few mechanisms of competition and cooperation have been identified and would be aided by the use of animal models. Little is known about the composition of the murine nasal microbiota; thus, we set out to improve assessment, including tissue sampling, composition, and comparison between mouse sources. Nasal washes were efficient in sampling the nasopharyngeal space but barely disrupted the nasal turbinates. Nasal tissue extraction increased the yield of cultivable bacterial compared to nasal washes, revealing distinct community compositions. Experimental pneumococcal colonization led to dominance by the colonizing pathogen in the nasopharynx and nasal turbinates, but the composition of the microbiota, and interactions with resident microbes, differed depending on the sampling method. Importantly, vendor source has a large impact on microbial composition. Bacterial interactions, including cooperation and colonization resistance, depend on the biogeography of the nose and should be considered during research design of experimental colonization with pathogens.IMPORTANCE The nasal microbiota is composed of species that play a role in the colonization success of pathogens, including Streptococcus pneumoniae and Staphylococcus aureus Murine models provide the ability to explore disease pathogenesis, but little is known about the natural murine nasal microbiota. This study established techniques to allow the exploration of the bacterial members of the nasal microbiota. The mouse nasal microbiota included traditional respiratory bacteria, including Streptococcus, Staphylococcus, and Moraxella species. Analyses were affected by different sampling methods as well as the commercial source of the mice, which should be included in future research design of infectious disease research.

Disruption of Physiological Rhythms Persist Following Cessation of Cigarette Smoke Exposure in Mice.

BACKGROUND: Physiological rhythms in mammals are essential for maintaining health, whereas disruptions may cause or exacerbate disease pathogenesis. As such, our objective was to characterize how cigarette smoke exposure affects physiological rhythms of otherwise healthy mice using telemetry and cosinor analysis. METHODS: Female BALB/c mice were implanted with telemetry devices to measure body temperature, heart rate, systolic blood pressure (SBP), and activity. Following baseline measurements, mice were exposed to cigarette smoke for approximately 50 min twice daily during weekdays over 24 weeks. Physiological parameters were recorded after 1, 4, 8, and 24 weeks of exposure or after 4 weeks cessation following 4 weeks of cigarette smoke exposure. RESULTS: Acute cigarette smoke exposure resulted in anapyrexia, and bradycardia, with divergent effects on SBP. Long term, cigarette smoke exposure disrupted physiological rhythms after just 1 week, which persisted across 24 weeks of exposure (as shown by mixed effects on mesor, amplitude, acrophase, and goodness-of-fit using cosinor analysis). Four weeks of cessation was insufficient to allow full recovery of rhythms. CONCLUSION: Our characterization of the pathophysiology of cigarette smoke exposure on physiological rhythms of mice suggests that rhythm disruption may precede and contribute to disease pathogenesis. These findings provide a clear rationale and guide for the future use of chronotherapeutics.

Estradiol Enhances Antiviral CD4+ Tissue-Resident Memory T Cell Responses following Mucosal Herpes Simplex Virus 2 Vaccination through an IL-17-Mediated Pathway.

Estradiol (E2) is a sex hormone which has been shown to be protective against sexually transmitted infections such as herpes simplex virus 2 (HSV-2). However, few studies have examined the underlying mechanisms by which this occurs. Here, we investigated the effect of E2 on the establishment of memory T cells post-intranasal immunization with HSV-2. CD4+ T cell responses first appeared in the upper respiratory tract (URT) within 3 days postimmunization before being detected in the female reproductive tract (FRT) at 7 days. E2 treatment resulted in greater and earlier Th17 responses, which preceded augmented Th1 responses at these sites. The CD4+ T cells persisted in the URT for up to 28 days, and E2 treatment resulted in higher frequencies of memory T cells. Intranasal immunization also led to the establishment of CD4+ tissue-resident memory T cells (TRM cells) in the FRT, and E2 treatment resulted in increased Th1 and Th17 TRM cells. When the migration of circulating T cells into the FRT was blocked by FTY720, immunized E2-treated mice remained completely protected against subsequent genital HSV-2 challenge compared to non-E2 controls, confirming that TRM cells alone are adequate for protection in these mice. Finally, the enhanced vaginal Th1 TRM cells present in E2-treated mice were found to be modulated through an interleukin 17 (IL-17)-mediated pathway, as E2-treated IL-17A-deficient mice had impaired establishment of Th1 TRM cells. This study describes a novel role for E2 in enhancing CD4+ memory T cells and provides insight on potential strategies for generating optimal immunity during vaccination.IMPORTANCE Herpes simplex virus 2 (HSV-2) is a highly prevalent sexually transmitted infection for which there is currently no vaccine available. Interestingly, the female sex hormone estradiol has been shown to be protective against HSV-2. However, the underlying mechanisms by which this occurs remains relatively unknown. Our study demonstrates that under the influence of estradiol treatment, intranasal immunization with an attenuated strain of HSV-2 leads to enhanced establishment of antiviral memory T cell responses in the upper respiratory tract and female reproductive tract. In these sites, estradiol treatment leads to greater Th17 memory cells, which precede enhanced Th1 memory responses. Consequently, the T cell responses mounted by tissue-resident memory cells in the female reproductive tract of estradiol-treated mice are sufficient to protect mice against vaginal HSV-2 challenge. This study offers important insights regarding the regulation of mucosal immunity by hormones and on potential strategies for generating optimal immunity during vaccination.

Dissociation between airway and systemic autoantibody responses in chronic obstructive pulmonary disease.

BACKGROUND: Autoimmune processes have been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). However, the relationship between airway and systemic autoantibody responses remains unclear. The aim of this study was to elucidate this relationship in patients with stable COPD by investigating the correlation patterns between sputum and serum autoantibodies. METHODS: In this cross-sectional study, sputum supernatant and serum obtained from 47 patients with stable COPD were assayed for the presence of IgG antibodies against ten autoantigens: Smith antigen (Sm), ribosomal phosphoprotein P0 (P0), Ro/Sjögren syndrome type A antigen (Ro/SSA), La/Sjögren syndrome type B antigen (La/SSB), DNA topoisomerase I (Scl-70), histidyl-tRNA synthetase (Jo-1), U1 small nuclear ribonucleoprotein (U1-SnRNP), thyroid peroxidase (TPO), proteinase-3 (PR3), and myeloperoxidase (MPO). A second cohort of 55 stable COPD patients was recruited for validation, and a group of 59 non-COPD controls and a group of 20 connective-tissue disease-associated interstitial lung disease (CTD-ILD) patients were also recruited for comparison. Hierarchical clustering and network analysis were used to evaluate the correlation patterns between sputum and serum autoantibody profiles. RESULTS: Both hierarchical clustering and network analysis showed that sputum and serum autoantibody profiles were distinct in either analytic COPD cohort or validation cohort. In contrast, the autoantibodies of the two compartments in non-COPD controls and CTD-ILD patients were inadequately distinguished using either hierarchical clustering or network analysis. Many autoantibodies in the sputum were found to have significant correlations with lung function, symptom score and frequency of prior exacerbations in COPD patients, but the antibodies in the serum were not. CONCLUSIONS: We observed a dissociation between sputum autoantibodies and serum autoantibodies in patients with stable COPD, suggesting that airway and systemic immune status may play very different roles in the disease. Sputum autoantibodies are more clinically relevant than serum autoantibodies. Focusing on airway autoimmunity may help improve understanding of the immunopathological mechanism of COPD.

A Refined View of Airway Microbiome in Chronic Obstructive Pulmonary Disease at Species and Strain-Levels.

Little is known about the underlying airway microbiome diversity in chronic obstructive pulmonary disease (COPD) at in-depth taxonomic levels. Here we present the first insights on the COPD airway microbiome at species and strain-levels. The full-length 16S rRNA gene was characterized from sputum in 98 COPD patients and 27 age-matched healthy controls, using the Pacific Biosciences sequencing platform. Individual species within the same genus exhibited reciprocal relationships with COPD and disease severity. Species dominant in health can be taken over by another species within the same genus but with potentially increasing pathogenicity in severe COPD patients. Ralstonia mannitolilytica, an opportunistic pathogen, was significantly increased in frequent exacerbators (fold-change = 4.94, FDR P = 0.005). There were distinct patterns of interaction between bacterial species and host inflammatory mediators according to neutrophilic or eosinophilic inflammations, two major airway inflammatory phenotypes in COPD. Haemophilus influenzae, Moraxella catarrhalis, Pseudomonas aeruginosa, and Neisseria meningitidis were associated with enhanced Th1, Th17 and pro-inflammatory mediators, while a group of seven species including Tropheryma whipplei were specifically associated with Th2 mediators related to eosinophilia. We developed an automated pipeline to assign strain-level taxonomy leveraging bacterial intra-genomic 16S allele frequency. Using this pipeline we further resolved three non-typeable H. influenzae strains PittEE, PittGG and 86-028NP with reasonable precision and uncovered strain-level variation related to airway inflammation. In particular, 86-028NP and PittGG strains exhibited inverse associations with Th2 chemokines CCL17 and CCL13, suggesting their abundances may inversely predict eosinophilic inflammation. A systematic comparison of 16S hypervariable regions indicated V1V3 instead of the commonly used V4 region was the best surrogate for airway microbiome. The full-length 16S data augmented the power of functional inference, which slightly better recapitulated the actual metagenomes. This led to the unique identification of butyrate-producing and nitrate reduction pathways as depleted in COPD. Our analysis uncovered finer-scale airway microbial diversity that was previously underappreciated, thus enabled a refined view of the airway microbiome in COPD.

Multi-omic meta-analysis identifies functional signatures of airway microbiome in chronic obstructive pulmonary disease.

The interaction between airway microbiome and host in chronic obstructive pulmonary disease (COPD) is poorly understood. Here we used a multi-omic meta-analysis approach to characterize the functional signature of airway microbiome in COPD. We retrieved all public COPD sputum microbiome datasets, totaling 1640 samples from 16S rRNA gene datasets and 26 samples from metagenomic datasets from across the world. We identified microbial taxonomic shifts using random effect meta-analysis and established a global classifier for COPD using 12 microbial genera. We inferred the metabolic potentials for the airway microbiome, established their molecular links to host targets, and explored their effects in a separate meta-analysis on 1340 public human airway transcriptome samples for COPD. 29.6% of differentially expressed human pathways were predicted to be targeted by microbiome metabolism. For inferred metabolite-host interactions, the flux of disease-modifying metabolites as predicted from host transcriptome was generally concordant with their predicted metabolic turnover in microbiome, suggesting a synergistic response between microbiome and host in COPD. The meta-analysis results were further validated by a pilot multi-omic study on 18 COPD patients and 10 controls, in which airway metagenome, metabolome, and host transcriptome were simultaneously characterized. 69.9% of the proposed "microbiome-metabolite-host" interaction links were validated in the independent multi-omic data. Butyrate, homocysteine, and palmitate were the microbial metabolites showing strongest interactions with COPD-associated host genes. Our meta-analysis uncovered functional properties of airway microbiome that interacted with COPD host gene signatures, and demonstrated the possibility of leveraging public multi-omic data to interrogate disease biology.

Cigarette smoke-initiated autoimmunity facilitates sensitisation to elastin-induced COPD-like pathologies in mice.

It is currently not understood whether cigarette smoke exposure facilitates sensitisation to self-antigens and whether ensuing auto-reactive T cells drive chronic obstructive pulmonary disease (COPD)-associated pathologies.To address this question, mice were exposed to cigarette smoke for 2 weeks. Following a 2-week period of rest, mice were challenged intratracheally with elastin for 3 days or 1 month. Rag1-/- , Mmp12-/- , and Il17a-/- mice and neutralising antibodies against active elastin fragments were used for mechanistic investigations. Human GVAPGVGVAPGV/HLA-A*02:01 tetramer was synthesised to assess the presence of elastin-specific T cells in patients with COPD.We observed that 2 weeks of cigarette smoke exposure induced an elastin-specific T cell response that led to neutrophilic airway inflammation and mucus hyperproduction following elastin recall challenge. Repeated elastin challenge for 1 month resulted in airway remodelling, lung function decline and airspace enlargement. Elastin-specific T cell recall responses were dose dependent and memory lasted for over 6 months. Adoptive T cell transfer and studies in T cells deficient Rag1-/- mice conclusively implicated T cells in these processes. Mechanistically, cigarette smoke exposure-induced elastin-specific T cell responses were matrix metalloproteinase (MMP)12-dependent, while the ensuing immune inflammatory processes were interleukin 17A-driven. Anti-elastin antibodies and T cells specific for elastin peptides were increased in patients with COPD.These data demonstrate that MMP12-generated elastin fragments serve as a self-antigen and drive the cigarette smoke-induced autoimmune processes in mice that result in a bronchitis-like phenotype and airspace enlargement. The study provides proof of concept of cigarette smoke-induced autoimmune processes and may serve as a novel mouse model of COPD.

Effect of long-acting ß-agonists/glucocorticoids on human airway epithelial cell cytokine, transcriptomic and oxidative stress responses to cannabis smoke.

LABA/GC intervention in airway epithelial cells exposed to cannabis smoke reduces levels of pro-inflammatory (CXCL8) and antiviral (CXCL10) mediators, while transcriptomic signatures of neutrophil-mediated immunity and oxidative stress remain elevated http://bit.ly/2qiSQhH.

Transcriptomic and barrier responses of human airway epithelial cells exposed to cannabis smoke.

Globally, many jurisdictions are legalizing or decriminalizing cannabis, creating a potential public health issue that would benefit from experimental evidence to inform policy, government regulations, and user practices. Tobacco smoke exposure science has created a body of knowledge that demonstrates the conclusive negative impacts on respiratory health; similar knowledge remains to be established for cannabis. To address this unmet need, we performed in vitro functional and transcriptomic experiments with a human airway epithelial cell line (Calu-3) exposed to cannabis smoke, with tobacco smoke as a positive control. Demonstrating the validity of our in vitro model, tobacco smoke induced gene expression profiles that were significantly correlated with gene expression profiles from published tobacco exposure datasets from bronchial brushings and primary human airway epithelial cell cultures. Applying our model to cannabis smoke, we demonstrate that cannabis smoke induced functional and transcriptional responses that overlapped with tobacco smoke. Ontology and pathway analysis revealed that cannabis smoke induced DNA replication and oxidative stress responses. Functionally, cannabis smoke impaired epithelial cell barrier function, antiviral responses, and increased inflammatory mediator production. Our study reveals striking similarities between cannabis and tobacco smoke exposure on impairing barrier function, suppressing antiviral pathways, potentiating of pro-inflammatory mediators, and inducing oncogenic and oxidative stress gene expression signatures. Collectively our data suggest that cannabis smoke exposure is not innocuous and may possess many of the deleterious properties of tobacco smoke, warranting additional studies to support public policy, government regulations, and user practices.