Leopoldo N. Segal

Enrichment of lung microbiome with supraglottic taxa is associated with increased pulmonary inflammation

BackgroundThe lung microbiome of healthy individuals frequently harbors oral organisms. Despite evidence that microaspiration is commonly associated with smoking-related lung diseases, the effects of lung microbiome enrichment with upper airway taxa on inflammation has not been studied. We hypothesize that the presence of oral microorganisms in the lung microbiome is associated with enhanced pulmonary inflammation. To test this, we sampled bronchoalveolar lavage (BAL) from the lower airways of 29 asymptomatic subjects (nine never-smokers, 14 former-smokers, and six current-smokers). We quantified, amplified, and sequenced 16S rRNA genes from BAL samples by qPCR and 454 sequencing. Pulmonary inflammation was assessed by exhaled nitric oxide (eNO), BAL lymphocytes, and neutrophils.ResultsBAL had lower total 16S than supraglottic samples and higher than saline background. Bacterial communities in the lower airway clustered in two distinct groups that we designated as pneumotypes. The rRNA gene concentration and microbial community of the first pneumotype was similar to that of the saline background. The second pneumotype had higher rRNA gene concentration and higher relative abundance of supraglottic-characteristic taxa (SCT), such as Veillonella and Prevotella, and we called it pneumotypeSCT. Smoking had no effect on pneumotype allocation, α, or β diversity. PneumotypeSCT was associated with higher BAL lymphocyte-count (P= 0.007), BAL neutrophil-count (P= 0.034), and eNO (P= 0.022).ConclusionA pneumotype with high relative abundance of supraglottic-characteristic taxa is associated with enhanced subclinical lung inflammation.

Enrichment of the lung microbiome with oral taxa is associated with lung inflammation of a Th17 phenotype.

Microaspiration is a common phenomenon in healthy subjects, but its frequency is increased in chronic inflammatory airway diseases, and its role in inflammatory and immune phenotypes is unclear. We have previously demonstrated that acellular bronchoalveolar lavage samples from half of the healthy people examined are enriched with oral taxa (here called pneumotypeSPT) and this finding is associated with increased numbers of lymphocytes and neutrophils in bronchoalveolar lavage. Here, we have characterized the inflammatory phenotype using a multi-omic approach. By evaluating both upper airway and acellular bronchoalveolar lavage samples from 49 subjects from three cohorts without known pulmonary disease, we observed that pneumotypeSPT was associated with a distinct metabolic profile, enhanced expression of inflammatory cytokines, a pro-inflammatory phenotype characterized by elevated Th-17 lymphocytes and, conversely, a blunted alveolar macrophage TLR4 response. The cellular immune responses observed in the lower airways of humans with pneumotypeSPT indicate a role for the aspiration-derived microbiota in regulating the basal inflammatory status at the pulmonary mucosal surface.

Lung Microbiome for Clinicians. New Discoveries about Bugs in Healthy and Diseased Lungs

Microbes are readily cultured from epithelial surfaces of the skin, mouth, and colon. In the last 10 years, culture-independent DNA-based techniques demonstrated that much more complex microbial communities reside on most epithelial surfaces; this includes the lower airways, where bacterial culture had failed to reliably demonstrate resident bacteria. Exposure to a diverse bacterial environment is important for adequate immunological development. The most common microbes found in the lower airways are also found in the upper airways. Increasing abundance of oral characteristic taxa is associated with increased inflammatory cells and exhaled nitric oxide, suggesting that the airway microbiome induces an immunological response in the lung. Furthermore, rhinovirus infection leads to outgrowth of Haemophilus in patients with chronic obstructive pulmonary disease, and human immunodeficiency virus-infected subjects have more Tropheryma whipplei in the lower airway, suggesting a bidirectional interaction in which the host immune defenses also influence the microbial niche. Quantitative and/or qualitative changes in the lung microbiome may be relevant for disease progression and exacerbations in a number of pulmonary diseases. Future investigations with longitudinal follow-up to understand the dynamics of the lung microbiome may lead to the development of new therapeutic targets.

HIV-1 and Bacterial Pneumonia in the Era of Antiretroviral Therapy

Community-acquired pneumonia affects approximately 4 million people in the United States, with 40,000 deaths per year. The incidence is increased about 35-fold in HIV-infected individuals, and this rate has decreased since the antiretroviral era has begun. Bacterial pneumonia has decreased from 5 to 20 cases per 100 person-years to less than 1 to 5 cases per 100 person-years in the era of antiretroviral therapy. HIV-1 infection impairs the function of neutrophils in the lung and infects CD4⁺ cells and alveolar macrophages. Opportunistic infections dramatically increase local HIV replication in the lung cells, especially alveolar macrophages and CD4⁺ cells. This enhanced replication increases viral mutations and provides opportunities for viral escape from latent reservoirs. Mortality is increased with more comorbidities in this highly susceptible population. Immunization with vaccines is recommended, especially pneumococcal vaccines, although the vaccine itself may stimulate viral replication. Recent studies show that the lower respiratory tract is a microbial reservoir in HIV-infected individuals rather than being a sterile environment, as originally thought. This may provide new opportunities for preventing opportunistic infections in HIV-infected subjects. Bacterial pneumonia presents an ongoing challenge in these high-risk individuals, particularly in studying the functions of the innate and acquired immune response.

A Brave New World: The Lung Microbiota in an Era of Change

The development of culture-independent techniques has revolutionized our understanding of how our human cells interact with the even greater number of microbial inhabitants of our bodies. As part of this revolution, data are increasingly challenging the old dogma that in health, the lung mucosa is sterile. To understand how the lung microbiome may play a role in human health, we identified five major questions for lung microbiome research: (1) Is the lung sterile? (2) Is there a unique core microbiome in the lung? (3) How dynamic are the microbial populations? (4) How do pulmonary immune responses affect microbiome composition? and (5) Are the lungs influenced by the intestinal immune responses to the gut microbiome? From birth, we are exposed to continuous microbial challenges that shape our microbiome. In our changing environment, perturbation of the gut microbiome affects both human health and disease. With widespread antibiotic use, the ancient microbes that formerly resided within us are being lost, for example, Helicobacter pylori in the stomach. Animal models show that antibiotic exposure in early life has developmental consequences. Considering the potential effects of this altered microbiome on pulmonary responses will be critical for future investigations.

The lung microbiota in early rheumatoid arthritis and autoimmunity

BackgroundAirway abnormalities and lung tissue citrullination are found in both rheumatoid arthritis (RA) patients and individuals at-risk for disease development. This suggests the possibility that the lung could be a site of autoimmunity generation in RA, perhaps in response to microbiota changes. We therefore sought to test whether the RA lung microbiome contains distinct taxonomic features associated with local and/or systemic autoimmunity.Methods16S rRNA gene high-throughput sequencing was utilized to compare the bacterial community composition of bronchoalveolar lavage fluid (BAL) in patients with early, disease-modifying anti-rheumatic drugs (DMARD)-naïve RA, patients with lung sarcoidosis, and healthy control subjects. Samples were further assessed for the presence and levels of anti-citrullinated peptide antibodies (including fine specificities) in both BAL and serum.ResultsThe BAL microbiota of RA patients was significantly less diverse and abundant when compared to healthy controls, but similar to sarcoidosis patients. This distal airway dysbiosis was attributed to the reduced presence of several genus (i.e., Actynomyces and Burkhordelia) as well as reported periodontopathic taxa, including Treponema, Prevotella, and Porphyromonas. While multiple clades correlated with local and systemic levels of autoantibodies, the genus Pseudonocardia and various related OTUs were the only taxa overrepresented in RA BAL and correlated with higher disease activity and erosions.ConclusionsDistal airway dysbiosis is present in untreated early RA and similar to that detected in sarcoidosis lung inflammation. This community perturbation, which correlates with local and systemic autoimmune/inflammatory changes, may potentially drive initiation of RA in a proportion of cases.

Randomised, double-blind, placebo-controlled trial with azithromycin selects for anti-inflammatory microbial metabolites in the emphysematous lung.

Introduction Azithromycin (AZM) reduces pulmonary inflammation and exacerbations in patients with COPD having emphysema. The antimicrobial effects of AZM on the lower airway microbiome are not known and may contribute to its beneficial effects. Here we tested whether AZM treatment affects the lung microbiome and bacterial metabolites that might contribute to changes in levels of inflammatory cytokines in the airways. Methods 20 smokers (current or ex-smokers) with emphysema were randomised to receive AZM 250 mg or placebo daily for 8 weeks. Bronchoalveolar lavage (BAL) was performed at baseline and after treatment. Measurements performed in acellular BAL fluid included 16S rRNA gene sequences and quantity; 39 cytokines, chemokines and growth factors and 119 identified metabolites. The response to lipopolysaccharide (LPS) by alveolar macrophages after ex-vivo treatment with AZM or bacterial metabolites was assessed. Results Compared with placebo, AZM did not alter bacterial burden but reduced α-diversity, decreasing 11 low abundance taxa, none of which are classical pulmonary pathogens. Compared with placebo, AZM treatment led to reduced in-vivo levels of chemokine (C-X-C) ligand 1 (CXCL1), tumour necrosis factor (TNF)-α, interleukin (IL)-13 and IL-12p40 in BAL, but increased bacterial metabolites including glycolic acid, indol-3-acetate and linoleic acid. Glycolic acid and indol-3-acetate, but not AZM, blunted ex-vivo LPS-induced alveolar macrophage generation of CXCL1, TNF-α, IL-13 and IL-12p40. Conclusion AZM treatment altered both lung microbiota and metabolome, affecting anti-inflammatory bacterial metabolites that may contribute to its therapeutic effects. Trial registration number NCT02557958.

Airway Dysfunction in Obesity: Response to Voluntary Restoration of End Expiratory Lung Volume

Introduction Abnormality in distal lung function may occur in obesity due to reduction in resting lung volume; however, airway inflammation, vascular congestion and/or concomitant intrinsic airway disease may also be present. The goal of this study is to 1) describe the phenotype of lung function in obese subjects utilizing spirometry, plethysmography and oscillometry; and 2) evaluate residual abnormality when the effect of mass loading is removed by voluntary elevation of end expiratory lung volume (EELV) to predicted FRC. Methods 100 non-smoking obese subjects without cardio-pulmonary disease and with normal airflow on spirometry underwent impulse oscillometry (IOS) at baseline and at the elevated EELV. Results FRC and ERV were reduced (44±22, 62±14% predicted) with normal RV/TLC (29±9%). IOS demonstrated elevated resistance at 20 Hz (R20, 4.65±1.07 cmH2O/L/s); however, specific conductance was normal (0.14±0.04). Resistance at 5–20 Hz (R5−20, 1.86±1.11 cmH2O/L/s) and reactance at 5 Hz (X5, −2.70±1.44 cmH2O/L/s) were abnormal. During elevation of EELV, IOS abnormalities reversed to or towards normal. Residual abnormality in R5−20 was observed in some subjects despite elevation of EELV (1.16±0.8 cmH2O/L/s). R5−20 responded to bronchodilator at baseline but not during elevation of EELV. Conclusions This study describes the phenotype of lung dysfunction in obesity as reduction in FRC with airway narrowing, distal respiratory dysfunction and bronchodilator responsiveness. When R5−20 normalized during voluntary inflation, mass loading was considered the predominant mechanism. In contrast, when residual abnormality in R5−20 was demonstrable despite return of EELV to predicted FRC, mechanisms for airway dysfunction in addition to mass loading could be invoked.

Neutrophils Activate Alveolar Macrophages by Producing Caspase-6–Mediated Cleavage of IL-1 Receptor-Associated Kinase-M

Alveolar macrophages (AMs) are exposed to respirable microbial particles. Similar to phagocytes in the gastrointestinal tract, AMs can suppress inflammation after exposure to nonpathogenic organisms. IL-1R–associated kinase-M (IRAK-M) is one inhibitor of innate immunity, normally suppressing pulmonary inflammation. During pneumonia, polymorphonuclear neutrophils (PMNs) are recruited by chemotactic factors released by AMs to produce an intense inflammation. We report that intact IRAK-M is strongly expressed in resting human AMs but is cleaved in patients with pneumonia via PMN-mediated induction of caspase-6 (CASP-6) activity. PMN contact is necessary and PMN membranes are sufficient for CASP-6 induction in macrophages. PMNs fail to induce TNF-α fully in macrophages expressing CASP-6 cleavage-resistant IRAK-M. Without CASP-6 expression, PMN stimulation fails to cleave IRAK-M, degrade IκBα, or induce TNF-α. CASP-6−/− mice subjected to cecal ligation and puncture have impaired TNF-α production in the lung and decreased mortality. LPS did not induce or require CASP-6 activity demonstrating that TLR2/4 signaling is independent from the CASP-6 regulated pathway. These data define a central role for CASP-6 in PMN-driven macrophage activation and identify IRAK-M as an important target for CASP-6. PMNs de-repress AMs via CASP-6–mediated IRAK-M cleavage. This regulatory system will blunt lung inflammation unless PMNs infiltrate the alveolar spaces.

Disparity Between Proximal and Distal Airway Reactivity During Methacholine Challenge

There is an increasing awareness of the role of distal airways in the pathophysiology of obstructive lung diseases including asthma and chronic obstructive pulmonary disease. We hypothesize that during induced bronchoconstriction: 1) disparity between distal and proximal airway reactivity may occur; and 2) changes in distal airway function may explain symptom onset in subjects with minimal FEV1 change. 185 subjects underwent methacholine challenge testing (MCT). In addition to spirometry, oscillometry was performed at baseline and after maximum dose of methacholine; 33/185 also underwent oscillometry after each dose. Oscillometric parameters included resistance at 5 and 20 Hz (R5, R20) and heterogeneity of distal airway mechanics assessed by frequency dependence of resistance 5–20 Hz (R5–20) and reactance area (AX). R5 varied widely during MCT (range -0.8 – 11.3 cmH2O/L/s) and correlated poorly with change in FEV1 (r = 0.17). Changes in R5 reflected changes in both R20 and R5–20 (r = 0.59, p<0.05; r = 0.87, p<0.0001). However, R20 increased only 0.3 cmH2O/L/s, while R5–20 increased 0.7 cmH2O/L/s for every 1cmH2O/L/s change in R5, indicating predominant effect of distal airway mechanics. 9/33 subjects developed symptoms despite minimal FEV1 change (<5%), while R5 increased 42% due to increased distal airway heterogeneity. These data indicate disparate behavior of proximal airway resistance (FEV1 and R20) and distal airway heterogeneity (R5–20 and AX). Distal airway reactivity may be associated with methacholine-induced symptoms despite absence of change in FEV1. This study highlights the importance of disparity between proximal and distal airway behavior, which has implications in understanding pathophysiology of obstructive pulmonary diseases and their response to treatment.