The Lung Microbiome, Immunity, and the Pathogenesis of Chronic Lung Disease.

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J Immunol. Author manuscript; available in PMC 2017 June 15. Published in final edited form as: J Immunol. 2016 June 15; 196(12): 4839–4847. doi:10.4049/jimmunol.1600279.

The Lung Microbiome, Immunity and the Pathogenesis of Chronic Lung Disease1 David N. O’Dwyer*, Robert P. Dickson*, and Bethany B. Moore*,† *Department

of Internal Medicine, Division of Pulmonary and Critical Care Medicine, University of Michigan Health System, Ann Arbor, MI, USA

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†Department

of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor,

MI, USA

Abstract

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The development of culture-independent techniques for microbiological analysis has uncovered the previously unappreciated complexity of the bacterial microbiome at various anatomic sites. The microbiome of the lung has relatively less bacterial biomass when compared to the lower gastrointestinal tract yet displays considerable diversity. The composition of the lung microbiome is determined by elimination, immigration and relative growth within its communities. Chronic lung disease alters these factors. Many forms of chronic lung disease demonstrate exacerbations that drive disease progression and are poorly understood. Mounting evidence supports ways in which microbiota dysbiosis can influence host defense and immunity, and in turn may contribute to disease exacerbations. Thus, the key to understanding the pathogenesis of chronic lung disease may reside in deciphering the complex interactions between the host, pathogen and resident microbiota during stable disease and exacerbations. In this brief review we discuss new insights into these labyrinthine relationships.

The microbiome shapes immunity

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The microbiome is defined as the “ecological community of commensal, symbiotic and pathogenic organisms that share our body space” (1). Most studies of host and microbiome interaction in the human have focused almost exclusively on bacteria, biotic factors and the host. These complex communities of microbiota that inhabit environments such as the lung, skin or gut are now appreciated for their role in maintaining organ, tissue and immune homeostasis. One striking example is the early observation that germ-free mice have absent/ impaired secondary lymphoid architecture with resulting loss of lymphoid cells (2). Additionally, commensal microbiota can have both systemic and site-specific autonomous immune effects. For example, Staphylococcus epidermidis colonization of the skin promotes CD4 cell IFN-γ production which protects against infection with the parasite Leishmania major. In contrast, colonization of the gut with S. epidermidis had no effect (3). In other

1This work was supported in part by NIH Grant HL115618 (to B.B.M), NIH Grant AI117229 (to B.B.M), NIH Grant HL130641 (to R.P.D) and UL1TR000433 (to R.P.D) Corresponding author: Bethany B. Moore, 4053 BSRB, 109 Zina Pitcher Place, Ann Arbor, MI 48109-2200, ; Email: [email protected], Office phone: 734-647-8378, Office fax: 734-615-2331

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situations, it is well established that alterations of the gut microbiome can influence immune responses at distal sites. Antibiotic treatment, which disrupts gut microbiota accompanied by increases in fungal colonization, can greatly exaggerate the allergic response to intranasal challenge with the mold spore Aspergillus fumigatus. With antibiotic treatment, mice showed increased levels of eosinophils, mast cells, IL-5, IL-13, IFN-γ, IgE, and mucussecreting cells (4). More recently, modulation of gut microbiota through the use of probiotics has been shown to increase the frequency of B cells expressing IgA in the colon and lymph nodes, likely secondary to increased lymph node T follicular helper (Tfh) cells and IL-23expressing dendritic cells (5), all changes that are likely to improve host defense at mucosal sites or response to vaccination. Conversely, antibiotic treatment can limit development of Tfh cells (6). The response of pathogen recognition receptors (PRRs) of the innate immune response in the lung is well described. We know that defective components of the innate response can predispose to overwhelming infection and mortality and in some cases reduce injury from pathogens (7–9). The relationship between resident microbiota and this flagship innate response and the subsequent adaptive immune response in the lung is poorly understood. With this backdrop, we have chosen to explore what is known about the potential role of the microbiota (both gut and lung) and host interaction in regulating the pathogenesis of several important lung diseases. Origins of lung microbiome and debunking lung sterility

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The lung is an organ constantly exposed to microbiota either through inhalation or subclinical micro aspiration from birth. Historically, medical texts allude to a sterile lung environment, and this dogma has persisted in contemporary medicine. In the last decade, a revolution of sorts has taken place in our understanding of how the lung and microbiota interact and exist. This revolution stems from new knowledge that the lung is not sterile (10) and in fact, harbors an abundance of diverse interacting microbiota. As mentioned above, the gut microbiome modulates host mucosal defense (11, 12); however, there is a paucity of information regarding the potential role of lung microbiota to regulate immunity and homeostasis.

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The lung is not sterile, contrary to centuries of dogma asserting the same. Throughout the 1900’s this inference was re-enforced by respiratory culture-based protocols that sought only to identify clinically significant pathogens and by a spurious conclusion that upper respiratory tract microbes cultured from the lung represented contamination (10). The lung is a warm environment exposed to 7000 liters of diverse microbe-ridden air every day (13). Microbes adapt and exist in hostile environments like polar ice sheets of Antarctica and hot sulfur springs of Japan, yet the belief the human lung existed in a sterile state was “cultivated” in the medical literature for decades (14, 15). The upper respiratory tract and oropharynx, where microbes are found in abundance (16), is in direct communication with the lung, and subclinical aspiration of oropharyngeal contents occurs universally in humans (17, 18). Thus, the lung is subject to a constant level of microbe immigration and elimination through host mucosal defense and mucociliary clearance. Modern studies employing sequence-based bacterial identification techniques support the presence of a consistentlydetected diverse bacterial signal in the lung of healthy humans (19–22). Therefore, the notion of lung sterility has been debunked.

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This new understanding was animated by the discovery of culture-independent techniques for bacterial identification. The most commonly used approach involves high-throughput sequencing of amplicons of the 16s rRNA gene, a highly conserved locus in the bacterial genome. Sequences are then categorized and classified according to publically available prior knowledge taxonomic databases to allow for measures of total and relative abundance and diversity. The first application of culture-independent techniques was undertaken in a cohort of healthy controls and asthma patients. Hilty et al reported healthy airways contain bacteria similar, but distinct, from the upper respiratory tract and airways of asthma patients were enriched with Proteobacteria phylum (20).

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Sampling the lung for microbiome sequencing is technically challenging given the relatively low biomass. Furthermore, sampling lower airways by bronchoscopy requires passage of the instrument through the oral or nasal route. This course allows for a theoretical risk of pharyngeal contamination of samples. Importantly, the mouth and nose microbiota are markedly different and studies have not identified any detectable influence on the reported microbiota based on scope insertion site (22, 23). In addition, if pharyngeal carryover from bronchoscope insertion site was heavily influencing the reported microbiota, then serial dilutions of samples should result in dilution of bacterial communities and signal. Several studies have established that this is not the case (19, 24). The evidence thus supports minimal contamination from pharyngeal sources acquired by bronchoscopic methods. Methodology and limitations in studies of the microbiota


Accurate and relevant studies of the microbiome require consideration of several principles. Representative samples must be acquired from the distal airways, and as discussed above, the possibility of contamination from other niche microbiota must be addressed. Furthermore, contamination can occur during sample processing; use of “no template” controls is essential in low-biomass studies to assess for the effects of reagent contamination. The extraction of nucleic acid requires lysis of species, some of whom are more susceptible to cell disruption than others. This discrepancy can lead to overrepresentation of some species over others. Further steps that may alter the accuracy and reliability of acquired data include the generation of appropriate PCR primers, data normalization, the choice of reference database, and divergent measures of diversity (25). The use of 16s rRNA sequencing remains a pillar step in the sample processing, however, 16s rRNA sequencing may not be able to differentiate between species with varying immunogenicity and pathogenicity (26). The microbiome is subject to a number of factors that are known to change its composition including age, diet, ethnicity and study design in humans requires careful management of these potential influences (27–29). However, in tandem, researchers are developing novel and ingenious methods to limit any possible error in microbiome studies. For example, as an alternate process to 16s rRNA sequencing and primer choice, metagenomic data is being generated through the sequencing of all DNA from a sample (shotgun sequencing) which may be even more informative (30). Culturebased techniques remain highly relevant as complements to culture-independent techniques; in determination of viability, in speciation, and in microbial phenotyping.


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Microbiome development and composition in healthy lung

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The human microbiota inhabits several organs and is primarily colonized by 6 phyla: Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Fusobacteria and Cyanobacteria (31–34). Murine studies have demonstrated bacterial load in the lungs increases over the first 2 weeks of life, and the phyla of organisms found in the lung shifted from Gammaproteobacteria and Firmicutes towards Bacteriodetes (35). Such developmental changes in the microbiota were associated with accumulation of a PD-L1-dependent T regulatory cell population that could promote tolerance to allergen challenge (35). These data suggest acquisition of a lung microbiome is an important early life event necessary to protect the lung from injurious responses to inhaled antigens. In humans, studies have largely focused on gut microbiota and shown that newborns acquire microbiota that resembles their mother quickly and in a manner specific to the method of delivery. Dominguez-Bello et al reported infants born by vaginal delivery acquired bacterial communities resembling their mother’s vaginal microbiota, dominated by Lactobacillus, Prevotella, or Sneathia species. Infants born by cesarean section acquired skin-predominant Staphylococcus, Corynebacterium, and Propionibacterium species (36). These communities were undifferentiated across multiple body habitats in the infants, in contrast to the diverse communities evident in the mothers. No studies to date have examined the dynamic changes that may occur in the lower respiratory tract microbiota as childhood progresses. However, it is likely from studies of the upper respiratory tract and intestinal microbiota that these bacterial communities are dynamic (37, 38). There is relatively low bacterial biomass in the human lung. Bacterial loads from bronchoalveolar lavage have reported ranges from 4.5 to 8.25 log copies per/ml (39, 40). Further analysis of lung tissue samples demonstrates some 10 – 100 bacterial cells per 1000 human cells (41). The healthy lung has been studied using culture-independent techniques and the predominant phyla are Bacteroidetes and Firmicutes (21, 24). While individuals exhibit some spatial variation in the microbiota of their respiratory tract, intra-subject variation is significantly less than that of inter-subject variation (39). Dynamic changes in lung microbiome in health and disease

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The lung is a relatively low nutrient resource compared to the intestinal tract for supporting microbiome development. Furthermore, physiological conditions are regionally variable even in healthy lungs. Conditions that affect bacterial proliferation include: oxygen tension, blood flow, local pH, temperature, effector inflammatory cell disposition, and epithelial cell architecture (42, 43). Coupled to this variable biogeography of the lung microbiome are the factors that influence microbe immigration and elimination from the lower respiratory tract. Taken together, these factors determine the dynamic change of the microbial ecosystem of the lung. Lung disease alters the population dynamics through effects on immigration/elimination and the local conditions of the microbial ecosystem of the lung (Table 1). Chronic lung disease in many forms, results in heterogeneous architectural distortion of the lung: upper lobe predominant destruction of the terminal bronchioles in emphysema and lower lobe predominant distortion of the parenchyma by honeycombing in Idiopathic Pulmonary Fibrosis (IPF) (44). Changes in the viscosity of the mucus and pH occur in Cystic Fibrosis


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(CF) (45). The resultant changes in oxygen tension, ventilation, perfusion, inflammatory cells and other local factors exert pressure on population dynamics. Immigration of microbiota from the upper respiratory to the lower respiratory tract is primarily promoted by subclinical aspiration which occurs in both health and disease and overt clinical infection occurs when local defense is blunted or overwhelmed (17, 18). Chronic lung disease is commonly associated with gastroesophageal reflux which may result in elevated volumes of micro aspiration (46, 47). Elimination is achieved by cough and mucociliary clearance. Host inflammatory cells are responsible for eradication of pathogens and the type and number of effector cells are associated with certain features of the microbiome. In a comparison of inflammatory cells and microbiota detected in bronchoalveolar lavage (BAL) fluid, Segal et al demonstrated increased community abundance of Prevotella and Veillonella (common anaerobic oral commensals) associated with higher levels of lymphocyte and neutrophil inflammation (24). Therefore the lung microbiome has a potential role to play in the pathogenesis of chronic lung disease through both the ability of lung microbiota to modulate local inflammatory responses and the influence of chronic lung diseases on the lung microbiome in turn.


Chronic lung diseases include asthma, Chronic Obstructive Pulmonary Disease (COPD), CF and IPF. Interestingly, these diseases are all characterized by natural histories that are punctuated by periods of acute exacerbations. Exacerbations are characterized by acute worsening of pulmonary symptoms and a decline in pulmonary function. Such exacerbations are responsible for significant mortality and morbidity. The onset of exacerbation may herald accelerated disease progression and many patients fail to return to their previous functional and physiological baseline (48). Studies of these events may reveal key data that re-animate our current understanding of the pathogenesis of chronic lung disease, and it is likely that these exacerbations are accompanied or induced by microbiota dysbiosis. COPD

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The relationship between COPD exacerbation and acute bacterial infection of the airway remains disputed. Potential pathogens cultured from sputum during COPD exacerbations are less frequently cultured during periods of clinical stability (49). Sethi et al identified similar culture densities of Haemophilus influenza, and lower densities of Moraxella catarrhalis and Streptococcus pneumoniae in sputum collected during acute exacerbations compared to samples during clinical stability (50). The use of antibiotic therapy in COPD exacerbations also lacks clarity. Recent work reported a clear role in reducing the rate of treatment failure for severe disease in hospitalized patients, but the role is unclear for mild to moderate disease (51). Culture-independent techniques have identified a diverse and abundant pulmonary microbiota in exacerbations from a variety of sampling types (41, 52–54) and exacerbations are definitively associated with changes in respiratory microbiota and airway inflammation. Millares et al analyzed sputum from COPD patients during exacerbations with paired sampling from periods of clinical stability and found increases in the relative abundance of bacteria associated with exacerbations, namely Haemophilus, Pseudomonas and Moraxella (52). Huang et al reported an alteration in community content towards the Proteobacteria phylum during COPD exacerbations, including non-typical COPD pathogens (53). Additionally, the influence of viral exposure may trigger COPD exacerbations (55) but


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the relationship between viral infection, microbiome composition and host defense is poorly understood. Patients experimentally infected with rhinovirus develop clinical features of COPD exacerbation and culprit viruses have been isolated in respiratory samples from 36– 56% of patients with exacerbations versus 6–19% of patients at clinical baseline (56–59). Interestingly, Molyneaux et al compared sputum in COPD patients at baseline and during exacerbations and noted that sputum acquired post-viral exposure demonstrated a shift towards the Proteobacteria phylum (60), a potential explanation for the increased presence of Pseudomonas spp noted in COPD exacerbations (52). Sequencing-based studies of tissue from COPD patients have demonstrated an increase in the Firmicutes community in severe disease (GOLD stage 4) attributable to an increase in the Lactobacillus genus (41). Phagocytosis of Lactobacillus spp by human macrophages reduces the effects of cigarette smoke-related inflammation, potentially suggesting these species are beneficial modifiers of smoking-related lung disease (61). Animal models of RSV infection have demonstrated that the anti-viral response within the lung mucosa can be modulated by the administration of Lactobacillus rhamnosus species prior to infection (62). Thus, changes in the microbiome may represent an adaptation to try to protect the lung from respiratory viral infection. However, we speculate that, as suggested above, once pathogenic infection does occur, these beneficial changes may be lost.

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Asthma


Studies of airway microbiota in asthma have established that composition is altered when compared to controls. Hilty et al identified asthma patients with more frequent Proteobacteria (particularly Haemophilus) in the bronchial tree compared to controls. The authors also noted a decrease in Bacteroidetes, especially Prevotella species, in asthmatic airways (20). Studies of asthma severity identified similar altered microbiomes with a predominance of Proteobacteria, and the observation that the airway microbiome of asthmatic patients was more diverse that non-asthmatic controls (63). Huang et al reported an association between bronchial hyper-responsiveness and community diversity and composition, secondary to an increased abundance of Proteobacteria (64). Bronchial hyperresponsiveness is accentuated during exacerbation and is an accurate predictor of future exacerbations (65). Alterations to the microbiome appear similar in both mild and severe disease and are specifically associated with features of the disease. No studies have analyzed the airway microbiome in asthma exacerbations. However, an estimated 80% of asthma exacerbations are associated with viral infection (66, 67). The host microbiome interaction may be crucial in the development of asthma. Ege et al have demonstrated that children with broad microbial exposures (i.e. traditional farming) were protected from asthma and atopy in childhood (68). Further studies reported an association with high fiber diet and a reduced risk of asthma (69). The proposed mechanism was related, in part, to an altered immune response. Mice fed a low fiber diet exhibited reduced levels of short chain fatty acids (SCFA) with increased allergic inflammation whereas mice with a high fiber diet had elevated levels of SCFA and were protected against allergic inflammation. SCFA propionate treatment of mice resulted in the generation of macrophages and dendritic cells with enhanced phagocytic properties but an attenuated capacity to initiate T helper type 2 response, a crucial component of allergic inflammation (69). Furthermore, studies have suggested resident microbiota may promote Th17-dependent neutrophil inflammation in a


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murine model of ovalbumin-induced asthma (70). Similarly, an experimental model of allergic airway inflammation is exacerbated by the administration of antibiotics during early life. This correlated with a reduction in microbial load and diversity (71). IPF

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IPF is a chronic fatal remodeling disease of the lung parenchyma of unknown etiology (72). The natural history of IPF is characterized by exacerbations that contribute greatly to disease related morbidity and mortality. Recent work has highlighted a potential role for both viral and bacterial infection in the pathogenesis of IPF (73–77). Unlike with asthma or COPD, the current definition of acute exacerbations of IPF excludes active infectious pathogens (78). Disease progression in IPF is characterized by an alteration in the microbiome with a relative increased abundance of Streptococcus and Staphylococcus taxonomic groups (79). This has particular relevance given recent work identifying pneumolysin, a Pneumococcus produced toxin, that mediates fibrotic progression in animal models via injury of the alveolar epithelium (73). Further study by Molyneaux et al describes an increased bacterial burden in the BAL fluid of IPF patients compared to controls using culture-independent techniques. These communities were enriched with Haemophilus, Streptococcus, Neisseria and Veillonella (76). The greater the bacterial burden in these patients, the greater the independent association with IPF disease progression. Recent trials of trimethoprimsulphamethtoxazole have demonstrated benefit with improved Medical Research Council dyspnea scores, quality of life and even all-cause mortality (HR 0.21; 95% CI 0.06 – 0.78: p=0.02)(80). This further supports a role for bacterial burden in disease progression. Aspects of host defense and innate immunity also have putative roles in IPF disease progression (81– 86). We have previously suggested defective toll-like receptor (TLR) 3 signaling promotes IPF disease progression (81). The L412F polymorphism (rs3775291) of TLR3 results in a functional defect in primary lung fibroblasts from IPF patients. This defect leads to aberrant inflammation and blunted interferon responses to TLR3 activation by synthetic dsRNA [and likely pathogen-associated molecular patterns (PAMPs) although this was not directly examined]. Genotyping studies of two independent cohorts of IPF patients confirmed an association between this polymorphism with increased mortality and functional decline. However, the interaction between the pulmonary microbiome, host defense, acute infection and IPF disease progression remains unclear. Ultimately, the questions of whether chronic lung disease promotes microbiome alterations or microbiome changes modify chronic lung disease remains to be answered.

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The manifestations of CF in the lung involve the progressive development of bronchiectasis and obstructive lung disease. Central to disease progression are exacerbations of CF bronchiectasis which are responsible for significant mortality, morbidity and accelerated disease progression (87). Exacerbations of disease are attributed to infection by specific pathogens that are cultured from sputum during exacerbations and clinical stability. These pathogens commonly include Staphylococcus aureus and Pseudomonas aeruginosa (88). The evidence to support the use of antibiotics directed against these pathogens is sparse. The bacterial density of sputum is not altered during CF exacerbations when antibiotics are administered (89). Clinical trials have not reported an association between the clinical J Immunol. Author manuscript; available in PMC 2017 June 15.

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response during antibiotic therapy and the in vitro susceptibility of the cultured bacteria to the administered antibiotics (90, 91). Indeed, culture independent analysis has primed a revision of our long held understanding of the bacterial pathogenesis of CF lung disease. Studies have consistently reported that CF exacerbations are not associated with increased bacterial density or attenuated diversity (92–94). However, evidence would support a loss of diversity with increasing age and disease severity, which was strongly associated with cumulative antibiotic exposure (95, 96). The emergence of new pathogens may have implications for our understanding of the microbiome and lung disease interaction in CF. Non-tuberculosis mycobacterium (NTM), in particular Abscessus, are associated with increased mortality and morbidity in CF (97–99). Guidelines for the management of Abscessus have been published and long term treatment with broad spectrum antibiotics is required in these cases (100). The consequences for the lung microbiome for this treatment remain unknown. This is of particular relevance given the limited treatment benefit for Abscessus infection in CF (100). The relationship between the microbiome, antibiotic exposure, exacerbation and ultimate disease progression will thus require further careful study in CF. Evidence and implications for a gut-lung axis and the regulation of host defense for chronic lung disease exacerbations


Exactly how microbiota may regulate innate immunity in health and disease is an area of active investigation, and very little is known about how the lung microbiota may specifically regulate lung immunity or the development of bronchial-associated lymphoid tissue. There is growing appreciation for the fact that the gut commensal microbiota is an important regulator of the innate immune system (101, 102). The bacterial biomass of the intestine dwarfs the relative mass seen in the lung (103). In healthy adults the intestine microbiota consists predominantly of 3 phyla: Bacteroides, Prevotella and Ruminococcus (104). There is evidence to support a crucial early period during life where intestinal microbiome development is important for the regulation of an appropriate immune response in the lung. CF and asthma are examples of chronic lung disease where disease course and susceptibility are influenced by shifts in the composition of the gut microbiota (105, 106). Furthermore, in the absence of normal gut biota, the host is more susceptible to pulmonary infections including Listeria monocytogenes (107), Klebsiella pneumoniae (108) and viruses (109). This raises the interesting possibility that exacerbations of chronic lung disease may arise from impaired innate and adaptive immune function secondary to alterations in the host gut microbiota. As mentioned above, patients with progressive IPF show evidence of enhanced burden of Streptococcus and Staphylococcus species in the lung, and previous studies have shown that the ability of neutrophils from microbiota-depleted mice to kill S. pneumoniae and S. aureus are reduced (110). Currently it is not known whether the accumulation of these species in the lung correlates with loss of gut microbial communities during IPF disease progression, but this is an interesting area for future study (Figure 1). Support for such a concept comes from recent work showing that bacterial activation of NLR receptors in the gut leads to enhanced production of reactive oxygen species within alveolar macrophages, the sentinel innate immune cell within the lung (101); implying that conditions associated with loss of gut bacterial homeostasis (e.g. antibiotic use) could provide a window of opportunity for lung immunity to be impaired. In COPD, exacerbations can occur due to


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viral infections (60, 111), and resulting pathogenesis could be the result of dysbiosis leading to altered airway microbiota and disproportionate inflammation. While loss of gut commensal signaling may impair lung innate immunity in this disorder, cigarette smoke directly and indirectly contributes to impaired innate immunity in the lung via alterations in ciliary function, mucus, innate immune cell phagocytosis and via direct enhancement of bacterial virulence (e.g. enhanced biofilm formation) [reviewed in (112)]. These changes could impact the ability of respiratory pathogens to exacerbate COPD.

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Given the propensity for viruses to precipitate lung disease exacerbations, it is interesting to note the potential impact of respiratory viral infection on the intestinal microbiota. Wang et al reported that influenza infection may lead to alterations in intestinal microbiota with a reduction in Lactobacillus and Lactococci and an outgrowth of Enterbacteriaceae. As noted above, this may lead to a loss of beneficial microbiota for smoking-related disease. The authors demonstrated that these shifts in intestinal microbiota were not secondary to lytic influenza gut infection. This injury was mediated by Th17 cells and IL-17 neutralization resulted in attenuated injury (113). In addition, antibiotic-mediated depletion of intestinal microbiota led to attenuated intestinal injury. Interestingly, this study also highlighted the importance of an effector T cell that arose in the lung post-infection then migrated to the small intestine to provide IFN-γ to alter the gut microbiome. Ultimately the alterations in the gut microbiota stimulated epithelial-derived IL-15 to promote the Th17 response. It is possible that IL-17 responses arising in the gut may further impact lung disease (114). IL-17 is involved in the elimination of certain pathogens (115) and is implicated in the pathogenesis of several pulmonary pathologies including asthma, Sarcoidosis, Obliterative Bronchiolitis, CF and bone marrow transplant related pneumonitis(116–120).


IL-17 may also play a central role in the dynamic change that occurs within the pulmonary microbiota of COPD. Yadava et al report the impact of experimental change on the lung microbiota in an emphysema animal model. Specific pathogen free and axenic mice were challenged with lipopolysaccharide (LPS)/elastase for 4 weeks. Microbiota diversity and abundance was decreased in the LPS/elastase model with an abundance of Pseudomonas, Lactobacillus and a depletion of Prevotella. Loss of bacterial load was associated with attenuated IL-17 production. The intranasal application of microbiota enriched fluid to axenic mice enhanced IL-17 production. The neutralization of IL-17 in mice harboring microbiota led to dampened inflammation and reduced disease burden (121). Several studies have implicated IL-17 in hepatic fibrosis, and certain experimental models of pulmonary fibrosis are IL-17A dependent (120, 122, 123). Furthermore, studies examining the development of intestinal fibrosis have reported an association with alterations in the microbiota and Th17 responses (124). The intestine is a known source of Th17 cells through binding of segmented filamentous bacteria (SFB) to intestinal epithelial cells (125). The case may be similar in the lung. Gauguet et al have demonstrated that intestinal SFB have the ability to promote pulmonary innate immunity through the induction of IL-17 to provide resistance to S. aureus pneumonia in animal models (126). This constitutes further evidence to support a gut-lung microbiome axis that may be pivotal in modulating the innate immune response of the lung. However, direct evidence of a gut-lung axis promoting exacerbation of chronic lung disease is limited to experimental data to date and requires further study.


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Conclusions

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In conclusion, growing evidence suggests alterations in the lung and/or gut microbiota characterize chronic lung diseases and may allow for exacerbations caused by endogenous microbiota alterations or susceptibility to new infection (Figure 1). We speculate that impairment in lung innate immunity caused by microbial dysbiosis may promote susceptibility of the host to infections that can exacerbate chronic lung diseases. Furthermore, shifts in cytokine profiles mediated by changes in the microbiota may also promote epithelial injury and fibrotic outcomes. Overall, there appears to be a vital crosstalk between the gut and lung mucosa and the microbial communities within. The device through which this cross talk may be achieved remains unknown. Possible instruments include translocation of gut microbiota (including potential pathogens) through blood or lymphatics, modulation of circulating or lung-resident effector immune and inflammatory cells or alterations in systemic cytokine profiles. These results highlight the need for careful future human studies that will characterize not only the lung, but also the gut microbiota during periods of disease stability vs. exacerbations. In addition, murine models may allow us to interrogate the PRRs and cytokine signaling pathways that promote exacerbations.

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Figure 1. Proposed regulation of disease exacerbation by the gut-lung axis

Author Manuscript

During normal homeostasis, the lung microbiome is primarily characterized by low biomass, but prominent diversity in microbial species. In contrast, the healthy gut microbiome is characterized by high diversity and high biomass. In homeostasis, the gut microbiome helps shape development of lymphoid architecture and appropriate immune responsiveness. Loss of gut diversity (e.g. as a result of viral infection or antibiotic use) may cause dysregulation of IL-17 or bacterial killing mechanisms systemically; potentially leading to impaired alveolar macrophage function and the overgrowth of selective organisms with pathogenic potential that may result in disease exacerbation. Alternatively, some forms of chronic lung disease exacerbations may be due to translocation and/or expansion of bacterial contents from the gut. Direct insults to the lung (e.g. viral infection or aspiration) may exacerbate disease in part via their effects on the lung or gut microbiota. Alterations in systemic cytokines (e.g. Th2 or Th17 induction) may promote pathologic fibrotic remodeling as well.


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Table 1

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Factors that influence the lung microbiota during acute and chronic disease Architectural: •

Airway obliteration (lung transplant, IPF)

•

Terminal bronchiole destruction (COPD)

•

Honeycombing and fibrosis (IPF)

•

Impaired mucociliary clearance (COPD, asthma)

Immunologic:

Author Manuscript

•

Innate immune cell impairment

•

Altered PRR signaling

•

Release of anti-microbial peptides

•

Apoptosis/Autophagy

•

Inflammation

•

Cytokine alterations

Microbiologic: •

Overgrowth of limited bacterial species (IPF, CF)

•

Antibiotic use (esp. in CF)

•

Lytic viral infection (COPD, asthma)

•

Latent viral infection (IPF?)

•

Biofilm formation (CF, COPD)

Pathologic: •

Osmotic changes (CF)

•

Thickened mucus (CF)

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•

Damaged cilia (COPD)

•

Changes in oxygen tension, ventilation and perfusion (IPF, COPD, CF, asthma)

•

Micro aspiration (IPF)

Author Manuscript J Immunol. Author manuscript; available in PMC 2017 June 15.

Microbiome effects on immunity, health and disease in the lung.

Role of the Lung Microbiome in the Pathogenesis of Chronic Obstructive Pulmonary Disease.

Influence of lung CT changes in chronic obstructive pulmonary disease (COPD) on the human lung microbiome.

The lung microbiome after lung transplantation.

The lung microbiome and viral-induced exacerbations of chronic obstructive pulmonary disease: new observations, novel approaches.

The Human Microbiome in the Lung: Are Infections Contributing to Lung Health and Disease?

ABCs of the lung microbiome.

The pediatric microbiome and the lung.

The cystic fibrosis lung microbiome.

Host Response to the Lung Microbiome in Chronic Obstructive Pulmonary Disease.

Spatial Variation in the Healthy Human Lung Microbiome and the Adapted Island Model of Lung Biogeography.

The role of the microbiome in exacerbations of chronic lung diseases.

Chronic lung inflammation primes humoral immunity and augments antipneumococcal resistance.

Pathogenesis and management of lung disease in cystic fibrosis.

The spatiotemporal cellular dynamics of lung immunity.

Pericytes in chronic lung disease.

Mucosal immunity and the microbiome.

The potential impact of the pulmonary microbiome on immunopathogenesis of Aspergillus-related lung disease.

Benjamin Felson lecture. Chronic interstitial lung disease of unknown cause: a new classification based on pathogenesis.

Chronic nonspecific lung disease and alcohol consumption.

Inflammation in cystic fibrosis lung disease: Pathogenesis and therapy.

Lung microbiome and disease progression in idiopathic pulmonary fibrosis: an analysis of the COMET study.

Strategies for Management of the Early Chronic Obstructive Lung Disease.

The spectrum of lung disease due to chronic occult aspiration.