STATE OF THE ART Viruses and Microbiome Alterations Susan V. Lynch1 1
Division of Gastroenterology, University of California San Francisco, San Francisco, California
Abstract Viral infection represents a common and problematic health care issue, particularly in younger and senior populations. The respiratory tract is a major portal for microbial exposure, where viral infection can result in nonsymptomatic, mild, and self-limiting or severe and sometimes fatal infection. Although it is well established that virus-specific properties, such as longevity and replication kinetics, impact clinical manifestations, it is less well understood why distinct infectious outcomes may occur across a population of individuals infected with the same strain of virus. Emerging evidence points to interpersonal variation in
pulmonary and gastrointestinal microbiome composition, and specifically to members of the Lactobacillus genus, as key components in defining respiratory viral infection outcomes. Moreover, human studies of airway microbiota after pH1N1 demonstrate that the composition of the respiratory microbiome can be modified by viral infection in a manner that enriches for pathogens associated with secondary bacterial infection. In this article, current knowledge in the field of human microbiome research, particularly as it pertains to respiratory viral infection, is reviewed. Keywords: microbiome; viral infection; respiratory; gastrointestinal
(Received in original form June 12, 2013; accepted in final form October 31, 2013 ) Correspondence and requests for reprints should be addressed to Susan V. Lynch, Ph.D., University of California San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143. E-mail: [email protected] Ann Am Thorac Soc Vol 11, Supplement 1, pp S57–S60, Jan 2014 Copyright © 2014 by the American Thoracic Society DOI: 10.1513/AnnalsATS.201306-158MG Internet address: www.atsjournals.org
The Human Microbiome Environmental microbial ecologists have long recognized that the true diversity of microbes in a given sample is poorly represented by those that are cultured under traditional laboratory conditions (1). This is due, in large part, to the myriad of metabolic capacities encoded by distinct microbial species and the relatively limited range of culture conditions under which microbes are typically grown in the laboratory. In addition, symbiotic and mutualistic microbial interactions are necessary for the viability of some microbial species, which precludes the ability to culture them easily under conventional conditions. As a result, until the recent application of culture-independent approaches to human samples, we have had a long-held, relatively restricted view of microbial diversity associated with the human host. Developed in the environmental microbial ecology field over Lynch: Viruses and Microbiome Alterations
the last several decades to identify the types of microbes in a given sample without recourse to culture, culture-independent methods have dramatically improved our appreciation of microbial diversity associated with humans. This is particularly true in recent years with the large advances in throughput afforded by nextgeneration sequencing and phylogenetic microarrays. Application of these tools to interrogate the diversity of microbial life that inhabits the human superorganism has revealed the presence of highly functional communities of organisms that represent up to 90% of all cells in the human body and as much as 99% of the total functional capacity of the human holobiont. Given the sheer scale of functional genetic capacity encoded by these communities, it is unsurprising that they are emerging as key contributors to critical processes, such as metabolism and maintenance of immune homeostasis. Although increased burden of particular
microbial species are clearly capable of inducing immune activation (2, 3), in healthy individuals the diverse communities of microbes found at a range of anatomical sites typically exist in a relatively quiescent state of symbiosis and immune homeostasis with their human host. What is even more interesting is that the distribution of bacteria in distinct sites distributed across the human host (e.g., the gut, skin, or oral cavity) house communities with distinct phylogenetic distributions, providing evidence for nichespecific selective pressures at each of these sites that influence microbial community composition and, presumably, the resulting site-specific functional attributes conferred by members of these assemblages.
The Respiratory Microbiome Although our view of the respiratory microbiome is not as well developed as that S57
STATE OF THE ART of the gastrointestinal tract, a number of recent studies have provided information on the patterns of microbial colonization in this system. The respiratory tract represents a major site of constant contact with the external (microbial) environment, and, as such, has developed innate and acquired immune responses to protect the mucosal surface from pathogenic infection (reviewed in References 4–6). As has been described in the healthy gastrointestinal tract, distinct sites along the respiratory tract (e.g., nares and oropharynx) exhibit discrete microbiome compositions (7), coupled with a gradient of bacterial burden that progresses from high levels in the upper, to reduced levels in the lower airways. Two independent studies, employing identical extraction and quantitative PCR assays to examine mucosal brushings of the sinuses and the right middle lobe mucosa of the lower airways, demonstrated the presence of high bacterial cell numbers in the upper airways (8), and little or no evidence of bacterial presence in the lower airways (9) of healthy individuals. On the contrary, patients with chronic airway disease, such as asthma, cystic fibrosis, or chronic obstructive pulmonary disease, possess large numbers of bacteria at this site and relatively diverse communities (9–13). Indeed, in a recent study, the extent of lower airway bacterial burden and diversity in the airways of subjects with asthma on inhaled corticosteroid treatment was significantly correlated with the degree of bronchial hyperresponsiveness. Patients with more reactive airways exhibited higher bacterial burden and diversity, indicating that variation in lower airway microbiome composition, and potentially the presence of specific members of these communities, was associated with the degree of bronchial hyperreactivity observed (9). These findings were largely concordant with an independent study of a distinct European corticosteroidusing population of patients with asthma (11). Collectively, these studies indicate that the composition of the mucosal microbiome in the lower airways of patients with chronic inflammatory pulmonary disease is associated with the degree of disease severity, which, by extension, implicates these communities in the immune activation status of these patients.
Respiratory Viral Infection and the Upper Airway Microbiome Respiratory viral infection represents a common occurrence, particularly in S58
pediatric and elderly populations, as well as immune-compromised individuals and those with underlying comorbidities. A recent review article by Hussell and colleagues (14) discusses how virus-specific properties, replication kinetics, and longevity affect the subsequent vigor of innate and adaptive immunity, which contribute to clinical manifestations. In addition, the timing of lung innate immune activation is different between individuals, and is determined by age, genetics, underlying conditions, and infection history—all factors that are also known to influence microbiome composition (10, 15, 16). Immune response to respiratory viral infection involves both the innate and adaptive arms of the immune system. Innate immune recognition of pathogenassociated molecular patterns results in rapid induction of type I IFNs, which signal through the IFN-a/b receptor to both the infected and local bystander cells to enhance IFN production and engage in a program of cellular gene expression aimed at inhibiting viral replication (17). In parallel with this IFN-driven response, expression of cytokines, such as IL-1b and IL-18, and chemokines, such as CC-chemokine ligand 2 (CCL2), result in activation of adjacent CD452 parenchymal cells (e.g., epithelial cells and fibroblasts), as well as local innate immune cells. These activated cells convert transforming growth factor-b to its active form, resulting in increased chemokine (CCL2 and CCL20) secretion by parenchymal stromal cells, and of cytokines, such TNF and IL12. This results in maturation of tissue-resident CD1031 and CD11b1 dendritic cells (DCs) and the recruitment and maturation of monocyte-derived DCs. Antigen-activated DCs then migrate to the draining lymph nodes, where they facilitate adaptive immune responses to the respiratory virus. Although these facets of innate and adaptive immune response to viral infection are well described, evidence is emerging that components of the microbiome, both at the local respiratory mucosa and in the gastrointestinal tract, may influence these host responses to viral infection. Although the healthy lower airways appear to possess a low burden of bacteria, the upper airways (i.e., the sinus cavity) represent a well established site of microbial colonization. Independent studies using fluorescent in situ hybridization to detect specific microbial species have
demonstrated the presence of polymicrobial biofilms (sessile microbial communities) attached to the sinus mucosa of subjects with chronic rhinosinusitis (CRS) and healthy control subjects (18, 19). Probes were generated to detect Haemophilus influenzae, Pseudomonas aeruginosa, Staphylococcus epidermidis, Staphylococcus aureus, and Streptococcus pneumonia, and demonstrated the presence of biofilms containing these known pathogenic species in the majority of both patients and healthy control subjects (18, 19). Another study demonstrated that patients with chronic sinusitus were characterized by an increased abundance of Staphylococcus aureus (20), indicating that this pathogen was involved in sinus pathology. Using a high-resolution microbiome profiling approach, Abreu and colleagues (8) examined the sinus microbiome of patients with CRS (n = 7) and healthy control subjects (n = 7), and demonstrated that known bacterial pathogens, such as H. influenza, P. aeruginosa, and S. aureus, were detected in both healthy and CRS sinuses, as was previously described. However, the sinus microbiome of patients with CRS exhibited characteristics of community collapse (i.e., loss of community membership), a more skewed distribution of species with these communities, and a significant reduction in lactic acid bacteria, including Lactobacillus sakei, which exhibited one of the most significant depletions in patients with CRS. In this state of disease-associated microbiome perturbation, only one species, Corynebacterium tuberculostearicum, was significantly enriched. C. tuberculostearicum is a skin commensal species not known to cause disease; therefore, to examine whether this species possessed pathogenic potential and if the intact microbiome of the sinonasal cavity could protect against this organism, a series of experiments was performed. Using a mouse model, the authors demonstrate that antibiotic depletion of the sinus microbiome before instillation of C. tuberculostearicum resulted in overt pathology that phenocopied that described in patients with CRS (i.e., mucin hypersecretion and goblet cell metaplasia). They also demonstrated that no airway pathology was observed in animals that received a coinstillation of L. sakei with C. tuberculostearicum, indicating that L. sakei is capable of mucosal surface protection.
AnnalsATS Volume 11 Supplement 1 | January 2014
STATE OF THE ART One of the mechanisms by which Lactobacillus species may protect the respiratory mucosa appears to involve modulation of host immune responses. A recent study demonstrated that nasal administration of specific Lactobacillus species protected mice against respiratory syncytial virus (RSV) infection (21). Instillation of two Lactobacillus rhamnosus strains, CRL1505 (Lr05) or CRL1506 (Lr06), afforded protection against RSV infection, albeit via distinct effects on the host immune response. Lr05 induced IFNg and IL-10, whereas Lr06 modulated production of IFN-a, IFN-b, and IL-6 expression. In both cases, Toll-like receptor 3/retinoic acid-inducible gene 1 activation was associated with protection of animals against RSV infection (21). In light of these data, it appears plausible that several members of the Lactobacillus genus, some of which are members of the healthy human sinonasal microbiota, afford broad-range protection of respiratory mucosal surfaces against both viral and bacterial pathogens, at least in part by effecting Toll-like receptor 3 signaling and inducing anti-inflammatory cytokine expression by the host (Figure 1).
Viral Infection Perturbs the Respiratory Microbiome Studies spanning back to the early 1930s demonstrated that viral infection enhanced bacterial pathogenesis in a variety of animal models, for example, influenza virus and Haemophilus influenza in a pig model (22), influenza virus and Staphylococcus species (23), Listeria monocytogenes (24), or group B Streptococcus species (25) in murine models of infection. The mechanisms by which viral infection may enhance bacterial pathogenesis are several, and include impairment of mucociliary clearance, increased bacterial adherence to
epithelial cells, and virus-induced epithelial damage, which facilitates bacterial translocation and dissemination and/or inhibition of antibacterial immune response. In addition, the presence of certain bacteria may also enhance viral pathogenesis. For example, influenza A virus gains entry to the mammalian cell via receptor-mediated endocytosis, which requires cleavage of its surface hemagglutinin (HA) glycoprotein. Viruses with the capacity for systemic infection do so because of the presence of HAs that can effectively be cleaved by enzymes that are widely distributed across a variety of mammalian cell types. Beyond mammalian host-encoded HA cleavage, it has also been demonstrated that particular bacterial species (e.g., Staphylococcus aureus [26], Streptomyces griseus, and Aerococcus viridans [27, 28]) possess the necessary proteolytic activity for effective viral replication and infectivity (28, 29). More recently, human microbiome studies have shed further light on the mechanisms by which viral–bacterial interactions may synergistically enhance pathogenesis, particularly in respiratory infections. Influenza A virus belongs to the Orthomyxoviridae family of viruses, a group of virions of approximately 80–120 nm in diameter containing an RNA genome of approximately 13.5 kb. The 2009 influenza A pdm09 virus (pH1N1) pandemic strain, although not considered a particularly lethal infectious agent, resulted in severe outcomes in some infected individuals. This was attributed to the capacity of this strain to replicate in the lower airways, cause extensive epithelial destruction, viral pneumonia, and secondary bacterial infections with increased lethality (30). The authors examined the oropharyngeal microbiome metagenome (sequence-based analysis of the microbial genomes present in the
Figure 1. Members of the Lactobacillus genus play a role in defining response to viral respiratory infection.
Lynch: Viruses and Microbiome Alterations
microbiomes of individuals) of patients with pneumonia with and without pH1N1 infection. At the phylum level, the relative abundance of Firmicutes and Proteobacteria were significantly enriched in patients with pH1N1. More specifically, a number of Pseudomonas (P. amygdali, P. fluorescens, and Pseudomonas sp. UK4) and Acinetobacter (e.g., A. baumanii and A. junii) were significantly increased in the pH1N1-infected group. Metagenomic sequencing of the samples permitted insights into the functional genes encoded by the organisms that were significantly enriched in the patients infected with pH1N1. pH1N1-associated airway microbiota were significantly enriched for motility, transcriptional regulation, metabolism, and signaling genes, as well as pathways involved in secondary metabolite biosynthesis and catabolism. In contrast, these functional attributes were notably absent from the genera that significantly decreased in relative abundance (e.g., Prevotella, Veillonella, and Neisseria species) in patients infected with pH1N1 (30). These data, generated with human samples, demonstrate that pH1N1 infection perturbs the respiratory microbiome (presumably through its induction of host immune response) in a manner that appears to select for metabolically flexible species capable of sensing and responding to a rapidly changing ecosystem, translocating to the lower airways, and producing secondary metabolites (which are frequently potent antimicrobial and immune-modulating molecules).
Gastrointestinal Microbiome Composition Influences Host Response to Respiratory Viral Infection Clearly, local mucosal microbiome composition in the respiratory tract influences immune responses. However, more recent studies have demonstrated that the composition of the gastrointestinal microbiome also plays a role in modulating immune responses to viral infection of the airways. Using a mouse model of influenza A infection, Ichinohe and colleagues (31) demonstrated that animals who received antibiotic treatment before respiratory viral infection mounted a significantly reduced anti-PR8 antibody response, were incapable of inducing CD4 T cell–mediated IFN-g S59
STATE OF THE ART response to PR8 antigen, and exhibited significantly fewer CD8 1 CD44 1 cells (effector memory T cells). These mice also demonstrated significantly increased viral titers in their lungs. Using neomycin, which resulted in a profound loss of Lactobacillus in both the gut and nares and concomitant enrichment of Sphingomonas species in the gut and Corynebacterium in the nares, exhibited the greatest deficit in effector memory T cells, again implicating Lactobacillus species as key components necessary to mount an appropriate antiviral response.
In conclusion, the upper respiratory tract represents a site of overt microbial colonization, where the composition of the microbiome in this niche appears to act as an ancillary barrier and define host response to respiratory pathogen invasion. Viral infection results in a perturbation to the respiratory microbiome; in the case of the recent pandemic influenza A virus strain, infection resulted in selection for a discrete subset of bacterial species with functional capacities consistent with survival and translocation to the lower airways, where they presumably contribute to secondary bacterial
References 1 Staley JT, Konopka A. Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats. Annu Rev Microbiol 1985;39:321–346. 2 Ivanov II, Atarashi K, Manel N, Brodie EL, Shima T, Karaoz U, Wei D, Goldfarb KC, Santee CA, Lynch SV, et al. Induction of intestinal Th17 cells by segmented filamentous bacteria. Cell 2009;139: 485–498. 3 Winter SE, Thiennimitr P, Winter MG, Butler BP, Huseby DL, Crawford RW, Russell JM, Bevins CL, Adams LG, Tsolis RM, et al. Gut inflammation provides a respiratory electron acceptor for Salmonella. Nature 2010;467:426–429. 4 Holt PG, Strickland DH, Wikstrom ¨ ME, Jahnsen FL. Regulation of immunological homeostasis in the respiratory tract. Nat Rev Immunol 2008;8:142–152. 5 Waffarn EE, Baumgarth N. Protective B cell responses to flu—no fluke! J Immunol 2011;186:3823–3829. 6 Braciale TJ, Sun J, Kim TS. Regulating the adaptive immune response to respiratory virus infection. Nat Rev Immunol 2012;12:295–305. 7 Lemon KP, Klepac-Ceraj V, Schiffer HK, Brodie EL, Lynch SV, Kolter R. Comparative analyses of the bacterial microbiota of the human nostril and oropharynx. MBio 2010;1:pii:e00129–10. 8 Abreu NA, Nagalingam NA, Song Y, Roediger FC, Pletcher SD, Goldberg AN, Lynch SV. Sinus microbiome diversity depletion and Corynebacterium tuberculostearicum enrichment mediates rhinosinusitis. Sci Transl Med 2012;4:151ra124. 9 Huang YJ, Nelson CE, Brodie EL, Desantis TZ, Baek MS, Liu J, Woyke T, Allgaier M, Bristow J, Wiener-Kronish JP, et al. Airway microbiota and bronchial hyperresponsiveness in patients with suboptimally controlled asthma. J Allergy Clin Immunol 2011;127:372–381.e1–e3. 10 Cox MJ, Allgaier M, Taylor B, Baek MS, Huang YJ, Daly RA, Karaoz U, Andersen GL, Brown R, Fujimura KE, et al. Airway microbiota and pathogen abundance in age-stratified cystic fibrosis patients. PLoS ONE 2010;5:e11044. 11 Hilty M, Burke C, Pedro H, Cardenas P, Bush A, Bossley C, Davies J, Ervine A, Poulter L, Pachter L, et al. Disordered microbial communities in asthmatic airways. PLoS ONE 2010;5:e8578. 12 Zhao J, Schloss PD, Kalikin LM, Carmody LA, Foster BK, Petrosino JF, Cavalcoli JD, VanDevanter DR, Murray S, Li JZ, et al. Decade-long bacterial community dynamics in cystic fibrosis airways. Proc Natl Acad Sci USA 2012;109:5809–5814. 13 Huang YJ, Kim E, Cox MJ, Brodie EL, Brown R, Wiener-Kronish JP, Lynch SV. A persistent and diverse airway microbiota present during chronic obstructive pulmonary disease exacerbations. OMICS 2010;14:9–59. 14 Hussell T, Godlee A, Salek-Ardakani S, Snelgrove RJ. Respiratory viral infections: knowledge based therapeutics. Curr Opin Immunol 2012; 24:438–443. 15 Koenig JE, Spor A, Scalfone N, Fricker AD, Stombaugh J, Knight R, Angenent LT, Ley RE. Succession of microbial consortia in the
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pneumonia. The gastrointestinal microbiome also appears to influence airway responses to viral infection, providing further support for an airway– gastrointestinal axis, and opening up the possibility that mucosal microbiome manipulation in both the sinus and gastrointestinal niches may represent a novel therapeutic approach for prevention, treatment, or management of acute respiratory infection, or indeed chronic pulmonary disease. n Author disclosures are available with the text of this article at www.atsjournals.org.
developing infant gut microbiome. Proc Natl Acad Sci USA 2011; 108:4578–4585. Li E, Hamm CM, Gulati AS, Sartor RB, Chen H, Wu X, Zhang T, Rohlf FJ, Zhu W, Gu C, et al. Inflammatory bowel diseases phenotype, C. difficile and NOD2 genotype are associated with shifts in human ileum associated microbial composition. PLoS One 2012;7:e26284. Moltedo B, Lopez ´ CB, Pazos M, Becker MI, Hermesh T, Moran TM. Cutting edge: stealth influenza virus replication precedes the initiation of adaptive immunity. J Immunol 2009;183:3569–3573. Healy DY, Leid JG, Sanderson AR, Hunsaker DH. Biofilms with fungi in chronic rhinosinusitis. Otolaryngol Head Neck Surg 2008;138:641–647. Sanderson AR, Leid JG, Hunsaker D. Bacterial biofilms on the sinus mucosa of human subjects with chronic rhinosinusitis. Laryngoscope 2006;116:1121–1126. Feazel LM, Robertson CE, Ramakrishnan VR, Frank DN. Microbiome complexity and Staphylococcus aureus in chronic rhinosinusitis. Laryngoscope 2012;122:467–472. Tomosada Y, Chiba E, Zelaya H, Takahashi T, Tsukida K, Kitazawa H, Alvarez S, Villena J. Nasally administered Lactobacillus rhamnosus strains differentially modulate respiratory antiviral immune responses and induce protection against respiratory syncytial virus infection. BMC Immunol 2013;14:40. Shope RE. Swine influenza: III. Filtration experiments and etiology. J Exp Med 1931;54:373–385. Jakab GJ, Warr GA, Knight ME. Pulmonary and systemic defenses against challenge with Staphylococcus aureus in mice with pneumonia due to influenza A virus. J Infect Dis 1979;140:105–108. Gardner ID. Effect of influenza virus infection on susceptibility to bacteria in mice. J Infect Dis 1980;142:704–707. Jones WT, Menna JH, Wennerstrom DE. Lethal synergism induced in mice by influenza type A virus and type Ia group B streptococci. Infect Immun 1983;41:618–623. Tashiro M, Ciborowski P, Klenk HD, Pulverer G, Rott R. Role of Staphylococcus protease in the development of influenza pneumonia. Nature 1987;325:536–537. Klenk HD, Rott R, Orlich M. Further studies on the activation of influenza virus by proteolytic cleavage of the haemagglutinin. J Gen Virol 1977;36:151–161. Scheiblauer H, Reinacher M, Tashiro M, Rott R. Interactions between bacteria and influenza A virus in the development of influenza pneumonia. J Infect Dis 1992;166:783–791. Tashiro M, Ciborowski P, Reinacher M, Pulverer G, Klenk HD, Rott R. Synergistic role of staphylococcal proteases in the induction of influenza virus pathogenicity. Virology 1987;157:421–430. Leung RK, Zhou JW, Guan W, Li SK, Yang ZF, Tsui SK. Modulation of potential respiratory pathogens by pH1N1 viral infection. Clin Microbiol Infect 2013;19:930–935. Ichinohe T, Pang IK, Kumamoto Y, Peaper DR, Ho JH, Murray TS, Iwasaki A. Microbiota regulates immune defense against respiratory tract influenza A virus infection. Proc Natl Acad Sci USA 2011;108:5354–5359.
AnnalsATS Volume 11 Supplement 1 | January 2014
Division of Gastroenterology, University of California San Francisco, San Francisco, California
Abstract Viral infection represents a common and problematic health care issue, particularly in younger and senior populations. The respiratory tract is a major portal for microbial exposure, where viral infection can result in nonsymptomatic, mild, and self-limiting or severe and sometimes fatal infection. Although it is well established that virus-specific properties, such as longevity and replication kinetics, impact clinical manifestations, it is less well understood why distinct infectious outcomes may occur across a population of individuals infected with the same strain of virus. Emerging evidence points to interpersonal variation in
pulmonary and gastrointestinal microbiome composition, and specifically to members of the Lactobacillus genus, as key components in defining respiratory viral infection outcomes. Moreover, human studies of airway microbiota after pH1N1 demonstrate that the composition of the respiratory microbiome can be modified by viral infection in a manner that enriches for pathogens associated with secondary bacterial infection. In this article, current knowledge in the field of human microbiome research, particularly as it pertains to respiratory viral infection, is reviewed. Keywords: microbiome; viral infection; respiratory; gastrointestinal
(Received in original form June 12, 2013; accepted in final form October 31, 2013 ) Correspondence and requests for reprints should be addressed to Susan V. Lynch, Ph.D., University of California San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143. E-mail: [email protected] Ann Am Thorac Soc Vol 11, Supplement 1, pp S57–S60, Jan 2014 Copyright © 2014 by the American Thoracic Society DOI: 10.1513/AnnalsATS.201306-158MG Internet address: www.atsjournals.org
The Human Microbiome Environmental microbial ecologists have long recognized that the true diversity of microbes in a given sample is poorly represented by those that are cultured under traditional laboratory conditions (1). This is due, in large part, to the myriad of metabolic capacities encoded by distinct microbial species and the relatively limited range of culture conditions under which microbes are typically grown in the laboratory. In addition, symbiotic and mutualistic microbial interactions are necessary for the viability of some microbial species, which precludes the ability to culture them easily under conventional conditions. As a result, until the recent application of culture-independent approaches to human samples, we have had a long-held, relatively restricted view of microbial diversity associated with the human host. Developed in the environmental microbial ecology field over Lynch: Viruses and Microbiome Alterations
the last several decades to identify the types of microbes in a given sample without recourse to culture, culture-independent methods have dramatically improved our appreciation of microbial diversity associated with humans. This is particularly true in recent years with the large advances in throughput afforded by nextgeneration sequencing and phylogenetic microarrays. Application of these tools to interrogate the diversity of microbial life that inhabits the human superorganism has revealed the presence of highly functional communities of organisms that represent up to 90% of all cells in the human body and as much as 99% of the total functional capacity of the human holobiont. Given the sheer scale of functional genetic capacity encoded by these communities, it is unsurprising that they are emerging as key contributors to critical processes, such as metabolism and maintenance of immune homeostasis. Although increased burden of particular
microbial species are clearly capable of inducing immune activation (2, 3), in healthy individuals the diverse communities of microbes found at a range of anatomical sites typically exist in a relatively quiescent state of symbiosis and immune homeostasis with their human host. What is even more interesting is that the distribution of bacteria in distinct sites distributed across the human host (e.g., the gut, skin, or oral cavity) house communities with distinct phylogenetic distributions, providing evidence for nichespecific selective pressures at each of these sites that influence microbial community composition and, presumably, the resulting site-specific functional attributes conferred by members of these assemblages.
The Respiratory Microbiome Although our view of the respiratory microbiome is not as well developed as that S57
STATE OF THE ART of the gastrointestinal tract, a number of recent studies have provided information on the patterns of microbial colonization in this system. The respiratory tract represents a major site of constant contact with the external (microbial) environment, and, as such, has developed innate and acquired immune responses to protect the mucosal surface from pathogenic infection (reviewed in References 4–6). As has been described in the healthy gastrointestinal tract, distinct sites along the respiratory tract (e.g., nares and oropharynx) exhibit discrete microbiome compositions (7), coupled with a gradient of bacterial burden that progresses from high levels in the upper, to reduced levels in the lower airways. Two independent studies, employing identical extraction and quantitative PCR assays to examine mucosal brushings of the sinuses and the right middle lobe mucosa of the lower airways, demonstrated the presence of high bacterial cell numbers in the upper airways (8), and little or no evidence of bacterial presence in the lower airways (9) of healthy individuals. On the contrary, patients with chronic airway disease, such as asthma, cystic fibrosis, or chronic obstructive pulmonary disease, possess large numbers of bacteria at this site and relatively diverse communities (9–13). Indeed, in a recent study, the extent of lower airway bacterial burden and diversity in the airways of subjects with asthma on inhaled corticosteroid treatment was significantly correlated with the degree of bronchial hyperresponsiveness. Patients with more reactive airways exhibited higher bacterial burden and diversity, indicating that variation in lower airway microbiome composition, and potentially the presence of specific members of these communities, was associated with the degree of bronchial hyperreactivity observed (9). These findings were largely concordant with an independent study of a distinct European corticosteroidusing population of patients with asthma (11). Collectively, these studies indicate that the composition of the mucosal microbiome in the lower airways of patients with chronic inflammatory pulmonary disease is associated with the degree of disease severity, which, by extension, implicates these communities in the immune activation status of these patients.
Respiratory Viral Infection and the Upper Airway Microbiome Respiratory viral infection represents a common occurrence, particularly in S58
pediatric and elderly populations, as well as immune-compromised individuals and those with underlying comorbidities. A recent review article by Hussell and colleagues (14) discusses how virus-specific properties, replication kinetics, and longevity affect the subsequent vigor of innate and adaptive immunity, which contribute to clinical manifestations. In addition, the timing of lung innate immune activation is different between individuals, and is determined by age, genetics, underlying conditions, and infection history—all factors that are also known to influence microbiome composition (10, 15, 16). Immune response to respiratory viral infection involves both the innate and adaptive arms of the immune system. Innate immune recognition of pathogenassociated molecular patterns results in rapid induction of type I IFNs, which signal through the IFN-a/b receptor to both the infected and local bystander cells to enhance IFN production and engage in a program of cellular gene expression aimed at inhibiting viral replication (17). In parallel with this IFN-driven response, expression of cytokines, such as IL-1b and IL-18, and chemokines, such as CC-chemokine ligand 2 (CCL2), result in activation of adjacent CD452 parenchymal cells (e.g., epithelial cells and fibroblasts), as well as local innate immune cells. These activated cells convert transforming growth factor-b to its active form, resulting in increased chemokine (CCL2 and CCL20) secretion by parenchymal stromal cells, and of cytokines, such TNF and IL12. This results in maturation of tissue-resident CD1031 and CD11b1 dendritic cells (DCs) and the recruitment and maturation of monocyte-derived DCs. Antigen-activated DCs then migrate to the draining lymph nodes, where they facilitate adaptive immune responses to the respiratory virus. Although these facets of innate and adaptive immune response to viral infection are well described, evidence is emerging that components of the microbiome, both at the local respiratory mucosa and in the gastrointestinal tract, may influence these host responses to viral infection. Although the healthy lower airways appear to possess a low burden of bacteria, the upper airways (i.e., the sinus cavity) represent a well established site of microbial colonization. Independent studies using fluorescent in situ hybridization to detect specific microbial species have
demonstrated the presence of polymicrobial biofilms (sessile microbial communities) attached to the sinus mucosa of subjects with chronic rhinosinusitis (CRS) and healthy control subjects (18, 19). Probes were generated to detect Haemophilus influenzae, Pseudomonas aeruginosa, Staphylococcus epidermidis, Staphylococcus aureus, and Streptococcus pneumonia, and demonstrated the presence of biofilms containing these known pathogenic species in the majority of both patients and healthy control subjects (18, 19). Another study demonstrated that patients with chronic sinusitus were characterized by an increased abundance of Staphylococcus aureus (20), indicating that this pathogen was involved in sinus pathology. Using a high-resolution microbiome profiling approach, Abreu and colleagues (8) examined the sinus microbiome of patients with CRS (n = 7) and healthy control subjects (n = 7), and demonstrated that known bacterial pathogens, such as H. influenza, P. aeruginosa, and S. aureus, were detected in both healthy and CRS sinuses, as was previously described. However, the sinus microbiome of patients with CRS exhibited characteristics of community collapse (i.e., loss of community membership), a more skewed distribution of species with these communities, and a significant reduction in lactic acid bacteria, including Lactobacillus sakei, which exhibited one of the most significant depletions in patients with CRS. In this state of disease-associated microbiome perturbation, only one species, Corynebacterium tuberculostearicum, was significantly enriched. C. tuberculostearicum is a skin commensal species not known to cause disease; therefore, to examine whether this species possessed pathogenic potential and if the intact microbiome of the sinonasal cavity could protect against this organism, a series of experiments was performed. Using a mouse model, the authors demonstrate that antibiotic depletion of the sinus microbiome before instillation of C. tuberculostearicum resulted in overt pathology that phenocopied that described in patients with CRS (i.e., mucin hypersecretion and goblet cell metaplasia). They also demonstrated that no airway pathology was observed in animals that received a coinstillation of L. sakei with C. tuberculostearicum, indicating that L. sakei is capable of mucosal surface protection.
AnnalsATS Volume 11 Supplement 1 | January 2014
STATE OF THE ART One of the mechanisms by which Lactobacillus species may protect the respiratory mucosa appears to involve modulation of host immune responses. A recent study demonstrated that nasal administration of specific Lactobacillus species protected mice against respiratory syncytial virus (RSV) infection (21). Instillation of two Lactobacillus rhamnosus strains, CRL1505 (Lr05) or CRL1506 (Lr06), afforded protection against RSV infection, albeit via distinct effects on the host immune response. Lr05 induced IFNg and IL-10, whereas Lr06 modulated production of IFN-a, IFN-b, and IL-6 expression. In both cases, Toll-like receptor 3/retinoic acid-inducible gene 1 activation was associated with protection of animals against RSV infection (21). In light of these data, it appears plausible that several members of the Lactobacillus genus, some of which are members of the healthy human sinonasal microbiota, afford broad-range protection of respiratory mucosal surfaces against both viral and bacterial pathogens, at least in part by effecting Toll-like receptor 3 signaling and inducing anti-inflammatory cytokine expression by the host (Figure 1).
Viral Infection Perturbs the Respiratory Microbiome Studies spanning back to the early 1930s demonstrated that viral infection enhanced bacterial pathogenesis in a variety of animal models, for example, influenza virus and Haemophilus influenza in a pig model (22), influenza virus and Staphylococcus species (23), Listeria monocytogenes (24), or group B Streptococcus species (25) in murine models of infection. The mechanisms by which viral infection may enhance bacterial pathogenesis are several, and include impairment of mucociliary clearance, increased bacterial adherence to
epithelial cells, and virus-induced epithelial damage, which facilitates bacterial translocation and dissemination and/or inhibition of antibacterial immune response. In addition, the presence of certain bacteria may also enhance viral pathogenesis. For example, influenza A virus gains entry to the mammalian cell via receptor-mediated endocytosis, which requires cleavage of its surface hemagglutinin (HA) glycoprotein. Viruses with the capacity for systemic infection do so because of the presence of HAs that can effectively be cleaved by enzymes that are widely distributed across a variety of mammalian cell types. Beyond mammalian host-encoded HA cleavage, it has also been demonstrated that particular bacterial species (e.g., Staphylococcus aureus [26], Streptomyces griseus, and Aerococcus viridans [27, 28]) possess the necessary proteolytic activity for effective viral replication and infectivity (28, 29). More recently, human microbiome studies have shed further light on the mechanisms by which viral–bacterial interactions may synergistically enhance pathogenesis, particularly in respiratory infections. Influenza A virus belongs to the Orthomyxoviridae family of viruses, a group of virions of approximately 80–120 nm in diameter containing an RNA genome of approximately 13.5 kb. The 2009 influenza A pdm09 virus (pH1N1) pandemic strain, although not considered a particularly lethal infectious agent, resulted in severe outcomes in some infected individuals. This was attributed to the capacity of this strain to replicate in the lower airways, cause extensive epithelial destruction, viral pneumonia, and secondary bacterial infections with increased lethality (30). The authors examined the oropharyngeal microbiome metagenome (sequence-based analysis of the microbial genomes present in the
Figure 1. Members of the Lactobacillus genus play a role in defining response to viral respiratory infection.
Lynch: Viruses and Microbiome Alterations
microbiomes of individuals) of patients with pneumonia with and without pH1N1 infection. At the phylum level, the relative abundance of Firmicutes and Proteobacteria were significantly enriched in patients with pH1N1. More specifically, a number of Pseudomonas (P. amygdali, P. fluorescens, and Pseudomonas sp. UK4) and Acinetobacter (e.g., A. baumanii and A. junii) were significantly increased in the pH1N1-infected group. Metagenomic sequencing of the samples permitted insights into the functional genes encoded by the organisms that were significantly enriched in the patients infected with pH1N1. pH1N1-associated airway microbiota were significantly enriched for motility, transcriptional regulation, metabolism, and signaling genes, as well as pathways involved in secondary metabolite biosynthesis and catabolism. In contrast, these functional attributes were notably absent from the genera that significantly decreased in relative abundance (e.g., Prevotella, Veillonella, and Neisseria species) in patients infected with pH1N1 (30). These data, generated with human samples, demonstrate that pH1N1 infection perturbs the respiratory microbiome (presumably through its induction of host immune response) in a manner that appears to select for metabolically flexible species capable of sensing and responding to a rapidly changing ecosystem, translocating to the lower airways, and producing secondary metabolites (which are frequently potent antimicrobial and immune-modulating molecules).
Gastrointestinal Microbiome Composition Influences Host Response to Respiratory Viral Infection Clearly, local mucosal microbiome composition in the respiratory tract influences immune responses. However, more recent studies have demonstrated that the composition of the gastrointestinal microbiome also plays a role in modulating immune responses to viral infection of the airways. Using a mouse model of influenza A infection, Ichinohe and colleagues (31) demonstrated that animals who received antibiotic treatment before respiratory viral infection mounted a significantly reduced anti-PR8 antibody response, were incapable of inducing CD4 T cell–mediated IFN-g S59
STATE OF THE ART response to PR8 antigen, and exhibited significantly fewer CD8 1 CD44 1 cells (effector memory T cells). These mice also demonstrated significantly increased viral titers in their lungs. Using neomycin, which resulted in a profound loss of Lactobacillus in both the gut and nares and concomitant enrichment of Sphingomonas species in the gut and Corynebacterium in the nares, exhibited the greatest deficit in effector memory T cells, again implicating Lactobacillus species as key components necessary to mount an appropriate antiviral response.
In conclusion, the upper respiratory tract represents a site of overt microbial colonization, where the composition of the microbiome in this niche appears to act as an ancillary barrier and define host response to respiratory pathogen invasion. Viral infection results in a perturbation to the respiratory microbiome; in the case of the recent pandemic influenza A virus strain, infection resulted in selection for a discrete subset of bacterial species with functional capacities consistent with survival and translocation to the lower airways, where they presumably contribute to secondary bacterial
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AnnalsATS Volume 11 Supplement 1 | January 2014
