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Viruses targeting mammalian cells can indirectly alter the gut microbiota, potentially compounding their phenotypic effects. Multiple studies have observed a disrupted gut microbiota in severe cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection that require hospitalization. Yet, despite demographic shifts in disease severity resulting in a large and continuing burden of non-hospitalized infections, we still know very little about the impact of mild SARS-CoV-2 infection on the gut microbiota in the outpatient setting. To address this knowledge gap, we longitudinally sampled 14 SARS-CoV-2-positive subjects who remained outpatient and 4 household controls. SARS-CoV-2 cases exhibited a significantly less stable gut microbiota relative to controls. These results were confirmed and extended in the K18-humanized angiotensin-converting enzyme 2 mouse model, which is susceptible to SARS-CoV-2 infection. All of the tested SARS-CoV-2 variants significantly disrupted the mouse gut microbiota, including USA-WA1/2020 (the original variant detected in the USA), Delta, and Omicron. Surprisingly, despite the fact that the Omicron variant caused the least severe symptoms in mice, it destabilized the gut microbiota and led to a significant depletion in Akkermansia muciniphila. Furthermore, exposure of wild-type C57BL/6J mice to SARS-CoV-2 disrupted the gut microbiota in the absence of severe lung pathology.IMPORTANCETaken together, our results demonstrate that even mild cases of SARS-CoV-2 can disrupt gut microbial ecology. Our findings in non-hospitalized individuals are consistent with studies of hospitalized patients, in that reproducible shifts in gut microbial taxonomic abundance in response to SARS-CoV-2 have been difficult to identify. Instead, we report a long-lasting instability in the gut microbiota. Surprisingly, our mouse experiments revealed an impact of the Omicron variant, despite producing the least severe symptoms in genetically susceptible mice, suggesting that despite the continued evolution of SARS-CoV-2, it has retained its ability to perturb the intestinal mucosa. These results will hopefully renew efforts to study the mechanisms through which Omicron and future SARS-CoV-2 variants alter gastrointestinal physiology, while also considering the potentially broad consequences of SARS-CoV-2-induced microbiota instability for host health and disease.

Maternal asthma status, prenatal exposures, and infant gut microbiota perturbation are associated with heightened risk of atopy and asthma risk in childhood, observations hypothetically linked by intergenerational microbial transmission. Using maternal vaginal (n = 184) and paired infant stool (n = 172) samples, we identify four compositionally and functionally distinct Lactobacillus-dominated vaginal microbiota clusters (VCs) that relate to prenatal maternal health and exposures and infant serum immunoglobulin E (IgE) status at 1 year. Variance in bacteria shared between mother and infant pairs relate to VCs, maternal allergy/asthma status, and infant IgE levels. Heritable bacterial gene pathways associated with infant IgE include fatty acid synthesis and histamine and tryptophan degradation. In vitro, vertically transmitted Lactobacillus jensenii strains induce immunosuppressive phenotypes on human antigen-presenting cells. Murine supplementation with L. jensenii reduces lung eosinophils, neutrophilic expansion, and the proportion of interleukin-4 (IL-4)+ CD4+ T cells. Thus, bacterial and atopy heritability are intimately linked, suggesting a microbial component of intergenerational disease transmission.

Resident microbes in skin and gut predominantly impact local immune cell function during homeostasis. However, colitis-associated neutrophilic skin disorders suggest possible breakdown of this compartmentalization with disease. Using a model wherein neonatal skin colonization by Staphylococcus epidermidis facilitates generation of commensal-specific tolerance and CD4+ regulatory T cells (Tregs), we ask whether this response is perturbed by gut inflammation. Chemically induced colitis is accompanied by intestinal expansion of S. epidermidis and reduces gut-draining lymph node (dLN) commensal-specific Tregs. It also results in reduced commensal-specific Tregs in skin and skin-dLNs and increased skin neutrophils. Increased CD4+ circulation between gut and skin dLN suggests that the altered cutaneous response is initiated in the colon, and resistance to colitis-induced effects in Cd4creIl1r1fl/fl mice implicate interleukin (IL)-1 in mediating the altered commensal-specific response. These findings provide mechanistic insight into observed connections between inflammatory skin and intestinal diseases.

Bacterial activation of T helper 17 (Th17) cells exacerbates mouse models of autoimmunity, but how human-associated bacteria impact Th17-driven disease remains elusive. We show that human gut Actinobacterium Eggerthella lenta induces intestinal Th17 activation by lifting inhibition of the Th17 transcription factor Rorγt through cell- and antigen-independent mechanisms. E. lenta is enriched in inflammatory bowel disease (IBD) patients and worsens colitis in a Rorc-dependent manner in mice. Th17 activation varies across E. lenta strains, which is attributable to the cardiac glycoside reductase 2 (Cgr2) enzyme. Cgr2 is sufficient to induce interleukin (IL)-17a, a major Th17 cytokine. cgr2+ E. lenta deplete putative steroidal glycosides in pure culture; related compounds are negatively associated with human IBD severity. Finally, leveraging the sensitivity of Cgr2 to dietary arginine, we prevented E. lenta-induced intestinal inflammation in mice. Together, these results support a role for human gut bacterial metabolism in driving Th17-dependent autoimmunity.

States of oral health and disease reflect the compositional and functional capacities of, as well as the interspecies interactions within, the oral microbiota. The oral cavity exists as a highly dynamic microbial environment that harbors many distinct substrata and microenvironments that house diverse microbial communities. Specific to the oral cavity, the nonshedding dental surfaces facilitate the development of highly complex polymicrobial biofilm communities, characterized not only by the distinct microbes comprising them, but cumulatively by their activities. Adding to this complexity, the oral cavity faces near-constant environmental challenges, including those from host diet, salivary flow, masticatory forces, and introduction of exogenous microbes. The composition of the oral microbiome is shaped throughout life by factors including host genetics, maternal transmission, as well as environmental factors, such as dietary habits, oral hygiene practice, medications, and systemic factors. This dynamic ecosystem presents opportunities for oral microbial dysbiosis and the development of dental and periodontal diseases. The application of both in vitro and culture-independent approaches has broadened the mechanistic understandings of complex polymicrobial communities within the oral cavity, as well as the environmental, local, and systemic underpinnings that influence the dynamics of the oral microbiome. Here, we review the present knowledge and current understanding of microbial communities within the oral cavity and the influences and challenges upon this system that encourage homeostasis or provoke microbiome perturbation, and thus contribute to states of oral health or disease.

BackgroundWhether microbiome characteristics of induced sputum or oral samples demonstrate unique relationships to features of atopy or mild asthma in adults is unknown.ObjectiveWe sought to determine sputum and oral microbiota relationships to clinical or immunologic features in mild atopic asthma and the impact on the microbiota of inhaled corticosteroid (ICS) treatment administered to ICS-naive subjects with asthma.MethodsBacterial microbiota profiles were analyzed in induced sputum and oral wash samples from 32 subjects with mild atopic asthma before and after inhaled fluticasone treatment, 18 atopic subjects without asthma, and 16 nonatopic healthy subjects in a multicenter study (NCT01537133). Associations with clinical and immunologic features were examined, including markers of atopy, type 2 inflammation, immune cell populations, and cytokines.ResultsSputum bacterial burden inversely associated with bronchial expression of type 2 (T2)-related genes. Differences in specific sputum microbiota also associated with T2-low asthma phenotype, a subgroup of whom displayed elevations in lung inflammatory mediators and reduced sputum bacterial diversity. Differences in specific oral microbiota were more reflective of atopic status. After ICS treatment of patients with asthma, the compositional structure of sputum microbiota showed greater deviation from baseline in ICS nonresponders than in ICS responders.ConclusionsNovel associations of sputum and oral microbiota to immunologic features were observed in this cohort of subjects with or without ICS-naive mild asthma. These findings confirm and extend our previous report of reduced bronchial bacterial burden and compositional complexity in subjects with T2-high asthma, with additional identification of a T2-low subgroup with a distinct microbiota-immunologic relationship.

The human body supports a thriving diversity of microbes which comprise a dynamic, ancillary, functional system that synergistically develops in lock-step with physiological development of its host. The human microbiome field has transitioned from cataloging this rich diversity to dissecting molecular mechanisms by which microbiomes influence human health. Early life microbiome development trains immune function. Thus, vertically, horizontally, and environmentally acquired microbes and their metabolites have the potential to shape developmental trajectories with life-long implications for health.

Neonates at risk of childhood atopy and asthma exhibit perturbation of the gut microbiome, metabolic dysfunction and increased concentrations of 12,13-diHOME in their faeces. However, the mechanism, source and contribution of this lipid to allergic inflammation remain unknown. Here, we show that intra-abdominal treatment of mice with 12,13-diHOME increased pulmonary inflammation and decreased the number of regulatory T (Treg) cells in the lungs. Treatment of human dendritic cells with 12,13-diHOME altered expression of PPARγ-regulated genes and reduced anti-inflammatory cytokine secretion and the number of Treg cells in vitro. Shotgun metagenomic sequencing of neonatal faeces indicated that bacterial epoxide hydrolase (EH) genes are more abundant in the gut microbiome of neonates who develop atopy and/or asthma during childhood. Three of these bacterial EH genes (3EH) specifically produce 12,13-diHOME, and treatment of mice with bacterial strains expressing 3EH caused a decrease in the number of lung Treg cells in an allergen challenge model. In two small birth cohorts, an increase in the copy number of 3EH or the concentration of 12,13-diHOME in the faeces of neonates was found to be associated with an increased probability of developing atopy, eczema or asthma during childhood. Our data indicate that elevated 12,13-diHOME concentrations impede immune tolerance and may be produced by bacterial EHs in the neonatal gut, offering a mechanistic link between perturbation of the gut microbiome during early life and atopy and asthma during childhood.

BackgroundIn infants, distinct nasopharyngeal bacterial microbiotas differentially associate with the incidence and severity of acute respiratory tract infection and childhood asthma development.ObjectiveWe hypothesized that distinct nasal airway microbiota structures also exist in children with asthma and relate to clinical outcomes.MethodsNasal secretion samples (n = 3122) collected after randomization during the fall season from children with asthma (6-17 years, n = 413) enrolled in a trial of omalizumab (anti-IgE) underwent 16S rRNA profiling. Statistical analyses with exacerbation as the primary outcome and rhinovirus infection and respiratory illnesses as secondary outcomes were performed. Using A549 epithelial cells, we assessed nasal isolates of Moraxella, Staphylococcus, and Corynebacterium species for their capacity to induce epithelial damage and inflammatory responses.ResultsSix nasal airway microbiota assemblages, each dominated by Moraxella, Staphylococcus, Corynebacterium, Streptococcus, Alloiococcus, or Haemophilus species, were observed. Moraxella and Staphylococcus species-dominated microbiotas were most frequently detected and exhibited temporal stability. Nasal microbiotas dominated by Moraxella species were associated with increased exacerbation risk and eosinophil activation. Staphylococcus or Corynebacterium species-dominated microbiotas were associated with reduced respiratory illness and exacerbation events, whereas Streptococcus species-dominated assemblages increased the risk of rhinovirus infection. Nasal microbiota composition remained relatively stable despite viral infection or exacerbation; only a few taxa belonging to the dominant genera exhibited relative abundance fluctuations during these events. In vitro, Moraxella catarrhalis induced significantly greater epithelial damage and inflammatory cytokine expression (IL-33 and IL-8) compared with other dominant nasal bacterial isolates tested.ConclusionDistinct nasal airway microbiotas of children with asthma relate to the likelihood of exacerbation, rhinovirus infection, and respiratory illnesses during the fall season.

Pneumonia is common and frequently fatal in HIV-infected patients, due to rampant, systemic inflammation and failure to control microbial infection. While airway microbiota composition is related to local inflammatory response, gut microbiota has been shown to correlate with the degree of peripheral immune activation (IL6 and IP10 expression) in HIV-infected patients. We thus hypothesized that both airway and gut microbiota are perturbed in HIV-infected pneumonia patients, that the gut microbiota is related to peripheral CD4+ cell counts, and that its associated products differentially program immune cell populations necessary for controlling microbial infection in CD4-high and CD4-low patients. To assess these relationships, paired bronchoalveolar lavage and stool microbiota (bacterial and fungal) from a large cohort of Ugandan, HIV-infected patients with pneumonia were examined, and in vitro tests of the effect of gut microbiome products on macrophage effector phenotypes performed. While lower airway microbiota stratified into three compositionally distinct microbiota as previously described, these were not related to peripheral CD4 cell count. In contrast, variation in gut microbiota composition significantly related to CD4 cell count, lung microbiota composition, and patient mortality. Compared with patients with high CD4+ cell counts, those with low counts possessed more compositionally similar airway and gut microbiota, evidence of microbial translocation, and their associated gut microbiome products reduced macrophage activation and IL-10 expression and increased IL-1β expression in vitro. These findings suggest that the gut microbiome is related to CD4 status and plays a key role in modulating macrophage function, critical to microbial control in HIV-infected patients with pneumonia.

Over the past decade, our view of human-associated microbes has expanded beyond that of a few species toward an appreciation of the diverse and niche-specialized microbial communities that develop in the human host with chronological age. The largest reservoir of microbes exists in the distal gastrointestinal tract, both in the lumen, where microbes facilitate primary and secondary metabolism, and on mucosal surfaces, where they interact with host immune cell populations. While local microbial-driven immunomodulation in the gut is well described, more recent studies have demonstrated a role for the gut microbiome in influencing remote organs and mucosal and hematopoietic immune function. Unsurprisingly, therefore, perturbation to the composition and function of the gut microbiota has been associated with chronic diseases ranging from gastrointestinal inflammatory and metabolic conditions to neurological, cardiovascular, and respiratory illnesses. Considerable effort is currently focused on understanding the natural history of microbiome development in humans in the context of health outcomes, in parallel with improving our knowledge of microbiome-host molecular interactions. These efforts ultimately aim to develop effective approaches to rehabilitate perturbed human microbial ecosystems as a means to restore health or prevent disease. This review details the role of the gut microbiome in modulating host health with a focus on immunomodulation and discusses strategies for manipulating the gut microbiome for the management or prevention of chronic inflammatory conditions.

The gut microbiome, the multispecies community of microbes that exists in the gastrointestinal tract, encodes several orders of magnitude more functional genes than the human genome. It also plays a pivotal role in human health, in part due to metabolism of environmental, dietary, and host-derived substrates, which produce bioactive metabolites. Perturbations to the composition and associated metabolic output of the gut microbiome have been associated with a number of chronic liver diseases, including nonalcoholic fatty liver disease (NAFLD). Here, we review the rapidly evolving suite of next-generation techniques used for studying gut microbiome composition, functional gene content, and bioactive products and discuss relationships with the pathogenesis of NAFLD.

Microbial dysbiosis commonly occurs in patients with inflammatory bowel diseases (IBD). Exogenous causes of dysbiosis such as antibiotics and diet are well described, but host derived causes are understudied. A20 is a potent regulator of signals triggered by microbial pattern molecules, and A20 regulates susceptibility to intestinal inflammation in mice and in humans. We now report that mice lacking A20 expression in dendritic cells, A20FL/FL CD11c-Cre mice (or A20dDC mice), spontaneously develop colitogenic intestinal dysbiosis that is evident upon weaning and precedes the onset of colitis. Intestines from A20dDC mice express increased amounts of Reg3β and Reg3γ, but not Ang4. A20 deficient DCs promote gut microbiota perturbation in the absence of adaptive lymphocytes. Moreover, A20 deficient DCs directly induce expression of Reg3β and Reg3γ but not Ang 4 in normal intestinal epithelial cell enteroid cultures in the absence of other cell types. These findings reveal a pathophysiological pathway in which defective expression of an IBD susceptibility gene in DCs drives aberrant expression of anti-bacterial peptides and luminal dysbiosis that in turn confers host susceptibility to intestinal inflammation.

BackgroundPerturbations to the composition and function of bronchial bacterial communities appear to contribute to the pathophysiology of asthma. Unraveling the nature and mechanisms of these complex associations will require large longitudinal studies, for which bronchoscopy is poorly suited. Studies of samples obtained by sputum induction and nasopharyngeal brushing or lavage have also reported asthma-associated microbiota characteristics. It remains unknown, however, whether the microbiota detected in these less-invasive sample types reflect the composition of bronchial microbiota in asthma.ResultsBacterial microbiota in paired protected bronchial brushings (BB; n = 45), induced sputum (IS; n = 45), oral wash (OW; n = 45), and nasal brushings (NB; n = 27) from adults with mild atopic asthma (AA), atopy without asthma (ANA), and healthy controls (HC) were profiled using 16S rRNA gene sequencing. Though microbiota composition varied with sample type (p

BackgroundPsoriasis impacts 1-3% of the world's population and is characterized by hyper-proliferation of keratinocytes and increased inflammation. At the molecular level, psoriasis is commonly driven by a Th17 response, which serves as a major therapeutic target. Microbiome perturbations have been associated with several immune-mediated diseases such as atopic dermatitis, asthma, and multiple sclerosis. Although a few studies have investigated the association between the skin microbiome and psoriasis, conflicting results have been reported plausibly due to the lack of standardized sampling and profiling protocols, or to inherent microbial variability across human subjects and underpowered studies. To better understand the link between the cutaneous microbiota and psoriasis, we conducted an analysis of skin bacterial communities of 28 psoriasis patients and 26 healthy subjects, sampled at six body sites using a standardized protocol and higher sequencing depth compared to previous studies. Mouse studies were employed to examine dermal microbial-immune interactions of bacterial species identified from our study.ResultsSkin microbiome profiling based on sequencing the 16S rRNA V1-V3 variable region revealed significant differences between the psoriasis-associated and healthy skin microbiota. Comparing the overall community structures, psoriasis-associated microbiota displayed higher diversity and more heterogeneity compared to healthy skin bacterial communities. Specific microbial signatures were associated with psoriatic lesional, psoriatic non-lesional, and healthy skin. Specifically, relative enrichment of Staphylococcus aureus was strongly associated with both lesional and non-lesional psoriatic skin. In contrast, Staphylococcus epidermidis and Propionibacterium acnes were underrepresented in psoriatic lesions compared to healthy skin, especially on the arm, gluteal fold, and trunk. Employing a mouse model to further study the impact of cutaneous Staphylcoccus species on the skin T cell differentiation, we found that newborn mice colonized with Staphylococcus aureus demonstrated strong Th17 polarization, whereas mice colonized with Staphylococcus epidermidis or un-colonized controls showed no such response.ConclusionOur results suggest that microbial communities on psoriatic skin is substantially different from those on healthy skin. The psoriatic skin microbiome has increased diversity and reduced stability compared to the healthy skin microbiome. The loss of community stability and decrease in immunoregulatory bacteria such as Staphylococcus epidermidis and Propionibacterium acnes may lead to higher colonization with pathogens such as Staphylococcus aureus, which could exacerbate cutaneous inflammation along the Th17 axis.

Our understanding of the link between the human microbiome and disease, including obesity, inflammatory bowel disease, arthritis and autism, is rapidly expanding. Improvements in the throughput and accuracy of DNA sequencing of the genomes of microbial communities that are associated with human samples, complemented by analysis of transcriptomes, proteomes, metabolomes and immunomes and by mechanistic experiments in model systems, have vastly improved our ability to understand the structure and function of the microbiome in both diseased and healthy states. However, many challenges remain. In this review, we focus on studies in humans to describe these challenges and propose strategies that leverage existing knowledge to move rapidly from correlation to causation and ultimately to translation into therapies.

Recent studies leveraging next-generation sequencing and functional approaches to understand the human microbiota have demonstrated the presence of diverse, niche-specific microbial communities at nearly every mucosal surface. These microbes contribute to the development and function of physiologic and immunological features that are key to host health status. Not surprisingly, several chronic inflammatory diseases have been attributed to dysbiosis of microbiota composition or function, including chronic rhinosinusitis (CRS). CRS is a heterogeneous disease characterized by inflammation of the sinonasal cavity and mucosal microbiota dysbiosis. Inflammatory phenotypes and bacterial community compositions vary considerably across individuals with CRS, complicating current studies that seek to address causality of a dysbiotic microbiome as a driver or initiator of persistent sinonasal inflammation. Murine models have provided some experimental evidence that alterations in local microbial communities and microbially-produced metabolites influence health status. In this perspective, we will discuss the clinical implications of distinct microbial compositions and community-level functions in CRS and how mucosal microbiota relate to the diverse inflammatory endotypes that are frequently observed. We will also describe specific microbial interactions that can deterministically shape the pattern of co-colonizers and the resulting metabolic products that drive or exacerbate host inflammation. These findings are discussed in the context of CRS-associated inflammation and in other chronic inflammatory diseases that share features observed in CRS. An improved understanding of CRS patient stratification offers the opportunity to personalize therapeutic regimens and to design novel treatments aimed at manipulation of the disease-associated microbiota to restore sinus health.

Gut microbiota dysbiosis and metabolic dysfunction in infancy precedes childhood atopy and asthma development. Here we examined gut microbiota maturation over the first year of life in infants at high risk for asthma (HR), and whether it is modifiable by early-life Lactobacillus supplementation. We performed a longitudinal comparison of stool samples collected from HR infants randomized to daily oral Lactobacillus rhamnosus GG (HRLGG) or placebo (HRP) for 6 months, and healthy (HC) infants. Meconium microbiota of HRP participants is distinct, follows a delayed developmental trajectory, and is primarily glycolytic and depleted of a range of anti-inflammatory lipids at 6 months of age. These deficits are partly rescued in HRLGG infants, but this effect was lost at 12 months of age, 6 months after cessation of supplementation. Thus we show that early-life gut microbial development is distinct, but plastic, in HR infants. Our findings offer a novel strategy for early-life preventative interventions.

BackgroundChronic rhinosinusitis (CRS) is a heterogeneous disease characterized by persistent sinonasal inflammation and sinus microbiome dysbiosis. The basis of this heterogeneity is poorly understood. We sought to address the hypothesis that a limited number of compositionally distinct pathogenic bacterial microbiota exist in CRS patients and invoke discrete immune responses and clinical phenotypes in CRS patients.ResultsSinus brushings from patients with CRS (n = 59) and healthy individuals (n = 10) collected during endoscopic sinus surgery were analyzed using 16S rRNA gene sequencing, predicted metagenomics, and RNA profiling of the mucosal immune response. We show that CRS patients cluster into distinct sub-groups (DSI-III), each defined by specific pattern of bacterial co-colonization (permutational multivariate analysis of variance (PERMANOVA); p = 0.001, r 2 = 0.318). Each sub-group was typically dominated by a pathogenic family: Streptococcaceae (DSI), Pseudomonadaceae (DSII), Corynebacteriaceae [DSIII(a)], or Staphylococcaceae [DSIII(b)]. Each pathogenic microbiota was predicted to be functionally distinct (PERMANOVA; p = 0.005, r 2 = 0.217) and encode uniquely enriched gene pathways including ansamycin biosynthesis (DSI), tryptophan metabolism (DSII), two-component response [DSIII(b)], and the PPAR-γ signaling pathway [DSIII(a)]. Each is also associated with significantly distinct host immune responses; DSI, II, and III(b) invoked a variety of pro-inflammatory, TH1 responses, while DSIII(a), which exhibited significantly increased incidence of nasal polyps (Fisher's exact; p = 0.034, relative risk = 2.16), primarily induced IL-5 expression (Kruskal Wallis; q = 0.045).ConclusionsA large proportion of CRS patient heterogeneity may be explained by the composition of their sinus bacterial microbiota and related host immune response-features which may inform strategies for tailored therapy in this patient population.

Many HIV-infected individuals on antiretroviral therapy (ART) exhibit persistent systemic inflammation, which predicts morbidity and mortality. ART-treated subjects concurrently exhibit marked compositional alterations in the gut bacterial microbiota and the degree of dysbiosis correlates with systemic inflammation. Whether interventions to modulate the microbiome can affect systemic inflammation is unknown. An open-label fecal microbial transplantation (FMT) was delivered by colonoscopy to asymptomatic HIV-infected ART-suppressed individuals without antibiotic pre-treatment. Stool was assessed before and after FMT for engraftment of donor microbes, and peripheral blood was assayed for immune activation biomarkers. Six participants received FMT and 2 participants served as controls. No serious adverse effects occurred during 24 weeks of follow-up. At baseline, HIV-infected individuals exhibited microbiota profiles distinct from uninfected donors. During the 8 weeks post-FMT, recipients demonstrated partial engraftment of the donor microbiome (P < 0.05). Recipient microbiota remained significantly distant from donors, unlike that observed following FMT for treatment of C. difficile infection. Systemic inflammatory markers showed no significant change post-FMT. FMT was well-tolerated in ART-treated, HIV-infected individuals. Engraftment was detectable but modest, and appeared to be limited to specific bacterial taxa. Whether antibiotic conditioning can enhance engraftment and the capacity of microbiota to modulate inflammation remains to be investigated.

BackgroundCompositional differences in the bronchial bacterial microbiota have been associated with asthma, but it remains unclear whether the findings are attributable to asthma, to aeroallergen sensitization, or to inhaled corticosteroid treatment.ObjectivesWe sought to compare the bronchial bacterial microbiota in adults with steroid-naive atopic asthma, subjects with atopy but no asthma, and nonatopic healthy control subjects and to determine relationships of the bronchial microbiota to phenotypic features of asthma.MethodsBacterial communities in protected bronchial brushings from 42 atopic asthmatic subjects, 21 subjects with atopy but no asthma, and 21 healthy control subjects were profiled by using 16S rRNA gene sequencing. Bacterial composition and community-level functions inferred from sequence profiles were analyzed for between-group differences. Associations with clinical and inflammatory variables were examined, including markers of type 2-related inflammation and change in airway hyperresponsiveness after 6 weeks of fluticasone treatment.ResultsThe bronchial microbiome differed significantly among the 3 groups. Asthmatic subjects were uniquely enriched in members of the Haemophilus, Neisseria, Fusobacterium, and Porphyromonas species and the Sphingomonodaceae family and depleted in members of the Mogibacteriaceae family and Lactobacillales order. Asthma-associated differences in predicted bacterial functions included involvement of amino acid and short-chain fatty acid metabolism pathways. Subjects with type 2-high asthma harbored significantly lower bronchial bacterial burden. Distinct changes in specific microbiota members were seen after fluticasone treatment. Steroid responsiveness was linked to differences in baseline compositional and functional features of the bacterial microbiome.ConclusionEven in subjects with mild steroid-naive asthma, differences in the bronchial microbiome are associated with immunologic and clinical features of the disease. The specific differences identified suggest possible microbiome targets for future approaches to asthma treatment or prevention.

RationaleThe potential role of the airway microbiota in dictating immune responses and infection outcomes in HIV-associated pneumonia is largely unknown.ObjectivesTo investigate whether microbiologically and immunologically distinct subsets of patients with HIV and pneumonia exist and are related to mortality.MethodsBronchoalveolar lavage samples from Ugandan patients with HIV and pneumonia (n = 182) were obtained at study enrollment (following antibiotic treatment); patient demographics including 8- and 70-day mortality were collected. Lower airway bacterial community composition was assessed via amplification and sequencing of the V4 region of the 16S ribosomal RNA gene. Host immune response gene expression profiles were generated by quantitative polymerase chain reaction using RNA extracted from bronchoalveolar lavage fluid. Liquid and gas chromatography mass spectrometry was used to profile serum metabolites.Measurements and main resultsBased on airway microbiome composition, most patients segregated into three distinct groups, each of which were predicted to encode metagenomes capable of producing metabolites characteristically enriched in paired serum samples from these patients. These three groups also exhibited differences in mortality; those with the highest rate had increased ceftriaxone administration and culturable Aspergillus, and demonstrated significantly increased induction of airway T-helper cell type 2 responses. The group with the lowest mortality was characterized by increased expression of T-cell immunoglobulin and mucin domain 3, which down-regulates T-helper cell type 1 proinflammatory responses and is associated with chronic viral infection.ConclusionsThese data provide evidence that compositionally and structurally distinct lower airway microbiomes are associated with discrete local host immune responses, peripheral metabolic reprogramming, and different rates of mortality.

BackgroundAs little is known of association(s) between gut microbiota profiles and host immunological markers, we explored these in children with and without multiple sclerosis (MS).MethodsChildren ≤18 years provided stool and blood. MS cases were within 2-years of onset. Fecal 16S rRNA gene profiles were generated on an Illumina Miseq platform. Peripheral blood mononuclear cells were isolated, and Treg (CD4+CD25hiCD127lowFoxP3+) frequency and CD4+ T-cell intracellular cytokine production evaluated by flow cytometry. Associations between microbiota diversity, phylum-level abundances and immune markers were explored using Pearson's correlation and adjusted linear regression.ResultsTwenty-four children (15 relapsing-remitting, nine controls), averaging 12.6 years were included. Seven were on a disease-modifying drug (DMD) at sample collection. Although immune markers (e.g. Th2, Th17, Tregs) did not differ between cases and controls (p > 0.05), divergent gut microbiota associations occurred; richness correlated positively with Th17 for cases (r = +0.665, p = 0.018), not controls (r = -0.644, p = 0.061). Bacteroidetes inversely associated with Th17 for cases (r = -0.719, p = 0.008), not controls (r = +0.320, p = 0.401). Fusobacteria correlated with Tregs for controls (r = +0.829, p = 0.006), not cases (r = -0.069, p = 0.808).ConclusionsOur observations motivate further exploration to understand disruption of the microbiota-immune balance so early in the MS course.

Homeostasis of the gastrointestinal (GI) tract is controlled by complex interactions between epithelial and immune cells and the resident microbiota. Here, we studied the role of Wnt signaling in GI homeostasis using Disheveled 1 knockout (Dvl1-/-) mice, which display an increase in whole gut transit time. This phenotype is associated with a reduction and mislocalization of Paneth cells and an increase in CD8+ T cells in the lamina propria. Bone marrow chimera experiments demonstrated that GI dysfunction requires abnormalities in both epithelial and immune cells. Dvl1-/- mice exhibit a significantly distinct GI microbiota, and manipulation of the gut microbiota in mutant mice rescued the GI transit abnormality without correcting the Paneth and CD8+ T cell abnormalities. Moreover, manipulation of the gut microbiota in wild-type mice induced a GI transit abnormality akin to that seen in Dvl1-/- mice. Together, these data indicate that microbiota manipulation can overcome host dysfunction to correct GI transit abnormalities. Our findings illustrate a mechanism by which the epithelium and immune system coregulate gut microbiota composition to promote normal GI function.

RationaleImproved understanding of the lung microbiome in HIV-infected individuals could lead to better strategies for diagnosis, therapy, and prophylaxis of HIV-associated pneumonias. Differences in the oral and lung microbiomes in HIV-infected and HIV-uninfected individuals are not well defined. Whether highly active antiretroviral therapy influences these microbiomes is unclear.ObjectivesWe determined whether oral and lung microbiomes differed in clinically healthy groups of HIV-infected and HIV-uninfected subjects.MethodsParticipating sites in the Lung HIV Microbiome Project contributed bacterial 16S rRNA sequencing data from oral washes and bronchoalveolar lavages (BALs) obtained from HIV-uninfected individuals (n = 86), HIV-infected individuals who were treatment naive (n = 18), and HIV-infected individuals receiving antiretroviral therapy (n = 38).Measurements and main resultsMicrobial populations differed in the oral washes among the subject groups (Streptococcus, Actinomyces, Rothia, and Atopobium), but there were no individual taxa that differed among the BALs. Comparison of oral washes and BALs demonstrated similar patterns from HIV-uninfected individuals and HIV-infected individuals receiving antiretroviral therapy, with multiple taxa differing in abundance. The pattern observed from HIV-infected individuals who were treatment naive differed from the other two groups, with differences limited to Veillonella, Rothia, and Granulicatella. CD4 cell counts did not influence the oral or BAL microbiome in these relatively healthy, HIV-infected subjects.ConclusionsThe overall similarity of the microbiomes in participants with and without HIV infection was unexpected, because HIV-infected individuals with relatively preserved CD4 cell counts are at higher risk for lower respiratory tract infections, indicating impaired local immune function.

Asthma is a common chronic disease without cure. Our understanding of asthma onset, pathobiology, classification, and management has evolved substantially over the past decade; however, significant asthma-related morbidity and excess healthcare use and costs persist. To address this important clinical condition, the NHLBI convened a group of extramural investigators for an Asthma Research Strategic Planning workshop on September 18-19, 2014, to accelerate discoveries and their translation to patients. The workshop focused on (1) in utero and early-life origins of asthma, (2) the use of phenotypes and endotypes to classify disease, (3) defining disease modification, (4) disease management, and (5) implementation research. This report summarizes the workshop and produces recommendations to guide future research in asthma.

Asthma and atopy, classically associated with hyper-activation of the T helper 2 (Th2) arm of adaptive immunity, are among the most common chronic illnesses worldwide. Emerging evidence relates atopy and asthma to the composition and function of the human microbiome, the collection of microbes that reside in and on and interact with the human body. The ability to interrogate microbial ecology of the human host is due in large part to recent technological developments that permit identification of microbes and their products using culture-independent molecular detection techniques. In this review we explore the roles of respiratory, gut, and environmental microbiomes in asthma and allergic disease development, manifestation, and attenuation. Though still a relatively nascent field of research, evidence to date suggests that the airway and/or gut microbiome may represent fertile targets for prevention or management of allergic asthma and other diseases in which adaptive immune dysfunction is a prominent feature.

BackgroundWheezing illnesses cause major morbidity in infants and are frequent precursors to asthma.ObjectiveWe sought to examine environmental factors associated with recurrent wheezing in inner-city environments.MethodsThe Urban Environment and Childhood Asthma study examined a birth cohort at high risk for asthma (n = 560) in Baltimore, Boston, New York, and St Louis. Environmental assessments included allergen exposure and, in a nested case-control study of 104 children, the bacterial content of house dust collected in the first year of life. Associations were determined among environmental factors, aeroallergen sensitization, and recurrent wheezing at age 3 years.ResultsCumulative allergen exposure over the first 3 years was associated with allergic sensitization, and sensitization at age 3 years was related to recurrent wheeze. In contrast, first-year exposure to cockroach, mouse, and cat allergens was negatively associated with recurrent wheeze (odds ratio, 0.60, 0.65, and 0.75, respectively; P ≤ .01). Differences in house dust bacterial content in the first year, especially reduced exposure to specific Firmicutes and Bacteriodetes, was associated with atopy and atopic wheeze. Exposure to high levels of both allergens and this subset of bacteria in the first year of life was most common among children without atopy or wheeze.ConclusionsIn inner-city environments children with the highest exposure to specific allergens and bacteria during their first year were least likely to have recurrent wheeze and allergic sensitization. These findings suggest that concomitant exposure to high levels of certain allergens and bacteria in early life might be beneficial and suggest new preventive strategies for wheezing and allergic diseases.

Specific bacterial species are implicated in the pathogenesis of exacerbations of chronic obstructive pulmonary disease (COPD). However, recent studies of clinically stable COPD patients have demonstrated a greater diversity of airway microbiota, whose role in acute exacerbations is unclear. In this study, temporal changes in the airway microbiome before, at the onset of, and after an acute exacerbation were examined in 60 sputum samples collected from subjects enrolled in a longitudinal study of bacterial infection in COPD. Microbiome composition and predicted functions were examined using 16S rRNA-based culture-independent profiling methods. Shifts in the abundance (≥ 2-fold, P < 0.05) of many taxa at exacerbation and after treatment were observed. Microbiota members that were increased at exacerbation were primarily of the Proteobacteria phylum, including nontypical COPD pathogens. Changes in the bacterial composition after treatment for an exacerbation differed significantly among the therapy regimens clinically prescribed (antibiotics only, oral corticosteroids only, or both). Treatment with antibiotics alone primarily decreased the abundance of Proteobacteria, with the prolonged suppression of some microbiota members being observed. In contrast, treatment with corticosteroids alone led to enrichment for Proteobacteria and members of other phyla. Predicted metagenomes of particular microbiota members involved in these compositional shifts indicated exacerbation-associated loss of functions involved in the synthesis of antimicrobial and anti-inflammatory products, alongside enrichment in functions related to pathogen-elicited inflammation. These trends reversed upon clinical recovery. Further larger studies will be necessary to determine whether specific compositional or functional changes detected in the airway microbiome could be useful indicators of exacerbation development or outcome.

Exposure to dogs in early infancy has been shown to reduce the risk of childhood allergic disease development, and dog ownership is associated with a distinct house dust microbial exposure. Here, we demonstrate, using murine models, that exposure of mice to dog-associated house dust protects against ovalbumin or cockroach allergen-mediated airway pathology. Protected animals exhibited significant reduction in the total number of airway T cells, down-regulation of Th2-related airway responses, as well as mucin secretion. Following dog-associated dust exposure, the cecal microbiome of protected animals was extensively restructured with significant enrichment of, amongst others, Lactobacillus johnsonii. Supplementation of wild-type animals with L. johnsonii protected them against both airway allergen challenge or infection with respiratory syncytial virus. L. johnsonii-mediated protection was associated with significant reductions in the total number and proportion of activated CD11c(+)/CD11b(+) and CD11c(+)/CD8(+) cells, as well as significantly reduced airway Th2 cytokine expression. Our results reveal that exposure to dog-associated household dust results in protection against airway allergen challenge and a distinct gastrointestinal microbiome composition. Moreover, the study identifies L. johnsonii as a pivotal species within the gastrointestinal tract capable of influencing adaptive immunity at remote mucosal surfaces in a manner that is protective against a variety of respiratory insults.

Sub-Saharan Africa represents 69% of the total number of individuals living with HIV infection worldwide and 72% of AIDS deaths globally. Pulmonary infection is a common and frequently fatal complication, though little is known regarding the lower airway microbiome composition of this population. Our objectives were to characterize the lower airway microbiome of Ugandan HIV-infected patients with pneumonia, to determine relationships with demographic, clinical, immunological, and microbiological variables and to compare the composition and predicted metagenome of these communities to a comparable cohort of patients in the US (San Francisco). Bronchoalveolar lavage samples from a cohort of 60 Ugandan HIV-infected patients with acute pneumonia were collected. Amplified 16S ribosomal RNA was profiled and aforementioned relationships examined. Ugandan airway microbiome composition and predicted metagenomic function were compared to US HIV-infected pneumonia patients. Among the most common bacterial pulmonary pathogens, Pseudomonas aeruginosa was most prevalent in the Ugandan cohort. Patients with a richer and more diverse airway microbiome exhibited lower bacterial burden, enrichment of members of the Lachnospiraceae and sulfur-reducing bacteria and reduced expression of TNF-alpha and matrix metalloproteinase-9. Compared to San Franciscan patients, Ugandan airway microbiome was significantly richer, and compositionally distinct with predicted metagenomes that encoded a multitude of distinct pathogenic pathways e.g secretion systems. Ugandan pneumonia-associated airway microbiome is compositionally and functionally distinct from those detected in comparable patients in developed countries, a feature which may contribute to adverse outcomes in this population.

RationaleLung infections caused by opportunistic or virulent pathogens are a principal cause of morbidity and mortality in HIV infection. It is unknown whether HIV infection leads to changes in basal lung microflora, which may contribute to chronic pulmonary complications that increasingly are being recognized in individuals infected with HIV.ObjectivesTo determine whether the immunodeficiency associated with HIV infection resulted in alteration of the lung microbiota.MethodsWe used 16S ribosomal RNA targeted pyrosequencing and shotgun metagenomic sequencing to analyze bacterial gene sequences in bronchoalveolar lavage (BAL) and mouths of 82 HIV-positive and 77 HIV-negative subjects.Measurements and main resultsSequences representing Tropheryma whipplei, the etiologic agent of Whipple's disease, were significantly more frequent in BAL of HIV-positive compared with HIV-negative individuals. T. whipplei dominated the community (>50% of sequence reads) in 11 HIV-positive subjects, but only 1 HIV-negative individual (13.4 versus 1.3%; P = 0.0018). In 30 HIV-positive individuals sampled longitudinally, antiretroviral therapy resulted in a significantly reduced relative abundance of T. whipplei in the lung. Shotgun metagenomic sequencing was performed on eight BAL samples dominated by T. whipplei 16S ribosomal RNA. Whole genome assembly of pooled reads showed that uncultured lung-derived T. whipplei had similar gene content to two isolates obtained from subjects with Whipple's disease.ConclusionsAsymptomatic subjects with HIV infection have unexpected colonization of the lung by T. whipplei, which is reduced by effective antiretroviral therapy and merits further study for a potential pathogenic role in chronic pulmonary complications of HIV infection.

RationaleResults from 16S rDNA-encoding gene sequence-based, culture-independent techniques have led to conflicting conclusions about the composition of the lower respiratory tract microbiome.ObjectivesTo compare the microbiome of the upper and lower respiratory tract in healthy HIV-uninfected nonsmokers and smokers in a multicenter cohort.MethodsParticipants were nonsmokers and smokers without significant comorbidities. Oral washes and bronchoscopic alveolar lavages were collected in a standardized manner. Sequence analysis of bacterial 16S rRNA-encoding genes was performed, and the neutral model in community ecology was used to identify bacteria that were the most plausible members of a lung microbiome.Measurements and main resultsSixty-four participants were enrolled. Most bacteria identified in the lung were also in the mouth, but specific bacteria such as Enterobacteriaceae, Haemophilus, Methylobacterium, and Ralstonia species were disproportionally represented in the lungs compared with values predicted by the neutral model. Tropheryma was also in the lung, but not the mouth. Mouth communities differed between nonsmokers and smokers in species such as Porphyromonas, Neisseria, and Gemella, but lung bacterial populations did not.ConclusionsThis study is the largest to examine composition of the lower respiratory tract microbiome in healthy individuals and the first to use the neutral model to compare the lung to the mouth. Specific bacteria appear in significantly higher abundance in the lungs than would be expected if they originated from the mouth, demonstrating that the lung microbiome does not derive entirely from the mouth. The mouth microbiome differs in nonsmokers and smokers, but lung communities were not significantly altered by smoking.

Repeated pulmonary exacerbation and progressive lung function decline characterize cystic fibrosis (CF) disease, and represents one of the leading causes of mortality in this patient population. Recent studies have shown, using culture-independent assays, that multiple microbial species can be detected in airway samples from CF patients. Moreover, specific groups of bacteria within these bacterial communities or microbiota, are highly associated with disease-associated factors such as antibiotic administration. This raises the possibility that, as in other human niches, pathogenic processes in the CF airways represent polymicrobial activities and that microbiome composition and perturbations to these communities define patient pulmonary health status. Airway samples are typically collected through the mouth, and are thus susceptible to contamination by upper airway secretions; hence, caution must be exercised in interpreting these data. Nonetheless, given the continuum of the upper and lower respiratory tract, understanding the contribution of these mixed-species assemblages to airway health is essential to improving CF patient care. This article aims to discuss recent advances in the field of CF airway microbiome research and interpret these findings in the context of CF pulmonary disease.

The composition of the gastrointestinal microbiome is increasingly recognized as a crucial contributor to immune and metabolic homeostasis-deficiencies in which are characteristic of cystic fibrosis (CF) patients. The murine model (CFTR (-/-) , CF), has, in previous studies, demonstrated characteristic CF gastrointestinal (GI) manifestations including slowed transit and significant upregulation of genes associated with inflammation. To determine if characteristics of the microbiome are associated with these phenotypes we used a phylogenetic microarray to compare small intestine bacterial communities of wild type and congenic CF mice. Loss of functional CFTR is associated with significant decreases in GI bacterial community richness, evenness and diversity and reduced relative abundance of putative protective species such as Acinetobacter lwoffii and a multitude of Lactobacilliales members. CF mice exhibited significant enrichment of Mycobacteria species and Bacteroides fragilis, previously associated with GI infection and immunomodulation. Antibiotic administration to WT and CF animals resulted in convergence of their microbiome composition and significant increases in community diversity in CF mice. These communities were characterized by enrichment of members of the Lactobacillaceae and Bifidobacteriaceae and reduced abundance of Enterobacteriaceae and Clostridiaceae. These data suggest that Enterobacteria and Clostridia species, long associated with small intestinal overgrowth and inflammatory bowel disease, may suppress both ileal bacterial diversity and the particular species which maintain motility and immune homeostasis in this niche. Thus, these data provide the first indications that GI bacterial colonization is strongly impacted by the loss of functional CFTR and opens up avenues for alternative therapeutic approaches to improve CF disease management.

Persistent mucosal inflammation and microbial infection are characteristics of chronic rhinosinusitis (CRS). Mucosal microbiota dysbiosis is found in other chronic inflammatory diseases; however, the relationship between sinus microbiota composition and CRS is unknown. Using comparative microbiome profiling of a cohort of CRS patients and healthy subjects, we demonstrate that the sinus microbiota of CRS patients exhibits significantly reduced bacterial diversity compared with that of healthy controls. In our cohort of CRS patients, multiple, phylogenetically distinct lactic acid bacteria were depleted concomitant with an increase in the relative abundance of a single species, Corynebacterium tuberculostearicum. We recapitulated the conditions observed in our human cohort in a murine model and confirmed the pathogenic potential of C. tuberculostearicum and the critical necessity for a replete mucosal microbiota to protect against this species. Moreover, Lactobacillus sakei, which was identified from our comparative microbiome analyses as a potentially protective species, defended against C. tuberculostearicum sinus infection, even in the context of a depleted sinus bacterial community. These studies demonstrate that sinus mucosal health is highly dependent on the composition of the resident microbiota as well as identify both a new sino-pathogen and a strong bacterial candidate for therapeutic intervention.