Faculty Bibliography

OBJECTIVES/HYPOTHESIS: Idiopathic subglottic stenosis (iSGS) is an unexplained obstruction involving the lower laryngeal and upper tracheal airway. Persistent mucosal inflammation is a hallmark of the disease. Epithelial microbiota dysbiosis is found in other chronic inflammatory mucosal diseases; however, the relationship between tracheal microbiota composition and iSGS is unknown. Given the critical role for host defense at mucosal barriers, we analyzed tissue specimens from iSGS patients for the presence of microbial pathogens. METHODS: Utilizing 30 human iSGS, 20 intubation-related tracheal stenosis (iLTS), and 20 healthy control specimens, we applied molecular, immunohistochemical, electron microscopic, immunologic, and Sanger-sequencing techniques. RESULTS: With unbiased culture-independent nucleic acid, protein, and immunologic approaches, we demonstrate that Mycobacterium species are uniquely associated with iSGS. Phylogenetic analysis of the mycobacterial virulence factor rpoB suggests that, rather than Mycobacterium tuberculosis, a variant member of the Mycobacterium tuberculosis complex or a closely related novel mycobacterium is present in iSGS specimens. CONCLUSION: These studies identify a novel pathogenic role for established large airway bacteria and provide new targets for future therapeutic intervention. LEVEL OF EVIDENCE: N/A. Laryngoscope, 2016.

OBJECTIVES/HYPOTHESIS: Idiopathic subglottic stenosis (iSGS) is a rare and devastating extrathoracic obstruction involving the lower laryngeal and upper tracheal airway. It arises without known antecedent injury or associated disease process. Persistent mucosal inflammation and a localized fibrotic response are hallmarks of the disease. Despite the initial clinical description of iSGS more than 40 year ago, there have been no substantive investigations into the pathogenesis of this enigmatic and progressive airway obstruction. In these studies, we present the initial characterization of the molecular pathogenesis underlying the fibrosing phenotype of iSGS. METHODS: Utilizing 20 human iSGS and healthy control specimens, we applied histologic, immunohistochemical, molecular, and immunologic techniques. RESULTS: We demonstrate significant activation of the canonical IL-23/IL-17A pathway in the tracheal mucosa of iSGS patients, as well as identify gammadelta T cells as the primary cellular source of IL-17A. CONCLUSION: Our results suggest that aberrant mucosal immune activation is a component in of the pathogenesis of iSGS. Most critically, our work offers new targets for future therapeutic intervention. LEVEL OF EVIDENCE: N/A. Laryngoscope, 2016.

Oral microbiome dysbiosis is associated with oral disease and potentially with systemic diseases; however, the determinants of these microbial imbalances are largely unknown. In a study of 1204 US adults, we assessed the relationship of cigarette smoking with the oral microbiome. 16S rRNA gene sequencing was performed on DNA from oral wash samples, sequences were clustered into operational taxonomic units (OTUs) using QIIME and metagenomic content was inferred using PICRUSt. Overall oral microbiome composition differed between current and non-current (former and never) smokers (P<0.001). Current smokers had lower relative abundance of the phylum Proteobacteria (4.6%) compared with never smokers (11.7%) (false discovery rate q=5.2 x 10-7), with no difference between former and never smokers; the depletion of Proteobacteria in current smokers was also observed at class, genus and OTU levels. Taxa not belonging to Proteobacteria were also associated with smoking: the genera Capnocytophaga, Peptostreptococcus and Leptotrichia were depleted, while Atopobium and Streptococcus were enriched, in current compared with never smokers. Functional analysis from inferred metagenomes showed that bacterial genera depleted by smoking were related to carbohydrate and energy metabolism, and to xenobiotic metabolism. Our findings demonstrate that smoking alters the oral microbiome, potentially leading to shifts in functional pathways with implications for smoking-related diseases.The ISME Journal advance online publication, 25 March 2016; doi:10.1038/ismej.2016.37.

The purpose of this study was to identify fecal bacterial microbiome changes in patients with chronic human immunodeficiency virus (HIV) infection in China. Bacterial 16S rRNA genes were amplified, sequenced (454 pyrosequencing), and clustered into operational taxonomic units using the QIIME software. Relative abundance at the phylum and genus levels were calculated. Alpha diversity was determined by Chao 1 and observed-species indices, and beta diversity was determined by double principal component analysis using the estimated phylogeny-based unweighted Unifrac distance matrices. Fecal samples of the patients with chronic HIV-infection tended to be enriched with bacteria of the phyla Firmicutes (47.20%+/-0.43 relative abundance) and Proteobacteria (37.21%+/-0.36) compared with those of the non-HIV infected controls (17.95%+/-0.06 and 3.81%+/-0.02, respectively). Members of the genus Bilophila were exclusively detected in samples of the non-HIV infected controls. Bacteroides and arabacteroides were more abundant in the chronic HIV-infected patients. Our study indicated that chronic HIV-infected patients in China have a fecal bacterial microbiome composition that is largely different from that found in non-HIV infected controls, and further study is needed to evaluate whether microbiome changes play a role in disease complications in the distal gut, including opportunistic infections.

OBJECTIVES: To evaluate the impact of HIV infection on colonization resistance in the proximal gut. DESIGN: It was a case-control study. METHODS: We contrasted microbiota composition between eight HIV-1-infected patients and eight HIV-negative controls to characterize community alteration and detect exogenous bacteria in the esophagus, stomach, and duodenum, as well as the mouth using a universal 16s ribosomal RNA gene survey and correlated the findings with HIV serostatus and peripheral blood T-cell counts. RESULTS: HIV infection was associated with an enrichment of Proteobacteria (P=0.020) and depletion of Firmicutes (P = 0.005) in the proximal gut. In particular, environmental species Burkholderia fungorum and Bradyrhizobium pachyrhizi colonized the duodenum of HIV patients who had abnormal blood CD4 T-cell counts but were absent in HIV-negative controls or HIV patients whose CD4 cell counts were normal. The two species coexisted and exhibited a decreasing trend proximally toward the stomach and esophagus and were virtually absent in the mouth. B. fungorum always outnumbered B. pachyrhizi in a ratio of approximately 15 to 1 regardless of the body sites (P < 0.0001, r = 0.965). Their abundance was inversely correlated with CD4 cell counts (P = 0.004) but not viral load. Overgrowth of potential opportunistic pathogens for example, Prevotella, Fusobacterium, and Ralstonia and depletion of beneficial bacteria, for example, Lactobacillus was also observed in HIV patients. CONCLUSIONS: The colonization of the duodenum by environmental bacteria reflects loss of colonization resistance in HIV infection. Their correlation with CD4 cell counts suggests that compromised immunity could be responsible for the observed invasion by exogenous microbes.

OBJECTIVE: Gender differences in immune response and the rate of disease progression in HIV-infected individuals have been reported but the underlying mechanism remains unclear, in part because of the lack of relevant animal models. Here, we report a novel nonhuman primate model for investigation of sex disparity in HIV disease progression. DESIGN/METHODS: Viral load and rate of disease progression were evaluated in rhesus macaques infected intrarectally with lineage-related subtype C R5 simian HIVs. Cytokine/chemokine levels in rectal swab eluates, and bacterial species adherent to the swabs and in the feces were determined. RESULTS: Simian HIV-infected female rhesus macaques progressed faster to AIDS than male macaques, recapitulating the sex bias in HIV-1 disease in humans. There were no significant differences in the levels of soluble immune mediators in the rectal mucosa of naive female and male macaques. However, an exploratory longitudinal study in six infected macaques indicates that the female macaques mounted an earlier and more robust proinflammatory skewed rectal immune response to infection. Moreover, expansion of Proteobacteria that increase in other intestinal inflammatory disorders was significantly higher in the rectal mucosa of female than male macaques during acute infection. CONCLUSION: These findings suggest that sex differences in local innate immune activation and compositional shifts in the gut microbiota could be the drivers of increased disease susceptibility in female macaques. Further studies with this novel nonhuman primate model of HIV infection could lead to innovative research on gender differences, which may have important therapeutic implications for controlling disease in infected men as well as women.

Increasing evidence suggests that the composition of the human gut microbiome is important in the etiology of human diseases; however, the personal factors that influence the gut microbiome composition are poorly characterized. Animal models point to sex hormone-related differentials in microbiome composition. In this study, we investigated the relationship of sex, body mass index (BMI) and dietary fiber intake with the gut microbiome in 82 humans. We sequenced fecal 16S rRNA genes by 454 FLX technology, then clustered and classified the reads to microbial genomes using the QIIME pipeline. Relationships of sex, BMI, and fiber intake with overall gut microbiome composition and specific taxon abundances were assessed by permutational MANOVA and multivariate logistic regression, respectively. We found that sex was associated with the gut microbiome composition overall (p=0.001). The gut microbiome in women was characterized by a lower abundance of Bacteroidetes (p=0.03). BMI (>25 kg/m2 vs. <25 kg/m2) was associated with the gut microbiome composition overall (p=0.05), and this relationship was strong in women (p=0.03) but not in men (p=0.29). Fiber from beans and from fruits and vegetables were associated, respectively, with greater abundance of Actinobacteria (p=0.006 and false discovery rate adjusted q=0.05) and Clostridia (p=0.009 and false discovery rate adjusted q=0.09). Our findings suggest that sex, BMI, and dietary fiber contribute to shaping the gut microbiome in humans. Better understanding of these relationships may have significant implications for gastrointestinal health and disease prevention.

With the development of culture-independent technique, a complex microbiome has been established and described in the distal esophagus. The incidence of esophageal adenocarcinoma (EAC) has increased dramatically in the United States. Studies documenting an altered microbiome associated with EAC and its precedents suggest that dysbiosis may be contributing to carcinogenesis, potentially mediated by interactions with toll-like receptors. Investigations attempting to associate viruses with EAC have not been as consistent. Currently available data are cross-sectional and therefore cannot prove causal relationships. Prospectively, microbiome studies open a new avenue to the understanding of the etiology and pathogenesis of reflux disorders and EAC.