IL-1β promotes chronic intestinal inflammation through recruitment of granulocytes, activation of ILCs, accumulation of pathogenic T cells, and promotion of Th17 responses.

Objectives: Traditionally, the urinary tract has been thought to be sterile in the absence of a clinically identifiable infection. However, recent evidence suggests that the urinary tract harbors a variety of bacterial species, known collectively as the urinary microbiome, even when clinical cultures are negative. Whether these bacteria promote urinary health or contribute to urinary tract disease remains unknown. Emerging evidence indicates that a shift in the urinary microbiome may play an important role in urgency urinary incontinence (UUI). The goal of this prospective pilot study was to determine how the urinary microbiome is different between women with and without UUI. We also sought to identify if characteristics of the urinary microbiome are associated with UUI severity.Methods: We collected urine from clinically well-characterized women with UUI (n = 10) and normal bladder function (n = 10) using a transurethral catheter to avoid bacterial contamination from external tissue. To characterize the resident microbial community, we amplified the bacterial 16S rRNA gene by PCR and performed sequencing using Illumina MiSeq. Sequences were processed using the workflow package QIIME. We identified bacteria that had differential relative abundance between UUI and controls using DESeq2 to fit generalized linear models based on the negative binomial distribution. We also identified relationships between the diversity of the urinary microbiome and severity of UUI symptoms with Pearson's correlation coefficient.Results: We successfully extracted and sequenced bacterial DNA from 95% of the urine samples and identified that there is a polymicrobial community in the female bladder in both healthy controls and women with UUI. We found the relative abundance of 14 bacteria significantly differed between control and UUI samples. Furthermore, we established that an increase in UUI symptom severity is associated with a decrease in microbial diversity in women with UUI.Conclusions: Our study provides further characterization of the urinary microbiome in both healthy controls and extensively phenotyped women with UUI. Our results also suggest that the urinary microbiome may play an important role in the pathophysiology of UUI and that the loss of microbial diversity may be associated with clinical severity.

Microbial communities are commonly studied using culture-independent methods, such as 16S rRNA gene sequencing. However, one challenge in accurately characterizing microbial communities is exogenous bacterial DNA contamination, particularly in low-microbial-biomass niches. Computational approaches to identify contaminant sequences have been proposed, but their performance has not been independently evaluated. To identify the impact of decreasing microbial biomass on polymicrobial 16S rRNA gene sequencing experiments, we created a mock microbial community dilution series. We evaluated four computational approaches to identify and remove contaminants, as follows: (i) filtering sequences present in a negative control, (ii) filtering sequences based on relative abundance, (iii) identifying sequences that have an inverse correlation with DNA concentration implemented in Decontam, and (iv) predicting the sequence proportion arising from defined contaminant sources implemented in SourceTracker. As expected, the proportion of contaminant bacterial DNA increased with decreasing starting microbial biomass, with 80.1% of the most diluted sample arising from contaminant sequences. Inclusion of contaminant sequences led to overinflated diversity estimates and distorted microbiome composition. All methods for contaminant identification successfully identified some contaminant sequences, which varied depending on the method parameters used and contaminant prevalence. Notably, removing sequences present in a negative control erroneously removed >20% of expected sequences. SourceTracker successfully removed over 98% of contaminants when the experimental environments were well defined. However, SourceTracker misclassified expected sequences and performed poorly when the experimental environment was unknown, failing to remove >97% of contaminants. In contrast, the Decontam frequency method did not remove expected sequences and successfully removed 70 to 90% of the contaminants. IMPORTANCE The relative scarcity of microbes in low-microbial-biomass environments makes accurate determination of community composition challenging. Identifying and controlling for contaminant bacterial DNA are critical steps in understanding microbial communities from these low-biomass environments. Our study introduces the use of a mock community dilution series as a positive control and evaluates four computational strategies that can identify contaminants in 16S rRNA gene sequencing experiments in order to remove them from downstream analyses. The appropriate computational approach for removing contaminant sequences from an experiment depends on prior knowledge about the microbial environment under investigation and can be evaluated with a dilution series of a mock microbial community.

The HLA-B27 gene is a major risk factor for clinical diseases including ankylosing spondylitis, acute anterior uveitis, reactive arthritis, and psoriatic arthritis, but its mechanism of risk enhancement is not completely understood. The gut microbiome has recently been shown to influence several HLA-linked diseases. However, the role of HLA-B27 in shaping the gut microbiome has not been previously investigated. In this study, we characterize the differences in the gut microbiota mediated by the presence of the HLA-B27 gene. We identified differences in the cecal microbiota of Lewis rats transgenic for HLA-B27 and human β2-microglobulin (hβ2m), compared with wild-type Lewis rats, using biome representational in situ karyotyping (BRISK) and 16S rRNA gene sequencing. 16S sequencing revealed significant differences between transgenic animals and wild type animals by principal coordinates analysis. Further analysis of the data set revealed an increase in Prevotella spp. and a decrease in Rikenellaceae relative abundance in the transgenic animals compared to the wild type animals. By BRISK analysis, species-specific differences included an increase in Bacteroides vulgatus abundance in HLA-B27/hβ2m and hβ2m compared to wild type rats. The finding that HLA-B27 is associated with altered cecal microbiota has not been shown before and can potentially provide a better understanding of the clinical diseases associated with this gene.

PurposeTo investigate the contribution of the gut microbiota to the pathogenesis of uveitis.MethodsExperimental autoimmune uveitis (EAU) in B10.RIII mice was induced using interphotoreceptor binding protein peptide. Mice were treated with oral or intraperitoneal (IP) antibiotics. Effector (Teff) and regulatory (Treg) T lymphocytes were identified using flow cytometry; 16S rRNA gene sequencing and qPCR were performed on gastrointestinal (GI) contents.ResultsBroad-spectrum (four antibiotics given simultaneously) oral, but not IP, antibiotics reduced mean uveitis clinical scores significantly compared with water-treated animals (0.5 vs. 3.0, P < 0.0001 for oral; 3.4 vs. 3.4, P > 0.99 for IP). Both oral metronidazole (P = 0.02) and vancomycin (P < 0.0001) alone decreased inflammation, whereas neomycin (P = 0.7) and ampicillin (P = 0.4) did not change mean uveitis scores. Oral broad-spectrum antibiotics increased Tregs in the GI lamina propria of EAU animals at 1 week, and in extraintestinal lymphoid tissues later, whereas Teff and inflammatory cytokines were reduced. 16S sequencing of GI contents revealed altered microbiota in immunized mice compared with nonimmunized mice, and microbial diversity clustering in EAU mice treated with uveitis-protective antibiotics. Experimental autoimmune uveitis mice also demonstrated gut microbial diversity clustering associated with clinical score severity.ConclusionsOral antibiotics modulate the severity of inducible EAU by increasing Tregs in the gut and extraintestinal tissues, as well as decreasing effector T cells and cytokines. 16S sequencing suggests that there may be protective and, conversely, potentially uveitogenic, gut microbiota. These findings may lead to a better understanding of how uveitis can be treated or prevented by modulating the gut microbiome.