Fecal transplants in spondyloarthritis and uveitis: ready for a clinical trial?

PURPOSE OF REVIEW: The intestinal microbiome is thought to play a role in the pathogenesis of inflammatory bowel disease (IBD). There are many shared clinical manifestations between IBD and spondyloarthritis (SpA), of which the most common are peripheral arthritis and uveitis. Clinical overlap along with similar genetics between these diseases suggests a possible shared pathogenetic mechanism, which might center on the intestinal microbiota. In this review, we discuss the available evidence that SpA is a microbiome-driven disease and indicate how SpA-associated uveitis could be tied to gut dysbiosis. We conclude by discussing different treatment paradigms targeting the intestinal microbiome for SpA. RECENT FINDINGS: Recent studies support the growing evidence of the intestinal microbiome as a crucial player in SpA disease pathogenesis. There is emerging evidence that the gut microbiome may play a causative role in uveitis. SUMMARY: The field is beginning to discover a new level of understanding how the intestinal microbiome is involved in SpA. Treatment methods to alter intestinal microbiota to treat SpA-related diseases are still in its infancy.

The interaction between host genetics and the microbiome in the pathogenesis of spondyloarthropathies.

PURPOSE OF REVIEW: The intestinal microbiome is increasingly implicated in the pathogenesis of ankylosing spondylitis, reactive arthritis, and other diseases collectively known as the spondyloarthropathies (SpAs). In common with other complex inflammatory diseases, SpAs have both a strong genetic and environmental component. Recent genetic studies have highlighted host pathways that may intersect the host-microbiota interaction and offer novel paradigms to understand the pathophysiology of these diseases. RECENT FINDINGS: Genetic association studies have identified genes such as RUNX3, PTPEN2, and IL-33 as susceptibility loci for SpAs. Functional studies in humans have extended knowledge of established genetic risk factors for ankylosing spondylitis that include ERAP1, ERAP2, and interleukin-23R. Recent basic research has identified new mechanisms that regulate host immune responses to the microbiota that conceivably may be dysregulated in SpA. SUMMARY: Intestinal barrier function, deletional tolerance, Th17 signature response, and endoplasmic reticulum stress pathways have been recently linked to SpA. Dysregulated immune responses to the gut microbiota and an altered microbial community structure are shared features of SpA. Although the cause-effect dynamic of this relationship remains equivocal, it nonetheless has major implications for both intestinal and extra-intestinal pathology observed in SpA.

The Microbiome: a Revolution in Treatment for Rheumatic Diseases?

PURPOSE OF REVIEW: The microbiome is the term that describes the microbial ecosystem that cohabits an organism such as humans. The microbiome has been implicated in a long list of immune-mediated diseases which include rheumatoid arthritis, ankylosing spondylitis, and even gout. The mechanisms to account for this effect are multiple. The clinical implications from observations on the microbiome and disease are broad. RECENT FINDINGS: A growing number of microbiota constituents such as Prevotella copri, Porphyromonas gingivalis, and Collinsella have been correlated or causally related to rheumatic disease. The microbiome has a marked effect on the immune system. Our understanding of immune pathways modulated by the microbiota such as the induction of T helper 17 (Th17) cells and secretory immunoglobulin A (IgA) responses to segmented filamentous bacteria continues to expand. In addition to the gut microbiome, bacterial communities of other sites such as the mouth, lung, and skin have also been associated with the pathogenesis of rheumatic diseases. Strategies to alter the microbiome or to alter the immune activation from the microbiome might play a role in the future therapy for rheumatic diseases.

The intestinal microbiome in spondyloarthritis.

PURPOSE OF REVIEW: Microbial dysbiosis in the gut is emerging as a common component in various inflammatory disorders including spondyloarthritis (SpA). The depth of this influence has begun to be realized with next-generation sequencing of the gut microbiome providing unbiased assessment of previously uncharted bacterial populations. RECENT FINDINGS: Decreased numbers of Firmicutes, a major phyla of gut commensals, especially the species Faecalibacterium prausnitzii and Clostridium leptum have been found in various inflammatory disorders including SpA and inflammatory bowel disease (IBD), and could be an important link between SpA and gut inflammation. Multiple studies in ankylosing spondylitis, psoriatic arthritis, juvenile SpA, and animal models of SpA are revealing common bacterial associations among these diseases as well as IBD. SUMMARY: We are beginning to appreciate the complex relationship between the gut microbiome and host immune regulation and dysregulation in health and disease. Potentially important differences have been revealed in SpA, but cause and effect relationships remain far from established. Many critical questions remain to be answered before we can apply new knowledge to improve therapeutics in SpA.

Genome-wide analysis of T cell responses during acute and latent simian varicella virus infections in rhesus macaques.

Varicella zoster virus (VZV) is the etiological agent of varicella (chickenpox) and herpes zoster (HZ [shingles]). Clinical observations suggest that VZV-specific T cell immunity plays a more critical role than humoral immunity in the prevention of VZV reactivation and development of herpes zoster. Although numerous studies have characterized T cell responses directed against select VZV open reading frames (ORFs), a comprehensive analysis of the T cell response to the entire VZV genome has not yet been conducted. We have recently shown that intrabronchial inoculation of young rhesus macaques with simian varicella virus (SVV), a homolog of VZV, recapitulates the hallmarks of acute and latent VZV infection in humans. In this study, we characterized the specificity of T cell responses during acute and latent SVV infection. Animals generated a robust and broad T cell response directed against both structural and nonstructural viral proteins during acute infection in bronchoalveolar lavage (BAL) fluid and peripheral blood. During latency, T cell responses were detected only in the BAL fluid and were lower and more restricted than those observed during acute infection. Interestingly, we identified a small set of ORFs that were immunogenic during both acute and latent infection in the BAL fluid. Given the close genome relatedness of SVV and VZV, our studies highlight immunogenic ORFs that may be further investigated as potential components of novel VZV vaccines that specifically boost T cell immunity.

Chronic ethanol consumption modulates growth factor release, mucosal cytokine production, and microRNA expression in nonhuman primates.

BACKGROUND: Chronic alcohol consumption has been associated with enhanced susceptibility to both systemic and mucosal infections. However, the exact mechanisms underlying this enhanced susceptibility remain incompletely understood. METHODS: Using a nonhuman primate model of ethanol (EtOH) self-administration, we examined the impact of chronic alcohol exposure on immune homeostasis, cytokine, and growth factor production in peripheral blood, lung, and intestinal mucosa following 12 months of chronic EtOH exposure. RESULTS: EtOH exposure inhibited activation-induced production of growth factors hepatocyte growth factor (HGF), granulocyte colony-stimulating factor (G-CSF), and vascular-endothelial growth factor (VEGF) by peripheral blood mononuclear cells (PBMC). Moreover, EtOH significantly reduced the frequency of colonic Th1 and Th17 cells in a dose-dependent manner. In contrast, we did not observe differences in lymphocyte frequency or soluble factor production in the lung of EtOH-consuming animals. To uncover mechanisms underlying reduced growth factor and Th1/Th17 cytokine production, we compared expression levels of microRNAs in PBMC and intestinal mucosa. Our analysis revealed EtOH-dependent up-regulation of distinct microRNAs in affected tissues (miR-181a and miR-221 in PBMC; miR-155 in colon). Moreover, we were able to detect reduced expression of the transcription factors STAT3 and ARNT, which regulate expression of VEGF, G-CSF, and HGF and contain targets for these microRNAs. To confirm and extend these observations, PBMC were transfected with either mimics or antagomirs of miR-181 and miR-221, and protein levels of the transcription factors and growth factors were determined. Transfection of microRNA mimics led to a reduction in both STAT3/ARNT as well as VEGF/HGF/G-CSF levels. The opposite outcome was observed when microRNA antagomirs were transfected. CONCLUSIONS: Chronic EtOH consumption significantly disrupts both peripheral and mucosal immune homeostasis, and this dysregulation may be mediated by changes in microRNA expression.

Network-Based Differences in the Vaginal and Bladder Microbial Communities Between Women With and Without Urgency Urinary Incontinence.

Background: Little is known about the relationship of proximal urogenital microbiomes in the bladder and the vagina and how this contributes to bladder health. In this study, we use a microbial ecology and network framework to understand the dynamics of interactions/co-occurrences of bacteria in the bladder and vagina in women with and without urgency urinary incontinence (UUI). Methods: We collected vaginal swabs and catheterized urine specimens from 20 women with UUI (cases) and 30 women without UUI (controls). We sequenced the V4 region of the bacterial 16S rRNA gene and evaluated using alpha and beta diversity metrics. We used microbial network analysis to detect interactions in the microbiome and the betweenness centrality measure to identify central bacteria in the microbial network. Bacteria exhibiting maximum betweenness centrality are considered central to the microbe-wide networks and likely maintain the overall microbial network structure. Results: There were no significant differences in the vaginal or bladder microbiomes between cases and controls using alpha and beta diversity. Silhouette metric analysis identified two distinct microbiome clusters in both the bladder and vagina. One cluster was dominated by Lactobacillus genus while the other was more diverse. Network-based analyses demonstrated that vaginal and bladder microbial networks were different between cases and controls. In the vagina, there were similar numbers of genera and subgroup clusters in each network for cases and controls. However, cases tend to have more unique bacterial co-occurrences. While Bacteroides and Lactobacillus were the central bacteria with the highest betweenness centrality in controls, Aerococcus had the highest centrality in cases and correlated with bacteria commonly associated with bacterial vaginosis. In the bladder, cases have less than half as many network clusters compared to controls. Lactobacillus was the central bacteria in both groups but associated with several known uropathogens in cases. The number of shared bacterial genera between the bladder and the vagina differed between cases and controls, with cases having larger overlap (43%) compared to controls (29%). Conclusion: Our study shows overlaps in microbial communities of bladder and vagina, with higher overlap in cases. We also identified differences in the bacteria that are central to the overall community structure.

Axial spondyloarthritis patients have altered mucosal IgA response to oral and fecal microbiota.

Axial spondyloarthritis (axSpA) is an inflammatory arthritis involving the spine and the sacroiliac joint with extra-articular manifestations in the eye, gut, and skin. The intestinal microbiota has been implicated as a central environmental component in the pathogenesis of various types of spondyloarthritis including axSpA. Additionally, alterations in the oral microbiota have been shown in various rheumatological conditions, such as rheumatoid arthritis (RA). Therefore, the aim of this study was to investigate whether axSpA patients have an altered immunoglobulin A (IgA) response in the gut and oral microbial communities. We performed 16S rRNA gene (16S) sequencing on IgA positive (IgA+) and IgA negative (IgA-) fractions (IgA-SEQ) from feces (n=17 axSpA; n=14 healthy) and saliva (n=14 axSpA; n=12 healthy), as well as on IgA-unsorted fecal and salivary samples. PICRUSt2 was used to predict microbial metabolic potential in axSpA patients and healthy controls (HCs). IgA-SEQ analyses revealed enrichment of several microbes in the fecal (Akkermansia, Ruminococcaceae, Lachnospira) and salivary (Prevotellaceae, Actinobacillus) microbiome in axSpA patients as compared with HCs. Fecal microbiome from axSpA patients showed a tendency towards increased alpha diversity in IgA+ fraction and decreased diversity in IgA- fraction in comparison with HCs, while the salivary microbiome exhibits a significant decrease in alpha diversity in both IgA+ and IgA- fractions. Increased IgA coating of Clostridiales Family XIII in feces correlated with disease severity. Inferred metagenomic analysis suggests perturbation of metabolites and metabolic pathways for inflammation (oxidative stress, amino acid degradation) and metabolism (propanoate and butanoate) in axSpA patients. Analyses of fecal and salivary microbes from axSpA patients reveal distinct populations of immunoreactive microbes compared to HCs using the IgA-SEQ approach. These bacteria were not identified by comparing their relative abundance alone. Predictive metagenomic analysis revealed perturbation of metabolites/metabolic pathways in axSpA patients. Future studies on these immunoreactive microbes may lead to better understanding of the functional role of IgA in maintaining microbial structure and human health.

Pathogenic and protective roles of MyD88 in leukocytes and epithelial cells in mouse models of inflammatory bowel disease.

BACKGROUND & AIMS: Toll-like receptors (TLR) are innate immune receptors involved in recognition of the intestinal microflora; they are expressed by numerous cell types in the intestine, including epithelial cells, myeloid cells, and lymphocytes. Little is known about the relative contributions of TLR signaling in distinct cellular compartments to intestinal homeostasis. We aimed to define the roles of TLR signals in distinct cell types in the induction and regulation of chronic intestinal inflammation. METHODS: We assessed the roles of the shared TLR signaling adaptor protein, MyD88, in several complementary mouse models of inflammatory bowel disease, mediated by either innate or adaptive immune activation. MyD88-deficient mice and bone marrow chimeras were used to disrupt TLR signals selectively in distinct cellular compartments in the intestine. RESULTS: MyD88-dependent activation of myeloid cells was required for the development of chronic intestinal inflammation. By contrast, although epithelial cell MyD88 signals were required for host survival, they were insufficient to induce intestinal inflammation in the absence of an MyD88-competent myeloid compartment. MyD88 expression by T cells was not required for their pathogenic and regulatory functions in the intestine. CONCLUSIONS: Cellular compartmentalization of MyD88 signals in the intestine allow the maintenance of host defense and prevent deleterious inflammatory responses.

TLR2-independent induction and regulation of chronic intestinal inflammation.

Interactions between the intestinal microflora and host innate immune receptors play a critical role in intestinal homeostasis. Several studies have shown that TLR2 can modulate inflammatory responses in the gut. TLR2 signals enhance tight junction formation and fortify the epithelial barrier, and may play a crucial role in driving acute inflammatory responses towards intestinal bacterial pathogens. In addition, TLR2 agonists can have direct effects on both Th1 cells and Treg. To define the role of TLR2 in the induction and regulation of chronic intestinal inflammation we examined the effects of TLR2 deletion on several complementary models of inflammatory bowel disease. Our results show that TLR2 signals are not required for the induction of chronic intestinal inflammation by either innate or adaptive immune responses. We further show that TLR2(-/-) mice harbor normal numbers of Foxp3(+) Treg that are able to suppress intestinal inflammation as effectively as their WT counterparts. We also did not find any intrinsic role for TLR2 for pathogenic effector T-cell responses in the gut. Thus, in contrast to their role in acute intestinal inflammation and repair, TLR2 signals may have a limited impact on the induction and regulation of chronic intestinal inflammation.

Preliminary Report on Interleukin-22, GM-CSF, and IL-17F in the Pathogenesis of Acute Anterior Uveitis.

Purpose:Anterior uveitis is the most common anatomic subset of uveitis. We developed a novel multi-parametric flow cytometry panel to identify immune dysregulation signatures in HLA B27-associated acute anterior uveitis (AAU) and axial spondyloarthritis (AxSpA).Methods: We used fluorescence activated cell sorting to characterize T cell cytokine expression in stimulated T cell subsets from patients with AAU (n = 4) compared to healthy controls (n = 14) or subjects with AxSpA (n = 6).Results: Positive findings among subjects with AAU included a statistically significant increase in stimulated granulocyte-macrophage colony stimulating factor (GM-CSF), IL-17, and IL-22 synthesized by CD8 cells, a trend for stimulated ILC (innate lymphoid cells)-3 cells to synthesize more IL-22 (p = .07), and stimulated MAIT (mucosa associated innate lymphoid cells)-like cells that express the T cell receptor V alpha 7.2 to express IL-17A, IL-17F, and IL-22 in a greater percentage of cells relative to controls. IL-17F, GM- CSF, and IL-22 represent potentially novel targets in AAU.Conclusion: Our report is arguably the first to implicate IL-17F or ILC-3 and MAIT cells in the pathogenesis of AAU.Abbreviations AAU: acute anterior uveitis; AxSpA: axial spondyloarthritis; BASDAI: Bath ankylosing spondylitis disease activity index; CCR: chemokine receptor; DMSO: dimethylsulfoxide; EULAR:European League Against Rheumatism; FACS: fluorescence activated cell sorter; FBS: fetal bovine serum; FSC: orward light scatter; GM-CSF: granulocyte-macrophage colony stimulating factor; HC: healthy control; ILC: innate lymphoid cell; KIR: killer immunoglobulin receptor; MAIT: mucosal associated immune T cell; ND: not detected; NK: natural killer cell; OHSU-Oregon Health & Science University; PBMC: peripheral blood mononuclear cell; SSC: side light scatter; TCR: T cell receptor.

Expression of Cytokines in Porcine Iris, Retina and Choroidal Tissues Stimulated by Microbe-associated Molecular Patterns.

PURPOSE: The innate immune system is strongly implicated in the pathogenesis of uveitis. This study was designed to clarify the responses of the innate immune system in uveal tissues. MATERIALS AND METHODS: We utilized quantitative, real-time RT-PCR to measure mRNA of innate immune system receptors from porcine iris, choroid, and retina tissues. We used RT-PCR for cytokines to evaluate the responses of these tissues to specific ligands or extracts of whole bacteria that activate the innate immune system. We used ELISA for IL-6 on selected choroidal supernatants to confirm that the mRNA measurement correlated with protein levels. RESULTS: In each of the studied tissues, we detected the expression of important receptors belonging to the innate immune system including dectin-1, TLR4, TLR8, and NOD2. Relative mRNA expression was generally lower in the retina compared to iris or choroid. All three tissues demonstrated upregulation of cytokine mRNA in response to a range of ligands that activate the innate immune system. The measurement of IL-6 protein was consistent with results based on mRNA. Notably, the expression of mRNA for IL-23 was more pronounced than IL-12 in all three tissues after stimulation with various innate immune system ligands. CONCLUSIONS: These data provide evidence of a potent innate immune response intrinsic to uveal tissues. Specific innate immune system ligands as well as bacterial extracts enhanced the production of several inflammatory cytokines. Furthermore, the observation of higher upregulation of IL-23 mRNA, compared to IL-12 in response to innate immune stimuli, suggested that a local TH17 response might be more robust than a local TH1 response in uveal tissues. Our results expand the understanding as to how the innate immune system may contribute to uveitis.

HLA-A alleles including HLA-A29 affect the composition of the gut microbiome: a potential clue to the pathogenesis of birdshot retinochoroidopathy.

Birdshot retinochoroidopathy occurs exclusively in individuals who are HLA-A29 positive. The mechanism to account for this association is unknown. The gut microbiome has been causally implicated in many immune-mediated diseases. We hypothesized that HLA-A29 would affect the composition of the gut microbiome, leading to a dysbiosis and immune-mediated eye disease. Fecal and intestinal biopsy samples were obtained from 107 healthy individuals from Portland, Oregon environs, 10 of whom were HLA-A29 positive, undergoing routine colonoscopy. Bacterial profiling was achieved via 16S rRNA metabarcoding. Publicly available whole meta-genome sequencing data from the Human Microbiome Project (HMP), consisting of 298 healthy controls mostly of US origin, were also interrogated. PERMANOVA and sparse partial least squares discriminant analysis (sPLSDA) demonstrated that subjects who were HLA-A29 positive differed in bacterial species composition (beta diversity) compared to HLA-A29 negative subjects in both the Portland (p = 0.019) and HMP cohorts (p = 0.0002). The Portland and HMP cohorts evidenced different subsets of bacterial species associated with HLA-A29 status, likely due to differences in the metagenomic techniques employed. The functional composition of the HMP cohort did not differ overall (p = 0.14) between HLA-A29 positive and negative subjects, although some distinct pathways such as heparan sulfate biosynthesis showed differences. As we and others have shown for various HLA alleles, the HLA allotype impacts the composition of the microbiome. We hypothesize that HLA-A29 may predispose chorioretinitis via an altered gut microbiome.

Performance of Microbiome Sequence Inference Methods in Environments with Varying Biomass.

Microbiome community composition plays an important role in human health, and while most research to date has focused on high-microbial-biomass communities, low-biomass communities are also important. However, contamination and technical noise make determining the true community signal difficult when biomass levels are low, and the influence of varying biomass on sequence processing methods has received little attention. Here, we benchmarked six methods that infer community composition from 16S rRNA sequence reads, using samples of varying biomass. We included two operational taxonomic unit (OTU) clustering algorithms, one entropy-based method, and three more-recent amplicon sequence variant (ASV) methods. We first compared inference results from high-biomass mock communities to assess baseline performance. We then benchmarked the methods on a dilution series made from a single mock community-samples that varied only in biomass. ASVs/OTUs inferred by each method were classified as representing expected community, technical noise, or contamination. With the high-biomass data, we found that the ASV methods had good sensitivity and precision, whereas the other methods suffered in one area or in both. Inferred contamination was present only in small proportions. With the dilution series, contamination represented an increasing proportion of the data from the inferred communities, regardless of the inference method used. However, correlation between inferred contaminants and sample biomass was strongest for the ASV methods and weakest for the OTU methods. Thus, no inference method on its own can distinguish true community sequences from contaminant sequences, but ASV methods provide the most accurate characterization of community and contaminants. IMPORTANCE Microbial communities have important ramifications for human health, but determining their impact requires accurate characterization. Current technology makes microbiome sequence data more accessible than ever. However, popular software methods for analyzing these data are based on algorithms developed alongside older sequencing technology and smaller data sets and thus may not be adequate for modern, high-throughput data sets. Additionally, samples from environments where microbes are scarce present additional challenges to community characterization relative to high-biomass environments, an issue that is often ignored. We found that a new class of microbiome sequence processing tools, called amplicon sequence variant (ASV) methods, outperformed conventional methods. In samples representing low-biomass communities, where sample contamination becomes a significant confounding factor, the improved accuracy of ASV methods may allow more-robust computational identification of contaminants.

Novel Inter-omic Analysis Reveals Relationships Between Diverse Gut Microbiota and Host Immune Dysregulation in HLA-B27-Induced Experimental Spondyloarthritis.

OBJECTIVE: To define inflammation-related host-microbe interactions in experimental spondyloarthritis (SpA) using novel inter-omic approaches. METHODS: The relative frequency of gut microbes was determined by 16S ribosomal RNA (rRNA) gene sequencing, and gene expression using RNA-Seq of host tissue. HLA-B27/human ß2 -microglobulin-transgenic (HLA-B27-transgenic) and wild-type rats from dark agouti, Lewis, and Fischer backgrounds were used. Inter-omic analyses using Cytoscape were employed to identify relevant relationships. PICRUSt was used to predict microbial functions based on known metagenomic profiles. RESULTS: Inter-omic analysis revealed several gut microbes that were strongly associated with dysregulated cytokines driving inflammatory response pathways, such as interleukin-17 (IL-17), IL-23, IL-17, IL-1, interferon-γ (IFNγ), and tumor necrosis factor (TNF). Many microbes were uniquely associated with inflammation in Lewis or Fischer rats, and one was relevant on both backgrounds. Several microbes that were strongly correlated with immune dysregulation were not differentially abundant in HLA-B27-transgenic compared to wild-type controls. A multi-omic network analysis revealed non-overlapping clusters of microbes in Lewis and Fischer rats that were strongly linked to overlapping dysregulated immune/inflammatory genes. Prevotella, Clostridiales, and Blautia were important in Lewis rats, while Akkermansia muciniphila and members of the Lachnospiraceae family dominated in Fischer rats. Inflammation-associated metabolic pathway perturbation (e.g., butanoate, propanoate, lipopolysaccharide, and steroid biosynthesis) was also predicted from both backgrounds. CONCLUSION: Inter-omic and network analysis of gut microbes and the host immune response in experimental SpA provides an unprecedented view of organisms strongly linked to dysregulated IL-23, IL-17, IL-1, IFNγ, and TNF. Functional similarities between these organisms may explain why animals of different genetic backgrounds exhibit common patterns of immune dysregulation, possibly through perturbation of similar metabolic pathways. These results highlight the power of linking analyses of gut microbiota with the host immune response to gain insights into the role of dysbiotic microbes in SpA beyond taxonomic profiling.

Disruption of Intestinal Homeostasis and Intestinal Microbiota During Experimental Autoimmune Uveitis.

Purpose: We determine the changes in intestinal microbiota and/or disruptions in intestinal homeostasis during uveitis. Methods: Experimental autoimmune uveitis (EAU) was induced in B10.RIII mice with coadministration of interphotoreceptor retinoid-binding protein peptide (IRBP) and killed mycobacterial antigen (MTB) as an adjuvant. Using 16S rRNA gene sequencing, we looked at intestinal microbial differences during the course of uveitis, as well as intestinal morphologic changes, changes in intestinal permeability by FITC-dextran leakage, antimicrobial peptide expression in the gastrointstinal tract, and T lymphocyte prevalence before and at peak intraocular inflammation. Results: We demonstrate that increased intestinal permeability and antimicrobial peptide expression in the intestinal tract coincide in timing with increased effector T cells in the mesenteric lymph nodes, during the early stages of uveitis, before peak inflammation. Morphologic changes in the intestine were most prominent during this phase, but also occurred with adjuvant MTB alone, whereas increased intestinal permeability was found only in IRBP-immunized mice that develop uveitis. We also demonstrate that the intestinal microbiota were altered during the course of uveitis, and that some of these changes are specific to uveitic animals, whereas others are influenced by adjuvant MTB alone. Intestinal permeability peaked at 2 weeks, coincident with an increase in intestinal bacterial strain differences, peak lipocalin production, and peak uveitis. Conclusions: An intestinal dysbiosis accompanies a disruption in intestinal homeostasis in autoimmune uveitis, although adjuvant MTB alone promotes intestinal disruption as well. This may indicate a novel axis for future therapeutic targeting experimentally or clinically.

Controlling for Contaminants in Low-Biomass 16S rRNA Gene Sequencing Experiments.

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.

Alpha-Glucosidase Inhibitors Alter Gut Microbiota and Ameliorate Collagen-Induced Arthritis.

Acarose is an anti-diabetic drug and exhibits anti-arthritic effects. We hypothesized that acarbose influences the gut microbiota to affect the course of arthritis and tested this hypothesis in a collagen-induced arthritis (CIA) murine model. Acarbose in drinking water was administered via gastric gavage started prior to or at the time of CIA induction. Gut microbiota were evaluated with 16S rRNA gene sequencing from fecal pellets collected prior to arthritis induction, during onset of arthritis, and after treatment. Immune response was evaluated by measuring changes in T helper-17 (Th17) and T regulatory (Treg) cells in the spleen and intestine, as well as serum cytokine levels. Before induction of CIA, acarbose significantly reduced the incidence of arthritis and attenuated clinical severity of arthritis. The frequency of Th17 cells was significantly decreased in the intestinal lamina propria in acarbose treated mice. Mice that were treated with acarbose showed significantly increased CD4+CD25+Foxp3+ Treg cells with elevation of Helios and CCR6. A remarkable alteration in microbial community was observed in acarbose treated mice. Bacterial diversity and richness in mice with arthritis were significantly lower than those in acarbose treated groups. The frequency of Firmicutes was significantly reduced after arthritis onset but was restored after treatment with acarbose. The frequency of Lactobacillus, Anaeroplasma, Adlercreutzia, RF39 and Corynebacterium was significantly higher in control groups than in acarbose treated, while Oscillospira, Desulfovibrio and Ruminococcus enriched in acarbose treated group. Miglitol, another α-glucosidase inhibitor showed a similar but less potent anti-arthritic effect to that of acarbose. These data demonstrate that acarbose alleviated CIA through regulation of Th17/Treg cells in the intestinal mucosal immunity, which may have resulted from the impact of acarbose on gut microbial community. Inexpensive antidiabetic drugs with an excellent safety profile are potentially useful for managing rheumatoid arthritis.

HLA Alleles Associated With Risk of Ankylosing Spondylitis and Rheumatoid Arthritis Influence the Gut Microbiome.

OBJECTIVE: HLA alleles affect susceptibility to more than 100 diseases, but the mechanisms that account for these genotype-disease associations are largely unknown. HLA alleles strongly influence predisposition to ankylosing spondylitis (AS) and rheumatoid arthritis (RA). Both AS and RA patients have discrete intestinal and fecal microbiome signatures. Whether these changes are the cause or consequence of the diseases themselves is unclear. To distinguish these possibilities, we examined the effect of HLA-B27 and HLA-DRB1 RA risk alleles on the composition of the intestinal microbiome in healthy individuals. METHODS: Five hundred sixty-eight stool and biopsy samples from 6 intestinal sites were collected from 107 healthy unrelated subjects, and stool samples were collected from 696 twin pairs from the TwinsUK cohort. Microbiome profiling was performed using sequencing of the 16S ribosomal RNA bacterial marker gene. All subjects were genotyped using the Illumina CoreExome SNP microarray, and HLA genotypes were imputed from these data. RESULTS: Associations were observed between the overall microbial composition and both the HLA-B27 genotype and the HLA-DRB1 RA risk allele (P = 0.0002 and P = 0.00001, respectively). These associations were replicated using the stool samples from the TwinsUK cohort (P = 0.023 and P = 0.033, respectively). CONCLUSION: This study shows that the changes in intestinal microbiome composition seen in AS and RA are at least partially due to effects of HLA-B27 and HLA-DRB1 on the gut microbiome. These findings support the hypothesis that HLA alleles operate to cause or increase the risk of these diseases through interaction with the intestinal microbiome and suggest that therapies targeting the microbiome may be effective in preventing or treating these diseases.

The microbiome and HLA-B27-associated acute anterior uveitis.

Acute anterior uveitis (AAU) and the spondyloarthritis (SpA) subtypes ankylosing spondylitis, reactive arthritis and psoriatic arthritis are among the inflammatory diseases affected by the biology of the intestinal microbiome. In this Review, the relationship between AAU, SpA and the microbiome is discussed, with a focus on the major SpA risk gene HLA-B*27 and how it is associated with both intestinal tolerance and the loss of ocular immune privilege that can accompany AAU. We provide four potential mechanisms to account for how dysbiosis, barrier function and immune response contribute to the development of ocular inflammation and the pathogenesis of AAU. Finally, potential therapeutic avenues to target the microbiota for the clinical management of AAU and SpA are outlined.