Faculty Bibliography

BACKGROUND: Perturbation of commensal intestinal microbiota has been associated with several autoimmune diseases. Mice deficient in interleukin-1 receptor antagonist (Il1rn -/- mice) spontaneously develop autoimmune arthritis and are susceptible to other autoimmune diseases such as psoriasis, diabetes, and encephalomyelitis; however, the mechanisms of increased susceptibility to these autoimmune phenotypes are poorly understood. We investigated the role of interleukin-1 receptor antagonist (IL-1Ra) in regulation of commensal intestinal microbiota, and assessed the involvement of microbiota subsets and innate and adaptive mucosal immune responses that underlie the development of spontaneous arthritis in Il1rn -/- mice. RESULTS: Using high-throughput 16S rRNA gene sequencing, we show that IL-1Ra critically maintains the diversity and regulates the composition of intestinal microbiota in mice. IL-1Ra deficiency reduced the intestinal microbial diversity and richness, and caused specific taxonomic alterations characterized by overrepresented Helicobacter and underrepresented Ruminococcus and Prevotella. Notably, the aberrant intestinal microbiota in IL1rn -/- mice specifically potentiated IL-17 production by intestinal lamina propria (LP) lymphocytes and skewed the LP T cell balance in favor of T helper 17 (Th17) cells, an effect transferable to WT mice by fecal microbiota. Importantly, LP Th17 cell expansion and the development of spontaneous autoimmune arthritis in IL1rn -/- mice were attenuated under germ-free condition. Selective antibiotic treatment revealed that tobramycin-induced alterations of commensal intestinal microbiota, i.e., reduced Helicobacter, Flexispira, Clostridium, and Dehalobacterium, suppressed arthritis in IL1rn -/- mice. The arthritis phenotype in IL1rn -/- mice was previously shown to depend on Toll-like receptor 4 (TLR4). Using the ablation of both IL-1Ra and TLR4, we here show that the aberrations in the IL1rn -/- microbiota are partly TLR4-dependent. We further identify a role for TLR4 activation in the intestinal lamina propria production of IL-17 and cytokines involved in Th17 differentiation preceding the onset of arthritis. CONCLUSIONS: These findings identify a critical role for IL1Ra in maintaining the natural diversity and composition of intestinal microbiota, and suggest a role for TLR4 in mucosal Th17 cell induction associated with the development of autoimmune disease in mice.

Optimal management of patients with both psoriasis and psoriatic arthritis (PsA) necessitates collaboration among dermatologists and rheumatologists. In this manuscript, we discuss challenges and opportunities for dual care models for patients with psoriasis and PsA and the results of a survey of combined clinics based in North America.

BACKGROUND: Oral vancomycin remains the mainstay of therapy for severe infections produced by Clostridium difficile, the most prevalent cause of healthcare-associated infectious diarrhoea in developed countries. However, its short- and long-term effects on the human intestinal microbiota remain largely unknown. METHODS: We utilized high-throughput sequencing to analyse the effects of vancomycin on the faecal human microbiota up to 22 weeks post-antibiotic cessation. The clinical relevance of the observed microbiota perturbations was studied in mice. RESULTS: During vancomycin therapy, most intestinal microbiota genera and operational taxonomic units (OTUs) were depleted in all analysed subjects, including all baseline OTUs from the phylum Bacteroidetes. This was accompanied by a vast expansion of genera associated with infections, including Klebsiella and Escherichia/Shigella. Following antibiotic cessation, marked differences in microbiota resilience were observed among subjects. While some individuals recovered a microbiota close to baseline composition, in others, up to 89% of abundant OTUs could no longer be detected. The clinical relevance of the observed microbiota changes was further demonstrated in mice, which developed analogous microbiota alterations. During vancomycin treatment, mice were highly susceptible to intestinal colonization by an antibiotic-resistant pathogen and, upon antibiotic cessation, a less-resilient microbiota allowed higher levels of pathogen colonization. CONCLUSIONS: Oral vancomycin induces drastic and consistent changes in the human intestinal microbiota. Upon vancomycin cessation, the microbiota recovery rate varied considerably among subjects, which could influence, as validated in mice, the level of susceptibility to pathogen intestinal colonization. Our results demonstrate the negative long-term effects of vancomycin, which should be considered as a fundamental aspect of the cost-benefit equation for antibiotic prescription.

BACKGROUND: Airway abnormalities and lung tissue citrullination are found in both rheumatoid arthritis (RA) patients and individuals at-risk for disease development. This suggests the possibility that the lung could be a site of autoimmunity generation in RA, perhaps in response to microbiota changes. We therefore sought to test whether the RA lung microbiome contains distinct taxonomic features associated with local and/or systemic autoimmunity. METHODS: 16S rRNA gene high-throughput sequencing was utilized to compare the bacterial community composition of bronchoalveolar lavage fluid (BAL) in patients with early, disease-modifying anti-rheumatic drugs (DMARD)-naive RA, patients with lung sarcoidosis, and healthy control subjects. Samples were further assessed for the presence and levels of anti-citrullinated peptide antibodies (including fine specificities) in both BAL and serum. RESULTS: The BAL microbiota of RA patients was significantly less diverse and abundant when compared to healthy controls, but similar to sarcoidosis patients. This distal airway dysbiosis was attributed to the reduced presence of several genus (i.e., Actynomyces and Burkhordelia) as well as reported periodontopathic taxa, including Treponema, Prevotella, and Porphyromonas. While multiple clades correlated with local and systemic levels of autoantibodies, the genus Pseudonocardia and various related OTUs were the only taxa overrepresented in RA BAL and correlated with higher disease activity and erosions. CONCLUSIONS: Distal airway dysbiosis is present in untreated early RA and similar to that detected in sarcoidosis lung inflammation. This community perturbation, which correlates with local and systemic autoimmune/inflammatory changes, may potentially drive initiation of RA in a proportion of cases.

Background/Purpose: High-throughput sequencing of intestinal microbiota recently revealed that the composition of intestinal microbiota is perturbed in patients with new onset untreated rheumatoid arthritis (RA). In mice, both germ-free condition and administration of oral antibiotics before the onset of arthritis modulate T cell differentiation and attenuate the disease. However, it is not known whether and how modulation of intestinal microbiota after the onset of arthritis may influence the disease. Here, we investigated the involvement of commensal intestinal microbiota in the progression of established arthritis in both T cell-dependent and - independent mouse models. Methods: Mice with established collagen-induced arthritis (CIA) as the most widely-used T cell-dependent model of RA, as well as mice with K/BxN serum-transfer arthritis as a T cell-independent model were treated orally with a cocktail of metronidazole, neomycin, ampicillin (1 g/l each) and vancomycin (0.5 g/l) for one week to partially eliminate intestinal microbiota. Arthritis was assessed macroscopically and by histologic analysis. Differentiation of Th1, Th17 and regulatory T (Treg) cells and production of their prototypic cytokines in intestinal lamina propria and joint-draining lymph nodes were assessed by flow cytometry and Luminex array. Development of anti-collagen type II antibodies was assessed in serum of CIA mice using ELISA. Intestinal and synovial expression of cytokines and serum amyloid A (SAA) isotypes was measured by qPCR. Results: Elimination of intestinal microbiota in mice with ongoing CIA specifically suppressed intestinal Th17 cell differentiation without affecting Th1 and Treg cells. Accordingly, production of interleukin-17 (IL-17), but not interferon gamma, IL-4 and IL-10, by lamina propria lymphocytes was significantly diminished in antibiotic-treated mice. Furthermore, intestinal expression of SAA isoforms and IL-22, known to promote lamina propria Th17 cell differentiation, was suppressed in antibiotic-treated CIA mice. Importantly, elimination of intestinal microbiota resulted in suppressed Th17 cell differentiation and IL-17 production in joint-draining lymph nodes and reduced the severity of established CIA. In contrast, antibiotic treatment did not influence disease severity in the T cellindependent K/BxN serum-transfer arthritis. Intriguingly, the abundance of intestinal Th17 cells strongly correlated with the severity of arthritis in the CIA mice. However, elimination of intestinal microbiota after disease onset did not affect the development of anti-collagen type II autoantibodies. Conclusion : These observations suggest that modulation of commensal intestinal microbiota during established arthritis specifically suppress Th17 differentiation and dampen T cell-mediated arthritic processes. While our study does not advocate the use of antibiotics as a treatment for RA, it supports the notion that inflammatory signals provided by the gut microbiota continue to promote arthritis after its onset. Understanding the exact mechanisms linking the intestinal T cell response with arthritis may help identifying novel therapeutic strategies for RA

Background/Purpose: Despite significant advances in the therapeutics of inflammatory arthritides, methotrexate (MTX) remains the mainstay of treatment for rheumatoid arthritis (RA) and related conditions worldwide. However, it has a hihgly variable inter-individual bioavailabity (ranging from 20 to 80%) and there is currently no mechanism to predict efficacy. One of the fundamental roles of the intestinal microbiome is to metabolize xenobiotics and synthetic drugs. Here we characterize the effects of oral methotrexate on the gut community composition of patients with RA and the potential role of baseline human microbiota in predicting response to MTX therapy. Methods: Demographic characteristics, drug use and disease activity scores-28 (DAS28) were recorded from new-onset rheumatoid arthritis (NORA) patients (n=33). For each participant, fecal samples were collected at baseline and at pre-established intervals for at least 3 months after initiation of oral methotrexate (range 3-48 months). 16S rDNA was extracted per protocol (MoBio, USA) and amplicons targeting the hypervariable V4 region were sequenced using 454 and MiSeq (Illumina) platforms to define the microbiota composition. The obtained 16S rRNA sequences were analyzed using the Quantitative Insights into Microbial Ecology (QIIME) pipeline. Taxonomic relative abundance at all hierarchical levels was determined to establish baseline microbiota composition prior to MTX initiation. Two-tailed Wilcoxon non-parametric test was applied to identify significant microbiota taxonomic changes that occur after MTX therapy. The False Discovery Rate (FDR) approach was applied to adjust for multiple hypothesis testing. Changes with a P<0.05 and FDR<0.2 were considered significant.Spearman correlations between baseline relative composition of intestinal microbiota and clinical response to MTX at each time point were also applied. Results: To quantify microbiota similarities among fecal samples we used unweighted UniFrac and hierarchical clustering. Samples from MTX-treated patients clustered with their respective baseline samples, indicating that the gut microbiota is stable with inter-individual taxonomic differences maintained for at least 6 months. NORA patients receiving MTX developed minimal changes over time. Intriguingly, however, baseline microbiome signatures in these patients predicted clinical response to MTX at 3 and 6 months, including the overabundance of unclassified Coriobacteriaceae (r=-0.756; P <0.01) and a Coprococcus-related OTU (r=-0.755; P =0.022). Conclusion: Although oral methotrexate does not induce significant changes in the over all structure of the human intestinal microbiota of NORA patients, the abundance of several taxa at baseline correlate with a significant improvement in clinical disease activity 3 and 6 months into therapy. Whether specific intestinal commensals can modulate the pharmacokinetics and bioavailability of methotrexate (and other DMARDs), remains to be elucidated. Better understanding of MTX pharamcomicrobiomics will be necessary to achieve precision medicine strategies in RA and related conditions

Background/Purpose: Psoriasis (PsO) is a chronic immune-mediated skin condition affecting ~3% of adults worldwide. Up to a third of PsO patients go on to develop psoriatic arthritis (PsA), a heterogeneous inflammatory arthritis characterized by concomitant bone erosion and osteoproliferation. Although multiple advances have been made in the pathogenesis and therapeutics of these disorders, it is currently not possible to predict which individuals will progress from PsO to PsA. The role of the microbiome as a potential trigger for autoimmunity and rheumatic disease has recently been implicated. The goal of this study was to characterize the cutaneous microbiota of patients with PsO and PsA (in both psoriatic plaques and unaffected skin) to determine if there are characteristic features related to disease phenotype. Methods: Skin swabs from subjects with PsO (n=29) and PsA (n=62) were collected from both psoriatic plaque lesions and contralateral unaffected skin. 16S rDNA was extracted per protocol (MoBio, USA) and amplicons targeting the hypervariable V4 region were sequenced using MiSeq (Illumina) to define the microbiota composition. The obtained 16S rRNA sequences were analyzed using the Quantitative Insights into Microbial Ecology (QIIME) pipeline. Taxonomic relative abundance was determined to compare their prevalence among different phenotypes using Kruskal-Wallis statistical analysis. Alpha diversity plots and weighted Unifrac analysis (beta diversity) of cutaneous bacterial communities were generated. False discovery rate analysis was applied to identify unique differentiating taxa. Results: Baseline characteristics were comparable in both groups. PsO samples had, on average, a similar number of operational taxonomic units as compared to PsA samples. Beta diversity plots did not demonstrate statistically distinct clustering of microbial communities between PsO and PsA subjects, PsO and PsA nonlesional skin, or PsO and PsA lesional skin. Staphylococcus and Corynebacterium were the most abundant genera across all samples. However, several genera were statistically more abundant in PsO compared to PsA lesions, including unclassified Bradyrhizobiaceae (p<0.0006), Rahnella (p<0.0006), unclassified Prevotellaceae (p<0.001), and Parvibaculum (p<0.002). Rothia was more abundant in PsA (p<0.02). Conclusion: Our results characterize, for the first time, the cutaneous microbial composition of individuals with PsO compared to those with PsA both in psoriatic lesions and unaffected skin. Although we did not find overall community differences among the various phenotypes, our preliminary observations point towards differences in specific genera, which are characteristically more abundant in PsO. Further in-depth analysis is required to better understand the significance of this dysbiotic process in PsA and whether it contributes to the pathogenesis of the psoriatic disease spectrum. Current efforts are devoted to incorporating healthy controls into our analysis, and analyzing the cutaneous microbiome (and metagenome) across multiple body sites, multiple visits, as well as pre- and post-immunosuppressive/biologic therapy