Faculty Bibliography

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

Background/Purpose: To describe characteristics & potential risk factors(including treatment exposure) for all-cause mortality & malignancies (excluding NMSC) in psoriasis (PsO) patients (pts) with psoriatic arthritis (PsA) from PSOLAR. Methods: PSOLAR is an international, disease-based, observational study in which pts eligible for or receiving conventional systemic & biologic agents for PsO are followed prospectively. Characteristics & adverse events for pts who reported PsA, including a subset with PsA confirmed by a joint-specialist are summarized. For characteristics & incidence rates, the cohorts were defined as & attribution was based on treatment exposure prior to/during registry in the following order(regardless of sequence & duration):(1)ustekinumab(UST) (2)other sponsor biologic(primarily infliximab[IFX])(3)non-sponsor biologic(primarily adalimumab/etanercept [ADA/ETN]),&(4)non-biologic therapies(NB)(including immunomodulators(IMM)[eg. MTX], phototherapy&topical therapy). Exposure to any therapy higher in the order precluded inclusion in the lower cohorts. Multivariate analyses using Cox hazard regression(without attribution bias)were used to identify factors associated with time to first malignancy&mortality[compared to no biologic use]&for IMM [compared to no IMM use]. Results: As of Aug 23, 2015, PSOLAR is fully enrolled with 12090 pts(48870 total pt-yrs [PY] of follow-up). Overall, 4315 pts reported having PsA:1551 UST, 754 IFX, 1650 ADA/ETN, 360 NB;of these, 1719 had confirmed PsA(689 UST, 346 IFX, 566 ADA/ETN,118 NB). Baseline demographics & medical history were generally balanced across cohorts; however, in overall PsA subgroup, more pts in NB cohort were >65yrs of age&had a medical history of cancer. In overall PsA subgroup, cumulative incidence rates/100PY for all-cause mortality: UST0.34, IFX0.37, ADA/ETN0.57, NB0.75; in statistical analysis: age, history of cardiovascular disease, history of diabetes&smoking were associated with increased risk of mortality(P<0.05). Cumulative incidence rates/100PY for malignancy:UST0.57, IFX0.71, ADA/ETN0.60,& NB0.96. Statistical analysis identified the following significant(p<0.05) risk factors for malignancy:severity of PsO, age, smoking,&history of malignancy. For confirmed PsA subset, cumulative incidence rates/100PY for all-cause mortality: UST0.29, IFX0.37, ADA/ETN0.44, NB0.44; age & smoking were associated with increased risk of mortality, Cumulative incidence rates/100PY for malignancy: UST0.53, IFX0.66, ADA/ETN0.96, NB 1.10;age was the only significant factor associated with increased risk of malignancy(p=<0.05). Inherent bias with observational data may apply. Variability in group size & clinical features was noted. Incidence rates are not adjusted for differences in demographics & disease characteristics (adjustment for such key factors are included in statistical analyses). Statistical analyses interpretation may be limited due to number of deaths in subset of confirmed PSA (24). Conclusion: Unadjusted rates of all-cause mortality & malignancies for biologics were generally comparable among both PsA subsets with the exception of generally numerically higher rates in pts in NB cohort. The common risk factors associated with mortality for the overall & confirmed PsA subsets were age & smoking; age alone was the common risk factor associated with malignancy. Biologics & IMM use were not associated with increased risk of mortality or malignancy

Background: Airway abnormalities, increased tissue citrullination and local inflammation are found in the lungs of early RA. Recent data suggest that the gut and oral microbiome might potentially contribute to the priming of aberrant systemic immune response in RA. Our objective was to study whether RA lung microbiome contains distinct taxonomic features associated with local and/or systemic autoimmunity. Materials: Bronchoalveolar lavage (BAL) from twenty RA, ten sarcoidosis and twenty-eight healthy individuals were obtained by research bronchoscopy. 16S rDNA sequencing was performed to define microbiota composition. Autoantibodies, including anti-CCP, RF and ACPAs were measured in sera of RA subjects. Results: The16S sequencing data showed similar alpha/beta diversity between RA and sarcoid groups, but significantly different from healthy. Taxonomic comparison between groups was performed using LEfSe, which revealed several significant differences. Multiple taxa, including Rhanella and Rhodanobacter were present only in the RA and sarcoid groups, but completely absent from healthy. While RA BAL samples were enriched with Sphingobacteria, sarcoidosis BAL was enriched with Bacteroidia, Rhizobiales, Nitrospirales, and Campylobacter. Raoultella and Barnesiella correlated with CCP2 levels in BAL. Serum levels of CCP-IgA had a negative correlation with Massilia and Tannerella, and a positive correlation with Vagococcus and Lactobacillus. Serum levels of anti-CCP2 antibodies had a positive correlation with Porphyromonas, Rahnella and Chryseobacterium. Conclusion: Despite the relatively small number of samples, several taxonomic differences were noted between groups. Further evaluation of functional aspects of this microbiome may provide insights into its possible contribution to RA

The role of the gut microbiome in animal models of inflammatory and autoimmune disease is now well established. The human gut microbiome is currently being studied as a potential modulator of the immune response in rheumatic disorders. However, the vastness and complexity of this host-microorganism interaction is likely to go well beyond taxonomic, correlative observations. In fact, most advances in the field relate to the functional and metabolic capabilities of these microorganisms and their influence on mucosal immunity and systemic inflammation. An intricate relationship between the microbiome and the diet of the host is now fully recognized, with the microbiota having an important role in the degradation of polysaccharides into active metabolites. This Review summarizes the current knowledge on the metabolic role of the microbiota in health and rheumatic disease, including the advances in pharmacomicrobiomics and its potential use in diagnostics, therapeutics and personalized medicine.

Background: Airway abnormalities and increased lung tissue citrullination is found in both RA patients and individuals at at-risk for the development of disease. Recent data suggest that the gut (Prevotella copri) and oral (Porphyromonas gingivalis) microbiome might potentially contribute to the priming of an aberrant systemic immune response characteristic of RA. Objectives: Our objective was to study whether RA lung microbiome contains distinct taxonomic features associated with local and/or systemic autoimmunity. Methods: Bronchoalveolar lavage (BAL) samples from 20 subjects with RA, 10 with sarcoidosis and 28 healthy controls were obtained by research bronchoscopy. 16S rRNA sequencing was performed to define microbiota composition. Autoantibodies, including anti-CCP, RF and ACPAs were also measured in sera of RA subjects. Results: The 16s sequencing data showed similar alpha/beta diversity between RA and sarcoid groups, but significantly different from healthy (ANOSIM test; p=0.002 and 0.021 for healthy vs RA and healthy vs sarcoid, respectively). Taxonomic comparison between groups was performed using LEfSe, which revealed several significant differences. RA BALF samples had a decrease in the families Actinomycetaceae (P<0.0001) and Spirochaetaceae (P=0.0009) compared to healthy. Burkholderia was significantly decreased in both RA and sarcoidosis compared to controls. The genus Treponema (also highly associated with periodontitis) was exclusively found in healthy subjects' BALF (P<0.01 vs RA). RA disease activity was positively correlated with Micrococcus and Renibaterium at the genus level, and with various OTUs belonging to Pseudonocardia and Xanthomonadaceae whereas, the genus Veillonella and unclassified Oxalobacteraceae had a negative correlation. Levels of local autoantibodies i.e., anti-CCP2 correlated positively with the genus Corynebacterium, Megasphera and unclassified Comamonadaceae. Circulating anti-CCP (IgA isotype), had a positive significant correlation with relative abundance of BALF Enhydrobacter and unclassified Bradyrhizobiaceae (P=0.015 and 0.004, respectively). A modest association with erosive disease was observed with the presence and abundance of Pseudonocardia in the RA BALF (85% of erosive RA patients vs. 23% of non-erosive RA; P=0.019). Conclusions: Despite the relatively small number of samples analyzed, several taxonomic differences were noted between groups. Correlations between relative abundance of specific taxa in RA BAL with serum autoantibodies (i.e., anti-CCP) support an association between the lung microbiome and the host immune phenotype in RA. Further evaluation of the functional aspects of this microbiome may provide insights into its possible contribution to RA