Joint-specific regulation of homeobox D10 expression in rheumatoid arthritis fibroblast-like synoviocytes.

Rheumatoid arthritis (RA) is a systemic immune-mediated disease characterized by joint inflammation and destruction. The disease typically affects small joints in the hands and feet, later progressing to involve larger joints such as the knees, shoulders, and hips. While the reasons for these joint-specific differences are unclear, distinct epigenetic patterns associated with joint location have been reported. In this study, we evaluated the unique epigenetic landscapes of fibroblast-like synoviocytes (FLS) from hip and knee synovium in RA patients, focusing on the expression and regulation of Homeobox (HOX) transcription factors. These highly conserved genes play a critical role in embryonic development and are known to maintain distinct expression patterns in various adult tissues. We found that several HOX genes, especially HOXD10, were differentially expressed in knee FLS compared with hip FLS. Epigenetic differences in chromatin accessibility and histone marks were observed in HOXD10 promoter between knee and hip FLS. Histone modification, particularly histone acetylation, was identified as an important regulator of HOXD10 expression. To understand the mechanism of differential HOXD10 expression, we inhibited histone deacetylases (HDACs) with small molecules and siRNA. We found that HDAC1 blockade or deficiency normalized the joint-specific HOXD10 expression patterns. These observations suggest that epigenetic differences, specifically histone acetylation related to increased HDAC1 expression, play a crucial role in joint-specific HOXD10 expression. Understanding these mechanisms could provide insights into the regional aspects of RA and potentially lead to therapeutic strategies targeting specific patterns of joint involvement during the course of disease.

Joint-specific rheumatoid arthritis fibroblast-like synoviocyte regulation identified by integration of chromatin access and transcriptional activity.

The mechanisms responsible for the distribution and severity of joint involvement in rheumatoid arthritis (RA) are not known. To explore whether site-specific FLS biology might be associated with location-specific synovitis and explain the predilection for hand (wrist/metacarpal phalangeal joints) involvement in RA, we generated transcriptomic and chromatin accessibility data from FLS to identify the transcription factors (TFs) and pathways. Networks were constructed by integration of chromatin accessibility and gene expression data. Analysis revealed joint-specific patterns of FLS phenotype, with proliferative, migratory, proinflammatory, and matrix-degrading characteristics observed in resting FLS derived from the hand joints compared with hip or knee. TNF-stimulation amplified these differences, with greater enrichment of proinflammatory and proliferative genes in hand FLS compared with hip and knee FLS. Hand FLS also had the greatest expression of markers associated with an 'activated' state relative to the 'resting' state, with the greatest cytokine and MMP expression in TNF-stimulated hand FLS. Predicted differences in proliferation and migration were biologically validated with hand FLS exhibiting greater migration and cell growth than hip or knee FLS. Distinctive joint-specific FLS biology associated with a more aggressive inflammatory response might contribute to the distribution and severity of joint involvement in RA.

Dysregulated NUB1 and Neddylation Enhances Rheumatoid Arthritis Fibroblast-Like Synoviocyte Inflammatory Responses.

OBJECTIVE: Fibroblast-like synoviocytes (FLS) contribute to the pathogenesis of rheumatoid arthritis (RA), in part due to activation of the proinflammatory transcription factor NF-κB. Neddylation is modulated by the negative regulator of ubiquitin-like protein (NUB) 1. We determined whether NUB1 and neddylation are aberrant in the models with RA FLS, thereby contributing to their aggressive phenotype. METHODS: Models with RA or osteoarthritis (OA) FLS were obtained from arthroplasty synovia. Real-time quantitative polymerase chain reaction and Western blot analysis assessed gene and protein expression, respectively. NUB1 was overexpressed using an expression vector. NF-κB activation was assessed by stimulating FLS with interleukin (IL)-1ß. Neddylation inhibitor (MLN4924) and proteasome inhibitor were used in migration and gene expression assays. MLN4924 was used in the model with K/BxN serum-transfer arthritis. RESULTS: Enhanced H3K27ac and H3K27me3 peaks were observed in the NUB1 promoter in the OA FLS compared with the RA FLS. NUB1 was constitutively expressed by FLS, but induction by IL-1ß was significantly greater in the OA FLS. The ratio of neddylated cullin (CUL) 1 to nonneddylated CUL1 was lower in the OA FLS than the RA FLS. NUB1 overexpression decreased NF-κB nuclear translocation and IL-6 messenger RNA (mRNA) in IL-1ß-stimulated the RA FLS. MLN4924 decreased CUL1 neddylation, NF-κB nuclear translocation, and IL-6 mRNA in IL-1ß-stimulated the RA FLS. MLN4924 significantly decreased arthritis severity in the model with K/BxN serum-transfer arthritis. CONCLUSION: CUL1 neddylation and NUB1 induction is dysregulated in the models with RA, which increases FLS activation. Inhibition of neddylation is an effective therapy in an animal model of arthritis. These data suggest that the neddylation system contributes to the pathogenesis of RA and that regulation of neddylation could be a novel therapeutic approach.

Multifaceted immune dysregulation characterizes individuals at-risk for rheumatoid arthritis.

Molecular markers of autoimmunity, such as antibodies to citrullinated protein antigens (ACPA), are detectable prior to inflammatory arthritis (IA) in rheumatoid arthritis (RA) and may define a state that is 'at-risk' for future RA. Here we present a cross-sectional comparative analysis among three groups that include ACPA positive individuals without IA (At-Risk), ACPA negative individuals and individuals with early, ACPA positive clinical RA (Early RA). Differential methylation analysis among the groups identifies non-specific dysregulation in peripheral B, memory and naïve T cells in At-Risk participants, with more specific immunological pathway abnormalities in Early RA. Tetramer studies show increased abundance of T cells recognizing citrullinated (cit) epitopes in At-Risk participants, including expansion of T cells reactive to citrullinated cartilage intermediate layer protein I (cit-CILP); these T cells have Th1, Th17, and T stem cell memory-like phenotypes. Antibody-antigen array analyses show that antibodies targeting cit-clusterin, cit-fibrinogen and cit-histone H4 are elevated in At-Risk and Early RA participants, with the highest levels of antibodies detected in those with Early RA. These findings indicate that an ACPA positive at-risk state is associated with multifaceted immune dysregulation that may represent a potential opportunity for targeted intervention.

Clonal associations of lymphocyte subsets and functional states revealed by single cell antigen receptor profiling of T and B cells in rheumatoid arthritis synovium.

Rheumatoid arthritis (RA) is an autoimmune disease initiated by antigen-specific T cells and B cells, which promote synovial inflammation through a complex set of interactions with innate immune and stromal cells. To better understand the phenotypes and clonal relationships of synovial T and B cells, we performed single-cell RNA and repertoire sequencing on paired synovial tissue and peripheral blood samples from 12 donors with seropositive RA ranging from early to chronic disease. Paired transcriptomic-repertoire analyses highlighted 3 clonally distinct CD4 T cells populations that were enriched in RA synovium: T peripheral helper (Tph) and T follicular helper (Tfh) cells, CCL5+ T cells, and T regulatory cells (Tregs). Among these cells, Tph cells showed a unique transcriptomic signature of recent T cell receptor (TCR) activation, and clonally expanded Tph cells expressed an elevated transcriptomic effector signature compared to non-expanded Tph cells. CD8 T cells showed higher oligoclonality than CD4 T cells, and the largest CD8 T cell clones in synovium were highly enriched in GZMK+ cells. TCR analyses revealed CD8 T cells with likely viral-reactive TCRs distributed across transcriptomic clusters and definitively identified MAIT cells in synovium, which showed transcriptomic features of TCR activation. Among B cells, non-naive B cells including age-associated B cells (ABC), NR4A1+ activated B cells, and plasma cells, were enriched in synovium and had higher somatic hypermutation rates compared to blood B cells. Synovial B cells demonstrated substantial clonal expansion, with ABC, memory, and activated B cells clonally linked to synovial plasma cells. Together, these results reveal clonal relationships between functionally distinct lymphocyte populations that infiltrate RA synovium.

Systems-biology analysis of rheumatoid arthritis fibroblast-like synoviocytes implicates cell line-specific transcription factor function.

Rheumatoid arthritis (RA) is an immune-mediated disease affecting diarthrodial joints that remains an unmet medical need despite improved therapy. This limitation likely reflects the diversity of pathogenic pathways in RA, with individual patients demonstrating variable responses to targeted therapies. Better understanding of RA pathogenesis would be aided by a more complete characterization of the disease. To tackle this challenge, we develop and apply a systems biology approach to identify important transcription factors (TFs) in individual RA fibroblast-like synoviocyte (FLS) cell lines by integrating transcriptomic and epigenomic information. Based on the relative importance of the identified TFs, we stratify the RA FLS cell lines into two subtypes with distinct phenotypes and predicted active pathways. We biologically validate these predictions for the top subtype-specific TF RARα and demonstrate differential regulation of TGFß signaling in the two subtypes. This study characterizes clusters of RA cell lines with distinctive TF biology by integrating transcriptomic and epigenomic data, which could pave the way towards a greater understanding of disease heterogeneity.

Improving Transcriptome Fidelity Following Synovial Tissue Disaggregation.

Objective: To improve the fidelity of the cellular transcriptome of disaggregated synovial tissue for applications such as single-cell RNA sequencing (scRNAseq) by modifying the disaggregation technique. Methods: Osteoarthritis (OA) and rheumatoid arthritis (RA) synovia were collected at arthroplasty. RNA was extracted from intact or disaggregated replicate pools of tissue fragments. Disaggregation was performed with either a proprietary protease, Liberase TL (Lib) as a reference method, Liberase TL with an RNA polymerase inhibitor flavopyridol (Flavo), or a cold digestion with subtilisin A (SubA). qPCR on selected markers and RNAseq were used to compare disaggregation methods using the original intact tissue as reference. Results: Disaggregated cell yield and viability were similar for all three methods with some viability improved (SubA). Candidate gene analysis showed that Lib alone dramatically increased expression of several genes involved in inflammation and immunity compared with intact tissue and was unable to differentiate RA from OA. Both alternative methods reduced the disaggregation induced changes. Unbiased analysis using bulk RNAseq and the 3 protocols confirmed the candidate gene studies and showed that disaggregation-induced changes were largely prevented. The resultant data improved the ability to distinguish RA from OA synovial transcriptomes. Conclusions: Disaggregation of connective tissues such as synovia has complex and selective effects on the transcriptome. We found that disaggregation with an RNA polymerase inhibitor or using a cold enzyme tended to limit induction of some relevant transcripts during tissue processing. The resultant data in the disaggregated transcriptome better represented the in situ transcriptome. The specific method chosen can be tailored to the genes of interest and the hypotheses being tested in order to optimize the fidelity of technique for applications based on cell suspensions such as sorted populations or scRNAseq.

Lipopolysaccharide Exposure Differentially Alters Plasma and Brain Inflammatory Markers in Adult Male and Female Rats.

Humans and rodents have sexually dimorphic immune responses, which could influence the brain's response to a systemic inflammatory insult. Lipopolysaccharide (LPS) is a stimulator of the innate immune system and is routinely used in animal models to study blood-brain barrier (BBB) dysfunction under inflammatory conditions. Therefore, we examined whether inflammatory response to LPS and the associated BBB disruption differed in male and female adult rats. Rats were treated with saline or two injections of 1 mg/kg LPS and studied 24 h after the second LPS injection. Plasma isolated from trunk blood and brain tissue homogenates of the prefrontal cortex (PFC), dorsal striatum (DS), hippocampus, and cerebellum were analyzed for cytokines and chemokines using a 9-plex panel from Meso Scale Discovery. BBB disruption was analyzed with tight junction proteins claudin-5 and VE-cadherin via Western blotting and VEGF by ELISA. This allowed us to compare sex differences in the levels of individual cytokines as well as associations among cytokines and expression of tight junction proteins between the plasma and specific brain regions. Higher levels of interferon-γ, interleukin-10 (IL-10), IL-13, IL-4, CXCL-1, and VEGF in the plasma were revealed compared to the brain homogenates, and higher levels of TNFα, IL-1ß, IL-6, and IL-5 in the PFC were seen compared with plasma and other brain regions in males. Females showed higher levels of plasma CXCL1 and VEGF compared to males, and males showed higher levels of PFC TNFα, IL-6, IL-4, and VEGF compared to females. LPS induced significant increases in plasma cytokines and VEGF in both sexes. LPS did not significantly alter cytokines in brain tissue homogenates, however, it increased chemokines in the PFC, DS, and hippocampus. In the PFC, LPS produced BBB disruption, which is evident as reduced expression of claudin-5 in males and reduced expression of VE-cadherin in both sexes. Taken together, our results reveal significant sex differences in pro-inflammatory cytokine and chemokine levels in plasma and brain that were associated with BBB disruption after LPS, and validate the use of multiplex assay for plasma and brain tissue samples.

Genetic regulation of OAS1 nonsense-mediated decay underlies association with COVID-19 hospitalization in patients of European and African ancestries.

The chr12q24.13 locus encoding OAS1-OAS3 antiviral proteins has been associated with coronavirus disease 2019 (COVID-19) susceptibility. Here, we report genetic, functional and clinical insights into this locus in relation to COVID-19 severity. In our analysis of patients of European (n = 2,249) and African (n = 835) ancestries with hospitalized versus nonhospitalized COVID-19, the risk of hospitalized disease was associated with a common OAS1 haplotype, which was also associated with reduced severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) clearance in a clinical trial with pegIFN-λ1. Bioinformatic analyses and in vitro studies reveal the functional contribution of two associated OAS1 exonic variants comprising the risk haplotype. Derived human-specific alleles rs10774671-A and rs1131454 -A decrease OAS1 protein abundance through allele-specific regulation of splicing and nonsense-mediated decay (NMD). We conclude that decreased OAS1 expression due to a common haplotype contributes to COVID-19 severity. Our results provide insight into molecular mechanisms through which early treatment with interferons could accelerate SARS-CoV-2 clearance and mitigate against severe COVID-19.

Crosstalk between CD4 T cells and synovial fibroblasts from human arthritic joints promotes hyaluronan-dependent leukocyte adhesion and inflammatory cytokine expression in vitro.

The content and organization of hyaluronan (HA) in the extracellular matrix (ECM) have been identified as strong indicators of inflammation in joint disease, although the source and role of HA as an effector of inflammation is not clear. In this study, we established co-cultures of activated human CD4 T cells with fibroblast-like synoviocytes (FLS) from osteoarthritis (OA) and rheumatoid arthritis (RA) subjects and examined the role of HA in promoting inflammatory events. Co-cultures of RA FLS with activated CD4 T cells generated an HA-enriched ECM that promoted enhanced monocyte adhesion compared to co-cultures of OA FLS with activated CD4 T cells. In addition, both OA FLS and RA FLS co-cultures with activated CD4 T cells elicited significant increases in the expression of IL1ß, TNF, and IL6, with the increase in IL6 expression most prominent in RA co-cultures. Blocking HA synthesis and accumulation with 4-methylumbelliferone reduced expression of IL6, IL1ß, and TNF in both OA FLS and RA FLS co-cultures. The increase in HA synthesis in the co-cultures was mimicked by IL6 trans-signaling of FLS in the absence of CD4 T cells. Inhibition of HA synthesis blocked the increase in IL6 by RA FLS mediated by IL6 trans-signaling, suggesting that the HA synthetic pathway may be a key mediator in IL6 expression by FLS. Overall, our study indicates that HA-enriched ECM generated by co-cultures of activated CD4 T cells with FLS from human joints creates a pathogenic microenvironment by promoting adhesion of leukocytes and expression of inflammatory cytokines including IL6.

Caspase-8 Variant G Regulates Rheumatoid Arthritis Fibroblast-Like Synoviocyte Aggressive Behavior.

OBJECTIVE: Fibroblast-like synoviocytes (FLS) play a pivotal role in rheumatoid arthritis (RA) by contributing to synovial inflammation and progressive joint damage. An imprinted epigenetic state is associated with the FLS aggressive phenotype. We identified CASP8 (encoding for caspase-8) as a differentially marked gene and evaluated its pathogenic role in RA FLSs. METHODS: RA FLS lines were obtained from synovial tissues at arthroplasty and used at passage 5-8. Caspase-8 was silenced using small interfering RNA, and its effect was determined in cell adhesion, migration and invasion assays. Quantitative reverse transcription PCR and western blot were used to assess gene and protein expression, respectively. A caspase-8 selective inhibitor was used determine the role of enzymatic activity on FLS migration and invasion. Caspase-8 isoform transcripts and epigenetic marks in FLSs were analyzed in FLS public databases. Crystal structures of caspase-8B and G were determined. RESULTS: Caspase-8 deficiency in RA FLSs reduced cell adhesion, migration, and invasion independent of its catalytic activity. Epigenetic and transcriptomic analyses of RA FLSs revealed that a specific caspase-8 isoform, variant G, is the dominant isoform expressed (~80% of total caspase-8) and induced by PDGF. The crystal structures of caspase-8 variant G and B were identical except for a unique unstructured 59 amino acid N-terminal domain in variant G. Selective knockdown of caspase-8G was solely responsible for the effects of caspase-8 on calpain activity and cell invasion in FLS. CONCLUSION: Blocking caspase-8 variant G could decrease cell invasion in diseases like RA without the potential deleterious effects of nonspecific caspase-8 inhibition.

Genetic regulation of OAS1 nonsense-mediated decay underlies association with risk of severe COVID-19.

Genomic regions have been associated with COVID-19 susceptibility and outcomes, including the chr12q24.13 locus encoding antiviral proteins OAS1-3. Here, we report genetic, functional, and clinical insights into genetic associations within this locus. In Europeans, the risk of hospitalized vs. non-hospitalized COVID-19 was associated with a single 19Kb-haplotype comprised of 76 OAS1 variants included in a 95% credible set within a large genomic fragment introgressed from Neandertals. The risk haplotype was also associated with impaired spontaneous but not treatment-induced SARS-CoV-2 clearance in a clinical trial with pegIFN-λ1. We demonstrate that two exonic variants, rs10774671 and rs1131454, affect splicing and nonsense-mediated decay of OAS1 . We suggest that genetically-regulated loss of OAS1 expression contributes to impaired spontaneous clearance of SARS-CoV-2 and elevated risk of hospitalization for COVID-19. Our results provide the rationale for further clinical studies using interferons to compensate for impaired spontaneous SARS-CoV-2 clearance, particularly in carriers of the OAS1 risk haplotypes.

Distinct DNA Methylation Patterns of Rheumatoid Arthritis Peripheral Blood and Synovial Tissue T Cells.

OBJECTIVE: To study epigenetic patterns in T lymphocytes that accumulate in the rheumatoid arthritis (RA) synovium, we characterized DNA methylation of CD3+ T cells in peripheral blood and synovial tissue in patients with RA and osteoarthritis (OA). METHODS: Genomic DNA of CD3+ T cells was isolated from patients with RA (n = 8) and OA (n = 5) from blood or the synovium at the time of an arthroplasty using antibodies and magnetic beads. Methylation was measured by using the Illumina Infinium MethylationEPIC Kit. Differentially methylated loci (DML) and differentially methylated genes (DMGs) were identified by using Welch's t-test. Principal component analysis, hierarchical clustering, and pathway analysis were used to determine relationships among groups. RESULTS: When we compared DNA methylation of CD3+ T cells between peripheral blood and synovial tissue within each disease, 4615 and 164 DML were identified in RA and OA samples, respectively, resulting in 832 and 36 DMGs. A principal component analysis showed that methylation differences in T cells were greater on the basis of on location (blood vs synovium) rather than disease (RA vs OA). Differentially modified pathways were significantly enriched between RA blood and synovial T cells, especially in genes related to complement, integrin cell surface interactions, and the P53 pathway. The limited number of DMGs identified between OA blood and synovial T cells did not conform to biologic pathways. CONCLUSION: The patterns of DNA methylation in RA show location-specific differences related to immune pathways, whereas methylation differences in OA are limited. The RA joint-specific signatures could be due to selective accumulation of T-cell populations or expansion of differentially marked adaptive immune cells. Understanding epigenetic patterns could provide clues to the types of T cells that accumulate in the RA joint and identify potential therapeutic targets.

IgG Epitopes Processed and Presented by IgG+ B Cells Induce Suppression by Human Thymic-Derived Regulatory T Cells.

We described a human regulatory T cell (Treg) population activated by IgG+ B cells presenting peptides of the heavy C region (Fc) via processing of the surface IgG underlying a model for B cell-Treg cooperation in the human immune regulation. Functionally, Treg inhibited the polarization of naive T cells toward a proinflammatory phenotype in both a cognate and a noncognate fashion. Their fine specificities were similar in healthy donors and patients with rheumatoid arthritis, a systemic autoimmune disease. Four immunodominant Fc peptides bound multiple HLA class II alleles and were recognized by most subjects in the two cohorts. The presentation of Fc peptides that stimulate Treg through the processing of IgG by dendritic cells (DC) occurred in myeloid DC classical DC 1 and classical DC 2. Different routes of Ag processing of the IgG impacted Treg expansion in rheumatoid arthritis patients.

Gastrointestinal Surgery for Inflammatory Bowel Disease Persistently Lowers Microbiome and Metabolome Diversity.

BACKGROUND: Many studies have investigated the role of the microbiome in inflammatory bowel disease (IBD), but few have focused on surgery specifically or its consequences on the metabolome that may differ by surgery type and require longitudinal sampling. Our objective was to characterize and contrast microbiome and metabolome changes after different surgeries for IBD, including ileocolonic resection and colectomy. METHODS: The UC San Diego IBD Biobank was used to prospectively collect 332 stool samples from 129 subjects (50 ulcerative colitis; 79 Crohn's disease). Of these, 21 with Crohn's disease had ileocolonic resections, and 17 had colectomies. We used shotgun metagenomics and untargeted liquid chromatography followed by tandem mass spectrometry metabolomics to characterize the microbiomes and metabolomes of these patients up to 24 months after the initial sampling. RESULTS: The species diversity and metabolite diversity both differed significantly among groups (species diversity: Mann-Whitney U test P value = 7.8e-17; metabolomics, P-value = 0.0043). Escherichia coli in particular expanded dramatically in relative abundance in subjects undergoing surgery. The species profile was better able to classify subjects according to surgery status than the metabolite profile (average precision 0.80 vs 0.68). CONCLUSIONS: Intestinal surgeries seem to reduce the diversity of the gut microbiome and metabolome in IBD patients, and these changes may persist. Surgery also further destabilizes the microbiome (but not the metabolome) over time, even relative to the previously established instability in the microbiome of IBD patients. These long-term effects and their consequences for health outcomes need to be studied in prospective longitudinal trials linked to microbiome-involved phenotypes.

Synoviocyte-targeted therapy synergizes with TNF inhibition in arthritis reversal.

Fibroblast-like synoviocytes (FLS) are joint-lining cells that promote rheumatoid arthritis (RA) pathology. Current disease-modifying antirheumatic agents (DMARDs) operate through systemic immunosuppression. FLS-targeted approaches could potentially be combined with DMARDs to improve control of RA without increasing immunosuppression. Here, we assessed the potential of immunoglobulin-like domains 1 and 2 (Ig1&2), a decoy protein that activates the receptor tyrosine phosphatase sigma (PTPRS) on FLS, for RA therapy. We report that PTPRS expression is enriched in synovial lining RA FLS and that Ig1&2 reduces migration of RA but not osteoarthritis FLS. Administration of an Fc-fusion Ig1&2 attenuated arthritis in mice without affecting innate or adaptive immunity. Furthermore, PTPRS was down-regulated in FLS by tumor necrosis factor (TNF) via a phosphatidylinositol 3-kinase-mediated pathway, and TNF inhibition enhanced PTPRS expression in arthritic joints. Combination of ineffective doses of TNF inhibitor and Fc-Ig1&2 reversed arthritis in mice, providing an example of synergy between FLS-targeted and immunosuppressive DMARD therapies.

Development of Gut-Selective Pan-Janus Kinase Inhibitor TD-1473 for Ulcerative Colitis: A Translational Medicine Programme.

BACKGROUND AND AIMS: Oral systemic pan-Janus kinase [JAK] inhibition is effective for ulcerative colitis [UC] but is limited by toxicities. We describe preclinical to clinical translation of TD-1473-an oral gut-selective pan-JAK inhibitor-from in vitro characterization through a Phase 1b study in patients with UC. METHODS: TD-1473 JAK inhibition potency was evaluated in vitro; plasma pharmacokinetics, safety and efficacy were assessed in mice. In a first-time-in-human study, plasma pharmacokinetics and safety were assessed after single and multiple [14 days] ascending doses administered orally to healthy subjects. The Phase 1b study randomized patients with moderately to severely active UC to receive once-daily oral TD-1473 20, 80 or 270 mg, or placebo for 28 days. Plasma and colonic tissue concentrations were measured; safety was assessed; and efficacy was evaluated by UC clinical parameters, disease-surrogate biomarkers, endoscopy, histology and colonic tissue JAK signalling. RESULTS: TD-1473 exhibited potent pan-JAK inhibitory activity in vitro. Oral TD-1473 administration to mice achieved high, biologically active colonic tissue concentrations with low plasma exposure and decreased oxazolone-induced colitis activity without reducing blood cell counts vs placebo. TD-1473 administration in healthy human subjects and patients with UC yielded low plasma exposure and was generally well tolerated; treatment in patients with UC resulted in biologically active colonic tissue concentrations and descriptive trends toward reduced clinical, endoscopic and histological disease activity vs placebo. CONCLUSION: Gut-selective pan-JAK inhibition with TD-1473 administration resulted in high intestinal vs plasma drug exposure, local target engagement, and trends toward reduced UC disease activity. [Clinicaltrials.gov NCT02657122, NCT02818686].

Differential DNA Methylation of Networked Signaling, Transcriptional, Innate and Adaptive Immunity, and Osteoclastogenesis Genes and Pathways in Gout.

OBJECTIVE: In gout, autoinflammatory responses to urate crystals promote acute arthritis flares, but the pathogeneses of tophi, chronic synovitis, and erosion are less well understood. Defining the pathways of epigenomic immunity training can reveal novel pathogenetic factors and biomarkers. The present study was undertaken to seminally probe differential DNA methylation patterns utilizing epigenome-wide analyses in patients with gout. METHODS: Peripheral blood mononuclear cells (PBMCs) were obtained from a San Diego cohort of patients with gout (n = 16) and individually matched healthy controls (n = 14). PBMC methylome data were processed with ChAMP package in R. ENCODE data and Taiji data analysis software were used to analyze transcription factor (TF)-gene networks. As an independent validation cohort, whole blood DNA samples from New Zealand Maori subjects (n = 13 patients with gout, n = 16 control subjects without gout) were analyzed. RESULTS: Differentially methylated loci clearly separated gout patients from controls, as determined by hierarchical clustering and principal components analyses. IL23R, which mediates granuloma formation and cell invasion, was identified as one of the multiple differentially methylated gout risk genes. Epigenome-wide analyses revealed differential methylome pathway enrichment for B and T cell receptor signaling, Th17 cell differentiation and interleukin-17 signaling, convergent longevity regulation, circadian entrainment, and AMP-activated protein kinase signaling, which are pathways that impact inflammation via insulin-like growth factor 1 receptor, phosphatidylinositol 3-kinase/Akt, NF-κB, mechanistic target of rapamycin signaling, and autophagy. The gout cohorts overlapped for 37 (52.9%) of the 70 TFs with hypomethylated sequence enrichment and for 30 (78.9%) of the 38 enriched KEGG pathways identified via TFs. Evidence of shared differentially methylated gout TF-gene networks, including the NF-κB activation-limiting TFs MEF2C and NFATC2, pointed to osteoclast differentiation as the most strongly weighted differentially methylated pathway that overlapped in both gout cohorts. CONCLUSION: These findings of differential DNA methylation of networked signaling, transcriptional, innate and adaptive immunity, and osteoclastogenesis genes and pathways suggest that they could serve as novel therapeutic targets in the management of flares, tophi, chronic synovitis, and bone erosion in patients with gout.

Joint Location-Specific JAK-STAT Signaling in Rheumatoid Arthritis Fibroblast-like Synoviocytes.

OBJECTIVE: Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) derived from hip and knee have distinctive DNA methylation and transcriptome patterns in interleukin (IL)-6 signaling and Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathways. To determine the functional effects of these joint-specific signatures, we evaluated how RA hip and knee FLS differ in their response to IL-6. METHODS: Hip or knee RA FLS were obtained after arthroplasty. Previously published datasets on epigenetic landscape of FLS were mined to identify joint-specific IL-6-related epigenomic differences. RNA sequencing was performed on five RA hip and five knee FLS treated with or without IL-6. Differential gene expression was determined using edgeR software. STAT3 phosphorylation was measured using bead assays. Sensitivity to tofacitinib was evaluated by measuring CCL2 inhibition using quantitative polymerase chain reaction. RESULTS: Assay for Transposase-Accessible Chromatin sequencing and histone chromatin immunoprecipitation sequencing datasets from RA FLS were analyzed to identify epigenomic differences between hip and knee. Differential chromatin accessibility was associated with IL-6,IL-6R, and JAK1 genes. H3K27ac was also differentially marked at other JAK-STAT-related genes, including STAT3-STAT5A region. Principal component analysis of RNA sequencing data confirmed segregation between RA hip and knee FLS under basal conditions, that persisted following IL-6 treatment. STAT3 phosphorylation after IL-6 was significantly higher in knee than hip FLS and was highly correlated with JAK1 protein levels. Knee FLS were less sensitive to the JAK inhibitor tofacitinib than hip FLS. CONCLUSION: RA hip and knee FLS have distinct transcriptomes, epigenetic marks, and STAT3 activation patterns in the IL-6 pathway. These joint-specific differences might contribute to a differential clinical response in individual joints to targeted therapies such as JAK inhibitors.

Defining inflammatory cell states in rheumatoid arthritis joint synovial tissues by integrating single-cell transcriptomics and mass cytometry.

To define the cell populations that drive joint inflammation in rheumatoid arthritis (RA), we applied single-cell RNA sequencing (scRNA-seq), mass cytometry, bulk RNA sequencing (RNA-seq) and flow cytometry to T cells, B cells, monocytes, and fibroblasts from 51 samples of synovial tissue from patients with RA or osteoarthritis (OA). Utilizing an integrated strategy based on canonical correlation analysis of 5,265 scRNA-seq profiles, we identified 18 unique cell populations. Combining mass cytometry and transcriptomics revealed cell states expanded in RA synovia: THY1(CD90)+HLA-DRAhi sublining fibroblasts, IL1B+ pro-inflammatory monocytes, ITGAX+TBX21+ autoimmune-associated B cells and PDCD1+ peripheral helper T (TPH) cells and follicular helper T (TFH) cells. We defined distinct subsets of CD8+ T cells characterized by GZMK+, GZMB+, and GNLY+ phenotypes. We mapped inflammatory mediators to their source cell populations; for example, we attributed IL6 expression to THY1+HLA-DRAhi fibroblasts and IL1B production to pro-inflammatory monocytes. These populations are potentially key mediators of RA pathogenesis.