TNFR2 is critical for TNF-induced rheumatoid arthritis fibroblast-like synoviocyte inflammation.

OBJECTIVES: TNF-induced activation of fibroblast-like synoviocytes (FLS) is a critical determinant for synovial inflammation and joint destruction in RA. The detrimental role of TNF-receptor 1 (TNFR1) has thoroughly been characterized. The contributions of TNFR2, however, are largely unknown. This study was performed to delineate the role of TNFR2 in human FLS activation. METHODS: TNFR2 expression in synovial tissue samples was determined by immunohistochemistry. Expression of TNFR2 was silenced using RNAi or CRISPR/Cas9 technologies. Global transcriptional changes were determined by RNA-seq. QPCR, ELISA and immunoblotting were used to validate RNA-seq results and to uncover pathways operating downstream of TNFR2 in FLS. RESULTS: TNFR2 expression was increased in RA when compared with OA synovial tissues. In particular, RA-FLS demonstrated higher levels of TNFR2 when compared with OA-FLS. TNFR2 expression in RA-FLS correlated with RA disease activity, synovial T- and B-cell infiltration. TNF and IL1ß were identified as inflammatory mediators that upregulate TNFR2 in RA-FLS. Silencing of TNFR2 in RA-FLS markedly diminished the TNF-induced expression of inflammatory cytokines and chemokines, including CXCR3-binding chemokines and the B-cell activating factor TNFSF13B. Immunobiochemical analyses revealed that TNFR2-mediated expression of inflammatory mediators critically depends on STAT1. CONCLUSION: Our results define a critical role for TNFR2 in FLS-driven inflammation and unfold its participation in the unresolved course of synovial inflammation in RA.

FOXO3 is involved in the tumor necrosis factor-driven inflammatory response in fibroblast-like synoviocytes.

Fibroblast-like synoviocytes (FLS) are major contributors to joint inflammation in rheumatoid arthritis (RA). Forkhead box O 3 (FOXO3) perturbations in immune cells are increasingly linked to RA pathogenesis. Here, we show that FOXO3 is distinctly inactivated/phosphorylated in the FLS of rheumatoid synovitis. In vitro, stimulation of FLS with tumor necrosis factor-alpha α (TNFα) induced a rapid and sustained inactivation of FOXO3. mRNA profiling revealed that the inactivation of FOXO3 is important for the sustained pro-inflammatory interferon response to TNFα (CXCL9, CXCL10, CXCL11, and TNFSF18). Mechanistically, our studies demonstrate that the inactivation of FOXO3 results from TNF-induced downregulation of phosphoinositide-3-kinase-interacting protein 1 (PIK3IP1). Thus, we identified FOXO3 and its modulator PIK3IP1 as a critical regulatory circuit for the inflammatory response of the resident mesenchymal cells to TNFα and contribute insight into how the synovial tissue brings about chronic inflammation that is driven by TNFα.

Targeted inhibition of Janus kinases abates interfon gamma-induced invasive behaviour of fibroblast-like synoviocytes.

Objectives: The aim was to explore the function of the T-cell cytokine IFNγ for mesenchymal tissue remodelling in RA and to determine whether IFNγ signalling controls the invasive potential of fibroblast-like synoviocytes (FLS). Methods: To assess architectural responses, FLS were cultured in three-dimensional micromasses. FLS motility was analysed in migration and invasion assays. Signalling events relevant to cellular motility were defined by western blots. Baricitinib and small interfering RNA pools were used to suppress Janus kinase (JAK) functions. Results: Histological analyses of micromasses revealed unique effects of IFNγ on FLS shape and tissue organization. This was consistent with accelerated migration upon IFNγ stimulation. Given that cell shape and cell motility are under the control of the focal adhesion kinase (FAK), we next analysed its activity. Indeed, IFNγ stimulation induced the phosphorylation of FAK-Y925, a phosphosite implicated in FAK-mediated cell migration. Small interfering RNA knockdown of JAK2, but not JAK1, substantially abrogated FAK activation by IFNγ. Correspondingly, IFNγ-induced FAK activation and invasion of FLS was abrogated by the JAK inhibitor, baricitinib. Conclusion: Our study contributes insight into the synovial response to IFNγ and reveals JAK2 as a potential therapeutic target for FLS-mediated joint destruction in arthritis, especially in RA.

Amino Acids Fueling Fibroblast-Like Synoviocyte Activation and Arthritis By Regulating Chemokine Expression and Leukocyte Migration.

OBJECTIVE: We analyzed the impact of amino acid (AA) availability on the inflammatory response in arthritis. METHODS: We stimulated rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs) with tumor necrosis factor (TNF) in the presence or absence of proteinogenic AAs and measured their response by QuantSeq 3' messenger RNA sequencing, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay. Signal transduction events were determined by Western blot. We performed K/BxN serum transfer arthritis in mice receiving a normal and a low-protein diet and analyzed arthritis clinically and histologically. RESULTS: Deprivation of AAs decreased the expression of a specific subset of genes, including the chemokines CXCL10, CCL2, and CCL5 in TNF-stimulated FLSs. Mechanistically, the presence of AAs was required for the TNF-induced activation of an interferon regulatory factor 1 (IRF1)-STAT1 signaling circuit that drives the expression of chemotactic factors. The expression of IRF1 and the IRF1-dependent gene set in FLSs was highly correlated with the presence of inflammatory cells in human RA, emphasizing the important role of this AA-dependent pathway in inflammatory cell recruitment to the synovial tissue. Finally, we show that mice receiving a low-protein diet expressed less IRF1 in the inflamed synovium and consequently developed reduced clinical and histologic signs of arthritis. CONCLUSION: AA deprivation reduces the severity of arthritis by suppressing the expression of IRF1-STAT1-driven chemokines, which are crucial for leukocyte recruitment to the arthritic joint. Overall, our study provides novel insights into critical determinants of inflammatory arthritis and may pave the way for dietary intervention trials in RA.

Interferon signals and monocytic sensitization of the interferon-γ signaling pathway in the peripheral blood of patients with rheumatoid arthritis.

OBJECTIVE: Both type I interferons (IFNα and IFNß) and type II IFN (IFNγ) signal via pSTAT-1. Immunohistochemistry and the gene expression signatures of rheumatoid arthritis (RA) synovial tissue suggest an activated IFN/STAT-1 signaling pathway. The aim of this study was to determine the systemic activity of the IFN/STAT-1 signaling pathway in the peripheral blood cells of patients with RA. METHODS: Fluorocytometry or quantitative polymerase chain reaction was used to measure the expression of STAT-1, pSTAT-1, and IFN-inducible genes (monokine induced by interferon-γ [MIG], interferon-γ-inducible protein 10 [IP-10], and 2',5'-oligoadenylate synthetase [OAS]) in the peripheral blood mononuclear cells (PBMCs) and purified CD14+ peripheral blood monocytes of patients with RA and healthy control subjects. PBMCs were also incubated for 48 hours with IFNs and several other cytokines to investigate influences on STAT-1 levels. To examine the significance of STAT-1 activation in RA monocytes after stimulation with IFNγ, the expression of pSTAT-1 and of the IFNγ-inducible chemokine MIG was measured using fluorocytometry. RESULTS: Levels of STAT-1 were significantly increased in peripheral lymphocytes and monocytes from patients with RA compared with those from healthy control subjects. STAT-1 levels correlated well with RA disease activity, as measured by the Disease Activity Score in 28 joints and the Clinical Disease Activity Index. Furthermore, STAT-1 messenger RNA expression in RA CD14+ monocytes correlated with the expression of other IFN-target genes, such as IP-10, OAS, or MIG. In RA PBMCs, STAT-1 expression was increased not only by IFNs but also by tumor necrosis factor. RA monocytes demonstrated a considerably higher increase in pSTAT-1 and MIG levels upon IFNγ stimulation when compared with monocytes from control subjects, indicating that RA monocytes are more sensitive to IFNγ stimulation. CONCLUSION: In addition to supporting the role of IFNs in systemic proinflammatory activity, the results of this study further suggest preactivation of the IFNγ/STAT-1 signaling pathway, especially in RA monocytes.

Quantitative and qualitative deficiencies of regulatory T cells in patients with systemic lupus erythematosus (SLE).

The objective of the study was that the regulatory T cells (Treg) that specialize in the suppression of immune responses might be critically involved in the pathogenesis of autoimmune disease. As for systemic lupus erythematosus (SLE), however, published data concerning Treg phenotype and function are partly conflicting. We therefore performed quantitative and qualitative analyses of naturally occurring CD4(+)CD25(+) Treg from SLE patients as compared with healthy controls (HC) in order to further elucidate the role of Treg in this systemic autoimmune disease. The phenotype of peripheral blood CD4(+)CD25(+) Treg was determined by flow cytometry (FACS) in SLE patients and HC. Treg were isolated from SLE patients and HC and their functional capacity was analyzed in suppression assays. Phenotypic and functional data were correlated with clinical data. Decreased proportions of CD4(+) Treg with high-level expression of CD25 (CD4(+)CD25(hi)) were observed in active and inactive SLE patients (0.96 +/- 0.08 and 1.17 +/- 0.08%, respectively) as compared with HC (2 +/- 0.1%). In contrast to HC, Treg from SLE patients displayed an activated phenotype as determined by the expression of CD69, CD71 and HLA-DR. The suppressive capacity of isolated Treg from SLE patients, however, was significantly reduced as compared with HC. Proportions of CD4(+)CD25(hi) T cells and the suppressive capacity of Treg were inversely correlated with the clinical disease activity in SLE patients. Our data describe quantitative and qualitative defects of Treg in SLE patients. These deficiencies might contribute to the breakdown of self-tolerance and the development of the autoimmune response in SLE patients.

IRF1 is critical for the TNF-driven interferon response in rheumatoid fibroblast-like synoviocytes : JAKinibs suppress the interferon response in RA-FLSs.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by persistent synovial inflammation. The major drivers of synovial inflammation are cytokines and chemokines. Among these molecules, TNF activates fibroblast-like synoviocytes (FLSs), which leads to the production of inflammatory mediators. Here, we show that TNF regulates the expression of the transcription factor interferon regulatory factor 1 (IRF1) in human FLSs as well as in a TNF transgenic arthritis mouse model. Transcriptomic analyses of IRF1-deficient, TNF-stimulated FLSs define the interferon (IFN) pathway as a major target of IRF1. IRF1 expression is associated with the expression of IFNß, which leads to the activation of the JAK-STAT pathway. Blocking the JAK-STAT pathway with the Janus kinase inhibitor (JAKinib) baricitinib or tofacitinib reduces the expression of IFN-regulated genes (IRGs) in TNF-activated FLSs. Therefore, we conclude that TNF induces a distinct inflammatory cascade, in which IRGs are key elements, in FLSs. The IFN-signature might be a promising biomarker for the efficient and personalized use of new treatment strategies for RA, such as JAKinibs.

mTOR Senses Environmental Cues to Shape the Fibroblast-like Synoviocyte Response to Inflammation.

Accumulating evidence suggests that metabolic master regulators, including mTOR, regulate adaptive and innate immune responses. Resident mesenchymal tissue components are increasingly recognized as key effector cells in inflammation. Whether mTOR also controls the inflammatory response in fibroblasts is insufficiently studied. Here, we show that TNF signaling co-opts the mTOR pathway to shift synovial fibroblast (FLS) inflammation toward an IFN response. mTOR pathway activation is associated with decreased NF-κB-mediated gene expression (e.g., PTGS2, IL-6, and IL-8) but increased STAT1-dependent gene expression (e.g., CXCL11 and TNFSF13B). We further demonstrate how metabolic inputs, such as amino acids, impinge on TNF-mTORC1 signaling to differentially regulate pro-inflammatory signaling circuits. Our results define a critical role for mTOR in the regulation of the pro-inflammatory response in FLSs and unfold its pathogenic involvement in TNF-driven diseases, such as rheumatoid arthritis (RA).

Activation of the interferon-gamma signaling pathway in systemic lupus erythematosus peripheral blood mononuclear cells.

OBJECTIVE: To investigate interferon-gamma (IFNgamma) signaling in peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE) by analyzing IFNgamma receptor (IFNgammaR) expression, STAT-1 expression and phosphorylation, and the regulation of IFNgamma-inducible genes. METHODS: Fluorocytometry was used to investigate expression of STAT-1, pSTAT-1, CD95, HLA-DR, class I major histocompatibility complex (MHC), IFNgamma-inducible 10-kd protein (IP-10), monokine induced by IFNgamma (Mig), and IFNgammaR in PBMCs from SLE patients and healthy individuals. STAT-1 phosphorylation was determined by fluorocytometry and Western blotting after stimulation with IFNalpha or IFNgamma. Quantitative polymerase chain reaction was used to assess messenger RNA (mRNA) expression of the IFNgamma-inducible genes IP-10 and Mig shortly after preparation or after stimulation with IFNgamma in monocytes. RESULTS: STAT-1 expression was increased in PBMCs from SLE patients and correlated significantly with disease activity and with the IFN-inducible expression of CD95 and HLA-DR. STAT-1 expression also showed a trend toward association with class I MHC expression. In addition, the expression of other IFNgamma-inducible genes, such as IP-10 or Mig, was increased in SLE monocytes. While STAT-1 phosphorylation in SLE PBMCs and PBMCs from healthy individuals was similar after IFNalpha stimulation, incubation with IFNgamma induced STAT-1 phosphorylation only in SLE lymphocytes. Moreover, SLE monocytes showed a considerably higher increase in pSTAT-1 expression upon IFNgamma stimulation than monocytes from healthy individuals. Increased responsiveness of SLE monocytes to IFNgamma was also confirmed on the mRNA level, where expression of the IFN-inducible, STAT-1-dependent genes IP-10 and Mig was more efficiently increased in SLE cells. However, IFNgammaR was similarly expressed on SLE lymphocytes and monocytes and those from healthy individuals. CONCLUSION: In addition to supporting the role of IFNs in SLE immunopathogenesis in general, the findings of the present study support a role of IFNgamma in this disease.