TL1A mediates fibroblast-like synoviocytes migration and Indian Hedgehog signaling pathway via TNFR2 in patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by joints inflammation. One of the aggressive characteristics of RA fibroblast-like synoviocytes (FLS) is the tendency for migration in the local environment, which plays a central role in the RA pathogenesis. Tumor Necrosis Factor (TNF)-like cytokine 1A (TL1A) is a member of TNF superfamily, which has a role in autoimmunity and influences the RA-FLS behavior through TNF receptor 2 (TNFR2).We investigated the effect of TNF-like cytokine 1A (TL1A) on RA-FLS migration using patients’ samples. Specifically, we examined the hedgehog signaling pathway which is a key regulator in chondrocyte growth and differentiation. We found that TL1A increased significantly the hedgehog homologue Indian hedgehog (IHH) and its receptor Patched 1, 2 (PTCH 1, 2) in RA-FLS. In addition, TL1A-stimulated RA-FLS promoted significantly IHH protein expression. However, both mRNA and protein levels decreased substantially after blocking TL1A with TNFR2 antagonist. The migratory property of RA-FLS was enhanced after stimulation of RA-FLS with TL1A, but was compromised following TL1A blockage. In conclusion, our study has revealed that TL1A modulated RA-FLS migration and Indian hedgehog signaling pathway using TNFR2.     

Functional signaling of membrane-bound TL1A induces IFN-γ expression

TL1A, a TNF member implicated in autoimmune diseases, is a transmembrane protein that is processed to release soluble TL1A (TL1A-S). TL1A-S induces a Th1 response, although the functional significance of membrane-bound TL1A (TL1A-M) remains unknown. We generated TL1A-M expression in HEK-293 cells capable of binding DR3-Fc. Co-incubating IL-12/IL-18-primed CD4⁺ T cells with HEK-293 cells expressing TL1A-M induced 3-fold increase in IFN-γ that was blocked by anti-TL1A Ab. These results demonstrate that TL1A-M can bind death domain receptor 3 (DR3) through cell-cell contact to induce downstream IFN-γ secretion enhancement. Anti-TL1A antibodies designed to treat immune diseases should be verified to block both endogenous TL1A forms.     

Cyr61 induces IL-6 production by fibroblast-like synoviocytes promoting Th17 differentiation in rheumatoid arthritis

Cysteine-rich protein 61 (Cyr61)/CCN1 is a product of an immediate early gene and functions in mediating cell adhesion and inducing cell migration. We previously showed that increased production of Cyr61 by fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) promotes FLS proliferation and participates in RA pathogenesis with the IL-17-dependent pathway. However, whether Cyr61 in turn regulates Th17 cell differentiation and further enhances inflammation of RA remained unknown. In the current study, we explored the potential role of Cyr61 as a proinflammatory factor in RA pathogenesis. We found that Cyr61 treatment dramatically induced IL-6 production in FLS isolated from RA patients. Moreover, IL-6 production was attenuated by Cyr61 knockdown in FLS. Mechanistically, we showed that Cyr61 activated IL-6 production via the αvβ5/Akt/NF-κB signaling pathway. Further, using a coculture system consisting of purified CD4(+) T cells and RA FLS, we found that RA FLS stimulated Th17 differentiation, and the pro-Th17 differentiation effect of RA FLS can be attenuated or stimulated by Cyr61 RNA interference or addition of exogenous Cyr61, respectively. Finally, using the collagen-induced arthritis animal model, we showed that treatment with the anti-Cyr61 mAb led to reduction of IL-6 levels, decrease of Th17 response, and attenuation of inflammation and disease progression in vivo. Taken together, our results reveal a novel role of Cyr61 in promoting Th17 development in RA via upregulation of IL-6 production by FLS, thus adding a new layer into the functional interplay between FLS and Th17 in RA pathogenesis. Our study also suggests that targeting of Cyr61 may represent a novel strategy in RA treatment.     

Gene polymorphisms and serum levels of TL1A in patients with rheumatoid arthritis

Recent findings showed elevated expression of tumor necrosis factor (TNF)-like ligand 1A (TL1A) in rheumatoid arthritis (RA) patients and arthritis mice. However, whether TL1A gene polymorphisms may correlate with RA susceptibility needs to be discussed. This case-control study was performed on 350 RA patients and 556 healthy subjects to identify TL1A genetic variants (rs3810936, rs6478109, and rs7848647) and their possible association with TL1A levels, susceptibility to and severity of RA. Odds ratio and 95% confidence interval were calculated to represent the correlation between TL1A polymorphisms and RA. The TL1A serum levels were evaluated. Results showed that frequencies of TC, TT + TC genotypes of rs3810936, rs7848647 in RA patients were significantly lower in RA patients compared with controls. Patients with C allele showed more severe disease course (disease activity index: erythrocyte sedimentation rate, rheumatoid factor) than in carriers of T allele. However, the allele or genotype frequencies of rs6478109 were not associated with RA. In addition, TL1A genetic variants conferred higher TL1A levels in RA patients compared with controls. In conclusion, these findings indicated an association between TL1A rs3810936, rs7848647 variation and the susceptibility of RA in a sample of Chinese individuals, and TL1A may correlate with severity of RA.     

IL-17A is involved in diabetic inflammatory pathogenesis by its receptor IL-17RA

Interleukin (IL)-17A, a proinflammatory cytokine produced by T-helper (Th)17 cells, has been associated with autoimmune diseases. Type 1 diabetes (T1D) is caused either due to mutation of insulin gene or developed as an autoimmune disease. Studies have shown that IL-17A expression is upregulated in the pancreas in T1D patients and animal models. However, role or importance of IL-17A in T1D pathogenesis needs elucidation. Particularly, evidence for a direct injury of IL-17A to pancreatic β cells through activating IL-17 receptor A (IL-17RA) is lacking. Ins2^Akita (Akita) mouse, a T1D model with spontaneous mutation in insulin 2 gene leading to β-cell apoptosis, was crossed with IL-17A-knockout mouse and male IL-17A-deficient Akita mice were used. Streptozotocin, a pancreatic β-cell-specific cytotoxin, was employed to induce a diabetic model in MIN6 cells, a mouse insulinoma cell line. IL-17A expression in the pancreas was upregulated in both Akita and streptozotocin-induced diabetic mice. IL-17A-knockout Akita mice manifested reduced blood glucose concentration and raised serum insulin level. IL-17A deficiency also decreased production of the proinflammatory cytokines tumor necrosis factor (TNF)-α, IL-1β, and interferon (IFN)-γ in Akita mice. IL-17RA expression in MIN6 cells was upregulated by IL-17A. IL-17A enhanced expression of TNF-α, IL-1β, IFN-γ, and inducible nitric oxide synthase (iNOS) and further increased streptozotocin-induced expression of the inflammatory factors in MIN6 cells. IL-17A exacerbated streptozotocin-induced MIN6 cell apoptosis and insulin secretion impairment. Blocking IL-17RA with anti-IL-17RA-neutralizing antibody reduced all these deleterious effects of IL-17A on MIN6 cells. Collectively, IL-17A deficiency alleviated hyperglycemia, hypoinsulinemia, and inflammatory response in Akita mice that are characteristic for T1D. IL-17A exerted an alone and synergistic destruction with streptozotocin to pancreatic β cells through IL-17RA pathway. Thus, the data suggest that targeting IL-17A and/or IL-17RA is likely to preserve remaining β-cell function and treat T1D.Impact statementThe participation of interleukin (IL)-17A in diabetic pathogenesis is suggested in animal models of autoimmune diabetes and in patients with type 1 diabetes (T1D), but with some contradictory results. Particularly, evidence for a direct injury of IL-17A to pancreatic β cells is lacking. We showed that IL-17A deficiency alleviated diabetic signs including hyperglycemia, hypoinsulinemia, and inflammatory response in Ins2^Akita (Akita) mice, a T1D model with spontaneous mutation in insulin 2 gene leading to β-cell apoptosis. IL-17A enhanced inflammatory reaction, oxidative stress, and cell apoptosis but attenuated insulin level in mouse insulin-producing MIN6 cells. IL-17A had also a synergistic destruction to MIN6 cells with streptozotocin (STZ), a pancreatic β-cell-specific cytotoxin. Blocking IL-17 receptor A (IL-17RA) reduced all these deleterious effects of IL-17A on MIN6 cells. The results demonstrate the role and the importance of IL-17A in T1D pathogenesis and suggest a potential therapeutic strategy for T1D targeting IL-17A and/or IL-17RA.     

TL1A/DR3 axis involvement in the inflammatory cytokine network during pulmonary sarcoidosis

Background

TNF-like ligand 1A (TL1A), a recently recognized member of the TNF superfamily, and its death domain receptor 3 (DR3), firstly identified for their relevant role in T lymphocyte homeostasis, are now well-known mediators of several immune-inflammatory diseases, ranging from rheumatoid arthritis to inflammatory bowel diseases to psoriasis, whereas no data are available on their involvement in sarcoidosis, a multisystemic granulomatous disease where a deregulated T helper (Th)1/Th17 response takes place.

Methods

In this study, by flow cytometry, real-time PCR, confocal microscopy and immunohistochemistry analyses, TL1A and DR3 were investigated in the pulmonary cells and the peripheral blood of 43 patients affected by sarcoidosis in different phases of the disease (29 patients with active sarcoidosis, 14 with the inactive form) and in 8 control subjects.

Results

Our results demonstrated a significant higher expression, both at protein and mRNA levels, of TL1A and DR3 in pulmonary T cells and alveolar macrophages of patients with active sarcoidosis as compared to patients with the inactive form of the disease and to controls. In patients with sarcoidosis TL1A was strongly more expressed in the lung than the blood, i.e., at the site of the involved organ. Additionally, zymography assays showed that TL1A is able to increase the production of matrix metalloproteinase 9 by sarcoid alveolar macrophages characterized, in patients with the active form of the disease, by reduced mRNA levels of the tissue inhibitor of metalloproteinase (TIMP)-1.

Conclusions

These data suggest that TL1A/DR3 interactions are part of the extended and complex immune-inflammatory network that characterizes sarcoidosis during its active phase and may contribute to the pathogenesis and to the progression of the disease.     

IL-17 receptor and its functional significance in psoriatic arthritis

To delineate the functional significance of IL-17 Receptor (IL-17RA) and characterize the IL-17 producing T cell (Th17) subpopulation in psoriatic arthritis (PsA). Mononuclear cells from blood and synovial fluid (SF) were obtained from PsA (n=20), rheumatoid arthritis (RA, n=20) and osteoarthritis (OA, n=20) patients. Synoviocytes (FLS) were isolated from the synovium of RA (n=5), PsA (n=5) and OA (n=5) patients. IL-17RA expression in FLS was identified by western blotting (WB) and flowcytometry. T lymphocytes derived from the SF of these patients were studied to identify and phenotype the Th17 cells. The functional significance of IL-17RA was determined by evaluating its regulatory role on the production of proinflammatory cytokines and endopeptidase. IL-17RA expression was found to be significantly higher in FLS of RA (15.7%±4.9) and PsA (4.5%±0.9) in comparison to OA (1.14%±0.9). Western blot analyses showed that the relative intensity (RI) of IL-17RA protein was higher in RA and PsA compared to OA (Fisher exact, P<0.01). A significant enrichment of IL-17-producing CD4+ T cells (7.9%±2.8) was observed in the SF of PsA patients compared to that of OA patients (P<.001). Compared to OA-FLS, recombinant IL-17 induced higher levels of IL-6, IL-8, and MMP-3 production in PsA-FLS. Blockage of IL-17RA with an anti-IL-17RA antibody inhibited the production of IL-6, IL-8, and MMP-3. This is the first report to demonstrate the functional significance of IL-17RA in PsA. Results of this study support the hypothesis that IL-17RA blocking antibodies have the potential to be a therapeutic option for psoriatic arthritis.     

The TNF-Family Cytokine TL1A Inhibits Proliferation of Human Activated B Cells

Death receptor (DR3) 3 is a member of the TNFR superfamily. Its ligand is TNF-like ligand 1A (TL1A), a member of the TNF superfamily. TL1A/DR3 interactions have been reported to modulate the functions of T cells, NK, and NKT cells and play a crucial role in driving inflammatory processes in several T-cell-dependent autoimmune diseases. However, TL1A expression and effects on B cells remain largely unknown. In this study, we described for the first time that B cells from human blood express significant amounts of DR3 in response to B cell receptor polyclonal stimulation. The relevance of these results has been confirmed by immunofluorescence analysis in tonsil and spleen tissue specimens, which showed the in situ expression of DR3 in antigen-stimulated B cells in vivo. Remarkably, we demonstrated that TL1A reduces B-cell proliferation induced by anti-IgM-antibodies and IL-2 but did not affect B-cell survival, suggesting that TL1A inhibits the signal(s) important for B-cell proliferation. These results revealed a novel function of TL1A in modulating B-cell proliferation in vitro and suggest that TL1A may contribute to homeostasis of effector B-cell functions in immune response and host defense, thus supporting the role of the TL1A/DR3 functional axis in modulating the adaptive immune response.     

Inhibition of TNF-induced IL-6 by the TWEAK-Fn14 interaction in rheumatoid arthritis fibroblast like synoviocytes

ObjectivesTNF-like weak inducer of apoptosis (TWEAK), a member of the TNF superfamily, has been shown to increase cytokine production by rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). In this study, we determined the effect of interaction between TWEAK and its receptor fibroblast growth factor-inducible-14 (Fn14) on cytokine expression in RAFLS.MethodsRAFLS were obtained from surgical synovial specimens and used at passage 5–10. Cytokine protein and mRNA expression were measured with ELISA and real time-PCR, respectively. Apoptotic cells were detected by TUNEL assay. RelB activation was detected by Western blot analysis.ResultsTWEAK inhibited IL-6 production from total synovial cells from RA. TWEAK weakly induced FLS IL-6 and IL-8, but in contrast TWEAK dose-dependently inhibited IL-6 and IL-8 production by TNFα-activated FLS. TWEAK did not induce apoptosis in FLS but inhibited proliferation of TNFα-activated FLS. TWEAK induced RelB activation and suppressed IL-6 mRNA expression in TNFα-activated FLS and both of these phenomenon were abolished by inhibition of new protein synthesis with cycloheximide.ConclusionsTWEAK has a previously unsuspected inhibitory effect on cytokine production by TNFα-activated RAFLS. This observation suggests that the effects of TWEAK on cytokine expression varies with the pro-inflammatory context, and that in TNFα-activated states such as RA TWEAK may have a net inhibitory effect.     

Structural basis for tumor necrosis factor blockade with the therapeutic antibody golimumab

Tumor necrosis factor α (TNFα) is a proinflammatory cytokine, and elevated levels of TNFα in serum are associated with various autoimmune diseases, including rheumatoid arthritis (RA), ankylosing spondylitis (AS), Crohn's disease (CD), psoriasis, and systemic lupus erythaematosus. TNFα performs its pleiotropic functions by binding to two structurally distinct transmembrane receptors, TNF receptor (TNFR) 1 and TNFR2. Antibody‐based therapeutic strategies that block excessive TNFα signaling have been shown to be effective in suppressing such harmful inflammatory conditions. Golimumab (Simponi®) is an FDA‐approved fully human monoclonal antibody targeting TNFα that has been widely used for the treatment of RA, AS, and CD. However, the structural basis underlying the inhibitory action of golimumab remains unclear. Here, we report the crystal structure of the Fv fragment of golimumab in complex with TNFα at a resolution of 2.73 Å. The resolved structure reveals that golimumab binds to a distinct epitope on TNFα that does not overlap with the binding residues of TNFR2. Golimumab exerts its inhibitory effect by preventing binding of TNFR1 and TNFR2 to TNFα by steric hindrance. Golimumab does not induce conformational changes in TNFα that could affect receptor binding. This mode of action is specific to golimumab among the four anti‐TNFα therapeutic antibodies currently approved for clinical use.     

The expression of the receptor for advanced glycation end-products (RAGE) in RA-FLS is induced by IL-17 via Act-1

Introduction

The receptor for advanced glycation end-products (RAGE) has been implicated in the pathogenesis of arthritis. We conducted this study to determine the effect of interleukin (IL)-17 on the expression and production of RAGE in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA). The role of nuclear factor-κB (NF-κB) activator 1 (Act1) in IL-17-induced RAGE expression in RA-FLS was also evaluated.

Methods

RAGE expression in synovial tissues was assessed by immunohistochemical staining. RAGE mRNA production was determined by real-time polymerase chain reaction. Act-1 short hairpin RNA (shRNA) was produced and treated to evaluate the role of Act-1 on RAGE production.

Results

RAGE, IL-17, and Act-1 expression increased in RA synovium compared to osteoarthritis synovium. RAGE expression and production increased by IL-17 and IL-1β (*P <0.05 vs. untreated cells) treatment but not by tumor necrosis factor (TNF)-α in RA-FLS. The combined stimuli of both IL-17 and IL-1β significantly increased RAGE production compared to a single stimulus with IL-17 or IL-1β alone (P <0.05 vs. 10 ng/ml IL-17). Act-1 shRNA added to the RA-FLS culture supernatant completely suppressed the enhanced production of RAGE induced by IL-17.

Conclusions

RAGE was overexpressed in RA synovial tissues, and RAGE production was stimulated by IL-17 and IL-1β. Act-1 contributed to the stimulatory effect of IL-17 on RAGE production, suggesting a possible inhibitory target for RA treatment.     

Inflammatory cytokines induced down-regulation of m-calpain mRNA expression in fibroblastic synoviocytes from patients with osteoarthritis and rheumatoid arthritis

Our previous reports revealed that calpain has proteoglycanase activity and exists in synovial fluid in osteoarthritis and rheumatoid arthritis. We examined the effects of cytokines on expression of the calpain-calpastatin system in fibroblastic synoviocytes (FLS). Primary cultures of human FLS from osteoarthritis (OA) and rheumatoid arthritis (RA) patients were stimulated with inflammatory cytokines and the amounts of m-calpain and calpastatin mRNAs expressed were determined by Northern blotting. Northern blots were subjected to computerized densitometer and band intensities were determined. Interleukin-1 (IL-1) down-regulated m-calpain and tissue-type calpastatin mRNA expression in OA and RA FLS. In RA FLS, although IL-6 did not alter m-calpain mRNA expression, IL-1 + tumor necrosis factor (TNF) and IL-1 + transforming growth factor (TGF) down-regulated m-calpain mRNA expression. These results provide new information about the effects of inflammatory cytokines on calpain and calpastatin system in OA and RA pathology.     

Dominant role for TL1A/DR3 pathway in IL-12 plus IL-18-induced IFN-gamma production by peripheral blood and mucosal CCR9+ T lymphocytes

The TNF-like cytokine TL1A augments IFN-gamma production by anti-CD3 plus anti-CD28 and IL-12/IL-18-stimulated peripheral blood (PB) T cells. However, only a small subset of PB T cells respond to TL1A stimulation with IFN-gamma production. PB CCR9+ T cells represent a small subset of circulating T cells with mucosal T cell characteristics and a Th1/Tr1 cytokine profile. In the current study, we show that TL1A enhanced IFN-gamma production by TCR- or CD2/CD28-stimulated CCR9(+)CD4+ PB T cells. However, TL1A had the most pronounced effect on augmenting IFN-gamma production by IL-12/IL-18-primed CCR9(+)CD4+ PB T cells. TL1A enhanced both the percentage and the mean fluorescence intensity of IFN-gamma in CCR9(+)CD4+ T cells as assessed by intracellular cytokine staining. IL-12 plus IL-18 up-regulated DR3 expression in CCR9(+)CD4+ T cells but had negligible effect on CCR9(-)CD4+ T cells. CCR9(+)CD4+ T cells isolated from the small intestine showed a 37- to 105-fold enhancement of IFN-gamma production when TL1A was added to the IL-12/IL18 cytokine combination. Cell membrane-expressed TL1A was preferentially expressed in CCR9(+)CD4+ PB T cells, and a blocking anti-TL1A mAb inhibited IFN-gamma production by cytokine-primed CCR9(+)CD4+ T cells by approximately 50%. Our data show that the TL1A/DR3 pathway plays a dominant role in the ultimate level of cytokine-induced IFN-gamma production by CCR9+ mucosal and gut-homing PB T cells and could play an important role in Th1-mediated intestinal diseases, such as Crohn's disease, where increased expression of IL-12, IL-18, TL1A, and DR3 converge in the inflamed intestinal mucosa.     

Deficiency of TNFR1 protects myocardium through SOCS3 and IL-6 but not p38 MAPK or IL-1beta

Tumor necrosis factor-alpha (TNF-alpha) plays an important role in the development of heart failure. There is a direct correlation between myocardial function and myocardial TNF levels in humans. TNF may induce local inflammation to exert tissue injury. On the other hand, suppressors of cytokine signaling (SOCS) proteins have been shown to inhibit proinflammatory signaling. However, it is unknown whether TNF mediates myocardial inflammation via STAT3/SOCS3 signaling in the heart and, if so, whether this effect is through the type 1 55-kDa TNF receptor (TNFR1). We hypothesized that TNFR1 deficiency protects myocardial function and decreases myocardial IL-6 production via the STAT3/SOCS3 pathway in response to TNF. Isolated male mouse hearts (n = 4/group) from wild-type (WT) and TNFR1 knockout (TNFR1KO) were subjected to direct TNF infusion (500 pg.ml(-1).min(-1) x 30 min) while left ventricular developed pressure and maximal positive and negative values of the first derivative of pressure were continuously recorded. Heart tissue was analyzed for active forms of STAT3, p38, SOCS3 and SOCS1 (Western blot analysis), as well as IL-1beta and IL-6 (ELISA). Coronary effluent was analyzed for lactate dehydrogenase (LDH) activity. As a result, TNFR1KO had significantly better myocardial function, less myocardial LDH release, and greater expression of SOCS3 (percentage of SOCS3/GAPDH: 45 +/- 4.5% vs. WT 22 +/- 6.5%) after TNF infusion. TNFR1 deficiency decreased STAT3 activation (percentage of phospho-STAT3/STAT3: 29 +/- 6.4% vs. WT 45 +/- 8.8%). IL-6 was decreased in TNFR1KO (150.2 +/- 3.65 pg/mg protein) versus WT (211.4 +/- 26.08) mice. TNFR1 deficiency did not change expression of p38 and IL-1beta following TNF infusion. These results suggest that deficiency of TNFR1 protects myocardium through SOCS3 and IL-6 but not p38 MAPK or IL-1beta.     

The T cell costimulator TL1A is induced by FcgammaR signaling in human monocytes and dendritic cells

The recently described TL1A/DR3 ligand/receptor pair mediates strong costimulation of Th1 cells. Activation of T and NK cells induces DR3 expression, permitting soluble recombinant TL1A to increase IFN-gamma production and proliferation of these cells. Gut T cells and macrophages express TL1A, especially in Crohn's disease (CD), and there is a strong association between CD and tl1a single nucleotide polymorphisms. Murine studies implicate TL1A in gut inflammation. To determine whether professional T cell-activating cells can express TL1A, fresh blood monocytes and monocyte-derived dendritic cells were stimulated with various activating ligands, including TLR agonists, IFN-gamma, and immune complexes. FcgammaR stimulation strongly induced TL1A mRNA in both cell types, which correlated with the detection of TL1A on the cell surface and in cell culture medium. TLR agonists capable of inducing IL-6 and TNF-alpha in monocytes and dendritic cells did not induce surface nor soluble TL1A. Furthermore, we demonstrate that TL1A production in monocytes leads to enhancement of T cell responses. The induction of TL1A on APCs via specific pathway stimulation suggests a role for TL1A in Th1 responses to pathogens, and in CD.