Critical role of IL-1α in IL-1ß-induced inflammatory responses: cooperation with NF-κBp65 in transcriptional regulation.

The IL-1 cytokines are considered among the first family of cytokines that orchestrate acute and chronic inflammatory diseases. Both IL-1ß and IL-1α are members of the IL-1 family; however, their distinct roles in the inflammatory processes remain poorly understood. We explored the role of IL-1α in IL-1ß-activated signaling pathways causing synovial inflammation in rheumatoid arthritis (RA). Using synovial fibroblasts isolated from RA joints, we found that IL-1ß significantly stimulated IL-1α expression, which was selectively inhibited by blocking the NF-κB pathway. Knockdown of IL-1α using small interfering RNA abolished IL-1ß-induced pro-IL-1α and pro-IL-1ß expression and suppressed inflammation. Native and chromatin immunoprecipitation studies showed that IL-1α cooperates in NF-κBp65 binding to the distal region of IL-1α promoter and to the proximal region of IL-1ß promoter upstream of the transcription start site to stabilize their gene transcription. Molecular dynamics simulation of IL-1α or IL-1ß binding to IL-1 receptor showed distinct interaction sites that corroborate with the ability of IL-1α to differentially activate phosphorylation of signaling proteins compared with IL-1ß. Our study highlights the importance of IL-1α in mediating IL-1ß-induced inflammation in addition to maintaining its expression and providing a rationale for targeting IL-1α to minimize the role of IL-1ß in inflammatory diseases like RA.-Singh, A. K., Fechtner, S., Chourasia, M., Sicalo, J., Ahmed, S. Critical role of IL-1α in IL-1ß-induced inflammatory responses: cooperation with NF-κBp65 in transcriptional regulation.

Role of cannabinoid receptor 2 in mediating interleukin-1ß-induced inflammation in rheumatoid arthritis synovial fibroblasts.

OBJECTIVES: Recent studies showed that the expression of cannabinoid receptor 2 (CB2), not CB1, is upregulated at both the mRNA and protein levels in rheumatoid arthritis synovial fibroblasts (RASFs), however, little is known about its endogenous role in pro-inflammatory cytokine signalling in RASFs. Our aim was to investigate the role of CB2 receptor in mediating IL-1ß-induced inflammation in human RASFs. METHODS: Human RASFs were pretreated with CB2 selective agonist (JWH-133), followed by stimulation with interleukin-1ß (IL-1ß, 10 ng/mL). The role of CB2 in IL-1ß signalling was examined using small interfering RNA (siRNA) or an overexpression plasmid specific for CB2. RESULTS: Pretreatment with JWH-133 did not reduce IL-1ß-induced IL-6 and IL-8 production and amplified the cellular expression of cyclooxygenase-2 (COX-2) by >2-fold in human RASFs. Furthermore, the knockdown of CB2 using siRNA markedly inhibited IL-1ß-induced IL-6, IL-8, ENA-78, and RANTES production by more than 50% and completely abrogated COX-2 expression in human RASFs. MMP-2 and MMP-9 activity was also reduced by 50% with CB2 knockdown. On the contrary, overexpression of CB2 in human RASFs further increased IL-1ß-induced IL-6, IL-8, and RANTES by approximately 3-fold whereas ENA-78 expression increased by 1.5-fold. Immunoprecipitation analysis to study the protein-protein interactions revealed that JWH-133 coordinates CB2 association with TGFß-activated kinase 1 (TAK1), a key signalling molecule, to increase IL-1ß-induced nuclear translocation of transcription factors nuclear factor-κBp65 (NF-κBp65) and activation protein-1 (AP-1). CONCLUSIONS: Overall, our results indicate for the first time that CB2 mediates IL-1ß-induced signalling pathways in RASFs and may serve as a potential target to manage pain and inflammation in RA.

Transforming growth factor ß activated kinase 1: a potential therapeutic target for rheumatic diseases.

Pro-inflammatory cytokines such as IL-1ß, IL-6 and TNF-α are central regulators of autoinflammatory diseases. While targeting these cytokines has proven to be a successful clinical strategy, the long-term challenges such as drug resistance, lack of efficacy and poor clinical outcomes in some patients are some of the limitations faced by these therapies. This has ignited strategies to reduce inflammation by potentially targeting a variety of molecules, including cell surface receptors, signalling proteins and/or transcription factors to minimize cytokine-induced inflammation and tissue injury. In this regard, transforming growth factor ß activated kinase 1 (TAK1) is activated in the inflammatory signal transduction pathways in response to IL-1ß, TNF-α or toll-like receptor stimulation. Because of its ideal position upstream of mitogen-activated protein kinases and the IκB kinase complex in signalling cascades, targeting TAK1 may be an attractive strategy for treating diseases characterized by chronic inflammation. Here, we discuss the emerging role of TAK1 in mediating the IL-1ß, TNF-α and toll-like receptor mediated inflammatory responses in diseases such as RA, OA, gout and SS. We also review evidence suggesting that TAK1 inhibition may have potential therapeutic value. Finally, we focus on the current status of the development of TAK1 inhibitors and suggest further opportunities for testing TAK1 inhibitors in rheumatic diseases.

Molecular insights into the differences in anti-inflammatory activities of green tea catechins on IL-1ß signaling in rheumatoid arthritis synovial fibroblasts.

In this study, we found that catechins found in green tea (EGCG, EGC, and EC) differentially interfere with the IL-1ß signaling pathway which regulates the expression of pro-inflammatory mediators (IL-6 and IL-8) and Cox-2 in primary human rheumatoid arthritis synovial fibroblasts (RASFs). EGCG and EGC inhibited IL-6, IL-8, and MMP-2 production and selectively inhibited Cox-2 expression. EC did not exhibit any inhibitory effects. When we looked at the expression of key signaling proteins in the IL-1ß signaling pathway, we found all the tested catechins could inhibit TAK-1 activity. Therefore, the consumption of green tea offers an overall anti-inflammatory effect. Molecular docking analysis confirms that EGCG, EGC, and EC all occupy the active site of the TAK1 kinase domain. However, EGCG occupies the majority of the TAK1 active site. In addition to TAK1 inhibition, EGCG can also inhibit P38 and nuclear NF-κB expression whereas EC and EGC were not effective inhibitors. Our findings suggest one of the main health benefits associated with the consumption of green tea are due to the activity of EGCG and EGC which are both present at higher amounts. Although EGCG is the most effective catechin at inhibiting downstream inflammatory signaling, its effectiveness could be hindered by the presence of EC. Therefore, varying EC content in green tea may reduce the anti-inflammatory effects of other potential catechins in green tea.

3,3-Dimethyl-1-Butanol and its Metabolite 3,3-Dimethylbutyrate Ameliorate Collagen-induced Arthritis Independent of Choline Trimethylamine Lyase Activity.

Conflicting data exist in rheumatoid arthritis and the collagen-induced arthritis (CIA) murine model of autoimmune arthritis regarding the role of bacterial carnitine and choline metabolism into the inflammatory product trimethylamine (TMA), which is oxidized in the liver to trimethylamine-N-oxide (TMAO). Using two published inhibitors of bacterial TMA lyase, 3,3-dimethyl-1-butanol (DMB) and fluoromethylcholine (FMC), we tested if TMA/TMAO were relevant to inflammation in the development of CIA. Surprisingly, DMB-treated mice demonstrated > 50% reduction in arthritis severity compared to FMC and vehicle-treated mice, but amelioration of disease was independent of TMA/TMAO production. Given the apparent contradiction that DMB did not inhibit TMA, we then investigated the mechanism of protection by DMB. After verifying that DMB acted independently of the intestinal microbiome, we traced the metabolism of DMB within the host and identified a novel host-derived metabolite of DMB, 3,3-dimethyl-1-butyric acid (DMBut). In vivo studies of mice treated with DMB or DMBut demonstrated efficacy of both molecules in significantly reducing disease and proinflammatory cytokines in CIA, while in vitro studies suggest these molecules may act by modulating secretion of proinflammatory cytokines from macrophages. Altogether, our study suggests that DMB and/or its metabolites are protective in CIA through direct immunomodulatory effects rather than inhibition of bacterial TMA lyases.

3,3-dimethyl-1-butanol and its metabolite 3,3-dimethylbutyrate ameliorate collagen-induced arthritis independent of choline trimethylamine lyase activity.

Previous studies have identified significant alterations in intestinal carnitine metabolism in mice with collagen-induced arthritis (CIA), potentially linking bacterial dysbiosis with autoimmunity. Bacterial trimethylamine (TMA) lyases metabolize dietary carnitine to TMA, which is oxidized in the liver to trimethylamine-N-oxide (TMAO). TMAO is associated with inflammatory diseases, such as atherosclerosis, whose immunologic processes mirror that of rheumatoid arthritis (RA). Therefore, we investigated the possibility of ameliorating CIA by inhibiting TMA lyase activity using 3,3-dimethyl-1-butanol (DMB) or fluoromethylcholine (FMC). During CIA, mice were treated with 1% vol/vol DMB, 100mg/kg FMC, or vehicle. DMB-treated mice demonstrated significant (>50%) reduction in arthritis severity compared to FMC and vehicle-treated mice. However, in contrast to FMC, DMB treatment did not reduce cecal TMA nor circulating TMAO concentrations. Using gas chromatography, we confirmed the effect of DMB is independent of TMA lyase inhibition. Further, we identified a novel host-derived metabolite of DMB, 3,3-dimethyl-1-butyric acid (DMBut), which also significantly reduced disease and proinflammatory cytokines in CIA mice. Altogether, our study suggests that DMB the immunomodulatory activity of DMB and/or its metabolites are protective in CIA. Elucidating its target and mechanism of action may provide new directions for RA therapeutic development.

Microbiota-dependent indole production is required for the development of collagen-induced arthritis.

Altered tryptophan catabolism has been identified in inflammatory diseases like rheumatoid arthritis (RA) and spondyloarthritis (SpA), but the causal mechanisms linking tryptophan metabolites to disease are unknown. Using the collagen-induced arthritis (CIA) model we identify alterations in tryptophan metabolism, and specifically indole, that correlate with disease. We demonstrate that both bacteria and dietary tryptophan are required for disease, and indole supplementation is sufficient to induce disease in their absence. When mice with CIA on a low-tryptophan diet were supplemented with indole, we observed significant increases in serum IL-6, TNF, and IL-1ß; splenic RORγt+CD4+ T cells and ex vivo collagen-stimulated IL-17 production; and a pattern of anti-collagen antibody isotype switching and glycosylation that corresponded with increased complement fixation. IL-23 neutralization reduced disease severity in indole-induced CIA. Finally, exposure of human colon lymphocytes to indole increased expression of genes involved in IL-17 signaling and plasma cell activation. Altogether, we propose a mechanism by which intestinal dysbiosis during inflammatory arthritis results in altered tryptophan catabolism, leading to indole stimulation of arthritis development. Blockade of indole generation may present a novel therapeutic pathway for RA and SpA.

Microbiota-dependent indole production stimulates the development of collagen-induced arthritis in mice.

Altered tryptophan catabolism has been identified in inflammatory diseases like rheumatoid arthritis (RA) and spondyloarthritis (SpA), but the causal mechanisms linking tryptophan metabolites to disease are unknown. Using the collagen-induced arthritis (CIA) model, we identified alterations in tryptophan metabolism, and specifically indole, that correlated with disease. We demonstrated that both bacteria and dietary tryptophan were required for disease and that indole supplementation was sufficient to induce disease in their absence. When mice with CIA on a low-tryptophan diet were supplemented with indole, we observed significant increases in serum IL-6, TNF, and IL-1ß; splenic RORγt+CD4+ T cells and ex vivo collagen-stimulated IL-17 production; and a pattern of anti-collagen antibody isotype switching and glycosylation that corresponded with increased complement fixation. IL-23 neutralization reduced disease severity in indole-induced CIA. Finally, exposure of human colonic lymphocytes to indole increased the expression of genes involved in IL-17 signaling and plasma cell activation. Altogether, we propose a mechanism by which intestinal dysbiosis during inflammatory arthritis results in altered tryptophan catabolism, leading to indole stimulation of arthritis development. Blockade of indole generation may present a unique therapeutic pathway for RA and SpA.

Antibody Responses to Epstein-Barr Virus in the Preclinical Period of Rheumatoid Arthritis Suggest the Presence of Increased Viral Reactivation Cycles.

OBJECTIVE: Patients with established rheumatoid arthritis (RA) demonstrate altered immune responses to Epstein-Barr virus (EBV), but the presence and roles of EBV have not been fully explored during the pre-clinical disease period. This study was undertaken to determine if EBV infection, as evidenced by an altered anti-EBV antibody response, either plays an important role in driving the development of RA or is a result of expanded RA-related autoimmunity. METHODS: A total of 83 subjects with RA according to the 1987 American College of Rheumatology (ACR) criteria and 83 age-, sex-, and race-matched control subjects without RA were included in our study. We collected sera from RA subjects and matched controls during the pre-RA and post-RA diagnosis periods and tested the sera for the presence of 5 anti-EBV antibodies (anti-EBV nuclear antigen 1 IgG isotype, anti-viral capsid antigen [anti-VCA] isotypes IgG and IgA, and anti-early antigen [EA] isotypes IgG and IgA), 7 RA-related autoantibodies (rheumatoid factor measured by nephelometry [RF-Neph] as well as isotype-specific IgA-RF, IgM-RF, and IgG-RF, and anti-cyclic citrullinated peptide [anti-CCP] antibodies, including anti-CCP2, anti-CCP3, and anti-CCP3.1), 22 cytokines/chemokines, 36 individual anti-citrullinated protein antibodies, and IgG-cytomegalovirus (CMV) antibodies. Random forest classification, mixed modeling, and joint mixed modeling were used to determine differences in anti-EBV antibody levels between RA subjects and controls. RESULTS: Random forest analysis identified the presence of preclinical EBV antibodies in the serum that differentiated RA subjects from controls without RA. Specifically, IgG-EA antibody levels were higher in RA subjects (mean ± SD 0.82 ± 0.72 international standardized ratio [ISR]) compared to controls (mean ± SD 0.49 ± 0.28 ISR). In subjects with RA, elevated serum IgG-EA levels in the preclinical period before seroconversion were significantly correlated with increased serum IgM-RF levels (P = 0.007), whereas this correlation was not seen in control subjects without RA (P = 0.15). IgG-CMV antibody levels did not differ between groups. CONCLUSION: Subjects whose serum IgG-EA antibody levels are elevated in the preclinical period will eventually develop RA, which suggests that EBV reactivation cycles are increased during the preclinical period of RA. A combination of RF and EBV reactivation may play an important role in the development of RA.

Cannabinoid Receptor 2 Agonist JWH-015 Inhibits Interleukin-1ß-Induced Inflammation in Rheumatoid Arthritis Synovial Fibroblasts and in Adjuvant Induced Arthritis Rat via Glucocorticoid Receptor.

Management of pain in the treatment of rheumatoid arthritis (RA) is a priority that is not fully addressed by the conventional therapies. In the present study, we evaluated the efficacy of cannabinoid receptor 2 (CB2) agonist JWH-015 using RA synovial fibroblasts (RASFs) obtained from patients diagnosed with RA and in a rat adjuvant-induced arthritis (AIA) model of RA. Pretreatment of human RASFs with JWH-015 (10-20 µM) markedly inhibited the ability of pro-inflammatory cytokine interleukin-1ß (IL-1ß) to induce production of IL-6 and IL-8 and cellular expression of inflammatory cyclooxygenase-2 (COX-2). JWH-015 was effective in reducing IL-1ß-induced phosphorylation of TAK1 (Thr184/187) and JNK/SAPK in human RASFs. While the knockdown of CB2 in RASFs using siRNA method reduced IL-1ß-induced inflammation, JWH-015 was still effective in eliciting its anti-inflammatory effects despite the absence of CB2, suggesting the role of non-canonical or an off-target receptor. Computational studies using molecular docking and molecular dynamics simulations showed that JWH-105 favorably binds to glucocorticoid receptor (GR) with the binding pose and interactions similar to its well-known ligand dexamethasone. Furthermore, knockdown of GR using siRNA abrogated JWH-015's ability to reduce IL-1ß-induced IL-6 and IL-8 production. In vivo, administration of JWH-015 (5 mg/kg, daily i.p. for 7 days at the onset of arthritis) significantly ameliorated AIA in rats. Pain assessment studies using von Frey method showed a marked antinociception in AIA rats treated with JWH-015. In addition, JWH-015 treatment inhibited bone destruction as evident from micro-CT scanning and bone analysis on the harvested joints and modulated serum RANKL and OPG levels. Overall, our findings suggest that CB2 agonist JWH-015 elicits anti-inflammatory effects partly through GR. This compound could further be tested as an adjunct therapy for the management of pain and tissue destruction as a non-opioid for RA.