MyD88 dimerization inhibitor ST2825 targets the aggressiveness of synovial fibroblasts in rheumatoid arthritis patients.

BACKGROUND: Dimerization of the myeloid differentiation primary response 88 protein (MyD88) plays a pivotal role in the exacerbated response to innate immunity-dependent signaling in rheumatoid arthritis (RA). ST2825 is a highly specific inhibitor of MyD88 dimerization, previously shown to inhibit the pro-inflammatory gene expression in peripheral blood mononuclear cells from RA patients (RA PBMC). In this study, we elucidated the effect of disrupting MyD88 dimerization by ST2825 on the pathological properties of synovial fibroblasts from RA patients (RA SFs). METHODS: RA SFs were treated with varying concentrations of ST2825 in the presence or absence of bacterial lipopolysaccharides (LPS) to activate innate immunity-dependent TLR signaling. The DNA content of the RA SFs was quantified by imaging cytometry to investigate the effect of ST2825 on different phases of the cell cycle and apoptosis. RNA-seq was used to assess the global response of the RA SF toward ST2825. The invasiveness of RA SFs in Matrigel matrices was measured in organoid cultures. SFs from osteoarthritis (OA SFs) patients and healthy dermal fibroblasts were used as controls. RESULTS: ST2825 reduced the proliferation of SFs by arresting the cells in the G0/G1 phase of the cell cycle. In support of this finding, transcriptomic analysis by RNA-seq showed that ST2825 may have induced cell cycle arrest by primarily inhibiting the expression of critical cell cycle regulators Cyclin E2 and members of the E2F family transcription factors. Concurrently, ST2825 also downregulated the genes encoding for pain, inflammation, and joint catabolism mediators while upregulating the genes required for the translocation of nuclear proteins into the mitochondria and members of the mitochondrial respiratory complex 1. Finally, we demonstrated that ST2825 inhibited the invasiveness of RA SFs, by showing decreased migration of LPS-treated RA SFs in spheroid cultures. CONCLUSIONS: The pathological properties of the RA SFs, in terms of their aberrant proliferation, increased invasiveness, upregulation of pain and inflammation mediators, and disruption of mitochondrial homeostasis, were attenuated by ST2825 treatment. Taken together with the previously reported anti-inflammatory effects of ST2825 in RA PBMC, this study strongly suggests that targeting MyD88 dimerization could mitigate both systemic and synovial pathologies in a variety of inflammatory arthritic diseases.

Targeting inflammasome-dependent mechanisms as an emerging pharmacological approach for osteoarthritis therapy.

Arthritic diseases have attracted enormous scientific interest because of increased worldwide prevalence and represent a significant socioeconomic burden. Osteoarthritis (OA) is the most prevalent form of arthritis. It is a disorder of the diarthrodial joints, characterized by degeneration and loss of articular cartilage associated with adjacent subchondral bone changes. Chronic and unresolving inflammation has been identified as a critical factor driving joint degeneration and pain in OA. Despite numerous attempts at therapeutic intervention, no effective disease-modifying agents targeting OA inflammation are available to the patients. Inflammasomes are protein complexes known to play a critical role in the inflammatory pathology of several diseases, and their roles in OA pathogenesis have become evident over the last decade. In this sense, it is relevant to evaluate the vital role of inflammasomes as potential modulators of pathogenic features in OA. This review will provide an overview and perspectives on why understanding inflammasome activation is critical for identifying effective OA therapies. We elaborate on the contribution of extracellular mediators from the circulatory system and synovial fluid as well as intracellular activators within the synovial fibroblasts and articular chondrocytes toward invoking the inflammasome in OA. We further discuss the merits of emerging inflammasome targeting therapies and speculate on the potential strategies for inflammasome blockade for OA therapy.

SOX4 and RELA Function as Transcriptional Partners to Regulate the Expression of TNF- Responsive Genes in Fibroblast-Like Synoviocytes.

SOX4 belongs to the group C of the SOX transcription factor family. It is a critical mediator of tumor necrosis factor alpha (TNF)-induced transformation of fibroblast-like s-ynoviocytes (FLS) in arthritis. In this study we investigated the genome wide association between the DNA binding and transcriptional activities of SOX4 and the NF-kappaB signaling transcription factor RELA/p65 downstream of TNF signaling. We used ChIP-seq assays in mouse FLS to compare the global DNA binding profiles of SOX4 and RELA. RNA-seq of TNF-induced wildtype and SoxC-knockout FLS was used to identify the SOX4-dependent and independent aspects of the TNF-regulated transcriptome. We found that SOX4 and RELA physically interact with each other on the chromatin. Interestingly, ChIP-seq assays revealed that 70.4% of SOX4 peak summits were within 50bp of the RELA peak summits suggesting that both proteins bind in close-proximity on regulatory sequences, enabling them to co-operatively regulate gene expression. By integrating the ChIP-seq results with RNA-seq from SoxC-knockout FLS we identified a set of TNF-responsive genes that are targets of the RELA-SOX4 transcriptional complex. These TNF-responsive and RELA-SOX4-depenedent genes included inflammation mediators, histone remodeling enzymes and components of the AP-1 signaling pathway. We also identified an autoregulatory mode of SoxC gene expression that involves a TNF-mediated switch from RELA binding to SOX4 binding in the 3' UTR of Sox4 and Sox11 genes. In conclusion, our results show that SOX4 and RELA together orchestrate a multimodal regulation of gene expression downstream of TNF signaling. Their interdependent activities play a pivotal role in the transformation of FLS in arthritis and in the inflammatory pathology of diverse tissues where RELA and SOX4 are co-expressed.

Targeting MyD88 Downregulates Inflammatory Mediators and Pathogenic Processes in PBMC From DMARDs-Naïve Rheumatoid Arthritis Patients.

MyD88-dependent intracellular signalling cascades and subsequently NF-kappaB-mediated transcription lead to the dynamic inflammatory processes underlying the pathogenesis of rheumatoid arthritis (RA) and related autoimmune diseases. This study aimed to identify the effect of the MyD88 dimerization inhibitor, ST2825, as a modulator of pathogenic gene expression signatures and systemic inflammation in disease-modifying antirheumatic drugs (DMARDs)-naïve RA patients. We analyzed bulk RNA-seq from peripheral blood mononuclear cells (PBMC) in DMARDs-naïve RA patients after stimulation with LPS and IL-1ß. The transcriptional profiles of ST2825-treated PBMC were analyzed to identify its therapeutic potential. Ingenuity Pathway Analysis was implemented to identify downregulated pathogenic processes. Our analysis revealed 631 differentially expressed genes between DMARDs-naïve RA patients before and after ST2825 treatment. ST2825-treated RA PBMC exhibited a gene expression signature similar to that of healthy controls PBMC by downregulating the expression of proinflammatory cytokines, chemokines and matrix metalloproteases. In addition, B cell receptor, IL-17 and IL-15 signalling were critically downregulated pathways by ST2825. Furthermore, we identified eight genes (MMP9, CXCL9, MZB1, FUT7, TGM2, IGLV1-51, LINC01010, and CDK1) involved in pathogenic processes that ST2825 can potentially inhibit in distinct cell types within the RA synovium. Overall, our findings indicate that targeting MyD88 effectively downregulates systemic inflammatory mediators and modulates the pathogenic processes in PBMC from DMARDs-naïve RA patients. ST2825 could also potentially inhibit upregulated genes in the RA synovium, preventing synovitis and joint degeneration.

Chronic exposure to TNF reprograms cell signaling pathways in fibroblast-like synoviocytes by establishing long-term inflammatory memory.

Fibroblast-like synoviocytes (FLS) play a critical role in the pathogenesis of rheumatoid arthritis (RA). Chronic inflammation induces transcriptomic and epigenetic modifications that imparts a persistent catabolic phenotype to the FLS, despite their dissociation from the inflammatory environment. We analyzed high throughput gene expression and chromatin accessibility data from human and mouse FLS from our and other studies available on public repositories, with the goal of identifying the persistently reprogrammed signaling pathways driven by chronic inflammation. We found that the gene expression changes induced by short-term tumor necrosis factor-alpha (TNF) treatment were largely sustained in the FLS exposed to chronic inflammation. These changes that included both activation and repression of gene expression, were accompanied by the remodeling of chromatin accessibility. The sustained activated genes (SAGs) included established pro-inflammatory signaling components known to act at multiple levels of NF-kappaB, STAT and AP-1 signaling cascades. Interestingly, the sustained repressed genes (SRGs) included critical mediators and targets of the BMP signaling pathway. We thus identified sustained repression of BMP signaling as a unique constituent of the long-term inflammatory memory induced by chronic inflammation. We postulate that simultaneous targeting of these activated and repressed signaling pathways may be necessary to combat RA persistence.

Downregulation of Inflammatory Cytokine Release from IL-1ß and LPS-Stimulated PBMC Orchestrated by ST2825, a MyD88 Dimerisation Inhibitor.

The inflammatory process implicates homeostasis disruption and increased production of inflammatory mediators. Myeloid differentiation primary response 88 (MyD88) is an essential protein recruited after lipopolysaccharide (LPS) and interleukin (IL)-1ß stimulation, a process that converges in nuclear factor kappa B (NF-κB) activation, as well as a transcription of several genes of both pro- and anti-inflammatory cytokines. The inhibition of MyD88 has shown efficacy by decrease inflammatory response, and has demonstrated potential application as a therapeutic target in chronic diseases. In this study, we investigate the effect of MyD88 dimerisation inhibitor ST2825 on cytokine production from rhIL-1ß and LPS-stimulated peripheral blood mononuclear cells (PBMC) from healthy blood donors (HBD). ST2825 significantly downregulates the production of IFN-γ, IL-6, IL-12, IL-2, IL-15, IL-7, VEGF, IL-1Ra, IL-4, IL-5, IL-13 and IL-9 (p < 0.05) in LPS-stimulated PBMC. Moreover, ST2825 had a relatively low impact on IL-1ß signalling pathway inhibition, showing that only a few specific cytokines, such as IFN-γ and IL-1Ra, are inhibited in rhIL-1ß-stimulated PBMC (p < 0.01). In conclusion, MyD88 dimerisation inhibitor ST2825 showed high efficacy by inhibiting pro- and anti-inflammatory cytokine production in LPS-stimulated PBMC. Moreover, although rhIL-1ß induced a sustained cytokine production (p < 0.05), ST2825 did not show a significant effect in the secretion of neither pro- nor anti-inflammatory cytokines in rhIL-1ß-stimulated PBMC.