Background/Purpose: Deregulation of Toll-Like Receptor 4 (TLR4) signaling is thought to play a role in the pathogenesis of certain autoimmune diseases. In rheumatoid arthritis (RA), increased expression of several endogenous TLR4 ligands has been reported in both blood and synovial fluids. We first confirmed that macrophages can be activated by immune complexes containing citrullinated fibrinogen (cFb-IC) via TLR4 and then demonstrated that this activation is inhibited by NI-0101, a new therapeutic anti-human TLR4 monoclonal antibody (mAb). The purpose of this study was 1) to investigate the ability of RA synovial fluids to induce cytokine production by fibroblasts and monocytes obtained from RA patients, 2) to analyze its relation with levels of endogenous TLR4 ligands and finally 3) to demonstrate the ability of NI-0101 to inhibit RA synovial fluids induced cytokine production.

Methods: cFb-IC were incubated with blood derived macrophages from healthy subjects and cytokine release determined by ELISA. The capacity of pooled and individual RA synovial fluids to stimulated cytokine production from RA blood-derived monocytes and RA joint-derived fibroblasts was analyzed by ELISA and multiplex analyses. The expression levels of S100A8/A9, High-mobility group protein B1 (HMGB1), tenascin C were measured by commercially available ELISA kits and anti-citrullinated protein antibodies (ACPA) were measured in RA synovial fluid samples by ELISA method developed for purpose. TLR4 antagonism was assessed using NI-0101.

Results: cFb-IC stimulated TNFα production in blood-derived macrophages was significantly inhibited by NI-0101 (p<0.001), confirming that the ability of cFb-IC to induce cytokine production is realized through TLR4-activation. Eleven out of fifteen individual RA synovial fluids stimulated IL-6 production from patient-derived synovial fibroblasts and 10 out of 14 from monocytes. NI-0101 significantly reduced the individual RA synovial fluids induced response (5/11 RA synovial fluids on fibroblasts and 10/10 RA synovial fluids on monocytes).  Analysis of RA synovial fluids composition demonstrated variable patterns of ACPA, S100A8/A9, High-mobility group protein B1 (HMGB1), tenascin C and cFb-IC levels.  These synovial fluids containing the highest combined level of ACPA, cFb-IC, HMBG1, S100A8/A9 were found to induce the more robust IL-6 response. Furthermore, the NI-0101 inhibitory response of IL-6 induction was also correlated with the combined presence of the afore mentioned TLR4 ligands.

Conclusion: The ability of RA synovial fluids to induce ex vivo inflammatory cytokine production is directly correlated to the levels of endogenous TLR4 ligands in RA synovial fluids (including S100A8/A9, HMGB1, ACPA and cFb-IC). NI-0101 effectively abrogates RA synovial fluids induced cytokine release in those samples with high levels of endogenous TLR4 ligands.  These results indicate that TLR4 blockade by NI-0101, currently in late Phase I of clinical development, is a promising strategy in RA treatment and endogenous TLR4 ligand levels might be used as a biomarker to identify patients for anti-TLR4 therapy.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/pathogenic-pro-inflammatory-cytokine-production-induced-by-synovial-fluid-from-ra-patients-is-related-to-levels-of-endogenous-tlr4-ligands-and-is-blocked-by-a-novel-therapeutic-anti-human-tlr4-monoclo/