Background/Purpose: Anti-TNF biologics are effective in the treatment of inflammatory diseases such as rheumatoid arthritis and psoriasis. However, they can lead to de novo inflammation occurrences, termed paradoxical adverse events, driven by inappropriate Th1/Th2/Th17 skewing and suboptimal function of regulatory T cells (Treg). Studies have reported that TNF blocking biologics downregulate both pro-inflammatory TNFR1 and anti-inflammatory TNFR2-mediated signaling. A TNF small molecule inhibitor that blocks pro-inflammatory TNFR1 signaling but spares beneficial TNFR2 signaling has been reported previously and may circumvent these shortcomings of biologic drugs. Herein, we describe the effects of TNF small molecule inhibitors on TNF-mediated inflammatory signaling, inhibition of TNFR1 vs TNFR2 signaling, Treg cell activity and control of murine arthritis.

Methods: NF-κB reporter-expressing HEK293 cells were used to measure cellular potency of TNF small molecule inhibitors. Soluble TNF trimer inhibition and IL-8 secretion were assessed in human umbilical vein endothelial (HUVEC) cells using ELISA and MSD assays. HeLa cells expressing a mutant non-cleavable TNF that allows expression of membrane TNF to activate TNFR2 signaling were generated. For measurement of TNFR1 and TNFR2-specific signaling, NF-κB reporter-expressing K562 cells were stimulated with soluble TNF and membrane TNF-expressing cells. Treg stability and function were evaluated using cytokine and phenotypic marker levels by flow cytometry. The collagen antibody-induced arthritis mouse model was used to demonstrate the activity of TNF signaling blockade.

Results: TNF small molecule inhibitors displayed a concentration-dependent reduction in soluble TNF/TNFR1-stimulated NF-κB activation in HEK293 cells and IL-8 production in HUVEC cells, equivalent to blockade using anti-TNF biologics. Using K562 cells pre-incubated with TNFR1-blocking antibody and co-cultured with membrane TNF-expressing cells, TNFR2-dependent NF-κB activation was retained with small molecule inhibitors but abolished using anti-TNF biologics. TNFR2 signaling has been established to promote Treg functionality in vitro. The proliferation of stable FoxP3highHelios+ Tregs, and expression of suppressive cytokines IL-10 and TGF-β was retained using TNF small molecule inhibitors but impaired using an anti-TNF biologic. In a murine collagen antibody-induced arthritis model, TNF small-molecule inhibition showed equivalent disease suppression to an anti-TNF biologic but, interestingly, anti-TNF treatment led to enhanced circulating IL-17 levels in the biologic-treated group but not with small-molecule inhibition.

Conclusion: Taken together, these data demonstrate that TNF small-molecule inhibition is effective at inhibiting pro-inflammatory signaling and providing control of murine arthritis like anti-TNF biologics. We also show that TNF small-molecule inhibition has the potential to promote Treg stability and potentially circumvent paradoxical adverse events observed following anti-TNF biologic therapy.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/tnf-inhibition-with-small-molecule-agents-results-in-amelioration-of-inflammation-similar-to-that-of-anti-tnf-biologics-while-preserving-tnfr2-signaling-and-maintaining-a-suppressive-regulatory-t-cell/