Exploring the role of S100A9 in mediating failure of anti-TNF therapy in RA

Rheumatoid arthritis (RA) is characterized by inflammation and bone destruction. Introduction of anti-TNF biologic agents have dramatically improved clinical outcome for RA. However, there are still 40-50% of patients discontinue anti-TNF agents because of treatment failure. It is essential to explore mechanism underlying failure of anti-TNF therapy and develop precision medicine-based treatment strategy.

Recently, we collected RA patients receiving an anti-TNF agent Adalimumab and classified them into responders and non-responders based on diagnostic criteria involving CRP, ESR, RF and DAS28. RNA sequencing using peripheral blood cells revealed that the non-responders had higher enrichment of neutrophils in the blood, along with increased S100A9 mRNA level, when compared to the responders. S100A9 is constitutively expressed in neutrophils and released into extracellular environment as part of neutrophil extracellular traps (NETs) under inflammatory conditions. NETs are networks of extracellular fibers composed of DNA-histone complexes and proteins. Bioinformatics analysis showed that neutrophils-related signaling pathways including NETs formation were upregulated in non-responders.

S100A9 has been reported to participate in a series of autoimmune disorders. We further revealed that sera of non-responders contained higher level of S100A9 than responders. To find out the main target cells of S100A9, we incubated several dominant effector cells in joint microenvironment, including T cells, monocytes, macrophages and fibroblast-like synoviocytes (FLSs), with recombinant human S100A9 (rhS100A9) and observed that rhS100A9 minimally induced activation of T cells and monocytes, macrophages, but dramatically promoted expression of inflammatory cytokines, chemokines, major
histocompatibility complex class (MHC) molecules in FLSs. In addition, S100A9 also significantly enhanced capacity of invasion, migration, apoptosis resistance and proliferation of FLSs, implying that FLSs was the main target cells of S100A9 released from neutrophil in RA.

These clinical findings were confirmed in collagen-induced arthritis (CIA) mouse model. We used an anti-Gr1 antibody to induce more S100A9 release from neutrophil in CIA mice and the mice developed more severe CIA. After treatment with Adalimumab, we observed the ineffectiveness to Adalimumab in these mice and boosted bone erosion. There were large amounts of co-localization between FLSs and S100A9 in the anti-Gr1 antibody-treated mice, whereas no obvious distribution of rhS100A9 in T cells and monocytes/macrophages was noticed. Besides, CIA mice were also directly injected with rhS100A9 and then administrated with Adalimumab. Similarly, we also observed the non-response to Adalimumab in these mice. These results lead us to hypothesize that the aberrant S100A9 targeting FLSs was a causal factor for the non-response to anti-TNF therapy and inhibition of S100A9 could reverse failure of anti-TNF therapy in RA.

Aptamers are RNA or ssDNA, which specifically bind to targets. We screened an aptamer APT012, which bound with S100A9 with high affinity and inhibited S100A9-induced expression of inflammatory cytokines, chemokines, major histocompatibility complex class (MHC) molecules and tumor-like phenotypes of FLSs in vitro. To examine our above hypothesis, we will examine the following objectives:
(1) to explore the mechanism of S100A9 in mediating activation of FLSs;
(2) to test whether the S100A9-specific aptamer APT012 could reverse failure of anti-TNF therapy in CIA mice injected with an anti-Gr1 antibody;
(3) to reveal why S100A9 and NETs was upregulated in RA patients who were non-responders to anti-TNF therapy.

This study will demonstrate S100A9 as a valuable diagnostic marker and therapeutic target for RA patients who do not response to anti-TNF therapy and propose a precision medicine-based therapeutic strategy.