Background/Purpose: During trained immunity, monocytes and macrophages undergo a functional and transcriptional reprogramming toward activation, which is induced by a priming stimulus and results in enhanced responsiveness to subsequent triggers. We aimed to investigate if citrullinated vimentin (c-vimentin), a damage-associated pattern in rheumatoid arthritis (RA), induces trained immunity in vitro in healthy individuals.

Methods: Monocytes isolated from the peripheral blood (EDTA blood, n=22; buffy coats, n=6) of healthy donors by Ficoll-paque centrifugation and negative selection, were stimulated with c-vimentin (0.1 μg/ml) for 24h and re-stimulated 5 days later with the lipopolysaccharide of E.coli (LPS) (10 ng/ml). Protein release (ELISA, Western blot) and gene expression levels (RNA sequencing) were measured. Chromatin accessibility changes were assessed by ATAC sequencing. The methylation of histone H3 at lysine 4 (H3K4) was examined by chromatin immunoprecipitation. The ligand-receptor glycocapture technology was used to identify candidate cell surface targets of c-vimentin.

Results: Priming with c-vimentin induced training in human monocytes, as suggested by the significantly increased levels of secreted interleukin-6 (IL-6), the chemokine CXCL1 and CCL20/Macrophage Inflammatory Protein 3a, upon restimulation with LPS (1.29-2.32-fold increase, n=22, all p< 0.001). A significant increase in proinflammatory cytokine and chemokine expression was also shown in RNA sequencing (n=4), beside up-regulation of genes related to glucose and lipid metabolism as well as epigenetic effectors. Exposition to c-vimentin induced chromatin rearrangement (n=4), with increased proportion of accessible promoters (14.4% on day 6 vs. 2.4% on day 1), and a positive correlation to gene expression (r=0.35). Besides, c-vimentin induced H3K4 methylation with increased levels of this mark in the promoter of the IL-6 gene (n=6, p=0.020). Interestingly, we observed an upregulation in the expression of histone methyltransferase PRDM8 (37-fold, SD +/-10.8). At the same time, by inhibiting histone methyltransferases with methylthioadenosine (1 mM), trained immunity was reversed (8.43-fold decrease in IL-6 release, n=6, p=0.031). A shift in metabolism was supported by high lactate production measured by ELISA in the trained cells (n=9, p=0.004), while by inhibiting the metabolic pathway of glycolysis with 2-deoxyglucose (11 mM), the induction of trained immunity could be counteracted (5.32-fold decrease in IL-6 release, n=6, p=0.030). Finally, STING was identified as cell surface receptor for the ligand c-vimentin. Indeed, c-vimentin induced activation of TBK1, which is implicated in the STING signaling pathway, by phosphorylation, while STING inhibition with the covalent small molecule H151 (2 μM) abolished this effect, and decreased IL-6 release (1.61-fold decrease, n=5, p=0.051).

Conclusion: C-vimentin induces epigenetic and metabolic changes in monocytes, probably through a STING and TBK1-dependent activation, resulting in enhanced cytokine and chemokine production upon restimulation. Inhibition of the STING signaling pathway may be a novel therapeutic target against myeloid activation in RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/citrullinated-vimentin-induces-epigenetic-memory-of-the-innate-immune-system/