Background: 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. Monocytes from patients with rheumatoid arthritis (RA) display features consistent with a trained immunity phenotype. Citrullinated proteins as citrullinated vimentin (c-vimentin), which function as damage-associated patterns in RA, may be implicated in the process of trained immunity.
Objectives: We aimed to investigate if c-vimentin induces trained immunity in vitro in healthy individuals.
Methods: Monocytes were isolated from the peripheral blood (EDTA blood, n=22; buffy coats, n=6) from healthy donors by Ficoll-paque centrifugation and negative selection using CD3/CD19/CD56 magnetic beads. The cells 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 as well as lactate release were estimated in cell culture supernatants at day 6 by ELISA. RT-PCR and/or Western Blotting were applied to measure mRNA and/or protein expression. The Ligand-receptor glycocapture technology LRC-TRiCEPS was used to identify candidate cell surface targets of c-vimentin. The methylation of histone H3 at lysine 4 (H3K4) was examined by chromatin immunoprecipitation.
Results: Priming with citrullinated vimentin induced training in human monocytes, as suggested by the significantly increased levels of secreted interleukin-6 (IL-6), upon restimulation with LPS (1.29-fold increase, n=22, p<0.001). Likewise, the release of chemokines CXCL1 and CCL20/Macrophage Inflammatory Protein 3a was significantly increased (1.81-fold and 2.32-fold increase, respectively, n=14, both p<0.001). LRC-TRiCEPS enabled the identification of STING 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 (2μM) abolished this effect. Besides, H151 inhibited trained immunity by decreasing IL-6 release and expression (1.61-fold and 1.93-fold decrease, respectively, n=5). Trained monocytes also displayed high lactate production (primed vs. unprimed cells, n=9, p=0.004), reflecting a shift in metabolism with an increase in glycolysis. By inhibiting the metabolic pathway of glycolysis by 2-deoxyglucose (11mM), the induction of trained immunity could be counteracted (5.32-fold decrease in IL-6 release, n=7, p=0.016). Finally, c-vimentin induced H3K4 methylation with increased levels of this mark in the promoter of the IL-6 gene. By modulating the function of epigenetic enzymes with methylthioadenosine (1mM), which specifically inhibits histone methyltransferase, trained immunity was reversed (8.43-fold decrease in IL-6 release, n=6, p=0.031).
Conclusion: Citrullinated vimentin induces epigenetic modifications 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 for myeloid activation in RA.
Disclosure of Interests: None declared
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