Background/Purpose: Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by monocyte (MN) infiltration into the synovium. Citrullination is a post-translational modification of arginine to citrulline mediated by peptidyl arginine deiminase (PAD). Previously, we have shown that citrullination converts epithelial-derived neutrophil-activating peptide 78 (ENA-78/CXCL5) from a neutrophil recruiter to a MN recruiter. In this study, we investigated the signaling pathways involved in MN recruitment by citrullinated ENA-78/CXCL5 (citENA-78/CXCL5).

Methods: Recombinant human (rh) ENA-78/CXCL5 was citrullinated by incubation with rhPAD enzyme. To confirm the citrullination sites, citENA-78/CXCL5 was analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). To determine the signaling pathways involved, MN chemotaxis assay was performed with citENA-78/CXCL5 in modified Boyden chambers with chemical signaling inhibitors. MNs were also transfected with JNK silencing RNA (siRNA). Three mutants (R45K, R48K and R45/48K) of rhENA-78/CXCL5 were generated by transmutation of arginines to lysines and were used in MN chemotaxis assays. To assess citENA-78/CXCL5-induced MN migration in vivo, a mouse subcutaneous air pouch model was used. The exudates from the air pouch were collected after 24 hours and immunofluorescence was performed to detect MNs/macrophages using anti-F4/80 antibody. MNs were stimulated with citENA-78/CXCL5 for 15 minutes and Western blots were performed to evaluate phosphorylation of signaling molecules. We also assessed the crosstalk between the signaling pathways after incubating with chemical signaling inhibitors before citENA-78/CXCL5 stimulation.

Results: LC-MS/MS analysis confirmed citrullination of citENA-78/CXCL5. CitENA-78/CXCL5-induced MN migration was significantly reduced by JNK and NFκB inhibitors, but not by inhibitors of Src, p38, and Erk1/2. JNK siRNA-transfected MNs displayed decreased citENA-78/CXCL5-induced MN migration compared to control transfected MNs. CitR45K and citR48K mutations significantly reduced MN chemotaxis, suggesting that citENA-78/CXCL5-induced MN migration is dependent, at least partially, on both citrulline residues. The mouse air pouch model showed marked MN recruitment in response to citENA-78/CXCL5 compared to the controls, suggesting that citENA-78/CXCL5 increases MN ingress in vivo. CitENA-78/CXCL5-stimulated MNs showed increased expression of phosphorylated-JNK and phosphorylated-NFκB (p-NFκB) which was inhibited by signaling inhibitors. Moreover, a JNK inhibitor reduced the level of p-NFκB, suggesting that JNK is upstream of NFκB in citENA-78/CXCL5-stimulated MNs.

Conclusion: We found that citENA-78/CXCL5 induces MN migration via JNK and NFκB signaling pathways. MN chemotaxis induced by citENA-78/CXCL5 is partially dependent on both citrulline residues. CitENA-78/CXCL5 plays an important role in MN migration in vivo in the air pouch inflammatory model. Our data suggest that citENA-78/CXCL5 induces JNK and NFκB signaling pathways with JNK upstream of NFκB. Targeting citENA-78/CXCL5 and its signaling pathways may be a novel approach to treat MN-dependent diseases.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/citrullinated-epithelial-derived-neutrophil-activating-peptide-78-ena-78cxcl5-induces-monocyte-migration-via-jnk-and-nfb-signaling-pathways/