Redundant role of PAD2 and PAD4 in the development of cardiovascular lesions in a mouse model of Kawasaki disease vasculitis

Abstract Kawasaki disease (KD) is the leading cause of acquired heart disease in children. While circulating neutrophils are increased and activated during acute KD, it is unclear whether neutrophils and neutrophil extracellular traps (NETs) contribute to the pathogenesis of KD. Peptidylarginine deiminase 4 (PAD4), an enzyme involved in protein citrullination and essential for NETs formation, is implicated in the pathogenesis of various diseases. Here, we used the Lactobacillus casei cell wall extract (LCWE)-induced mouse model of KD vasculitis to determine the contribution of PAD4 in KD vasculitis. We found that the pan-PADs inhibitor, Cl-amidine, significantly reduced LCWE-induced cardiovascular lesions, but neutrophil-specific Padi4 KO mice did not impact the development of KD vasculitis. While in vitro treatment of macrophages, which highly express Padi4, with Cl-amidine inhibited IL-1β secretion, macrophage-specific Padi4 KO mice did not reduce the lesions. Padi4−/− mice also developed KD vasculitis, AFM30a, a PAD2 inhibitor, significantly reduced KD vasculitis in Padi4−/− mice, indicating a compensatory role of PAD2 in PAD4 deficiency. We also identified several citrullinated proteins in macrophages with constitutively active NLRP3 inflammasome that were inhibited by Cl-amidine treatment, suggesting that protein citrullination participates in NLRP3 inflammasome activation. These data indicate a dispensable role for PAD4-dependent NETs formation, and a redundant role of PAD2 and PAD4 in this murine KD vasculitis. The cardioprotective effects of Cl-amidine to reduce the severity of murine KD vasculitis are not limited to PAD4 inhibition and may include decreased citrullination in the inflammasome pathway.