The S100A8/A9-NLRP3 inflammasome axis in HFpEF development

Abstract Background Heart failure with preserved ejection fraction (HFpEF) develops in an outside-in mechanism where comorbidities drive systemic low-grade inflammation damaging the heart. The innate immunity member S100A8/A9 is increased in comorbidities underlying HFpEF. Though, its involvement in the pathogenesis of HFpEF has not been studied before. Purpose Therefore, the objective of this study was to determine the role of S100A8/A9 and its downstream signaling via the NLRP3 inflammasome in HFpEF across patients, porcine and mouse models. Methods Serum S100A8/A9 levels were measured in HFpEF patients and controls from the SFB 1470 HFpEF and TYPE-HF cohort, respectively. Experimental HFpEF was induced in female 13–14-months-old Göttingen mini pigs via a western diet and DOCA implant for 5 months, whereas controls received a standard diet. Blood was collected and peripheral blood mononuclear cells (PBMCs) isolated for subsequent flow cytometry analyses. The 2-hit HFpEF mouse model, induced by a high-fat diet (HFD) and L-NAME, was applied to male C57BL6 mice for 15 weeks in both wild-type (WT) and S100A9 knockout (-/-) mice. In a separate experiment, HFD+L-NAME-treated mice were administered the S100A9 inhibitor Paquinimod or vehicle during the final 4 weeks. Blood, bone marrow, spleens, adipose tissues and hearts were collected for subsequent analyses. Results HFpEF patients (HFA-PEFF score ≥ 5) exhibited higher S100A8/A9 serum levels compared to control patients. Female HFpEF pigs also showed elevated S100A8/A9 levels accompanied by higher percentages of S100A9+, NLRP3+ and IL-1ß+ PBMCs compared to controls. Additionally, monocyte (CD68+) and neutrophil (Ly6G+) populations were expanded in the blood. HFD+L-NAME mice depicted increased systemic and cardiac inflammation evident – among others - by elevated C-reactive protein levels and higher S100A9 protein content in the heart compared to healthy mice. Cardiac fibroblasts isolated from HFD+L-NAME WT mice had a proinflammatory phenotype by expressing more NLRP3, IL-1ß and alpha-SMA compared to fibroblasts from control WT mice. Left ventricular diastolic dysfunction was less pronounced in S100A9-/- mice subjected to HFD+L-NAME compared with their WT littermates. This improvement was accompanied by reduced cardiomyocyte passive force, restored titin phosphorylation, and decreased NLRP3+ and IL-1ß+ cells in the heart and spleen. Paquinimod treatment attenuated the abundance of NLRP3+ and IL-1ß+ cell populations in the heart and extra-cardiac organs of HFD+L-NAME mice. It also largely restored total titin and side-specific titin phosphorylation, thereby alleviating cardiomyocyte stiffness. Conclusions These findings support an important role of S100A8/A9 and downstream NLRP3/IL-1ß signaling in the inflammatory phenotype of HFpEF on a systemic, organ and cellular level.