Gut Microbiota Metabolites and Neutrophil Signaling in the Pathogenesis of Calcific Aortic Valve Disease

Abstract

Calcific aortic valve disease (CAVD) is a progressive and increasingly common disorder for which no disease-modifying drugs are currently available (Zheng et al., 2020). Although inflammation and calcification driven by macrophages, lymphocytes, and valvular interstitial cells are well documented, the roles of neutrophils and the impact of gut microbiota-derived metabolites on these cells remain incompletely understood (Akahori et al., 2018; Li et al., 2019; Raddatz et al., 2019). This study addresses this gap by exploring how short-chain fatty acids (SCFAs) generated by intestinal bacteria regulate neutrophil function relevant to CAVD pathogenesis.<br/> The first hypothesis was that butyrate and propionate attenuate neutrophil inflammatory activation. It was then determined whether SCFAs suppressed the expression of mediators by neutrophils that promote endothelial-to-mesenchymal transition (EndMT), fibrosis, calcification, and angiogenesis. Ultimately, it was crucial to ascertain whether neutrophils from patients with severe CAVD exhibited an exaggerated pro-inflammatory, pro-fibrotic, and pro-calcific activation state that could be mitigated by ex vivo SCFA treatment. <br/> Optimized non-cytotoxic SCFA concentrations were applied to LPS-stimulated MOLM-20 cells and primary human neutrophils from healthy donors. SCFAs reduced the relative gene expression and protein levels of canonical cytokines (TNF, IL1B, ICAM1, and CXCL8), while simultaneously downregulating the osteogenic marker BMP2 and pro-fibrotic mediators (TGFB1 and MMP9). Thus, one metabolic stimulus curtailed both the pro-inflammatory and osteoinductive signaling pathways in neutrophils. <br/> Neutrophils from patients with severe CAVD showed elevated basal expression of inflammatory and osteogenic genes compared with those from healthy individuals and those with coronary artery disease. The levels of pro-inflammatory cytokines and osteogenic gene expression increased progressively from healthy controls to CAD, then to CAVD, and finally to CAD + CAVD, indicating the additive effects of both conditions. <br/> The convergent transcriptional signature suggests that SCFAs reprogram neutrophils, leading to a decreased release of signals that promote inflammation, EndMT, matrix remodeling, and valve calcification. Although the current study did not establish a direct causal link between neutrophil signaling and calcification, it outlines a plausible mechanistic connection that warrants further testing. <br/> This thesis demonstrates that gut microbiota-derived SCFAs significantly reduce neutrophil inflammatory responses and osteogenic programming. Positioning the gut-neutrophil axis as a modifiable factor in CAVD, these findings open new pathways for pharmacological intervention in valvular heart disease.