Prothymosin Alpha: A Novel Contributor to Estradiol Receptor Alpha-Mediated CD8+T-Cell Pathogenic Responses and Recognition of Type 1 Collagen in Rheumatic Heart Valve Disease

Abstract

Background: Rheumatic heart valve disease (RHVD) is a leading cause of cardiovascular death in low-and middle-income countries and affects predominantly women. The underlying mechanisms of chronic valvular damage remain unexplored and regulators of sex predisposition are unknown. Methods: Proteomics analysis of human heart valves (nondiseased aortic valves, nondiseased mitral valves [NDMVs], valves from patients with rheumatic aortic valve disease, and valves from patients with rheumatic mitral valve disease; n=30) followed by system biology analysis identified ProTa (prothymosin alpha) as a protein associated with RHVD. Histology, multiparameter flow cytometry, and enzyme-linked immunosorbent assay confirmed the expression of ProTa. In vitro experiments using peripheral mononuclear cells and valvular interstitial cells were performed using multiparameter flow cytometry and quantitative polymerase chain reaction. In silico analysis of the RHVD and Streptococcus pyogenes proteomes were used to identify mimic epitopes. Results: A comparison of NDMV and nondiseased aortic valve proteomes established the baseline differences between nondiseased aortic and mitral valves. Thirteen unique proteins were enriched in NDMVs. Comparison of NDMVs versus valves from patients with rheumatic mitral valve disease and nondiseased aortic valves versus valves from patients with rheumatic aortic valve disease identified 213 proteins enriched in rheumatic valves. The expression of the 13 NDMV-enriched proteins was evaluated across the 213 proteins enriched in diseased valves, resulting in the discovery of ProTa common to valves from patients with rheumatic mitral valve disease and valves from patients with rheumatic aortic valve disease. ProTa plasma levels were significantly higher in patients with RHVD than in healthy individuals. Immunoreactive ProTa colocalized with CD8+T cells in RHVD. Expression of ProTa and estrogen receptor alpha correlated strongly in circulating CD8+T cells from patients with RHVD. Recombinant ProTa induced expression of the lytic proteins perforin and granzyme B by CD8+T cells as well as higher estrogen receptor alpha expression. In addition, recombinant ProTa increased human leukocyte antigen class I levels in valvular interstitial cells. Treatment of CD8+T cells with specific estrogen receptor alpha antagonist reduced the cytotoxic potential promoted by ProTa. In silico analysis of RHVD and S pyogenes proteomes revealed molecular mimicry between human type 1 collagen epitope and bacterial collagen-like protein, which induced CD8+T-cell activation in vitro. Conclusions: ProTa-dependent CD8+T-cell cytotoxicity was associated with estrogen receptor alpha activity, implicating ProTa as a potential regulator of sex predisposition in RHVD. ProTa facilitated recognition of type 1 collagen mimic epitopes by CD8+T cells, suggesting mechanisms provoking autoimmunity.