Increased M1 Macrophages Infiltration Is Associated with Thrombogenesis in Rheumatic Mitral Stenosis Patients with Atrial Fibrillation

Abstract

Atrial fibrillation (AF) is the most common arrhythmia. In patients with AF, the role of macrophage subsets in thrombogenesis is unclear. In the present study, we analyzed the role of M1 and M2 macrophages and related cytokines in thrombogenesis of AF. Immunohistochemistry, Western blot, and TUNEL assay were used to detect M1/M2 macrophage infiltration, the expression pattern of IL-1β and inflammasome components, and apoptosis of cardiomyocytes in 71 specimens obtained from the left atrial appendage of patients with rheumatic mitral stenosis (MS) with or without thrombosis. We demonstrated that proinflammatory M1 macrophages were predominant in the atrium of MS patients with AF and thrombus. NLRP3 inflammasomes and IL-1β, which are primarily functional in macrophages, were activated in those patients. We also showed that increased cell death was associated with thrombogenesis in MS patients. These data indicate that infiltration of M1 macrophages and over-activation of NLRP3 inflammasomes may play a role in progressive atrial inflammation and thrombogenesis in rheumatic mitral stenosis patients with AF.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. Immunostaining of monoclonal antibodies with anti-HLA-DR for M1 and anti-CD163 for M2 cells.

(A-F) M1 cells were predominantly increased and localized in the atrial endothelium of patients with thrombus formation. (G-I) M2 cells were similar in all the three groups.

Fig 2. Immunoblot of inflammasome components in lysates of atrium.

(A) Representative plot showing expression of NLRP3, pro- and cleaved caspase-1, pro- and cleaved IL-1β. GAPDH represents the loading control. (B-F) Semiquantitative analysis of inflammasome expression; NLRP3, pro- and cleaved caspase-1, and pro- and cleaved IL-1β expressions were significantly increased in patients with AF and thrombosis. * denotes P < 0.05, ** denotes P < 0.01, *** denotes P < 0.0001.

Fig 3. Increased cardiomyocyte death and thrombogenesis in MS patients.

(A) TUNEL-positive cells are primarily located in the atrial endothelium. (arrowhead) (B) Frequency of apoptotic cells in the atrium of MS patients as indicated by TUNEL-positive cells per 100 DAPI in three groups. ** denotes P < 0.01.