Cardiac fibroblast cytokine profiles induced by proinflammatory or profibrotic stimuli promote monocyte recruitment and modulate macrophage M1/M2 balance in vitro

Abstract

Macrophage polarization plays an essential role in cardiac remodeling after injury, evolving from an initial accumulation of proinflammatory M1 macrophages to a greater balance of anti-inflammatory M2 macrophages. Whether cardiac fibroblasts themselves influence this process remains an intriguing question. In this work, we present evidence for a role of cardiac fibroblasts (CF) as regulators of macrophage recruitment and skewing. Adult rat CF, were treated with lipopolysaccharide (LPS) or TGF-β1, to evaluate ICAM-1 and VCAM-1 expression using Western blot and proinflammatory/profibrotic cytokine secretion using LUMINEX. We performed in vitro migration and adhesion assays of rat spleen monocytes to layers of TGF-β1- or LPS-pretreated CF. Finally, TGF-β1- or LPS-pretreated CF were co-cultured with monocyte, to evaluate their effects on macrophage polarization, using flow cytometry and cytokine secretion.There was a significant increase in monocyte adhesion to LPS- or TGF-β1-stimulated CF, associated with increased CF expression of ICAM-1 and VCAM-1. siRNA silencing of either ICAM-1 or VCAM-1 inhibited monocyte adhesion to LPS-pretreated CF; however, monocyte adhesion to TGF-β1-treated CF was dependent on only VCAM-1 expression. Pretreatment of CF with LPS or TGF-β1 increased monocyte migration to CF, and this effect was completely abolished with an MCP-1 antibody blockade. LPS-treated CF secreted elevated levels of TNF-α and MCP-1, and when co-cultured with monocyte, LPS-treated CF stimulated increased macrophage M1 polarization and secretion of proinflammatory cytokines (TNF-α, IL-12 and MCP-1). On the other hand, CF stimulated with TGF-β1 produced an anti-inflammatory cytokine profile (high IL-10 and IL-5, low TNF-α). When co-cultured with monocytes, the TGF-β1 stimulated fibroblasts skewed monocyte differentiation towards M2 macrophages accompanied by increased IL-10 and decreased IL-12 levels. Taken together, our results show for the first time that CF can recruit monocytes (via MCP-1-mediated chemotaxis and adhesion to ICAM-1/VCAM-1) and induce their differentiation to M1 or M2 macrophages (through the CF cytokine profile induced by proinflammatory or profibrotic stimuli).