Effect of CSE on M1/M2 polarization in alveolar and peritoneal macrophages at different concentrations and exposure in vitro

Abstract

Cigarette smoke exposure is one of the main etiologies for chronic obstructive pulmonary disease. Moreover, cigarette smoke participates in disease progression by inducing abnormal macrophage polarization; however, the effects of cigarette smoke on M1/M2 macrophage polarization have not been established. The aim of the current study was to determine the effects of cigarette smoke extract (CSE) on M1/M2 macrophage polarization in alveolar and peritoneal macrophages (AM and PM, respectively) at different concentrations and exposure times. Rat AM and PM were cultured with CSE at different concentrations. CCK-8 was used as an indicator of cell viability, and mRNA expression of M1 (iNOS, TNF-α, and IL-1β) and M2 markers (arg-1, CD206, and TGF-β1) were measured at 3, 6, 9, 12, and 24h using qPCR. Expressions of CD86 and CD206 proteins at 12h were determined using flow cytometry, and the iNOS/arg-1 ratio was used to determine the polarization dominance of M1 and M2. M2 subtypes were detected at 12h using qPCR and flow cytometry. CSE increased the expression of iNOS, TNF-α, and IL-1β mRNA, and the proportion of CD86-positive cells in AM and PM promoted M1 polarization, and M1 polarization was continuously enhanced as exposure time and concentration increased. CSE reduced the expression of arg-1, CD206, and TGF-β1 mRNA and the proportion of CD206-positive cells in AM and PM and inhibited M2 polarization. At 9-24h of CSE exposure, the expression of arg-1 in AM and PM gradually increased, showing tendency towards activation of M2 polarization. Besides, CSE might induce M2b and M2d polarization at 12h. After 12h of CSE exposure, transformation from M1 to M2 polarization dominance was shown in AM; however, M1 polarization was continuously enhanced in PM within 24h of CSE exposure. CSE promoted M1 polarization in macrophages, exhibiting dynamic regulatory effects on M2 polarization, first as a suppressor and then as a promoter. The polarization change induced by CSE on AM was more sensitive than PM.