Diesel exhaust particles activate macrophages and IRF5 expression in atherosclerosis

Abstract

Abstract Introduction Over 20% of global deaths related to cardiovascular disease have been attributed in part to air pollution, particularly fine particulate matter (PM2.5) (1). In highly trafficked urban areas, smaller ultrafine diesel exhaust particles (DEP) represent a significant proportion of PM2.5. DEP is associated with the progression of atherosclerosis and increased plaque susceptibility to rupture (2). We have previously shown that interferon regulatory factor 5 (IRF5) has a key role in necrotic core formation, a distinguishing feature of vulnerable atherosclerotic plaques, by rendering macrophages defective at efferocytosis (3). Pronounced elevation of IRF5 is also seen in symptomatic carotid plaques, particularly in the rupture-prone shoulder regions (4). Purpose We examined the effect of DEP on macrophage phenotype and IRF5 expression in atherosclerosis. Methods Bone marrow-derived macrophages (BMDMs) from C57BL/6 mice, differentiated with GM-CSF (to mimic inflammatory macrophages) or M-CSF (mimicking homeostatic resident macrophages) were incubated with PBS or DEP (10 µg/mL) in vitro for 18 hours. Expression of inflammatory cytokines (IL-6, IL-12α), macrophage polarisation markers CD11c (itgax, inflammatory) and CD206 (mrc1, resident anti-inflammatory) and IRF5 was quantified by real-time PCR. In addition, high fat-fed ApoE-/- mice were exposed to saline or DEP (35 µL at 1 mg/mL) twice weekly for 5 weeks by pulmonary administration. The pan-macrophage marker CD64, CD206 and IRF5 were quantified in paraffin-embedded sections of brachiocephalic artery lesions by immunohistochemistry. Results In M-CSF-differentiated murine macrophages incubated with DEP, significant fold increases in gene expression (relative to PBS control) were observed: IRF5 (1.4-fold), IL-6 (31.5-fold) and IL-12α (5.8-fold) while CD206 (0.8-fold) decreased (Figure 1a, n = 5, p<0.05, two-tailed paired t-tests). CD11c expression was unchanged. No statistically significant differences were detected in GM-CSF-differentiated macrophages. In the brachiocephalic arteries of DEP-exposed ApoE-/- mice we observed increases in CD64+ macrophages (28.2±7.4 vs 15.5±5.6) and IRF5 (16.6±3.6 vs 10.1±3.4) expression, while CD206 expression decreased (4.1±0.9 vs 11.0±1.2) in comparison to saline-exposed mice (Figure 1b mean±SD, n = 5, p<0.05, two-tailed unpaired t-tests, data expressed as % of intima area). Conclusion DEP promotes CD64+ macrophages and IRF5 expression in murine atherosclerosis, while inflammation and IRF5 is induced in vitro, but only in M-CSF-differentiated murine BMDMs. This suggests that DEP has no impact on inflammatory macrophages but could switch homeostatic resident macrophages to a more inflammatory phenotype via the activation of IRF5. Therefore, IRF5 is a promising candidate for further investigation into the initial macrophage response to inhaled particles that leads to the exacerbation of vascular disease.