CXCL5 limits macrophage foam cell formation in atherosclerosis

Abstract

The ELR(+)-CXCL chemokines have been described typically as potent chemoattractants and activators of neutrophils during the acute phase of inflammation. Their role in atherosclerosis, a chronic inflammatory vascular disease, has been largely unexplored. Using a mouse model of atherosclerosis, we found that CXCL5 expression was upregulated during disease progression, both locally and systemically, but was not associated with neutrophil infiltration. Unexpectedly, inhibition of CXCL5 was not beneficial but rather induced a significant macrophage foam cell accumulation in murine atherosclerotic plaques. Additionally, we demonstrated that CXCL5 modulated macrophage activation, increased expression of the cholesterol efflux regulatory protein ABCA1, and enhanced cholesterol efflux activity in macrophages. These findings reveal a protective role for CXCL5, in the context of atherosclerosis, centered on the regulation of macrophage foam cell formation.

Figure 1. Upregulation of CXCL5 in atherosclerosis.

(A) Assessment of CXCL5 mRNA (blue triangles) and protein (red triangles) expression during the progression of atherosclerosis in aortas and plasma of Apoe^–/– mice, respectively (n = 6–8 per time point), fed a WD for the indicated time. Apoe^–/– mice fed a CD were used as controls. (B) Aortic Cxcl1, Cxcl2, and Cxcl5 mRNA and (C) plasma CXCL5 protein expression was measured in Apoe^–/– mice fed CD or WD for 12 weeks (n = 8 mice per group). WT mice fed CD for 12 weeks were used as controls. (D) Assessment of Cxcl5 mRNA in aortas of WT mice (n = 6–8 per time point) fed WD. WT mice fed CD were used as controls. (E–G) HUVECs were subjected to high or low LSS and stimulated with (E) oxidized LDL (n = 3) or (F and G) IL-1β (n = 6). (E and F) CXCL8 and CXCL6 mRNA expression and (G) protein release were determined. (A–G) mRNA and protein levels were measured by qPCR and ELISA, respectively. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2. Blockade of CXCL5 is associated with macrophage foam cell accumulation in atherosclerotic plaques.

Apoe^–/– mice were fed a WD and treated with either IgG isotype control (IgG) or anti-CXCL5 Ab (CXCL5 Ab) for 12 weeks. (A and B) Representative images from brachiocephalic artery lesions of picrosirius red staining for collagen detection. Black asterisks indicate some foam cells. (C) Representative images of macrophages (Mac2 immunostaining) containing lipid droplets (adipophilin immunostaining) from anti-CXCL5 Ab-treated brachiocephalic artery lesions. White asterisks indicate double-positive cells. (D–F) Quantification of (D) sirius red staining, (E) Mac2, and (F) adipophilin immunostaining in brachiocephalic artery lesions. Representative images from aortic root lesions of (G) Mac2 and (H) adipophilin immunostaining. Quantification of (I) Mac2 and (J) adipophilin immunostaining in aortic root lesions. (K) Quantification of plasma IL-6 by ELISA. Data in D–F and I–K represent mean ± SEM. n =7–8. **P < 0.01. Scale bars: 100 μm (A, G, and H); 50 μm (B); 10 μm (C).

Figure 3. CXCL5 modulates macrophage activation and induces ABCA1 expression and cholesterol efflux in macrophages.

(A) Apoe^–/– mice were fed a WD and treated with CXCL5 for 12 days. By flow cytometry, PMs (CD11b⁺CD115⁺) were gated and foam cells were identified as high forward scatter (FSChi) and high side scatter (SSChi) cells. Data are mean ± SEM. *P < 0.05. n = 3 animals per group. (B and D–G) BMDMs or (C and H) PMs were cholesterol loaded (gray bars) or not (white bars) with acLDL and treated with or without CXCL5 in vitro. (B) Macrophage activation was determined by Il6 mRNA expression in BMDMs using qPCR. n = 6. (C) Foam cell formation was assessed in cholesterol-loaded PMs as the percentage of Oil Red O–positive cells. Scale bar: 50 μm. n = 4. (D) Expression of genes involved in cholesterol trafficking was measured by qPCR in BMDMs. n = 6. (E) ABCA1 protein expression was measured by Western blotting in BMDMs. Lanes separated by a white line were run on the same gel but were noncontiguous. n = 3–5. (F) CXCL5-induced ABCA1 expression was blocked by anti-CXCL5 Ab and anti-CXCR2 Ab in cholesterol-loaded BMDMs. n = 4. (G) Expression of CXCL5-induced Abca1 in naive (M0), classically activated (M1), and alternatively activated (M2) macrophages from BMDM was determined by qPCR. n = 4. (H) Cholesterol efflux was assessed in cholesterol-loaded PMs treated or not with CXCL5. n = 6. (I) Proposed mechanism of action for CXCL5 in atherosclerosis. EC, endothelial cell; Mac, macrophage. Data are mean ± SEM. *P < 0.05, **P < 0.01.