Deletion of Talin1 in Myeloid Cells Facilitates Atherosclerosis in Mice

Abstract

Background: Integrin-regulated monocyte recruitment and cellular responses of monocyte-derived macrophages are critical for the pathogenesis of atherosclerosis. In the canonical model, talin1 controls ligand binding to integrins, a prerequisite for integrins to mediate leukocyte recruitment and induce immune responses. However, the role of talin1 in the development of atherosclerosis has not been studied. Our study investigated how talin1 in myeloid cells regulates the progression of atherosclerosis.

Methods: On an Apoe^-/- background, myeloid talin1-deficient mice and the control mice were fed with a high-fat diet for 8 or 12 weeks to induce atherosclerosis. The atherosclerosis development in the aorta and monocyte recruitment into atherosclerotic lesions were analyzed.

Results: Myeloid talin1 deletion facilitated the formation of atherosclerotic lesions and macrophage deposition in lesions. Talin1 deletion abolished integrin β2-mediated adhesion of monocytes but did not impair integrin α4β1-dependent cell adhesion in a flow adhesion assay. Strikingly, talin1 deletion did not prevent Mn²⁺- or chemokine-induced activation of integrin α4β1 to the high-affinity state for ligands. In an in vivo competitive homing assay, monocyte infiltration into inflamed tissues was prohibited by antibodies to integrin α4β1 but was not affected by talin1 deletion or antibodies to integrin β2. Furthermore, quantitative polymerase chain reaction and ELISA (enzyme-linked immunosorbent assay) analysis showed that macrophages produced cytokines to promote inflammation and the proliferation of smooth muscle cells. Ligand binding to integrin β3 inhibited cytokine generation in macrophages, although talin1 deletion abolished the negative effects of integrin β3.

Conclusions: Integrin α4β1 controls monocyte recruitment during atherosclerosis. Talin1 is dispensable for integrin α4β1 activation to the high-affinity state and integrin α4β1-mediated monocyte recruitment. Yet, talin1 is required for integrin β3 to inhibit the production of inflammatory cytokines in macrophages. Thus, intact monocyte recruitment and elevated inflammatory responses cause enhanced atherosclerosis in talin1-deficient mice. Our study provides novel insights into the roles of myeloid talin1 and integrins in the progression of atherosclerosis.

Keywords: atherosclerosis; cell adhesion; inflammation; integrins; myeloid cells.

Figure 1.. Myeloid talin1 deficiency enhances atherosclerosis and macrophage burden in atherosclerotic lesion.

(A) Representative images of en face Oil Red-stained aortas from Control (Apoe^−/−/LysMCre⁻;Tln1^f/f) and Mye Tln1^−/−(Apoe^−/−/LysMCre⁺;Tln1^f/f) mice that were fed with a high-fat diet for 8 and 12 weeks. In each genotype, n = 8. Red dashed line demarcated the aortic arch and thoracic aorta. (B) Quantification of the area (%) of Oil Red-positive lesion in aortic arch and thoracoabdominal aorta in aortas shown in A. (C) Representative immunostaining images of transverse sections of aortic sinus from Control and Mye Tln1^−/− mice that were fed with a high-fat diet for 8 and 12 weeks. (D) Quantification of the MOMA-2-positive area (%) in transverse sections of aortic sinus shown in C. (E) Quantification of the lesion area in transverse sections of aortic sinus. All data are acquired from three independent experiments, n = 8 male mice per genotype, and results are expressed as mean ± sd. Scale bar, 200 μm.

Figure 2.. Integrin α4β1 dominantly contributes to monocyte adhesion on inflamed vessels and extravasation into tissues in vivo.

Adherent and extravasated monocytes from Control (green) and Mye Tln1^−/− (red) mice at 2 hours after IV injection into TNF-α-challenged WT mice that were pre-injected with (A) isotype Abs, and inhibitory Abs to (C) integrin β2 or (E) α4β1. Blood vessel endothelial cells were stained with anti-CD31 mAb (blue). The images are representative of 4 independent experiments. Scale bar, 200 μm. (B, D and F) Quantification of adherent luminal and extravascular monocytes at 2 hours after injection was presented in the right of the images. The data represent mean ± sd of 50 to 300 monocytes counted in 7–8 venules from each of 4 independent experiments. Each dot represents a mean from one mouse.

Figure 3.. Integrin β1-mediated adhesion and integrin α4β1 activation are intact in talin1-deficient monocytes.

Adhesion of (A) monocytes and (B) macrophages of the indicated genotype on plates coated with P-selectin, ICAM-1 in the presence or absence of CXCL12 at 1 dyne/cm². In some experiments, cells were pretreated with anti-integrin β2 antibodies before perfusing over the plates. Adhesion of (C) monocytes and (D) macrophages of the indicated genotype on plates coated with VCAM-1 in the presence or absence of CXCL12 at 1 dyne/cm². In some experiments, cells were pre-treated with Bio1211, an antagonist to integrin β1 before adhesion. Flow cytometry measurement of LDV binding to (E) Jurkat cells and (F) monocytes of the indicated genotype treated with Ca²⁺ or Mn²⁺/EGTA. (G) Flow cytometry measurement of LDV binding to monocytes of the indicated genotype treated with the vehicle control or CXCL12. (H) Representative images of LDV-stained monocytes adhered on plates coated with poly-lysine and VCAM-1 with or without CXCL-12. All the results are the representative from 5 independent experiments, and the data represent mean ± sd. Scale bar, 10 μm.

Figure 4.. Myeloid talin1 deficiency increases inflammation at the early phase of atherosclerosis.

Measurement of the plasma levels of (A) TNF-α, (B) IL-1b, (C) IL-6, (D) IFN-γ, and (E) IL-10 in Control and Mye Tln1^−/− mice at 0 week, 8 weeks and 12 weeks after feeding with the high-fat diet. Plasma samples at each time point were from 8 mice of each genotype. Immunofluorescent staining of the transverse sections of aortic sinus from control mice and Mye Tln1^−/− mice after feeding with a high-fat diet for 12 weeks with antibodies to (F) TNF-α and (G) IL-6 and quantification of the signals. (H) Quantitative analysis of gene expression in macrophages from mice of the indicated genotypes. (I) Quantitative analysis of gene expression in Control macrophages treated with the vehicle control or RGD peptide. The dotted line represents the mean expression of the control gene, GAPDH. The results are from at least 4 independent experiments, and the data represent mean ± sd.

Figure 5.. Myeloid talin1 deficiency increases smooth muscle cells in atherosclerotic aortas.

(A) Representative images of transverse sections of aortic sinus from Control and Mye Tln1^−/− mice that were fed with a high-fat diet for 8 and 12 weeks. The cryosections were stained with antibodies to α-smooth muscle actin (SMA). Medial SMCs were demarcated with white dotted line, and SMCs in lesions were indicated with white arrow heads. In each genotype, n = 8. Quantification of (B) the size of SMC-positive area and (C) the percentage of SMC-positive area in the lesions in transverse sections of aortic sinus. (D) Quantitative analysis of gene expression in macrophages that were derived from monocytes of the indicated genotypes. (E) Quantitative analysis of gene expression in control macrophages treated with the vehicle control or RGD peptide. (F) Quantitative analysis of gene expression in myeloid talin1-deficient macrophages treated with the vehicle control or RGD peptide. The dotted line represents the mean expression of the control gene, GAPDH. All the results are the representative from 4 independent experiments, and the data represent mean ± sd. Scale bar, 200 μm.