Functional role of CD11c+ monocytes in atherogenesis associated with hypercholesterolemia

Abstract

Background: Monocyte activation and migration into the arterial wall are key events in atherogenesis associated with hypercholesterolemia. CD11c/CD18, a beta2 integrin expressed on human monocytes and a subset of mouse monocytes, has been shown to play a distinct role in human monocyte adhesion on endothelial cells, but the regulation of CD11c in hypercholesterolemia and its role in atherogenesis are unknown.

Methods and results: Mice genetically deficient in CD11c were generated and crossbred with apolipoprotein E (apoE)-/- mice to generate CD11c-/-/apoE-/- mice. Using flow cytometry, we examined CD11c on blood leukocytes in apoE-/- hypercholesterolemic mice and found that compared with wild-type and apoE-/- mice on a normal diet, apoE-/- mice on a Western high-fat diet had increased CD11c+ monocytes. Circulating CD11c+ monocytes from apoE-/- mice fed a high-fat diet exhibited cytoplasmic lipid vacuoles and expressed higher levels of CD11b and CD29. Deficiency of CD11c decreased firm arrest of mouse monocytes on vascular cell adhesion molecule-1 and E-selectin in a shear flow assay, reduced monocyte/macrophage accumulation in atherosclerotic lesions, and decreased atherosclerosis development in apoE-/- mice on a high-fat diet.

Conclusions: CD11c, which increases on blood monocytes during hypercholesterolemia, plays an important role in monocyte recruitment and atherosclerosis development in an apoE-/- mouse model of hypercholesterolemia.

Figure 1. CD11c targeting construct and homologous recombination

A: Partial restriction map of murine CD11c gene. Boxes represent exons 1-4. The 5′ probe was generated from flanking DNA not used in the targeting construct. B: The CD11c replacement construct results in the replacement of a 0.8-kb fragment of the CD11c gene that contains exons 2 and 3 and the coding sequence of exon 1 with a neomycin resistance cassette (NEO). C: Map of the predicted homologous recombination event. D: HindIII digestion results in a 4.0-kb fragment in mutant and a 9.4-kb fragment in wild-type (WT). E: Genotyping of WT (+/+), mutant (-/-), and heterozygous (+/-) mice by Southern blot analysis using the probe indicated in (A).

Figure 2. Increased CD11c⁺ monocytes in blood of hypercholesterolemic mice

A: representative FACS of CD204 and CD115 expression on blood leukocytes of WT and apoE^-/- HFD. B: Representative FACS of CD11c and CD204 expression on blood leukocytes of WT and apoE^-/- on HFD, indicating CD11c⁺/CD204⁺ and CD11c^-/CD204⁺ monocyte subsets. C: Compared to WT or apoE^-/- on ND, apoE^-/- mice on HFD (12 weeks) showed significantly increased CD11c⁺/CD204⁺ and CD11c^-/CD204⁺ monocytes. n=9-12 mice/group.

Figure 3. Characteristics of monocytes from blood of apoE^-/- mice on HFD

A and B: Representative scatter pattern (cell size [FSC] versus granularity [SSC]) of total blood leukocytes and some phenotypic characteristics of blood CD11c⁺ cells from WT and apoE^-/- mice on HFD analyzed by FACS. C: Relative ratio of CD11c⁺ to CD11c^- monocytes in apoE^-/- mice on HFD (R1/granulocyte and R2/monocyte regions) or WT, n=9-12 mice/group; *P<0.001 vs. WT, #P<0.001 vs. apoE^-/- R2. D and E: CD11c⁺ monocytes in R1 from apoE^-/- mice on HFD had higher levels of CD11b and CD29 than CD11c⁺ monocytes in R2; n=5 for each of WT and apoE^-/- on HFD. F and G: Representative images of CD11c⁺ cells isolated from blood of WT or apoE^-/- on HFD by magnetic separation (F), and CD11c⁺ and CD11c^- monocytes isolated from R1 and R2 regions of apoE^-/- on HFD by cell sorting (G). Original magnification: ×600 (Neat Stain), ×100 (Oil Red O).

Figure 3. Characteristics of monocytes from blood of apoE^-/- mice on HFD

A and B: Representative scatter pattern (cell size [FSC] versus granularity [SSC]) of total blood leukocytes and some phenotypic characteristics of blood CD11c⁺ cells from WT and apoE^-/- mice on HFD analyzed by FACS. C: Relative ratio of CD11c⁺ to CD11c^- monocytes in apoE^-/- mice on HFD (R1/granulocyte and R2/monocyte regions) or WT, n=9-12 mice/group; *P<0.001 vs. WT, #P<0.001 vs. apoE^-/- R2. D and E: CD11c⁺ monocytes in R1 from apoE^-/- mice on HFD had higher levels of CD11b and CD29 than CD11c⁺ monocytes in R2; n=5 for each of WT and apoE^-/- on HFD. F and G: Representative images of CD11c⁺ cells isolated from blood of WT or apoE^-/- on HFD by magnetic separation (F), and CD11c⁺ and CD11c^- monocytes isolated from R1 and R2 regions of apoE^-/- on HFD by cell sorting (G). Original magnification: ×600 (Neat Stain), ×100 (Oil Red O).

Figure 3. Characteristics of monocytes from blood of apoE^-/- mice on HFD

A and B: Representative scatter pattern (cell size [FSC] versus granularity [SSC]) of total blood leukocytes and some phenotypic characteristics of blood CD11c⁺ cells from WT and apoE^-/- mice on HFD analyzed by FACS. C: Relative ratio of CD11c⁺ to CD11c^- monocytes in apoE^-/- mice on HFD (R1/granulocyte and R2/monocyte regions) or WT, n=9-12 mice/group; *P<0.001 vs. WT, #P<0.001 vs. apoE^-/- R2. D and E: CD11c⁺ monocytes in R1 from apoE^-/- mice on HFD had higher levels of CD11b and CD29 than CD11c⁺ monocytes in R2; n=5 for each of WT and apoE^-/- on HFD. F and G: Representative images of CD11c⁺ cells isolated from blood of WT or apoE^-/- on HFD by magnetic separation (F), and CD11c⁺ and CD11c^- monocytes isolated from R1 and R2 regions of apoE^-/- on HFD by cell sorting (G). Original magnification: ×600 (Neat Stain), ×100 (Oil Red O).

Figure 3. Characteristics of monocytes from blood of apoE^-/- mice on HFD

A and B: Representative scatter pattern (cell size [FSC] versus granularity [SSC]) of total blood leukocytes and some phenotypic characteristics of blood CD11c⁺ cells from WT and apoE^-/- mice on HFD analyzed by FACS. C: Relative ratio of CD11c⁺ to CD11c^- monocytes in apoE^-/- mice on HFD (R1/granulocyte and R2/monocyte regions) or WT, n=9-12 mice/group; *P<0.001 vs. WT, #P<0.001 vs. apoE^-/- R2. D and E: CD11c⁺ monocytes in R1 from apoE^-/- mice on HFD had higher levels of CD11b and CD29 than CD11c⁺ monocytes in R2; n=5 for each of WT and apoE^-/- on HFD. F and G: Representative images of CD11c⁺ cells isolated from blood of WT or apoE^-/- on HFD by magnetic separation (F), and CD11c⁺ and CD11c^- monocytes isolated from R1 and R2 regions of apoE^-/- on HFD by cell sorting (G). Original magnification: ×600 (Neat Stain), ×100 (Oil Red O).

Figure 4. ox-LDL increases CD11c expression on mouse MNCs

A: Proportion of CD11c⁺ monocytes in mouse MNCs after incubation with native LDL or ox-LDL (20 □g/ml) for 24 hours in vitro (n=8/group). B: Representative FACS of monocytes in apoE^-/- mice on ND at different timepoints after intravenous injection of DiI-ox-LDL or DiI-native LDL with PBS as negative control. C: Relative ratio of CD11c⁺/DiI⁺ to CD11c^-/DiI⁺ cells in blood (total DiI⁺ cells assumed to be 100%) of apoE^-/- mice at 1 hour and 24 hours after DiI-ox-LDL or DiI-native LDL injection. n=5 for DiI-ox-LDL; n=3 for DiI-native LDL; *P=0.0018 vs. 1 hr DiI-ox-LDL.

Figure 4. ox-LDL increases CD11c expression on mouse MNCs

A: Proportion of CD11c⁺ monocytes in mouse MNCs after incubation with native LDL or ox-LDL (20 □g/ml) for 24 hours in vitro (n=8/group). B: Representative FACS of monocytes in apoE^-/- mice on ND at different timepoints after intravenous injection of DiI-ox-LDL or DiI-native LDL with PBS as negative control. C: Relative ratio of CD11c⁺/DiI⁺ to CD11c^-/DiI⁺ cells in blood (total DiI⁺ cells assumed to be 100%) of apoE^-/- mice at 1 hour and 24 hours after DiI-ox-LDL or DiI-native LDL injection. n=5 for DiI-ox-LDL; n=3 for DiI-native LDL; *P=0.0018 vs. 1 hr DiI-ox-LDL.

Figure 4. ox-LDL increases CD11c expression on mouse MNCs

A: Proportion of CD11c⁺ monocytes in mouse MNCs after incubation with native LDL or ox-LDL (20 □g/ml) for 24 hours in vitro (n=8/group). B: Representative FACS of monocytes in apoE^-/- mice on ND at different timepoints after intravenous injection of DiI-ox-LDL or DiI-native LDL with PBS as negative control. C: Relative ratio of CD11c⁺/DiI⁺ to CD11c^-/DiI⁺ cells in blood (total DiI⁺ cells assumed to be 100%) of apoE^-/- mice at 1 hour and 24 hours after DiI-ox-LDL or DiI-native LDL injection. n=5 for DiI-ox-LDL; n=3 for DiI-native LDL; *P=0.0018 vs. 1 hr DiI-ox-LDL.

Figure 5. β₁ and β₂integrin cooperativity in monocyte arrest on E-selectin/VCAM-1 in shear flow

MNCs from blood of CD11c^+/+/apoE^-/- and CD11c^-/-/apoE^-/- mice were perfused over E-selectin/VCAM-1 in a microfluidic flow chamber at 2 dyne/cm². A and B: Bivariate plots of the intensity of Gr-1 and CD204 staining on firmly arrested MNCs from CD11c^+/+/apoE^-/- (A) and CD11c^-/-/apoE^-/- (B) mice. C: Number of firmly arrested MNCs per field as a function of the presence or absence of blocking antibodies to VLA-4 and CD18. D: MNCs firmly arrested at 2 dynes/cm² were subjected to a stepped increase in shear stress to 10 dynes/cm². Percentage of the arrested cells remaining 1 minute after this stepped increase is presented. Data represent mean ± standard error from 3-8 independent experiments.

Figure 6. CD11c and atherosclerosis in apoE^-/- mice

All samples were collected from mice on HFD for 12 weeks. A-D: Representative immunofluorescence staining on atherosclerotic lesions of CD11c^+/+/apoE^-/- aortas, with (A) PE-anti-mouse CD11c (red), (B) PE-anti-mouse CD11c and FITC-anti-mouse CD205 (green), or (C, D) PE-anti-mouse CD11c and FITC-anti-mouse MOMA-2 (green) mAbs (yellow indicates the overlapping of the red and green), with counterstaining of DAPI (blue) for nuclei. E: Representative Sudan IV staining of mouse aortas (original magnification, × 6.5). F and G: quantification of atherosclerotic lesions in whole aortas (F) and aortic arch (G) (n=20 for CD11c^+/+/apoE^-/-, n=30 for CD11c^-/-/apoE^-/-). H: Representative macrophage staining (original magnification, × 100), and I: quantification of macrophage contents in atherosclerotic lesions of aortic sinus (n=13 for CD11c^+/+/apoE^-/-, n=11 for CD11c^-/-/apoE^-/-).

Figure 6. CD11c and atherosclerosis in apoE^-/- mice

All samples were collected from mice on HFD for 12 weeks. A-D: Representative immunofluorescence staining on atherosclerotic lesions of CD11c^+/+/apoE^-/- aortas, with (A) PE-anti-mouse CD11c (red), (B) PE-anti-mouse CD11c and FITC-anti-mouse CD205 (green), or (C, D) PE-anti-mouse CD11c and FITC-anti-mouse MOMA-2 (green) mAbs (yellow indicates the overlapping of the red and green), with counterstaining of DAPI (blue) for nuclei. E: Representative Sudan IV staining of mouse aortas (original magnification, × 6.5). F and G: quantification of atherosclerotic lesions in whole aortas (F) and aortic arch (G) (n=20 for CD11c^+/+/apoE^-/-, n=30 for CD11c^-/-/apoE^-/-). H: Representative macrophage staining (original magnification, × 100), and I: quantification of macrophage contents in atherosclerotic lesions of aortic sinus (n=13 for CD11c^+/+/apoE^-/-, n=11 for CD11c^-/-/apoE^-/-).

Figure 6. CD11c and atherosclerosis in apoE^-/- mice

All samples were collected from mice on HFD for 12 weeks. A-D: Representative immunofluorescence staining on atherosclerotic lesions of CD11c^+/+/apoE^-/- aortas, with (A) PE-anti-mouse CD11c (red), (B) PE-anti-mouse CD11c and FITC-anti-mouse CD205 (green), or (C, D) PE-anti-mouse CD11c and FITC-anti-mouse MOMA-2 (green) mAbs (yellow indicates the overlapping of the red and green), with counterstaining of DAPI (blue) for nuclei. E: Representative Sudan IV staining of mouse aortas (original magnification, × 6.5). F and G: quantification of atherosclerotic lesions in whole aortas (F) and aortic arch (G) (n=20 for CD11c^+/+/apoE^-/-, n=30 for CD11c^-/-/apoE^-/-). H: Representative macrophage staining (original magnification, × 100), and I: quantification of macrophage contents in atherosclerotic lesions of aortic sinus (n=13 for CD11c^+/+/apoE^-/-, n=11 for CD11c^-/-/apoE^-/-).

Figure 7. A model for the involvement of CD11c in atherogenesis in hypercholesterolemia

In severe hypercholesterolemia, uptake of ox-LDL by circulating monocytes (through scavenger receptors and undefined pathways) activates monocytes, and drives CD11c^--to-CD11c⁺ monocyte differentiation, forming CD11c⁺ “foamy monocytes.” These CD11c⁺ “foamy monocytes” express high levels of VLA-4 and CD11b, and adhere efficiently to ECs through interactions of CD11c and VLA-4 with VCAM-1 on activated ECs, thereby playing an active role in development of atherosclerosis and xanthomas associated with severe hypercholesterolemia.