Deletion of hematopoietic Dectin-2 or CARD9 does not protect against atherosclerotic plaque formation in hyperlipidemic mice

Abstract

Inflammatory reactions activated by pattern recognition receptors (PRRs) on the membrane of innate immune cells play an important role in atherosclerosis. Whether the PRRs of the C-type lectin receptor (CLR) family including Dectin-2 may be involved in the pathogenesis of atherosclerosis remains largely unknown. Recently, the CLR-adaptor molecule caspase recruitment domain family member 9 (CARD9) has been suggested to play a role in cardiovascular pathologies as it provides the link between CLR activation and transcription of inflammatory cytokines as well as immune cell recruitment. We therefore evaluated whether hematopoietic deletion of Dectin-2 or CARD9 reduces inflammation and atherosclerosis development. Low-density lipoprotein receptor (Ldlr)-knockout mice were transplanted with bone marrow from wild-type, Dectin-2- or Card9-knockout mice and fed a Western-type diet containing 0.1% (w/w) cholesterol. After 10 weeks, lipid and inflammatory parameters were measured and atherosclerosis development was determined. Deletion of hematopoietic Dectin-2 or CARD9 did not influence plasma triglyceride and cholesterol levels. Deletion of hematopoietic Dectin-2 did not affect atherosclerotic lesion area, immune cell composition, ex vivo cytokine secretion by peritoneal cells or bone marrow derived macrophages. Unexpectedly, deletion of hematopoietic CARD9 increased atherosclerotic lesion formation and lesion severity. Deletion of hematopoietic CARD9 did also not influence circulating immune cell composition and peripheral cytokine secretion. Besides a tendency to a reduced macrophage content within these lesions, plasma MCP-1 levels decreased upon WTD feeding. Deletion of hematopoietic Dectin-2 did not influence atherosclerosis development in hyperlipidemic mice. The absence of CARD9 unexpectedly increased atherosclerotic lesion size and severity, suggesting that the presence of CARD9 may protect against initiation of atherosclerosis development.

Figure 1

Deletion of hematopoietic Dectin-2 or CARD9 does not influence meta bolic parameters. (A) Lethally irradiated Ldlr^−/− mice were reconstituted with control (WT), Dectin-2^−/− or Card9^−/− BM. After 10 weeks of recovery, mice were fed a Western-type diet containing 0.1% cholesterol. Just before and after 4 and 10 weeks of WTD feeding, (B) triglyceride and (C) cholesterol levels were measured in plasma. (D) Total cholesterol exposure were determined at the end of the study and (E) body weight before and after 4 and 10 weeks of WTD feeding. Data are presented as mean ± SEM. n = 13–14/group.

Figure 2

Deletion of hematopoietic CARD9, but not Dectin-2, increased atherosclerotic plaque formation. After 10 weeks of Western-type diet (WTD) feeding, hearts were collected from Ldlr^−/− mice transplanted with BM from control (WT), Dectin-2^−/− or Card9^−/− and aortic roots were analyzed by Immunohistochemistry. (A) Aortic valves were stained with hematoxylin-phloxine-saffron (HPS) and representative pictures are shown (scale bar represents 100 μm). (B) Total atherosclerotic lesion area was assessed in 4 sections of the aortic root and (C) average lesion area was calculated. (D) Lesion severity of was accessed following the guidelines of the American Heart Association adapted to mice and shown as percentage of total lesions. Data for atherosclerotic plaque composition are presented as mean ± SEM. n = 13–14/group. *p < 0.05.

Figure 3

Hematopoietic deletion of CARD9, decreased plasma MCP-1 levels, tended to decrease macrophage content but does not altered the distribution of M1/M2 markers in the plaque. After 10 weeks of Western-type diet (WTD) feeding, hearts were collected from Ldlr^−/− mice transplanted with BM from control (WT), Dectin-2^−/− or Card9^−/− and aortic roots were analyzed by Immunohistochemistry. (A) Macrophage area was measured by MAC3 staining, (B) quantified in 4 sections and (C) average was calculated. T-cells were determined by CD3 staining, (D) counted in 4 sections and (E) average was calculated (F). M1 and M2 macrophages were determined by (G) iNos and (J) Arginase staining and (H) quantified in 12 sections and presented as ratio between M1/M2 marker. (I) RT-qPCR was used to quantify the expression of markers for M1 and M2 macrophages. (K) Plasma levels of monocyte attractant protein 1 (MCP-1) were measured at the beginning (T = 0) of WTD feeding and at the end of the study (T = 10). (K). Representative pictures for the staining are shown with  sclae bars representing 100 µm. Data for atherosclerotic plaque composition are presented as mean ± SEM. n = 13–14/group. Plasma MCP-1 levels. n = 7–11/group, *p < 0.05.

Figure 4

Hematopoietic deletion of Dectin-2 or CARD9 hardly affects immune cell composition in the circulation or in bone marrow. At the end of the study, after 10 weeks of Western-type diet feeding, immune cells subsets in the blood of Ldlr^−/− mice transplanted with BM from control (WT), Dectin-2^−/− or Card9^−/− were determined by flow cytometry. The amount of innate immune cell subsets are shown for (A) total Ly6C monocytes, which were subdivided into (B) Ly6C^hi-, (C) Ly6C^med-, (D) Ly6C^lo-monocytes. Further, the percentage of (E) neutrophils, (F) eosinophils and (G) dendritic cells is shown. The percentage of adaptive immune cell subsets is determined for (H) B-cells, (I) total T-cells and T-cell subsets such as (J) cytotoxic T-cells and (K) T-helper cells. Data are presented as mean ± SEM. n = 7–8/group.

Figure 5

Deletion of hematopoietic Dectin-2 or CARD9 does not influence cytokine secretion. At the end of the study, after 10 weeks of Western-type diet feeding bone marrow-derived macrophages (BMDMs, A–D) and peritoneal cells (E–H) from Ldlr^−/− mice transplanted with BM from control (WT), Dectin-2^−/− or Card9^−/− were isolated and ex vivo stimulated. (A) Tumor necrosis factor (TNF)α and (B) Interleukin (IL-)6 cytokine secretion were measured after stimulation with (A,B) lipopolysaccharide (LPS-EB, 10 ng/mL) or (C,D) Pam3Cys (10 µg/mL) in BMDMs. Similarly, (E,G) TNFα and (F,H) IL-6 were measured after stimulation with (E,F) LPS-EB or (G,H) Pam3Cys. Data are presented as mean ± SEM. n = 8/group. N.D., values are below detection limit. Detection limit is indicated with the black dotted line.