Deficiency of Dab2 (Disabled Homolog 2) in Myeloid Cells Exacerbates Inflammation in Liver and Atherosclerotic Plaques in LDLR (Low-Density Lipoprotein Receptor)-Null Mice-Brief Report

Abstract

Objective: Inflammatory macrophages promote the development of atherosclerosis. We have identified the adaptor protein Dab2 (disabled homolog 2) as a regulator of phenotypic polarization in macrophages. The absence of Dab2 in myeloid cells promotes an inflammatory phenotype, but the impact of myeloid Dab2 deficiency on atherosclerosis has not been shown.

Approach and results: To determine the role of myeloid Dab2 in atherosclerosis, Ldlr^-/- mice were reconstituted with either Dab2-positive or Dab2-deficient bone marrow and fed a western diet. Consistent with our previous finding that Dab2 inhibits NFκB (nuclear factor κ-light-chain-enhancer of activated B cells) signaling in macrophages, Ldlr^-/- mice reconstituted with Dab2-deficient bone marrow had increased systemic inflammation as evidenced by increased serum IL-6 (interleukin-6) levels and increased inflammatory cytokine expression levels in liver. Serum lipid levels were significantly lower in Ldlr^-/- mice reconstituted with Dab2-deficient bone marrow, and further examination of livers from these mice revealed drastically increased inflammatory tissue damage and massive infiltration of immune cells. Surprisingly, the atherosclerotic lesion burden in Ldlr^-/- mice reconstituted with Dab2-deficient bone marrow was decreased compared with Ldlr^-/- mice reconstituted with wild-type bone marrow. Further analysis of aortic root sections revealed increased macrophage content and evidence of increased apoptosis in lesions from Ldlr^-/- mice reconstituted with Dab2-deficient bone marrow but no difference in collagen or α-smooth muscle actin content.

Conclusions: Dab2 deficiency in myeloid cells promotes inflammation in livers and atherosclerotic plaques in a mouse model of atherosclerosis. Nevertheless, decreased serum lipids as a result of massive inflammatory liver damage may preclude an appreciable increase in atherosclerotic lesion burden in mice reconstituted with Dab2-deficient bone marrow.

Keywords: atherosclerosis; inflammation; liver; macrophage; myeloid cell.

Figure 1. Myeloid Dab2 deficiency exacerbates hepatic inflammation

A, Serum IL6 levels were measured by ELISA in WT-tp and MɸDab2^−/−-tp female mice were fed a western diet for 9 weeks (n=8 for both groups) and 20 weeks (n=12, n=10). Data are presented as mean +/− s.e.m. *p=0.037 by Student’s t-test with Welch’s correction. B, Expression of inflammatory cytokines Il1β, Il6, Tnfα, and Ifnγ was assessed by qRT-PCR in liver tissue from WT-tp and MɸDab2^−/−-tp mice fed a western diet for 9 (n=8) and 20 weeks (n=12, 10). Data is normalized to 9 week WT-tp samples and presented as mean +/− s.e.m. *p<0.001 by ANOVA with Tukey’s multiple comparison test. For A and B data points greater or less than 2 standard deviations from the mean were excluded. C, Representative images of hematoxylin and eosin (H&E) stained section of liver from WT-tp and MɸDab2^−/−-tp mice fed a western diet for 9 and 20 weeks. Scale bar represents 100 μm. Note aggregate of purple staining immune cells in the center of the 20 week MɸDab2^{−/−−/−}-tp liver image. Quantification of relative area of aggregates in livers from mice fed western diet for 20 weeks is shown at right. Data is presented as mean +/− s.e.m. Each point represents one mouse n=12 and 10.*p=0.01 by Mann-Whitney test. D, Immunohistochemistry was performed on liver sections from WT-tp and MɸDab2^−/−-tp mice. Representative images stained for T cells (CD3), B cells (B220), and macrophages (Mac2) are shown with positive staining appearing brown. Scale bar represents 200 μm for CD3 and B220 images and 500 μm for Mac2 images. Quantification is shown on right. Presence of infiltrating T cells and B cells is shown as the percentage of positive (brown) cells of the total cell number per field of view. Quantification of Mac2 histological staining is shown as percentage of positive (brown) area. Data is presented as mean +/− s.e.m. Each point represents one mouse n=7. *p<0.006 by Student’s unpaired t-test with Welch’s correction for unequal variance. E, Expression of Cxcl13, Lta, and Ccl20 genes involved in formation of tertiary lymphoid organs was measured by qRT-PCR in liver tissue from WT-tp and MɸDab2^−/−-tp mice fed a western diet for 9 (n=8) and 20 weeks (n=12, 10). Data is normalized to 9 week WT-tp samples and presented as mean +/− s.e.m. *p<0.05 by Student’s t-test. Data points greater or less than 2 standard deviations from the mean were excluded. F, Liver weight as a percentage of total body weight was not different between WT-tp and MɸDab2^−/−-tp mice (n=12,10) fed a western diet for 20 weeks. Data is presented as mean +/− s.e.m.

Figure 2. Decreased serum VLDL and liver triglycerides in MΦDab2^−/−-tp mice

A, Serum lipids were measured in WT-tp and MɸDab2^−/−-tp mice before western diet (week 0, n=20) and then 9 (n=8) and 20 (n=12 and 10) weeks after western diet. Data are presented as mean +/− s.e.m. *p< 0.004 by Student’s t-test. B, FPLC analysis of serum from WT-tp and MɸDab2^−/−-tp mice fed western diet for 20 weeks. Each line represents pooled serum samples from 9 mice. C, Liver triglyceride content was measured in WT-tp and MɸDab2^−/−-tp mice fed a western diet 20 weeks (n=12, 10). Data is presented as mean +/− s.e.m. *p=0.036 by Student’s t-test. D, Oil Red O staining of liver sections from WT-tp and MɸDab2^−/−-tp mice fed western diet for 9 and 20 weeks. Quantification at right, n=5 mice with at least 6 sections analyzed per mouse. Data are presented as mean mean +/− s.d. *p=0.02 by 1 way ANOVA with Tukey’s multiple comparison test. E, Expression of genes involved in liver lipid metabolism Ppara, Abca1, Abcg1, Lpl, and Vldlr was determined by qRT-PCR in liver tissue from WT-tp and MɸDab2^−/−-tp mice fed a western diet 20 weeks (n=12, 10). Data is presented as mean +/− s.e.m. *p<0.001 by ANOVA with Tukey’s multiple comparison test for Ppara.*p=0.0006 for Abcg1 and *p=0.03 for Lpl by Student’s t-test with Welch’s correction. F, Western blot for Abcg1 and Abca1 protein in liver homogenates from representative WT-tp and MɸDab2^−/−-tp mice fed a western diet 20 weeks with actin as loading control. Each lane represents one mouse. G, Quantitation of Abcg1 and Abca1 protein in liver homogenates from representative WT-tp and MɸDab2^−/−-tp mice fed a western diet 20 weeks. Normalized to Actin loading control. Each point represents one mouse. (n=12, 10) Data points greater or less than 2 standard deviations from the mean were excluded. Data are presented as mean +/− s.e.m. *p=0.014by Student’s t-test.

Figure 3. MΦDab2^−/−-tp mice have smaller but more inflamed atherosclerotic lesions compared to WT-tp mice

A, WT-tp and MɸDab2^−/−-tp female mice were fed a western diet for 9 (n=6,8) and 20 weeks (n=12, 10). Atherosclerotic lesion area in the aortic arch region was determined by quantification of Sudan IV staining. Data are presented as mean +/− s.e.m. *p<0.05 by one-way ANOVA with Tukey’s multiple comparison test. Representative images from mice fed western diet for 20 weeks are shown at right. Scale bar represents 2mm. B, Representative images of aortic root section from WT-tp and MՓDab2^−/−-tp mice stained with H&E. Quantification of lesion area in each group of mice is shown in bar graph. Representative images of necrotic regions (N) in the aortic root lesions. Quantification of necrotic regions is shown in bar graph. C, Atherosclerotic lesion area from WT-tp (n=12) and MɸDab2^−/−-tp (n=10) female mice fed a western diet for 20 weeks was quantified in aortic root sections stained with Oil Red O. Representative images shown on left. Scale bar represents 500 μm. Quantification is shown on right. Data are presented as mean +/− s.e.m. *p=0.06 by Student’s t-test. D, Representative images for aortic root sections at 20 weeks of WT-tp and MɸDab2^−/−-tp (n=12, 10) with picrosirius red staining for detection of collagen and smooth muscle α-actin immunostaining to determine smooth muscle cell content. Quantification of collagen and SMC content per vessel based on picrosirius red and SM α-actin staining, respectively, showed no significant differences between the two chimeric groups. Data are presented as mean +/− s.e.m. E, Representative images for aortic root sections at 20 weeks of WT-tp and MɸDab2^−/−-tp (n=12, 10) with Mac2 immunostaining for detection of macrophages and caspase immunostaining to measure apoptosis within the lesions. Quantification of percent Mac2 staining per lesion and percent caspase staining of vessel area are shown at left. p<0.02 and p<0.05, respectively by 2-tailed Student’s t-test. Data are presented as mean +/− s.e.m.