Immunoglobulin M is required for protection against atherosclerosis in low-density lipoprotein receptor-deficient mice - PubMed (original) (raw)

Immunoglobulin M is required for protection against atherosclerosis in low-density lipoprotein receptor-deficient mice

Myles J Lewis et al. Circulation. 2009.

Abstract

Background: Immunoglobulin M (IgM) natural antibodies bind oxidatively-modified low-density lipoprotein (LDL) and apoptotic cells and have been implicated as being important for protection against atherosclerosis. We have directly investigated the requirement for IgM by studying the effects of IgM deficiency in LDL receptor-deficient (Ldlr(-/-)) mice.

Methods and results: Mice deficient in serum IgM (sIgM) or complement C1q were crossed with Ldlr(-/-) mice and studied on both low-fat and high-fat semisynthetic diets. On both diets, en face and aortic root atherosclerotic lesions in sIgM.Ldlr(-/-) mice were substantially larger and more complex, with accelerated cholesterol crystal formation and increased smooth muscle cell content in aortic root lesions. Combined C1q and IgM deficiency had the same effect as IgM deficiency alone. Increased apoptosis was observed in aortic root lesions of both sIgM.Ldlr(-/-) and C1qa.Ldlr(-/-) mice. Because lesions were significantly larger in IgM-deficient mice than in the absence of C1q, IgM protective mechanisms appear to be partially independent of classical pathway activation and apoptotic cell clearance. Levels of IgG antibodies against copper-oxidized LDL were lower in sIgM.Ldlr(-/-) mice fed a high-fat diet, suggesting compensatory consumption of IgG in the absence of IgM.

Conclusions: This study provides direct evidence that IgM antibodies play a central role in protection against atherosclerosis. The mechanism appears to be at least partly independent of classical pathway complement activation by C1q.

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Figures

Figure 1

Figure 1. Deposition of IgM in atherosclerotic lesions

Confocal images of aortic root sections double-immunostained for CD68 (green) and IgM (red) and counterstained with TOPRO nuclear dye (blue). (A) Low power view of aortic root lesion from LF-fed Ldlr−/− mouse showing IgM deposition in the acellular base of the lesion and its relationship to lesional macrophages. (B) Low power view of similar lesion from LF-fed sIgM.Ldlr−/− mouse showing complete absence of IgM.

Figure 2

Figure 2. Accelerated atherosclerotic lesion formation in aortic roots of sIgM deficient mice

(A) Representative sections from Ldlr−/−, C1qa.Ldlr−/−, sIgM.Ldlr−/− and C1qa.sIgM.Ldlr−/− mice stained with Oil Red O and haematoxylin. Scale bars 500μm. (B, C) Cross-sectional aortic root lesion area fraction (%) on (B) LF or (C) HF diet. Each point represents the mean of 5 sections per mouse, and bars show overall median. *P<0.05, **P<0.01, ***P<0.001 by Kruskal-Wallis test with Dunn's post-test.

Figure 3

Figure 3. Accelerated lesion formation in en face aorta preparations of sIgM deficient mice

(A, C) Representative Sudan IV-stained en face aorta preparations from Ldlr−/−, C1qa.Ldlr−/−, sIgM.Ldlr−/− and C1qa.sIgM.Ldlr−/− mice following 12 weeks of (A) LF or (C) HF diet. Scale bars 0.5mm. (B, D) Quantification of en face aorta lesion area expressed as % lesion area fraction on (B) LF or (D) HF diet, with bars showing the median. *P<0.05, **P<0.01, ***P<0.001 by Kruskal-Wallis test with Dunn's post-test.

Figure 4

Figure 4. Increased complexity of aortic root lesions in sIgM deficient mice

MOMA-2 positive macrophages, α-smooth muscle actin positive VSMC and CD3+ T cells expressed as % of lesional cells on (A) low fat (Ldlr−/− n=12, sIgM.Ldlr−/− n=14) and (B) high fat diet (Ldlr−/− n=12, sIgM.Ldlr−/− n=15). Density of aortic root lesion IgG and C3 deposition measured as mean fluorescence intensity per pixel on (A) low fat and (B) high fat diet (n numbers as before), expressed as relative fluorescence units (RFU) per pixel. Bars represent median, and statistical analysis was by Mann-Whitney test. (C) Representative photomicrographs of aortic root lesions showing cholesterol crystals under polarizing microscopy (age 22 wk, LF diet), smooth muscle fibrous cap formation (blue), lesional IgG and C3 deposition using fluorescence microscopy on HF diet in sIgM.Ldlr−/− compared to Ldlr−/− mice. Autofluorescence using a FITC-conjugated isotype control is shown below. Scale bars 100μm.

Figure 5

Figure 5. Increased apoptosis in atherosclerotic lesions of LF-fed C1qa.Ldlr−/−, sIgM.Ldlr−/− and C1qa.sIgM.Ldlr−/− mice

(A) Representative photomicrographs of TUNEL staining (brown) from LF-fed mice, counterstained with haematoxylin, showing few apoptotic cells in Ldlr−/− mice, but increased lesional apoptotic cells in C1qa.Ldlr−/−, sIgM.Ldlr−/− and C1qa.sIgM.Ldlr−/− mice. Scale bars 50μm. Quantification of lesional apoptotic cells on basis of TUNEL positive staining and morphological nuclear changes consistent with apoptosis on (B) LF diet and (C) HF diet, expressed as % of total lesional nuclei. Bars represent median. **P<0.01 by Kruskall-Wallis post-test.

Figure 6

Figure 6. IgG and IgM anti-oxLDL antibodies in Ldlr−/− and sIgM.Ldlr−/− mice

Serial dilution curves showing titres of (A) anti-MDA-LDL and (B) anti-CuOxLDL antibodies in Ldlr−/− and sIgM.Ldlr−/− mice on low and high fat diets at 22 weeks of age. Non-specific binding to native LDL is shown at a single dilution. Error bars show SEM. †P<0.05 sIgM.Ldlr−/− (LF) vs. Ldlr−/− (LF). **P<0.01 sIgM.Ldlr−/− (HF) vs. Ldlr−/− (HF). §P<0.05, §§P<0.01, §§§P<0.001 Ldlr−/− (LF) vs. Ldlr−/− (HF). Statistical analysis by unpaired t-test with Bonferroni correction for multiple comparisons.

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