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Anti-inflammatory immune skewing is atheroprotective: Apoe−/−FcγRIIb−/− mice develop fibrous carotid plaques.

Harmon EY, Fronhofer V, Keller RS, Feustel PJ, Zhu X, Xu H, Avram D, Jones DM, Nagarajan S, Lennartz MR - J Am Heart Assoc (2014)

Bottom Line: Similarly, in vitro stimulation produced higher IL‐10 and Arg‐1 and lower iNOS, IL‐1β, and TNF‐α in DKO versus Apoe−/− macrophages.These results define a systemic anti‐inflammatory phenotype.However, we found that deletion of FcγRIIb on a congenic C57BL/6 background induces an anti‐inflammatory Treg/M2 polarization that is atheroprotective.

View Article: PubMed Central - PubMed

Affiliation: Centers for Cell Biology and Cancer Research, Albany Medical College, Albany, NY

ABSTRACT

Background: Stroke, caused by carotid plaque rupture, is a major cause of death in the United States. Whereas vulnerable human plaques have higher Fc receptor (FcγR) expression than their stable counterparts, how FcγR expression impacts plaque histology is unknown. We investigated the role of FcγRIIb in carotid plaque development and stability in apolipoprotein (Apo)e−/− and Apoe−/−FcγRIIb−/− double knockout (DKO) animals.

Methods and results: Plaques were induced by implantation of a shear stress‐modifying cast around the carotid artery. Plaque length and stenosis were followed longitudinally using ultrasound biomicroscopy. Immune status was determined by flow cytometry, cytokine release, immunoglobulin G concentration and analysis of macrophage polarization both in plaques and in vitro. Surprisingly, DKO animals had lower plaque burden in both carotid artery and descending aorta. Plaques from Apoe−/− mice were foam‐cell rich and resembled vulnerable human specimens, whereas those from DKO mice were fibrous and histologically stable. Plaques from DKO animals expressed higher arginase 1 (Arg‐1) and lower inducible nitric oxide synthase (iNOS), indicating the presence of M2 macrophages. Analysis of blood and cervical lymph nodes revealed higher interleukin (IL)‐10, immune complexes, and regulatory T cells (Tregs) and lower IL‐12, IL‐1β, and tumor necrosis factor alpha (TNF‐α) in DKO mice. Similarly, in vitro stimulation produced higher IL‐10 and Arg‐1 and lower iNOS, IL‐1β, and TNF‐α in DKO versus Apoe−/− macrophages. These results define a systemic anti‐inflammatory phenotype.

Conclusions: We hypothesized that removal of FcγRIIb would exacerbate atherosclerosis and generate unstable plaques. However, we found that deletion of FcγRIIb on a congenic C57BL/6 background induces an anti‐inflammatory Treg/M2 polarization that is atheroprotective.

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DKO plaques contain M2‐polarized macrophages. Representative images of CD68 (macrophages), iNOS (M1), and Arg‐1 (M2) staining in DKO and Apoe−/− plaques; scale bar=100 μm. Staining score: 3 sections from each plaque were stained for iNOS or Arg‐1. Staining was scored on a 0 to 3 scale (see Methods) for each protein and the averages plotted; each symbol represents the average score from 1 plaque. Group ranks were significantly different (iNOS lower, and Arg‐1 higher, in DKO plaques) by Mann–Whitney's nonparametric test; *P<0.05. (B) Expression of IL‐10, TGF‐β, and TNF‐α in plaques was quantified by qRT‐PCR. Data are presented as mean±SEM for 4 (Apoe−/−) or 5 (DKO) plaques. There were no significant differences between genotypes (independent sample t test). Apo indicates apolipoprotein; Arg‐1, arginase 1; DKO, double knockout; IL, interleukin; iNOS, inducible nitric oxide synthase; qRT‐PCR, quantitative real time‐polymerase chain reaction; TGF‐β, transforming growth factor beta; TNF‐α, tumor necrosis factor alpha.
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fig06: DKO plaques contain M2‐polarized macrophages. Representative images of CD68 (macrophages), iNOS (M1), and Arg‐1 (M2) staining in DKO and Apoe−/− plaques; scale bar=100 μm. Staining score: 3 sections from each plaque were stained for iNOS or Arg‐1. Staining was scored on a 0 to 3 scale (see Methods) for each protein and the averages plotted; each symbol represents the average score from 1 plaque. Group ranks were significantly different (iNOS lower, and Arg‐1 higher, in DKO plaques) by Mann–Whitney's nonparametric test; *P<0.05. (B) Expression of IL‐10, TGF‐β, and TNF‐α in plaques was quantified by qRT‐PCR. Data are presented as mean±SEM for 4 (Apoe−/−) or 5 (DKO) plaques. There were no significant differences between genotypes (independent sample t test). Apo indicates apolipoprotein; Arg‐1, arginase 1; DKO, double knockout; IL, interleukin; iNOS, inducible nitric oxide synthase; qRT‐PCR, quantitative real time‐polymerase chain reaction; TGF‐β, transforming growth factor beta; TNF‐α, tumor necrosis factor alpha.

Mentions: Histologically, large macrophages (MØ) with expanded ORO‐positive cytoplasm (foam cells) and smaller MØ have been associated with pro‐ (M1) and anti‐inflammatory (M2, Mhem, and Mox) polarization, respectively.25–26 M1 polarized MØ express iNOS, whereas M2 synthesize Arg‐1 (reviewed previously27). If the MØ in Apoe−/− plaques are M1 polarized, they should preferentially express iNOS. Conversely, DKO MØ should be enriched for Arg‐1. Three sections of each plaque were stained for iNOS or Arg‐1. The slides were given arbitrary numbers and scored by a lab mate blinded to both the genotype and identity of the stained protein. When the code was broken and the averages calculated, Apoe−/− plaques had significantly higher iNOS and lower Arg‐1 (Figure 6A), suggesting M1 and M2 polarization of Apoe−/− and DKO plaque MØ, respectively.


Anti-inflammatory immune skewing is atheroprotective: Apoe−/−FcγRIIb−/− mice develop fibrous carotid plaques.

Harmon EY, Fronhofer V, Keller RS, Feustel PJ, Zhu X, Xu H, Avram D, Jones DM, Nagarajan S, Lennartz MR - J Am Heart Assoc (2014)

DKO plaques contain M2‐polarized macrophages. Representative images of CD68 (macrophages), iNOS (M1), and Arg‐1 (M2) staining in DKO and Apoe−/− plaques; scale bar=100 μm. Staining score: 3 sections from each plaque were stained for iNOS or Arg‐1. Staining was scored on a 0 to 3 scale (see Methods) for each protein and the averages plotted; each symbol represents the average score from 1 plaque. Group ranks were significantly different (iNOS lower, and Arg‐1 higher, in DKO plaques) by Mann–Whitney's nonparametric test; *P<0.05. (B) Expression of IL‐10, TGF‐β, and TNF‐α in plaques was quantified by qRT‐PCR. Data are presented as mean±SEM for 4 (Apoe−/−) or 5 (DKO) plaques. There were no significant differences between genotypes (independent sample t test). Apo indicates apolipoprotein; Arg‐1, arginase 1; DKO, double knockout; IL, interleukin; iNOS, inducible nitric oxide synthase; qRT‐PCR, quantitative real time‐polymerase chain reaction; TGF‐β, transforming growth factor beta; TNF‐α, tumor necrosis factor alpha.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4338708&req=5

fig06: DKO plaques contain M2‐polarized macrophages. Representative images of CD68 (macrophages), iNOS (M1), and Arg‐1 (M2) staining in DKO and Apoe−/− plaques; scale bar=100 μm. Staining score: 3 sections from each plaque were stained for iNOS or Arg‐1. Staining was scored on a 0 to 3 scale (see Methods) for each protein and the averages plotted; each symbol represents the average score from 1 plaque. Group ranks were significantly different (iNOS lower, and Arg‐1 higher, in DKO plaques) by Mann–Whitney's nonparametric test; *P<0.05. (B) Expression of IL‐10, TGF‐β, and TNF‐α in plaques was quantified by qRT‐PCR. Data are presented as mean±SEM for 4 (Apoe−/−) or 5 (DKO) plaques. There were no significant differences between genotypes (independent sample t test). Apo indicates apolipoprotein; Arg‐1, arginase 1; DKO, double knockout; IL, interleukin; iNOS, inducible nitric oxide synthase; qRT‐PCR, quantitative real time‐polymerase chain reaction; TGF‐β, transforming growth factor beta; TNF‐α, tumor necrosis factor alpha.
Mentions: Histologically, large macrophages (MØ) with expanded ORO‐positive cytoplasm (foam cells) and smaller MØ have been associated with pro‐ (M1) and anti‐inflammatory (M2, Mhem, and Mox) polarization, respectively.25–26 M1 polarized MØ express iNOS, whereas M2 synthesize Arg‐1 (reviewed previously27). If the MØ in Apoe−/− plaques are M1 polarized, they should preferentially express iNOS. Conversely, DKO MØ should be enriched for Arg‐1. Three sections of each plaque were stained for iNOS or Arg‐1. The slides were given arbitrary numbers and scored by a lab mate blinded to both the genotype and identity of the stained protein. When the code was broken and the averages calculated, Apoe−/− plaques had significantly higher iNOS and lower Arg‐1 (Figure 6A), suggesting M1 and M2 polarization of Apoe−/− and DKO plaque MØ, respectively.

Bottom Line: Similarly, in vitro stimulation produced higher IL‐10 and Arg‐1 and lower iNOS, IL‐1β, and TNF‐α in DKO versus Apoe−/− macrophages.These results define a systemic anti‐inflammatory phenotype.However, we found that deletion of FcγRIIb on a congenic C57BL/6 background induces an anti‐inflammatory Treg/M2 polarization that is atheroprotective.

View Article: PubMed Central - PubMed

Affiliation: Centers for Cell Biology and Cancer Research, Albany Medical College, Albany, NY

ABSTRACT

Background: Stroke, caused by carotid plaque rupture, is a major cause of death in the United States. Whereas vulnerable human plaques have higher Fc receptor (FcγR) expression than their stable counterparts, how FcγR expression impacts plaque histology is unknown. We investigated the role of FcγRIIb in carotid plaque development and stability in apolipoprotein (Apo)e−/− and Apoe−/−FcγRIIb−/− double knockout (DKO) animals.

Methods and results: Plaques were induced by implantation of a shear stress‐modifying cast around the carotid artery. Plaque length and stenosis were followed longitudinally using ultrasound biomicroscopy. Immune status was determined by flow cytometry, cytokine release, immunoglobulin G concentration and analysis of macrophage polarization both in plaques and in vitro. Surprisingly, DKO animals had lower plaque burden in both carotid artery and descending aorta. Plaques from Apoe−/− mice were foam‐cell rich and resembled vulnerable human specimens, whereas those from DKO mice were fibrous and histologically stable. Plaques from DKO animals expressed higher arginase 1 (Arg‐1) and lower inducible nitric oxide synthase (iNOS), indicating the presence of M2 macrophages. Analysis of blood and cervical lymph nodes revealed higher interleukin (IL)‐10, immune complexes, and regulatory T cells (Tregs) and lower IL‐12, IL‐1β, and tumor necrosis factor alpha (TNF‐α) in DKO mice. Similarly, in vitro stimulation produced higher IL‐10 and Arg‐1 and lower iNOS, IL‐1β, and TNF‐α in DKO versus Apoe−/− macrophages. These results define a systemic anti‐inflammatory phenotype.

Conclusions: We hypothesized that removal of FcγRIIb would exacerbate atherosclerosis and generate unstable plaques. However, we found that deletion of FcγRIIb on a congenic C57BL/6 background induces an anti‐inflammatory Treg/M2 polarization that is atheroprotective.

Show MeSH
Related in: MedlinePlus