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PI3K p110γ deletion attenuates murine atherosclerosis by reducing macrophage proliferation but not polarization or apoptosis in lesions.

Zotes TM, Arias CF, Fuster JJ, Spada R, Pérez-Yagüe S, Hirsch E, Wymann M, Carrera AC, Andrés V, Barber DF - PLoS ONE (2013)

Bottom Line: Atherosclerosis is an inflammatory disease regulated by infiltrating monocytes and T cells, among other cell types.We analyzed atherosclerosis development in LDLR(-/-)p110γ(+/-) and LDLR(-/-)p110γ(-/-) mice, and performed expression and functional assays in tissues and primary cells from these and from p110γ(+/-) and p110γ(-/-) mice.Lack of p110γ in LDLR(-/-) mice reduces the atherosclerosis burden.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Oncology, Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas (CNB-CSIC), Madrid, Spain.

ABSTRACT
Atherosclerosis is an inflammatory disease regulated by infiltrating monocytes and T cells, among other cell types. Macrophage recruitment to atherosclerotic lesions is controlled by monocyte infiltration into plaques. Once in the lesion, macrophage proliferation in situ, apoptosis, and differentiation to an inflammatory (M1) or anti-inflammatory phenotype (M2) are involved in progression to advanced atherosclerotic lesions. We studied the role of phosphoinositol-3-kinase (PI3K) p110γ in the regulation of in situ apoptosis, macrophage proliferation and polarization towards M1 or M2 phenotypes in atherosclerotic lesions. We analyzed atherosclerosis development in LDLR(-/-)p110γ(+/-) and LDLR(-/-)p110γ(-/-) mice, and performed expression and functional assays in tissues and primary cells from these and from p110γ(+/-) and p110γ(-/-) mice. Lack of p110γ in LDLR(-/-) mice reduces the atherosclerosis burden. Atherosclerotic lesions in fat-fed LDLR(-/-)p110γ(-/-) mice were smaller than in LDLR(-/-)p110γ(+/-) controls, which coincided with decreased macrophage proliferation in LDLR(-/-)p110γ(-/-) mouse lesions. This proliferation defect was also observed in p110γ(-/-) bone marrow-derived macrophages (BMM) stimulated with macrophage colony-stimulating factor (M-CSF), and was associated with higher intracellular cyclic adenosine monophosphate (cAMP) levels. In contrast, T cell proliferation was unaffected in LDLR(-/-)p110γ(-/-) mice. Moreover, p110γ deficiency did not affect macrophage polarization towards the M1 or M2 phenotypes or apoptosis in atherosclerotic plaques, or polarization in cultured BMM. Our results suggest that higher cAMP levels and the ensuing inhibition of macrophage proliferation contribute to atheroprotection in LDLR(-/-) mice lacking p110γ. Nonetheless, p110γ deletion does not appear to be involved in apoptosis, in macrophage polarization or in T cell proliferation.

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M1 and M2 macrophage populations are similar in LDLR−/−p110γ+/− and LDLR-/−p110γ−/− mice.(A) Representative photomicrographs of immunofluorescent-stained M1 (iNOS+) and M2 (arginase1+) macrophages in aortic sinus sections from LDLR−/−p110γ+/− and LDLR−/−p110γ−/− mice fed a two-month high-fat diet (n = 5/genotype). Bar = 50 μm. (B) Quantification of the percentage of M1+ (iNOS+), M2+ (arginase1+) and M1+M2+ (iNOS+arginase1+) macrophage subsets relative to total macrophages in aortic plaques. Mean ± SD; Student’s t-test. Expression of M1 (iNOS, IL-12) and M2 markers (arginase1, YM1, IL-10) was analyzed by qRT-PCR in BMM stimulated with (C) IFNγ+LPS (M1) (n = 4 experiments, 3 mice/genotype) or (D) IL-4 (M2) (n = 4 experiments, 3 mice/genotype); marker expression is shown as RQ values. RQ = 2–ΔCt.
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pone-0072674-g005: M1 and M2 macrophage populations are similar in LDLR−/−p110γ+/− and LDLR-/−p110γ−/− mice.(A) Representative photomicrographs of immunofluorescent-stained M1 (iNOS+) and M2 (arginase1+) macrophages in aortic sinus sections from LDLR−/−p110γ+/− and LDLR−/−p110γ−/− mice fed a two-month high-fat diet (n = 5/genotype). Bar = 50 μm. (B) Quantification of the percentage of M1+ (iNOS+), M2+ (arginase1+) and M1+M2+ (iNOS+arginase1+) macrophage subsets relative to total macrophages in aortic plaques. Mean ± SD; Student’s t-test. Expression of M1 (iNOS, IL-12) and M2 markers (arginase1, YM1, IL-10) was analyzed by qRT-PCR in BMM stimulated with (C) IFNγ+LPS (M1) (n = 4 experiments, 3 mice/genotype) or (D) IL-4 (M2) (n = 4 experiments, 3 mice/genotype); marker expression is shown as RQ values. RQ = 2–ΔCt.

Mentions: cAMP is linked to macrophage transition to the M2 phenotype [36]. As we observed increased cAMP levels in LDLR−/−p110γ−/− BMM, we analyzed macrophage polarization in atherosclerotic lesions. Immunofluorescence experiments showed no significant differences in the percentage of M1 (iNOS+) and M2 (arginase1+) macrophages in aortic sinus cross-sections from LDLR−/−p110γ+/− and LDLR-/−p110γ−/− mice (Figure 5A, 5B), although there was a tendency toward more M2 macrophages in LDLR−/−p110γ−/− mice. Consistent with this finding, qRT-PCR studies of p110γ+/− and p110γ−/− mouse BMM stimulated in vitro (24 h) towards the M1 (IFNγ+LPS) or M2 phenotypes (IL-4) showed no significant differences in M1 (iNOS, IL-12) and M2 (arginase1, IL-10, YM1) marker expression (Figure 5C, 5D).


PI3K p110γ deletion attenuates murine atherosclerosis by reducing macrophage proliferation but not polarization or apoptosis in lesions.

Zotes TM, Arias CF, Fuster JJ, Spada R, Pérez-Yagüe S, Hirsch E, Wymann M, Carrera AC, Andrés V, Barber DF - PLoS ONE (2013)

M1 and M2 macrophage populations are similar in LDLR−/−p110γ+/− and LDLR-/−p110γ−/− mice.(A) Representative photomicrographs of immunofluorescent-stained M1 (iNOS+) and M2 (arginase1+) macrophages in aortic sinus sections from LDLR−/−p110γ+/− and LDLR−/−p110γ−/− mice fed a two-month high-fat diet (n = 5/genotype). Bar = 50 μm. (B) Quantification of the percentage of M1+ (iNOS+), M2+ (arginase1+) and M1+M2+ (iNOS+arginase1+) macrophage subsets relative to total macrophages in aortic plaques. Mean ± SD; Student’s t-test. Expression of M1 (iNOS, IL-12) and M2 markers (arginase1, YM1, IL-10) was analyzed by qRT-PCR in BMM stimulated with (C) IFNγ+LPS (M1) (n = 4 experiments, 3 mice/genotype) or (D) IL-4 (M2) (n = 4 experiments, 3 mice/genotype); marker expression is shown as RQ values. RQ = 2–ΔCt.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3750002&req=5

pone-0072674-g005: M1 and M2 macrophage populations are similar in LDLR−/−p110γ+/− and LDLR-/−p110γ−/− mice.(A) Representative photomicrographs of immunofluorescent-stained M1 (iNOS+) and M2 (arginase1+) macrophages in aortic sinus sections from LDLR−/−p110γ+/− and LDLR−/−p110γ−/− mice fed a two-month high-fat diet (n = 5/genotype). Bar = 50 μm. (B) Quantification of the percentage of M1+ (iNOS+), M2+ (arginase1+) and M1+M2+ (iNOS+arginase1+) macrophage subsets relative to total macrophages in aortic plaques. Mean ± SD; Student’s t-test. Expression of M1 (iNOS, IL-12) and M2 markers (arginase1, YM1, IL-10) was analyzed by qRT-PCR in BMM stimulated with (C) IFNγ+LPS (M1) (n = 4 experiments, 3 mice/genotype) or (D) IL-4 (M2) (n = 4 experiments, 3 mice/genotype); marker expression is shown as RQ values. RQ = 2–ΔCt.
Mentions: cAMP is linked to macrophage transition to the M2 phenotype [36]. As we observed increased cAMP levels in LDLR−/−p110γ−/− BMM, we analyzed macrophage polarization in atherosclerotic lesions. Immunofluorescence experiments showed no significant differences in the percentage of M1 (iNOS+) and M2 (arginase1+) macrophages in aortic sinus cross-sections from LDLR−/−p110γ+/− and LDLR-/−p110γ−/− mice (Figure 5A, 5B), although there was a tendency toward more M2 macrophages in LDLR−/−p110γ−/− mice. Consistent with this finding, qRT-PCR studies of p110γ+/− and p110γ−/− mouse BMM stimulated in vitro (24 h) towards the M1 (IFNγ+LPS) or M2 phenotypes (IL-4) showed no significant differences in M1 (iNOS, IL-12) and M2 (arginase1, IL-10, YM1) marker expression (Figure 5C, 5D).

Bottom Line: Atherosclerosis is an inflammatory disease regulated by infiltrating monocytes and T cells, among other cell types.We analyzed atherosclerosis development in LDLR(-/-)p110γ(+/-) and LDLR(-/-)p110γ(-/-) mice, and performed expression and functional assays in tissues and primary cells from these and from p110γ(+/-) and p110γ(-/-) mice.Lack of p110γ in LDLR(-/-) mice reduces the atherosclerosis burden.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Oncology, Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas (CNB-CSIC), Madrid, Spain.

ABSTRACT
Atherosclerosis is an inflammatory disease regulated by infiltrating monocytes and T cells, among other cell types. Macrophage recruitment to atherosclerotic lesions is controlled by monocyte infiltration into plaques. Once in the lesion, macrophage proliferation in situ, apoptosis, and differentiation to an inflammatory (M1) or anti-inflammatory phenotype (M2) are involved in progression to advanced atherosclerotic lesions. We studied the role of phosphoinositol-3-kinase (PI3K) p110γ in the regulation of in situ apoptosis, macrophage proliferation and polarization towards M1 or M2 phenotypes in atherosclerotic lesions. We analyzed atherosclerosis development in LDLR(-/-)p110γ(+/-) and LDLR(-/-)p110γ(-/-) mice, and performed expression and functional assays in tissues and primary cells from these and from p110γ(+/-) and p110γ(-/-) mice. Lack of p110γ in LDLR(-/-) mice reduces the atherosclerosis burden. Atherosclerotic lesions in fat-fed LDLR(-/-)p110γ(-/-) mice were smaller than in LDLR(-/-)p110γ(+/-) controls, which coincided with decreased macrophage proliferation in LDLR(-/-)p110γ(-/-) mouse lesions. This proliferation defect was also observed in p110γ(-/-) bone marrow-derived macrophages (BMM) stimulated with macrophage colony-stimulating factor (M-CSF), and was associated with higher intracellular cyclic adenosine monophosphate (cAMP) levels. In contrast, T cell proliferation was unaffected in LDLR(-/-)p110γ(-/-) mice. Moreover, p110γ deficiency did not affect macrophage polarization towards the M1 or M2 phenotypes or apoptosis in atherosclerotic plaques, or polarization in cultured BMM. Our results suggest that higher cAMP levels and the ensuing inhibition of macrophage proliferation contribute to atheroprotection in LDLR(-/-) mice lacking p110γ. Nonetheless, p110γ deletion does not appear to be involved in apoptosis, in macrophage polarization or in T cell proliferation.

Show MeSH
Related in: MedlinePlus