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Deficient CD40-TRAF6 signaling in leukocytes prevents atherosclerosis by skewing the immune response toward an antiinflammatory profile.

Lutgens E, Lievens D, Beckers L, Wijnands E, Soehnlein O, Zernecke A, Seijkens T, Engel D, Cleutjens J, Keller AM, Naik SH, Boon L, Oufella HA, Mallat Z, Ahonen CL, Noelle RJ, de Winther MP, Daemen MJ, Biessen EA, Weber C - J. Exp. Med. (2010)

Bottom Line: The CD40-CD40 ligand (CD40L) signaling axis plays an important role in immunological pathways.Conceivably, more targeted intervention strategies in CD40 signaling will have less deleterious side effects.Mice with defective CD40-TRAF6 signaling display a reduced blood count of Ly6C(high) monocytes, an impaired recruitment of Ly6C(+) monocytes to the arterial wall, and polarization of macrophages toward an antiinflammatory regulatory M2 signature.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, Cardiovascular Research Institute Maastricht, University of Maastricht, Maastricht 6200 MD, Netherlands. E.Lutgens@path.unimaas.nl

ABSTRACT
The CD40-CD40 ligand (CD40L) signaling axis plays an important role in immunological pathways. Consequently, this dyad is involved in chronic inflammatory diseases, including atherosclerosis. Inhibition of CD40L in apolipoprotein E (Apoe)-deficient (Apoe(-/-)) mice not only reduced atherosclerosis but also conferred a clinically favorable plaque phenotype that was low in inflammation and high in fibrosis. Blockade of CD40L may not be therapeutically feasible, as long-term inhibition will compromise systemic immune responses. Conceivably, more targeted intervention strategies in CD40 signaling will have less deleterious side effects. We report that deficiency in hematopoietic CD40 reduces atherosclerosis and induces features of plaque stability. To elucidate the role of CD40-tumor necrosis factor receptor-associated factor (TRAF) signaling in atherosclerosis, we examined disease progression in mice deficient in CD40 and its associated signaling intermediates. Absence of CD40-TRAF6 but not CD40-TRAF2/3/5 signaling abolishes atherosclerosis and confers plaque fibrosis in Apoe(-/-) mice. Mice with defective CD40-TRAF6 signaling display a reduced blood count of Ly6C(high) monocytes, an impaired recruitment of Ly6C(+) monocytes to the arterial wall, and polarization of macrophages toward an antiinflammatory regulatory M2 signature. These data unveil a role for CD40-TRAF6, but not CD40-TRAF2/3/5, interactions in atherosclerosis and establish that targeting specific components of the CD40-CD40L pathway harbors the potential to achieve therapeutic effects in atherosclerosis.

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Deficiency in CD40-TRAF6 interactions induces polarization of macrophages toward a regulatory M2 macrophage phenotype. (a–c) BM-derived macrophages of CD40-Twt, CD40-T2/3/5−/−, CD40-T6−/−, and CD40-T2/3/5/6−/− mice were cultured, stimulated with 1 µg/ml of the CD40-clustering antibody FGK45, and analyzed for expression of different M1 (a) and M2 (b, wound-healing macrophages; c, regulatory macrophages) subset markers by real-time PCR. n = 6 per group of two independent experiments. (d) The levels of IκBα in FGK45-stimulated macrophages were decreased in BM-derived macrophages of CD40-T6−/− and CD40-T2/3/5/6−/− mice (n = 6). Error bars represent mean ± SEM. *, P < 0.05.
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fig8: Deficiency in CD40-TRAF6 interactions induces polarization of macrophages toward a regulatory M2 macrophage phenotype. (a–c) BM-derived macrophages of CD40-Twt, CD40-T2/3/5−/−, CD40-T6−/−, and CD40-T2/3/5/6−/− mice were cultured, stimulated with 1 µg/ml of the CD40-clustering antibody FGK45, and analyzed for expression of different M1 (a) and M2 (b, wound-healing macrophages; c, regulatory macrophages) subset markers by real-time PCR. n = 6 per group of two independent experiments. (d) The levels of IκBα in FGK45-stimulated macrophages were decreased in BM-derived macrophages of CD40-T6−/− and CD40-T2/3/5/6−/− mice (n = 6). Error bars represent mean ± SEM. *, P < 0.05.

Mentions: BM-derived macrophages of CD40-T6−/− mice stimulated with FGK45 produced less IL-12 and iNOS but increased amounts of IL-10 (Fig. 8), indicating that CD40-TRAF6 deficiency skews macrophage polarization toward an antiinflammatory M2 phenotype. Further subtyping of the M2 macrophages into wound-healing and regulatory macrophages (Mosser and Edwards, 2008) showed that CD40–TRAF6 interactions did not affect the wound-healing macrophage markers YM-1, RELM-α, arginase I, IGF1, or DCIR but markedly dampened the M1 polarization response and, rather, induced a regulatory macrophage subtype that produces high amounts of IL-10. Importantly, the process of monocyte recruitment driven by CD40-TRAF6 was mediated by IL-10, as treatment of CD40-T6−/− mice with a blocking antibody to IL-10 reversed the protective effect on Ly6C+ cell numbers and adhesion (Fig. 7 d). The phenotype that occurs in CD40-T6−/− mice is likely associated with an impaired NF-κB signaling because IκBα levels in macrophages were markedly decreased (Fig. 8 d). Consistent with findings in CD40-deficient macrophages, CD40-T6−/− macrophages displayed a reduced gelatinolytic activity and increased expression of TIMP-1 (Fig. S5, c and d). Flow cytometry analysis further revealed reduced numbers of CD4+CD44highCD62Llow promigratory effector memory T lymphocytes and plasmacytoid DCs (Figs. S3, c and d).


Deficient CD40-TRAF6 signaling in leukocytes prevents atherosclerosis by skewing the immune response toward an antiinflammatory profile.

Lutgens E, Lievens D, Beckers L, Wijnands E, Soehnlein O, Zernecke A, Seijkens T, Engel D, Cleutjens J, Keller AM, Naik SH, Boon L, Oufella HA, Mallat Z, Ahonen CL, Noelle RJ, de Winther MP, Daemen MJ, Biessen EA, Weber C - J. Exp. Med. (2010)

Deficiency in CD40-TRAF6 interactions induces polarization of macrophages toward a regulatory M2 macrophage phenotype. (a–c) BM-derived macrophages of CD40-Twt, CD40-T2/3/5−/−, CD40-T6−/−, and CD40-T2/3/5/6−/− mice were cultured, stimulated with 1 µg/ml of the CD40-clustering antibody FGK45, and analyzed for expression of different M1 (a) and M2 (b, wound-healing macrophages; c, regulatory macrophages) subset markers by real-time PCR. n = 6 per group of two independent experiments. (d) The levels of IκBα in FGK45-stimulated macrophages were decreased in BM-derived macrophages of CD40-T6−/− and CD40-T2/3/5/6−/− mice (n = 6). Error bars represent mean ± SEM. *, P < 0.05.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2822598&req=5

fig8: Deficiency in CD40-TRAF6 interactions induces polarization of macrophages toward a regulatory M2 macrophage phenotype. (a–c) BM-derived macrophages of CD40-Twt, CD40-T2/3/5−/−, CD40-T6−/−, and CD40-T2/3/5/6−/− mice were cultured, stimulated with 1 µg/ml of the CD40-clustering antibody FGK45, and analyzed for expression of different M1 (a) and M2 (b, wound-healing macrophages; c, regulatory macrophages) subset markers by real-time PCR. n = 6 per group of two independent experiments. (d) The levels of IκBα in FGK45-stimulated macrophages were decreased in BM-derived macrophages of CD40-T6−/− and CD40-T2/3/5/6−/− mice (n = 6). Error bars represent mean ± SEM. *, P < 0.05.
Mentions: BM-derived macrophages of CD40-T6−/− mice stimulated with FGK45 produced less IL-12 and iNOS but increased amounts of IL-10 (Fig. 8), indicating that CD40-TRAF6 deficiency skews macrophage polarization toward an antiinflammatory M2 phenotype. Further subtyping of the M2 macrophages into wound-healing and regulatory macrophages (Mosser and Edwards, 2008) showed that CD40–TRAF6 interactions did not affect the wound-healing macrophage markers YM-1, RELM-α, arginase I, IGF1, or DCIR but markedly dampened the M1 polarization response and, rather, induced a regulatory macrophage subtype that produces high amounts of IL-10. Importantly, the process of monocyte recruitment driven by CD40-TRAF6 was mediated by IL-10, as treatment of CD40-T6−/− mice with a blocking antibody to IL-10 reversed the protective effect on Ly6C+ cell numbers and adhesion (Fig. 7 d). The phenotype that occurs in CD40-T6−/− mice is likely associated with an impaired NF-κB signaling because IκBα levels in macrophages were markedly decreased (Fig. 8 d). Consistent with findings in CD40-deficient macrophages, CD40-T6−/− macrophages displayed a reduced gelatinolytic activity and increased expression of TIMP-1 (Fig. S5, c and d). Flow cytometry analysis further revealed reduced numbers of CD4+CD44highCD62Llow promigratory effector memory T lymphocytes and plasmacytoid DCs (Figs. S3, c and d).

Bottom Line: The CD40-CD40 ligand (CD40L) signaling axis plays an important role in immunological pathways.Conceivably, more targeted intervention strategies in CD40 signaling will have less deleterious side effects.Mice with defective CD40-TRAF6 signaling display a reduced blood count of Ly6C(high) monocytes, an impaired recruitment of Ly6C(+) monocytes to the arterial wall, and polarization of macrophages toward an antiinflammatory regulatory M2 signature.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, Cardiovascular Research Institute Maastricht, University of Maastricht, Maastricht 6200 MD, Netherlands. E.Lutgens@path.unimaas.nl

ABSTRACT
The CD40-CD40 ligand (CD40L) signaling axis plays an important role in immunological pathways. Consequently, this dyad is involved in chronic inflammatory diseases, including atherosclerosis. Inhibition of CD40L in apolipoprotein E (Apoe)-deficient (Apoe(-/-)) mice not only reduced atherosclerosis but also conferred a clinically favorable plaque phenotype that was low in inflammation and high in fibrosis. Blockade of CD40L may not be therapeutically feasible, as long-term inhibition will compromise systemic immune responses. Conceivably, more targeted intervention strategies in CD40 signaling will have less deleterious side effects. We report that deficiency in hematopoietic CD40 reduces atherosclerosis and induces features of plaque stability. To elucidate the role of CD40-tumor necrosis factor receptor-associated factor (TRAF) signaling in atherosclerosis, we examined disease progression in mice deficient in CD40 and its associated signaling intermediates. Absence of CD40-TRAF6 but not CD40-TRAF2/3/5 signaling abolishes atherosclerosis and confers plaque fibrosis in Apoe(-/-) mice. Mice with defective CD40-TRAF6 signaling display a reduced blood count of Ly6C(high) monocytes, an impaired recruitment of Ly6C(+) monocytes to the arterial wall, and polarization of macrophages toward an antiinflammatory regulatory M2 signature. These data unveil a role for CD40-TRAF6, but not CD40-TRAF2/3/5, interactions in atherosclerosis and establish that targeting specific components of the CD40-CD40L pathway harbors the potential to achieve therapeutic effects in atherosclerosis.

Show MeSH
Related in: MedlinePlus