Macrophage Plasticity and Polarization Are Altered in the Experimental Model of Multiple Sclerosis

Abstract

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system. MS is characterized by infiltrations of leukocytes such as T and B lymphocytes and macrophages. Macrophages have been identified as major effectors of inflammation and demyelination in both MS and its animal model, experimental autoimmune encephalomyelitis (EAE). However, the activation and heterogeneity of macrophages in MS has been poorly investigated. Thus, in this study, we evaluated M1 and M2 macrophages immunophenotype from EAE and control mice by analyzing over 30 surface and intracellular markers through polychromatic flow cytometry, qRT-PCR, and ELISA assay. We showed that M1 macrophages possessed a higher proinflammatory profile in EAE compared to control mice, since they expressed higher levels of activation/co-stimulatory markers (iNOS, CD40, and CD80) and cytokines/chemokines (IL-6, IL-12, CCL2, and CXCL10), whereas M2 lost their M2-like phenotype by showing a decreased expression of their signature markers CD206 and CCL22, as well as a concomitant upregulation of several M1 makers. Furthermore, immunization of M1 and M2 macrophages with MOG35-55 led to a significant hyperactivation of M1 and a concomitant shift of anti-inflammatory M2 to pro-inflammatory M1 macrophages. Overall, we provide evidence for a phenotypic alteration of M1/M2 balance during MS, which can be of crucial importance not only for a better understanding of the immunopathology of this neurodegenerative disease but also to potentially develop new macrophage-centered therapeutic strategies.

Keywords: EAE; chemokines; cytokines; macrophages; multiple sclerosis; toll-like receptors.

Figure 1

Immunophenotype of M1 and M2 macrophages obtained from CFA mice. (A) Monocytes obtained from spleen of CFA mice were differentiated into macrophages using M-CSF (50 ng/mL) for 6 days and then polarized into M1 with IFN-γ (10 ng/mL) plus LPS (1 μg/mL) or into M2 with IL-4 (20 ng/mL) for two more days. (B) Representative flow cytometry plot showing the gating strategy to identify monocyte-derived macrophages (C) Expression of markers by flow cytometry upon staining cells at cell surface (CD40, CD11b, MHC-II, CD80, CD86, CD25, CD206, CD11c, CD62L, CD44, CD115, and CCR5) or intracellularly (iNOS). Data are reported as percentage of positive cells and are representative of 10 independent experiments ± SEM * p < 0.05; ** p < 0.01.

Figure 2

Expression of cytokines/chemokines and TLRs in M1 and M2 macrophages obtained from CFA mice. (A) ELISA of cytokines (IL-1β, IL-6, IL-12, TNF-α, IL-10) and chemokines (CCL2, CCL3, CCL4, CCL22, CXCL9, and CXCL10) in supernatants of M1 or M2 macrophages. Data are reported as pg/mL and represent 10 independent experiments means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. (B,C) qRT-PCR analysis of the expression of both bacterial (TLR1, TLR2, TLR4, TLR5, TLR6) (B) and viral (TLR3, TLR7, TLR8, TLR9) (C) Toll-like receptors in M1 and M2 macrophages. Cycling threshold values are normalized to those of mRNA encoding β2-microglobulin and data are normalized to the maximum value obtained for each donor. Data are expressed as arbitrary units (A.U.) and are shown as means ± SEM of eight independent experiments each in duplicate. * p < 0.05.

Figure 3

Immunophenotype of M1 macrophages obtained from CFA and EAE mice. Expression of markers by flow cytometry upon staining cells at cell surface (CD40, CD80 and CD206) or intracellularly (iNOS) in M1 and M2 macrophages obtained from CFA and EAE mice. Data are reported as percentage of positive cells and are representative of eight independent experiments ± SEM * p < 0.05, ** p < 0.01.

Figure 4

Expression of cytokines/chemokines and TLRs in M1 obtained from CFA and EAE mice. (A) ELISA of cytokines (IL-1β, IL-6, IL-12, TNF-α, IL-10) and chemokines (CCL2, CCL3, CXCL9, and CXCL10) in supernatants of M1 macrophages. Data are reported as pg/mL and represent 10 independent experiments means ± SEM. * p < 0.05 (B,C) qRT-PCR analysis of the expression of both bacterial (TLR1, TLR2, TLR4, TLR5, TLR6) (B) and viral (TLR3, TLR7, TLR8, TLR9) (C) Toll-like receptors in M1 macrophages. Cycling threshold values are normalized to those of mRNA encoding β2-microglobulin, and data are normalized to the maximum value obtained for each donor. Data are expressed as arbitrary units (A.U.) and are shown as means ± SEM of eight independent experiments each in duplicate. * p < 0.05.

Figure 5

Immunophenotype of M2 macrophages obtained from CFA and EAE mice. Monocytes obtained from spleen of CFA and EAE mice were differentiated into macrophages using M-CSF (50 ng/mL) for 6 days and then polarized into M2 with IL-4 (20 ng/mL) for 2 more days. Expression of markers by flow cytometry upon staining cells at cell surface (CD40, MHC-II, CD80, CD86, CD206, CD11c, and CD44) or intracellularly (iNOS). Data are reported as percentage of positive cells and are representative of eight independent experiments ± SEM. * p < 0.05, ** p < 0.01.

Figure 6

Expression of cytokines/chemokines and TLRs in M2 obtained from CFA and EAE mice. (A) ELISA of chemokines (CCL2, CCL3, CCL4, and CCL22) in supernatants of M2 macrophages. Data are reported as pg/mL and represent eight independent experiments means ± SEM. * p < 0.05 (B,C) qRT-PCR analysis of the expression of both bacterial (TLR1, TLR2, TLR4, TLR5, TLR6) (B) and viral (TLR3, TLR7, TLR8, TLR9) (C) Toll-like receptors in M2 macrophages. Cycling threshold values are normalized to those of mRNA encoding β2-microglobulin, and data are normalized to the maximum value obtained for each donor. Data are expressed as arbitrary units (A.U.) and are shown as means ± SEM of eight independent experiments each in duplicate. * p < 0.05.

Figure 7

Immunophenotype of MOG-immunized M1 and M2 macrophages obtained from CFA mice. Monocytes obtained from spleen of CFA mice were differentiated into macrophages using M-CSF (50 ng/mL) for 6 days and then polarized into M1 with IFN-γ (10 ng/mL) plus LPS (1 μg/mL) or into M2 with IL-4 (20 ng/mL) for 2 more days in the presence of myelin oligodendrocyte glycoprotein epitope (MOG35-55). (A) Expression of markers by flow cytometry upon staining cells at cell surface (CD40 and CD86) or intracellularly (iNOS). Data are reported as percentage of positive cells and are representative of four independent experiments ± SEM. * p < 0.05, ** p < 0.01 (B) ELISA of cytokines and chemokines (IL-6, IL-12, TNF-α and CCL2) in supernatants of MOG-immunized M1 macrophages obtained from CFA mice. Data are reported as pg/mL and represent eight independent experiments means ± SEM. * p < 0.05 (C) Expression of markers by flow cytometry upon staining cells at cell surface (CD40, CD80 and CD86). Data are reported as percentage of positive cells and are representative of eight independent experiments ± sem. * p < 0.05, (D) ELISA of chemokines (CCL2, CCL3, CCL4, and CCL22) in supernatants of M2 macrophages. Data are reported as pg/mL and represent eight independent experiments means ± SEM. * p < 0.05, *** p < 0.001.

Figure 8

Summary of M1/M2-Mϕ immunophenotype in CFA and EAE mice. Induction of EAE delineates an ex vivo immunophenotypic alteration of M1/M2-Mϕ, with M1 being hyperactive and more pro-inflammatory while M2 being less protective.