Cyclic AMP Pathway Suppress Autoimmune Neuroinflammation by Inhibiting Functions of Encephalitogenic CD4 T Cells and Enhancing M2 Macrophage Polarization at the Site of Inflammation

Abstract

Although it has been demonstrated that cAMP pathway affect both adaptive and innate cell functions, the role of this pathway in the regulation of T-cell-mediated central nervous system (CNS) autoimmune inflammation, such as in experimental autoimmune encephalomyelitis (EAE), remains unclear. It is also unclear how cAMP pathway affects the function of CD4 T cells in vivo at the site of inflammation. We found that adenylyl cyclase activator Forskolin besides inhibition of functions autoimmune CD4 T cells also upregulated microRNA (miR)-124 in the CNS during EAE, which is associated with M2 phenotype of microglia/macrophages. Our study further established that in addition to direct influence of cAMP pathway on CD4 T cells, stimulation of this pathway promoted macrophage polarization toward M2 leading to indirect inhibition of function of T cells in the CNS. We demonstrated that Forskolin together with IL-4 or with Forskolin together with IL-4 and IFNγ effectively stimulated M2 phenotype of macrophages indicating high potency of this pathway in reprogramming of macrophage polarization in Th2- and even in Th1/Th2-mixed inflammatory conditions such as EAE. Mechanistically, Forskolin and/or IL-4 activated ERK pathway in macrophages resulting in the upregulation of M2-associated molecules miR-124, arginase (Arg)1, and Mannose receptor C-type 1 (Mrc1), which was reversed by ERK inhibitors. Administration of Forskolin after the onset of EAE substantially upregulated M2 markers Arg1, Mrc1, Fizz1, and Ym1 and inhibited M1 markers nitric oxide synthetase 2 and CD86 in the CNS during EAE resulting in decrease in macrophage/microglia activation, lymphocyte and CD4 T cell infiltration, and the recovery from the disease. Forskolin inhibited proliferation and IFNγ production by CD4 T cells in the CNS but had rather weak direct effect on proliferation of autoimmune T cells in the periphery and in vitro, suggesting prevalence of indirect effect of Forskolin on differentiation and functions of autoimmune CD4 T cells in vivo. Thus, our data indicate that Forskolin has potency to skew balance toward M2 affecting ERK pathway in macrophages and indirectly inhibit pathogenic CD4 T cells in the CNS leading to the suppression of autoimmune inflammation. These data may have also implications for future therapeutic approaches to inhibit autoimmune Th1 cells at the site of tissue inflammation.

Thymoma Associated Myasthenia Gravis (TAMG): Differential Expression of Functional Pathways in Relation to MG Status in Different Thymoma Histotypes

“…Apart from the above pathways related to neurodegeneration (Parkinson and Huntington disease) and metabolism (Oxidative phosphorylation) (bold/italics in Table 1), one of the eight identified MG-associated inflammatory pathways (9) showed the "counter-enrichment" pattern as well: The MacTh1 clusterassociated gene set was significantly upregulated in TAMGassociated B2 thymomas and downregulated in AB thymomas (bold/italics in Table 2). Imbalanced macrophage polarization is well-known to play an important role in T cell-and autoantibody-mediated autoimmune and allergic diseases (25)(26)(27)(28), and can affect T cell apoptosis (29), i.e., a key feature of normal thymic tolerance induction and abnormal thymopoiesis inside TAMG-associated thymomas (30). Furthermore, analyses of MHC class II expression levels and the step-wise maturation of thymocytes inside different thymoma subtypes already gave strong hints that the mechanisms shaping the autoimmune CD4+ T cell repertoire are different in AB and B2 thymomas (31).…”

Neuronal extracellular microRNAs miR‐124 and miR‐9 mediate cell–cell communication between neurons and microglia

“…Ex vivo isolated microglia also expressed miR‐9, which was not detected in cultured adult microglia (not shown). We also investigated the expression of miR‐155, which was upregulated in classically activated M1 macrophages and often opposes the pattern of expression of miR‐124, which is expressed more in alternatively activated M2 macrophages (Ponomarev et al, ; Veremeyko et al, ). We found that NCM induced miR‐155 in YS‐, but not in BM‐derived macrophages.…”

“…Rat anti‐mouse monoclonal anti‐CD11b‐AF488 antibodies (clone M1/70) that recognize macrophages marker CD11b (Veremeyko et al, ) were purchased from BD Biosciences (0.2 mg/ml, dilution 1:100; RRID: AB_396784). Goat polyclonal anti‐human/rat/mouse antibodies for Iba1 (NP_116573.1; NP_001614.3) were purchased from BioRad (cat#AHP2024; 0.5 mg/ml, dilution 1:200; RRID: AB_2224406) and were used with secondary donkey anti‐goat IgG antibodies conjugated with AF488 (Jackson ImmunoResearch, cat#705‐546‐147, RRID: AB_2340430).…”

“…For analysis of BMDMs, the cells were obtained from DsRed tg mice and stained with rat anti‐mouse monoclonal anti‐MHC class II‐PE‐Cy5 (eBioscience; clone MS/114.15.2; 0.2 mg/ml, dilution 1:200; RRID: AB_467560), anti‐CD45‐APC‐Cy7, (BioLegend; clone 30‐F11; 0.2 mg/ml, dilution 1:100; RRID: AB_312980), and anti‐F4/80‐AF488 (BioLegend; clone BM8; 0.5 mg/ml, dilution 1:200; RRID: AB_893479) as described earlier (Ponomarev, Tarasenko, & Sapozhnikov, ; Starossom, Veremeyko, Dukhinova, Yung, & Ponomarev, ; Veremeyko et al, ). F4/80 + DsRed + gated macrophages were sorted from neuronal co‐cultures or analyzed for the expression of MHC class II and CD45.…”

“…Western blot analysis was performed according to a standard protocol as previously reported (Ponomarev, Veremeyko, Barteneva et al, ; Veremeyko et al, ). Rabbit polyclonal antibodies for human/mouse/rat HDLs (immunogen: KLH conjugated synthetic peptide from a 41–125 amino acid sequence of human HDL) were purchased from Biorbyt (cat#orb1003; 0.5 mg/ml, dilution 1:1,000; RRID: AB_2737031).…”