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.
Highlights
Various pathways are known to affect differentiation and function of CD4 T cells in vivo during inflammation associated with autoimmunity or infection
We hypothesized that administration of Forskolin would lead to activation of cAMP pathway in various cell types that might be beneficial for suppression of experimental autoimmune encephalitis (EAE) by inhibiting autoimmune CD4 T cells and/or changing balance of macrophage polarization from M1 to M2
This study showed that cAMP pathway is more important to modulate function of macrophages rather than T cells during EAE
Summary
Various pathways are known to affect differentiation and function of CD4 T cells in vivo during inflammation associated with autoimmunity or infection. More detailed and recent studies demonstrated that cAMP-inducing agents Cholera Toxin, 8-bromo-cAMP, and FDA-approved drug Forskolin inhibited proliferation of Th1 but not Th2 effector cells in vitro [2]. It was shown that Cholera Toxin-induced expansion rather than inhibition of Th17 cells in vivo [3]. Pertussis toxin stimulated rather than inhibited expansion of Th1 cells in vivo leading to development of CNS autoimmune inflammation [5]. Exact role of cAMP pathway in the modulation of function of effector T cells during CNS autoimmune inflammation remains unclear. An important factor that could affect functions of T cells in the tissues during inflammation are tissue-resident and blood-derived macrophages that are recruited to the sited of inflammation and could be affected by cAMP-inducing agents
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