CONICET | Buscador de Institutos y Recursos Humanos

Astrocytes and microglia are the main immunomodulatory cells within the CNS. During damage or infection they react by shifting their activation state in a stimulus-dependent manner. Two main activation states have been described for microglial cells. The classic activation state (also termed M1), typically induced by pro-inflammatory stimuli such as LPS, is characterized by an increased production of IL-1beta and TNF-alpha. Conversely, anti-inflammatory stimuli such as IL-4, IL-13, IL-10 and TGF-beta drive microglia towards an alternative activation state (M2) with a main anti-inflammatory or immunosuppressive role. The M2 phenotype is characterized by the production of IL-10 and TGF-beta and by increased expression of genes with immunomodulatory functions such as Arginase-1 (AG1) and IL-4Ralpha. alpha-melanocyte-stimulating hormone (alpha-MSH) is an anti-inflammatory neuropeptide that binds to the melanocortin receptor 4 (MC4R). We have previously shown that primary cultured Wistar rat astrocytes and microglia express MC4R and that the alpha-MSH analogue Nle4-D-Phe7 (NDP-MSH) induces PPAR-gamma expression in these cells, stimulates IL-10 release from microglia and TGF-beta release from astrocytes. Since PPAR-gamma is known to participate in the induction of the M2 profile in macrophages and IL-10 and TGF-beta are hallmark M2 cytokines, we hypothesized that NDP-MSH may be acting as an M2-like-profile inductor and therefore decided to study the effect of this neuropeptide on the expression of other M2 markers in astrocytes and microglia. We found that AG1 mRNA levels are significantly increased after incubation with 100 nM NDP-MSH for 24 h in both astrocytes and microglia. Additionally, incubation with NDP-MSH for 24 h decreases microglial mRNA expression levels for the LPS receptor TLR4, inhibits LPS-induced TNF-alpha release from these cells and reduces HMGB1 protein expression (a pro-inflammatory molecule and late mediator of sepsis) while not affecting its mRNA levels. On the other hand, NDP-MSH does not modify mRNA levels for the IL-4Ralpha subunit or IL-4 release into the culture supernatant in both cell types. We conclude that NDP-MSH drives microglial cells in vitro to the induction of an anti-inflammatory M2-like phenotype. Some of the M2 markers are also increased in astrocytes, suggesting that these cells may be undergoing a similar shift in their activation state. These observations point to a role for endogenous alpha-MSH in the induction and/or maintenance of the immunosuppressive microenvironment that exists within the healthy CNS, in which both microglial cells and astrocytes are likely to be key players.