Anti-inflammatory effects of α-MSH through p-CREB expression in sarcoidosis like granuloma model

Lung inflammation due to sarcoidosis is characterized by a complex cascade of immunopathologic events, including leukocyte recruitment and granuloma formation. alpha -melanocyte stimulating hormone (alpha -MSH) is a melanocortin signaling peptide with anti-inflammatory properties. We aimed to evaluate the effects of alpha -MSH in a novel in vitro sarcoidosis model. An in vitro sarcoidosis-like granuloma model was developed by challenging peripheral blood mononuclear cells (PBMCs) derived from patients with confirmed treatment-naive sarcoidosis with microparticles generated from Mycobacterium abscessus cell walls. Unchallenged PBMCsand developed granulomas were treated daily with 10 mu M alpha -MSH or saline as control. Cytokine concentrations in supernatants of culture and in cell extracts were measured using Illumina multiplex Elisa and western blot, respectively. Gene expression was analyzed using RNA-Seq and RT-PCR. Protein secretion and gene expression of IL-7, IL-7R, IFN-gamma, MC1R, NF-kappa B, phosphorylated NF-kappa B (p-NF-kappa B), MARCO, and p-CREB were measured with western blot and RNAseq. A significant increase in IL-7, IL-7R, and IFN-gamma protein expression was found in developed granulomas comparing to microparticle unchallenged PBMCs. IL-7, IL-7R, and IFN-gamma protein expression was significantly reduced in developed granulomas after exposure to alpha -MSH compared with saline treated granulomas. Compared with microparticle unchallenged PBMCs, total NF-kappa B and p-NF-kappa B were significantly increased in developed granulomas, while expression of p-CREB was not changed. Treatment with alpha -MSH promoted a significantly higher concentration of p-CREB in granulomas. The anti-inflammatory effects of alpha -MSH were blocked by specific p-CREB inhibition. alpha -MSH has anti-inflammatory properties in this in vitro granuloma model, which is an effect mediated by induction of phosphorylation of CREB.