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. α-melanocyte stimulating hormone (α-MSH) is a melanocortin signaling peptide with anti-inflammatory properties. We aimed to evaluate the effects of α-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-naïve sarcoidosis with microparticles generated from Mycobacterium abscessus cell walls. Unchallenged PBMCsand developed granulomas were treated daily with 10 µM α-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-γ, MC1R, NF-κB, phosphorylated NF-κB (p-NF-κB), MARCO, and p-CREB were measured with western blot and RNAseq. A significant increase in IL-7, IL-7R, and IFN-γ protein expression was found in developed granulomas comparing to microparticle unchallenged PBMCs. IL-7, IL-7R, and IFN-γ protein expression was significantly reduced in developed granulomas after exposure to α-MSH compared with saline treated granulomas. Compared with microparticle unchallenged PBMCs, total NF-κB and p-NF-κB were significantly increased in developed granulomas, while expression of p-CREB was not changed. Treatment with α-MSH promoted a significantly higher concentration of p-CREB in granulomas. The anti-inflammatory effects of α-MSH were blocked by specific p-CREB inhibition. α-MSH has anti-inflammatory properties in this in vitro granuloma model, which is an effect mediated by induction of phosphorylation of CREB.

Growth Hormone-Releasing Hormone Receptor Antagonist Modulates Lung Inflammation and Fibrosis due to Bleomycin.

PURPOSE: Growth hormone-releasing hormone (GHRH) is a 44-amino acid peptide that regulates growth hormone (GH) secretion. We hypothesized that a GHRH receptor (GHRH-R) antagonist, MIA-602, would inhibit bleomycin-induced lung inflammation and/or fibrosis in C57Bl/6J mice. METHODS: We tested whether MIA-602 (5 µg or vehicle given subcutaneously [SC] on days 1-21) would decrease lung inflammation (at day 14) and/or fibrosis (at day 28) in mice treated with intraperitoneal (IP) bleomycin (0.8 units on days 1, 3, 7, 10, 14, and 21). Bleomycin resulted in inflammation and fibrosis around airways and vessels evident histologically at days 14 and 28. RESULTS: Inflammation (histopathologic scores assessed blindly) was visibly less evident in mice treated with MIA-602 for 14 days. After 28 days, lung hydroxyproline (HP) content increased significantly in mice treated with vehicle; in contrast, lung HP did not increase significantly compared to naïve controls in mice treated with GHRH-R antagonist. GHRH-R antagonist increased basal and maximal oxygen consumption of cultured lung fibroblasts. Multiple genes related to chemotaxis, IL-1, chemokines, regulation of inflammation, and extracellular signal-regulated kinases (ERK) were upregulated in lungs of mice treated with bleomycin and MIA-602. MIA-602 also prominently suppressed multiple genes related to the cellular immune response including those for T-cell differentiation, receptor signaling, activation, and cytokine production. CONCLUSIONS: MIA-602 reduced lung inflammation and fibrosis due to bleomycin. Multiple genes related to immune response and T-cell functions were downregulated, supporting the view that MIA-602 can modulate the cellular immune response to bleomycin lung injury.