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

Abstract

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.

Activity of the GHRH Antagonist MIA602 and its Underlying Mechanisms of Action in sarcoidosis

“…As previously presented in details (14), microparticles were generated from a rough colony of a clinical strain of mycobacterium abscessus (MAB) with sonicating and heating live bacilli. High quality images of non-infectious, MAB particles were obtained by scanning electron microscope (SEM).…”

“…In vitro granuloma was developed from challenging PBMC to microparticles as previously described (14).…”

“…As we previously presented (14), PBMCs were lysed in lysis buffer (Cell Signaling Technology, Beverly, MA) with protease inhibitor cocktail (Cell Signaling Technology, Beverly, MA) and sonicated three times for 2 s each with at least 1-min rest on ice between each 2-s pulse. Samples were centrifuged at 10,000 × g for 5 min at 4°C and the supernatant was collected.…”

“…In this study, we investigated the anti-inflammatory properties of MIA602 by measuring cytokines and gene expression in both an in vitro and an in vivo granuloma models. We developed the in vitro model by exposing human peripheral blood mononuclear cells (PBMCs) to microparticles generated from mycobacterial cell walls (14). We expose mouse lungs to the same microparticles to study lung granulomatous disease in vivo (15).…”

Novel Three-Dimensional Organoid Sarcoidosis Granuloma Model

“…Peripheral blood mononuclear cells (PMBCs) were isolated from peripheral blood samples of patients with confirmed sarcoidosis taken from University of Miami Sarcoidosis biobank as previously described [23]. A quantity of 2 x10 6 PBMC was cultured in a 12-well tissue culture dish and challenged with in-house microparticles developed to induce granuloma formation (Figure 1) [24].…”