Anti-inflammatory Properties of the Alpha-Melanocyte-Stimulating Hormone in Models of Granulomatous Inflammation

Abstract

Purpose: Alpha-melanocyte stimulating hormone (α-MSH) is known to have anti-inflammatory effects. However, the anti-inflammatory properties of α-MSH on normal bronchial epithelial cells are largely unknown, especially in the context of in vitro sarcoidosis models.

Methods: We evaluated the anti-inflammatory effects of α-MSH on two different in vitro sarcoidosis models (lung-on-membrane model; LOMM and three-dimensional biochip pulmonary sarcoidosis model; 3D-BSGM) generated from NBECs and an in vivo sarcoidosis mouse model.

Results: Treatment with α-MSH decreased inflammatory cytokine levels and downregulated type I interferon pathway genes and related proteins in LOMM and 3D-BSGM models. Treatment with α-MSH also significantly decreased macrophages and cytotoxic T-cells counts in a sarcoidosis mice model.

Conclusion: Our results confirm the direct role of type I IFNs in the pathogenesis of sarcoid lung granulomas and highlight α-MSH as a potential novel therapeutic agent for treating pulmonary sarcoidosis.

Keywords: Bronchial epithelial cells; In vitro granuloma model; Mice model; Sarcoidosis; Type I interferons; α-MSH.

Fig. 1

Shows the level of different cytokines measured from 3 different LOMM, including Control, Challenged LOMM, and Challenged LOMM + α-MSH. Two groups of LOMM were challenged with MAB microparticles (Challenged LOMM and Challenged LOMM + α-MSH). The challenged LOMM model received saline as treatment while the Challenged LOMM + α-MSH received α-MSH as treatment. Each LOMM was generated from the NBECs of 5 different donors, and each donor had three replicates. The mean value of the replicates (n = 3) for each donor was used to generate the graphs. In total, five LOMM were included in each group—the mean value of replicates. Significant variations are highlighted for each plot. ns: no significant variation; *indicates significant variations (*: < 0.05, ** < 0.005 and ***: < 0.0005)

Fig. 2

Heat map of RNA-Seq transcriptome analysis of challenged LOMM (P1-5; no α-MSH treatment) and challenged LOMM + α-MSH (M1-5; after α-MSH treatment). a Genes with significant variation in their expression level between two groups (> 2.5-fold increase; green or decrease; red) are highlighted. The sample size was 5 for each group. b Bar graphs also show significant changes in type I IFN genes among three LOMM groups; control, challenged, and challenged + α-MSH

Fig. 3

Type I IFN proteins (MX1, OSA1, and ISG15) were expressed in 4 LOMM groups; control challenged LOMM and LOMM + α-MSH and α-MSH only. In total, four LOMM (from four different human subjects) were included in each group. Only two groups, including challenged LOMM and challenged LOMM + α-MSH, were exposed to MAB microparticles. Control received only saline, while the α-MSH group was only treated with α-MSH and were used as controls for this experiment. Significant variations are highlighted for each plot

Fig. 4

Shows representative western blotting for three groups: control, 3D-BSGM, and 3D-BSGM + α-MSH. Two 3D-BSGM groups were challenged with MAB microparticles to generate granuloma, and a group only received saline to serve as the control. In addition, one of the 3D-BSGM groups was treated with α-MSH, but the other only received saline as treatment. Four 3D-BSGM were included in each group, generated from 4 different donors. Significant variations are highlighted for each plot

Fig. 5

Flow cytometry results of isolated single pulmonary cells from 3 groups of mice, control, granuloma (challenged with MAB microparticles and treated with saline), and granuloma + α-MSH (challenged with MAB microparticles and treated with daily α-MSH). AM alveolar macrophage, DCS dendritic cells. Each Model had four mice. Alveolar macrophage and monocytes with internalized IFNγ were also presented. Siglec F⁺ CD11b⁻ CD11c⁺ CD64⁺ cells were identified as alveolar macrophage while CD11b⁺ MHC II⁻ CD64^+/− Ly6Clo⁺ were grouped as monocytes/undifferentiated. DCs were defined as CD11c⁺ CD103⁺ CD24⁺. Significant variations are highlighted for each plot