Meteorin-like protein/METRNL/Interleukin-41 ameliorates atopic dermatitis-like inflammation

Abstract

Background: Meteorin-like protein (METRNL)/Interleukin-41 (IL-41) is a novel immune-secreted cytokine/myokine involved in several inflammatory diseases. However, how METRNL exerts its regulatory properties on skin inflammation remains elusive. This study aims to elucidate the functionality and regulatory mechanism of METRNL in atopic dermatitis (AD).

Methods: METRNL levels were determined in skin and serum samples from patients with AD and subsequently verified in the vitamin D3 analogue MC903-induced AD-like mice model. The cellular target of METRNL activity was identified by multiplex immunostaining, single-cell RNA-seq and RNA-seq.

Results: METRNL was significantly upregulated in lesions and serum of patients with dermatitis compared to healthy controls (p <.05). Following repeated MC903 exposure, AD model mice displayed elevated levels of METRNL in both ears and serum. Administration of recombinant murine METRNL protein (rmMETRNL) ameliorated allergic skin inflammation and hallmarks of AD in mice, whereas blocking of METRNL signaling led to the opposite. METRNL enhanced β-Catenin activation, limited the expression of Th2-related molecules that attract the accumulation of Arginase-1 (Arg1)^hi macrophages, dendritic cells, and activated mast cells.

Conclusions: METRNL can bind to KIT receptor and subsequently alleviate the allergic inflammation of AD by inhibiting the expansion of immune cells, and downregulating inflammatory gene expression by regulating the level of active WNT pathway molecule β-Catenin.

Keywords: Meteorin‐like protein (METRNL); WNT signaling pathways; atopic dermatitis; cytokines; skin inflammation.

FIGURE 1

METRNL is increased in patients with dermatitis and MC903‐induced atopic dermatitis (AD) model. (A) Protein levels of METRNL in the serum of healthy controls (CT) and patients with AD were measured (n = 20) by ELISA. (B) The percentage of cells expressing Metrnl assessed by spatial scRNA‐seq (n = 6–7). (C) Representative immunohistochemistry images for METRNL in the skin of healthy controls and lesions from AD patients (n = 2). Positive stains were defined as yellow‐brown in color. Scale bars, 500 μm. (D) Representative images of multiplex immunofluorescent staining of METRNL (green), CD11b (red) and CD11c (yellow) in the skin of healthy controls and lesions from AD patients (n = 3). Scale bar, 50 μm. (E) Representative immunohistochemistry images for METRNL in the skin of vehicle‐treated control mice and MC903‐induced AD model (n = 3). Scale bars, 50 μm. (F, G) The concentrations of METRNL in ear lysates and serum of vehicle‐treated control mice and MC903‐induced AD model were measured by ELISA (n = 3–4). (H) Representative images of multiplex immunofluorescent staining of METRNL (yellow), SIGLEC‐F (red), CD163 (green), and CD4 (purple) in the ear skin of mouse model with or without MC903 or METRNL treatment. Scale bar, 100 μm. Data were compared by two‐tailed unpaired Student's t‐test (A,F,G) or one‐way ANOVA with Tukey's multiple comparison test (B) indicated by asterisks (*p <.05; **p <.01; ***p <.001). All data are presented as mean ± SD. CT, control; AD, atopic dermatitis.

FIGURE 2

rmMETRNL restrains allergic skin inflammation. (A) Protocol outline for administering recombinant murine METRNL in the MC903‐induced AD model. Mice treated with MC903 were intradermally injected with rmMETRNL or PBS. (B) Ear thickness of mice treated in (A). (C) Representative hematoxylin and eosin staining images. Scale bars are 50 μm. (D) QPCR quantification of Il4, Il6, and Tnf transcript levels in mice ear skin. Data are presented as fold change expression normalized to the average expression in sham mice. (E) Western blots analysis of mouse ear. Data are from one experiment with 3–4 mice per group. Each data point represents the mean ± SD of each group at the indicated time points (B) or one mouse (D). Statistical significance was determined using two‐way ANOVA (B, D). *p <.05, **p <.01, ***p <.001.

FIGURE 3

METRNL provokes remodeling of the immune landscape in MC903‐inflamed skin, spleen and lymph nodes. (A) Left: Representative immunofluorescence co‐staining of METRNL (yellow) and c‐kit (KIT; red), CD11c (green), CD4 (purple), and DAPI (blue) in ear sections of mice treated with MC903 with or without rmMETRNL (n = 5–7). Scale bar, 100 μm. Right: Percentage of respective positive cells in the whole tissues as quantified by HALO software. (B) Spatial analysis by HALO software showing the number of CD4+ cells in proximity (<100 μM radical distance) of METRNL+ and METRNL‐DCs (CD11c+), macrophages (CD163+) and eosinophils (SiglecF+) in MC903 treated ears. n = 5 per group. Scale bars, 100 μm. (C) Quantification of Th2 cells and eosinophils in the spleen (n = 3–4). (D‐E) Unsupervised analysis of CD45+ cells from concatenated samples using the FlowSOM algorithm (D) and quantification of pop2 and pop6 (E) (n = 3–4). Each data point in (A, C, E) represents one animal. Data are presented as mean ± SD. Statistical significance was determined by one‐way ANOVA with Tukey's multiple comparison test (A, C, E) and paired t‐test (B) indicated by asterisks (*p <.05; **p <.01; ***p <.001). DAPI, 4′‐6‐Diamidino‐2‐phenylindole, dihydrochloride.

FIGURE 4

rmMETRNL represses Arg1 ^hi macrophages in MC903‐induced AD‐like disease. (A) Single‐cell RNA‐seq UMAP of annotated cell type clusters from all mice. (B) Metrnl and Kit mRNA expression in different cell types in mouse ears was assessed by single‐cell RNA‐seq. (C) Quantification of the percentage of cell clusters in each group. (D) Functional enrichment of biological terms of fibroblasts and macrophages comparing the MC903 and MC903 + rmMETRNL group. (E) UMAP plots with macrophage subsets in mice of sham, MC903, and MC903 plus rmMETRNL group. Fb, fibroblasts; HF, hair follicle keratinocytes; SG sebaceous gland keratinocytes; MAC, macrophages; Mono, monocytes; DC, dendritic cells; EC, endothelial cells; LV, lymphatic vessel endothelial cells.

FIGURE 5

METRNL blockade increases MC903‐induced AD‐like severity. The ear skin of mice was epicutaneously exposed to MC903 or ethanol as a control, injected intradermally with anti‐METRNL or Isotype IgG every other day, and analyzed on day 14. (A) Experimental schedule. (B) Ear thickness kinetics. (C) H&E staining of mouse ear tissue. Scale bars are 50 μm. (D) Bulk ear skin RNA was sequenced and ssGSEA analysis was performed. Each column is one mouse. Each row is a significantly enriched pathway. The color implies the pathway scoring. (E) The concentration of cytokines measured by LEGENDplex™ MU Th2 Panel kit. IL‐5 and IL‐13 were under the detection limits. (F) Western blot analysis of active β‐Catenin, ARG1 and MMP12 of mice ear with indicated treatment. Data are from one experiment with 4 mice per group. Each data point represents the mean ± SD of each group at the indicated time points (B) or one mouse (E). Statistical significance was determined using two‐way ANOVA (B, E). *p <.05, **p <.01, ***p <.001, ****p <.0001.

FIGURE 6

METRNL is upregulated by cAMP and VDR. (A) Data were obtained from a public data set (GEO accession number GSE167248) and analyzed with a web‐based tool easyGEO. ****p <.0001. (B) CD4+ T cells were isolated from human buffy coats and re‐stimulated with α‐CD3 antibodies (5 μg/mL), α‐CD28 antibodies (1 μg/mL), and rhIL‐2 (10 ng/mL). Th2/Th17 cells were induced to undergo differentiation with IL‐4 (20 ng/mL) or IL‐6 (240 ng/mL) and TGF‐β (2.5 ng/mL). On day 5, cells were stimulated with db‐cAMP (100 nM) for 24 h. METRNL concentration of cell culture supernatants was assessed by ELISA. (C) Human keratinocyte cell line HaCaT cells were treated with db‐cAMP (100 nM) for 24 h and METRNL expression was analyzed by Western blot. (D, E) The amount of METRNL produced by the human monocyte cell line THP1 was analyzed by ELISA (D) and qPCR (E) upon the stimulation of the cells for 24 h with db‐cAMP (100 nM). METRNL levels in pg/mL from four independent experiments are shown as mean ± SD. t test, *p <.01. **p <.001. (F) The binding profile of VDR in the Metrnl promoters. y‐axis shows the number of mapped reads at each genomic position.