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.

METRNL was significantly upregulated in lesions and serum of patients with dermatitis compared to healthy controls. Administration of rmMETRNL reduced 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 attracted the accumulation of Arg1hi‐macrophages, dendritic cells, and activated mast cells.Abbreviations: AD, atopic dermatitis; Arg1, Arginase‐1; DC, dendritic cell; EOS, eosinophil; IL, interleukin; MC, mast cell; METRNL, meteorin‐like protein; MO, monocyte; MΦ, macrophage; rmMETRNL, recombinant murine METRNL protein; MMP‐12, matrix metallopeptidase 12; TNF, tumor necrosis factor.

Abbreviations

AD

atopic dermatitis

Arg1

Arginase‐1

DC

dendritic cell

EOS

eosinophil

IL

interleukin

MC

mast cell

METRNL

meteorin‐like protein

MO

monocyte

MΦ

macrophage

rmMETRNL

recombinant murine METRNL protein

MMP‐12

matrix metallopeptidase 12

TNF

tumor necrosis factor

1. INTRODUCTION

The skin, the body's largest organ, serves as a vital physical barrier against stimuli with immunological properties, including both humoral and cellular immune responses. In response to both external and internal stimuli, skin‐resident cells release substances that attract immune effector cells and facilitate skin inflammation. Atopic dermatitis (AD) is a common chronic inflammatory skin disorder with a complex pathophysiology, involving barrier dysfunction and immune dysregulation. Individuals diagnosed with AD exhibit pruritic eczematous plaques and may also experience other atopic conditions such as allergic asthma and allergic rhinitis. Classically, AD is driven by type 2 cytokines, including IL‐4, IL‐5, and IL‐13, which originate from T helper 2 (Th2) cells, type 2 innate lymphoid cells, basophils, mast cells, and eosinophils. These Type 2 cytokines promote IgE synthesis to activate mast cells, leading to subsequent IgE‐mediated allergic inflammation. Thymic stromal lymphopoietin (TSLP) secreted by keratinocytes may further amplify the Type 2 cytokines‐mediated response. Besides, growing evidence shows that Th17 cells are also involved in the aetiology of AD. ¹ , ² Therefore, it is crucial to understand the immunopathogenesis and key mediators that regulate the progress of AD.

Meteorin‐like protein (METRNL), is a secretory protein ³ that exhibits an anti‐inflammatory phenotype during muscle regeneration, ⁴ , ⁵ endothelial inflammation, ⁶ , ⁷ and sepsis‐induced kidney injury. ⁸ Physiological factors that can regulate the increase of METRNL include inflammation, exercise, ⁹ cold exposure, ¹⁰ metabolic status ¹¹ and hormones. A recent striking work has identified KIT, receptor tyrosine kinase type III, as the receptor for METRNL. ¹² Metrnl knockout mice develop inflammatory lesions in the uterus. ¹³ Our previous study showed that METRNL, derived from epithelial cells stimulated by house dust mites, eosinophils and macrophages, attenuates dendritic cells (DCs)‐mediated type 2 inflammation in allergic asthma ¹⁴ and exhibits anti‐inflammatory activity in COVID‐19 patients. ¹⁵ Chen et al. demonstrated that METRNL influences the host's ability to fight off sepsis by dramatically influencing the recruitment of macrophages and modifying the balance of Treg/Th17 immune cells. ¹⁶ While it has been documented that myeloid cells are the primary origin of METRNL, ⁵ it has been suggested that hypo‐responsive Th2 cells with an impaired capacity to produce Th2 cytokines may have higher levels of Metrnl mRNA expression. ¹⁷ In light of that METRNL is highly expressed in normal skin and overexpressed in lesions of patients with AD and psoriasis, ³ , ¹⁸ , ¹⁹ we postulate that METRNL might have a regulatory role in skin inflammation.

In this study, we employed an experimental murine model reproducing important clinical features of AD and assessed how METRNL blockade and supplement affect the immune status, respectively. We also delineated the anti‐inflammatory function of METRNL in vitro, and ex vivo using a native skin model. Our findings therefore elucidate its novel anti‐inflammatory mechanisms and highlight a potential therapeutic role for METRNL in AD.

2. MATERIALS AND METHODS

2.1. Human samples

Formalin‐fixed, paraffin‐embedded (FFPE) tissues of human skin samples and serum were obtained from the Department of Dermatology, Sun Yat‐sen Memorial Hospital, Guangzhou and the Department of Paediatrics, Prince of Wales Hospital, Hong Kong. Four NativeSkin models, containing skin biopsies from donated surgical discards, were purchased from the Genoskin Company, Toulouse, France. The study was approved by the Clinical Research Ethics Committee, The Chinese University of Hong Kong‐New Territories East Cluster Hospitals and conducted strictly according to the ethical principles outlined in the Declaration of Helsinki. All the enrolled participants provided informed written consent.

2.2. Animals

Female C57BL/6 mice aged 6–8 weeks were employed throughout the entire study and housed in the animal facility of the Prince Wales Hospital, Hong Kong. All experimental procedures were approved by the Animal Ethics Committee of the Chinese University of Hong Kong. AD‐like model was set up converted with standard procedures, ²⁰ , ²¹ mice received 10 μL of absolute ethanol with or without 1 nM MC903 (calcipotriol; Tocris Bioscience, Bristol, United Kingdom) per side (dorsal and ventral) of both ears every 2 days from day 0 to day 6, 12, 20 as indicated. Recombinant murine METRNL protein (rmMETRNL) (R&D Systems, Minneapolis, MN, USA) was injected intradermally or intraperitoneally every other day from day −1 to day 7, 13, 21 (1 μg per dose). Mice were sacrificed on day 8, 14, and 22. To study the effects of rmMETRNL applied after MC903 application, rmMETRNL was injected intradermally for seven consecutive days and mice were sacrificed on day 22. Anti‐murine METRNL antibody (anti‐METRNL) (R&D Systems, Minneapolis, MN, USA) was injected intradermally or every other day from day −1 to day 13 (5 μg per dose), and mice were sacrificed on day 14. Ear thickness was measured at the respective time points using a dial thickness gauge (Model G, Peacock, OZAKI MFG. CO., LTD, Tokyo, Japan).

For the following Methodologies including Cell lines; 10x Genomics single‐cell RNA sequencing (scRNA‐seq), RNA‐seq and quantitative polymerase chain reaction (qPCR); Immunoblotting, multiplex immunoassays, and Enzyme‐linked immunosorbent assay (ELISA); Histology; Flow cytometry; Microarray and chromatin immunoprecipitation sequencing (ChIP‐Seq) analysis and Th cells induction, please refers to the Data S1.

2.3. Statistics

Results are presented as mean ± standard deviation (SD) unless otherwise stated. Student's t‐tests or one‐way ANOVA followed by the Tukey test for three groups were performed using Graphpad Prism v9.0 (La Jolla, CA, USA). Statistically significant differences were defined as p‐values less than .05.

3. RESULTS

3.1. METRNL expression is enhanced in patients and mice with AD

We first anticipated determining the METRNL levels of human and mouse tissues with or without AD. Remarkably, individuals with moderate to severe AD manifested statistically higher serum METRNL (Figure 1A). We used an online tool ²² to analyze the spatial single RNA‐sequencing data of lesions and non‐lesions of AD patients as well as healthy control skin upload by Prof. Akdis's group, ²³ and found the percentage of Metrnl positive cells increased significantly in AD lesions compared with non‐lesions and healthy control skin (Figure 1B). By immunohistochemical (IHC) staining, we observed intense brown signals in keratinocytes and a subset of infiltrating immune cells within the lesions of AD patients. By contrast, we observed only a few stains in keratinocytes for skin samples from healthy controls (Figure 1C). Using immunofluorescence (IF) staining, we confirmed CD11b + myeloid cells expressed METRNL and some of these cells are CD11c double‐positive DCs (Figure 1D). To further investigate the role of METRNL in AD, we employed a MC903‐induced AD mouse model, which involved the upregulation of Th2‐TSLP in keratinocytes and the initiation of the Th2 immune response cascade of the local inflammatory sites of skin. ²⁴ Consistently, we showed METRNL was upregulated in the ear tissue (Figure 1E, F) and serum (Figure 1G) of mice upon repeated exposure to MC903 when compared to control mice. In addition, multiplexed immunofluorescent staining of skin sections suggested that METRNL was expressed by macrophages (CD163+ cells), eosinophils (Siglec‐F+ cells) and keratinocytes (Figure 1H). These findings suggest that METRNL participates in AD development.

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.

3.2. rmMETRNL supplement leads to diminished inflammatory responses in AD model

To delineate METRNL‐dependent events in the skin following MC903 treatment, recombinant murine METRNL protein (rmMETRNL) was administered intradermally into ears at 48 h intervals before and during the topical application of MC903 (Figure 2A). The thickening of ear skin caused by MC903, an indicator of an inflammatory reaction, was reduced by treatment with rmMETRNL (Figure 2B). The phenotype of mice injected with rmMETRNL without MC903 application was similar to the sham group. Based on histology, we observed AD‐like mice treated with rmMETRNL showed decreased ear thickness and inflammatory infiltrates compared to the MC903‐alone group (Figure 2C), thereby indicating that METRNL suppressed skin inflammation. MC903 increased the mRNA expression of Il4, Il6, Tnf and the protein expression of ARG1, MMP12, while the supplement of rmMETRNL decreased their expression measured by qPCR (Figure 2D) and Western blot (Figure 2E), respectively. Western blot results also showed that rmMETRNL upregulated the level of active β‐Catenin and Tyr568/570 phosphorylation of KIT. Together, these data demonstrated that the addition of rmMETRNL activated the WNT pathway, downregulated type 2 inflammatory molecules, and ameliorated AD‐like inflammation.

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.

3.3. METRNL shapes the inflammatory immune response in MC903‐inflamed skin and peripheral immune organs

We also investigated the effect of intraperitoneal injection of rmMETRNL on the MC903 model. We performed multiplex staining of AD‐like mouse ear tissues to demonstrate that the application of MC903 increased the number of METRNL+ cells, DCs (CD11c+), mast cells (KIT+), and CD4+ cells in ear tissue, as quantified by the HALO software. Significantly, co‐administration of rmMETRNL reduced the levels of CD11c, KIT and CD4 (Figure 3A). Spatial interaction analysis of MC903‐treated ear sections shows that METRNL+ DCs (CD11c+), macrophages (CD163+) and eosinophils (SiglecF+) are closely interacting with CD4 + Th cells when compared to their negative counterparts (Figure 3B). In order to understand the effect of METRNL+ cells on CD4+ cells, we sought to characterize the phenotypes of CD4+ cells in close proximity to METRNL+ cells by mIF (Figure S1A). Results showed that upon administration with rmMETRNL, Th2 cells (GATA3 + CD4+) decreased, while Treg cells (FOXP3 + CD4+) and exhausted CD4 (PD1 + CD4+) cells increased significantly (Figure S1B). Having identified the effect of rmMETRNL on the inflammatory responses of the skin in AD mice, we sought to explore the immunological misfiring in spleen and skin‐draining lymph nodes by flow cytometry. Notably, co‐injection of rmMETRNL reduced IL‐4 + CD4+ Th2 cells and CD11b + Siglec‐F+ eosinophils (Figure 3C) in the spleens of MC903‐treated mice. We profiled the skin‐draining lymph node infiltrate by using a 10‐parameter flow cytometry analysis and entailed FlowSOM Plugins of FlowJo software to self‐organize CD45+ cells in draining lymph nodes into 12 populations (Pop). A heatmap visualization was used to quantify and summarize each marker's median intensity for each cluster. We found that the proportion of Pop two (LY6c + monocytes) and six (CD11b + Siglec‐F+ eosinophils) altered significantly (all p <.05). All of them were elevated in the MC903 group while rmMETRNL suppressed their expansions (Figure 3D,E). These results demonstrate that METRNL attenuates the development of MC903‐induced AD at multiple sites via the downregulation of the Th2 inflammatory responses.

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.

3.4. scRNA‐seq data reveal that METRNL increases the WNT pathway activity of Kit‐expressing cells

Single‐cell RNA‐seq is a valuable but expensive tool for studying the immune microenvironment. We combined the ear cell suspensions of two mice from the sham group, MC903 group and MC903 + rmMETRNL group, in other words, six mice together, for one single‐cell RNA‐seq reaction. Cell clusters were manually curated from transcriptomic profiles based on canonical cell‐type markers and we identified 14 distinct cell clusters in the ears of all mice, including fibroblasts (Fb), four clusters of keratinocytes (basal, spinous, hair follicle, and sebaceous gland), immune cells and stromal cells (Figure 4A). Besides, macrophages and DCs can be derived from monocytes, thus prompting us to first classify them inside the same cluster denoted as “Mac./Mono./DC.” As shown in Figure 4B, Metrnl exhibited a broad expression pattern in mice ear cells including but not limited to spinous keratinocytes, Fb, and Mac./Mono./DC while Kit was widely expressed in mast cell, Fb, Mac./Mono./DC, endothelial cells and so on. Upon induction of AD, the percentage of Mac./Mono./DC and neutrophils were increased, whereas the addition of rmMETRNL reduced this leukocytic influx (Figure 4C). As Fb and macrophages are the predominant cell types in ears expressing Kit, we performed decoupleR analysis of functional enrichment of biological terms for these two cell types and we found their WNT pathway activity was upregulated in MC903 plus rmMETRNL group contrasting with MC903 group (Figure 4D). We further categorized macrophages into eight clusters and found that cluster 5, characterized by high Arg1 expression, was most abundant in the MC903 group, higher than that in the rmMETRNL‐treated group (Figure 4E). This cluster was also rich in Mmp12, Mmp14, Mmp19, Cxcl3, Il‐1a, and Spp1. Together, these results show that the administration of rmMETRNL can potentially suppress skin inflammation by upregulating the WNT pathway and reducing the number of infiltrated immune cells especially Arg1 ^hi macrophages in inflammatory sites.

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.

3.5. METRNL blockade promotes the development of MC903‐induced AD‐like disease

To investigate the effects of METRNL in AD, a neutralizing antibody against METRNL was injected intradermally into ears every other day for 2 weeks and MC903 was applied 1 day after the injection (Figure 5A). Anti‐METRNL antibody injection in the absence of an inflammatory stimulus did not cause ear thickening but led to the most apparent thickening combined with MC903 among the four groups (Figure 5B). H&E staining revealed that MC903 application resulted in immune cell infiltration in the dermis and many more immune cells including eosinophils were present in this layer when MC903 was administered together with anti‐METRNL antibody (Figure 5C). To get a comprehensive understanding of METRNL‐regulated genes and pathways in skin inflammation, RNA sequencing of mouse ear tissues was performed. Single‐sample Gene Set Enrichment Analysis (ssGSEA) verified that there were minimal transcriptional variations between the sham group and the anti‐METRNL antibody injection‐only group. However, upon challenge with MC903, the administration of anti‐METRNL antibody resulted in a significant amplification of various inflammatory pathways, including eosinophils migration and mast cell activation. Interestingly, positive regulation WNT pathway, methylation and monoubiquitinated protein deubiquitination were most downregulated in the anti‐METRNL in conjunction with MC903 group (Figure 5D). We confirmed the differential expression of some type 2 inflammation molecules including IL‐4, IL‐6 and TNF‐α by ELISA (Figure 5E), ARG1 and MMP12 by Western blot (Figure 5F). In addition, Western blot proved that active β‐Catenin, an important WNT pathway component, was most inhibited in the anti‐METRNL plus MC903 group. In short, these results provided evidence that the neutralization of METRNL suppressed the WNT pathway, upregulated type 2 inflammation molecules, and exacerbated AD‐like inflammation.

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.

3.6. METRNL expression is regulated by cyclic adenosine monophosphate (cAMP) and vitamin D receptor (VDR)

Being curious about the upstream events leading to METRNL expression, we examined the expression of METRNL in different cell types by analyzing publicly available microarray and RNA sequencing data. We observed an upregulation of METRNL in Th17 cells that were stimulated with the membrane‐permeable cAMP analogue db‐cAMP (Figure 6A). We then verified this result by using ELISA, which showed the amount of METRNL produced by CD4+ T cells under a Th17 condition was lower than the amount produced under a Th2 condition upon db‐cAMP addiction (Figure 6B). An inhibitor of cyclic nucleotide phosphodiesterase 4 (PDE4) that increases the intracellular cAMP level through blocking cAMP degradation is an FDA‐approved drug for AD. The increase of the second messenger cAMP for a variety of inflammatory mediators such as prostaglandin E2, histamine and adenosine, ²⁵ tend to have anti‐inflammatory effects. ²⁶ In addition, db‐cAMP also facilitated METNRL production in human keratinocyte cell lines (HaCaT) as well as in matured human monocyte cell line THP‐1 cells (Figure 6C–E). We also analyzed the CHIP‐seq data (GSE89431) and found that the level of VDR at the METRNL promoter region of unstimulated THP‐1 cells was lower than that in vitamin D‐treated cells, indicating that METRNL is targeted by VDR (Figure 6F). Overall, the regulation of METRNL is influenced by cAMP and VDR.

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. ⁶⁰ ****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.

According to the Immgen database, ²⁷ the expression level of Kit, the receptor of METRNL, in mast cells is significantly greater, ranging from 4 to 19 times when compared to DCs; the difference is even more pronounced, ranging from 100 to 200 times higher, when compared to macrophages (Figure S2A), Consequently, we investigated the in vitro effect of rmMETRNL on murine mast cell line MC/9 cells. We found that METRNL activates KIT (Y568/Y570) of MC/9 cells (Figure S2B). The mixture of anti‐dinitrophenyl‐IgE (anti‐DNP‐IgE), dinitrophenyl‐human serum albumin (DNP‐HSA) and ionomycin, has been approved as simulators to induce mast cell activation. ²⁸ Indeed, this mixture resulted in elevated production of IL‐6 and TNFα by MC/9 cells. Upregulation of these pro‐inflammatory molecules was counterbalanced by pretreatment with rmMETRNL. Furthermore, XVA‐939, a WNT signaling pathways inhibitor, effectively abolished the anti‐inflammatory effects of rmMETRNL (Figure S2C). These results demonstrate that METRNL is implicated in mast cell inflammation, at least partly, through the activation of the WNT pathway.

To address the translational relevance of METRNL in human skin diseases, we assessed the role played by METRNL in skin inflammation ex vivo using four full‐thickness healthy human skin explants. As reported by another group, individuals who are frequently exposed to detergent compounds like SDS tend to develop AD, ²⁹ we employed a 10% concentration of SDS combined with heat‐killed Staphylococcus aureus (HKSA) an exacerbating factor for AD, to set up AD model. Recombinant human METRNL protein (rhMETRNL) reversed the occurrence of skin pigmentation and SDS‐ and HKSA‐induced IL‐1β production (Figure S3).

4. DISCUSSION

This study is the first to investigate whether novel anti‐inflammatory METRNL could attenuate the onset and progression of AD. We found that METRNL was significantly upregulated in the lesioned skin and serum of AD mice and patients. We identified several cell types involved in the pathogenesis of AD as potential responders to METRNL, including but not limited to macrophages, DCs, and mast cells. We also demonstrated that METRNL limited the pro‐inflammatory signals of Th2 molecules in part by activating WNT pathway in the AD model. In addition, our in vitro findings demonstrated that keratinocytes and immune cells stimulated with db‐cAMP could upregulate METNRL expression, implying that METRNL plays an anti‐inflammatory role in maintaining local immune homeostasis.

METRNL has been observed to exhibit elevated expression levels in keratinocytes, which serve as the primary barrier against epicutaneous stimuli. This enhanced expression is also noted within myeloid cells, notably macrophages, eosinophils and DCs, which are integral to the pathophysiological development of AD. The upregulation of METRNL in these cell types suggests a potential modulatory function in the inflammatory and immune responses characteristic of AD.

Applying MC903 on alternat days to induce an AD model showed that in the first week, the inflammation was not pronounced (Figure S6). By the third week, there might be instances of individual mice experiencing desquamation of the stratum corneum, leading to alterations in ear thickness (Figure S7). Therefore, our investigations primarily focused on assessments conducted in the second week. We found METRNL showed a pronounced impact on type 2 molecules, such as IL‐4, IL‐6, TNF‐α, but we didn't find rmMETRNL decreased the regulatory cytokine including IL‐10, IL‐35, and TGFβ (Figure S4). We also experimented with applying MC903 for 14 days to induce dermatitis, followed by the cessation of MC903 and daily intradermal injections of rmMETRNL. It was observed that METRNL tended to decrease IL‐4 and IL‐6 levels (Figure S5). Type 2 cytokines contribute to diminished innate immune defenses by impairing the function of skin‐resident immune cells. The recruitment of eosinophils and DCs to the skin, a hallmark of allergic inflammatory responses, is facilitated by the chemotactic properties of these cytokines. Additionally, IL‐4 is critical for inducing antibody class switching to IgE in B cells, a process central to AD's pathogenesis. Our findings are consistent with previous studies showing that METRNL inhibited IL‐4 expression in sepsis ⁸ and METRNL was associated with a notable suppression of DCs accumulation, maturation, and polarization of naïve T cells to Th2 cells. ¹⁴

Our result showed that ARG1 was upregulated in MC903‐treated murine ear skin, which is in concordance with previous literature. ³⁰ In the MC903‐induced murine model of AD, lesional Arg1 was significantly increased when compared to that of control mice, whereas basophil depletion by applying Diphtheria Toxin to Mcpt8 ^DTR mice decreased Arg1 expression, accompanied by the amelioration of MC903‐induced itch responses. ³¹ Inhibition of arginase with an arginase‐specific inhibitor, N (omega)‐hydroxy‐nor‐L‐arginine, ameliorated the skin inflammatory response caused by repeated application of 2, 4‐Dinitrochlorobenzene. ³² While ARG1 is commonly regarded as an indicator of type 2 macrophage (M2) activation, it is worth noting that its expression is not exclusively limited to M2 macrophages. ³³ ARG1 expression is also not always consistent with other M2 markers. ³⁴ In addition, Vu et al. discovered that Arg1^hi macrophages present in wounded skin produced high levels of pro‐inflammatory chemokine/cytokines (Cxcl1/2/3, Il‐1a), suggesting an inflammatory M1/M2 hybrid identity. ³⁵ We found that the Arg1^hi macrophage cluster downregulated by METRNL also produced Cxcl2, Cxcl3, and Il‐1a, similar to the cluster identified by Vu et al. IL‐1α and CXCL3 exhibit strong chemotactic properties, attracting neutrophils to the site of inflammation ³⁶ , ³⁷ Besides, the Arg1^hi macrophage cluster highly expressed Mmp12, a general inflammatory marker, which exacerbates contact hypersensitivity. ³⁸ Therefore, the downregulation of Arg1^hi macrophage by METRNL also contributes to its anti‐inflammation effects.

Despite prior discoveries indicating that the regulatory role of METRNL in inflammation may be attributed to its broad immunosuppressive effects, the molecule itself remains perplexing, partly due to its receptor. METRNL has been shown to induce KIT (Y568/Y570) phosphorylation in human coronary artery endothelial cells, similar to the effects of stem cell factor (SCF). ¹² , ³⁹ It is worth mentioning that the activation of KIT is regulated by negative feedback loops, including ubiquitination, dephosphorylation, and protein kinase C‐dependent serine phosphorylation. ³⁹ , ⁴⁰ , ⁴¹ The observed phenomena of decreasing numbers of KIT+ cells may be a consequence of negative feedback loops involving METRNL, which downregulates KIT expression. DCs and mast cells with KIT expression are central players in AD. ⁴² According to literature precedent, DCs that possess functioning KIT can modulate the differentiation of Th cells in the context of allergic asthma. ⁴³ Sc‐RNA Seq data from mononuclear phagocytes in skin‐draining lymph nodes indicated the presence of Kit transcript in resident DCs, inflammatory conventional DCs, and cycling myeloid cells, ⁴⁴ , ⁴⁵ which may be targeted by METRNL to regulate skin inflammation. An integrative proteomics approach showed a direct interaction between KIT and β‐Catenin in prostate cancer cells. ⁴⁶ Activation of β‐Catenin signaling initiates the differentiation and expansion of CD103+ DCs ⁴⁷ and inhibits the infiltration of effector T cells. ⁴⁸ KIT also mediates the allergic inflammatory activity of mast cells. Type 2 cytokines, such as IL‐4, ⁴⁹ have been demonstrated to impact the secretion of inflammatory mediators from mast cells by their interaction with SCF/KIT. ⁵⁰ Mast cells polarize T cells into Th1 and Th2 subsets by interacting with DCs ⁵¹ or directly activate effector T cells. ⁵² Additionally, MCs secrete a range of cytokines, including TNF‐α, which serves a pivotal function in the recruitment of neutrophils to sites of inflammation. ⁵³ KIT is also expressed by eosinophils ⁵⁴ , ⁵⁵ macrophages, ⁵⁶ endothelial cells, ⁵⁷ innate lymphoid cells, and γδT17 cells ⁵⁸ that are involved in the cutaneous hyper‐inflammatory response. KIT/Gsk3β/β‐Catenin signaling in endothelial cells induces neovascularization through the enhancement of the nuclear translocation of β‐Catenin and the transcription of β‐Catenin target genes related to angiogenesis. ⁵⁹ Overall, METRNL activates KIT and β‐Catenin but the precise mechanisms in various cell types under diverse conditions have yet to be fully understood.

In conclusion, this study provides molecular and immunological evidence that METRNL may have an important function in the modulation of skin inflammation. Results therefore provide a biochemical basis for the development of a novel treatment for inflammatory pathogenesis and allergic diseases using regulatory cytokines.

AUTHOR CONTRIBUTIONS

DH and C‐KW designed the experiments. C‐KW obtained the funding. XL and T‐FL recruited the patients and performed clinical study. DH, JL, PF‐YC, XL, XG, C‐KC, GG, LF, KC‐YW, LL‐YK and JW‐HC performed the experiments and analyzed the data. DH, C‐KW, Q‐JM, JL and PF‐YC drafted and revised the manuscript.

FUNDING INFORMATION

This research project was supported by the Funding for Regenerative Immunology Research (The Chinese University of Hong Kong/CUHK), the State Key Laboratory of Research on Bioactivities and Clinical Applications of Medicinal Plants (The Chinese University of Hong Kong/CUHK) from the Innovation and Technology Commission, Hong Kong, and the National Natural Science Foundation of China (82302609) and Natural Science Foundation of Jiangsu Province (BK20230840).

CONFLICT OF INTEREST STATEMENT

Ms. Huang, Ms. Liu, Dr. Gao, Dr. Choi, Mr. Giglio, Mr. Farah, Dr. Leung, Ms. Wong, Dr. Kan, Mr. Chong, Dr. Meng, Dr. Liao, Dr. Cheung and Dr. Wong have nothing to disclose.

Supporting information

Data S1.

Huang D, Liu X, Gao X, et al. Meteorin‐like protein/METRNL/Interleukin‐41 ameliorates atopic dermatitis‐like inflammation. Allergy. 2025;80:474‐488. doi: 10.1111/all.16150

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.