METEORIN-LIKE is a cytokine associated with barrier tissues and alternatively activated macrophages

Abstract

Cytokines are involved in many functions of the immune system including initiating, amplifying and resolving immune responses. Through bioinformatics analyses of a comprehensive database of gene expression (BIGE: Body Index of Gene Expression) we observed that a small secreted protein encoded by a poorly characterized gene called meteorin-like (METRNL), is highly expressed in mucosal tissues, skin and activated macrophages. Further studies indicate that Metrnl is produced by Alternatively Activated Macrophages (AAM) and M-CSF cultured bone marrow macrophages (M2-like macrophages). In the skin, METRNL is expressed by resting fibroblasts and IFNγ-treated keratinocytes. A screen of human skin-associated diseases showed significant over-expression of METRNL in psoriasis, prurigo nodularis, actinic keratosis and atopic dermatitis. METRNL is also up-regulated in synovial membranes of human rheumatoid arthritis. Taken together, these results indicate that Metrnl represents a novel cytokine, which is likely involved in both innate and acquired immune responses.

Keywords: Cytokine; M2 macrophages; Meteorin-like; Psoriasis; Rheumatoid arthritis; Skin.

Figure 1. Metrn and Metrnl expression in human tissues (BIGE database)

(A) BIGE expression profiles of METRN and METRNL indicating that METRN is mainly expressed in the CNS while METRNL is not expressed in the CNS but is highly expressed in activated monocytes, skin and mucosal tissues. (B) Q-PCR was used to confirm the microarray data (BIGE database) pattern of Metrnl expression.

Figure 2. Metrnl is expressed by alternatively activated macrophages (AAM/M2)

(A) Peritoneal exudate macrophages from C57BL mice were cultured under either M1 (IFNγ) or M2 (IL-4) polarizing conditions. 8 hours post stimulation Metrnl expression was measured by Q-PCR. Metrnl expression was elevated in response to IL-4 and it was suppressed by IFNγ. M1 marker CXCL10 and M2 markers Arginase I were measured in the polarized macrophages to confirm successful polarization. (B) Metrnl protein levels were measured by ELISA in supernatants from macrophages cultured in the presence of IL-4, IL-13, IFNγ and LPS for 24, 48 and 72hrs. Production of Metrnl was increased in peritoneal cavity macrophages in response to IL-4 (*P < 0.001) and IL-13 (**P < 0.02). (C) The Metrnl expression levels were measured Q-PCR in GM-CSF -or M-CSF– derived BMM (GM-BMM and BMM, respectively). Metrnl was elevated in BMM and paralleled expression of another BMM cytokine (IL-10). (D) Metrnl levels were measured by ELISA in GM-BMM and BMM. Metrnl levels were elevated in supernatants from BMM when compared to GM-BMM (*P < 0.0001). Culturing BMM in the presence of IL-4 led to an increase of Metrnl production whereas IFNγ suppressed Metrnl production (**P < 0.02). Each experiment was done in triplicate and performed three times. A representative experiment (out of three) is shown.

Figure 3. In skin, METRNL is expressed in IFNγ-treated keratinocytes and is associated with several skin disorders

(A) Analysis of METRNL expression in various human diseases indicates strong over-expression in psoriasis (p<0.002) (B) Comparison of METRNL expression in human keratinocytes, fibroblasts, PBMC and endothelial cells indicate that the highest levels are expressed by fibrobasts. (C) Human keratinocytes were cultured with a range of activating agents prior to measuring METRNL expression levels. METRNL expression was highest in IFNγ-induced keratinocytes.

Figure 4. METRNL is over-expressed in Rheumatoid Arthritis (RA)

Microarray data from Affymetrix U133 2.0 Plus genearray analyses of synovial membranes of patients with RA or normal controls indicate that METRNL is over-expressed in RA lesions (*P<0.047). N=5 for both patients and controls (from ref 18).