The expression and regulation of chemerin in the epidermis.

Chemerin is a protein ligand for the G protein-coupled receptor CMKLR1 and also binds to two atypical heptahelical receptors, CCRL2 and GPR1. Chemerin is a leukocyte attractant, adipokine, and antimicrobial protein. Although chemerin was initially identified as a highly expressed gene in healthy skin keratinocytes that was downregulated during psoriasis, the regulation of chemerin and its receptors in the skin by specific cytokines and microbial factors remains unexplored. Here we show that chemerin, CMKLR1, CCRL2 and GPR1 are expressed in human and mouse epidermis, suggesting that this tissue may be both a source and target for chemerin mediated effects. In human skin cultures, chemerin is significantly downregulated by IL-17 and IL-22, key cytokines implicated in psoriasis, whereas it is upregulated by acute phase cytokines oncostatin M and IL-1β. Moreover, we show that human keratinocytes in vitro and mouse skin in vivo respond to specific microbial signals to regulate expression levels of chemerin and its receptors. Furthermore, in a cutaneous infection model, chemerin is required for maximal bactericidal effects in vivo. Together, our findings reveal previously uncharacterized regulators of chemerin expression in skin and identify a physiologic role for chemerin in skin barrier defense against microbial pathogens

Chemerin is an antimicrobial agent in human epidermis.

Chemerin, a chemoattractant ligand for chemokine-like receptor 1 (CMKLR1) is predicted to share similar tertiary structure with antibacterial cathelicidins. Recombinant chemerin has antimicrobial activity. Here we show that endogenous chemerin is abundant in human epidermis, and that inhibition of bacteria growth by exudates from organ cultures of primary human skin keratinocytes is largely chemerin-dependent. Using a panel of overlapping chemerin-derived synthetic peptides, we demonstrate that the antibacterial activity of chemerin is primarily mediated by Val(66)-Pro(85), which causes direct bacterial lysis. Therefore, chemerin is an antimicrobial agent in human skin

Chemerin and chemerin receptor expression in mouse skin.

<p>Chemerin mRNA expression (A), chemerin protein expression in tissue lysates (B), chemerin receptor (CMKLR1, CCRL2, GPR1) mRNA expression (C-E), or chemerin protein expression in skin homogenates (F) and chemerin expression in plasma (G) was measured in the indicated tissues isolated from C57BL/6 mice (A-E) or the indicated mice on BalbC backround (F-G) by RT-QPCR and ELISA. The expression data of the indicated genes was normalized to cyclophilin A and RPL13A, and presented relative to liver as the mean ± SEM, n = 4–5 different mice (A, C-E). The amount of chemerin protein in plasma, or skin lysates and homogenates normalized to total protein is shown as the mean ± SEM, n = 4 (B), or n≥6 mice (F-G). Statistical significance is indicated by asterisk(s); *p<0.05, **p<0.01, by ANOVA followed by a Bonferroni post hoc test.</p

Chemerin in healthy human skin is primarily expressed in the epidermis.

<p>Chemerin mRNA expression (A), chemerin protein expression in tissue lysates (B), and chemerin receptor (CMKLR1, CCRL2, GPR1) mRNA expression (C-E), was measured in the indicated tissues isolated from healthy human donors. Total RNA was subjected to RT-QPCR. The expression data of the indicated genes was normalized to B2M and GAPDH, and presented relative to skin (A, C-E). The amount of chemerin in skin lysates, normalized to total protein was determined by ELISA (B). The mean of n = 6 (A, C-E) or n = 8 (B) different donors ± SEM is shown. Statistical significance between epidermis and dermis is shown by asterisk(s); *p<0.05, **p<0.01, by ANOVA followed by a Bonferroni post hoc test.</p

Chemerin is bactericidal <i>in vivo</i>.

<p>Chemerin–deficient (ChemKO) and WT mice were ectopically treated with <i>S. aureus</i>. Bacteria were retrieved from skin 24h later, and presented as a % of input inoculum. Each data point represents one experiment and a horizontal line indicate the mean value in each group. *p<0.05, by <i>t</i> test.</p

Expression of chemerin receptors in human skin equivalents treated with bacteria.

<p>Keratinocytes were treated with the indicated factors for 24h (A) or 48 h (B). RT-QPCR was performed and the expression data were normalized to cyclophilin A and expressed relative to unstimulated cells. Mean ± SD of 5–7 independent experiments is shown. Statistical significance comparing cytokine-treated cells vs. untreated cells (* p<0.05) was determined by ANOVA followed by a Bonferroni post hoc test.</p

Bacteria upregulate chemerin expression in epidermis.

<p>Keratinocytes were treated with the indicated factors for 24 (A, B, E) or 48h (C, D). Total RNA was subjected to RT-QPCR. Relative expression of stimulated cells over control is shown as the mean ±SD from five-nine independent experiments (A, C). Levels of secreted chemerin were determined in parallel in conditioned media by ELISA. Data show the mean ±SD from five-nine independent experiments (B, D). Statistical significance between control and the treated cells is shown by asterisk; *p<0.05 by ANOVA followed by a Bonferroni post hoc test. E. c., <i>E. coli</i>; HK, heat-killed; SN, supernatant; S. a., <i>S. aureus</i>; A, ampicillin-treated. Microscope images of keratinocytes stained with hematoxilin and eosin (H&E) and fluorescence microscope images of keratinocytes stained for chemerin (chem) or control rabbit Abs (cAb) (red), with Hoechst counterstain to detect cell nuclei (blue). Scale bar = 10 μm. Data are representative of three different donors. SC, stratum corneum, SG, stratum granulosum, SS, stratum spinosum, SB stratum basale, TM transwell membrane (E).</p

Psoriasis-associated cytokines downregulate chemerin and acute phase cytokines upregulate chemerin expression in epidermis.

<p>Normal keratinocytes grown in 3D culture were treated with the indicated factors for 24 (A-B) or 48h (C-D). Total RNA was subjected to RT-QPCR. Relative expression of stimulated cells over control is shown as the mean ±SD from five-nine independent experiments (A, C). Levels of secreted chemerin were determined in parallel in conditioned media by ELISA. Data show the mean ±SD from five-nine independent experiments (B, D). Statistical significance between control and the treated cells is shown by asterisk; *p<0.05 by ANOVA followed by a Bonferroni post hoc test.</p