Interleukin 17 Promotes Expression of Alarmins S100A8 and S100A9 During the Inflammatory Response of Keratinocytes

Abstract

Psoriasis is one of the most common immune-mediated inflammatory skin diseases. Expression and secretion of two pro-inflammatory molecules of the S100-alarmin family, S100A8 and S100A9, in keratinocytes is a hallmark of psoriasis, which is also characterized by an altered differentiation of keratinocytes. Dimers of S100A8/S100A9 (calprotectin) bind to Toll-like receptor 4 and induce an inflammatory response in target cells. Targeted deletion of S100A9 reduced the inflammatory phenotype of psoriasis-like inflammation in mice. A role of S100-alarmins in differentiation and activation of keratinocytes was suggested but has been never shown in primary keratinocytes. We now confirm that induction of S100-alarmins in an imiquimod-induced murine model of psoriasis-like skin inflammation was associated with an increased expression of interleukin (IL)-1α, IL-6, IL-17A, or TNFα. This association was confirmed in transcriptome data obtained from controls, lesional and non-lesional skin of psoriasis patients, and a down-regulation of S100-alarmin expression after IL-17 directed therapy. However, analyzing primary S100A9^-/- keratinocytes we found that expression of S100A8/S100A9 has no significant role for the maturation and inflammatory response pattern of keratinocytes. Moreover, keratinocytes are no target cells for the pro-inflammatory effects of S100A8/S100A9. However, different cytokines, especially IL-17A and F, highly abundant in psoriasis, strongly induced expression of S100-alarmins preferentially during early maturation stages of keratinocytes. Our data indicate that expression of S100A8 and S100A9 does not primarily influence maturation or activation of keratinocytes but rather represents the inflammatory response of these cells during psoriasis.

Keywords: S100A8; MRP14; MRP8; S100A9; calprotectin; keratinocytes; myeloid-related proteins; psoriasis.

Figure 1

S100A8 and S100A9 are up-regulated in psoriasis like skin inflammation. Naïve BALB/c mice were treated with imiquimod (IMQ) or placebo cream for 7 consecutive days. (A) Typical skin appearance of IMQ- or placebo cream treated BALB/c mice (n = 5 per group). (B) Quantitative measurements of epidermal thickness are shown as means ± SEM. Student’s t-test ***p < 0.001. (C) Representative paraffin skin sections stained with anti-S100A8, anti-S100A9, IgG control and HE. One representative image for each condition is shown (original magnification 10 x, scale bar = 100 µm). (D) Serum concentrations of S100A8/S100A9 in mice was analyzed via ELISA after 7 days of IMQ treatment (▪) and controls (▼) (Student’s t-test **p < 0.01). (E) Differential expression pattern of S100A8, S100A9 and various cytokines on transcriptional level in IMQ (▪)- and placebo (▼)- treated skin sections. Transcripts were measured by quantitative real-time PCR and normalized to RPL housekeeping gene. Values are expressed as mean ± SEM (n ≥ 3 mice per group; Student’s t-test *p < 0.05).

Figure 2

Imiquimod (IMQ) treatment induced inflammatory gene expression pattern. Naïve BALB/c mice were treated with IMQ or placebo cream for 7 consecutive days (n = 5 per group). (A, B) Cytokine/chemokines concentrations in serum of mice was analyzed at day 7 of IMQ (▪)- and placebo (▼)-treated mice with LEGENDplex multianalyte flow assay kit. In addition to individual data points, values are expressed as mean ± SEM. Student’s t-test *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

Morphology and differentiation of naïve primary keratinocytes of wildtype and S100A9^−/− mice. Primary epidermal keratinocytes were isolated from naïve wildtype C57BL/6 (WT)- and S100A9^−/− mice. (A) Representative phase contrast images of primary keratinocytes from WT- and S100A9^−/− mice at indicated stages of maturation (original magnification 10 x, scale bar = 100 µm). (B) Quantitative real-time PCR analysis of proliferation and maturation markers in keratinocytes from WT (white column)- and S100A9^−/− (black column) mice at indicated stages of maturation. Columns represent means ± SEM of at least eight independent experiments. Values are normalized to RPL housekeeping gene. Time course and cell culture conditions of primary keratinocytes are illustrated in Supplementary Figure 1 . Student’s t-test *p < 0.05.

Figure 4

Expression of S100A8 and S100A9 in naïve primary keratinocytes of wildtype and S100A9^−/− mice. Primary keratinocytes were isolated from wildtype C57BL/6 (WT)- and S100A9^−/− mice and matured for indicated time points ex vivo. (A) Representative immunofluorescence images of cell nuclei (DAPI, blue), S100A8, S100A9, or IgG control (Alexa Fluor 546, red) in primary WT- and S100A9^−/− keratinocytes at indicated stages of maturation (original magnification 20 x, scale bar = 100 µm). (B) Transcriptional expression patterns of S100A8 and S100A9 were determined by quantitative real-time PCR analysis in primary WT (white column)- and S100A9^−/− (black column) keratinocytes at indicated stages of maturation. Columns represent the means ± SEM of at least eight independent experiments. Values are normalized to RPL housekeeping gene. Student’s t-test showed no significances.

Figure 5

Inflammatory response patterns of primary keratinocytes of wildtype and S100A9^−/− mice. Primary keratinocytes were isolated from naïve wildtype C57BL/6 (WT, white column)- and S100A9^−/− (black column) mice, matured for indicated time points and stimulated for 24 h with indicated reagents [IL-17A, IL-17F, IL-1α, TNFα, S100A8, flagellin (FLA)]. Supernatants were collected for chemokine evaluation via flow cytometry associated bead-based immunoassay. Columns represent the means ± SEM of at least two independent experiments. Student’s t-test showed no significances between wildtype and S100A9^−/− mice.

Figure 6

Stimulation of S100A8 and S100A9 protein expression in naïve primary keratinocytes. Primary keratinocytes were isolated from wildtype C57BL/6 mice, matured, and stimulated for 24 h ex vivo. (A) Messenger RNA (mRNA) expression of S100A8 and S100A9 were analyzed by quantitative real-time PCR. Columns represent means ± SEM of at least five independent experiments. Values were standardized to RPL housekeeping gene and represent the n-fold to their respective unstimulated WT keratinocytes at each maturation time point (w/o = 1). Asterisks indicate significant differences to unstimulated controls based on the original expression data (ANOVA *p < 0.05; **p < 0.01; ***p < 0.001). (B) Release of S100A8/S100A9 into culture supernatants by WT keratinocytes during maturation was analyzed after incubation with different stimuli via ELISA. Columns represent the means ± SEM of at least three independent experiments. Values represent the n-fold to unstimulated WT keratinocytes (w/o = 1). Student’s t-test revealed no significant differences. (C) Expression levels of S100A8 and S100A9 in primary WT keratinocytes were determined by Western blotting. β-actin served as loading control. One representative experiment out of two independent experiments is shown.

Figure 7

Human gene expression analysis. (A) Microarray expression data from psoriatic patients and normal controls. Top left: principle component analysis (PCA) of 180 samples with skin samples of healthy controls (CTRL, n = 64) and 58 paired samples of psoriasis patients (PN = uninvolved skin, PL = involved skin). Top right: contributions of selected gene probes toward the variance in first and second principle components from PCA. Bottom left: boxplot of selected gene probes in the separate groups. Bottom right: Spearman correlation coefficient of expression between selected gene probes, where correlations were crossed out if they were not found to be significant with p <0.05. Data taken from GEO accession ID GSE13355. (B) Microarray expression data from psoriatic patients during IL-17 directed treatment. The different groups represent 25 patients with lesional (PL) and non-lesional (PN) skin. Additionally, biopsies were taken of lesional skin after 43 days of placebo (PL_placebo) or 700 mg of brodalumab treatment (PL_treatment). Data were taken from GEO accession ID GSE53552.Boxplot of selected gene probes based on expression data run on Affymetrix HU133 Plus 2.0 microarrays.