Fig 1: IFNa-induced CXCL11 and CCL5 expression involves STAT2 and IRF9, but not STAT1.Cultured human keratinocytes were transfected with siRNA directed against STAT2 (siSTAT2), STAT1 (siSTAT1), IRF9 (siIRF9), or control siRNA (siCon) before IFNa (1000 U/ml) stimulation for 24 hours. (A) Protein extracts were isolated from the cells and the protein level of STAT2, STAT1, and IRF9 examined by western blotting. ß-actin was used as a loading control. One representative gel out of three is shown. (B, C) The protein level of (B) CXCL11 and (C) CCL5 in the cell culture medium was analyzed by ELISA (n = 3). Results are expressed as mean ± standard deviation. *P < 0.05.
Fig 2: The expression of CXCL11 and CCL5 is positively correlated with IFN? expression in lesional psoriatic skin.(A) Chemotaxis assays were performed using a 24-well plate-based assay. PBMCs were isolated from blood taken from psoriatic patients and loaded on the upper chamber containing a 5-µm pore-size filter. Wells (lower chamber) were added vehicle or 10 nM recombinant CXCL11 or CCL20 and incubated for 20 hours. Cells migrating to the lower chamber were analyzed by flow cytometry (n = 9). Results are expressed as mean ± standard deviation. *P < 0.01. (B-E) RNA from punch biopsies taken from 16 psoriatic patients was isolated and the mRNA expression of CXCL11, CCL5, IFN?, and IL-17A analyzed by qPCR. The correlation between the expression levels of these cytokines was analyzed as indicated in the figure.
Fig 3: Serum levels of CXCL9, CXCL10, and CXCL11 in melanoma patients treated with CPIs. Chemokine levels were analyzed by ELISA in serum samples of melanoma patients before (T0) and after 1 month (T1) and 3 months (T2) of treatment. Best overall response according to iRECIST criteria: iPD, progressive disease (n. 13 patients); iSD, stable disease (n. 16 patients); iPR, partial response (n. 17 patients); complete response, iCR (n. 11 patients). (a,b) CXCL9, (c,d) CXCL10, (e,f) CXCL11 amount (pg/ml). Data are indicated as mean value ± standard error of the mean (SEM). *p < 0.05, as assessed by Mann–Whitney U test to compare between-group differences; or by Wilcoxon signed-rank test to evaluate before–after treatment differences.
Fig 4: Increased mRNA expression of CXCL11 and CCL5 in lesional psoriatic skin.Total RNA was isolated from biopsies obtained from normal healthy volunteers as well as lesional and nonlesional psoriatic skin. mRNA expression of (A) CXCL11 and (B) CCL5 was analyzed by qPCR. RPLP0 mRNA expression was used for normalization. Scatterplot shows the result from 6 healthy volunteers and 16 psoriatic patients. *P < 0.01.
Fig 5: STAT2-mediated regulation of CXCL11 and CCL5 expression.(A) Cultured human keratinocytes were stimulated with IFNa (1000 U/ml) for the indicated time points, and the phosphorylation level of STAT2 analyzed by western blotting (n = 3). (B) Keratinocytes were transfected with STAT2 siRNA (siSTAT2), control siRNA (siCon) or transfection reagent alone (Mock) before being stimulated with IFNa (1000 U/ml) for 24 hours. Protein extracts were isolated from the cells and STAT2 and P-STAT2 expression analyzed by western blotting (n = 3). ß-actin was used as a loading control. (C) Human keratinocytes were transfected with STAT2 siRNA (siSTAT2) or control siRNA (siCon) before stimulation with IFNa (1000 U/ml) for 24 hours. Then the cell culture medium was screened for 102 proinflammatory cytokines and chemokines using a proteome profile array. Numbers on the membrane marks the following targets: 1 (CXCL11), 2 (IL-1ra), 3 (IL-1a), 4 (CCL5), 5 (MCP-1), 6 (IL-8), 7 (CXCL1) and 8 (ENA-78). (D, E) Human keratinocytes were stimulated as in (C) and the expression level of CXCL11 and CCL5 analyzed by ELISA (n = 3). Results are expressed as mean ± standard deviation. *P < 0.05.
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