Expression of IL-23/Th17-related cytokines in basal cell carcinoma and in the response to medical treatments

Abstract

Several immune-related markers have been implicated in basal cell carcinoma (BCC) pathogenesis. The BCC inflammatory infiltrate is dominated by Th2 cytokines, suggesting a specific state of immunosuppression. In contrast, regressing BCC are characterized by a Th1 immune response with IFN-γ promoting a tumor suppressive activity. IL-23/Th17-related cytokines, as interleukin (IL)-17, IL-23 and IL-22, play a significant role in cutaneous inflammatory diseases, but their involvement in skin carcinogenesis is controversial and is poorly investigated in BCC. In this study we investigated the expression of IFN-γ, IL-17, IL-23 and IL-22 cytokines in BCC at the protein and mRNA level and their modulation during imiquimod (IMQ) treatment or photodynamic therapy (PDT). IFN-γ, IL-17, IL-23 and IL-22 levels were evaluated by immunohistochemistry and quantitative Real Time PCR in 41 histopathologically-proven BCCs (28 superficial and 13 nodular) from 39 patients. All BCC samples were analyzed at baseline and 19 of 41 also during medical treatment (9 with IMQ 5% cream and 10 with MAL-PDT). Association between cytokines expression and clinico-pathological variables was evaluated. Higher levels of IFN-γ, IL-17, IL-23 and IL-22 were found in BCCs, mainly in the peritumoral infiltrate, compared to normal skin, with the expression being correlated to the severity of the inflammatory infiltrate. IFN-γ production was higher in superficial BCCs compared to nodular BCCs, while IL-17 was increased in nodular BCCs. A significant correlation was found between IFN-γ and IL-17 expression with both cytokines expressed by CD4+ and CD8+ T-cells. An increase of all cytokines occurred during the inflammatory phase induced by IMQ and at the early time point of PDT treatment, with significant evidence for IFN-γ, IL-23, and IL-22. Our results confirm the role of IFN-γ and support the involvement of IL-23/Th17-related cytokines in BCC pathogenesis and in the inflammatory response during IMQ and MAL-PDT treatments.

Fig 1. Immunohistochemical and mRNA expression of IL-17, IL-23, IFN-γ and IL-22 in normal skin and BCC.

(A) Bar graphs showing cytokines+ inflammatory cells per mm² in normal skin compared to BCC. *p<0.05; **p<0.01; ***p<0.001; (B) Immunodetection of all cytokines was significantly higher in BCC samples compared to normal skin, and mainly expressed in inflammatory cells of peritumoral infiltrate; (C) Significantly elevated IL-23 and IFN-γ mRNA expression in BCCs as compared to normal skin. Increased expression of IL-17 and IL-22 mRNA in BCCs, as compared to control samples.**p<0.01; ***p<0.001.

Fig 2. Immunohistochemical and mRNA expression of IL-17, IL-23, IFN-γ and IL-22 varies according to severity of the inflammatory infiltrate in BCC.

(A) Graph showing cytokine levels in BCC, considering the severity of the inflammatory infiltrate. Statistically significant higher levels of IL-17, IL-23 and IFN-γ expression were observed with the increasing amount of peritumoral inflammatory infiltrate and the trend was similar for IL-22 expression. **p<0.01; ***p<0.001; (B) Exemplificative images of increasing IL-23 immunostaining in BCC samples associated to absent/mild, moderate and severe inflammation; (C) Variation of IL-23 mRNA expression levels according to the amount of the peritumoral inflammatory infiltrate.

Fig 3. Double staining of IL-17 or IFN-γ with CD4 or CD8.

(A) Double-staining of IL-17 (red) and CD4+ (brown) inflammatory cells and (B) of IL-17+ (red) and CD8+ (brown) cells. (C) Double-staining of IFN-γ (red) and CD4+ (brown) inflammatory cells and (D) of IFN-γ (red) and CD8+ (brown) inflammatory cells. Arrowheads indicate double-positive cells. Left panel magnification: 200×; right panel magnification: 400×.

Fig 4. Inflammatory cells and expression of IL-17, IL-23, IL-22 and IFN-γ before and during treatment with IMQ 5% cream or MAL-PDT.

(A) Number of inflammatory cells increased significantly during the inflammatory phase of IMQ treatment (p<0.01) and the early time point of MAL-PDT treatment, while decreased at the late time point (*p<0.05). (B) Cytokine levels in BCC before treatment and during the inflammatory phase of IMQ 5% treatment. ANOVA, *p<0.05; **p<0.01 (C) Cytokine levels in BCC at baseline, at the early and late timepoints of MAL-PDT. *p<0.05. TP1, early time point; TP2, late time point.