TLR4-mediated skin carcinogenesis is dependent on immune and radioresistant cells

Abstract

Skin cancers are the most commonly diagnosed cancers. Understanding what are the factors contributing to skin tumour development can be instrumental to identify preventive therapies. The myeloid differentiation primary response gene (MyD)88, the downstream adaptor protein of most Toll-like receptors (TLR), has been shown to be involved in several mouse tumourigenesis models. We show here that TLR4, but not TLR2 or TLR9, is upstream of MyD88 in skin tumourigenesis. TLR4 triggering is not dependent on lipopolysaccharide associated to skin-colonizing bacteria, but on the high mobility group box-1 protein (HMGB1), an endogenous ligand of TLR4. HMGB1 is released by necrotic keratinocytes and is required for the recruitment of inflammatory cells and for the initiation of inflammation. The expression of TLR4 on both bone marrow-derived and radioresistant cells is necessary for carcinogenesis. Consistently, a human tissue microarray analysis showed that melanoma and colon cancer display an over-expression of TLR4 and its downstream adaptor protein MyD88 within tumours. Together, our results suggest that the initial release of HMGB1 triggers a TLR4-dependent inflammatory response that leads to tumour development.

Figure 1

TLR4 and MyD88 KO mice are resistant to chemical-induced skin carcinogenesis. (A) Schematic representation of carcinogenesis protocol. Mice were followed up to a year and mice bearing a tumour >15 mm or sick were killed. (B) Average number of papillomas per mouse after DMBA/CO two-stage chemical carcinogenesis in function of time (weeks). ^*P<0.05 between WT, TLR2 KO and TLR9 KO versus TLR4 and MyD88 KO after 52 weeks. (C) Graph displaying percentage of mice carrying at least one carcinoma (in black) in the different genetic backgrounds; 15 mice (MyD88, TLR9, TLR2), 17 mice (TLR4) and 20 mice (WT) were used. The experiment was repeated twice using the same numerosity of mice with similar results. (D) Tumour incidence, showing the percentage of tumour-bearing mice over time. P1 (<0.01) represents statistical significance value between TLR4 and WT and P2 (<0.01) represents statistical significance value between MyD88 and WT. (E) Real-time PCR analysis for COX2 and MMP9 in the size-matched papillomas from WT and TLR4 KO mice. Data represents mRNA levels expressed relative to ribosomal protein L32. Values represent means±s.d. of the mean from at least five papillomas from each genotype. ^*P<0.05. The experiment was repeated twice with similar results.

Figure 2

TLR4 is important for CO-induced skin inflammation. The skin from WT and different KO mice was taken after 12 or 24 h of CO or acetone treatment and analysed for gene expression or production of different cytokines. (A) Real-time PCR analysis of different inflammatory mediators on mRNA isolated from skin extracts after 12 h of CO treatment. The results are expressed as fold induction over acetone-treated skins in each group after normalization to L32 mRNA. (B) Production of inflammatory cytokines IL-6, IL-17 and IL1-β in the skins as measured by ELISA after 24 h of CO or acetone treatment. Values represent means±s.d. of the mean from at least three skin samples from each genotype and each experiment was repeated at least twice. ^*P<0.05, ^**P<0.01, NS, not significant.

Figure 3

Recruitment of inflammatory cells in the skin after croton oil treatment. The skin from WT and TLR4 KO mice was collected 24 h after single treatment with CO and analysed for immune infiltrates. (A) Representative immunohistochemistry showing Gr1 staining of granulocytes in the skin of WT and TLR4 KO mice. Arrows show immune cell infiltrates. Scale bar, 100 μm. (B) FACS analysis showing the recruitment of granulocytes (Gr1) and monocytes/phagocytes (CD11b) in the skin. Values represent means±s.d. of the mean from at least three skin samples from each genotype and each experiment was repeated at least twice. ^*P<0.05, NS, not significant. (C) Real-time PCR analysis for S100A8 and S100A9 in the skins from WT and TLR4 KO mice 12, 24 and 48 h from CO treatment. Data represents fold induction of mRNA levels relative to acetone-treated animals after normalization to ribosomal protein L32. Values represent means±s.d. of the mean from at least five skins from each genotype. ^*P<0.05. The experiment was repeated twice with similar results.

Figure 4

Radioresistant and BM-derived cells both contribute to skin tumourigenesis. (A) Schematic representation of skin carcinogenesis protocol for chimeras. Mice were lethally irradiated (R=9.5 Gy), and 18 h later, injected i.v. with 2 × 10⁶ BM cells. Eight weeks later, mice were treated after the carcinogenesis protocol as shown in Figure 1A. (B) Average number of papillomas per mouse in different chimeras group is shown over time. (C) Tumour incidence, showing percentage of tumour-bearing mice over time. (D) Graph displaying percentage of mice carrying at least one carcinoma (in black) in the different genetic backgrounds. N=21 for WT → WT and N=15 for the other groups. One experiment was carried out. (E) Fold induction of IL-6 gene expression in the skin from chimeras taken 12 h after CO treatment and normalized to L32 mRNA. Values represent means±s.d. of the mean from five mice per each group and the experiment was repeated three times. ^*P<0.05. NS, not significant.

Figure 5

Carcinogen-induced inflammation is not dependent on skin bacteria or LPS. (A) Total number of bacteria/cm² of the back skin in mice treated or not (control) with ethanol (EtOH 90%). One representative experiment of three is shown. (B) Real-time PCR analysis of mRNA isolated from skin extracts showing expression of inflammatory cytokine genes in the group of WT mice treated or not with antiseptics along with croton oil in combination or not with polymyxin B (PB). As a control, mice were treated topically with LPS in acetone. ^*P<0.05, ^**P<0.01, ND, not determined.

Figure 6

Croton oil or TPA treatment induces release of HMGB1, LDH and necrosis of keratinocytes. (A) Release of LDH (left panel) and HMGB1 (right panel) in skin culture supernatants. The skins were removed at the indicated time points after single treatment with TPA and cultured in medium to reach 24 h from the initial TPA treatment. Values represent means±s.d. of the mean from at least three mice from each group and the experiment was repeated at least twice. (B) Immunofluorescence staining of HMGB1 (red) after 18 h croton oil treatment of WT (left, top) and TLR4 KO mice (right, top) in comparison with the control skin from respective mice (bottom). Scale bar=250 μm. Insets show higher magnification of the areas reported in the dashed line boxes ( × 2.5). Nuclei are stained with dapi (blue). Images are representative of at least five mice from each genotype. One representative image is shown per mouse. One experiment of three is shown. (C) Primary mouse keratinocytes were isolated and kept in culture as described in Materials and methods section. A total of 1.5 × 10⁵ cells were treated with DMBA (1 μM) or croton oil (0.5 mg/ml) and cell supernatant was collected after 16 h. Cells were collected for FACS analysis 4 h after treatment with DMBA or croton oil and stained with Annexin V and propium iodide. Supernatant was analysed for the release of LDH and HMGB1. Lysis buffer from a cytotoxicity assay kit was used as a positive control to measure total LDH. The graph shows the percentage of HMGB1 and LDH release (left y axis), and the percentage of necrotic cells (right y axis) compared with cells treated with lysis buffer (positive control) from the same experiment. ^*P<0.05 between CO or DMBA+CO groups versus control, acetone (Ac) or DMBA groups.

Figure 7

Blocking of HMGB1 in the skin reduces inflammatory cytokine production and inflammatory cell recruitment. The skin from WT mice was taken after 12 h for gene expression and 24 h for recruitment of inflammatory cells. (A) Real-time PCR analysis of mRNA isolated from skin extracts showing expression of inflammatory cytokine genes in the group of WT mice treated or not with ethanol (EtOH 90%) or HMGB1 inhibitors (glycyrrhizin or Box-A) along with CO. Values represent means±s.d. of the mean from at least three mice from each group and the experiment was repeated at least twice. P<0.05 (^*) represents statistical significant difference between croton oil-treated group and the other groups. NS, not significant, ND, not determined. (B) Representative images of immunofluorescence staining for Gr1 (red) showing reduced infiltration of granulocytes in the skin of WT mice treated with HMGB1 inhibitor glycyrrhizin (middle). Nuclei staining: DAPI, blue. The results are representative of at least three independent experiments. Scale bar, 200 μm. (C) Quantification of Gr1-positive cells in the skin from WT mice treated or not with the HMGB1 inhibitor glycyrrhizin (panel B). P<0.05 (^*) represents statistical significant difference between croton oil-treated group and CO + glycyrrhizin.

Figure 8

TLR4 and MyD88 expression in the progression of colon and melanoma tumours. (A, B) Tissue microarray analysis showing the expression of MyD88 (top) and TLR4 (bottom) during colon tumour progression in normal (N), hyperplasia (H), adenoma (A) and carcinoma (C) tissue samples. (A) Bar graphs showing marker expression (colour code as in the legend); (B) representative immunohistochemical images of MyD88 (top) and TLR4 (bottom) staining. (C) Tissue microarray analysis showing the expression of MyD88 (top) and TLR4 (bottom) during melanoma tumour progression as staged using Breslow (left) or Clark parameters (right). Breslow parameter refers to the depth of melanoma invasion and Clark refers to the anatomical invasion of the melanoma in the skin. (D) Representative immunohistochemistry images of MyD88 (top) and TLR4 (bottom) staining in samples staged using the Clark parameter. A melanoma-specific microarray composed of 34 benign lesions (nevi), 135 melanoma and 61 metastatic melanoma and a colon-specific microarray composed of 39 normal colon epithelium, 5 hyperplasia, 23 adenoma and 104 carcinoma were used. From each sample were arrayed two representative areas.