Interleukin‐38 promotes skin tumorigenesis in an IL‐1Rrp2‐dependent manner
2022; Springer Nature; Volume: 23; Issue: 6 Linguagem: Inglês
10.15252/embr.202153791
ISSN1469-3178
AutoresHong Zhou, Qixiang Zhao, Chengcheng Yue, Jiadong Yu, Huaping Zheng, Jing Hu, Zhonglan Hu, Haozhou Zhang, Xiu Teng, Xiao Liu, Xiaoqiong Wei, Yuxi Zhou, Fanlian Zeng, Yan Hao, Yawen Hu, Xiaoyan Wang, Chen Zhang, Linna Gu, Wenling Wu, Yifan Zhou, Kaijun Cui, Nongyu Huang, Wei Li, Zhen Wang, Jiong Li,
Tópico(s)Dermatology and Skin Diseases
ResumoArticle17 May 2022free access Transparent process Interleukin-38 promotes skin tumorigenesis in an IL-1Rrp2-dependent manner Hong Zhou Hong Zhou orcid.org/0000-0002-2785-5135 State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Conceptualization, Software, Investigation, Methodology, Writing - original draft, Project administration Search for more papers by this author Qixiang Zhao Qixiang Zhao State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Investigation, Methodology Search for more papers by this author Chengcheng Yue Chengcheng Yue State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Investigation, Project administration Search for more papers by this author Jiadong Yu Jiadong Yu State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Investigation, Project administration Search for more papers by this author Huaping Zheng Huaping Zheng State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Search for more papers by this author Jing Hu Jing Hu State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Investigation, Project administration Search for more papers by this author Zhonglan Hu Zhonglan Hu State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Investigation, Project administration Search for more papers by this author Haozhou Zhang Haozhou Zhang orcid.org/0000-0002-6321-2727 State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Investigation Search for more papers by this author Xiu Teng Xiu Teng orcid.org/0000-0002-2576-9495 State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Investigation Search for more papers by this author Xiao Liu Xiao Liu State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Investigation Search for more papers by this author Xiaoqiong Wei Xiaoqiong Wei orcid.org/0000-0001-7576-4820 State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Investigation Search for more papers by this author Yuxi Zhou Yuxi Zhou orcid.org/0000-0002-9231-2794 Department of Dermatovenereology, West China Hospital, Sichuan University, Chengdu, China Contribution: Investigation Search for more papers by this author Fanlian Zeng Fanlian Zeng State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Investigation Search for more papers by this author Yan Hao Yan Hao State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Investigation Search for more papers by this author Yawen Hu Yawen Hu State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Investigation Search for more papers by this author Xiaoyan Wang Xiaoyan Wang State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Investigation Search for more papers by this author Chen Zhang Chen Zhang orcid.org/0000-0001-9873-7235 State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Investigation Search for more papers by this author Linna Gu Linna Gu State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Investigation Search for more papers by this author Wenling Wu Wenling Wu State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Resources Search for more papers by this author Yifan Zhou Yifan Zhou State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Resources Search for more papers by this author Kaijun Cui Kaijun Cui Department of Cardiology, West China Hospital, Sichuan University, Chengdu, China Contribution: Resources Search for more papers by this author Nongyu Huang Nongyu Huang State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Resources Search for more papers by this author Wei Li Wei Li Department of Dermatovenereology, West China Hospital, Sichuan University, Chengdu, China Contribution: Resources Search for more papers by this author Zhen Wang Corresponding Author Zhen Wang [email protected] orcid.org/0000-0002-2516-4752 State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Department of Liver Surgery & Liver Transplantation, West China Hospital, Sichuan University and Collaborative Innovation Center of Biotherapy, Chengdu, China Contribution: Writing - review & editing Search for more papers by this author Jiong Li Corresponding Author Jiong Li [email protected] orcid.org/0000-0003-2320-9387 State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Search for more papers by this author Hong Zhou Hong Zhou orcid.org/0000-0002-2785-5135 State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Conceptualization, Software, Investigation, Methodology, Writing - original draft, Project administration Search for more papers by this author Qixiang Zhao Qixiang Zhao State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Investigation, Methodology Search for more papers by this author Chengcheng Yue Chengcheng Yue State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Investigation, Project administration Search for more papers by this author Jiadong Yu Jiadong Yu State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Investigation, Project administration Search for more papers by this author Huaping Zheng Huaping Zheng State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Search for more papers by this author Jing Hu Jing Hu State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Investigation, Project administration Search for more papers by this author Zhonglan Hu Zhonglan Hu State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Investigation, Project administration Search for more papers by this author Haozhou Zhang Haozhou Zhang orcid.org/0000-0002-6321-2727 State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Investigation Search for more papers by this author Xiu Teng Xiu Teng orcid.org/0000-0002-2576-9495 State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Investigation Search for more papers by this author Xiao Liu Xiao Liu State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Investigation Search for more papers by this author Xiaoqiong Wei Xiaoqiong Wei orcid.org/0000-0001-7576-4820 State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Investigation Search for more papers by this author Yuxi Zhou Yuxi Zhou orcid.org/0000-0002-9231-2794 Department of Dermatovenereology, West China Hospital, Sichuan University, Chengdu, China Contribution: Investigation Search for more papers by this author Fanlian Zeng Fanlian Zeng State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Investigation Search for more papers by this author Yan Hao Yan Hao State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Investigation Search for more papers by this author Yawen Hu Yawen Hu State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Investigation Search for more papers by this author Xiaoyan Wang Xiaoyan Wang State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Investigation Search for more papers by this author Chen Zhang Chen Zhang orcid.org/0000-0001-9873-7235 State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Investigation Search for more papers by this author Linna Gu Linna Gu State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Investigation Search for more papers by this author Wenling Wu Wenling Wu State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Resources Search for more papers by this author Yifan Zhou Yifan Zhou State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Resources Search for more papers by this author Kaijun Cui Kaijun Cui Department of Cardiology, West China Hospital, Sichuan University, Chengdu, China Contribution: Resources Search for more papers by this author Nongyu Huang Nongyu Huang State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Contribution: Resources Search for more papers by this author Wei Li Wei Li Department of Dermatovenereology, West China Hospital, Sichuan University, Chengdu, China Contribution: Resources Search for more papers by this author Zhen Wang Corresponding Author Zhen Wang [email protected] orcid.org/0000-0002-2516-4752 State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Department of Liver Surgery & Liver Transplantation, West China Hospital, Sichuan University and Collaborative Innovation Center of Biotherapy, Chengdu, China Contribution: Writing - review & editing Search for more papers by this author Jiong Li Corresponding Author Jiong Li [email protected] orcid.org/0000-0003-2320-9387 State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China Search for more papers by this author Author Information Hong Zhou1,†, Qixiang Zhao1,†, Chengcheng Yue1,†, Jiadong Yu1,†, Huaping Zheng1, Jing Hu1, Zhonglan Hu1, Haozhou Zhang1, Xiu Teng1, Xiao Liu1, Xiaoqiong Wei1, Yuxi Zhou2, Fanlian Zeng1, Yan Hao1, Yawen Hu1, Xiaoyan Wang1, Chen Zhang1, Linna Gu1, Wenling Wu1, Yifan Zhou1, Kaijun Cui3, Nongyu Huang1, Wei Li2, Zhen Wang *,1,4 and Jiong Li *,1 1State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China 2Department of Dermatovenereology, West China Hospital, Sichuan University, Chengdu, China 3Department of Cardiology, West China Hospital, Sichuan University, Chengdu, China 4Department of Liver Surgery & Liver Transplantation, West China Hospital, Sichuan University and Collaborative Innovation Center of Biotherapy, Chengdu, China † These authors contributed equally to this work *Corresponding author. Tel: +86 028 85164059; E-mail: [email protected] author. Tel: +86 028 85401949; E-mail: [email protected] EMBO Reports (2022)23:e53791https://doi.org/10.15252/embr.202153791 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract Interleukin-38 (IL-38) is strongly associated with chronic inflammatory diseases; however, its role in tumorigenesis is poorly understood. We demonstrated that expression of IL-38, which exhibits high expression in the skin, is downregulated in human cutaneous squamous cell carcinoma and 7,12-dimethylbenzanthracene/12-O-tetradecanoyl phorbol-13-acetate-induced mouse skin tumorigenesis. IL-38 keratinocyte-specific knockout mice displayed suppressed skin tumor formation and malignant progression. Keratinocyte-specific deletion of IL-38 was associated with reduced expression of inflammatory cytokines, leading to reduced myeloid cell infiltration into the local tumor microenvironment. IL-38 is dispensable for epidermal mutagenesis, but IL-38 keratinocyte-specific deletion reduces proliferative gene expression along with epidermal cell proliferation and hyperplasia. Mechanistically, we first demonstrated that IL-38 activates the c-Jun N-terminal kinase (JNK)/activator protein 1 signal transduction pathway to promote the expression of cancer-related inflammatory cytokines and proliferation and migration of tumor cells in an IL-1 receptor-related protein 2 (IL-1Rrp2)-dependent manner. Our findings highlight the role of IL-38 in the regulation of epidermal cell hyperplasia and pro-tumorigenic microenvironment through IL-1Rrp2/JNK and suggest IL-38/IL-1Rrp2 as a preventive and potential therapeutic target in skin cancer. Synopsis Decreased inflammation and epidermal cell proliferation in Il-38 cKO mice suppress skin tumor formation and malignant progression. IL-1Rrp2/JNK pathway is crucial for IL-38 mediated expression of inflammatory cytokines and proliferation and migration of tumor cells. Interleukin-38 (IL-38) is downregulated in human cutaneous squamous cell carcinoma and DMBA/TPA-induced mouse skin tumorigenesis. IL-38 keratinocyte-specific deletion ameliorates DMBA/TPA- induced skin tumors, reduces the number of immune cells and expression of inflammatory cytokines. IL-38 forms a complex with IL-1Rrp2 and activates the JNK/AP-1 signal transduction pathway in an IL-1Rrp2-dependent manner. The proliferation and migration of tumor cells and expression of inflammatory cytokines are induced by IL-38 via IL-1Rrp2/JNK. Introduction Skin cancer is the most common cancer in the United States of America (USA) (Rogers et al, 2010; Ming et al, 2014). Approximately, 2–3 million cases of non-melanoma skin cancer occur annually worldwide, and its incidence has been rising rapidly over the past several decades (Samarasinghe & Madan, 2012). 7,12-dimethylbenzanthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin cancer has remarkable phenotypic and genotypic homology to human cutaneous squamous cell carcinoma (cSCC) development. This model has been widely used to study how exogenous chemically induced inflammation triggers epithelial transformation and promotes subsequent tumor development (Wang et al, 2010b; Cataisson et al, 2012; Modi et al, 2012). The core of skin carcinogenesis lies in the activation of the RAS-mitogen-activated protein kinase (MAPK) pathway in early tumor cells and related inflammatory processes that seem to enhance tumor growth (Rundhaug & Fischer, 2010; Cataisson et al, 2012; Hayashi et al, 2015). The interleukin (IL)-1 family of cytokines has been widely studied for its pro- and anti-tumor functions in cancer. Several members of this family, such as IL-1β and IL-18, have been extensively investigated in cancer to decipher their role in immune cell recruitment as well as tumor cell proliferation, migration, invasion, and metastasis (Guo et al, 2016; Nakamura et al, 2018; Baker et al, 2019). IL-38 [molecular weight 16.9 kDa, as predicted using ProtParam tool (Yuan et al, 2015)], is the most recently identified member of the IL-1 family (Bensen et al, 2001; Lin et al, 2001). The main soluble form of IL-38 is considered to be a secreted ligand, and it is observed mainly in two forms: the full-length form (1–152 aa) and the truncated form (20–152 aa) (Mora et al, 2016). However, the true nature of IL-38 is still controversial, and its co-receptor has not yet been determined. Although several studies have explored the anti-inflammatory effect of IL-38, there are some contradictions that require elucidation. Reportedly, IL-38 can promote the production of several pro-inflammatory cytokines in different cell types in response to different stimuli (Huard et al, 2021; Mora et al, 2016; van de Veerdonk et al, 2012). As an IL-1 family receptor antagonist, IL-38 has been shown to limit IL-17 production in multiple disease models (Yuan et al, 2016; Boutet et al, 2017; Han et al, 2019). Nevertheless, a recent study has shown that local inflammation was reduced in IL-38 knockout mice subjected to the experimental autoimmune encephalomyelitis (EAE) model. Increasing evidence suggests that IL-38 is a crucial element in the pathogenesis of inflammatory autoimmune diseases, such as spondyloarthritis (Xia et al, 2021), psoriasis (Mercurio et al, 2018), psoriatic arthritis (Li et al, 2017), rheumatoid arthritis (Boutet et al, 2016), and systemic lupus erythematosus (Rudloff et al, 2015; Takeuchi et al, 2018). However, few studies to date have investigated the role of IL-38 in cancer. In the present study, we aimed to investigate the molecular and cellular targets of IL-38 during skin tumorigenesis. The study reveals a novel mechanism of IL-38 in skin cancer and highlights the essential role of keratinocyte-derived cytokines in cutaneous disease. Our results indicate that IL-38 keratinocyte-specific deletion dramatically ameliorates DMBA/TPA-induced skin tumors accompanied by a reduction in the number of immune cells and expression of cancer-related inflammatory cytokines. We have also shown that IL-38 forms a complex with IL-1 receptor-related protein type 2 (IL-1Rrp2) and activates the JNK/AP-1 signal transduction pathway in an IL-1Rrp2-dependent manner. The proliferation and migration of tumor cells and expression of cancer-related inflammatory cytokines are induced by IL-38 via an IL-1Rrp2/JNK-mediated pathway. Our findings provide in-depth insights into cytokine functions in cancer and offer a potential new preventive and therapeutic strategy. Results Expression of IL-38 is decreased in human cutaneous squamous cell carcinomas (cSCC) and DMBA/TPA-induced mouse tumorigenesis We analyzed IL-38 expression using the Genotype-Tissue Expression (GTEx) project (V8 dbGaP Accession phs000424.v8. p2) to determine the landscape of expression of IL-38. The analysis revealed the lowest expression of IL-38 in most tissues, while the highest expression was observed in the skin (Fig EV1A). Further, to determine IL-38 levels in skin tumors, we analyzed the expression of IL-38 in human skin cancer tissues and found that the protein was significantly lower in cSCC than in normal human skin tissues (Figs 1A and B, and EV1B). Analysis of IL-38 expression in normal mouse skin and DMBA/TPA-induced tumors revealed weak levels of IL-38 in tumors, consistent with findings in human patients (Fig 1C–F). Collectively, these results suggest a possible role of IL-38 in skin tumorigenesis. Click here to expand this figure. Figure EV1. Expression of IL-38 in different tissues and the expression of IL-1 family members in cSCC and DMBA/TPA-induced mouse tumors IL-38 expression in various tissues sourced from control subjects (n = 6–860) using the GTEx database. Representative immunohistochemical staining micrographs of IL-38 and rabbit IgG in human normal skin. Scale bars represent 200 μm. Relative expression of IL-1 family members in human normal tissues (n = 9) and cSCC (n = 18) were analyzed using Geo Datasets (GSE98767). Relative expression of IL-1 family members in normal tissues (n = 9) and cSCC (n = 38) of mice analyzed using Geo Datasets (GSE63967). Representative micrographs of human skin sections stained with anti-IL-18 antibody from normal patients (n = 11) and tumors of cSCC patients (n = 13). Scale bars represent 100 μm. The graph shows the quantification of mean IL-18 expression per high-powered field in tissues. The dorsal hair of normal C57/BL6 mice was shaved and treated with DMBA/TPA twice a week for 32 weeks to induce skin tumors. Representative micrographs of mouse normal skin (n = 6) and tumor (n = 6) sections stained with anti-IL-18 antibody. Scale bars represent 100 μm. The graph shows the quantification of mean IL-38 expression in tissues. Representative western blot bands indicating IL-18 in mouse normal skin (n = 3) and DMBA/TPA-induced tumors (n = 3). The graph shows the quantification of mean IL-18 expression in tissues. β-actin blots of Figs EV1G and EV3F are derived from the same experiment. Data information: Error bars represent the mean ± SD. All data are biological replicates. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; P values were calculated using Student’s t-test. Download figure Download PowerPoint Figure 1. IL-38 is closely related to the occurrence and development of skin tumors A. Representative micrographs of human skin sections stained with hematoxylin-eosin (H&E) (left) and anti-IL-38 antibody (right) from normal patients (n = 11) and tumors of cSCC patients (n = 13). Scale bars represent 100 μm. The graph shows the quantification of mean IL-38 expression per high-powered field in tissues. B. Relative expression of IL-38 in human normal tissues (n = 9) and cSCC (n = 18) was analyzed using Geo Datasets (GSE98767). C, D. The dorsal hair of normal C57/BL6 mice was shaved and treated with DMBA/TPA twice a week for 32 weeks to induce skin tumors. (C) Representative micrographs of mouse normal skin (n = 6) and tumor (n = 6) sections stained with anti-IL-38 antibody. The graph shows the quantification of mean IL-38 expression per high-powered field in tissues. Scale bars represent 100 μm. (D) Relative expression levels of Il-38 in normal skin (n = 5) and tumors (n = 5) of mice were quantified using qPCR. E. Relative expression levels of Il-38 in normal tissues (n = 9) and cSCC tissues (n = 38) of mice were analyzed using Geo Datasets (GSE63967). F. Representative western blot bands indicating IL-38 in mouse normal skin (n = 3) and DMBA/TPA-induced tumors (n = 3). The graph shows the quantification of mean IL-38 expression in tissues. Data information: Error bars represent the mean ± SD. All data are biological replicates. *P < 0.05; **P < 0.01; ***P < 0.001; P values were calculated using Student’s t-test. Download figure Download PowerPoint At the same time, we searched the Gene Expression Omnibus (GEO) database of the National Center for Biotechnology Information (NCBI) and identified multiple IL-1 family members exhibiting differential expression in skin cancer compared with normal skin searching (Fig EV1C and D). Further, experiments to detect IL-18 expression in normal human and mouse skin and tumors demonstrated that the expression of IL-18 decreased in human cutaneous squamous cell carcinoma (cSCC) and DMBA/TPA-induced mouse tumors compared with its expression in normal skin tissues, which is consistent with the findings in the GEO database (Fig EV1E–G). IL-38 keratinocyte-specific deletion suppresses the development of tumors To analyze the function of IL-38 in skin, we crossed mice with floxed alleles of Il-38 (Il-38f/f) with keratin14-Cre knock-in mice (K14Cre/+-Il-38+/+) to generate mice lacking Il-38 in keratinocytes (K14Cre/+-Il-38f/f) (Fig EV2A–D). Quantitative polymerase chain reaction (qPCR) and western blot analysis confirmed the deletion of Il-38 in epidermal splits from K14Cre/+-Il-38f/f mice (Fig EV2E and F). Studies have shown that the epidermis acts as a barrier to prevent water loss and exclude foreign substances and microorganisms (Franzke et al, 2012). Therefore, we analyzed the effect of IL-38 deletion in keratinocytes on the epidermal barrier in the steady state. Our results showed that keratinocyte-specific IL-38 deficiency had no significant effect on the epidermal barrier (Fig EV2G–M). Further, to examine whether IL-38 has a potential function in tumorigenesis, we applied a two-stage chemical skin carcinogenesis protocol to generate cutaneous squamous cell carcinoma (cSCC). K14Cre/+-Il-38f/f mice and control littermates were treated with a single dose of DMBA, followed by weekly application of TPA to allow skin tumor development (Fig 2A). IL-38 was completely lost or under detectable in DMBA/TPA-treated skin and DMBA/TPA-induced tumors of K14Cre/+-Il-38f/f mice (Fig EV3A–D). Unexpectedly, both the number and volume of tumors were significantly lower in K14Cre/+-Il-38f/f mice than in Il-38f/f mice (Fig 2B–D). The first tumors were observed in both Il-38f/f mice as well as in the K14Cre/+-Il-38f/f mice 8 weeks after beginning the DMBA/TPA treatment. After 15 weeks, all Il-38f/f mice developed tumors on their back skin, while approximately 37.5% K14Cre/+-Il-38f/f mice remained tumor-free (Fig 2E). In addition, the vast majority of tumors were collected 32 weeks after initiation. Several tumors in Il-38f/f mice already contained sites of high-grade dysplasia and foci of microinvasion (Fig 2F; arrow). To further investigate malignant conversion, we performed keratin8 (K8) staining in tumors. Multiple tumors in Il-38f/f mice were positive for the presence of K8 (Fig 2G). The percentage of tumors that progressed to malignancy was slightly decreased in K14Cre/+-Il-38f/f mice compared to that in Il-38f/f mice. Most importantly, the percentage of mice with malignant tumors was significantly lower in IL-38 keratinocyte-specific deletion mice (Fig 2H). These results suggest that IL-38 not only accelerates tumorigenesis, but also the malignant progression of tumors. Click here to expand this figure. Figure EV2. Construction and identification of keratinocytes IL-38-specific knockout mice and effect of keratinocyte-specific IL-38 deficiency on epidermal barrier Construction of Il-38-loxP (Il-38f/f) mice. Typical genetic cross scheme of keratinocyte IL-38-specific knockout (K14Cre/+-Il-38f/f) mice. PCR analysis of WT mice (at 317 and 245 bp) and Il-38-loxP mice (at 387 and 373 bp). PCR analysis of keratin 14 promoter directing expression of Cre recombinase(left) and deletion of IL-38 (right). Relative expression of IL-38 in the epidermal splits of Il-38f/f (n = 4) and K14Cre/+-Il-38f/f (n = 4) mice. Representative western blot bands indicating IL-38 in the epidermal splits of Il-38f/f and K14Cre/+-Il-38f/f mice. Appearance of Il-38f/f and K14Cre/+-Il-38f/f mice. Cumulative body weight changes in Il-38f/f (n = 12) and K14Cre/+-Il-38f/f (n = 12) mice in 60 days. Representative histological photographs of skin stained with hematoxylin-eosin (H&E) from Il-38f/f (n = 5) and K14Cre/+-Il-38f/f (n = 5) mice. Scale bars represent 100 μm. The graph shows the quantification of epidermal thickness. Skin barrier-dependent dye exclusion assay using toluidine blue in Il-38f/f mice (n = 5) and K14Cre/+-Il-38f/f littermate (n = 5)
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