CXCL1 Inhibition Regulates UVB-Induced Skin Inflammation and Tumorigenesis in Xpa- Deficient Mice  Makoto Kunisada, Chieko Hosaka, Chihiro Takemori, Eiji Nakano, Chikako Nishigori  Journal of Investigative Dermatology  Volume 137, Issue 9, Pages 1975-1983 (September 2017) DOI: 10.1016/j.jid.2017.04.034 Copyright © 2017 The Authors Terms and Conditions

Figure 1 Intense and prolonged reaction to UVB exposure in Xpa-deficient mice. (a) Kinetic UVB-induced erythema and ear swelling in Xpa-deficient mice (Xpa) and wild-type mice (WT). Three mice of each genotype were observed after 1.0 kJ/m2 UVB exposure. (b) Ear swelling after UVB exposure. Both ears from three mice of each genotype were measured. Mean thickness and error bars were calculated. Error bars represent mean ± standard deviation. (c) Histological analysis by hematoxylin and eosin staining of skin for each genotype of mouse after UVB exposure. Inset of Xpa at 48 hours shows abundant neutrophil infiltration. Scale bar = 50 μm. Journal of Investigative Dermatology 2017 137, 1975-1983DOI: (10.1016/j.jid.2017.04.034) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Systemic CXCL1 up-regulation after UVB exposure in Xpa-deficient mice. (a) Systemic and local UVB exposure on mice. Local exposure was limited to 1 cm2 area of the back of mice, with other parts of body shielded. (b) The blood level of CXCL1 after UVB exposure, along with that of no UVB exposure, was measured by ELISA. (Left) 1.0 kJ/m2 UVB, (right) 0.5 kJ/m2 UVB, respectively. Each group analyzed consisted of three mice, and the mean value is shown. ∗P < 0.02, ∗∗P < 0.005. Error bars represent mean ± standard deviation. (c) The level of CXCL1 in supernatants of mouse embryonic fibroblasts established from Xpa-deficient mice and wild-type mice after 0.2 kJ/m2 UVB irradiation was assayed by ELISA. The supernatant of samples of no-UVB control was collected immediately after cells attached. ∗∗P < 0.005. Error bars represent mean ± standard deviation. h, hour; MEF, mouse embryonic fibroblast. Journal of Investigative Dermatology 2017 137, 1975-1983DOI: (10.1016/j.jid.2017.04.034) Copyright © 2017 The Authors Terms and Conditions

Figure 3 The anti-inflammatory effects of CXCL1 antibody and NAC on Xpa-deficient mice. (a) Ear swelling was measured in the same mice with or without peritoneal administration of CXCL1 (50 ng/body) neutralizing antibody (CXCL1 Ab) after 96 hours of 0.5 kJ/m2 UVB exposure. CXCL1 Ab was injected immediately and 24 hours after UVB exposure. (b) The blood level of CXCL1 48 hours after 0.5 kJ/m2 UVB exposure was measured by ELISA in the same mice as in a. (c) Representative features of redness and roughness of the skin of Xpa-deficient mice 96 hours after 0.5 kJ/m2 UVB with or without CXCL1 Ab administration. Identification numbers and sex of each mouse are indicated. (d) The blood levels of CXCL1 of Xpa-deficient mice 48 hours after 0.5 kJ/m2 UVB with or without orally administered N-acetylcysteine (NAC). NAC was administered 4 weeks before measurement of CXCL1 by ELISA. (e) Ear swelling of the same Xpa-deficient mice 96 hours after 0.5 kJ/m2 UVB with or without peritoneally administered CXCL1 recombinant protein (CXCL1 Rec). CXCL1 Rec was injected right after and 24 hours after UVB exposure. Journal of Investigative Dermatology 2017 137, 1975-1983DOI: (10.1016/j.jid.2017.04.034) Copyright © 2017 The Authors Terms and Conditions

Figure 4 Anti-tumor effects of CXCL1 neutralizing antibody (CXCL1 Ab) and N-acetylcysteine (NAC) on chronic UVB-irradiated Xpa-deficient mice. (a) The three top panels show the average number of skin tumors/mouse (mean ± standard deviation). The three middle panels show cumulative volume of tumors. The three bottom panels show skin tumor incidence by treatment with CXCL1 Ab (n = 10), recombinant CXCL1 (CXCL1 Rec) (n = 9), NAC (n = 10), and UVB-only control (n = 10). Xpa-deficient mice were irradiated weekly with 0.25 kJ/m2 UVB for 10 weeks along with either treatment followed by observation of skin tumor development with no UVB exposure or treatment for 15 weeks. The peritoneal injections of CXCL1 Ab (50 ng/mouse) and CXCL1 Rec (100 ng/mouse) were administered twice a week, right after and 24 hours after UVB exposure for 10 weeks. A diet supplemented with NAC (200 mg/10kg chow) was supplied to mice of the NAC group throughout experiments. Each statistical analysis was performed with Student t test. (b) Representative features of skin tumors in three Xpa-deficient mice with different treatments at the end of observation. wk, week. Journal of Investigative Dermatology 2017 137, 1975-1983DOI: (10.1016/j.jid.2017.04.034) Copyright © 2017 The Authors Terms and Conditions

Figure 5 Immunohistochemical study of p53 and 8-oxoGua. (Upper panel) After 0.5 kJ/m2 UVB exposure, the skin of wild type, Xpa-deficient mice, and Xpa-deficient mice with peritoneal injection of CXCL1 Ab or CXCL1 Rec and oral intake of NAC were conducted and subjected to immunohistochemical staining using mouse monoclonal p53 antibody at 3 and 24 hours. Follicular epidermal cells that tested positive for the expression are indicated with arrowheads. Scale bar = 50 μm. (Lower panel) Immunohistochemical analysis using a mouse monoclonal antibody against 8-oxoGua was performed for Xpa-deficient mice and wild-type mice skin after 0.5 kJ/m2 UVB exposure. The magnified epidermis of each group 3 hours after UVB is shown with insets. 8-oxoGua, 7,8-dihydro-8-oxoguanine; Ab, antibody; NAC, N-acetylcysteine; Rec, recombinant; WT, wild type. Journal of Investigative Dermatology 2017 137, 1975-1983DOI: (10.1016/j.jid.2017.04.034) Copyright © 2017 The Authors Terms and Conditions