Volume 64, Issue 1, Pages 160-170 (January 2016) CXC chemokine receptor 3 promotes steatohepatitis in mice through mediating inflammatory cytokines, macrophages and autophagy  Xiang Zhang, Juqiang Han, Kwan Man, Xiaoxing Li, Jinghua Du, Eagle S.H. Chu, Minnie Y.Y. Go, Joseph J.Y. Sung, Jun Yu  Journal of Hepatology  Volume 64, Issue 1, Pages 160-170 (January 2016) DOI: 10.1016/j.jhep.2015.09.005 Copyright © 2015 European Association for the Study of the Liver Terms and Conditions

Journal of Hepatology 2016 64, 160-170DOI: (10. 1016/j. jhep. 2015. 09 Copyright © 2015 European Association for the Study of the Liver Terms and Conditions

Fig. 1 CXCR3 is upregulated in liver tissues of human NAFLD and murine steatohepatitis. (A) CXCR3 mRNA expression was significantly increased in human NAFLD tissues compared to normal liver tissues by quantitative real time PCR (qRT-PCR); (B) Representative immunohistochemistry images of CXCR3 protein expression in human normal liver tissues and NAFLD tissues. (C) Hepatic CXCR3 mRNA expression (qRT-PCR) in liver tissues of WT and CXCR3−/− mice, fed with control or MCD; (D) Representative immunohistochemistry images of CXCR3 protein expression in WT mice fed with control or MCD diet; (E) Representative H&E staining from db/db mice fed with control or MCD diet; (F) Hepatic CXCR3 mRNA expression (qRT-PCR) in db/db mice fed control or MCD diet. Values are mean±SD. Journal of Hepatology 2016 64, 160-170DOI: (10.1016/j.jhep.2015.09.005) Copyright © 2015 European Association for the Study of the Liver Terms and Conditions

Fig. 2 Deletion of CXCR3 protects against MCD diet-induced steatohepatitis. (A) Representative H&E staining (arrows, inflammatory cells) from 4-week liver sections of C57BL/6 WT (upper panel) and CXCR3−/− (lower panel) mice fed control (left panel) or MCD diet (right panel) were shown. (B) Serum ALT; (C) Total hepatic lipoperoxide and (D) liver triglyceride content in WT and CXCR3−/− mice fed control or MCD diet. Values are mean±SD (n=5/group). *p<0.05, **p<0.001 vs. same genotype mice fed control diet. Journal of Hepatology 2016 64, 160-170DOI: (10.1016/j.jhep.2015.09.005) Copyright © 2015 European Association for the Study of the Liver Terms and Conditions

Fig. 3 Hepatic chemokines expression, NF-κB activity and macrophage recruitment were decreased in liver tissues of CXCR3−/− mice. (A) Hepatic TNF-α, MCP-1 and IL-5 protein levels in the livers from WT and CXCR3−/− mice fed with control or MCD diet for 4weeks were quantified by cytokine profiling assay. (B) NF-κB nuclear binding activity was determined by an ELISA-based assay, protein levels of phosphorylated NF-κB subunits p50 were measured by Western blot (GAPDH served as loading control) and mRNA levels of ICAM-1 by qRT-PCR in WT and CXCR3−/− mice fed control or MCD diet. (C) F4/80 protein (Immunohistochemistry) and (D) mRNA expression (qRT-PCR) was determined in WT and CXCR3−/− mice fed control or MCD diet. (E) MCP-1 and its receptor CCR2 mRNA levels in liver tissues of WT and CXCR3−/− mice were measured by qRT-PCR. (F) Association of CXCR3 and MCP-1 expression in human liver tissues. Data were expressed as mean±SD. n=5–8/group. *p<0.05, **p<0.01, ***p<0.0001 vs. same genotype mice fed control diet. Journal of Hepatology 2016 64, 160-170DOI: (10.1016/j.jhep.2015.09.005) Copyright © 2015 European Association for the Study of the Liver Terms and Conditions

Fig. 4 CXCR3 mediates Th1 and Th17 immune response in liver tissues. Representative fluorescence-activated cell sorter analysis of (A) CD3+ T cells, (B) IFN-γ positive T cells (Th1 cells) and (C) IL-17 positive Th17 cells in the liver tissues of WT mice fed with control diet, WT mice fed with MCD diet, CXCR3−/− mice fed with MCD diet and MCD-fed CXCR3−/− mice adoptively transferred with wild-type T cells. The graphs represent percentage of CD45+ cells in the liver tissues. Data are shown as mean±SD. *p<0.05, **p<0.01, ***p<0.0001 vs. WT mice fed with control diet. Journal of Hepatology 2016 64, 160-170DOI: (10.1016/j.jhep.2015.09.005) Copyright © 2015 European Association for the Study of the Liver Terms and Conditions

Fig. 5 CXCR3 induced steatohepatitis through mediating lipogenic genes. (A) mRNA expression of hepatic lipogenic genes by qRT-PCR; (B) Hepatic SREBP-1c DNA binding activity was performed by an ELISA-based assay from MCD fed WT and CXCR3−/− mice liver tissues; (C) Hepatic mRNA expression of lipolytic genes (qRT-PCR); (D) Association of CXCR3 and SREBP-1c expression in human liver tissues of 24 NAFLD patients and 20 healthy control subjects. Specific mRNA expression values were normalized to the expression of GAPDH. Data are mean±SD, n=5/group. *p<0.05, **p<0.01, ***p<0.0001 compared with corresponding mice fed control diet. #p<0.05, ##p<0.0001 compared with WT mice fed MCD diet. Journal of Hepatology 2016 64, 160-170DOI: (10.1016/j.jhep.2015.09.005) Copyright © 2015 European Association for the Study of the Liver Terms and Conditions

Fig. 6 Autophagy is induced in the absence of CXCR3. (A) Hepatic protein expression of LC3 and p62 by Western blot. GAPDH served as loading control; and (B) Representative images of p62 protein expression and localization by immunohistochemistry in liver tissues from WT and CXCR3−/− mice fed control or MCD diet; (C) Hepatic protein expression of LAMP-1, LAMP-2; (D) polyubiquitinated proteins expression and (E) ER stress markers GRP78, PDI, p-PERK, PERK, p-eIF2α and eIF2α by Western blot in WT and CXCR3−/− mice fed control or MCD diet. (F) Apoptosis was determined by TUNEL staining in liver tissues from WT and CXCR3−/− mice fed control or MCD diet. Ten random fields from five slides per group were examined, and the TUNEL-positive brown nuclei within the hepatocytes were counted. Data were expressed as the number of TUNEL-positive cells/1000 cells. Data were expressed as mean±SD, n=5/group. Journal of Hepatology 2016 64, 160-170DOI: (10.1016/j.jhep.2015.09.005) Copyright © 2015 European Association for the Study of the Liver Terms and Conditions

Fig. 7 Deletion of CXCR3 protects against HFHC diet-induced steatohepatitis. (A) Representative H&E staining (arrows, inflammatory cells) from 12-week liver sections of C57BL/6 WT and CXCR3−/− mice, fed control or HFHC diet are shown, protein levels of phosphorylated NF-κB subunits p105 (p-p105), total p105, phosphorylated p65 (p-p65) and total p65 were detected by Western blot in HFHC-fed WT and CXCR3−/− mice. (B) Schematic diagram for the mechanisms of CXCR3 in the promotion of dietary steatohepatitis. CXCR3 plays a pivotal role in the pathogenesis of NASH by inducing production of cytokines, macrophage infiltration, fatty acid synthesis and causing autophagy deficiency and ER stress. Journal of Hepatology 2016 64, 160-170DOI: (10.1016/j.jhep.2015.09.005) Copyright © 2015 European Association for the Study of the Liver Terms and Conditions