Figure 4

Ccl5 is secreted by hepatic stellate cells isolated from mice with CDAHFD induced steatohepatitis and induces hepatocyte steatosis. (A,B) Expression of Ccl5 and Acta2 co-localized to hepatic stellate cells in both mouse steatohepatitis and human NASH assessed by immunofluorescence (7 out of 11 human samples tested positive). (C) Hepatic stellate cells isolated from mice with steatohepatitis continued to express Ccl5 at a greater level than initially healthy hepatic stellate cells, even after further activation on dish, measured with qPCR. (D) Recombinant Ccl5 protein induced steatosis in freshly isolated primary mouse hepatocytes, observed with Bodipy staining. (E) Hepatocytes that became steatotic with recombinant Ccl5 upregulated pro-inflammatory cytokines and chemokines, measured with qPCR. (F,G) The conditioned media from Ccl5 overexpressing hepatic stellate cells induced steatosis, detected with Bodipy stain, and caused increased expression of several inflammatory cytokines and chemoattractants in primary hepatocytes, measured with qPCR. (H) Hepatic stellate cells overexpressing Ccl5 also had increased expression of other pro-inflammatory mediators besides Ccl5, measured with qPCR. All data are presented as mean +/− SD (*P < 0.05). (I) Neutralizing Ccl5 in HSC conditioned media with a blocking antibody reduced steatosis in hepatocytes treated with the media. NASH, non-alcoholic steatohepatitis; HSC, hepatic stellate cell; CDAHFD, choline-deficient L-amino acid defined high fat diet; Hep, hepatocyte; qHSC, quiescent hepatic stellate cell; acHSC, activated hepatic stellate cell; Rc, recombinant; OE, overexpression; CM, conditioned media; ns, not significant.