We found no effect of liposomal clodronate on HSC viability, thereby excluding the possibility that liposomal clodronate directly induces HSC apoptosis in fibrotic livers (Fig. 2I). We also observed reduced IL-1β and TNF-α mRNA in fibrotic livers from clodronate-treated mice (Fig. 4C). To test the in vivo relevance of this pathway, we first investigated how deficiency of IL-1β, the predominant activator of NF-κB in our coculture experiments, affects liver fibrosis. In contrast to previously published studies, we found no statistically significant difference in BDL-induced liver fibrosis between IL-1R1 knockout and wild-type mice, and further confirmed

this data in the learn more CCl4 and thioacetamide models of liver fibrosis (Supporting Fig. 6). If IL-1 signaling promoted

liver fibrosis by increasing NF-κB–dependent HSC survival rather than direct HSC activation, it would be likely that TNF-α, the other major NF-κB–activating cytokine produced by macrophages, could still achieve NF-κB activation in HSCs and thus compensate for the loss of IL-1 signaling in this model. Based on the hypothesis that absence of both IL-1 and TNF signaling would be required to reduce HSC survival and liver fibrosis, we performed BDL in TNFR1/IL1R1 double knockout mice (dko) and wild-type control mice. Compared with wild-type mice, dko mice showed significantly reduced hepatic fibrosis after 5 or 15 days of BDL (Fig. 5A-B) and a five-fold increase in apoptotic TUNEL and desmin double-positive HSCs without significant differences in hepatic injury (Fig. 5B), supporting Bafilomycin A1 our hypothesis that suppression selleck kinase inhibitor of both IL-1 and TNF signaling are required to affect HSC survival and liver fibrosis. Moreover, we found a significant reduction of NF-κB–dependent genes—including Il6, Cxcl5, Saa3, Serpinb2, and Timp1—in ultrapure unplated HSCs from dko mice, thus confirming that NF-κB activation in HSCs was mediated by TNF

and IL-1 (Fig. 5C). Our microarray analysis revealed an up-regulation of two Trail decoy receptors, murine Trail decoy receptor 1 (Tntrsf23) and murine Trail decoy receptor 2 (Tnfrs22), in HSCs cocultured with HMs and in HSCs from BDL and CCl4 livers (Fig. 5D and Supporting Table 2). Notably, Trail-mediated apoptosis is a major contributor to HSC cell death induced by hepatic natural killer cells in vitro and in vivo.[11, 25] Neutralization of TNF or IL-1 prevented the up-regulation of Tnfrsf22 and Tnfrsf23 mRNA by HMs in coculture experiments (Supporting Fig. 7A). Moreover, depletion of HMs by liposomal clodronate or dko of TNFR1 and IL1R1 reduced Tnfrsf22 and Tnfrsf23 expression in vivo (Supporting Fig. 7B). Liposomal clodronate does not affect HSC number[13] or biology (Fig. 2H,I), but may deplete DCs, a highly endocytotic cell population, as demonstrated by our FACS analysis (Fig. 6A).