1D and Supporting Table 2). We confirmed these results via real-time qRT-PCR and found that coculture

with HMs for 24 hours or for 5 days increased the expression of known NF-κB–regulated genes, including Il6, Saa3, Cxcl5, Cxcl14, Serpinb2, Ch25h, and Mmp13 (Fig. 2A,C). Surprisingly, HMs did not induce classical HSC activation markers such as Col1a1, Col1a2, or Acta2 messenger RNA (mRNA) and did not change α-smooth muscle actin (α-SMA) protein levels in HSCs (Fig. 2C). We confirmed that all NF-κB–dependent genes, including Timp1, were suppressed in the presence of adenoviral IκB superrepressor (Fig. 2A) or by short-term treatment with IKK inhibitor Bay 11-7085 at very low nontoxic concentrations (Supporting Fig. 3). NF-κB activation was further confirmed via VX-809 in vivo p65 immunohistochemistry (Fig. 2D) and immunoblot (Fig. 2E) demonstrating p65 translocation, p65-S536 phosphorylation, and IκBα degradation in HSCs treated with conditioned media from HMs but not after treatment with control media. Similar observations were made in an NF-κB reporter assay, in which coculture with HMs induced a >15-fold increase in NF-κB–driven luciferase activity (Fig. 2F). Based on these results, we focused on the NF-κB pathway in subsequent analyses of mechanisms by which HMs affect HSCs and fibrogenesis. Next, we determined whether HMs alter NF-κB–dependent

gene expression in HSCs in the fibrotic liver by employing a depletion approach. check details For this purpose, we analyzed gene expression in fluorescence-activated selleck chemicals llc cell sorting (FACS) ultrapure HSCs isolates that were immediately lysed after isolation and thus provide a “snapshot” of HSC gene expression in the fibrotic liver. NF-κB–dependent gene expression was highly up-regulated in HSCs activated in vivo compared with quiescent HSCs (Fig. 2G). Macrophage depletion by repeated liposomal clodronate injection efficiently reduced F4/80-positive and CD11b- and F4/80-double positive macrophages and ameliorated liver fibrosis following BDL and CCl4 treatment (Supporting

Fig. 4). Notably, macrophage depletion strongly suppressed the expression of the NF-κB–dependent genes that were up-regulated by HMs in our coculture system (Fig. 2G). We further excluded that liposomal clodronate directly affects NF-κB via NF-κB reporter assay and or cell death in cultured HSCs (Fig. 2H,I). Next, we investigated mechanisms through which HMs induce NF-κB activation in HSCs. First, we tested the contribution of interleukin (IL)−1 and TNF to HM-induced NF-κB in HSCs based on their known potent activation of NF-κB, the presence of the IL-1 receptor in the NF-κB network identified by IPA analysis (Fig. 1C), and up-regulated M1 markers inducible nitric oxide synthase and Cox2 in HMs from BDL mice (Supporting Fig. 1C). HMs induced NF-κB to the same degree as rmIL-1β, and to a higher degree than rmTNFα (Fig. 3A).