Recent work has identified the NALP3 inflammasome as a critical pathway in the generation of proinflammatory signals during liver injury. Moreover, IL-1 generated through this pathway exerts profibrogenic activities through the modulation of TIMP-1 and MMP9. Aim of this study was to evaluate the role of CCR5

in mediating inflammasome activation in response to gp120, in cells involved in liver tissue repair, including HSC. Myofibroblastic human HSCs were isolated from normal human liver tissue and cultured on plastic until fully activated. PBMC were separated from human whole blood. Gene expression was measured by qPCR. Protein IL-1 β protein levels were assayed by ELISA. HSCs or PBMC were exposed to 500 ng/ml recombinant M-tropic gp120 PD98059 concentration (CN54) for 2, 8 and 24 hours showed a time-dependent, significant up-regulation of pycard and NALP3, critical proteins for the assembly

of NALP3-dependent inflammasome, and of cas-pase-1 and IL-1 β. gp120 efficiently induced secretion of mature IL-1β, as shown by ELISA tests performed on the supernatants of both cell types. Notably, pre-incubation of HSCs with TAK779, a CCR5 receptor antagonist or with neutralizing α-CCR5 antibody reverted gp120-mediated IL-1 β production, indicating a primary role for this receptor in the activation of inflammasome Natural Product Library complex. Interestingly, when HSC were stimulated with CCL5 (RANTES), a natural agonist of CCR5, we also found a significant up-regulation of IL-1 b, confirming that CCR5 may mediate activation of this inflammatory complex in HSC. In conclusion, HIV-gp1 20 significantly increases the expression of components of the NALP3 Carbachol inflammasome pathway in human HSC and PBMC, through activation of CCR5. These data identify a novel mechanism by which HIV-gp1 20 may directly influence hepatic

necroinflammation and fibrosis during HCV/HIV coinfection, i.e. through increased production of IL-1 β. Moreover, these data establish for the first time a direct link between the inflammasome complex, HIV proteins and CCR5. We thank aid-sreagent.org for the kind gift of gp120. Disclosures: Fabio Marra – Advisory Committees or Review Panels: Abbott; Consulting: Bayer Healthcare, Gilead; Grant/Research Support: ViiV The following people have nothing to disclose: Andrea Cappon, Raffaele Bruno, Sandra Gessani, Andrea Masotti Matrix metalloproteinases (MMPs) are involved in various processes such as modulation of inflammation, tissue remodeling and collagen turnover. MMP-8 plays a yet ill-defined role in liver fibrogenesis and fibrolysis. Thus, we investigated the role of MMP-8 in toxin-induced liver fibrosis and spontaneous fibrosis regression using MMP-8 knock-out mice. Six week old female MMP-8 KO mice (n=10/group,C57/BL6 background) were treated according to an optimized CCl4 and TAA fibrosis-induction protocol for 4 and 8 weeks. For fibrosis regression, mice were harvested 5 days, 2 weeks, and 4 weeks after discontinuation of toxin treatment.

The appearance of the bands representing cleaved caspase-3 was prominent in cytoplasmic liver samples 120 minutes after TNFα/D-galN treatment in WT mice but only after 480 minutes in NS3/4A-Tg mice

to a much lower degree (Fig. 2A). This could be confirmed in liver sections from LPS/D-galN–treated mice using immunohistochemistry. Whereas a significant staining for cleaved caspase-3 was visible in WT mice 6 hours after application of LPS/D-galN, only a weak staining for cleaved caspase-3 was evident in NS3/4A-Tg mice (Fig. 2B). Furthermore, we analyzed the degree of apoptosis and necrosis in murine liver samples taken at different time points after LPS/D-galN administration through TUNEL staining. Again, liver sections from WT mice taken 6 hours after LPS/D-galN injection

had a significantly (P < 0,0001) higher PD0332991 research buy number of TUNEL-positive cells per 10 mm2 of liver compared with NS3/4A-Tg mice (Fig. 3A, upper right). Additionally, WT mice showed extensive and severe changes in the liver parenchyma with a high infiltration of inflammatory cells 6 hours after LPS/D-galN application (Fig. 3A, lower right). Thus, NS3/4A-Tg mice are less sensitive to TNFα-induced apoptosis compared with WT mice. These antiapoptotic effects might be exerted by the NS3/4A-related increase in NFκB activation. Activated NFκB is known both to protect hepatocytes from apoptosis and to PF-562271 purchase promote liver regeneration.13 We therefore determined the total number of hepatocyte nuclei and the number of dividing hepatocyte nuclei per 10 mm2 of liver in hematoxylin-eosin–stained liver sections from WT and NS3/4A-Tg mice left untreated or treated with LPS/D-galN. The total number of hepatocyte nuclei decreased during LPS/D-galN treatment in both WT and NS3/4A-Tg mice, with a more pronounced reduction in WT mice, compared with NS3/4A-Tg mice illustrating LPS/D-galN–mediated liver injury (Fig. 3B, left). In order to investigate liver regeneration, the amount of actively dividing hepatocyte nuclei was determined. Interestingly, this showed that, whereas the number of dividing hepatocyte

Orotic acid nuclei decreased in WT mice, NS3/4A-Tg mice revealed an increase in the number of dividing hepatocyte nuclei during LPS/D-galN treatment (Fig. 3B, right). Furthermore, after staining for the proliferation marker Ki67, liver sections from NS3/4A-Tg mice treated with LPS/D-galN had a significantly higher number of Ki67-positive nuclei compared with WT mice treated the same way (19.20 ± 2.49 versus 5.60 ± 1.65 positive nuclei per 10 mm2 of liver; P < 0.0001 [Mann-Whitney]) (Fig. 3C). Thus, NS3/4A not only exerts antiapoptotic effects but also promotes hepatocyte regeneration, most likely by increasing NFκB activation. NFκB regulates the transcription of an exceptionally large number of genes, many of which participate in immune and inflammatory responses, including TNFα, IL-1α, and IL-6.