C57BL/6J mice and IFN-γ−/− mice were purchased from the Jackson L

C57BL/6J mice and IFN-γ−/− mice were purchased from the Jackson Laboratory (Bar Harbor, ME).

IFN-γ−/− suppressor of cytokine signaling 1 (SOCS1)−/− mice were kindly provided by James Ihle (St. Jude Children’s Research Hospital, Memphis, TN). All mice used in the present study were housed in a specific pathogen-free facility and were cared for in accordance with National Institutes of Health guidelines. All animal experiments were approved by the Institutional Animal Care and Use Committee of the National Institute on Alcohol Abuse and Alcoholism. Hepatic fibrosis in mice was induced experimentally through intraperitoneal injection of CCl4 as described.6 Mice were injected with CCl4 for 2 weeks or 8 weeks and cotreated INK128 with or BEZ235 datasheet without poly I:C (2 μg/g, 3 times a week intraperitoneally) or IFN-γ (2,000 IU/g, 7 times per week subcutaneously) for an additional 2 or 4 weeks. Control mice received CCl4 plus saline. For acute poly I:C treatment, mice were injected intraperitoneally with poly I:C once. Data are expressed as the mean ± SEM. To compare values obtained from two or more groups, Student t test or one-way analysis of variance was performed. A value of P < 0.01 or 0.05 was considered significant. All other materials and methods are described in the Supporting Information. To investigate

functions of NK cells on early stage (2-week CCl4) or advanced (10-week CCl4) liver fibrosis, mice were injected with CCl4 for 2 or 10 weeks without or with cotreatment of poly I:C (for the final 2 weeks). In the 2-week CCl4 group, serum IFN-γ levels were significantly increased after poly I:C treatment, but such elevation was not observed in the 10-week CCl4 group (Fig. 1A). In addition, Sorafenib purchase basal levels of serum IFN-γ from the 10-week CCl4 mice without poly I:C treatment were lower than those from 2-week CCl4 mice. Flow cytometry analyses showed

the basal levels of liver NK cell population and natural killer group 2 member D (NKG2D) expression on these cells were significantly lower in 10-week CCl4 mice compared with those in 2-week CCl4 mice (Fig. 1B). Interestingly, poly I:C treatment resulted in approximately two-fold induction of liver NK cell population and NKG2D expression in both 2-week and 10-week CCl4 mice (Fig. 1B). Furthermore, poly I:C induction of several NK cell-associated genes in the livers of 10-week CCl4 mice was diminished compared with those of 2-week CCl4 mice (Fig. 1C). Cytotoxicity assay demonstrated that poly I:C treatment significantly increased cytotoxicity of NK cells isolated from 2-week CCl4 mice against Yac-1 cells (NK-sensitive target cells) but not those of NK cells isolated from 10-week CCl4 mice (Fig. 1D).

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