2). Liver damage during conA-induced hepatitis (CIH) depends mainly AZD2014 manufacturer on the activities of liver T- and NKT-cells and their production of a variety of cytokines, including TNF-α and IFN-γ.14, 15 To examine the effect of VSIG4 on cytokine production by liver T- and NKT-cells, we performed intracellular cytokine staining after ConA challenge. The frequency of NKT-cells producing intracellular proinflammatory cytokines such as IFN-γ, TNF-α, and IL-17A

was significantly higher in VSIG4 KO mice than in WT mice (Fig. 2A; Supporting Table 1). A similar pattern of intracellular cytokine production was observed in liver T-cells from VSIG4 WT and KO mice. Furthermore, in vivo administration of soluble VSIG4.Ig to B6 WT mice 2 hours before ConA challenge greatly decreased the frequency of proinflammatory cytokine-producing NKT-cells compared to those given control Ig (Fig. 2B; Supporting Table 1). A similar pattern of cytokine production was observed in liver T-cells from mice given control Ig and VSIG4.Ig. We found that VSIG4.Ig bound naïve liver T- and NKT-cells in a binding assay, which was not blocked by 14G8 mAb,

an antibody that blocks C3b PF-01367338 chemical structure binding to VSIG4 (Supporting Fig. 3),16 suggesting that VSIG4 may directly suppress liver T- and NKT-cells by way of unidentified receptor(s). To demonstrate the role of VSIG4+ KCs in the regulation of liver inflammation in vivo, we adoptively transferred VSIG4 WT or Diflunisal KO KCs into mice lacking VSIG4 7 days before ConA injection. Our preliminary study using CFSE-labeled KCs showed that ∼45% of adoptively transferred KCs were localized in liver until 1 week after adoptive transfer (Supporting Fig. 4). The purified KCs expressed comparable levels of CD80, CD86, MHC class II, and B7-H1, and produced similar levels of IL-10 and TGF-β between KCs from VSIG4 WT and KO mice (Supporting Fig. 5A,B). Serum ALT levels were significantly reduced in VSIG4 KO mice that received VSIG4+

WT KCs compared to those that received VSIG4 KO KCs (P < 0.001; Fig. 3A). Similar results were obtained for serum IFN-γ. Consistently, VSIG4 KO mice were largely free of hepatic parenchymal necrosis after receiving VSIG4+ WT KCs compared to those given VSIG4 KO KCs (Fig. 3B). To examine whether VSIG4+ KCs directly regulate liver T- and NKT-cell cytokine production in vitro, we cocultured WT liver NKT-cells with KCs isolated from VSIG4 WT or KO mice in the presence of α-GalCer (KRN 7000) for 2 days. Liver NKT-cells produced more IFN-γ and IL-4 when cocultured with VSIG4 KO KCs than with WT KCs (at KC:NKT ratio of 1:1; IFN-γ, P < 0.01; IL-4, P < 0.001; Fig. 4A). NKT-cells did not produce detectable levels of IFN-γ or IL-4 in a coculture without α-GalCer stimulation (data not shown). Intracellular TNF-α was increased more in liver NKT-cells cocultured with VSIG4 KO KCs than in counterpart NKT-cells (Fig. 4B).