ConA-induced hepatitis is dependent on NKT cell activity.21 Using an adoptive transfer approach, we found, as expected, that SCID mice that received liver NKT cells from PBS-treated mice exhibited typical liver necrosis (Fig. 6A) and elevation of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (Fig. 6B). In marked contrast, the histologic evidence of necrosis in the liver (Fig. 6A) and the ConA-induced elevation of the levels of serum ALT and AST

(Fig. 6B) were reduced significantly in the SCID mice that received liver NKT cells from IDEN-treated mice, indicating that IDENs have a direct effect on induction of anergy in liver NKT cells. APCs play an essential role in Pictilisib molecular weight liver NKT cell activation by presenting lipid-related antigen on an APC CD1d molecule–dependent and independent manner.22 First, we tested whether DCs take up IDENs. Both CD11c+ DCs and F4/80+ macrophages from the livers of naïve mice took up IDENs rapidly (as

early as 2 hours) and continued to take up IDENs over 24 hours (Fig. 7A, Supporting Fig. 7). We then tested whether IDEN treatment has an effect on NKT cell activation of mice. FACS analysis of liver leukocytes suggested click here that the expression of MHCII and CD86 by the DCs (Fig. 7B) was reduced in mice that had been administered IDENs. On coculture of DCs purified from the livers of mice that had been administered IDENs or vehicle with carboxyfluorescein succinimidyl ester–labeled liver NKT cells isolated from mice that had been administered IDENs or vehicle in the presence of α-GalCer, DCs from the livers of mice that had been administered IDENs exhibited a reduced ability to stimulate the proliferation of NKT cells, regardless of the source of the NKT cells (Fig. 7C). RT-PCR analysis further indicated that

the expression of IL-12 and tumor necrosis factor α, which are critical for activation of DCs and DC-mediated activation of NKT cells, was significantly lower in DCs sorted from the livers of α-GalCer–injected mice that had been administered IDENs (Fig. 7D). Additionally, the levels of the immunosuppressive cytokine IL-10 were higher. DCs also can activate NKT cells in a CD1d-independent manner through Toll-like receptor (TLR)-induced release of soluble mediators, including IL-12 and type I IFNs.23,24 We found that IDENs suppressed Cetuximab the expression of IL-12 and IFN-β in TLR-stimulated DCs (Fig. 7E) and that IFN-γ release was reduced greatly when TLR ligand–treated DCs were cocultured with liver NKT cells in the presence of IDENs (Fig. 7F). Thus, IDENs can also induce NKT cell anergy through modification of the ability of DCs to stimulate NKT cell anergy in the context of both glycolipid presentation and TLR-mediated pathways. To further determine whether IDEN-associated PGE2 plays a role in the inhibition of production of IL-12, the effects of IDENs isolated from indomethacin-treated mice on the production of TLR-stimulated DCs was evaluated. ELISA results (Fig.