the mechanisms by which TLR4-mediated activation of APCs regulates iNKT cell functions [21]. Therefore, it is generally accepted that iNKT cells are activated indirectly by TLR4- dependently activated DCs rather than directly by self-expressed TLR4 expressed. Although the expression pattern and functions of TLR4 in iNKT cells remain controversial, TLR4 is expressed on the cell surface of na¨ıve CD4+ T cells in mice and activated T cells in humans [24]. Moreover, TLR4 regulates subsequent TCR- dependent CD4+ T cell responses in experimental colitis in mice [25,26]. These findings led us to hypothesize that engagement of TLR4 might directly activate iNKT cells, resulting in modulation of immune responses in various diseases. Therefore, to address this hypothesis, we investigated whether (i) TLR4 is expressed in iNKT cells and (ii) direct engagement of TLR4 modulates the function of iNKT cells in vitro and in vivo. Our data demonstrate that direct engagement of TLR4, expressed on the cell surface and localized in the early endosome, enhances IFN-c production and reduces IL-4 production by iNKT cells, which modulates immune responses in various iNKT cell-mediated immune diseases. Results iNKT Cells Constitutively Express TLR4 on the Surface and in the Early Endosome To explore whether iNKT cells express TLR4, liver mono- nuclear cells were obtained from wild type (WT) B6 or TLR42/2 mice and TLR4 expression was examined both on the cell surface and in the cytoplasm of iNKT cells, macrophages, and conven- tional T cells. Flow cytometric and confocal microscopic analysis revealed that TLR4 was significantly expressed both on the cell surface and in the cytoplasm of macrophages and iNKT cells from WT, but not TLR42/2 mice, whereas none of T cells from WT and TLR42/2 mice expressed TLR4 (Fig. 1A–C). Moreover, TLR4 was co-localized with the early endosome maker EEA-1 in the cytoplasm of a-GalCer/CD1d tetramer+ iNKT cells from WT B6 mice, indicating that TLR4 is localized in the endosomal compartment of the cytoplasm in iNKT cells. Like as TLR4, both F4/80+ macrophages and iNKT cells also expressed CD14 on their surface (Fig. 1D). These findings indicate that a-GalCer/ CD1d tetramer+ iNKT cells express TLR4 on the cell surface and in the cytoplasmic endosomal compartment. Direct Engagement of TLR4 in iNKT Cells Enhances IFN-c Production, but Suppresses IL-4 Production To investigate the functional roles of TLR4 in iNKT and NK1.1+TCR-b+ NKT cells, the production of IL-4 and IFN-c by sorted iNKT NK1.1+TCR-b+ NKT cells was measured after LPS and/or TCR stimulation. LPS-mediated TLR4 engagement reduced IL-4 production in B6 mouse iNKT and NK1.1+TCR- b+ NKT cells activated by anti-CD3+ CD28 mAbs, whereas LPS enhanced IFN-c production (Fig. 2A and Fig. S1A, B). Moreover, LPS modulated IL-4 and IFN-c production by activated iNKT cells in dose-dependent and TLR2-independent manners (Fig. S1C, D). Unlike WT NKT cells, LPS did not alter the production of these cytokines by TLR4-deficient iNKT and NK1.1+TCR-b+ NKT cells in the presence of anti-CD3+ CD28 mAbs (Fig. 2A and Fig. S1B). These results suggest that TLR4 engagement in TCR- mediated activated iNKT and NK1.1+TCR-b+ NKT cells enhanced IFN-c production, but reduced IL-4 production. T-bet and GATA-3 are key transcription factors that regulate the expression of Th1/Th2 cytokine genes, respectively [28,29,30]. Therefore, it has been hypothesized that T-bet and/or GATA-3 might contribute to the differential cytokine production by iNKT TLR4 Directly Activates Invariant NKT Cells cells through TLR4 engagement. To address this hypothesis, we measured the transcriptional levels of GATA-3 and T-bet in gated iNKT cells from B6 or TLR42/2 mice stimulated with anti-CD3+ CD28 mAbs and/or LPS (Fig. 2B). iNKT cells from B6 and TLR42/2 mice stimulated with anti-CD3+ CD28 mAbs showed an increase in both GATA-3 and T-bet mRNA levels as compared with naive iNKT cells. In contrast, LPS-mediated TLR4 engagement reduced GATA-3 transcriptional levels and enhanced T-bet expression in WT iNKT cells, but not TLR4-deficient iNKT cells in the presence of TCR signals as compared with iNKT cells stimulated with anti-CD3+ CD28 mAbs alone. Although transcrip- tional levels of GATA-3 and T-bet do not completely reflect protein expression, these findings suggest that TLR4 engagement modulates the expression of GATA-3 and T-bet, which in turn differentially regulates Th1/Th2 cytokine production. To confirm TLR4-mediated differential regulation of IL-4 and IFN-c production by iNKT cells in vivo, we injected B6, CD1d2/2, or TLR42/2 mice with a-GalCer and/or LPS and determined serum IFN-c and IL-4 levels. a-GalCer treatment of B6 and TLR42/2 mice significantly enhanced the production of IL-4 and IFN-c, whereas CD1d2/2 mice showed no increases in the production of these cytokines (Fig. 2C). However, injection of a- GalCer and LPS into B6 mice reduced IL-4 production, but enhanced IFN-c production in the sera, whereas the differential IL-4 and IFN-c production by LPS and a-GalCer were not found in sera of TLR42/2 mice. These results indicate that TLR4 engagement in iNKT cells enhances IFN-c production and reduces IL-4 pro- duction in the presence of TCR stimulation in vivo. Collectively, TLR4 engagement in iNKT cells regulates T-bet and GATA3 expression, which in turn affects the production of IL-4 and IFN-c. Next, to investigate whether this differential cytokine production affect DC function, IL-12 production and CD1d expression on DCs were measured during co-culture of iNKT cells and bone marrow- derived DCs (BMDCs) from WT or TLR42/2 mice in the presence of LPS and/or a-GalCer. Treatment with a-GalCer + LPS did not alter IL-12 production in culture supernatant of iNKT cells and WT or TLR4-deficient BMDCs as compared with that of a-GalCer. However, IL-12 production in a culture containing irradiated BMDCs from WT or TLR42/2 mice and iNKT cells was minimal. Furthermore, LPS and/or a-GalCer did not affect the expression of CD1d by TLR4-deficient BMDCs, whereas WT BMDC displayed enhancement of CD1d expression after LPS stimulation (Fig. 2D). These findings indicate that differential IFN-c and IL-4 production by TLR4-dependently activated iNKT cells might not affect IL-12 production and CD1d expression by DCs during TLR4-mediated interaction. TLR4 Signals Mediated via MyD88 and TRIF Modulate Cytokine Production by iNKT Cells, which is Dependent on Endocytosis and Endosome Formation Upon engagement of TLR4, activation signals are delivered into cells via the MyD88-dependent and TRIF-dependent pathways [15]. Therefore, to investigate whether TLR4-mediated signals in iNKT cells depend on MyD88 or TRIF, sorted iNKT cells were stimulated with LPS, anti-CD3+ CD28 mAbs, or LPS + anti-CD3+ CD28 mAbs in the presence of MyD88 or TRIF inhibitors, or a control peptide. MyD88 and TRIF inhibitors suppressed IFN-c production, but enhanced IL-4 production by iNKT cells stimulated with anti-CD3+ CD28 mAbs and LPS as compared with the control peptide (Fig. 3A). These data suggest that signaling via TLR4 in iNKT cells depends on MyD88 and TRIF. It has been reported that LPS triggers endocytosis of membrane TLR4 via the dynamin-dependent pathway, and TLR4 engages MyD88 and TRIF sequentially at the surface and in the PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e45348