TLR4 Directly Activates Invariant NKT Cells Figure 2. LPS-mediated direct engagement in iNKT cells enhances IFN-c production, but reduces IL-4 production in the presence of TCR engagement. (A, B) Sorted iNKT cells from B6 or TLR42/2 mice (16105/well) were stimulated using coated anti-CD3 (5 mg mL21) + CD28 mAbs (5 mg mL21) in culture plates, LPS (5 mg mL21), or LPS (5 mg mL21) + anti-CD3 (5 mg mL21) + CD28 mAbs (5 mg mL21) for 24 h. (A) The amounts of IL-4 and IFN-c in the culture supernatant were measured by ELISA. (B) T-bet or GATA-3 mRNA expression were analyzed by real-time PCR. (C) B6, CD1d2/2, and TLR42/2 mice were injected i.p. with a-GalCer (1 mg in 300 ml PBS), LPS (25 mg in 300 ml PBS), or a-GalCer (1 mg) + LPS (25 mg in 300 ml PBS). Serum IL-4 levels were monitored 2 h later, and serum IFN-c levels were measured by ELISA 24 h after injection of these reagents. (D) iNKT cells were co-cultured with irradiated or un-irradiated bone marrow-derived DCs (BMDCs) from WT or TLR42/2 mice in the presence of LPS and/or a- GalCer for 24 h. The levels of IL-12 in culture supernatant and CD1d expression on BMDCs were estimated. Numbers in diagrams represent mean fluorescence intensity. (A–D) Data are presented as the means 6 SD of three mice in each group. Similar results were obtained from either two (D) or three (A–C) independent experiments. (*p,0.05, **p,0.01 and ***p,0.001). doi:10.1371/journal.pone.0045348.g002 the levels of SR-specific IgG in serum and reduced hydroxyproline levels in the lungs of CD1d2/2 mice as compared with untreated WT iNKT cells (Fig. 4B and 5B). Real-time PCR revealed that adoptive transfer of LPS-pretreated WT iNKT cells increased IFN-c production and reduced IL-4 production in the lungs of CD1d2/2 mice as compared with those mice adoptively trans- ferred with untreated iNKT cells in the HP and BIPF models (Fig. 4C, D and 5C). In contrast, pretreatment of TLR4-deficient iNKT cells with LPS did not affect SR-specific IgG levels or hydroxyproline levels in the lungs in SR-induced HP and BIPF as compared with that of untreated TLR4-deficient iNKT cells. These findings suggest that exogenous but not endogenous TLR4 ligands affect iNKT cell function in SR-induced HP and BIPF. Endogenous Ligand-mediated TLR4 Signals in iNKT Cells Promote Antibody-induced Arthritis To investigate whether TLR4-mediated differential production of IL-4 and IFN-c by iNKT cells regulates iNKT cell-mediated immune diseases, we compared joint inflammation in CD1d2/2 mice adoptively transferred with either WT or TLR4-deficient iNKT cells in the K/BxN serum transfer model. In a previous report, we demonstrated that NKT cells promoted antibody- induced arthritis by producing IL-4 and IFN-c [5]. WT iNKT cells promoted joint inflammation in CD1d2/2 mice as much as in B6 mice, whereas TLR4-deficient iNKT cells did not (Fig. 6A), which was consistent with histological alteration in the joints (Fig. 6C). Moreover, WT iNKT cells increased production of IL-4 and IFN-c and suppressed TGF-b production in the joint tissues of CD1d2/2 mice, whereas TLR4-deficient iNKT cells did not alter cytokine production (Fig. 6B). To explain the failure of TLR4- deficient iNKT cells to promote arthritis, it is possible that TLR4- deficient iNKT cells might be functionally defective with respect to activation in the serum transfer arthritis model, thereby contrib- uting to the failure to promote joint inflammation in CD1d2/2 mice. However, the ability of TLR4-deficient iNKT cells stimulated with a-GalCer to produce cytokines in vitro and in vivo indicates that TLR4-deficient iNKT cells are functionally intact in terms of activation by a-GalCer/CD1d complexes (Fig. 2A and C). Therefore, it is unlikely that the failure to promote antibody- induced joint inflammation in CD1d2/2 mice given TLR4- PLOS ONE | www.plosone.org 4 September 2012 | Volume 7 | Issue 9 | e45348