TLR4 Directly Activates Invariant NKT Cells Figure 5. LPS-mediated engagement of TLR4 in iNKT cells suppresses bleomycin-induced pulmonary fibrosis. (A) Lungs were removed from B6 or TLR42/2 mice 7 or 21 days after an intratracheal injection of bleomycin (2 mg/kg), and the levels of hydroxyproline, and IL-4 and IFN-c transcripts were determined. (B) Hydroxyproline content in the lungs of B6, CD1d2/2, and CD1d2/2 mice adoptively transferred with sorted WT, TLR4-deficient iNKT cells, LPS-pretreated WT iNKT, or LPS-pretreated TLR4-deficient iNKT cells was determined 21 days after bleomycin injection. The increased hydroxyproline content in the lungs of experimental groups are expressed as a percentage. Data are indicated as mean 6 SEM of six mice in each group. (C) The transcript levels of TGF-b1, IFN-c, and IL-4 were determined by quantitative analysis relative to GAPDH using real-time PCR in the lungs of B6, CD1d2/2, and CD1d2/2 mice adoptively transferred with sorted WT iNKT or TLR4-deficient iNKT cells seven days after intratracheal injection of bleomycin. Data are representative of three repeated experiments. (n = 3 in each group; *p,0.05, **p,0.01, ***p,0.001). doi:10.1371/journal.pone.0045348.g005 cells enhanced IFN-c production but reduced IL-4 levels in the lungs of mice with HP or BIPF, resulting in the regulation of these pulmonary diseases. Consistent with these results, iNKT cells have been reported to promote the development of LPS-induced lethal shock syndrome by producing IFN-c [43]. Therefore, these findings suggest that signaling via TLR4 enhances IFN-c pro- duction by iNKT cells in the presence of TCR engagement both in vitro and in vivo. Taken together, these data suggest that direct engagement of TLR4 in iNKT cells regulates immune responses by affecting the production of IFN-c and IL-4 in the presence of TCR signaling. In this study, TLR4 engagement directly activated iNKT cells, triggering differential production of IFN-c and IL-4. Based on these findings, we suggest a novel TLR4-mediated direct activation pathway of iNKT cells during interactions between APCs and iNKT cells, in addition to the indirect pathways suggested by previous studies [20,21]. However, it is unlikely that this differential cytokine production sequentially activates DCs to enhance IL-12 production and CD1d expression during in- teraction between DCs and iNKT cells. Therefore, it is conceiv- able that DAMPs (damage associated molecular pattern molecules) and PAMPs (pathogen-associated molecular patterns), released from damaged cells during tissue injury and inflammation, activate both DCs and iNKT cells by direct engagement of TLR4 expressed on these cells, and interactions between activated NKT cells and DCs further augments their activation status. Therefore, iNKT cells can be activated by either direct engagement of TLR4 and/or indirectly via IL-12 production (cytokine-driven) and enhanced presentation of endogenous glycolipid (self-antigen- driven) by TLR4-dependently activated DCs. This may contribute to the regulation of immune responses in various immune diseases including autoimmunity, transplantation, and tumor surveillance. Meanwhile, several groups have demonstrated that NKT cells directly recognize cell-wall glycosylceramides of the Gram- negative, LPS-negative Sphingomonas [44,45,46]. Therefore, in the case of bacterial infection, iNKT cells are activated not only via TLR4-mediated iNKT cell activation pathways that occur during PLOS ONE | www.plosone.org 7 September 2012 | Volume 7 | Issue 9 | e45348