TLR4 Directly Activates Invariant NKT Cells Figure 4 LPS-mediated engagement of TLR4 in iNKT cells aggravates Saccharopolyspora rectivirgula (SR)-induced hypersensitivity pneumonitis (HP). HP was induced by inoculating the SR antigen nasally. (A) The levels of SR-specific IgG in serum, and IL-4 and IFN-c transcripts in the lungs of B6 or TLR42/2 mice were analyzed seven days after the first nasal inoculation of SR antigen using ELISA and real-time PCR, respectively. (B–D) B6, CD1d2/2, and CD1d2/2 mice adoptively transferred with sorted iNKT cells (16105 cells) from WT B6 or TLR42/2 mice were inoculated nasally with SR antigens. Sorted iNKT cells from B6 or TLR42/2 mice were incubated with LPS or PBS for 30 min before adoptive transfer into CD1d2/ 2 mice. (B) These mice were sacrificed three weeks after induction of HP, and SR-specific IgG levels in serum were determined. (C) IL-4 and (D) IFN-c levels were measured in the bronchoalveolar lavage fluid from B6, CD1d2/2, and CD1d2/2 mice adoptively transferred with sorted iNKT cells from B6 or TLR42/2 mice seven days after inoculation of the first SR antigen by ELISA. (E) Histological examination of the lungs was performed 7 days after first SR treatment (6100). (A–D) Data are from a representative of three repeated experiments. (n = 4 in A, n = 3 in B, C, D; *p,0.05, **p,0.01, ***p,0.001). doi:10.1371/journal.pone.0045348.g004 intracellular compartments of several types of cells including intestinal epithelial cells, placental syncytiotrophoblasts, dendritic cells, and neuroblastoma cells [36,37,38]. Moreover, LPS-induced TLR4 signaling in the cytoplasm of intestinal epithelial cells requires internalization of LPS [36,39,40]. Consistent with these findings, LPS triggers endocytosis of TLR4 on the surface, which is dependent on dynamin and early endosome formation. Furthermore, TLR4 sequentially engages MyD88 in the cell membrane and TRIF in the endosome [31]. In our experiments, TLR4-mediated differential IL-4 and IFN-c production by iNKT cells was attenuated by treatment with anti-TLR4 or CD14 mAb, or inhibitors of dynamin, MyD88, TRIF, or the endosome. These data suggest that LPS binds TLR4 expressed on the cell surface and triggers endocytosis of TLR4, which in turn is re-localized to the early endosome, resulting in modulation of the functions of iNKT cells in MyD88- and TRIF-dependent manners. Askenase et al. reported that stimulation of iNKT cells with LPS alone induced rapid, preferential, and transient production of IL- 4, but not IFN-c in vitro and in vivo [34]. In contrast, our experiments demonstrated that treatment of LPS alone did not alter production of IL-4 and IFN-c by iNKT cells in vitro. Furthermore, LPS-mediated TLR4 signaling in iNKT cells enhanced IFN-c production, but reduced IL-4 production in the presence of TCR signals. Consistent with our in vitro results, two independent studies demonstrated that stimulation of iNKT cells or CD1d-restricted T cell clones with LPS enhanced IFN-c but not IL-4 production in the presence of DCs [41,42]. Furthermore, in our in vivo experiments, LPS-mediated TLR4 signaling in iNKT PLOS ONE | www.plosone.org 6 September 2012 | Volume 7 | Issue 9 | e45348