TLR4 Directly Activates Invariant NKT Cells Figure 1. iNKT cells constitutively express TLR4 on the cell surface and in the endosomal compartment. (A) TLR4 expression was analyzed on gated a-GalCer/CD1d tetramer-CD3+ T cells, a-GalCer/CD1d tetramer+ iNKT cells, and F4/80+ macrophages from B6 (solid line) or TLR42/2 mice (gray) compared with an isotype-matched control IgG (dotted line) by flow cytometric analysis. Numbers in diagrams represent mean fluorescence intensity (top for control, middle for TLR42/2 mice, bottom for B6 mice). (B) Sorted iNKT cells and F4/80+ macrophages were stained with anti-TLR4 mAb (green) or isotype-matched control IgG, and DAPI (blue) (C) Sorted iNKT cells were stained with anti-TLR4 mAb or isotype- matched control IgG (red), and EEA-1 (early endosome marker; green) and DAPI (blue). (D) CD14 expression was analyzed on gated a-GalCer/CD1d tetramer+ iNKT cells and F4/80+ macrophages from B6 mice (solid line) as compared with an isotype-matched IgG control (gray). Data are representative of three independent experiments. doi:10.1371/journal.pone.0045348.g001 endosome, respectively [31]. Therefore, to explore whether these events occur in TLR4 in NKT cells, iNKT cells were incubated with dynamin or endosome (Baflilomycin A [Balfi] and Chloro- quine [Chlo]) inhibitors, and co-cultured with irradiated spleno- cytes of WT mice in the presence of LPS, a-GalCer, or LPS + a- GalCer. Treatment with dynamin or endosome inhibitors di- minished LPS-induced cytokine modulation of IFN-c and IL-4 by WT iNKT cells in the presence of TCR engagement (Fig. 3B, C). Furthermore, bead-bound LPS or blockade of surface TLR4 or CD14 using mAbs neither enhanced IFN-c production and nor reduced IL-4 production by sorted iNKT cells in the presence of TCR signals as compared with unbound LPS or isotype-matched control mAbs, respectively (Fig. 3D, E). Consistent with these findings, CD14-independent (rough) LPS could not alter IL-4 and IFN-c production by activated iNKT cells, suggesting that LPS- mediated effect on iNKT cells also depends on CD14 (Fig. S1E). These findings indicate that TLR4-mediated cytokine modulation by iNKT cells in the presence of TCR signals depends on endocytosis of LPS and TLR4, and early endosome formation. LPS-mediated Engagement of TLR4 in iNKT Cells Aggravates Saccharopolyspora Rectivirgula (SR)-induced Hypersensitivity Pneumonitis, but Attenuates Bleomycin- induced Pulmonary Fibrosis Previously, we reported that IL-4-producing NKT cells play a protective role in SR-induced hypersensitivity pneumonitis (HP) by suppressing the IFN-c-producing neutrophils, thereby de- creasing SR-specific IgG levels, while NKT cells attenuate bleomycin-induced pulmonary fibrosis (BIPF) by producing IFN- c [7,32]. Therefore, based on TLR4-mediated differential IL-4 and IFN-c production by NKT cells, we hypothesized that TLR4 engagement in iNKT cells reduces BIPF and aggravates SR- induced HP. To confirm this, we examined SR-induced HP and BIPF in WT and TLR42/2 mice. However, there was no difference in the phenotypes of SR-induced HP and BIPF between WT and TLR42/2 mice (Fig. 4A and 5A). Moreover, adoptive transfer of TLR4-deficient iNKT cells reduced both serum SR- specific IgG levels in HP and hydroxyproline levels in the lungs of CD1d2/2 mice in BIPF as much as did WT iNKT cells (Fig. 4B and 5B). Histological alteration in the lungs from mice with HP was consistent with serum SR-specific IgG levels, although histological alteration was not homogenous in the lungs with HP (Fig. 4E). These findings indicate that TLR4-deficient iNKT cells are functionally similar to WT iNKT cells in terms of their regulatory effects on HP and BIPF. These findings suggest that endogenous TLR4 ligands in iNKT cells are only minimally involved in the regulation of HP and BIPF. Therefore, to examine whether exogenous ligand LPS-mediated TLR4 engagement in iNKT cells modulates immune responses in SR-induced HP and BIPF, sorted WT or TLR4-deficient iNKT cells were pre- incubated with LPS in vitro and adoptively transferred into CD1d2/2 mice. LPS pretreatment of WT iNKT cells enhanced PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e45348