TLR4 Directly Activates Invariant NKT Cells Figure 6. TLR4 in iNKT cells plays a crucial role in promoting antibody-induced joint inflammation. (A) Sorted WT or TLR4-deficient iNKT cells (36105cells mouse21) were adoptively transferred into CD1d2/2 mice one day before K/BxN serum transfer (n = 3 per group). Clinical scores and ankle thickness were then monitored (*p,0.05 and **p,0.01, ***p,0.001). (B) The amounts of IL-4, IFN-c and TGF-b1 were measured relative to GAPDH by real-time PCR in the joint tissues of B6, CD1d2/2, and CD1d2/2 mice administered sorted WT or TLR4-deficient iNKT cells 10 days after K/ BxN serum transfer. (C) Histological examination of the joints was performed 7 days after K/BxN serum transfer (6100). Data are representative of three independent experiments. (n = 4 in each group; *p,0.05, **p,0.01, ***p,0.001). doi:10.1371/journal.pone.0045348.g006 tissue injury but also via direct recognition of bacterial glycolipids using the TCR. With respect to the regulation of HP and BIPF, there was no functional difference between TLR4-deficient and WT iNKT cells in CD1d2/2 mice in adoptive transfer experiments. However, pretreatment of iNKT cells with LPS suppressed BIPF, but worsened SR-induced HP to a greater degree than did untreated- iNKT cells, indicating that exogenous rather than endogenous TLR4 ligands likely contribute to iNKT cell-mediated immune modulation in HP and BIPF. Unlike HP and BIPF, TLR4- deficient iNKT cells could not restore joint inflammation in CD1d2/2 mice in antibody-induced arthritis, whereas WT iNKT cells did. These findings suggest that endogenous ligands trigger TLR4-mediated signals in iNKT cells, resulting in the modulation of joint inflammation. Therefore, endogenous TLR4 ligand- mediated signals in iNKT cells promote inflammatory arthritis, whereas exogenous TLR4 ligands regulate iNKT cell functions in HP and BIPF. Consistent with these findings, endogenous ligand- mediated engagement of TLR4 is critical for the induction of rheumatoid arthritis [47,48,49,50]. Taken together, exogenous and/or endogenous ligands directly engage TLR4 on iNKT cells in vivo and trigger TLR4-mediated immune modulation by iNKT cells, depending on the microenvironment of the disease model. In conclusion, our experiments demonstrate that TLR4 is expressed on the surface and localized in the early endosome of iNKT cells. Direct engagement of TLR4 by either endogenous or exogenous ligands enhances IFN-c production and reduces IL-4 production by iNKT cells. Moreover, TLR4-mediated differential cytokine production by iNKT cells modulates immune responses in various iNKT cell-mediated immune diseases such as SR- induced HP, BIPF, and antibody-induced joint inflammation in mice. PLOS ONE | www.plosone.org 8 September 2012 | Volume 7 | Issue 9 | e45348