Rehovot, Israel) for 5–8 d. BMDC differentiation was determined by flow cytometric analysis using a PE-conjugated anti-mouse CD11c mAb (.70% purity). Normal or irradiated BMDCs were co-cultured with iNKT cells stimulated with a-GalCer and/or LPS for 24 h. ELISA For measuring cytokine production, sera and culture super- natants of sorted iNKT cells were collected and analyzed by ELISA. All cytokine ELISA kits were obtained from BD Biosciences, and ELISA was performed according to the manufacturer’s instructions. Standard curves were generated using known amounts of purified murine recombinant (r) IL-4, rIFN-c, and rIL-12 (BD Biosciences, San Diego, CA, USA). The reaction was stopped with 3 N hydrochloric acid and the absorbance at 450 and 570 nm was read. Blood collected from the lateral tail veins was centrifuged, and SR-specific IgG levels in the serum (dilution 1/10) were determined by ELISA. Induction of hypersensitivity Pneumonitis (HP) SR antigen was prepared from an SR strain obtained from the American Type Culture Collection (ATCC), and re-suspended in pyrogen-free saline. The SR antigen contained less than 20 ng/mg of endotoxin, estimated using a limulus amebocyte lysate assay (Sigma Chemical Co., St. Louis, MO). HP was induced by intranasally instilling 150 mg of SR antigen in PBS into mice under light anesthesia. This procedure was performed on three consecutive days per week for three weeks. Mice were sacrificed four days after the final treatment. Bronchoalveolar lavage (BAL) was performed by delivering 0.8 ml of ice-cold PBS into the cannulated trachea and gently aspirating the fluid. Induction of Bleomycin-induced Pulmonary Fibrosis (BIPF) and Hydroxyrproline Assays Mice were anesthetized with 2, 2, 2-tribromomethanol, and intratracheally injected with 2 mg/kg of bleomycin (Nippon Kayaku, Tokyo, Japan) in PBS (50 ml). The total hydroxyproline level of the lung was determined as described previously [7]. Serum Transfer and Arthritis Scoring Arthritic adult K/BxN mice were bled and sera were pooled. Recipient mice were administered a 150 ml intraperitoneal injection of pooled K/BxN sera on days zero and two. Ankle thickness was measured with a caliper (Manostat, Switzerland). Joint swellings were monitored and scored as follows: 0, no joint swelling; 1, swelling of one finger joint; 2, mild swelling of wrist or ankle; 3, severe swelling of the wrist or ankle. Histological Examination To evaluate histological alterations of the lungs and ankles, whole lungs or joint tissues were fixed in 10% formalin 7 days after References 1. Lantz O, Bendelac A (1994) An invariant T cell receptor alpha chain is used by a unique subset of major histocompatibility complex class I-specific CD4+ and CD4–8- T cells in mice and humans. J Exp Med 180: 1097–1106. 2. Bendelac A (1995) CD1: presenting unusual antigens to unusual T lymphocytes. Science 269: 185–186. 3. Sharif S, Arreaza GA, Zucker P, Mi QS, Sondhi J, et al. (2001) Activation of natural killer T cells by alpha-galactosylceramide treatment prevents the onset and recurrence of autoimmune Type 1 diabetes. Nat Med 7: 1057–1062. 4. Singh AK, Wilson MT, Hong S, Olivares-Villagomez D, Du C, et al. (2001) Natural killer T cell activation protects mice against experimental autoimmune encephalomyelitis. J Exp Med 194: 1801–1811. TLR4 Directly Activates Invariant NKT Cells first SR treatment or K/BxN serum transfer. Paraffin-embedded tissues were cut and stained with hematoxylin and eosin (H&E). Adoptive Transfer of iNKT Cells into CD1d2/2 Mice Sorted iNKT cells (16105 cells per mouse) were adoptively transferred into CD1d2/2 mice by intravenous injection 1 day prior to the first intranasal instillation of SR antigen, bleomycin, or K/BxN serum transfer. Statistical Analysis Statistical significance was analyzed using Prism ver. 5.0 (GraphPad Software, Inc., San Diego, CA, USA). A t-test was performed to compare two groups. To compare multiple groups, a one-way analysis of variance (ANOVA) using Turkey’s post hoc test was used. For all analyses, P,0.05 was considered statistically significant. Supporting Information Figure S1 (A) Purity of sorted iNKT cells is .99% using FACS Aria. (B) LPS-mediated direct engagement in NK1.1+TCR-b+ NKT cells enhances IFN-c production, but reduces IL-4 pro- duction in the presence of TCR engagement. Sorted NK1.1+TCR-b+ NKT 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. (C) Sorted iNKT cells were stimulated using coated anti- CD3 (5 mg mL21) + CD28 mAbs (5 mg mL21) in culture plates in the presence of various amount of LPS for 24 h. (D) Sorted iNKT cells were stimulated using coated anti-CD3 (5 mg mL21) + CD28 mAbs (5 mg mL21) in culture plates and LPS (5 mg mL21) in the presence of isotype-matched control IgG or anti-TLR2 mAb (10 mg/ml) for 24 h. (E) Sorted iNKT cells were stimulated using coated anti-CD3 (5 mg mL21) + CD28 mAbs (5 mg mL21) in culture plates and CD14-independent LPS (5 mg mL21) for 24 h. (B–E) The amounts of IL-4 and IFN-c in the culture supernatant were measured by ELISA. (TIF) Acknowledgments The authors thank all members of the Department of Experimental Animals at CRI SNUH for animal management and the NIH Tetramer Facility for providing CD1d tetramers loaded with PBS-57 for this work. We also are thankful for Dr. Seong Yong Sung’s support obtaining TLR42/2 mice and Dr. Su Jin Hwang for her technical support. Author Contributions Conceived and designed the experiments: JHK HSK HYK DHC. Performed the experiments: JHK HSK HYK SJO. Analyzed the data: JHK HSK HYK SJO DHC. Wrote the paper: DHC. 5. Kim HY, Kim HJ, Min HS, Kim S, Park WS, et al. (2005) NKT cells promote antibody-induced joint inflammation by suppressing transforming growth factor beta1 production. J Exp Med 201: 41–47. 6. Cui J, Shin T, Kawano T, Sato H, Kondo E, et al. (1997) Requirement for Valpha14 NKT cells in IL-12-mediated rejection of tumors. Science 278: 1623– 1626. 7. Kim JH, Kim HY, Kim S, Chung JH, Park WS, et al. (2005) Natural killer T (NKT) cells attenuate bleomycin-induced pulmonary fibrosis by producing interferon-gamma. Am J Pathol 167: 1231–1241. 8. Dieli F, Taniguchi M, Kronenberg M, Sidobre S, Ivanyi J, et al. (2003) An anti- inflammatory role for V alpha 14 NK T cells in Mycobacterium bovis bacillus Calmette-Guerin-infected mice. J Immunol 171: 1961–1968. PLOS ONE | www.plosone.org 10 September 2012 | Volume 7 | Issue 9 | e45348