Materials and Methods Mice C57BL/6 mice were purchased from Orient Company (Seoul, Korea). CD1d2/2 mice were obtained from the NIAID-Taconic facility of the National Institutes of Health (Dr. Hua Gu, Columbia University, New York, NY). TLR42/2 mice were a generous gift from Dr. Akira S (Osaka University, Osaka, Japan). KRN TCR transgenic mice and NOD mice were a gift from Drs. D. Mathis and C. Benoist (Harvard Medical School, Boston, MA) and from the Institute de Genetique et de Biologie Moleculaire et Cellulare (Strasbourg, France), and were maintained in a C57BL/6(B6) background (K/B). Arthritic mice (K/BxN) were produced by crossing K/B with NOD (N) mice. These mice were bred and maintained under specific pathogen-free conditions at the Clinical Research Institute Seoul National University Hospital, Seoul, Korea. All animal experiments were approved from the In- stitutional Animal Care and Use Committee at the Clinical Research Institute Seoul National University Hospital (IACUC No. 06212). Real-time PCR Analysis To perform real time PCR, total RNA was isolated using an RNeasy kit (Qiagen, Courtaboeuf, France) according to the manufacturer’s instructions. RNA was reverse transcribed into cDNA using a MMLV-RT Taq polymerase (Promega, Madison, Korea) prior to PCR. For quantitative real-time PCR, gene- specific PCR products were measured using an Applied Biosys- tems 7500 Sequence Detection System (Perkin-Elmer Biosystem), and results for each cytokine were normalized versus GAPDH expression. The following primers and probe were synthesized by Applied Biosystems: GAPDH (TaqMan pre-developed Assay Reagent: 4352339E), IFN-c (TaqMan pre-developed Assay Re- agent: Mm 00801778 m1); TGF-b1, GCAACATGTGGAACTC- TACCAGAA (forward), GACGTCAAAAGACAGCC ACTCA (reverse), and FAM-ACCTTGGTAACCGGCTGCTGACCC- TAMRA; IL-4 (TaqMan pre-developed Assay Reagent: Mm 00445259 m1); T-bet (TaqMan pre-developed Assay Reagent: Mm01299452_g1); and GATA-3 (TaqMan pre-developed Assay Reagent: Mm00484683_m1). Preparation of Mononuclear Cells from the Livers To hepatic mononuclear cells, the livers were homogenized and resuspended in loading buffer (PBS plus 10% FBS and 1 mM EDTA) and overlaid onto lympholyte-M (Cedarlane, Ontario, Canada). After centrifugation for 20 min at 9006g at 15uC, hepatic mononuclear cells were isolated from the interface. Flow Cytometry For flow cytometric analysis, mononuclear cells (16106) isolated from the liver were stained with antibodies for 30 min at 4uC. To perform intracellular staining, cells were surface stained, fixed, and permeabilized with Cytofix/Cytoperm according to the manufac- turer’s instructions (BD Biosciences). PE-cy5-conjugated anti- mouse F4/80 (eBioscience, San Diego, CA, USA), APC-conju- gated a-galactosylceramide (a-GalCer)/CD1d tetramer (NIH Tetramer Facility, NIH, Bethesda, MD), FITC-conjugated anti- mouse CD3e (BD Pharmingen, San Diego, CA, USA), FITC- conjugated anti-mouse CD14 (BD Pharmingen), and PE-conju- gated anti-mouse CD1d (BD Pharmingen) antibodies were used. Confocal Microscopic Examination Sorted iNKT cells from WT or TLR4-deficient mice were fixed with 4% formaldehyde for 30 min. After washing with phosphate TLR4 Directly Activates Invariant NKT Cells buffered saline (PBS) for 5 min at 1500 rpm, cells were incubated with permeable/fixation solution (BD Pharmingen, San Diego, CA, USA) for 1 h at RT. After permeabilization, cells were incubated with APC-conjugated anti-mouse TLR4 (5 mg, eBioscience, San Diego, CA) and goat anti-mouse EEA-1 (1:500; Santa Cruz, CA, USA) for 1 h at 30 min at RT, and then incubated with secondary antibody conjugated with Alexa 488 (10 mg; Molecular Probes, Eugene, OR). Slides were then stained with DAPI (49.6-diamidino-2-phenylindole, 1:2000, Invitrogen, Carlsbad, CA) for 5 min at RT and viewed at 1006magnification. Sorting and Activation of iNKT Cells Liver a-GalCer/CD1d tetramer+CD3+ iNKT and NK1.1+TCR-b+ NKT cells were sorted using FACS Aria (BD Bioscience, San Diego, CA) and purity of sorted NKT cells was .99% (Figure S1A). Sorted iNKT or NK1.1+TCR-b+ NKT cells were incubated with aCD3+aCD28 mAb or aCD3+aCD28 mAb + LPS in RPMI media for 48 h. Twenty-four-well culture plates were coated with aCD3 mAbs in PBS (5 mg/ml) at 37uC for 2 h prior to incubation. To block TLR4 signaling, cells were pre- treated with Myd88 or TRIF inhibitors or control peptide (20 mM, Invivo Gen, San Diego, CA, USA) for 1 h and then washed with PBS. To inhibit surface TLR4 and/or CD14, sorted iNKT cells were preincubated with anti-TLR4 mAb (25 mg/ml) or anti-CD14 mAb (BD Pharmingen San Diego, CA, USA) (50 mg/ml) for 30 min at 4uC before stimulation with anti-CD3+anti-CD28 mAb. Inhibition of Endocytosis and Endosomal Compartments in iNKT Cells The irradiated splenocytes (1000 rad) and iNKT cells were co- cultured with a-GalCer or a-GalCer+LPS for 24 h. Before co- culture, iNKT cells were preincubated with 100 mM/ml of dynamin inhibitor (Dynasore, Merck, Darmstadt, Germany), 100 nM/ml of bafilomycinA1 (Sigma, St. Louis, Mo., USA) for 30 min or 100 mM/ml of chloroquine (Sigma, St. Louis, Mo., USA) for 2 h. a-GalCer was synthesized using the method developed by Kim et al. [27]. Reagents and Antibody Smooth (CD14-dependent, catalog number; L8274) and rough (CD14-independent, catalog number; L9641) lipopolysaccharide (LPS; Sigma Chem. Co., St. Louis, MO) were used. Anti-TLR2 mAb was purchased from BD bioscience (San Diego, CA, USA), which neutralizes TLR2. Coating Agarose Beads with LPS To inhibit internalization of LPS into the cells, LPS-coated beads were prepared using the AminoLinkH plus Immobilization kit (Thermo Scientific Inc., Bremen, Germany) according to the manufacturer’s instructions. Briefly, LPS (2 mg) was diluted in 1 ml pH 7.2 coupling buffer, and end-over-end mixed with 4% beaded agarose supplied as 50% slurry in 0.02% sodium azide. Final LPS concentrations were determined using a Limulus ameboycte lysate (LAL) assay kit (Lonza, Walkersville, MD, USA). Preparation of Bone Marrow Dendritic Cells (BMDC) and Co-culture with iNKT Cells To obtain BM cells, the tibia and femurs of WT and TLR42/2 mice were flushed. CD4+, CD8+ T, B220+, and I-Ab+ cells in BM were depleted using a magnetic bead separation kit (Miltenyi Biotec, GmbH, Bergisch-Gladbach, Germany). BM cells were incubated with 10 ng/ml of IL-4 (eBioscience, San Diego, CA, USA) and 20 ng/ml of GM-CSF (ProSpec-Tany TechnoGene, 51 PLOS ONE | www.plosone.org 9 September 2012 | Volume 7 | Issue 9 | e45348