Obesity and type 2 diabetes are characterized by decreased insulin sensitivity, elevated concentrations of free fatty acids (FFAs), and increased macrophage infiltration in adipose tissue (AT). Here, we show that FFAs can cause activation of RAW264.7 cells primarily via the JNK signaling cascade and that TLR2 and TLR4 are upstream of JNK and help transduce FFA proinflammatory signals. We also demonstrate that F4/80+CD11b+CD11c+ bone marrow-derived dendritic cells (BMDCs) have heightened proinflammatory activity compared with F4/80+CD11b+CD11c− bone marrow-derived macrophages and that the proinflammatory activity and JNK phosphorylation of BMDCs, but not bone marrow-derived macrophages, was further increased by FFA treatment. F4/80+CD11b+CD11c+ cells were found in AT, and the proportion and number of these cells in AT is increased in ob/ob mice and by feeding wild type mice a high fat diet for 1 and 12 weeks. AT F4/80+CD11b+CD11c+ cells express increased inflammatory markers compared with F4/80+CD11b+CD11c− cells, and FFA treatment increased inflammatory responses in these cells. In addition, we found that CD11c expression is increased in skeletal muscle of high fat diet-fed mice and that conditioned medium from FFA-treated wild type BMDCs, but not TLR2/4 DKO BMDCs, can induce insulin resistance in L6 myotubes. Together our results show that FFAs can activate CD11c+ myeloid proinflammatory cells via TLR2/4 and JNK signaling pathways, thereby promoting inflammation and subsequent cellular insulin resistance. Obesity and type 2 diabetes are characterized by decreased insulin sensitivity, elevated concentrations of free fatty acids (FFAs), and increased macrophage infiltration in adipose tissue (AT). Here, we show that FFAs can cause activation of RAW264.7 cells primarily via the JNK signaling cascade and that TLR2 and TLR4 are upstream of JNK and help transduce FFA proinflammatory signals. We also demonstrate that F4/80+CD11b+CD11c+ bone marrow-derived dendritic cells (BMDCs) have heightened proinflammatory activity compared with F4/80+CD11b+CD11c− bone marrow-derived macrophages and that the proinflammatory activity and JNK phosphorylation of BMDCs, but not bone marrow-derived macrophages, was further increased by FFA treatment. F4/80+CD11b+CD11c+ cells were found in AT, and the proportion and number of these cells in AT is increased in ob/ob mice and by feeding wild type mice a high fat diet for 1 and 12 weeks. AT F4/80+CD11b+CD11c+ cells express increased inflammatory markers compared with F4/80+CD11b+CD11c− cells, and FFA treatment increased inflammatory responses in these cells. In addition, we found that CD11c expression is increased in skeletal muscle of high fat diet-fed mice and that conditioned medium from FFA-treated wild type BMDCs, but not TLR2/4 DKO BMDCs, can induce insulin resistance in L6 myotubes. Together our results show that FFAs can activate CD11c+ myeloid proinflammatory cells via TLR2/4 and JNK signaling pathways, thereby promoting inflammation and subsequent cellular insulin resistance. Chronic inflammation is a well described feature of insulin resistance and obesity characterized by elevated proinflammatory JNK 2The abbreviations used are: JNK, c-Jun N-terminal kinase; FA, fatty acid; FFA, free fatty acid; TLR, Toll-like receptor; AT, adipose tissue; BM, bone marrow; BMDC, bone marrow-derived dendritic cell; BMDM, bone marrow-derived macrophage; HF, high fat; HFD, high fat diet; IL, interleukin; TNF, tumor necrosis factor; FBS, fetal bovine serum; BSA, bovine serum albumin; RT, reverse transcription; ELISA, enzyme-linked immunosorbent assay; siRNA, small interfering RNA; WT, wild type; GM-CSF, granulocyte macrophage-colony-stimulating factor; FACS, fluorescence-activated cell sorter; PBS, phosphate-buffered saline; ITT, insulin tolerance test; SVF, stromal vascular fraction; SVC, stromal vascular cell; NC, normal chow; MNC, mononuclear cells; CM, conditioned medium; KD, knockdown; qPCR, quantitative PCR. and IKKβ kinase activity (1Yuan M. Konstantopoulos N. Lee J. Hansen L. Li Z.W. Karin M. Shoelson S.E. Science. 2001; 293: 1673-1677Crossref PubMed Scopus (1652) Google Scholar, 2Hirosumi J. Tuncman G. Chang L. Gorgun C.Z. Uysal K.T. Maeda K. Karin M. Hotamisligil G.S. Nature. 2002; 420: 333-336Crossref PubMed Scopus (2679) Google Scholar) and increased cyto/chemokine expression in insulin target tissues (3Dandona P. Aljada A. Bandyopadhyay A. Trends Immunol. 2004; 25: 4-7Abstract Full Text Full Text PDF PubMed Scopus (1672) Google Scholar). In white adipose tissue, chronic inflammation is associated with an increase in macrophage infiltration (4Weisberg S.P. McCann D. Desai M. Rosenbaum M. Leibel R.L. Ferrante Jr., A.W. J. Clin. Investig. 2003; 112: 1796-1808Crossref PubMed Scopus (7637) Google Scholar, 5Xu H. Barnes G.T. Yang Q. Tan G. Yang D. Chou C.J. Sole J. Nichols A. Ross J.S. Tartaglia L.A. Chen H. J. Clin. Investig. 2003; 112: 1821-1830Crossref PubMed Scopus (5275) Google Scholar, 6Bouloumie A. Curat C.A. Sengenes C. Lolmede K. Miranville A. Busse R. Curr. Opin. Clin. Nutr. Metab. Care. 2005; 8: 347-354Crossref PubMed Scopus (228) Google Scholar). Surgically induced weight loss (7Cancello R. Henegar C. Viguerie N. Taleb S. Poitou C. Rouault C. Coupaye M. Pelloux V. Hugol D. Bouillot J.L. Bouloumie A. Barbatelli G. Cinti S. Svensson P.A. Barsh G.S. Zucker J.D. Basdevant A. Langin D. Clement K. Diabetes. 2005; 54: 2277-2286Crossref PubMed Scopus (894) Google Scholar), diet and exercise (8Bruun J.M. Helge J.W. Richelsen B. Stallknecht B. Am. J. Physiol. 2006; 290: E961-E967Crossref PubMed Scopus (343) Google Scholar), and treatment with rosiglitazone, an insulin-sensitizing drug (5Xu H. Barnes G.T. Yang Q. Tan G. Yang D. Chou C.J. Sole J. Nichols A. Ross J.S. Tartaglia L.A. Chen H. J. Clin. Investig. 2003; 112: 1821-1830Crossref PubMed Scopus (5275) Google Scholar), all reduce macrophage infiltration in white adipose tissue (AT) and decrease the expression of proinflammatory markers in white AT and plasma. A definitive role for immune cells in metabolic dysregulation was recently demonstrated in mice with myeloid cell-specific knock-out of IKKβ (9Arkan M.C. Hevener A.L. Greten F.R. Maeda S. Li Z.W. Long J.M. Wynshaw-Boris A. Poli G. Olefsky J. Karin M. Nat. Med. 2005; 11: 191-198Crossref PubMed Scopus (1505) Google Scholar), indicating that macrophage activation can cause systemic insulin resistance. Macrophages are a heterogeneous population of phagocytic cells found throughout the body that originate from the mononuclear phagocytic system (10Mantovani A. Sica A. Locati M. Immunity. 2005; 23: 344-346Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar). These are highly plastic cells that arise from circulating myeloid-derived blood monocytes that have entered target tissues and gained the phenotypic and functional attributes of their tissue of residence. Like for other immune cells, the distribution and function of tissue macrophages have been largely characterized using monoclonal antibodies to cell surface proteins. In mice, the most commonly used monocyte/macrophage and myeloid cell surface markers are F4/80 and CD11b, although F4/80 and CD11b antibodies have been reported to react with eosinophils and dendritic cells and NK and other T and B cell subtypes, respectively (11Lai L. Alaverdi N. Maltais L. Morse H.C.R. J. Immunol. 1998; 160: 3861-3868PubMed Google Scholar). In AT, resident macrophages are surrounded by adipocytes that constantly release free fatty acids (FFAs) via lipolysis. FFAs thus have the potential to activate AT macrophages and consequently alter their function. FFAs can cause activation of JNK and IKKβ inflammatory pathways in adipose tissue, liver, and skeletal muscle (1Yuan M. Konstantopoulos N. Lee J. Hansen L. Li Z.W. Karin M. Shoelson S.E. Science. 2001; 293: 1673-1677Crossref PubMed Scopus (1652) Google Scholar, 2Hirosumi J. Tuncman G. Chang L. Gorgun C.Z. Uysal K.T. Maeda K. Karin M. Hotamisligil G.S. Nature. 2002; 420: 333-336Crossref PubMed Scopus (2679) Google Scholar, 12Wellen K.E. Hotamisligil G.S. J. Clin. Investig. 2005; 115: 1111-1119Crossref PubMed Scopus (3247) Google Scholar, 13Nguyen M.T. Satoh H. Favelyukis S. Babendure J.L. Imamura T. Sbodio J.I. Zalevsky J. Dahiyat B.I. Chi N.W. Olefsky J.M. J. Biol. Chem. 2005; 280: 35361-35371Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar), leading to cellular inflammation and insulin resistance. In man, elevated FFA levels are a feature of obesity and type 2 diabetes (12Wellen K.E. Hotamisligil G.S. J. Clin. Investig. 2005; 115: 1111-1119Crossref PubMed Scopus (3247) Google Scholar, 14Boden G. Curr. Opin. Clin. Nutr. Metab. Care. 2002; 5: 545-549Crossref PubMed Scopus (180) Google Scholar). In animal models, high fat (HF) feeding and direct lipid infusion can increase plasma FFA levels and cause tissue and systemic inflammation and insulin resistance (15Kraegen E.W. Cooney G.J. Ye J.M. Thompson A.L. Furler S.M. Exp. Clin. Endocrinol. Diabetes. 2001; 109: S189-S201Crossref PubMed Scopus (117) Google Scholar, 16Boden G. Diabetes. 1997; 46: 3-10Crossref PubMed Scopus (0) Google Scholar). Toll-like receptors (TLRs) are thought to participate in sensing extracellular FFAs. TLRs belong to the Toll/interleukin-1 receptor superfamily and are widely expressed on cells of the immune system (17Takeda K. Akira S. Int. Immunol. 2005; 17: 1-14Crossref PubMed Scopus (2752) Google Scholar, 18Liew F.Y. Xu D. Brint E.K. O'Neill L.A. Nat. Rev. Immunol. 2005; 5: 446-458Crossref PubMed Scopus (1272) Google Scholar). TLRs recognize bacteria-associated molecular patterns with high specificity, and TLR-mediated signal transduction leads to the activation of JNK and NFκB signaling pathways (17Takeda K. Akira S. Int. Immunol. 2005; 17: 1-14Crossref PubMed Scopus (2752) Google Scholar, 19Guha M. Mackman N. Cell Signal. 2001; 13: 85-94Crossref PubMed Scopus (2005) Google Scholar), initiating the innate immune response. The saturated FA, lauric acid, was shown to activate TLR2 in 293T cells as well as NFκB-mediated, TLR4-dependent signaling pathways in RAW264.7 cells (20Lee J.Y. Zhao L. Youn H.S. Weatherill A.R. Tapping R. Feng L. Lee W.H. Fitzgerald K.A. Hwang D.H. J. Biol. Chem. 2004; 279: 16971-16979Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar). Similarly, an oleate/palmitate mixture can induce NFκB-dependent TLR4 signaling in 293T cells and cause inflammation in wild type (WT) but not TLR4-deficient adipocytes. The saturated FA palmitate was also shown to cause TLR4-dependent IκBα degradation in elicited peritoneal macrophages (21Shi H. Kokoeva M.V. Inouye K. Tzameli I. Yin H. Flier J.S. J. Clin. Investig. 2006; 116: 3015-3025Crossref PubMed Scopus (2768) Google Scholar). In the present study, we examined the effects of saturated and unsaturated FFAs in cultured RAW264.7 cells, primary bone marrow (BM)-derived cells and adipose tissue macrophages. We demonstrate that FFAs signal through both TLR2 and TLR4 to activate JNK and stimulate inflammatory pathways in CD11c+ myeloid cells. We also show that in AT, F4/80+CD11b+CD11c+ cells are the direct targets for FFAs and that HF feeding increases the number of these cells in AT. As such, these results provide a novel mechanistic link between lipid metabolism, inflammation, and insulin resistance. Reagents—Antibodies to IL-6, MCP-1,TNF-α, and horseradish peroxidase-linked secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and all other antibodies were from Cell Signaling (Beverly, MA). Tissue culture reagents were purchased from Invitrogen and Hyclone (Logan, UT). Cell Culture and Treatment—Mouse monocyte/macrophage RAW264.7 cells were obtained from American Type Culture Collection (Manassas, VA) and cultured in Dulbecco's modified Eagle's medium (1g/liter glucose) supplemented with 10% low endotoxin FBS. In pretreated cells, 1 μm rosiglitazone or vehicle Me2SO was added at least 24 h prior to FFA treatment. RAW cells were then incubated for 3 h with either 500 μm of an FFA mixture containing equimolar amounts of tissue culture-grade arachidonic, lauric, linoleic, oleic, and myristic acids or with individual FFAs (500 μm) or ethanol vehicle in Dulbecco's modified Eagle's medium supplemented with FFA-free BSA (Sigma-Aldrich). All of the FFA solutions were pre-equilibrated with BSA at 37 °C for 1-1.5 h (22Spector A.A. John K. Fletcher J.E. J. Lipid Res. 1969; 10: 56-67Abstract Full Text PDF PubMed Google Scholar), and a 5:1 FFA:BSA ratio was used to simulate elevated FFAs levels. We ensured that all components for cell culture and treatment experiments contained very low or undetectable amounts of contaminating lipopolysaccharide by measuring endotoxin levels in the serum, culture medium, BSA preparation, and FFA stock solutions (LAL assay kit, Cambrex, East Rutherford, NJ). All of the solutions used contained undetectable amounts of endotoxin except for the BSA solution, which contained 0.3 EU/ml (0.03 ng/ml) of endotoxin. This amount of contaminating lipopolysaccharide was not sufficient to activate RAW264.7 cells and BMDCs. We found that treating RAW264.7 cells and BMDCs with 0.03 ng/ml of endotoxin for 3 h did not cause phosphorylation of JNK and secretion of TNF-α and JNK phosphorylation in these cells (data not shown). Western Blotting—The cells were lysed in cold buffer containing 50 mm HEPES, pH 7.4, 150 mm NaCl, 200 mm NaF, 20 mm sodium pyrophosphate, 10% glycerol, 1% Triton X-100, 4 mm sodium orthovanadate, 2 mm phenylmethylsulfonyl fluoride, and 1 mm EDTA. Whole cell lysates (25 μg) or conditioned medium (30 μl) were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA). Following blocking with 5% milk in TBST, the membranes were probed with primary antibodies as indicated and subsequently incubated with horseradish peroxidase-linked secondary antibodies for chemiluminescent detection (Pierce). The blots were stripped in Restore™ Western blot stripping buffer (Pierce) and reprobed as necessary. Semi-quantitative RT-PCR—Total RNA was isolated and purified using RNeasy columns and RNase-free DNase according to the manufacturer's instructions (Qiagen). One-step RT-PCR kits (Qiagen) and sequence-specific primers (see supplemental Fig. S3) were used to generate RT-PCR products, which were then run on 8% acrylamide gels, stained with ethidium bromide, visualized, and quantitated using the Kodak ID Imaging station and software (Kodak Scientific Imaging Systems, New Haven, CT). Real Time PCR—Total RNA was isolated from cells as outlined above. First strand cDNA was synthesized using Super-Script III and random hexamers (Invitrogen). The samples were run in 20-μl reactions using an AB1 7300 (Applied Biosystems, Foster City, CA); SYBR Green oligonucleotides were used for detection, and sequence-specific primers are listed in supplemental Fig. S3. Gene expression levels were calculated after normalization to the standard housekeeping gene GAPDH using the ΔΔCT method as described by the manufacturer (Invitrogen) and expressed as relative mRNA levels compared with control. ELISA—Conditioned medium was assayed for mouse IL-1β, IL-6, MCP-1, and TNF-α using ELISA kits (BIOSOURCE, Camarillo, CA) following the manufacturer's protocol. Protein Knockdown—2 × 107 RAW264.7 cells were electroporated with 2.5 nmol of high pressure liquid chromatography-purified siRNA oligonucleotides (IDT, Coralville, IA) to mouse JNK1, JNK2, p65-NFκB, TLR2, or TLR4 or with luciferase control siRNA (oligonucleotide sequences in supplemental Fig. S3) using the XCell Gene Pulser (Bio-Rad). The cells were plated into 24-well tissue culture plates, and 24-48 h post-electroporation, they were treated with FFAs as described above. Conditioned medium and cell lysates were analyzed by immunoblotting. Isolation and Treatment of Bone Marrow-derived Cells—All of the animal experiments were performed humanely under protocols approved by the University of California, San Diego. TLR2/4 DKO male mice on a C57BL/6 background (kindly provided by Dr. Shizuo Akira, University of Osaka, Japan) and WT C57BL/6 male mice were maintained under specific pathogen-free conditions. Bone marrow cells were isolated from the femurs and tibias of 10-12-week-old homozygous and WT mice by flushing the medullary cavity with RPMI medium. After washing, the cells were seeded in tissue culture plates and differentiated into either bone marrow-derived macrophages (BMDMs) or bone marrow-derived dendritic cells (BMDCs) in RPMI medium containing 30% of L-929 conditioned medium or 10 or 40 ng/ml of recombinant GM-CSF, respectively, and supplemented with 20% low endotoxin FBS and streptomycin/penicillin. We observed that differentiating precursor cells in 40 ng/ml of GM-CSF yielded more BMDCs than when using 10 ng/ml of GM-CSF. However, this higher GM-CSF concentration did not affect BMDC proinflammatory gene expression (IL-6, TNF-α, IL-1β, and COX-2), differentiation and cell surface antigen expression (F4/80, CD11b, and CD11c), as determined by qPCR and FACS analysis (data not shown). We were also able to differentiate BMDMs from bone marrow precursor cells using murine recombinant M-CSF (data not shown). BMDM and BMDC differentiation was complete 8 days after cell plating; this was confirmed by the expression of F4/80, a marker preferentially expressed by mature macrophages, and of CD11c, a cell surface marker for dendritic cells, using FACS (data not shown). For FFA experiments, day 8 or 9 BMDMs and BMDCs were stimulated with 500 μm FFA for 3 h, and conditioned medium and cell lysates were analyzed as indicated. Immunohistocytochemistry—Paraffin-embedded adipose tissue was sectioned, deparaffinized, and rehydrated prior to endogenous peroxidase and biotin removal using 0.03% H2O2 and 0.1% avidin. The slides were heated in 0.1 m citrate buffer for antigen retrieval, cooled, washed, and then blocked with 1% BSA in TBST. The sections were incubated overnight with CD11c primary antibody (eBioscience, San Diego, CA). Biotin-conjugated secondary and horseradish peroxidase-conjugated streptavidin tertiary antibodies were applied for detection, and the sections were developed in substrate chromogen, counterstained in Mayers Hematoxylin, and mounted in Gelmount Biomedia. Frozen quad muscle tissue was sectioned (5 um), air-dried, and fixed in acetone. After endogenous peroxidase removal using 0.03% H2O2, the sections were washed in PBS and blocked in 0.01% biotin for 15 min and then in 1% BSA in PBS for 30 min. Biotin-conjugated CD11c antibody was overlaid for 1 h, and the sections were further processed as outlined above. Animal Studies—Wild type male C57BL/6 and ob/obJ male mice were purchased from Harlan (Indianapolis, IN). The animals were housed in a pathogen-free facility with a 12-h light/12-h dark cycle and given free access to food and water. C57BL/6 mice were placed on a HFD consisting of 40% of calories from fat (TD.96132; Harlan Teklad, Madison, WI) starting at 12 weeks of age for 1, 12, or 20 weeks. Control C57BL/6 mice were fed a standard diet with 12% of calories from fat (LabDiet, 5001; Richmond, IN). The body weights and food intakes were recorded every week (data not shown). An insulin tolerance test (ITT) was performed on mice after 1 and 12 weeks of chow or HFD feeding. The mice were fasted for 6 h. After 4 h of fasting, basal plasma glucose was measured. At 6 h, the mice were injected intraperitoneal with 0.6 units of insulin/kg of body weight. Blood glucose was measured through the tail tip at the indicated times, using a OneTouch glucose-monitoring system (Lifescan, Milpitas, CA). Stromal Vascular Fraction (SVF) Isolation and FACS Analysis—Epididymal fat pads were excised from male C57BL/6 mice fed normal chow (NC) or HFD, weighed, rinsed three times in PBS, and then minced in FACS buffer (PBS + 1% low endotoxin BSA). Tissue suspensions were centrifuged at 500 × g for 5 min and then collagenase-treated (1 mg/ml; Sigma-Aldrich) for 30 min at 37 °C with shaking. The cell suspensions were filtered through a 100-μm filter and centrifuged at 500 × g for 5 min. SVF pellets were then incubated with RBC lysis buffer (eBioscience) for 5 min prior to centrifugation (300 × g for 5 min) and resuspension in FACS buffer. Stromal vascular cells (SVCs) were incubated with Fc Block (BD Biosciences, San Jose, CA) for 20 min at 4 °C prior to staining with fluorescently labeled primary antibodies or control IgGs for 25 min at 4 °C. F4/80-allophycocyanin FACS antibody was purchased from AbD Serotec (Raleigh, NC); all other fluorescein isothiocyanate- and phycoerythrin-conjugated FACS antibodies were from BD Biosciences. The cells were gently washed twice and resuspended in FACS buffer with propidium iodide (Sigma-Aldrich). SVCs were analyzed using FACSCalibur and FACSAria flow cytometers (BD Biosciences). Unstained, single stains, and fluorescence minus one controls were used for setting compensation and gates. Forcellsorting,F4/80+CD11b+CD11c−andF4/80+CD11b+-CD11c+ cells from lean, NC SVFs were sorted into FBS. Sorted cells were allowed to recover for 2 h. The cells were then washed and treated with FFA for subsequent RNA extraction and real time PCR analysis. Glucose Uptake Assay—BMDCs from WT and TLR2/4 DKO mice were treated with 500 μm FFA or vehicle for 3 h and then washed twice with RPMI medium to remove the FFAs. The cells were subsequently incubated with fresh culture medium containing 2% FBS. After 6 h of incubation, CM was harvested for ELISA analysis and co-culture experiments. L6 myocytes were cultured in α-minimal essential medium supplemented with 10% FBS and differentiated into myotubes in α-minimal essential medium containing 2% FBS for ∼6-7 days. The L6 myotube culture medium was then replaced with CM from BMDCs diluted 1:1 in fresh L6 differentiation medium. L6 myotubes were incubated with BMDC-derived CM for 24-48 h prior to assaying glucose uptake. For glucose uptake assays, L6 myotubes were serum starved for 3 h in α-minimal essential medium with 0.5% FFA-free BSA and then glucose-starved for 30 min in HEPES salt buffer containing 0.5% FFA-free BSA. The cells were stimulated with insulin (5 nm) at 37 °C for 20 min; tracer glucose was then added for 10 min. After 30 min of insulin stimulation, glucose uptake was assayed in quadruplicate wells for each condition using 1,2-3H-2-deoxy-d-glucose (0.2 μCi, 0.1 mm, 10 min) in four independent experiments. Data Analysis—Densitometric quantification and normalization were performed using the NIH Image 1.63 software. The values presented are expressed as the means ± S.E. The statistical significance of the differences between various treatments was determined by one-way analysis of variance with the Bonferroni correction. FFAs Cause a Proinflammatory Response in RAW264.7 Cells—Recent studies suggest that chronic inflammation in AT is an important mechanism underlying the insulin resistance associated with obesity, HF feeding, and diabetes and that infiltrating macrophages may be responsible for the activation of proinflammatory pathways (6Bouloumie A. Curat C.A. Sengenes C. Lolmede K. Miranville A. Busse R. Curr. Opin. Clin. Nutr. Metab. Care. 2005; 8: 347-354Crossref PubMed Scopus (228) Google Scholar, 23Kolb H. Mandrup-Poulsen T. Diabetologia. 2005; 48: 1038-1050Crossref PubMed Scopus (345) Google Scholar). Because FFAs are released in AT, we studied the effects of FFAs on AT macrophage function. We first treated RAW264.7 murine monocyte/macrophage cells with a mixture of saturated and unsaturated FFAs and surveyed various stress/inflammatory signaling responses. FFA treatment broadly activated the JNK and IKKβ signaling pathways (Fig. 1A), in a time- and concentration-dependent manner, with JNK activation being observed as early as 5 min after FFA treatment (data not shown). After washing to remove FFAs, followed by a 12-h recovery period, inflammatory markers returned to base-line levels, and the cells were once again responsive to lipopolysaccharide stimulation (data not shown). Thus, the proinflammatory effects of FFAs were specific and reversible and did not result from cell toxicity. When the cells were treated with 500 μm FFA for 3 h, we observed induction of the proinflammatory genes IL-1β, IL-6, MCP-1, and MMP-9, an increase in intracellular MCP-1 and TNF-α levels, and an increase in secreted proinflammatory chemo/cytokines IL-1β, IL-6, MCP-1, and TNF-α (supplemental Fig. S1, A-C). When we tested individual FAs, each FA in the mixture, except for myristic acid, was able to activate the proinflammatory kinase pathways in RAW264.7 cells and increased intracellular and secreted TNF-α levels, albeit to different degrees (supplemental Fig. S1, D and E). Overall, the unsaturated FA arachidonic acid was most potent in causing these effects. We assessed whether a TZD, rosiglitazone, could inhibit FFA-induced inflammation in RAW264.7 cells and found that all the FFA-induced effects were attenuated by pretreatment with rosiglitazone (supplemental Fig. S2, A-C). Contribution of JNK and IKKβ-NFκB to the FFA Proinflammatory Effects—Previous reports have documented that FFAs induce mostly NFκB-dependent effects, and we observed that FFA treatment activated both JNK and IKKβ signaling pathways. Therefore, we assessed the contribution of these two kinases to the overall FFA effect using siRNAs targeted specifically against JNK1, JNK2, and p65-NFκB. JNK1 and JNK2 siRNAs decreased levels of both JNK protein isoforms by >70% (13Nguyen M.T. Satoh H. Favelyukis S. Babendure J.L. Imamura T. Sbodio J.I. Zalevsky J. Dahiyat B.I. Chi N.W. Olefsky J.M. J. Biol. Chem. 2005; 280: 35361-35371Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar), and p65-NFκB siRNA decreased p65 protein levels by ∼75% (Fig. 1B). JNK knockdown (KD) significantly reduced basal MMP-9, IL-1β, TNF-α, and COX-2 gene expression levels, and inhibited the FFA-induced up-regulation in IL-1β and MMP-9 gene expression (Fig. 1C). In contrast, knocking down p65 produced smaller inhibitory effects on gene expression. Moreover, JNK KD resulted in decreased FFA-induced intracellular and secreted TNF-α levels (Fig. 1D), whereas p65 KD, while having the expected effect to decrease phosphorylation of NFκB, had little or no effect on intracellular and secreted TNF-α (Fig. 1E). This suggested that JNK plays a more central role in mediating the FFA-induced proinflammatory effects in RAW264.7 cells. We thus focused on examining the role of JNK kinase in mediating the effects of FFAs. TLR2 and TLR4 Mediate FA Signaling in RAW264.7 Cells—TLRs are expressed on monocytes and macrophages, and it has been reported that bacterial lipopolysaccharide, which contains an FA moiety, can induce proinflammatory effects through TLR4 (17Takeda K. Akira S. Int. Immunol. 2005; 17: 1-14Crossref PubMed Scopus (2752) Google Scholar, 24Poltorak A. He X. Smirnova I. Liu M.Y. Van Huffel C. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6514) Google Scholar, 25Hoshino K. Takeuchi O. Kawai T. Sanjo H. Ogawa T. Takeda Y. Takeda K. Akira S. J. Immunol. 1999; 162: 3749-3752Crossref PubMed Google Scholar). Thus, we hypothesized that TLR4, and possibly TLR2, another member of the TLR family, could mediate the FFA proinflammatory signals. To test this idea, siRNAs were used to knockdown TLR4 and TLR2 proteins in RAW264.7 cells (Fig. 2A), achieving ∼50% and ∼75% KD efficiency for TLR2 and TLR4, respectively (bar graph in Fig. 2A). TLR2 KD significantly reduced basal expression of MMP-9, IL-1β, and TNF-α genes, whereas TLR4 KD reduced the basal expression of these genes and that of COX-2 (Fig. 2B). TLR2 and TLR4 KD also inhibited the FFA-induced up-regulation in IL-1β and MMP-9 gene expression. Fig. 2C shows that in the TLR2 KD cells, FFA-induced JNK phosphorylation was inhibited, as was the increase in intracellular and secreted TNF-α levels. When TLR4 was depleted, these proinflammatory responses were also inhibited, but to a greater extent (Fig. 2D). These results indicate that both TLR4 and TLR2 play a role in mediating FA signaling via JNK activation. Studies in Bone Marrow-derived Cells—RAW264.7 cells have been extensively used as a model cell line for studies of macrophage biology. We found that RAW264.7 cells express high levels of the macrophage and myeloid cell surface markers F4/80 and CD11b but also high levels of a cell surface marker more typical of dendritic and T cells, CD11c (data not shown). With this in mind, and given the heterogeneity and plasticity of the macrophage population, we further examined the effects of FFAs in two types of primary myeloid-derived cell types: BMDMs, which express F4/80, CD11b but not CD11c, and BMDCs, which express all three markers. BMDMs and BMDCs were prepared as described in detail under “Experimental Procedures.” According to previously established methods, the phenotype of these primary cells was confirmed by FACS using fluorescently labeled antibodies showing that BMDMs express F4/80 and CD11b, whereas BMDCs are positive for F4/80, CD11b, and CD11c (data not shown). To characterize these two cell types, we examined the expression of inflammatory genes by qPCR (Fig. 3A). The relative expression of COX-2, CCR2, IL-1β, IL-6, and TNF-α genes was strikingly higher in BMDCs than BMDMs (p < 0.05 or less), suggesting that BMDCs possess greater proinflammatory activity than BMDMs. Consistent with this, BMDMs expressed high levels of anti-inflammatory cytokine IL-10, whereas BMDCs exhibited no detectable levels of IL-10. BMDMs and BMDCs have been shown to express TLR2 and TLR4. Because BMDCs have heightened proinflammatory activity and because FFAs exert their inflammat