TLR1/2-mediated responses are dependent on JNK activity. (A) RAW264.7 cells were cotransfected with a plasmid containing the luciferase gene under the influence of AP-1 (A) or NF-κB (B) response elements plus a plasmid containing the dnJNK form or a plasmid control (SK). The cells were then stimulated with a B. burgdorferi lysate (Bb) for 16 h, and luciferase activity was assayed. The results shown are the average ± standard error of four and three independent experiments, respectively. *, P < 0.001 (AP-1-luc) and P < 0.05 (NF-κB-luc). (C) RAW264.7 cells were transfected with a plasmid containing the dnJNK form or a plasmid control (SK) and stimulated with a B. burgdorferi lysate for the indicated times. The cells were then lysed and assessed for IκBα content by immunoblotting (left). Band quantitation was performed with the Java-based software ImageJ (right). The experiment shown is representative of three performed. (D) RAW264.7 cells were transfected with a plasmid containing the dnJNK form or a plasmid control (SK) and stimulated with recombinant murine IFN-γ for 30 min. Phospho-STAT1 and total STAT-1 levels were determined by immunoblotting. The results shown are representative of two experiments performed with similar results.

Structure of the human tlr1 gene. The 1-kb proximal promoter and the gene encoding TLR1 are shown. The proximal promoter sequence is shown in the magnified box, with the three putative AP-1 binding sites highlighted. The sequence corresponds to nucleotides 5956071 to 5955050 of contig NT016297 (NCBI/NIH).

JNK activity regulates TNF-α production in response to B. burgdorferi. (A) RAW264.7 (top) and primary CD11b⁺ (bottom) cells were stimulated with a B. burgdorferi lysate for the indicated times (top) or 40 min (bottom) prior to assaying JNK activity in vitro with c-Jun as a substrate. Phospho-c-Jun was then detected by immunoblotting. The results are representative of three experiments performed. (B) RAW264.7 cells were transfected with a plasmid containing dnJNK1 or a plasmid control (SK) and stimulated with a B. burgdorferi lysate (Bb) or PAM₃-CSK₄ for 16 h. The TNF-α levels in the stimulation supernatants were then quantified by ELISA. The results shown are the average plus standard error (SE) of three independent experiments. *, P < 0.001 and P < 0.01 for B. burgdorferi and PAM₃-CSK₄ stimulation, respectively. (C) RAW264 cells were stimulated with 10 μg/ml of a B. burgdorferi lysate (Bb) or 1 μg/ml of PAM₃-CSK₄ in the presence of increasing concentrations of the JNK inhibitor SP100625. TNF-α was quantified in the stimulation supernatants after 16 h. The results are the average plus SE of three independent experiments.

JNK1 regulates the expression of the tlr1 gene. (A) (Left) RAW264.7 cells were transfected with a plasmid containing a dnJNK form or a plasmid control (SK), and total RNA was extracted and assessed by RT-PCR (left) or real-time RT-PCR (right) for the expression of tlr1 and tlr2 mRNAs. Equal input of RNA (left) was assessed by the amplification of the housekeeping gene gapdh. (B) The cells were also used to determine the levels of TLR1 and TLR2 by Western blotting. Protein input was assessed by immunobloting with an anti-actin Ab. (C) CD11b⁺ cells were purified from B6 (WT) and JNK1- and JNK2-deficient mice. RNA was extracted and subjected to RT-PCR for the expression of tlr1 and tlr2. RNA input was assessed by the amplification of gapdh mRNA. (D) RAW264.7 cells were transfected with JNK1 siRNA or a control (siNC) and used to extract RNA and to determine the mRNA levels of jnk1, tlr1, and tlr2. Equal input was determined by amplification of gapdh mRNA. (E) Purified CD11b⁺ cells from B6 (WT) and JNK1- and JNK2-deficient mice were unstimulated (Unst.) or stimulated with a B. burgdorferi lysate (Bb). The levels of TNF-α were determined in the stimulation supernatants after 16 h. (F) dnJNK1-transfected RAW264.7 cells were stimulated with lipopolysaccharide (LPS), poly(dI-dC), and flagellin, and TNF-α was detected 16 h later. (G) RAW264.7 cells were transfected with siRNA oligonucleotides specific for JNK1 or control oligonucleotides (siNC). Forty-eight hours later, the cells were stimulated for 16 h with a B. burgdorferi lysate (Bb) or PAM₃-CSK₄ and assessed for TNF-α by ELISA. The results represent the average plus standard error (SE) of three independent experiments. *, P < 0.05. (H) RAW264.7 cells were cotransfected with a plasmid encoding TLR1 under the influence of a constitutively expressed promoter (UNO) or a control plasmid (GL3) plus a plasmid containing the dnJNK form or the empty plasmid (SK). All cells were transfected with the same amount of plasmid. The cells were then stimulated with a B. burgdorferi lysate (Bb) or PAM₃CysK₄. TNF-α levels in the supernatants were determined 16 h after stimulation. The results shown represent the average plus SE of three independent experiments. *, P < 0.05 compared to control-, UNO plus control-, or UNO plus dnJNK-transfected cells stimulated with B. burgdorferi and PAM₃-CSK₄.

JNK1 regulates tlr1 promoter activity. (A) (Top) RAW264.7 cells were cotransfected with a plasmid containing the luciferase gene downstream of the 1-kb proximal tlr1 promoter and the dnJNK plasmid or a plasmid control (SK) and stimulated with a B. burgdorferi lysate (Bb) or PAM₃-CSK₄ or left unstimulated (Unst.). Luciferase activity (AU, arbitrary units) was measured after 16 h of stimulation. The data presented correspond to the average plus standard deviation of triplicate determinations of one of four experiments performed. *, P < 0.05. (Bottom) Western blot of RAW264.7 cells stimulated with 10 μg/ml of a B. burgdorferi lysate for 16 h using anti-TLR1 Ab. Equal protein input was assessed with anti-Actin Ab. (B) EMSA showing nuclear extract binding to oligonucleotides (oligo) containing putative AP-1 binding sites in the proximal 1-kb tlr1 promoter. The reactions were performed in the absence or presence of an excess (100×) of unlabeled (cold) oligonucleotides. (C) EMSA of nuclear extracts of RAW264.7 cells unstimulated and stimulated with a B. burgdorferi lysate for 30 min. The reactions were performed in the absence or the presence of Abs against c-Jun, JunD, c-Fos, CREB, and ATF-2 and in the absence or presence of unlabeled (cold) oligonucleotides corresponding to the tlr1 promoter or the AP-1 consensus binding sequence. The arrows indicate the constitutive (top) and B. burgdorferi-enhanced (bottom) complexes formed. (D) EMSA of tlr1-derived and consensus AP-1 binding oligonucleotides with nuclear extracts from unstimulated or B. burgdorferi-stimulated RAW264.7 cells. (E) RAW264.7 cells were cotransfected with plasmids containing tlr1- or tlr1Δ(−502,−508) [tlr1(mut)]-luciferase and a plasmid containing dnJNK or a plasmid control (SK). After 48 h, the cells were stimulated with a B. burgdorferi lysate (Bb) or PAM₃-CSK₄ or left unstimulated (Unst.). Luciferase activity was measured 16 h poststimulation. The results shown are the average plus standard error of three independent experiments. *, P < 0.05.