Activation of TLR9s by CpG-ODN developed for rabbits.

<p>HEK 293 cells were transfected with expression vector for (A, C) rabTLR9 and for (B) rabbit, mouse, and human TLR9, as indicated, along with an NF-κB controlled luciferase-reporter gene, and treated with 2µM of CpG-ODN (A, B) or with different concentrations of CpG-ODN (C) for 7h. Relative luciferase activities were then determined. Data shown represent mean ± SD (n = 3 independent experiments). The sequences of CpG-ODN used in this study are shown on the right of panel A.</p

Activation of mouse splenocytes by CpG-ODN developed for rabbits.

<p>Mouse splenocytes were stimulated with 2µM of different CpG-ODN: CpG-2006 and CpG-2007, optimized for humans; CpG-1826, optimized for mice; and CpG-C46 and CpG-C4609, developed for rabbits. Induction of (A) IL-6 and (B) IFN-γ, and production of (C) IgM, were determined by ELISA. Data shown represent mean ± SD (n = 3 independent experiments). *<i>P</i><0.05 <i>vs.</i> the induction by CpG-1826.</p

Development of CpG-ODN for activation of rabTLR9.

<p>Activation of rabTLR9 by (A) CpG-ODN derived from CpG-2007 and CpG-1826, (B) CpG-ODN derived from CpG-1826C, (C) CpG-ODN derived from CpG-C4, and (D) CpG-ODN derived from CpG-C43. HEK 293 cells were co-transfected with expression vector for rabTLR9 and an NF-κB drivened luciferase-reporter gene, and treated with 2µM of different CpG-ODN as indicated for 7h. Relative luciferase activities were then determined. Data shown represent mean ± SD (n = 3 independent experiments). The sequences of CpG-ODN used in this study are shown at the bottom of the plots.</p

Activation of antigen-specific immune responses by CpG-ODN developed for rabbits.

<p>Rabbits were immunized with 100µg of RHDV NJ85 peptide mixed with or without 50µg of CpG-ODN, as indicated. (A) The cycle of immunization and serum collection followed a 14-day-interval schedule, as shown. (B) Inductions of IFN-γ from CD8 T cells by NJ85 were measured by ELISA. (C) Titers of anti-NJ85 antibody in serum samples were measured by ELISA. Data shown represent mean ± SD (n = 3 independent experiments). *<i>P</i><0.05 <i>vs.</i> the induction by CpG-C4609.</p

Activation of antigen-nonspecific immune responses by CpG-ODN developed for rabbits.

<p>Rabbit splenocytes were stimulated with 2µM of different CpG-ODN. (A) Induction of cytokines was analyzed by RT-PCR. (B) Production of IgM was determined by ELISA. (C) Proliferation of splenocytes was measured by MTS assay. Data shown represent mean ± SD (n = 3 independent experiments).*<i>P</i><0.05 <i>vs.</i> the induction by CpG-C4609.</p

Proposed model of NF-κB and p38 via C/EBPβ regulating the transcription of <i>Tnfaip3</i> in LPS-induced response.

<p>TLR4 engagement leads to activation of p38 MAPK and IKK/NF-κB. p38 MPAK subsequently through a yet-to-be-determined mechanism upregulates C/EBPβ, which induces A20 (TNFAIP3) transcription in conjunction with NF-κB.</p

NF-κB and C/EBPβ are required for induction of <i>Tnfaip3</i> in LPS-activated macrophages.

<p>(A) Schematic map of predicted NF-κB p65 and C/EBPβ DNA binding sites in the promoter of <i>Tnfaip3</i>. oPOSSUM (<a href="http://www.cisreg.ca/oPOSSUM/" target="_blank">http://www.cisreg.ca/oPOSSUM/</a>) was used as the prediction tool. The JASPAR CORE vertebrate database was selected for transcription factor binding site matrices. Two arrows depict the PCR primers. (B) p65 and C/EBPβ bind to the promoter of <i>Tnfaip3</i> after LPS stimulation. RAW264.7 cells were treated with LPS (100 ng/ml) for the indicated times. Chromatin was immunoprecipitated with anti-p65 and anti-C/EBPβ antibodies. Rabbit IgG was a negative control. Precipitated DNA or 1% of the chromatin input was amplified with primers for the <i>Tnfaip3</i> promoter (−89 ∼ −410). The PCR products were loaded and separated on a 2% agarose gel. One of two independent experiments is shown. (C) LPS-induced association of p65 and C/EBPβ with <i>Tnfaip3</i> was reduced in the presence of p38 inhibition. Chromatin isolated from RAW264.7 cells treated with LPS (100 ng/ml) for 4 h in the absence or presence of SB202190 (10 µM) were subjected to ChIP assay as described above. The relative quantity of promoter enriched by ChIP was quantified by real-time PCR and expressed as the fold enrichment of untreated control samples after normalization to rabbit IgG. Data represent the means of two independent experiments. (D) LPS-induced expression of A20 (TNFAIP3) was decreased in C/EBPβ-depleted RAW264.7 cells. Cells were infected with lentiviruses encoding shRNA against luciferase (<i>shLuc</i>) or C/EBPβ (<i>shCebpb</i>), and treated with LPS (100 ng/ml) for the indicated times. Cell lysates were collected and analyzed by immunoblotting using the indicated antibodies.</p

Depletion of IKKβ expression and inhibition of p38 signaling pathway in <i>Ikkβ</i>^Δ and SB202190-treated bone marrow-derived macrophages (BMDMs).

<p>(A) Immunoblotting of IKKβ and p38 from BMDMs isolated from wild-type (wt; <i>Ikkβ</i>^F/F) and <i>Ikkβ</i>^Δ mice. (B) Immunoblotting of p38 and phosphorylated p38 from wt BMDMs treated with LPS (100 ng/mL) in the absence or presence of SB202190 (10 µM) for 2 h. (C & D) mRNA expression levels of IL-1β and IL-6 were inhibited in SB202190-treated BMDMs after LPS treatment. The expression levels of <i>Il1b</i> (C) and <i>Il6</i> (D) were determined from wt BMDMs treated with LPS (100 ng/mL) for 4 h in the absence or presence of SB202190 (10 µM) for 2 h using real-time RT-PCR. Data represent the mean ± SEM for three independent experiments. ***, <i>P</i><0.005.</p

Prediction of transcription factor binding sites of NF-κB and p38-dependent genes using opossum.

§<p>TF: transcription factor.</p>†<p>Background TFBS rate: rate of transcription factor binding site in genome.</p>#<p>TF Abbreviation: HLF: hepatic leukemia factor; SRF: serum response factor; USF1: Upstream stimulatory factor 1.</p

Top 10 enriched canonical pathways of the NF-κB and p38-dependent genes.

<p>^aThe significance level of each canonical pathway was determined by in Ingenuity Pathway Analysis.</p