Data are expressed while mean SD of triplicates from 1 representative experiment. in the TLR4?/? and MyD88?/? mice (4.2 and 2.0 ng/mL; = .04 and .002) (Fig. 1 .001) (Fig. 1sepsis. Open in a separate windowpane Fig. 1. TLR4-deficient and MyD88-deficient mice are safeguarded from lethal Gram-negative bacterial sepsis. (O18 and treated with antibiotics as explained in .005 for TLR4?/? or MyD88?/? vs. WT or TLR2?/?, = .15 for TLR4?/? vs. MyD88?/?, and = .48 for WT vs. TLR2?/?; IL-6: .05 and .005 for TLR4?/? and MyD88?/? vs. WT or TLR2?/?, = .13 for TLR4?/? vs. MyD88?/?, and = .31 for WT vs. TLR2?/?). ( .001). Data points are from 1 experiment (= 6 to 7 mice per treatment organizations). Chimeric Mouse TLR4CHuman Fc Fusion Protein. TLR4 Forskolin is composed of N-terminal, NFKBIA central, and C-terminal domains. MD-2 binds to the concave surface of the N-terminal and central TLR4 domains (12, 18). To obtain anti-TLR4 antibodies, we 1st generated a soluble recombinant chimeric protein composed of the N-terminal half of the mouse TLR4 ectodomain (amino acids 1C334) fused to the Fc website of human being IgG1 (mTLR4-Fc). The recombinant mTLR4-Fc protein was produced in HEK 293T cells to ensure posttranscriptional modifications. In the presence of serum like a source of soluble MD-2, LPS was shown to Forskolin bind to mTLR4-Fc (Fig. S1and data not shown). In contrast, anti-TLR4 antibodies did not affect signal transduction or cytokine production by macrophages or by whole blood stimulated with additional TLR ligands, such as Pam3CSK4 (Fig. 2and peritonitis and pneumonia models (Fig. S2). Collectively, these results provide compelling evidence that anti-TLR4 antibodies identify membrane-bound TLR4 and inhibit innate immune reactions of cells stimulated with LPS or Gram-negative bacteria in vitro and in vivo. Open in a separate windowpane Fig. 2. Anti-TLR4 antibodies bind to TLR4 and inhibit the activation of macrophages induced by LPS. (= .001 for anti-TLR4 versus control antibodies. (O18 for 4 h. Data are indicated as mean SD of triplicates from 1 representative experiment. *.005 .05 and ** .005for Forskolin anti-TLR4 versus control antibodies. Anti-TLR4 Antibodies Protect Against Lethal Endotoxemia. Affording safety against lethal endotoxemia is definitely important in individuals with fulminant meningococcemia associated with high levels of circulating endotoxin (19). We explored the protecting capacity of the anti-TLR4 antibodies inside a model of endotoxemia in D-galactosamineCsensitized mice. Consistent with the results observed in vitro, anti-TLR4 antibodies given i.p. 15 min before an LPS challenge almost completely eliminated TNF production ( .0001) (Fig. 3= .005) (Fig. 3= .0001) (Fig. 3= .025). Anti-TLR4 antibodies did not guard mice from Forskolin harmful shock induced by Pam3CSK4, a Gram-positive lipopeptide and activator of TLR1-TLR2 heterodimers (Fig. S3), providing evidence of TLR4 specificity. Open in a separate windowpane Fig. 3. Anti-TLR4 antibodies inhibit cytokine production and guard mice from lethal endotoxemia when given prophylactically and therapeutically. (O111:B4 LPS. Plasma concentrations of TNF ( .0001 for TNF and = .005 for IL-6. (= 19 and 21 mice per treatment group; = .0001) and TLR4?/? mice (= 8). Data points are from 4 self-employed experiments for antibody evaluation. (O111:B4 LPS (= .025). Data points are from 1 experiment (= 8 mice per treatment group). Anti-TLR4 Antibodies Protect Against Lethal Live Sepsis. We analyzed the effect of anti-TLR4 antibodies inside a classical Gram-negative bacterial sepsis model induced by an i.p. injection of live .
