Ed and assayed for IL-6 (b) and IL-12p70 (c) by ELISA. Data shown are representative of three independently performed experiments.a250 200 IL-6 (pg/ml) 150 100Percentage of totalTLRb0Medium Flagellin Profilin LPSIL-12p70 (pg/ml)c0 Manage vector TLR5 siRNAJ Innate Immun 2014;six:68594 DOI: ten.1159/Salazar Gonzalez et al.ten,000 Percentage of maximum IL-6 (pg/ml) 1,000 100 ten 1 CTDC C14 CTDC C42 TLR2 Antagonist medchemexpress IL-12p40 (pg/ml) 100 TLR5 (MFI) 80 60 40 20 0 100 101 102 103 104 FL2-MFI 400 IL-6 (pg/ml) 300 200 one hundred 0 60 40 20LPS Flagellin Profilin1 CTDC C14 CTDC CCTDC CCTDC Cabcd1,500 IL-8 (pg/ml) Isotype handle Anti-huTLR5 mAb 1,IL-12p40 (pg/ml)CTDC C14 CTDC CCTDC C14 CTDC C300 200 10010 01 01 1 0 0 101 1 ten 01 0 0 1 1 0. 00 00 10 0. 00 0. 0.Tachyzoites:monocyte (m.o.i.)0 01 10 10 00 0. 0.Tachyzoites:monocyte (m.o.i.)0.0.1,0.1,1,ef0.Tachyzoites:monocyte (m.o.i.)gFig. 5. TLR5 (R392X) mutation abolishes monocyte cytokine pro-duction right after T. gondii profilin stimulation. Peripheral blood CD14+ monocytes were purified and stained as described in figure three. a Histogram overlays of isotype control versus TLR5 staining of CD14+ monocytes obtained from two donors (CTDC C42, gray histogram; CTDC C14, black histogram). Empty histograms represent isotype handle Ab staining (black line for CTDC C14 and gray line for CTDC C42). b Imply fluorescence intensity (MFI) of your samples. c, d Cell suspensions from the exact same donors had been then cultured in the presence of medium alone, LPS, flagellin or profilin (all at 1 g/ml) for 24 h. Supernatants have been harvested and assayed forIL-6 (c) and IL-12p40 (d) by ELISA. e, f Peripheral blood monocytes from donors CTDC C14 and CTDC C42 have been incubated inside the presence of reside T. gondii Rh strain tachyzoites (m.o.i. ranging from 1,000 to 0.0001); 24 h later supernatants were harvested and assayed for IL-6 (e) and IL-12p40 (f). g HEK293 cells were plated and incubated within the presence of isotype control Ab or anti-huTLR5 mAb and live tachyzoites as indicated in e and f. Culture supernatants had been harvested and assayed for IL-8 by ELISA. Information shown are representative of triplicate samples from 2 independent experiments. p 0.05 in between donors or between isotype handle and anti-huTLR5 mAb therapy as determined by t test.that expressed low and higher levels of TLR5. Figure 5b shows the imply fluorescence intensity of such samples and also the low/high profiles of TLR5 expression inside CD14+ cells. We then confirmed by real-time genotyping that the cells that showed low levels of TLR5 staining also showed high detection using primers containing the R392X mutation (online suppl. fig. 1, karger/ doi/10.1159/000362367). We then examined their cytokine profile in response to LPS, flagellin and profilin. Figure 5c and d show IL-6 and IL-12p40 levels induced above background (N-type calcium channel Inhibitor Purity & Documentation unstimulated manage) values. LPS stimulation triggered increased production of all cytokines tested in cells from both donors. On the other hand, flagellin and profilin triggered IL-6 and IL-12p40 production from TLR5high but not from TLR5 R392X cells (fig. 5c, d), hence offering proof that a totally functional TLR5 is expected for any monocyte response to T. gondii profilin. To additional establish the biological relevance of TLR5-mediated recognition of T. gondii profilin, we exposed TLR5WT and TLR5R392X peripheralProfilin Triggers Human TLRblood monocytes to reside T. gondii Rh strain tachyzoites at a number of multiplicities of infection (m.o.i.’s) and assayed for IL-6 and IL-12p40 by ELISA. Fi.