LRa1,Flagellin IL-8 production ( ) Profilin LPS Flagellin HEK125 100 75 50 25 0 r2 = 0.7963 IC50 = 31.54 ng/ml ? 0 2 4 FlagellinIL-8 (pg/ml)Profilin LPSTHP-0.0.1,b10,PAMP (ng/ml) Flagelline125 IL-8 production ( ) one hundred 75 50 25Anti-HuTLR5 mAb (log ng/ml)IL-8 (pg/ml)1,000 one hundred ten 0.01 0.10 1 10 one hundred 1,Profilin LPS Flagellin Profilin LPSHuTLRProfilinMoTLRr2 = 0.9465 IC50 = 3.32 ng/ml ? 0 2c1,000 ControlPAMP (ng/ml)f125 IL-8 production ( ) 100 75 50 25Anti-HuTLR5 mAb (log ng/ml)IL-8 (pg/ml)HuTLR5 MoTLR5LPS10 0.01 0.ten 1 10 100 1,?dProfilin (ng/ml)gAnti-HuTLR5 mAb (log ng/ml)Fig. 2. Endogenously expressed human TLR5 in HEK293 cells mediates IL-8 responses to flagellin and T. gondii-derived profilin. a HEK293 or THP-1 cells have been suspended in FACS buffer with PElabeled anti-huTLR5 antibody.2,4,5-Trichloroquinoline In stock Cells have been then washed and acquired for flow cytometry. Data shown are histogram overlays of samples stained with isotype antibody handle (IgG2a-PE) and anti-TLR5. b Subsequently, cells have been stimulated with quite a few concentrations of recombinant flagellin C (b), recombinant T. gondii profilin (c) orLPS (d) for 24 h. e HEK293 cells were plated and incubated in the presence of medium alone or with anti-TLR5 mAb, as indicated, followed by stimulation with flagellin (e), profilin (f) or LPS (g) for 24 h.3-Acrylamidobenzoic acid In stock Supernatants have been harvested and assayed for the presence of IL-8 by ELISA.PMID:24856309 Regression curves and r2 and median inhibitory concentration (IC50) values for anti-TLR5 mAb-mediated inhibition of IL-8 responses to flagellin, profilin or LPS are shown. Information shown are representative of no less than three independent experiments.Profilin Triggers Human TLRJ Innate Immun 2014;6:685?94 DOI: 10.1159/Color version readily available onlineMedium IL-6 (pg/ml) 200 150 100 50PAMPPAMP + proteinase K 200 150 100 50PAMP + anti-TLR5 800 600 400 200Percentage of totalbIL-12p70 (pg/ml)df150 100 50 0 0.01 0.1150 one hundred 50 0 0.01 0.1aHuTLR100 80 60 40 200.0.cFlagellin (g/ml)eProfilin (g/ml)gLPS (g/ml)Fig. 3. T. gondii-derived profilin triggers a TLR5-sensitive humanperipheral blood-derived monocyte proinflammatory cytokine production. a Peripheral blood monocytes have been purified and stained for intact (solid line) and permeabilized cells (dotted line). CD14-MACS bead-purified peripheral blood monocytes were suspended in FACS buffer with or with out PermWash buffer followed by incubation with PE-conjugated isotype manage IgG2a (gray histogram) or anti-TLR5 mAb. b Cells have been plated and incubated with medium alone, flagellin (b, c), profilin (d, e) or LPS (f, g) in PBS (PAMP), PAMP predigested with proteinase K (PAMP + proteinase K) or anti-huTLR5 mAb (PAMP + anti-TLR5; 1?.1 g/ ml). After incubation for 24 h, supernatants were harvested and assayed for IL-6 (b, d, f) or IL-12p70 (c, e, g) by ELISA. Data shown are representative of three independently performed experiments. Fig. 4. siRNA-mediated silencing of human TLR5 inhibits profilinand flagellin-mediated proinflammatory cytokine production by human peripheral blood-derived monocytes. Peripheral blood-derived monocytes had been isolated and electroporated inside the presence of medium alone, control siRNA or human TLR5 siRNA oligos. Cells were then stained for TLR5 as described in figure 3. a Transfected cells had been gated and TLR5 expression was analyzed. A histogram overlay of isotype control-stained cells (gray histogram), manage siRNA-transfected cells (dotted line) or TLR5 siRNAtransfected cells (strong line) is shown. b, c Cells had been plated and incubated in the p.