Article Figures & Data
Figures
- Fig. 1.
Soluble recombinant TLR4, TLR4(s), can inhibit TLR4-activated proinflammatory IL-8 secretion to distinct agonists in HEK293 hTLR4 cells. (A) IL-8 secretion of HEK293-hTLR4 cells pretreated with 0.1 nM soluble recombinant human TLR4 and subsequently treated with either 1 ng/mL LPS, 200 μM Ni2+, or 25 μM cisplatin. Data are shown as the percentage of TLR4 activity in the absence of soluble hTLR4. Nil representative of cells treated without agonists (n = 4 independent biologic replicates for all conditions). (B) IL-8 secretion of HEK293-hTLR4 cells pretreated with 0.1 nM soluble recombinant mouse TLR4 and subsequently treated with either 1 ng/mL LPS, 200 μM Ni2+, or 25 μM cisplatin. Data are shown as the percentage of TLR4 activity in the absence of soluble mTLR4 (n = 4 for all conditions). Nil representative of cells treated without agonists. For all panels, all individual data points (technical replicates) from each experiment are plotted with a central bar for the mean and whiskers denoting S.D. Statistical analyses were performed through two-way ANOVA and Bonferroni multiple-testing correction. ****P < 0.0001. ns, not significant.
- Fig. 2.
Human TLR4 can directly bind nickel and cisplatin. Microscale thermophoresis analysis showing normalized fluorescence of hTLR4 plotted against the indicated concentrations of (A) LPS (n = 5 independent replicates), (B) Ni2+ (n = 3 independent replicates), (C) Ca2+ (n = 6 independent replicates), or (D) cisplatin (n = 3–6 independent replicates), respectively. For all panels, data fitted to nonlinear best-fit curves following three parameters with a standardized slope. (A) Apparent Kd unavailable/undetectable. (B) Apparent Kd = 2.72 μM, R2 = 0.695. (C) Apparent Kd unavailable/undetectable. (D) Apparent Kd = 50.57 μM, R2 = 0.927.
- Fig. 3.
Histidine 456 and 458 mutations (H456A-H458L) reduce, but do not abrogate, TLR4 activation by cisplatin. (A) HEK293-Null2 cells were transfected with either empty vector (EV), hTLR4, or hTLR4 mutant constructs, and immunoblotting of lysate was used to analyze relative TLR4 protein levels. Images from different parts of the same gel are shown. IL-8 secretion from HEK293-Null2 cells transfected with either nothing (control), EV, hTLR4, or hTLR4 mutant constructs and subsequently treated with (B) 1 ng/mL LPS, (C) 200 μM Ni2+, (D) 100 μM platinum (II) [Pt(II)], (E) 100 μM platinum (IV) [Pt(IV)], or (F) 25 μM cisplatin displayed as a percentage of the response elicited by wild-type hTLR4 [n = 4 independent biologic replicates for experiments performed with LPS, Ni2+, and Pt(II); n = 3, independent biologic replicates for experiments involving Pt(IV) and cisplatin]. Cells treated with LPS were also cotransfected with an MD-2 construct. For (B–F), individual data (technical replicates) from each experiment are plotted with the mean and S.D. displayed. Statistical analyses were performed through one-way ANOVA (for all remaining experiments) with Bonferroni multiple-testing correction. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, hemagglutinin; n.s., nonsignificant.
- Fig. 4.
Mouse TLR4 directly binds cisplatin but not nickel. Microscale thermophoresis analysis showing normalized fluorescence of mTLR4 plotted against the indicated concentrations of (A) Ni2+ (n = 3 independent replicates) or (B) cisplatin (n = 3 to 4 independent replicates). (A) Apparent Kd unavailable/undetectable. (B) Apparent Kd = 104.9 μM; R2 = 0.721.
- Fig. 5.
TLR4 is activated by LPS, nickel, and cisplatin by distinct mechanisms. (Left panel) LPS requires MD-2 for TLR4 activation and cannot directly bind soluble TLR4 species. (Middle panel) Nickel can directly bind human TLR4, and this requires His456/His458. Soluble hTLR4 can block nickel activation of TLR4 (denoted by black line). Nickel cannot bind to mouse TLR4, which lacks these histidine residues. (Right panel) Cisplatin can directly bind both human and mouse TLR4, and this is not strictly dependent on His456/His458. Cisplatin has higher affinity for soluble hTLR4, which is more effective at blocking cisplatin activation of TLR4 compared with soluble mTLR4 (denoted by solid black versus gray dashed lines). The additional TLR4 residues that contribute to cisplatin binding remain to be elucidated (denoted by “?”). Figure was created with BioRender.com.
Additional Files
Supplemental Data
- Supplemental Figure -
SUPPLEMENTAL FIGURE 1. Cisplatin binds to hTLR4 with higher affinity than mTLR4.
- Supplemental Figure -
