Fig 2: Effects of CAP37 and CAP37-derived peptides on TLR4. (A) CAP37 and peptide 20-44 are partial agonists of TLR4. The ability of increasing concentrations of S100A9, CAP37, and CAP37-derived peptides to stimulate TLR4 was measured in HEK-hTLR4 cells. SEAP production in cell medium was measured following 24-hour incubation with ligands. Data shown are mean percent activation ± SEM relative to activation induced by S100A9 at 50 nM, arbitrarily defined as 100%. Results are from three experiments, each done in triplicate. First arrow, partial activation of TLR4 by CAP37; second arrow, partial activation of TLR4 by peptide 20-44. (B–F) Inhibitory effects of CAP37 protein and peptides on the activation of TLR4 by S100A9. Cells were treated with S100A9 at 10 nM alone or in combination with increasing concentrations of CAP37 (B) or CAP37 peptides (C–F). Filled symbols denote preincubation of CAP37 or the indicated peptide with 0- or 10-nM S100A9 for 1 hour before treatment. Open symbols denote co-treatment with 0- or 10-nM S100A9 without preincubation before treatment. Following 24-hour treatment, SEAP production was quantified. Results show the 50% activation of TLR4, induced by 10-nM S100A9 alone, and activations below 50% with indicated concentrations of CAP37 or CAP37 peptides. Data shown are mean ± SEM from three experiments, each done in triplicate. A two-way ANOVA, followed by Dunnett's multiple comparisons test, compared the percent activation of hTLR4 by S100A9 alone with that of S100A9 combined with CAP37 or peptide. Statistical significances of inhibitions are indicated on the graphs as follows: *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001.

Fig 3: Potential roles of the DNA-mediated proteolytic processing of HMGB1 by neutrophil elastase in NETs. Due to the enhanced binding activities of the processed HMGB1 protein, this processing may promote (1) TLR4 signaling, (2) binding to biofilm DNA, and (3) DNA sensing by cGAS. Due to the loss of residues 177–215, the processing of HMGB1 may diminish (4) RAGE signaling and (5) nuclear localization. NET, neutrophil extracellular trap.

Fig 4: CAP37 and four CAP37-derived peptides directly bind TLR4/MD-2 in vitro. ELISA plates were coated with the indicated proteins and peptides. (A, C) His-tagged TLR4/MD-2 (at 0 or 10 nM) was added to the coated wells and binding was quantified using anti-His tag antibody. Data are mean ± SEM of OD values from three independent experiments, after values with 0-nM TLR4/MD-2 were removed as background. A one-way ANOVA, using Dunnett's multiple comparisons test, compared the binding of each protein (A) or peptide (B) to the binding of BSA. Significances of *P < 0.05, **P < 0.01, and ***P < 0.001 are shown. (B) Dose-dependent binding of TLR4/MD-2 to indicated proteins. Data (mean ± SEM) are fitted to a curve of nonlinear regression. A two-way ANOVA was used with Dunnett's multiple comparisons test to compare the binding of each protein to that of BSA at the same concentration of TLR4/MD-2. LPS shows a significant binding of **P < 0.01 to TLR4/MD-2 at 50 nM and ****P < 0.0001 at all higher doses. CAP37 shows significance with **P < 0.01 at 10 nM and ****P < 0.0001 at all higher doses of TLR4/MD-2. S100A8 showed significant binding to TLR4/MD-2 at 75 nM (***P < 0.001) and at 100 nM (****P < 0.0001). Binding of NE and CG was not significant. (D) Dose-dependent binding of TLR4/MD-2 to peptides. Results are plotted and analyzed as described in (B). At all tested doses of TLR4/MD-2, the binding of peptide 120-146WH 5R-MP showed maximum significance of ****P < 0.0001. Peptides 20-44, 20-44 5R-MP, and 120-146WH significantly bind TLR4/MD-2 at 25 nM and reach maximum significance (****P < 0.0001) at 50 nM. Binding of peptides 95-122 and 95-122 5R-MP was not significant at any tested dose.

Fig 5: MicroRNA 130b-3p mimic attenuates rmCIRP's binding to TLR4/MD2 complexUsing surface plasmon resonance analysis, TLR4/MD2 complex was immobilized onto a CM5 series chip (GE Healthcare). AVarying concentrations of rmCIRP (31.25–500 nM) were injected alone as the analyte (K d of 116.5 ± 0.8 nM).BIC50 (55 nM) was analyzed by injecting a steady concentration of rmCIRP (0.25 µM) combined with varying concentrations of miRNA 130b-3p (16–500 nM) over immobilized TLR4/MD2 complex.C, DVarying concentrations of rmCIRP (31.25–1,000 nM) with a constant concentration of 50 nM and 100 nM of miRNA 130b-3p were injected as analytes over immobilized TLR4/MD2 complex (K d of 420 nM and 40 µM, respectively).EExperiments (A, C, and D) plotted to compare response units and K d values.FTo determine that miRNA 130b-3p does not bind TLR4/MD2 complex directly, varying concentrations of miRNA 130b-3p (31.25 nM up to 1,000 nM) were injected as the analyte with TLR4/MD2 complex immobilized onto a CM5 series chip. At least three independent BIAcore experiments were performed.