NF-κB activation by rFliC.

<p>(A) HEK-Blue^TM-hTLR5 cells (1.4 x 10⁵cells/ml) in HEK-Blue™ Detection medium were incubated with 1, 10, and 100 ng/ml rFliC in three independent experiments. The cells stimulated with flagellin purified from <i>S</i>. Typhimurium (FLA-ST) were used for comparison. After incubation for 24 h, NF-κB activation was determined by monitoring SEAP production. (B) THP1-Dual^TM cells (5.6 x10⁵cells/ml) were added into a 96-well plate containing 20 μl of rFliC at indicated concentrations. The supernatant was collected at 24 h after incubation and SEAP activity was measured using the Quanti-Blue assay. The result of THP-1 cell assay was obtained from three independent experiments. Data represent the mean, and error bars represent the standard deviation of the results of three independent experiments conducted in triplicate.</p

ELISA results of flagellin-specific IgM and IgG antibodies.

<p>ELISAs were evaluated for IgM and IgG antibodies using sera from melioidosis patients (N = 45) and healthy donors (N = 45) on the pre-coated plate with 15 μg/ml of rFliC. Box plots show OD at 450 nm of rFliC-specific IgG (A) and IgM (B) in different groups of subjects. All data in box plots are presented as 25^th and 75^th percentile boundaries in the box with the median line within the box; the whiskers indicate the 10^th and 90^th percentiles. (C) Receiver Operating Characteristics (ROC) plots.</p

Humoral responses to rFliC.

<p>ELISAs using HRP-conjugated rabbit anti-human IgM (A-C) and IgG (D-F) were conducted on prepared plates coated with 15 μg/ml of rFliC. Serum samples from 200 melioidosis patients were used at a dilution of 1:300. Box plots show OD at 450 nm for different groups of patients. All data in box plots are presented as 25^th and 75^th percentile boundaries in the box with the median line within the box; the whiskers indicate the 10^th and 90^th percentiles. Mann–Whitney test was used to assess for statistically significant difference of median OD values between different serum groups.</p

Differential cytokine profiles from individual subjects.

<p>Whole blood from healthy subjects (N = 14) was stimulated with rFliC at a final concentration of 500 ng/ml. The supernatants were collected at 6 h and 24 h after incubation and pro-inflammatory cytokine (IL-1β, IL-6 and TNF-α) productions were evaluated by ELISA. Each line represents an individual subject. The differences of medians between 6h and 24h were tested by the Mann-Whitney test.</p

Cytochalasin D decreases pyroptosis in TLR ligand-activated Raw264.7 cells.

Raw264.7 cells were pretreated with 2 μg/ml cytochalasin D (Cyt D) for 2 h before stimulation with TLR ligands for 18 h. (A, C) At the indicated time, the cultured supernatant of treated cells was collected and analyzed by the LDH assay. Data are means ± SEM from at least three independent experiments. All data were determined by one-way ANOVA followed by Tukey’s multiple comparison test. *p p p < 0.001. (B, D) The cell lysates were prepared and analyzed by immunoblotting. Representative bands are shown from three independent experiments.</p

S1 Raw images -

BackgroundCleistanthin A (CA), extracted from Phyllanthus taxodiifolius Beille, was previously reported as a potential V-ATPase inhibitor relevant to cancer cell survival. In the present study, ECDD-S16, a derivative of cleistanthin A, was investigated and found to interfere with pyroptosis induction via V-ATPase inhibition.ObjectiveThis study examined the ability of ECDD-S16 to inhibit endolysosome acidification leading to the attenuation of pyroptosis in Raw264.7 macrophages activated by both surface and endosomal TLR ligands.MethodsTo elucidate the activity of ECDD-S16 on pyroptosis-induced inflammation, Raw264.7 cells were pretreated with the compound before stimulation with surface and endosomal TLR ligands. The release of lactate dehydrogenase (LDH) was determined by LDH assay. Additionally, the production of cytokines and the expression of pyroptosis markers were examined by ELISA and immunoblotting. Moreover, molecular docking was performed to demonstrate the binding of ECDD-S16 to the vacuolar (V-)ATPase.ResultsThis study showed that ECDD-S16 could inhibit pyroptosis in Raw264.7 cells activated with surface and endosomal TLR ligands. The attenuation of pyroptosis by ECDD-S16 was due to the impairment of endosome acidification, which also led to decreased Reactive Oxygen Species (ROS) production. Furthermore, molecular docking also showed the possibility of inhibiting endosome acidification by the binding of ECDD-S16 to the vacuolar (V-)ATPase in the region of V0.ConclusionOur findings indicate the potential of ECDD-S16 for inhibiting pyroptosis and prove that vacuolar H+ ATPase is essential for pyroptosis induced by TLR ligands.</div

ECDD-S16 inhibits pyroptosis induced by TLR ligands in Raw264.7 cells.

Raw264.7 cells were pretreated with ECDD-S16 (0.5 μM) for 1 h before stimulation with TLR ligands for 18 h. (A, C) At the indicated time, the cultured supernatant of treated cells was collected and subjected to analysis by the LDH assay. Data are means ± SEM from at least three independent experiments. All data were determined by one-way ANOVA followed by Tukey’s multiple comparison test. **p p p < 0.0001. (B, D) The cell lysates were prepared and analyzed by immunoblotting. Representative bands are shown from three independent experiments.</p

Effect of ECDD-S16 on Pam2CSK4-induced ROS production in Raw264.7 cells.

Raw264.7 cells were pretreated with ECDD-S16 (0.5 μM) for 1 h or 30 μM chloroquine (CQ) for 15 min before stimulation with Pam2CSK4 (1 μg/ml) for 16 h. Then, the treated cells were washed and incubated with 2 μM of DCFH-DA for 20 min at 37°C. ROS production was analyzed by flow cytometry. The percentage of positive cells (A), mean fluorescent intensity (MFI) (B) and representative histograms of DCFH-DA (C) are shown. The results are presented as means ± SEM of three independent experiments. All data were determined by one-way ANOVA followed by Tukey’s multiple comparison test. *p p p < 0.001.</p

Synthesis of ECDD-S16, a benzoyl ester derivative of cleistanthin.

A. The esterification reaction between cleistanthin A and 4-fluorobenzoic acid in the presence of dicyclohexylcarbodiimide (DCC) and catalytic 4-dimethylaminopyridine (DMAP) affords ECDD-S16 in 36% yield.</p

Chloroquine inhibits pyroptosis in TLR ligand-activated Raw264.7 cells.

Raw264.7 cells were pretreated with 30 μM chloroquine (CQ) for 15 min before stimulation with TLR ligands for 18 h. (A, C) At the indicated time, the cultured supernatant of treated cells was collected and subjected to the LDH assay. Data are means ± SEM from at least three independent experiments. All data were determined by one-way ANOVA followed by Tukey’s multiple comparison test. *p p p < 0.0001. (B, D) The cell lysates were prepared and analyzed by immunoblotting. Representative bands are shown from three independent experiments.</p