Journal: bioRxiv

Article Title: Dickkopf1 is a Novel Endogenous Ligand for priming NLRP3 Inflammasome in Macrophages via TLR4

doi: 10.1101/2025.03.11.642660

Figure Lengend Snippet: (A) mBMDM from WT and and LysMCre-Lrp6 fl/fl mice were treated with Vehicle control (NC) or 30 ng/ml DKK1 for 6 hr and Hif1α, Arg1, Il1r1, Il1β, Cd274 and Marco mRNA levels were measured by qPCR. Tbp was used as housekeeping gene. (B) WT THP1 macrophages were treated with Vehicle control (NC), 100 ng/ml LPS or 30 ng/ml DKK1 for 15 min, 30 min and 1 hr. Cell lysates were immunoprecipitated with anti-MyD88 antibody and immunoblotted with anti-TLR4 antibody. GAPDH was used as a loading control. (C) WT and MyD88 KO THP1 macrophages were treated with Vehicle control (NC) or 30 ng/ml DKK1 for 15 min, 30 min and 1 hr. Cell lysates were immunoprecipitated with anti-MyD88 antibody and immunoblotted with anti-TLR4 antibody. GAPDH was used as a loading control. (D) WT THP1 macrophages were pretreated with 100 nM TAK-242 for 1 hr, followed by 100 ng/ml LPS or 30 ng/ml DKK1 for 4 hr, 24 hr and probed with anti-HIF1α, anti-NLRP3, anti-pro-IL-1β and anti-GAPDH antibodies. (E) WT THP1 macrophages were treated with 30 ng/ml DKK1 for 15 min, 30 min, 1 hr, 3 hr after pretreated with or without 100 nM TAK-242 for 1 hr. Cell lysates were probed with anti-p-TAK1 Ser412 , anti-TAK1, anti-p-IKKα/β, anti-IKKα, anti-IKKβ, anti-p-IκBα, anti-IκBα, anti-p-p65 Ser536 , anti-p65, anti-p-TBK1, anti-GAPDH antibodies. (F) HEK 293 cells were transfected with plasmids encoding hTLR4, hMD2 and hCD14 cDNAs. NFkB luciferase activity was measured after stimulating with 20 ng/ml LPS or 75 ng/ml DKK1 for 10 hr using Renilla luciferase (pRL-CMV) as a control. Statistically significant differences were analyzed by one-way ANOVA and Bonferroni’s multiple comparisons test (ns, not significant, * p<0.05, *** p<0.001). A representative of two independent experiments is shown.

Article Snippet: After blocking a human FC Receptor Blocking Ab (ThermoFisher, Cat# 14-9161-73), the cells were stained with Zombie Aqua Fixable Viability Kit and then incubated with fluorescent-conjugated antibodies against TLR4 and CD14.

Techniques: Control, Immunoprecipitation, Transfection, Luciferase, Activity Assay

Journal: bioRxiv

Article Title: Dickkopf1 is a Novel Endogenous Ligand for priming NLRP3 Inflammasome in Macrophages via TLR4

doi: 10.1101/2025.03.11.642660

Figure Lengend Snippet: (A) BMDMs from WT and LysMCre-Lrp6 fl/fl mice were treated with 100 ng/ml LPS or 30 ng/ml DKK1 for 3 hr, followed by 10 μM Nigericin for 1.5 hr. (B) WT THP1 macrophages were pretreated with 2 μM WAY-262611 for 1 hr, followed by treatment as in (A) . (C) BMDMs from WT and Tlr4 KO mice were treated as in ( A ). (D, E) BMDMs from WT mice and WT THP1 macrophages were treated as in (A) with or without 100 nM TAK-242 for 1 hr. (F, G) WT and MyD88 KO mBMDMs (F) and THP1 macrophages (G) were treated as in (A) . Cell death and IL-1β secretion were measured by LDH assay and ELISA, respectively. (H) Western blot analysis of IL-1β, cleaved IL-1β, caspase-1 and cleaved caspase-1 in WT vs MyD88 KO THP1 cell lysates or supernatants after stimulating with Vehicle control (NC), 100 ng/ml LPS or 30 ng/ml DKK1 for 3 hr, followed by 10 μM Nigericin for 1.5 hr. Statistically significant differences were analyzed by one-way or two-way ANOVA and Bonferroni’s multiple comparisons test (ns, not significant, * p<0.05, ** p<0.005, *** p<0.001, **** p<0.0001). A representative of two independent experiments is shown.

Article Snippet: After blocking a human FC Receptor Blocking Ab (ThermoFisher, Cat# 14-9161-73), the cells were stained with Zombie Aqua Fixable Viability Kit and then incubated with fluorescent-conjugated antibodies against TLR4 and CD14.

Techniques: Lactate Dehydrogenase Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: bioRxiv

Article Title: Dickkopf1 is a Novel Endogenous Ligand for priming NLRP3 Inflammasome in Macrophages via TLR4

doi: 10.1101/2025.03.11.642660

Figure Lengend Snippet: DKK1 binds to TLR4, recruits MyD88 and IRAK4 to induce TAK1 Ser412 , IKKα/β, IκBα and p65 Ser536 phosphorylation. DKK1-mediated NFκB pathway activation primes macrophages to upregulate NLRP3, pro-IL-1β and HIF1α protein expressions. Activation signals (Nigericin, Nano-SiO 2 , MSU) induces NLRP3 inflammasome activation, leading to ASC oligomerization and recruiting caspase-1. NLRP3 inflammasome activation cleaves pro-Caspase-1, which further cleaves pro-IL-1β and GSDMD. Cleaved GSDMD forms membrane pores and releases IL-1β via pyroptosis.

Article Snippet: After blocking a human FC Receptor Blocking Ab (ThermoFisher, Cat# 14-9161-73), the cells were stained with Zombie Aqua Fixable Viability Kit and then incubated with fluorescent-conjugated antibodies against TLR4 and CD14.

Techniques: Activation Assay, Membrane