Journal: Frontiers in Immunology

Article Title: A flagellin-conjugate protein induces dual NLRC4- and NLRP3-inflammasome activation which modulates inflammatory cytokine secretion from macrophages

doi: 10.3389/fimmu.2023.1136669

Figure Lengend Snippet: Both NLRP3- and NLRC4-inflammasome activation contributes to rFlaA:Betv1 induced IL-1β and pro-inflammatory cytokine secretion from THP-1 macrophages. PMA-differentiated wild-type (WT), NLRP3-, NLRC4-, or ASC-deficient THP-1 macrophages were stimulated with either LPS as a positive control, or equimolar amounts of rFlaA + rBet v 1, rFlaA:Betv1, rFlaA *D1 :Betv1, or rFlaA ΔDC0 :Betv1 for 24 h (A) . Supernatants were collected and checked for the secretion of IL-1β, IL-6, IL-12, and TNF-α by ELISA (B) . Data are the mean results of three independent experiments ± SD, and statistical significances are indicated as ns: p-value > 0.05, *: p-value < 0.05, **: p-value < 0.01, ***: p-value < 0.001.

Article Snippet: PMA-differentiated, THP-1 macrophages were pre-incubated with the indicated amounts of either the IKK-β inhibitors TPCA-1 (Abcam, Cambridge, UK) or BMS-345541 (Abcam), the unspecific inflammasome inhibitor VX-765 ( Invivo Gen), which inhibits caspase-1 activity, the specific NLRP3-inflammasome inhibitor MCC950 ( Invivo Gen), or the MAPK inhibitors SP600125 (SAP/JNK MAPK inhibitor, Invivo Gen), SB202190 (p38α/β MAPK inhibitor, Invivo Gen), or U0126 (ERK MAPK inhibitor, Cell Signaling Technologies, Leiden, The Netherlands) for 90 min and subsequently stimulated with rFlaA:Betv1 for 24 h. The target molecules of the used inhibitors are summarized in .

Techniques: Activation Assay, Positive Control, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: A flagellin-conjugate protein induces dual NLRC4- and NLRP3-inflammasome activation which modulates inflammatory cytokine secretion from macrophages

doi: 10.3389/fimmu.2023.1136669

Figure Lengend Snippet: NFκB- and SAP/JNK MAP kinase-signaling pathways contribute to inflammasome activation by regulating pro-Caspase-1, pro-IL-1β-, and NLRP3-expression in rFlaA:Betv1-stimulated THP-1 macrophages. PMA-differentiated THP-1 macrophages were pre-treated with the indicated inhibitors (500 nM TPCA-1, 5 µM BMS-345541, 25 µM SP600125, or 10 µM SB202190) for 90 minutes, followed by stimulation with 27.4 µg/mL rFlaA:Betv1 for additional 24 h (A) . Proteins in supernatant (Sup) and cell lysate (Lysate) were examined by Western Blot (B) . The intensity of Western Blot bands from three independent experiments was analyzed, first normalized to the loading control β-Tubulin and then again normalized to the unstimulated group (shown as value of “1”) (C) . Data are either representative (B) or mean results of three independent experiments ± SD (C) . Statistical significances are indicated as ns: p-value > 0.05, *: p-value < 0.05, **: p-value < 0.01, ***: p-value < 0.001.

Article Snippet: PMA-differentiated, THP-1 macrophages were pre-incubated with the indicated amounts of either the IKK-β inhibitors TPCA-1 (Abcam, Cambridge, UK) or BMS-345541 (Abcam), the unspecific inflammasome inhibitor VX-765 ( Invivo Gen), which inhibits caspase-1 activity, the specific NLRP3-inflammasome inhibitor MCC950 ( Invivo Gen), or the MAPK inhibitors SP600125 (SAP/JNK MAPK inhibitor, Invivo Gen), SB202190 (p38α/β MAPK inhibitor, Invivo Gen), or U0126 (ERK MAPK inhibitor, Cell Signaling Technologies, Leiden, The Netherlands) for 90 min and subsequently stimulated with rFlaA:Betv1 for 24 h. The target molecules of the used inhibitors are summarized in .

Techniques: Activation Assay, Expressing, Western Blot

Journal: Frontiers in Physiology

Article Title: Mitochondrial DNA Promotes NLRP3 Inflammasome Activation and Contributes to Endothelial Dysfunction and Inflammation in Type 1 Diabetes

doi: 10.3389/fphys.2019.01557

Figure Lengend Snippet: Cumulative concentration-response curves to Acetylcholine (ACh) (A) , endothelium-dependent vasodilator, in resistance mesenteric arteries from C57BL/6 non-diabetic (control, white circles) or diabetic (T1D, black circles) and Nlrp3 – / – non-diabetic ( Nlrp3 – / – , white squares) or diabetic ( Nlrp3 – / – T1D, black squares) mice in the presence of (B) vehicle (white diamonds) and a NLRP3 inhibitor (MCC950, black diamonds), (C) vehicle (white triangles) and superoxide scavenger Tiron (black triangles), and (D) vehicle (white triangles down) and catalase mimetic peg-catalase (black triangles down). Generation of (E) superoxide anion and (F) hydrogen peroxide in mesenteric bed from non-diabetic (control, white circles) or diabetic (T1D, black circles) C57BL/6 mice and non-diabetic ( Nlrp3 – / – , white squares) or diabetic ( Nlrp3 – / – T1D, black squares) Nlrp3 – / – mice. Each point/column represents the mean ±SEM. ∗ P < 0.05 vs. control, & P < 0.05 vs. T1D. N = 3–7, two-way ANOVA followed by Tukey’s post hoc test (A–F) .

Article Snippet: Phenylephrine hydrochloride, acetylcholine chloride, LPS, lucigenin (N,N′-Dimethyl-9,9′-biacridinium dinitrate) and peg-catalase (Catalase-polyethylene glycol) were obtained from Sigma-Aldrich (St. Louis, MO, United States), MCC950 from Avistron ® (Bude, Cornwall, United Kingdom), Tiron from Santa Cruz Biotechnology ® (San Juan, CA, United States) and all reagents used in RT-PCR and murine primers from Invitrogen ® (Carlsbad, CA, United States).

Techniques: Concentration Assay, Control

Journal: Frontiers in Physiology

Article Title: Mitochondrial DNA Promotes NLRP3 Inflammasome Activation and Contributes to Endothelial Dysfunction and Inflammation in Type 1 Diabetes

doi: 10.3389/fphys.2019.01557

Figure Lengend Snippet: On top, representative images of western blot and, at the bottom, densitometric analysis of the expression of (A) caspase-1 and pro-caspase-1 and (B) IL-1β and pro-IL-1β in endothelial cells primed with lipopolysaccharide (LPS) and incubated with vehicle (white squares) and mitochondrial DNA from control (cmDNA, white circles) and diabetic (dmDNA, black circles) mice. (C) Cumulative concentration-response curves to acetylcholine (ACh), endothelium-dependent vasodilator, in resistance mesenteric arteries from C57BL/6 incubated with vehicle (white circles) or mitochondrial DNA from diabetic mice (dmDNA, black circles) in the presence of vehicle (white triangles) or a NLRP3 inhibitor (MCC950, black triangles). Each point/column represents the mean ±SEM. ∗ P < 0.05 vs. vehicle. N = 3–5, one-way ANOVA (A–C) .

Article Snippet: Phenylephrine hydrochloride, acetylcholine chloride, LPS, lucigenin (N,N′-Dimethyl-9,9′-biacridinium dinitrate) and peg-catalase (Catalase-polyethylene glycol) were obtained from Sigma-Aldrich (St. Louis, MO, United States), MCC950 from Avistron ® (Bude, Cornwall, United Kingdom), Tiron from Santa Cruz Biotechnology ® (San Juan, CA, United States) and all reagents used in RT-PCR and murine primers from Invitrogen ® (Carlsbad, CA, United States).

Techniques: Western Blot, Expressing, Incubation, Control, Concentration Assay

Journal: Veterinary Research

Article Title: Staphylococcus aureus mediates pyroptosis in bovine mammary epithelial cell via activation of NLRP3 inflammasome

doi: 10.1186/s13567-022-01027-y

Figure Lengend Snippet: NLRP3 inflammasome regulates ASC speck formation during S. aureus . Fluorescence microscopy images of MAC-T cells immunoassayed for ASC (red) with or without MCC950 after 4 h of S. aureus treatment.

Article Snippet: Caspase-1 inhibitor VX765 and NLRP3 inflammasome inhibitor MCC950 were obtained from Glpbio (USA) and used at final concentrations of 100 and 15 μM.

Techniques: Fluorescence, Microscopy

Journal: Veterinary Research

Article Title: Staphylococcus aureus mediates pyroptosis in bovine mammary epithelial cell via activation of NLRP3 inflammasome

doi: 10.1186/s13567-022-01027-y

Figure Lengend Snippet: NLRP3 inflammasome activation by S. aureus is essential for the generation of GSDMD-N and the release of IL-1β and IL-18. A Activated caspase-1 and B GSDMD-N or released C IL-1β and D IL-18 of MAC-T cells treated with or without NLRP3 inhibitor MCC950 or caspase-1 inhibitor VX765 for 1.5 h prior to treatment with S. aureus for 4 h. E Levels of IL-1β and F IL-18 released after S. aureus treatment of MAC-T cells for 4 h in the presence or absence of 25, 50, or 75 mM KCl.

Article Snippet: Caspase-1 inhibitor VX765 and NLRP3 inflammasome inhibitor MCC950 were obtained from Glpbio (USA) and used at final concentrations of 100 and 15 μM.

Techniques: Activation Assay

Journal: Frontiers in Immunology

Article Title: Zika Virus Infection Induces Acute Kidney Injury Through Activating NLRP3 Inflammasome Via Suppressing Bcl-2

doi: 10.3389/fimmu.2019.01925

Figure Lengend Snippet: ZIKV infection induced NLRP3 inflammasome activation in the kidneys. (A,B) The relative mRNA levels (vs. mock group) of NLRP1, NLRP3, IPAF, and AIM2 in the kidneys of mock and ZIKV-infected newborn (A) and adult (B) mice at 7 days post-infection were detected by qRT-PCR. (C–I) Expression levels of NLRP3 (D) , ASC (E) , pro-caspase-1 (F) , cleaved-caspase-1 (G) , pro-IL-1β (H) , and mature IL-1β (I) in the kidneys of newborn and adult mice at 7 days post-infection were assessed by western blot. (J,K) Examination of mature IL-1β release in the supernatants of mouse kidneys at 7 days post-infection ( n = 6 per group) were determined by an enzyme-linked immunoadsorbent assay (ELISA). Data were presented as mean ± SEM ( n = 6), * p < 0.05 vs. mock group; # p < 0.05 vs. ZIKV group ( *** p < 0.001 vs. mock group or ZIKV+MCC950 group in Figure J and K).

Article Snippet: To inhibit NLRP3 and IL-1β activation, mice were i.p. injected with 10 μg MCC950 per gram body weight (BW) or 35 μg/g BW Anakinra (143090-92-0, ChemLeader, CN) or PBS (vehicle control) ever 2 days beginning from 2 days post-infection until tissue harvest.

Techniques: Infection, Activation Assay, Quantitative RT-PCR, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Zika Virus Infection Induces Acute Kidney Injury Through Activating NLRP3 Inflammasome Via Suppressing Bcl-2

doi: 10.3389/fimmu.2019.01925

Figure Lengend Snippet: Overexpression of Bcl-2 attenuated ZIKV-induced NLRP3 inflammasome activation in HK-2 cells. (A–C) Expression level of Bcl-2 in the kidneys of newborn (A,B) and adult mice (A,C) at 7 days post-infection were assessed by western blot. (D–K) HK-2 cells were transfected with control vector or pSG5-FLAG-Bcl-2 plasmid (2 μg/ml) for 24 h or pre-treated with MCC950 (10 μM) for 30 min followed by incubation with PBS (mock group) or ZIKV (MOI = 2), and cultured with complete medium in presence of MCC950 or PBS at 37°C for 24 h. The protein expression levels of Bcl-2, NLRP3, ASC, pro-caspase-1, cleaved-caspase-1, pro-IL-1β, and mature IL-1β protein were assessed by western blot. (L) The mRNA expression level of NLRP3 was detected by qRT-PCR. Data were presented as mean ± SEM, * p < 0.05 vs. Control group; # p < 0.05 vs. mock group.

Article Snippet: To inhibit NLRP3 and IL-1β activation, mice were i.p. injected with 10 μg MCC950 per gram body weight (BW) or 35 μg/g BW Anakinra (143090-92-0, ChemLeader, CN) or PBS (vehicle control) ever 2 days beginning from 2 days post-infection until tissue harvest.

Techniques: Over Expression, Activation Assay, Expressing, Infection, Western Blot, Transfection, Control, Plasmid Preparation, Incubation, Cell Culture, Quantitative RT-PCR