Journal: The EMBO Journal

Article Title: Poxvirus dsDNA genomes differentially activate AIM2 or NLRP3 inflammasomes in human primary cells

doi: 10.1038/s44318-025-00690-z

Figure Lengend Snippet: ( A – C ) Human monocyte-derived macrophages (hMDMs) differentiated in GM-CSF, ( D – F ) normal human epidermal keratinocytes (NHEK), ( G – I ) CD14 + monocytes, and ( J ) PMA-differentiated THP-1 macrophages were left untreated or pretreated with IFN-γ overnight. Cells were infected with indicated recombinant VACV strains at an MOI of 5 ( A – F , J ) or MOI 10 ( G – I ), in the presence ( A , C , D , F , G , I , J ) or absence ( B , E , H ) of VX, and where indicated, in the presence of 2.5 µM CRID3. Cells were harvested and fixed 6 h post infection. Infection (EGFP + cells) and C1C-EGFP speck assembly in infected or mock-treated cells was quantified by flow cytometry ( A , D , G , J ); infection data (EGFP + cells) corresponding to the experiment shown in panel ( J ) is displayed in Fig. . Membrane permeabilization was quantified by the uptake of DNA dye DRAQ7 over 6 h in an Incucyte live-cell imaging system ( B , E , H ). Scale bar = 100 μm. Samples for confocal microscopy were fixed and stained with 5 μg/mL wheat germ agglutinin (WGA)-Alexa Fluor (AF) 647 and 4 μM Hoechst 33342 ( C , F , I ). Scale bar = 5 μm. ( K ) PMA-differentiated WT or indicated knockout THP-1 cells were left untreated or pretreated with IFN-γ overnight and infected with the indicated reporter VACV strains at an MOI of 5 in the presence of VX. C1C-EGFP speck assembly was quantified as before. The corresponding infection data (EGFP + cells) is shown in Fig. . ( L , M ) NHEK ( L ) or PMA-differentiated THP-1 cells ( M ) constitutively expressing C1C-EGFP were left untreated or pretreated with IFN-γ overnight and infected with VACV WT or monkeypox virus (MPXV) at an MOI of 5, and where indicated, in the presence of CRID3. Cells were harvested and fixed 6 h post infection. VACV- or MPXV-infected cells were stained with rabbit polyclonal anti-H5 serum and AF647-labeled goat anti-rabbit IgG antibodies, and measured by flow cytometry. Corresponding infection data (αH5-AF647 + cells) is shown in Fig . C1C-EGFP speck assembly was analyzed in infected or mock-treated cells. Data represent average values (with individual data points) from N = 3 independent donors (primary cells) ± SEM, or from N = 3 independent experiments ± SEM (THP-1 cells). .

Article Snippet: CRID3 (MCC950) , Tocris , 5479.

Techniques: Derivative Assay, Infection, Recombinant, Flow Cytometry, Membrane, Live Cell Imaging, Confocal Microscopy, Staining, Knock-Out, Expressing, Virus, Labeling

Journal: The EMBO Journal

Article Title: Poxvirus dsDNA genomes differentially activate AIM2 or NLRP3 inflammasomes in human primary cells

doi: 10.1038/s44318-025-00690-z

Figure Lengend Snippet: ( A – I ) Human monocyte-derived macrophages (hMDMs) differentiated in GM-CSF ( A , D , E ), normal human epidermal keratinocytes (NHEK) ( B , F , G ), or human primary CD14 + monocytes ( C , H , I ), were left untreated or pretreated with IFN-γ overnight. Cells were infected with VACV C1C-EGFP at an MOI of 5 ( A , B ) or MOI 10 ( C ), in the absence of VX, and where indicated in the presence of 2.5 µM CRID3. Membrane permeabilization was quantified by the uptake of DNA dye DRAQ7 over 6 h in an Incucyte Live-Cell Imaging system ( A – C ). DRAQ7 uptake normalized to cell confluency are shown. IL-1β in the supernatant was measured by homogeneous time resolved fluorescence (HTRF) ( D , F , H ). Cell death was measured by LDH release and normalized to cells lysed in 1% Triton X-100 ( E , G , I ). ( J , K ) PMA-differentiated WT ( J ) or indicated knockout THP-1 cells ( K ) were left untreated or pretreated with IFN-γ overnight and infected with indicated VACV strains in the presence of VX, and where indicated, CRID3. Infection levels are displayed here; specking data from the same experiments are shown in Fig. . ( L , M ) NHEK ( L ) and PMA-differentiated THP-1 cells ( M ) constitutively expressing C1C-EGFP were left untreated or pretreated with IFN-γ overnight and infected with VACV or monkeypox virus (MPXV) at an MOI of 5 in the presence of VX, and where indicated, CRID3. Cells were fixed 6 h post infection and stained with rabbit polyclonal anti-H5 serum and AF647-labeled goat anti-rabbit IgG antibodies. Infected cells (EGFP + ) were analyzed in infected or mock-treated cells by flow cytometry as described before. Infection levels are displayed here; specking data from the same experiments are displayed in Fig. . Data represent average values (with individual data points) from N = 3 independent donors (primary cells) ± SEM, or from N = 3 independent experiments ± SEM (THP-1 cells).

Article Snippet: CRID3 (MCC950) , Tocris , 5479.

Techniques: Derivative Assay, Infection, Membrane, Live Cell Imaging, Fluorescence, Knock-Out, Expressing, Virus, Staining, Labeling, Flow Cytometry

Journal: The EMBO Journal

Article Title: Poxvirus dsDNA genomes differentially activate AIM2 or NLRP3 inflammasomes in human primary cells

doi: 10.1038/s44318-025-00690-z

Figure Lengend Snippet: ( A ) Genome structure of recombinant VACV strains expressing C1C-EGFP from the J2R early promoter (pE) and bivalent nanobodies from a synthetic early/late promoter (pE/L). Transgenes were inserted into the VACV TK locus by homologous recombination. ( B ) Scheme of AIM2 inflammasome components indicating the VACV-encoded nanobodies used to perturb inflammasome activation. ( C – E ) Human MDMs differentiated with GM-CSF ( C ), NHEKs ( D ), and CD14 + monocytes ( E ) were left untreated or treated with IFN-γ overnight. Cells were infected with VACV C1C-EGFP expressing the indicated nanobodies at an MOI of 5 ( C , D ) or 10 ( E ), in the presence of VX and, where indicated, CRID3. Six hours post infection, cells were harvested, fixed, and infection and C1C-EGFP speck assembly was analyzed by flow cytometry. Average values (with individual data points) from N = 3 independent donors ± SEM are displayed. .

Article Snippet: CRID3 (MCC950) , Tocris , 5479.

Techniques: Recombinant, Expressing, Homologous Recombination, Activation Assay, Infection, Flow Cytometry

Journal: Inflammation

Article Title: Induction of Exaggerated Cytokine Production in Human Peripheral Blood Mononuclear Cells by a Recombinant SARS-CoV-2 Spike Glycoprotein S1 and Its Inhibition by Dexamethasone

doi: 10.1007/s10753-021-01464-5

Figure Lengend Snippet: Effects of pre-treatment with BAY11-7082 (1 μM), SKF 86002 (1 μM) and CRID3 (1 μM) on TNFα ( a ), IL-6 ( b ), IL-1β ( c ) and IL-8 ( d ) production in human PBMCs. Culture supernatants were collected following stimulation for 24 h and analysed using human ELISA kits for TNFα, IL-6, IL-1β and IL-8. Values are mean ± SEM for at least 3 independent experiments (ns: not significant; ### p <0.001 versus unstimulated control; #### p < 0.0001 versus unstimulated control; * p < 0.05; ** p < 0.01; *** p < 0.001, compared with spike glycoprotein S1 stimulation; one-way ANOVA with post-hoc Tukey’s test).

Article Snippet: The following drugs were used: BAY11-7082 (Sigma), CRID3 sodium salt (Tocris), SKF 86002 dihydrochloride (Tocris) and dexamethasone (Sigma).

Techniques: Enzyme-linked Immunosorbent Assay, Control