Journal: Journal of Neuroinflammation

Article Title: Dopamine-driven increase in IL-1β in myeloid cells is mediated by differential dopamine receptor expression and exacerbated by HIV

doi: 10.1186/s12974-025-03403-9

Figure Lengend Snippet: Dopamine increases IL-1β and inflammasome expression in HIV-infected primary macrophages. A Representative immunofluorescent images at 20X objective (DAPI, blue; Phalloidin, red; p24, green) show human monocyte-derived macrophages (hMDM) infected with 0.5 ng/mL HIV ADA and treated with dopamine (10 –6 M) for 7 days. Higher levels of cell fusion and giant cell formation as well as p24 expression (white arrows) are seen in infected dopamine-treated cultures relative to cultures only infected with HIV. hMDM were also infected with 0.5 ng/mL HIV ADA for 7 days and then treated with dopamine (10 –6 M) for 3 h and examined for (B) IL-1β, (C) NLRP3, (D) NLRC4, and (E) AIM2 expression by qPCR. There was a significant increase in IL-1β, NLRP3, and NLRC4 and a trending increase in AIM2 expression with HIV + Dopamine relative to HIV alone. With these same samples we simultaneously extracted protein and examined (F) NLRP3, (G) NLRC4, and (H) AIM2 expression by Western Blot. There was only a significant increase in NLRP3 expression with HIV + Dopamine relative to HIV alone. Significance was determined by paired t-tests and Wilcoxon tests, *p < 0.05 and **p < 0.01

Article Snippet: Revert stain was then removed and then membranes were blocked in 5% BSA for 2 h at RT and then incubated overnight at 4 °C in either anti-NLRP3 antibody (15101S, 1:1000 in 5% BSA, Cell Signaling), anti-NLRC4 antibody (12421, 1:1000 in 5% BSA, Cell Signaling) or anti-AIM2 antibody ( MA5-38442, 1:1000 in 5% BSA, Thermo Fisher Scientific).

Techniques: Expressing, Infection, Derivative Assay, Western Blot

Journal: bioRxiv

Article Title: Streptococcus anginosus Activates the NLRP3 Inflammasome to Promote Inflammatory Responses from Macrophages

doi: 10.1101/2025.03.12.642696

Figure Lengend Snippet: a) western blot of NLRP3, NLRC4 and AIM2 from BMMs uninfected or infected for 24h with S. anginosus at a MOI of 10. b) ELISA of TNFα and IL-1β from WT and Asc −/− BMM supernatant uninfected or infected for 24h. Quantification of three biological replicates. c) ELISA of TNFα and IL-1β from WT and Nlrc4 −/− BMM supernatant uninfected or infected for 24h with S. anginosus . Quantification of three biological replicates. d) ELISA of TNFα and IL-1β from WT and Aim2 −/− BMM supernatant infected or uninfected for 24h. 1h LPS prime followed by 6h treatment of poly dA:dT was used as a positive control. Quantification of three biological replicates. e) ELISA of TNFα and IL-1β from WT and Nlrp3 −/− BMM supernatant infected or uninfected for 24h. 1h LPS prime followed by 3h nigericin treatment was used as a positive control. Quantification of three biological replicates. f) ELISA of IL-1β from WT BMM supernatant uninfected or infected for 24h with or without 10mM NLRP3 inhibitor, MCC950. Data is presented as mean +/− SEM. Stats quantified using an unpaired T test through GraphPad. *P<.05, **P<0.001 in comparison to uninfected conditions. Data without stats are ns.

Article Snippet: Primary antibodies used were rabbit mAb anti-COX2 (Cell Signaling 12282S), rabbit mAb anti-NLRC4 (Cell Signaling 12421T), rabbit mAb anti-NLRP3 (Cell Signaling 1510S), rabbit mAb phopsho-p65 (S536) (Cell Signaling 3033T), rabbit mAb anti-p65 (Cell Signaling 8242S), rabbit mAb anti-iNOS (abcam ab283655), rabbit mAb anti-AIM2 E518Z (Cell Signaling 53491S), and anti-β-Actin-HRP (Santa Cruz Biotech A1723).

Techniques: Western Blot, Infection, Enzyme-linked Immunosorbent Assay, Positive Control, Comparison

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: TLR priming licenses NAIP inflammasome activation by immunoevasive ligands.

doi: 10.1073/pnas.2412700121

Figure Lengend Snippet: Fig. 1. C-terminal glutamine residues in flagellin disable recognition by the NAIP/NLRC4 inflammasome. (A and B) WT, Nlrc4−/−, and Casp1/11−/− murine BMDMs were infected with ΔyopJ Yp expressing either YopE1-100 or YopE1-100 fused to the C-terminal D0 portion of FliC from S. Tm or EPEC for 4 h at an MOI of 20. (A) % cytotoxicity was measured via lactate dehydrogenase (LDH) release. (B) IL-1β release (ng/mL) was measured by ELISA. (C) BMDMs were infected with the indicated strains for 2 h at an MOI of 20. Combined supernatants and cellular lysates were analyzed by immunoblotting for Casp1, GSDMD, and β-actin (loading control). Representative of three independent experiments. (D and E) WT murine BMDMs were infected with the indicated strains for 4 h at an MOI of 20. (D) % cytotoxicity was measured via LDH release. (E) IL-1β release (ng/mL) was measured by ELISA. (F) BMDMs were infected with the indicated strains for 1 h at an MOI of 20. Combined supernatants and whole cell lysates were analyzed by immunoblotting for Casp1, GSDMD, and β-actin (loading control). Representative of three independent experiments. (A and D) Data shown are the pooled means ± SEM from three independent experiments. An unpaired t test was performed to assess statistical significance. (B and E) Data shown are the pooled means ± SEM from three independent experiments. Statistical significance was measured by performing an unpaired t test. ND = not detected; ns = not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The following primary antibodies were used for immunoblot analysis: 1:5,000 anti- β- Actin (Sigma clone AC- 74), 1:500 anti- Caspase- 1 (Genentech), 1:1,000 anti- GSDMD (Abcam [EPR19828]), 1:1,000 anti- mouse NLRC4 (Abcam [EPR19733]), 1:1,000 anti- human NLRC4 (Cell Signaling Technology, clone D5Y8E), 1:1,000 phospho- p38 (Invitrogen, clone C.7.8), and 1:1,000 p38 (Invitrogen, clone p38- 3F11).

Techniques: Infection, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: TLR priming licenses NAIP inflammasome activation by immunoevasive ligands.

doi: 10.1073/pnas.2412700121

Figure Lengend Snippet: Fig. 2. TLR priming licenses NAIP/NLRC4 inflammasome activation by immunoevasive NAIP ligands. (A and B) WT, Nlrc4−/−, Casp1/11−/−, and Naip5−/−murine unprimed BMDMs or primed with 0.5 μg/mL Pam3CSK4 (Pam3) for 16 h were infected with the indicated strains for 4 h at an MOI of 20. (A) Cell death (% cytotoxicity) was measured via LDH release. (B) IL-1β release (ng/mL) was measured by the ELISA. (C) BMDMs were infected with the indicated strains for 4 h at an MOI of 20, and TCA-precipitated supernatants and whole cell lysates were collected and combined. Samples were analyzed by immunoblot for Casp1, GSDMD, and β-actin (loading control). Image representative of three independent experiments. (D and E) WT murine unprimed BMDMs or primed with 0.5 μg/ mL Pam3CSK4 for 16 h were infected with the indicated strains for 2 h at an MOI of 20. (D) % cytotoxicity was measured via LDH release. (E) IL-1β release (ng/ mL) was measured by the ELISA. (F) BMDMs were infected with the indicated strains for 4 h at an MOI of 20. Combined supernatants and whole cell lysates were analyzed by immunoblotting for Casp1, GSDMD, and β-actin (loading control). Image representative of three independent experiments. (A and D) Data shown are pooled means ± SEM from three independent experiments. A paired t test was performed to assess statistical significance. (B and E) Data shown are representative of three independent experiments and are the combined means ± SEM. Statistical significance was measured by performing an unpaired t test. ns = not significant; *P < 0.05; **P < 0.01; *** P < 0.001; ****P < 0.001.

Article Snippet: The following primary antibodies were used for immunoblot analysis: 1:5,000 anti- β- Actin (Sigma clone AC- 74), 1:500 anti- Caspase- 1 (Genentech), 1:1,000 anti- GSDMD (Abcam [EPR19828]), 1:1,000 anti- mouse NLRC4 (Abcam [EPR19733]), 1:1,000 anti- human NLRC4 (Cell Signaling Technology, clone D5Y8E), 1:1,000 phospho- p38 (Invitrogen, clone C.7.8), and 1:1,000 p38 (Invitrogen, clone p38- 3F11).

Techniques: Activation Assay, Infection, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: TLR priming licenses NAIP inflammasome activation by immunoevasive ligands.

doi: 10.1073/pnas.2412700121

Figure Lengend Snippet: Fig. 3. TLR priming up-regulates NLRC4 expression via p38 MAPK signaling. (A and B) WT murine BMDMs were unprimed or primed for 16 h with (A) 0.5 μg/mL Pam3CSK4 or (B) 0.25 μg/mL, 0.5 μg/mL, or 1 μg/mL Pam3CSK4. (A) Nlrc4, Naip1, Naip2, Naip5, and Naip6 transcripts were assessed via qRT-PCR, and fold change was assessed relative to unstimulated BMDMs. (B) NLRC4 protein levels were assessed via immunoblot. Data are representative of three independent experiments. (C–E) WT murine BMDMs were unprimed or primed with 0.5 μg/mL Pam3CSK4. One hour prior to priming, the BMDMs were treated with 2.5 μM, 5 μM, or 10 μM p38 MAPK inhibitor SB202190 or Dimethyl sulfoxide (DMSO) vehicle control. (C) Nlrc4 transcript levels relative to Gapdh were assessed via qRT-PCR. Fold change was determined relative to unstimulated control. (D) NLRC4 protein levels were analyzed via immunoblot. NLRC4 protein intensity was quantified relative to loading controls, and average fold change ± SEM in NLRC4 protein levels was assessed relative to unstimulated DMSO-treated cells. Data are representative of three independent experiments. (E) % cytotoxicity was measured via LDH release 2 h postinfection with the indicated strains at an MOI of 20. (F) Immortalized ER-Hoxb8 Nlrc4−/− murine macrophages were transduced with the retroviral mscv2.2 vector alone (mscv2.2) or mscv2.2 overexpressing Nlrc4 (mscv2.2 Nlrc4). WT macrophages, Nlrc4−/− macrophages transduced with mscv2.2, or Nlrc4−/− macrophages transduced with mscv2.2 Nlrc4 were infected with indicated Yp strains expressing YopE1-100, S. Tm FliCD0, and EPEC FliCD0 for 2 h at an MOI of 20. % cytotoxicity was measured via LDH release. Only WT ER-Hoxb8 cells were primed for 16 h with Pam3CSK4. (A, C, E, and F) Data shown are representative of three experiments. Means ± SEM are shown (n = 3) and statistical significance was measured by performing an unpaired t test (A and F) or 1-way ANOVA (C–E). ND = not detected; ns = not significant; **P < 0.01; ***P < 0.001; ****P < 0.0001

Article Snippet: The following primary antibodies were used for immunoblot analysis: 1:5,000 anti- β- Actin (Sigma clone AC- 74), 1:500 anti- Caspase- 1 (Genentech), 1:1,000 anti- GSDMD (Abcam [EPR19828]), 1:1,000 anti- mouse NLRC4 (Abcam [EPR19733]), 1:1,000 anti- human NLRC4 (Cell Signaling Technology, clone D5Y8E), 1:1,000 phospho- p38 (Invitrogen, clone C.7.8), and 1:1,000 p38 (Invitrogen, clone p38- 3F11).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Transduction, Retroviral, Plasmid Preparation, Infection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: TLR priming licenses NAIP inflammasome activation by immunoevasive ligands.

doi: 10.1073/pnas.2412700121

Figure Lengend Snippet: Fig. 4. The human NAIP/NLRC4 inflammasome does not respond to TLR priming to detect evasive flagellin. (A) WT, Nlrc4−/−, or Naip−/− THP-1 macrophages were primed with 0.1 μg/mL Pam3CSK4 for 16 h and infected with WT Yp expressing either YopE1-100, S. Tm FliCD0, or EPEC FliCD0 at an MOI of 60 for 6 h. IL-1β release (ng/mL) was measured by the ELISA. (B and C) WT THP-1 macrophages were primed with 0.1 μg/mL (B and C) or 0.5 μg/mL (C) Pam3CSK4, 0.05 μg/mL Pam2CSK4, 100 ng/mL LPS, or 100 ng recombinant human IFN-γ for 16 h. (B) Nlrc4, Naip, and Il6 transcript levels relative to Hprt were assessed via qRT-PCR. Fold change in transcript levels was analyzed relative to unstimulated control. (C) NLRC4 protein levels were analyzed via immunoblot. NLRC4 protein intensity was quantified relative to loading controls, and fold change in NLRC4 protein levels was assessed relative to unstimulated cells. Data are representative of three independent experiments. (D) Primary hMDMs from three healthy human donors were primed with 0.1 μg/mL Pam3CSK4 for 16 h and infected with indicated strains at an MOI of 60 for 6 h. IL-1β release (pg/mL) was measured by the ELISA. Each dot represents the mean of each donor derived from triplicate wells. Bars depict the mean of three donors. (E) Immortalized ER-Hoxb8 Nlrc4−/− murine macrophages were transduced with the retroviral mscv2.2 vector alone or mscv2.2 overexpressing human NLRC4 (mscv2.2hNLRC4). WT, Nlrc4−/−mscv2.2, or Nlrc4−/− mscv2.2hNLRC4 macrophages were infected with indicated strains for 2 h at an MOI of 20. % cytotoxicity was measured via LDH release. Only WT Hoxb8 cells were primed for 16 h with Pam3CSK4. (F) THP-1 hMDMs transduced with the MigR1 retroviral vector alone or overexpressing hNLRC4 were primed with 0.1 μg/mL Pam3CSK4 for 16 h and infected with WT Yp expressing either YopE1-100, S. Tm FliCD0, or EPEC FliCD0 at an MOI of 60 for 6 h. IL-1β release (ng/mL) was measured by the ELISA. (A–F) Data shown are representative of three independent experiments. Means ± SEM are shown (n = 3) and statistical significance was measured by performing an unpaired t test (A, C, E, and F) or paired t test (D) or 1-way ANOVA (B). ND = not detected; ns = not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: The following primary antibodies were used for immunoblot analysis: 1:5,000 anti- β- Actin (Sigma clone AC- 74), 1:500 anti- Caspase- 1 (Genentech), 1:1,000 anti- GSDMD (Abcam [EPR19828]), 1:1,000 anti- mouse NLRC4 (Abcam [EPR19733]), 1:1,000 anti- human NLRC4 (Cell Signaling Technology, clone D5Y8E), 1:1,000 phospho- p38 (Invitrogen, clone C.7.8), and 1:1,000 p38 (Invitrogen, clone p38- 3F11).

Techniques: Infection, Expressing, Enzyme-linked Immunosorbent Assay, Recombinant, Quantitative RT-PCR, Control, Western Blot, Derivative Assay, Transduction, Retroviral, Plasmid Preparation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: TLR priming licenses NAIP inflammasome activation by immunoevasive ligands.

doi: 10.1073/pnas.2412700121

Figure Lengend Snippet: Fig. 5. TLR priming overcomes evasion of the NAIP/NLRC4 inflammasome during infection. (A) WT murine unprimed BMDMs or primed with 0.5 μg/mL Pam3CSK4 for 16 h were infected with WT, ΔfljB, or ΔfljB fliCR475QG S. Tm for 1 h at an MOI of 20. % cytotoxicity was measured via LDH release. (B) WT or Naip−/− THP-1 human macrophages were primed with 0.1 μg/mL Pam3CSK4 for 16 h and infected with WT, ΔfljB, or ΔfljB fliCR475QG S. Tm for 6 h at an MOI of 60. IL-1β release (pg/mL) was measured by the ELISA. (C and D) Total peritoneal exudate cells were harvested from WT C57Bl/6J mice intraperitoneally injected with heat-killed S. Tm (green bars) or PBS (gray bars) and then infected with the indicated Yp strains for 2 h at an MOI of 20. (C) % cytotoxicity was measured via LDH release. (D) IL-1β release (pg/mL) was measured by the ELISA. Each dot represents the mean of one mouse derived from triplicate wells. n = 7 for PBS-treated mice and n = 4 for heat-killed S. Tm-treated mice. Bars represent the means of all mice and statistical analyses were performed using a Mann–Whitney test. (E) WT C57Bl/6J mice were intraperitoneally infected with 500 CFUs of either ΔfljB or ΔfljB fliCR475QG S. Tm for 48 h. Serum IL-18 levels (pg/mL) were measured by the ELISA. Data shown are combined ± SEM from two independent experiments. An unpaired t test was performed to assess statistical significance. (A) Data shown are means ± SEM pooled from three independent experiments, and a paired t test was performed to assess statistical significance. (B) Data shown are representative of one out of at least three independent experiments. Means ± SEM are shown (n = 3), and statistical significance was measured by performing an unpaired t test. ns = not significant; *P < 0.05; **P < 0.01.

Article Snippet: The following primary antibodies were used for immunoblot analysis: 1:5,000 anti- β- Actin (Sigma clone AC- 74), 1:500 anti- Caspase- 1 (Genentech), 1:1,000 anti- GSDMD (Abcam [EPR19828]), 1:1,000 anti- mouse NLRC4 (Abcam [EPR19733]), 1:1,000 anti- human NLRC4 (Cell Signaling Technology, clone D5Y8E), 1:1,000 phospho- p38 (Invitrogen, clone C.7.8), and 1:1,000 p38 (Invitrogen, clone p38- 3F11).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Injection, Derivative Assay, MANN-WHITNEY

Journal: European journal of immunology

Article Title: Atg16l2 augments Nlrc4 inflammasome activation by facilitating NAIPs-NLRC4 association.

doi: 10.1002/eji.202451078

Figure Lengend Snippet: Figure 1. ATG16L2 deficiency impairs NLRC4 inflammasome activation. (A, B) WT and ATG16L2-KO BMDMs were pretreated with LPS (500 ng/mL) for 4 h and then transfected with flagellin (1 μg/mL). Cell lysates and culture supernatants (Sup) were collected and immunoblotted with the indicated antibodies. ELISA of IL-1β in cell-free supernatants. (C, D) WT and ATG16L2-KO BMDMs were pretreated with LPS (500 ng/mL) for 4 h, followed by infection with STM at an MOI of 20 for 2 h. Cell lysates and culture supernatants were collected and immunoblotted with the indicated antibodies. ELISA of IL-1β in cell-free supernatants. (E) ELISA of IL-6 (H) and LDH in cell-free supernatants from WT and ATG16L2-KO BMDMs that were treated as stated in (A) or (C). Data are presented as means ± SEM; ***p < 0.001 (Student’s t-test) all conditions were performed in technical triplicate. In each panel, data are representative of at least three independent experiments.

Article Snippet: Caspase-11(14340), anti-rat IgG-HRP-linked Antibody (7077), anti-Myc-Tag (9B11) and anti-NLRC4 (D5Y8E) antibodies were purchased from Cell Signaling Technology.

Techniques: Activation Assay, Transfection, Enzyme-linked Immunosorbent Assay, Infection

Journal: European journal of immunology

Article Title: Atg16l2 augments Nlrc4 inflammasome activation by facilitating NAIPs-NLRC4 association.

doi: 10.1002/eji.202451078

Figure Lengend Snippet: Figure 2. ATG16L2 is required for the consummate activation of the NLRC4 inflammasome in iBMDMs and Raw264.7. (A, B) ATG16L2-KO iBMDMs were generated through transfection with lentivirus carrying ATG16L2-SH plasmids (C, D) Cells were primed with LPS and transfected with 1 μg/mL flagellin for 6 h or infected with STM at an MOI of 20 for 2 h; cell lysate and culture supernatants were collected and immunoblotted with the indicated antibodies. ELISA of IL-1β and (E) LDH release from the cell-free supernatants from iBMDMs that were treated as stated in (A) or (C). ATG16L2-knockdown Raw264.7 cells were generated through transfection with lentivirus carrying ATG16L2-SH plasmids. These cells were primed with LPS followed by transfected with 1 μg/mL flagellin for 6 h (F) or infected with STM at an MOI of 20 for 2 h (G). Cell lysates were collected and immunoblotted with the indicated antibodies. Data are presented as means ± SEM; ***p < 0.001 (Student’s t-test). All the results are representative of at least three independent experiments. Each condition was performed in technical triplicate.

Article Snippet: Caspase-11(14340), anti-rat IgG-HRP-linked Antibody (7077), anti-Myc-Tag (9B11) and anti-NLRC4 (D5Y8E) antibodies were purchased from Cell Signaling Technology.

Techniques: Activation Assay, Generated, Transfection, Infection, Enzyme-linked Immunosorbent Assay, Knockdown

Journal: European journal of immunology

Article Title: Atg16l2 augments Nlrc4 inflammasome activation by facilitating NAIPs-NLRC4 association.

doi: 10.1002/eji.202451078

Figure Lengend Snippet: Figure 3. ATG16L2 deficiency attenuates ASC speck formation upon NLRC4 inflammasome activation. (A) LPS-primed WT and ATG16L2-knockdown BMDMs were either transfected in the cytosol with 1 μg/mL flagellin or infected with STM at an MOI of 20. Endogenous ASC specks (arrows) were visualised and quantified by immunofluorescence images (DAPI/blue, ASC/red). The data show representative results from three independent experiments: scale bar = 20 μm. (B) WT and ATG16L2 BMDMs following cytosolic delivery of 1 μg/mL flagellin or infection with STM at an MOI of 20 for 2 h. Cells were dissolved with Triton X-100-containing buffer followed by cross-linkage of insoluble fractions with DSS to capture ASC oligomers. Immunoblots of the insoluble fractions (insoluble + DSS) and soluble fractions were detected with an antibody to ASC. Data are presented as the mean ± SEM; determined using Student’s t-test; ***p < 0.001. All results are representative of at least three independent experiments.

Article Snippet: Caspase-11(14340), anti-rat IgG-HRP-linked Antibody (7077), anti-Myc-Tag (9B11) and anti-NLRC4 (D5Y8E) antibodies were purchased from Cell Signaling Technology.

Techniques: Activation Assay, Knockdown, Transfection, Infection, Immunofluorescence, Western Blot

Journal: European journal of immunology

Article Title: Atg16l2 augments Nlrc4 inflammasome activation by facilitating NAIPs-NLRC4 association.

doi: 10.1002/eji.202451078

Figure Lengend Snippet: Figure 4. ATG16L2 interacts with NAIPs and enhances NAIPs–NLRC4 complex formation. (A–C) Flag-ATG16L2 was co-expressed with Myc-NLRC4, HA-Naip2 or HA-Naip5 in HEK293T cells, respectively. The proteins were then immunoprecipitated and analysed via immunoblotting using the indicated antibodies. Whole-cell lysates were used as the input. (D, E) HEK293T cells were transfected with Myc-NLRC4 and Flag-ATG16L2, along with Myc-PrgJ and HA-Naip2 (D) or Myc-Flagellin and HA-Naip5 (E).

Article Snippet: Caspase-11(14340), anti-rat IgG-HRP-linked Antibody (7077), anti-Myc-Tag (9B11) and anti-NLRC4 (D5Y8E) antibodies were purchased from Cell Signaling Technology.

Techniques: Immunoprecipitation, Western Blot, Transfection