Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Overexpression of NLRP12 enhances macrophage immune response and alleviates herpes simplex keratitis

doi: 10.3389/fcimb.2024.1416105

Figure Lengend Snippet: NLRP12 mediates pyroptosis and promotes IL-18 following HSV-1 infection. LV-Ctrl and LV-NLRP12 macrophages were either mock or HSV-1 infected and assessed at specified time points. (A) Proteins within the pyroptosis pathway were detected after mock infection and 4h and 8h post-infection. (B) Western blot bands were normalized to β-actin and compared against controls (n=3 per group). (C) Immunofluorescence images showing NLRP12 (green), ASC (red) and their co-localization (yellow) post mock infection and at 8h post-infection. (D) LV-NLRP12 cells were treated with caspase-1 inhibitor VX-765 to inhibit pyroptosis and IL-18 binding protein (IL-18BP) to block active IL-18. Protein levels of HSV-gB and HSV-gD were measured at 8h post-infection. All of the data are representative of at least three independent experiments. Data are presented as the mean ± SEM. Statistical differences were determined using two-way ANOVA (B) . ns, not significnat, *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001.

Article Snippet: RAW264.7 cells were pre-treated with 20 μM VX-765 (T6090, TargetMol, China) to inhibit pyroptosis and with 50 ng/ml IL-18 binding protein (IL-18BP) (HY-P75841, MCE, China) to inhibit IL-18 activity.

Techniques: Infection, Western Blot, Immunofluorescence, Binding Assay, Blocking Assay

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Overexpression of NLRP12 enhances macrophage immune response and alleviates herpes simplex keratitis

doi: 10.3389/fcimb.2024.1416105

Figure Lengend Snippet: NLRP12 enhances antiviral functions through IL-18 downstream JAK-STAT signaling. (A) Protein levels of STAT1, p-STAT1, STAT4, pSTAT4 were assessed at 8h, 12h and 24h post-infection in both groups. Western blot bands were normalized to β-actin and compared against controls (n=3 per group). (B) Immunofluorescence images demonstrating MHC II + cell staining. All of the data are representative of at least three independent experiments. Data are presented as the mean ± SEM. Statistical differences were determined using two-way ANOVA (A) . ns, not significnat, *P <0.05, ***P <0.001, ****P <0.0001.

Article Snippet: RAW264.7 cells were pre-treated with 20 μM VX-765 (T6090, TargetMol, China) to inhibit pyroptosis and with 50 ng/ml IL-18 binding protein (IL-18BP) (HY-P75841, MCE, China) to inhibit IL-18 activity.

Techniques: Infection, Western Blot, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Microglia-derived IL-18 remodels hippocampal plasticity to constrain traumatic fear memory

doi: 10.64898/2026.05.04.721266

Figure Lengend Snippet: a , Schematic of PTSD model establishment. The SPS&S (Single Prolonged Stress & Shock) procedure, consisting of sequential restraint, forced swim, anesthesia, and foot shock. b–c , Contextual fear freezing in SPS&S-exposed and control mice at day 8 ( b ) and day 15 ( c ) post-stress. **P < 0.01, unpaired two-tailed t-test; n = 10 mice per group. d–e , Representative images showing IL-18-GFP fluorescence in sagittal brain sections from IL-18-GFP reporter mice at day 3 post-SPS&S. Images show whole-brain overview with magnified insets of the hippocampus and medial prefrontal cortex (mPFC) ( d ), and basolateral amygdala (BLA). IL-18-GFP specks are represented by green dots generated using IMARIS software. ( e ), scale bars,1mm (overview), 200 μm (inset, hippocampus), 100 μm (inset, mPFC and BLA). f , Quantification of mean IL-18-GFP fluorescent intensity across hippocampus, mPFC, and BLA in control and SPS&S groups. **P < 0.01, ns, not significant; two-way ANOVA with Bonferroni’s post hoc test; n = 3 mice per group. g , Schematic of the IL-18 luciferase reporter assay for quantifying IL-18 bioactivity in dissected brain regions. h , Luciferase activity in hippocampus, mPFC, and BLA from control and SPS&S mice. **P < 0.01, ns, not significant; two-way ANOVA with Bonferroni’s post hoc test; n = 3 per group. i , Schematic of hippocampal tissue collection and processing for IL-18 mRNA quantification by qPCR and protein quantification by ELISA at multiple post-stress time points. n = 3 mice per group. j , Temporal profile of hippocampal IL-18 mRNA levels from 0 to 15 days post-SPS&S. *P < 0.05, **P < 0.01 vs. 0 h; one-way ANOVA with Dunnett’s post hoc test; n = 7-11 per time point. k , Temporal profile of hippocampal mature IL-18 protein levels (normalized to total protein) from 0 to 15 days post-SPS&S. **P < 0.01 vs. 0 h; one-way ANOVA with Dunnett’s post hoc test; n = 7-11 per time point. l , Experimental timeline for intrahippocampal cannula implantation, SPS&S exposure, and daily PBS or IL-18 binding protein (IL-18BP) infusion, with fear memory assessed at days 8 and 15 post-stress. IL-18BP infusion began on day −3 to allow accumulation of antagonist before stress onset, ensuring complete neutralization of stress-induced IL-18. m–n , Contextual fear freezing at day 8 ( m ) and day 15 ( n ) in mice receiving intrahippocampal IL-18BP or PBS following SPS&S. *P < 0.05, **P < 0.01, ***P < 0.001; two-way ANOVA with Bonferroni’s post hoc test; n = 5-9 per group. o , Experimental timeline for intrahippocampal cannula implantation, SPS&S exposure, and daily IL18 infusion, with fear memory assessed at days 8 and 15 post-stress. p–q , Contextual fear freezing at day 8 ( p ) and day 15 (q ) in mice receiving intrahippocampal IL18 or PBS following SPS&S. *P < 0.05, **P < 0.01, ***P < 0.001; two-way ANOVA with Bonferroni’s post hoc test; n = 6–10 per group. Complete statistics are provided in Supplementary Table 1.

Article Snippet: IL-18 binding protein (IL-18BP; MCE) was dissolved in sterile PBS and stored at −80°C.

Techniques: Control, Two Tailed Test, Fluorescence, Generated, Software, Luciferase, Reporter Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Binding Assay, Neutralization

Journal: bioRxiv

Article Title: Microglia-derived IL-18 remodels hippocampal plasticity to constrain traumatic fear memory

doi: 10.64898/2026.05.04.721266

Figure Lengend Snippet: a, Representative confocal images of hippocampal sections from IL-18-GFP reporter mice at day 3 post-SPS&S. Scale bars, 10 μm. b, Experimental timeline for bilateral hippocampal AAV injection (day −21), SPS&S exposure (day 1), and contextual fear memory assessment (days 8 and 15). c, Contextual fear freezing at day 8 in mice receiving cell-type-specific AAV-shRNA targeting IL-18 in microglia (Iba1-shIL18), astrocytes (GFAP-shIL18), neurons (hSyn-shIL18 and CaMKII-shIL18), or scramble control (EF1-shCon), following SPS&S or control conditions. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant Two-way ANOVA with post-hoc Bonferroni correction, n=7-11 mice per group. d, Same as c at day 15. e, Representative confocal images of hippocampal sections stained for IL-18R1. Scale bars, 10 μm. f, Experimental timeline for bilateral hippocampal AAV injection (day −21), SPS&S exposure (day 1), and contextual fear memory assessment (days 8 and 15) for IL-18R1 knockdown experiments. g, Contextual fear freezing at day 8 in mice receiving cell-type-specific AAV-shRNA targeting IL-18R1 in microglia (Iba1-shIL18R1), astrocytes (GFAP-shIL18R1), or neurons (hSyn-shIL18R1), or scramble control (EF1-shCon), following SPS&S or control conditions. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant Two-way ANOVA with post-hoc Bonferroni correction, n=8-13 mice per group. h, Same as g at day 15. Complete statistics are provided in Supplementary Table 1.

Article Snippet: IL-18 binding protein (IL-18BP; MCE) was dissolved in sterile PBS and stored at −80°C.

Techniques: Injection, shRNA, Control, Staining, Knockdown

Journal: bioRxiv

Article Title: Microglia-derived IL-18 remodels hippocampal plasticity to constrain traumatic fear memory

doi: 10.64898/2026.05.04.721266

Figure Lengend Snippet: a, Experimental timeline for infusion tube installation (day −14), SPS&S exposure (day 1), daily intrahippocampal IL-18 or PBS infusion, and tissue collection for immunostaining at day 15. b, Representative confocal images of synaptophysin (yellow) immunostaining in hippocampal CA1 from PBS- and IL-18-treated mice under control and SPS&S conditions. Scale bars, 10 μm. c, Quantification of percent area synaptophysin staining across groups. ***P < 0.001, *P < 0.05, Two-way ANOVA with post-hoc Holm-Šídák, n=9-11 fields of view (FOVs) per group, from 3 mice per group. d, Quantification of normalized synaptophysin⁺ puncta density. Two-way ANOVA with post-hoc Holm-Šídák, n=9-11 fields of view (FOVs) per group, from 3 mice per group. e, Representative confocal images of VGLUT1 (green), Homer1 (red), and DAPI (blue) co-immunostaining in hippocampal CA1 from PBS- and IL-18-treated mice under control and SPS&S conditions. Scale bars, 10 μm. f, Quantification of normalized VGLUT1–Homer1 co-localization puncta. ***P < 0.001, Two-way ANOVA with post-hoc Bonferroni correction, n=18-21 fields of view (FOVs) per group, from 3 mice per group. g, Quantification of normalized VGLUT1⁺ puncta density. ***P < 0.001, Two-way ANOVA with post-hoc Bonferroni correction, n=18-21 fields of view (FOVs) per group, from 3 mice per group. h, Representative confocal images of WFA (yellow) and DAPI (blue) staining in hippocampal CA1 from PBS- and IL-18-treated mice under control and SPS&S conditions. Scale bars, 10 μm. i, Quantification of percent area WFA staining. **P < 0.01, *P < 0.05, Two-way ANOVA with post-hoc Bonferroni correction, n=6-13 fields of view (FOVs) per group, from 3 mice per group. Data are presented as mean ± SEM. Complete statistics are provided in Supplementary Table 1.

Article Snippet: IL-18 binding protein (IL-18BP; MCE) was dissolved in sterile PBS and stored at −80°C.

Techniques: Immunostaining, Control, Staining

Journal: bioRxiv

Article Title: Microglia-derived IL-18 remodels hippocampal plasticity to constrain traumatic fear memory

doi: 10.64898/2026.05.04.721266

Figure Lengend Snippet: a, Schematic of the TRAP2 engram-labeling system. AAV9-DIO-GFP was locally injected into the hippocampus of Fos-CreER T2 mice, enabling activity-dependent fluorescent labeling of SPS&S-activated neurons upon 4-OHT administration. b, Experimental timeline. DIO-GFP AAV was injected at day −14; mice received intrahippocampal IL-18 or PBS infusion 0.5h before SPS&S at day 1 and daily after, followed by intraperitoneal 4-OHT injection 1 hour later to complete engram labeling at day1. Brains were collected at day 15 for immunostaining. c, Representative confocal images of GFP (green, engram cells) and DAPI (blue) fluorescence in hippocampal sections from control and SPS&S-exposed mice under PBS or IL-18 treatment at day 15. Scale bars, 200 μm. d, Quantification of number of eGFP⁺ cells in hippocampus. ***P < 0.001, Two-way ANOVA with post-hoc Bonferroni correction, n=13 fields of view (FOVs) per group, from 3 mice per group. e, Schematic illustrating the synaptic markers (VGLUT1, Homer1, synaptophysin) analyzed within GFP⁺ engram cells. f, Representative confocal images of synaptophysin (yellow), DAPI (blue), and GFP (engram cell, white) immunostaining in hippocampal engram cells from PBS- and IL-18-treated mice under SPS&S conditions, scale bars, 10 μm. The magnified insets. Scale bars, 5 μm. g, Quantification of synaptophysin⁺ puncta contacts in engram cells per unit area (log scale). ns, not significant; unpaired two-tailed t-test; n=9 fields of view (FOVs) per group, from 3 mice per group. The extremely low baseline of engram cells in the control group precluded reliable quantification and thus precluded valid statistical comparisons. h, Representative confocal images of VGLUT1 (green), Homer1 (red), DAPI (blue), and GFP (engram cell, white) immunostaining within hippocampal engram cells from PBS- and IL-18-treated under control and SPS&S conditions, scale bars, 10 μm. The magnified insets. Scale bars, 5 μm. i, Quantification of VGLUT1⁺/Homer1⁺ co-localized puncta contacts in engram cells per unit area (log scale). ***P < 0.001, Unpaired Student’s t-test, n=14 fields of view (FOVs) per group, from 3 mice per group. j, Quantification of VGLUT1⁺ puncta contacts in engram cells per unit area (log scale). ***P < 0.001, Unpaired t-test with Welch’s correction, n=14 fields of view (FOVs) per group, from 3 mice per group. Extremely low baseline engram counts in the control group in panels i and j preclude reliable quantification (see also panel g ). Data are presented as mean ± SEM. Complete statistics are provided in Supplementary Table 1.

Article Snippet: IL-18 binding protein (IL-18BP; MCE) was dissolved in sterile PBS and stored at −80°C.

Techniques: Labeling, Injection, Activity Assay, Immunostaining, Fluorescence, Control, Two Tailed Test