Journal: Journal of Neuroinflammation

Article Title: STING activates ZBP1-mediated PANoptosis to defend against HSV-1 retinal infection

doi: 10.1186/s12974-025-03595-0

Figure Lengend Snippet: IVT HSV-1 injection induces acute retinitis and retinal inflammation. A Schematic diagram of HSV-1 injection. B paFundus photograph obtained 3 days post‑infection (dpi) demonstrating retinal lesions. Upper nels: fundus imaging reveals yellow-white lesions (arrows). Middle and lower panels: fluorescein angiography determines vascular permeability (arrowheads) (n=6 mice/group). C IF analysis of retinal whole mounts. Top: representative bright-field micrograph showing hemorrhagic foci (arrows), scale bar: 2 mm. Middle: IF staining of retinal vasculature (IB4, red) and red blood cells (TER119, green); scale bar: 200 μm. Bottom: High-magnification of boxed regions; scale bar: 20 μm; n=4 eyes/group. D OCT images showing immune cell infiltration in the vitreous cavity (n=4 mice/group). E Slit-lamp examination and HE staining demonstrate HSV-1 dose-dependent retinal damage. Upper panels: Slit-lamp examination reveals increased corneal opacity with higher HSV-1 dosage at 3 dpi. Synechiae were observed in both low and high virus dosage groups, (n=6 mice/group). Lower panels: HE staining of mouse eyes. Enlarged views show the peripheral, central, and optic nerve regions. Immune cell infiltration, retinal folds, and iritis were observed, with severity increasing at higher viral doses. Note no retinal detachments were seen in controls (PBS injected) (0/16 eyes). In eyes infected with 4x10^4 PFU virus, 13/14 (93%) showed detachment at 3 dpi and 14/14 (100%) at 9 dpi. Scale bars: overview 500 μm; magnified 100 μm, n=5 eyes/group. F Histopathological scoring of retinal damage. G HE stained sections at 40× and 63× original magnification. The viral inclusion bodies are indicated by white arrows. Scale bars: Scale bars: 200 μm (40×), 100 μm (63×); magnified 5 μm, n=5 eyes/group. H qPCR detection of HSV-1 DNA in retina, each dot represents a value from one retina. I Vitreous humor viral titers measured by plaque assay. Left: representative plaque images; right: quantification (PFU/mL).Each data point represents one pooled sample (3–4 eyes); n = 3–5 mice per group. J - M IF analysis of indicated proteins in retinal cryosections. Nuclei were counterstained with DAPI. Scale bars: 100 μm. Each dot represents a value from one capture fled. J IBA1+ microglia/macrophages. Right panel: quantification of microglia/macrophages number per captured field. GCL: ganglion cell layer, IPL: inner plexiform layer, INL: inner nuclear layer, OPL: outer plexiform layer, ONL: outer nuclear layer. K GFAP + Müller cell and astrocytes. Right panel: qualification of GFAP intensity per captured field. L Brn3a+ retinal ganglion cells (RGCs). Right panel: quantification of RGC cell number per captured field. M PKCα + bipolar cells. Data are presented as mean ± SD. Statistical significance for panels J–M was determined using unpaired Student’s t-test, and for panels F, H, and I using two-way ANOVA with Tukey’s post hoc test. (ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001)

Article Snippet: For the mock infection, HSV-1 was first inactivated by incubating it at 100 °C for 1 h. For combination treatment, 10 mg of CBL0137 (Topscience) or quinacrine (MedChemExpress, USA) was dissolved in 5 mL of PBS via ultrasonication (37 °C, 1 min) and mixed with HSV-1 to achieve a final concentration of 2 mg/mL CBL0137 or quinacrine and 4 × 104 PFU/mL HSV-1.

Techniques: Injection, Imaging, Permeability, Staining, Virus, Infection, Plaque Assay

Journal: Journal of Neuroinflammation

Article Title: STING activates ZBP1-mediated PANoptosis to defend against HSV-1 retinal infection

doi: 10.1186/s12974-025-03595-0

Figure Lengend Snippet: STING deficiency exacerbates retinal necrosis and increases viral burden WT and STING-/- mice were IVT injected with PBS (CTRL) or 4×104 PFU HSV-1 and analyzed at 3 and 9 dpi. A Slit-lamp examination showing corneal ulcers at 9 dpi in STING-/- mice, while WT mice exhibit moderate opacity; n=6 eyes/group; representative images from 2 independent experiments. B HE staining showing eye morphology. In STING-/- mice, severe retinal folding was observed at 3 dpi, and at 9 dpi, extensive retinal necrosis and complete loss of retinal lamination were detected. Scale bars: overview 500 μm; magnified 100 μm; n=5 eyes/group. C Histopathological scoring of retinal damage, n=5 eyes/group. D Vitreous humor viral titers measured by plaque assay. Left: representative plaque images; right: quantification (PFU/mL). Each data point represents one pooled sample (3–4 eyes), n = 3–5 mice per group. E IF analysis showing HSV-1 in mouse retinas. Virus were labeled by anti-HSV1 antibody, and nuclei were counterstained by DAPI. Scale bars: overview 100 μm; magnified 10 μm. F qPCR analysis of retinal HSV-1 DNA copy numbers. Each dot represents a value from one retina. G Representative three-dimensional images of retinal whole mounts showing HSV-1 (gB; magenta) distribution. Confocal z-stack images were captured using Zen 2.3 SP1 software (71 μm z-stack depth); scale bar: 50 μm; n=3 eyes/group. H - L IF analysis of indicated proteins in retinal cryosections. HSV-1 was labeled by anti-glycoprotein B (gB), nuclei were counterstained with DAPI. Scale bars: Top 100 μm; middle 10 μm; bottom 5 μm, n=3 eyes/group. H Left: Isotype IgG negative control co-stained with IBA1 in STING−/− retinas, demonstrating the specificity of HSV-1 labeling. Right: Co-staining of HSV-1 with IBA1+ microglia/macrophages. I HSV-1 co-staining with Brn3a+ retinal ganglion cells. J HSV-1 co-staining with PKCα+ bipolar cells. K HSV-1 co-staining with GFAP+ Müller glia/astrocytes. L HSV-1 co-staining with Tfap2α+ Amacrine cells. Data are presented as mean ± SD. Statistical significance for panels (C, D, and F) was determined using two-way ANOVA followed by Tukey’s post hoc test. (ns, not significant; *p < 0.05; ***p < 0.001; ****p < 0.0001)

Article Snippet: For the mock infection, HSV-1 was first inactivated by incubating it at 100 °C for 1 h. For combination treatment, 10 mg of CBL0137 (Topscience) or quinacrine (MedChemExpress, USA) was dissolved in 5 mL of PBS via ultrasonication (37 °C, 1 min) and mixed with HSV-1 to achieve a final concentration of 2 mg/mL CBL0137 or quinacrine and 4 × 104 PFU/mL HSV-1.

Techniques: Injection, Staining, Plaque Assay, Virus, Labeling, Software, Negative Control

Journal: Journal of Neuroinflammation

Article Title: STING activates ZBP1-mediated PANoptosis to defend against HSV-1 retinal infection

doi: 10.1186/s12974-025-03595-0

Figure Lengend Snippet: STING depletion attenuates multiple anti-virus signaling pathways upon HSV-1 retinal infection. A - D RNA-seq analysis was performed in WT-CTRL (n=5), STING-/--CTRL (n=5), WT-HSV1(n=4), STING-/--HSV1(n=4) retinas at 3 dpi (4x10^4 PFU). Gene set enrichment analysis (GSEA) showing HSV-1 infection pathway, False discovery rate (FDR) <0.05. B Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showing STING-dependent downregulated genes. FDR<0.05, log2FC<(−1). C Gene ontology (GO) analysis of biological process (BP) showing STING-dependent downregulated genes, FDR<0.05, log2FC<(−1). D Heatmap analysis of the top 10 deferentially expressed genes ranked by descending log2 Fold Change in the WT-HSV-1 group within the indicated signaling pathways. E - H Flow cytometry analysis of retinal immune populations at 3, 6 and 9 dpi (4x10^4 PFU); n=4 mice/group. E Representative flow cytometry plots depict myeloid (CD11b+) and leukocytes (CD45+). F Dot plots show the percentage of resident microglia (CD11b+CD45low) shown in (E), each dot represents one mouse. G Dot plots show the percentage of infiltrating monocytes/macrophages (CD11b+CD45high) shown in (E), each dot represents one mouse. H Dot plots show the percentage of infiltrating leukocytes (CD11blowCD45high) shown in (E), each dot represents one mouse. Data are presented as mean ± SD. Statistical analysis: panels A–C, significance thresholds for GSEA/KEGG/GO as indicated; panels F–H, two-way ANOVA with Tukey’s post hoc test (ns, not significant; ****p < 0.0001)

Article Snippet: For the mock infection, HSV-1 was first inactivated by incubating it at 100 °C for 1 h. For combination treatment, 10 mg of CBL0137 (Topscience) or quinacrine (MedChemExpress, USA) was dissolved in 5 mL of PBS via ultrasonication (37 °C, 1 min) and mixed with HSV-1 to achieve a final concentration of 2 mg/mL CBL0137 or quinacrine and 4 × 104 PFU/mL HSV-1.

Techniques: Virus, Protein-Protein interactions, Infection, RNA Sequencing, Flow Cytometry

Journal: Journal of Neuroinflammation

Article Title: STING activates ZBP1-mediated PANoptosis to defend against HSV-1 retinal infection

doi: 10.1186/s12974-025-03595-0

Figure Lengend Snippet: STING is required for PANoptosis activation following HSV-1 retinal infection. WT and STING-/- mice were IVT injected with PBS (CTRL) or 4×104 PFU HSV-1 and analyzed at 3 dpi. A GSEA of indicated pathways, FDR <0.05. B - D WB showing representative protein expression data from three independent experiments. E IF analysis of indicated proteins and TUNEL staining in retinal cryosections. Overlap of IBA1+ microglia/macrophages and TUNEL+ dead cells was observed in both the vitreous cavity (a) and retina (b) and (c) of WT mice. Negative control for TUNEL staining: HSV-1–infected wild-type retinal sections processed without terminal deoxynucleotidyl transferase (TdT) to assess background/nonspecific labeling. Scale bars: 100 μm and 5 μm. F Quantification of indicated cell numbers. Each dot represents one captured field, n = 3–4 eyes/group. G IF analysis of macrophages/microglia (IBA1+) and HSV-1 in WT retinal flat mounts. Scale bars: 10 μm. H WB analysis of indicated proteins in mouse retinas. I - K IF staining of macrophages/microglia (IBA1+) and Pyroptosis (I), Apoptosis (J) and Necroptosis (K) marker proteins in retinal flat mounts. Right panel: Quantification of indicated cell numbers and PANoptosis marker intensity. Each data point represents one captured field (n = 3–4 eyes/group). Scale bars: overview, 100 μm; magnified, 20 μm. L Isotype IgG negative control co-incubated with anti-IBA1 antibody, scale bar: 100 μm, n=3 eyes/group. Results are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.0005, **** P < 0.0001, ns: not significant; Figure F,two-way ANOVA, Figure I-K, unpaired t-test

Article Snippet: For the mock infection, HSV-1 was first inactivated by incubating it at 100 °C for 1 h. For combination treatment, 10 mg of CBL0137 (Topscience) or quinacrine (MedChemExpress, USA) was dissolved in 5 mL of PBS via ultrasonication (37 °C, 1 min) and mixed with HSV-1 to achieve a final concentration of 2 mg/mL CBL0137 or quinacrine and 4 × 104 PFU/mL HSV-1.

Techniques: Activation Assay, Infection, Injection, Expressing, TUNEL Assay, Staining, Negative Control, Labeling, Marker, Incubation

Journal: Journal of Neuroinflammation

Article Title: STING activates ZBP1-mediated PANoptosis to defend against HSV-1 retinal infection

doi: 10.1186/s12974-025-03595-0

Figure Lengend Snippet: STNG promotes Z-NA accumulation and ZBP1-mediated PANoptosis. A Venn diagram showing shared regulatory genes in the indicated pathways. Gene list derived from RNA-seq data in Figure . B RNA-seq Transcripts per million (TPM) values showing the expression of selected RNAs. ZBP1 is the most significantly upregulated gene involved in PANoptosome formation, comparing CTRL and HSV-1-infected groups in WT retinas. C WB shows indicated protein levels in mouse retinas. Mice were IVT injected with 4x10^4 PFU HSV-1 and analyzed at 3 dpi. D IF of primary microglia (PMs) infected with HSV-1 (multiplicity of infection (MOI) = 10, 6 h) showing HSV-1 gB (red) and Z-NA (purple). Scale bars: 100 μm. n = 4 independent cultures. E Left: Percentage of HSV-1-positive cells. Right: Percentage of Z-NA-positive cells. Each dot represents an independent experiment.Left: Percentage of HSV-1-positive cells. Right: Percentage of Z-NA-positive cells. Each dot represents an independent experiment. F - I BV2 Cells were treated without or with STING agonist DMXAA (20 μg/mL) in combination with PBS, or DNase I (10 μg/mL), or RNase A (100 U/mL) for 6 h before analysis. F IF analysis showing Z-NA and ZBP1. Scale bars: 5 μm. G Quantification of Z-NA specks per cell. Each dot represents a value from randomly selected cells in two independent experiments. H Propidium iodide (PI) staining shows dead cells under the indicated treatment. Scale bar: 50 μm. I Percentage of dead cells. Each dot represents an independent experiment. J ScRNA-seq showing ISG genes with A-I high editing potential in retinal microglia. K IF analysis of STING, ZBP1 and Z-NA in BV2 cells. Cells were infected without or with HSV-1 ((MOI=10) and analyzed 2 h post-infection. Scale bars: 5 μm L Intensity profiles in each channel along the white line in the merged image. M Quantification of STING-Z-NA-ZBP1 speck area. Each dot represents a value from a randomly selected speck in two independent experiments. Results are presented as mean ± SD. Statistical analysis: panels (B, E, G, I, M), two-way ANOVA followed by Tukey’s post hoc test. (*p < 0.05; **p < 0.01; ****p < 0.0001)

Article Snippet: For the mock infection, HSV-1 was first inactivated by incubating it at 100 °C for 1 h. For combination treatment, 10 mg of CBL0137 (Topscience) or quinacrine (MedChemExpress, USA) was dissolved in 5 mL of PBS via ultrasonication (37 °C, 1 min) and mixed with HSV-1 to achieve a final concentration of 2 mg/mL CBL0137 or quinacrine and 4 × 104 PFU/mL HSV-1.

Techniques: Derivative Assay, RNA Sequencing, Expressing, Infection, Injection, Staining

Journal: Journal of Neuroinflammation

Article Title: STING activates ZBP1-mediated PANoptosis to defend against HSV-1 retinal infection

doi: 10.1186/s12974-025-03595-0

Figure Lengend Snippet: ZBP1 deficiency exacerbates HSV-1 retinal pathogenesis and reduced PANoptosis. A – D WT and ZBP1-/- mice received IVT injections of PBS (CTRL) or HSV-1 (4X10^3 PFU) and were analyzed at 3 dpi (n = 6 mice/group). A Fundus photography (top) and fluorescein angiography (bottom) showing eye morphology. B HE staining of retinal sections. Scale bars: overview 500 μm; magnified 100 μm. C Histological scoring of HE-stained retinal tissues (n = 5 eyes/group). D OCT analysis showing hyperreflective inflammatory cells within the vitreous cavity following infection, and disruption of retinal structure was evident in ZBP1-/- retina. E – M WT and ZBP1-/- mice received IVT injections of PBS (CTRL) or HSV-1 (4X10^4 PFU) and were analyzed at 3 dpi (n = 6 mice/group). E Slit-lamp examination (top) and HE staining (bottom). Scale bars: overview 500 μm; magnified 100 μm. F Histological scoring of HE-stained retinal tissues (n = 5 eyes/group). G Plaque assay of viral titers in vitreous humor. Left: representative plaque formation; right: quantification, each data point represents one pooled sample (2–3 eyes), n = 3–6 mice per group. H qPCR quantification of retinal HSV-1 DNA copies (n = 3 retinas/group).I. BMDM cell death analysis. Left: PI staining (dead cells, red). Scale bar: 50 μm. Right: quantification of PI⁺ cells (% total cells; n = 4 independent experiments). J – L WB analysis of PANoptosis-associated proteins in mouse retinas. Representative blots from three independent biological replicates (n = 3 retinas/group). J Pyroptosis markers: caspase-1/cleaved caspase-1, GSDMD/cleaved GSDMD. K Apoptosis markers: caspase-3/cleaved caspase-3, caspase-7/cleaved caspase-7. L Necroptosis markers: ZBP1, p-MLKL, MLKL.M. WB analysis of cGAS-STING pathway proteins and ZBP1. Representative of three independent experiments. Data are presented as mean ± SD. Statistical significance for panels (C, F–I) was determined using two-way ANOVA followed by Tukey’s post hoc test; ns, not significant; ***p < 0.001; ****p < 0.0001

Article Snippet: For the mock infection, HSV-1 was first inactivated by incubating it at 100 °C for 1 h. For combination treatment, 10 mg of CBL0137 (Topscience) or quinacrine (MedChemExpress, USA) was dissolved in 5 mL of PBS via ultrasonication (37 °C, 1 min) and mixed with HSV-1 to achieve a final concentration of 2 mg/mL CBL0137 or quinacrine and 4 × 104 PFU/mL HSV-1.

Techniques: Staining, Infection, Disruption, Plaque Assay

Journal: Journal of Neuroinflammation

Article Title: STING activates ZBP1-mediated PANoptosis to defend against HSV-1 retinal infection

doi: 10.1186/s12974-025-03595-0

Figure Lengend Snippet: CBL0137 protects against HSV-1 retinal infection in both WT and STING-/- mice. A CBL0137 (CBL) reduces HSV-1 replication in BMDMs. BMDMs were either mock-infected or infected with HSV-1 (MOI = 10) and treated with or without CBL (1 μM) 2 h post-infection. Virus DNA was measured by qPCR 6 h after infection. Each point represents an independent experiment. B Plaque assay demonstrating that CBL (1 μM) decreased viral titers in both WT and STING-/-BMDMs. Left: representative plaque formation in Vero cells; right: quantification (PFU/mL; n = 3 independent experiments). C CBL induced BMDM death during HSV-1 infection. BMDMs were treated as in (B). Left: representative image of live cells (Calcein-AM, green) and dead cells (PI, red). Scale bar: 50 μm. Right: quantification of PI⁺ cells. Each dot represents an independent experiment (n = 3). D – I CBL defends against HSV-1 retinal infection. Mice were IVT injected with PBS (CTRL) or HSV-1(4×10⁴ PFU). CBL (2 μg) or PBS (mock) was co-injected with HSV-1. Analysis was performed at 3 or 9 dpi, n= 6 mice/group. E Slit-lamp examination showing eye morphology. CBL reduced corneal opacity in both WT and STING-/- mice and inhibited corneal ulcer formation in STING-/- mice at 9 dpi. E HE staining showing eye morphology. CBL preserved retinal structure in both WT and STING-/- mice following HSV-1 infection and prevented retinal necrosis in STING-/- mice at 9 dpi. Scale bars: overview 1 mm; magnified 100 μm. F Histopathological scoring of HE-stained retinal tissues, n = 5 eyes/group. G – H Vitreous viral load assessment. G.Representative plaque assay images. H Quantified titers,each data point represents one pooled sample (2–3 eyes), n = 3–6 mice per group.I. qPCR quantification of retinal HSV-1 DNA copies, n = 3 eyes/group. Data are presented as mean ± SD. Statistical significance was determined using two-way ANOVA followed by Tukey’s post hoc test. (ns, not significant; *p < 0.05;**p < 0.01; ***p < 0.001; ****p < 0.00001)

Article Snippet: For the mock infection, HSV-1 was first inactivated by incubating it at 100 °C for 1 h. For combination treatment, 10 mg of CBL0137 (Topscience) or quinacrine (MedChemExpress, USA) was dissolved in 5 mL of PBS via ultrasonication (37 °C, 1 min) and mixed with HSV-1 to achieve a final concentration of 2 mg/mL CBL0137 or quinacrine and 4 × 104 PFU/mL HSV-1.

Techniques: Infection, Virus, Plaque Assay, Injection, Staining

Journal: Journal of Neuroinflammation

Article Title: STING activates ZBP1-mediated PANoptosis to defend against HSV-1 retinal infection

doi: 10.1186/s12974-025-03595-0

Figure Lengend Snippet: STING-ZBP1-PANoptosis signaling defends against HSV-1 retinal infection. Diagram showing that STING activates ZBP1-mediated PANoptosis in microglia and macrophages to inhibit HSV-1 virus load and retinal necrosis

Article Snippet: For the mock infection, HSV-1 was first inactivated by incubating it at 100 °C for 1 h. For combination treatment, 10 mg of CBL0137 (Topscience) or quinacrine (MedChemExpress, USA) was dissolved in 5 mL of PBS via ultrasonication (37 °C, 1 min) and mixed with HSV-1 to achieve a final concentration of 2 mg/mL CBL0137 or quinacrine and 4 × 104 PFU/mL HSV-1.

Techniques: Infection, Virus