Journal: The Journal of Experimental Medicine

Article Title: Role for NF-κB in herpes encephalitis pathology in mice genocopying an inborn error of IRF3-IFN immunity

doi: 10.1084/jem.20250064

Figure Lengend Snippet: The hIRF3 R285Q/mIRF3 R278Q mutation impairs type I IFN responses in microglia and confers susceptibility to HSE-like disease. (A) Human iPSC-derived microglia, neurons, and astrocytes were generated from patient fibroblasts or control iPSC. Created with BioRender. (B–D) IFNB1 expression in (B) microglia, (C) astrocytes, and (D) cortical neurons 24 h after infection with HSV-1 at MOI 1.0. HSE pt., HSE patient. (E) Alignment of human and murine IRF3 around the region harboring R285 in WT human IRF3. (F–I) Ifnb1 and Isg15 expression after HSV-1 infection in murine microglia and neurons from WT and transgenic mice carrying the IRF3 R278Q mutation. Microglia (F and G) and neurons (H and I) 24 h after infection with HSV-1 at MOI 1.0. All in vitro experiments were performed in triplicates and independently repeated at least three times. Expression data were normalized to β-actin and shown as fold change compared with the UI control. (J–O) Mice were infected in the cornea with HSV-1 McKrea (2 × 10 6 PFU/eye), and HSE-like disease development was followed over time until reaching humane endpoint or recovering 100% of starting weight. (J) % weight change. (K) Symptom score. (L) Survival curve (UI, n = 7; WT, n = 15; Irf3 WT/R278Q , n = 15; Irf3 R278Q/R278Q , n = 16; Irf3 −/− n = 10). Dead animals were censored in the graphs and thus represented in the graphs with weight and symptom score at time of death. (M) Representative MR images performed on day 5 after infection. Red dotted line and white arrows indicate lesions. (N) Lesion volumes quantified blinded. (O and P) BBB disruption/integrity was assessed visibly by Evans blue perfusion of mice 5 days after HSV-1 infection. Representative microscope images of Evans blue dye leakage in brain stems from UI and HSV-1–infected WT and IRF3 R278Q/R278Q mice were obtained from (O) uncut ventral position (2× objective) and (P) coronal slides cut in 5 mm thickness (3.2× objective). Red circles indicate area of Evans blue passive diffusion into lesion sites. n = 3–7 mice per group. In vivo survival experiments were independently repeated three times, and MR-imaging experiment was repeated two times. Statistical analyses of cell culture experiments (B–D and F–I) were analyzed by two-tailed two-way ANOVA for difference of means, followed by two-tailed unpaired t test of means, error bars; SD. Disease development (weight change and symptom score) were compared between the groups using a mixed-effects analysis with Geisser-Greenhouse correction for multiple interacting variables (time and genotype). Survival was analyzed using log-rank Mantel–Cox test (L). Error bars; SEM. Lesion volumes (N) were analyzed by two-tailed one-way ANOVA followed by unpaired t test, error bars; SD. P values <0.05 were considered statistically significant, **P < 0.01, and ***P < 0.001.

Article Snippet: Quantitative PCR was performed using the following TaqMan Gene Expression Assays (Applied Biosystems): ACTB (Hs01060665_g1), 18S (Hs03003631_g1), IFNB (Hs01077958_s1), IL6 (Hs00174131_m1), TNFA (Hs00174128_m1), IL1B (Hs01555410), MX1 (Hs00895598_m1), CXCL10 (Hs00171042), and ISG15 (Hs01921425). mRNA levels of interest were normalized to the housekeeping gene ACTB or 18S (as indicated) using the ΔΔCt method.

Techniques: Mutagenesis, Derivative Assay, Generated, Control, Expressing, Infection, Transgenic Assay, In Vitro, Disruption, Microscopy, Diffusion-based Assay, In Vivo, Imaging, Cell Culture, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Role for NF-κB in herpes encephalitis pathology in mice genocopying an inborn error of IRF3-IFN immunity

doi: 10.1084/jem.20250064

Figure Lengend Snippet: Generation of human iPSC-derived microglia, cortical neurons, and astrocytes. (A–H) Confocal microscopy images of iPSCs and derived astrocytes, microglia, and cortical neurons from a healthy control donor and a pediatric HSE patient heterozygous for the IRF3 R285Q amino acid substitution stained with cell type–specific markers. (A and D) iPSCs: NANOG and OCT4 (both green); (B and E) astrocytes: S100 (red) and GFAP (green); (C and F) microglia: TREM2 and Iba1 (both green); (G and H) cortical neurons: MAP2, β-III tubulin (both green), synaptophysin, and TBR1 (both red); nuclei were identified by DAPI staining. Scale bar, 50 μm. (I and J) Comparison of IFNB response to HSV-1 infection in microglia from two different iPSC lines and two different patient-derived iPSC clones measured by RT-qPCR. Commercially available iPSC lines 015A and BIONC (I) and patient-derived iPSC clones C4 and C6 (J). (K) IFNB expression in microglia after stimulation with polyIC (25 μg/ml) and cGAMP (100 μg/ml). (L and M) ISG15 expression in microglia 4 and 24 h after infection with HSV-1 at MOI 1.0. (N and O) ISG15 expression in cortical neurons infected 24 h with HSV-1 at MOI 1.0 or stimulated for 4 h with polyIC (25 μg/ml) and cGAMP (100 μg/ml). Expression data were normalized to β-actin and shown as fold change compared with the UI control. Statistical analyses of gene expression in CNS cell cultures (K–O) were analyzed by two-tailed two-way ANOVA for difference of means, followed by an unpaired t test of means, error bars; SD. P values <0.05 were considered statistically significant, **P < 0.01, and ***P < 0.001.

Article Snippet: Quantitative PCR was performed using the following TaqMan Gene Expression Assays (Applied Biosystems): ACTB (Hs01060665_g1), 18S (Hs03003631_g1), IFNB (Hs01077958_s1), IL6 (Hs00174131_m1), TNFA (Hs00174128_m1), IL1B (Hs01555410), MX1 (Hs00895598_m1), CXCL10 (Hs00171042), and ISG15 (Hs01921425). mRNA levels of interest were normalized to the housekeeping gene ACTB or 18S (as indicated) using the ΔΔCt method.

Techniques: Derivative Assay, Confocal Microscopy, Control, Staining, Comparison, Infection, Clone Assay, Quantitative RT-PCR, Expressing, Gene Expression, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Role for NF-κB in herpes encephalitis pathology in mice genocopying an inborn error of IRF3-IFN immunity

doi: 10.1084/jem.20250064

Figure Lengend Snippet: Generation and characterization of transgenic mice carrying the IRF3 R278Q allele and susceptibility to infections with HSV-2 and IAV. (A) Mice carrying the patient-specific mIRF3 R278Q amino acid substitution were made using CRISPR microinjection in C57Bl6/J zygotes. The CRISPR guide used was 5′-GTG​GGA​GTG​GCC​TAG​GCG​CTG​GG-3′. (B) Litter size of Irf3 R278Q/R278Q and Irf3 −/− pubs bred at the Aarhus University animal core facility in 2024 compared with average litter size of C57Bl6/JRj mice bred by Janvier. (C) Weight of C57Bl6/JRj, Irf3 WT/R278Q , Irf3 R278Q/R278Q , and Irf3 −/− mice at experiment start (square, female; triangle, male). (D) WB analysis of IRF3 protein and phosphorylation of IRF3 Ser379 in lysates of BMDMs from WT, Irf3 WT/R278Q , Irf3 R278Q/R278Q , and Irf3 −/− mice stimulated with 100 μg/ml cGAMP for 2 h. (E and F) Ifnb and Isg15 gene expression response of murine astrocyte cultures to HSV-1 infection at MOI 1.0 for 24 h. (G–L) Ifnb and Isg15 gene expression response to stimulation with PRR agonists poly-IC (25 μg/ml) or cGAMP (100 μg/ml) for 4 h. Murine astrocyte (G and H), murine microglia (I and J), and murine neurons (K and L). CNS cell culture gene expression was measured by RT-qPCR, and data were normalized to β-actin ( Actb ) and are represented as fold change normalized to expression in UI control. All in vitro experiments were performed in triplicates and independently repeated at least three times. Statistical analyses of gene expression in CNS cell cultures (E–L) were analyzed by two-tailed two-way ANOVA for difference of means, followed by an unpaired t test of means, error bars; SD. (M–X) WT, Irf3 WT/R278Q , Irf3 R278Q/R278Q , and Irf3 −/− mice were infected with (M–T) HSV-2 by the vaginal route or (U–X) IAV via the nasal route and were followed for disease development over time until reaching humane endpoint or recovering 100% of starting weight. (M, Q, and U) % weight change. (N and R) Symptom score. (O, S, and V) Survival curve. Dead animals were censored in the graphs and thus represented in the graphs with weight and symptom score at time of death. HSV-2 longitudinal (M–O) (WT, n = 8; Irf3 R278Q/R278Q , n = 8; Irf3 −/− n = 8), (Q–S) (WT, n = 8; Irf3 WT/R278Q , n = 8; Irf3 R278Q/R278Q , n = 8), and IAV longitudinal (U and V) (WT, n = 11; Irf3 R278Q/R278Q , n = 11; Irf3 −/− , n = 7; UI, n = 6). Viral load was assessed by (P and T) HSV-2 TCID50% assay of vaginal washes on day 2 after infection; (P) WT, n = 8; Irf3 R278Q/R278Q , n = 7; Irf3 −/− n = 7; and (T) WT, n = 8; Irf3 WT/R278Q , n = 8; or (X) IAV M-Protein gene transcripts in lung homogenates on day 4 postnasal inhalation infection measured by RT-PCR (WT, n = 5; Irf3 R278Q/R278Q , n = 5; Irf3 −/− n = 5). Disease development (M, N, Q, R, and U) was compared between the groups using a mixed-effects analysis with Geisser-Greenhouse correction for multiple interacting variables (time and genotype). Error bars; SEM. Survival (O, S, and V) was analyzed using log-rank Mantel–Cox test. Viral load (P, T, and X) was analyzed by two-tailed two-way ANOVA for difference of means, followed by an unpaired t test of means, error bars; SD. P values <0.05 were considered statistically significant, *P < 0.05, **P < 0.01, and ***P < 0.001. Source data are available for this figure: .

Article Snippet: Quantitative PCR was performed using the following TaqMan Gene Expression Assays (Applied Biosystems): ACTB (Hs01060665_g1), 18S (Hs03003631_g1), IFNB (Hs01077958_s1), IL6 (Hs00174131_m1), TNFA (Hs00174128_m1), IL1B (Hs01555410), MX1 (Hs00895598_m1), CXCL10 (Hs00171042), and ISG15 (Hs01921425). mRNA levels of interest were normalized to the housekeeping gene ACTB or 18S (as indicated) using the ΔΔCt method.

Techniques: Transgenic Assay, CRISPR, Microinjection, Phospho-proteomics, Gene Expression, Infection, Cell Culture, Quantitative RT-PCR, Expressing, Control, In Vitro, Two Tailed Test, TCID50 Assay, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of Experimental Medicine

Article Title: Role for NF-κB in herpes encephalitis pathology in mice genocopying an inborn error of IRF3-IFN immunity

doi: 10.1084/jem.20250064

Figure Lengend Snippet: Comparative analysis of systemic and CNS responses to HSV-1 infection in vivo and in vitro . (A–C) Analysis of viral replication on day 4 after HSV-1 ocular infection (2 × 10 6 PFU/eye) of WT ( n = 12) and Irf3 WT/R278Q heterozygous ( n = 12) mice. UI controls were included (WT UI, n = 3; Irf3 WT/R278Q UI, n = 3). Viral titer in (A) eyes, (B) TG, and (C) brain stem homogenates quantified by plaque assay. (D–I) Systemic cytokine levels measured in serum from WT and Irf3 R278Q/R278Q mice on day 5 after HSV-1 infection. Data points represent cytokine level (pg/ml) in serum from individual mice measured by mesoscale. (J–N) CNS response in WT and Irf3 R278Q/R278Q mice to systemic LPS stimulation in vivo . Gene expression of Ifnb , Tnfa , Il1b , Il6 , and Ccl2 in whole-brain homogenates from mice treated with 5 mg/kg LPS or saline by i.p. injection. Gene expression was measured by RT-qPCR, and data were normalized to β-actin ( Actb ). (O) Validation of microglia depletion by qPCR of Iba1 expression in MBCs treated with 0.5 μM PLX5622 (PLX) or untreated (UT). (P and Q) Expression of Ccl2 in WT mixed brain cultures treated with (P) 0.5 μM PLX5622 (PLX) or mock treated for microglia depletion, or (Q) NF-κB activation inhibitors BMS-345541 2 µM, PDTC 25 µM, or saline, and infected with HSV-1 at MOI 1.0 for 24 h. (R and S) Isg15 and Cxcl10 infection-dose response analysis in murine microglia analyzed by RT-qPCR 24 h after infection with HSV-1 at increasing MOI. For all panels where statistical analyses were performed, the analysis was two-tailed two-way ANOVA for difference of means, followed by two-tailed unpaired t test of means, error bars; SD. P values <0.05 were considered statistically significant. *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: Quantitative PCR was performed using the following TaqMan Gene Expression Assays (Applied Biosystems): ACTB (Hs01060665_g1), 18S (Hs03003631_g1), IFNB (Hs01077958_s1), IL6 (Hs00174131_m1), TNFA (Hs00174128_m1), IL1B (Hs01555410), MX1 (Hs00895598_m1), CXCL10 (Hs00171042), and ISG15 (Hs01921425). mRNA levels of interest were normalized to the housekeeping gene ACTB or 18S (as indicated) using the ΔΔCt method.

Techniques: Infection, In Vivo, In Vitro, Plaque Assay, Gene Expression, Saline, Injection, Quantitative RT-PCR, Biomarker Discovery, Expressing, Activation Assay, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Role for NF-κB in herpes encephalitis pathology in mice genocopying an inborn error of IRF3-IFN immunity

doi: 10.1084/jem.20250064

Figure Lengend Snippet: Elevated proinflammatory cytokine levels in the brain of Irf3 R278Q/R278Q mice upon HSV-1 infection. (A–F) Expression of Ifnb , Mx1 , Tnfa , Il1b , Il6 , and Ccl2 in brain stems from WT or Irf3 R278Q/R278Q mice on days 1–4 after infection, measured by RT-qPCR. UI, uninfected control. WT, n = 5–7; Irf3 R278Q/R278Q , n = 5–12. (G–J) Mesoscale analysis of cytokine levels (TNFα, CCL2, and IFNγ) (G–I) and HSV-1 titer in brain stem homogenates on day 5 after infection quantified by plaque assay (J) (WT UI, n = 4, WT, n = 9; Irf3 R278Q/R278Q UI, n = 4; Irf3 R278Q/R278Q , n = 8). (K) Expression of Tnfa in brain stems from WT ( n = 7), Irf3 −/− ( n = 5), Irf3 R278Q/R278Q ( n = 9), and Irf3 WT/R278Q ( n = 6) mice on day 5 after HSV-1 infection, measured by RT-qPCR. (L–N) Ifnb, Tnfa , and Il6 response 16 h after infection with 1.0 MOI HSV-1 in MBCs treated with 0.5 μM PLX5622 (PLX) for microglia depletion. (O and P) Tnfa and Il6 response 16 h after infection with 1.0 MOI HSV-1 in MBCs treated with NF-κB inhibitors BMS-345541 (2 μM), PDTC (25 μM), or saline. UI controls were included. (Q and S) Ifnb and Tnfa mRNA levels in murine microglia from WT, Irf3 WT/R278Q , and Irf3 R278Q/R278Q mice 24 h after HSV-1 infection at increasing virus MOI as indicated. (R and T) IFNB1 and TNFA mRNA levels in iPSC-derived microglia from the IRF3 WT/R285Q HSE patient and controls 24 h after HSV-1 infection at increasing virus MOI as indicated. Expression data were normalized to β-actin and shown as fold change compared with the UI control. All in vitro experiments were performed in triplicates and independently repeated at least three times. Cytokine expression kinetics were compared between the groups using a mixed-effects analysis with Geisser-Greenhouse correction for multiple interacting variables (time and genotype) (A–F), error bars; SEM. Statistical analyses of cytokine and virus levels in brain stem homogenates (G–J and L–T) were analyzed by two-tailed two-way ANOVA for difference of means, followed by an unpaired t test of means, error bars; SD. CNS cell experiments (K) were analyzed with a two-tailed one-way ANOVA for difference of means followed by an unpaired t test of means. P values <0.05 were considered statistically significant, *P < 0.05, **P < 0.01, and ***P < 0.001. UT, untreated; MOI, multiplicity of infection.

Article Snippet: Quantitative PCR was performed using the following TaqMan Gene Expression Assays (Applied Biosystems): ACTB (Hs01060665_g1), 18S (Hs03003631_g1), IFNB (Hs01077958_s1), IL6 (Hs00174131_m1), TNFA (Hs00174128_m1), IL1B (Hs01555410), MX1 (Hs00895598_m1), CXCL10 (Hs00171042), and ISG15 (Hs01921425). mRNA levels of interest were normalized to the housekeeping gene ACTB or 18S (as indicated) using the ΔΔCt method.

Techniques: Infection, Expressing, Quantitative RT-PCR, Control, Plaque Assay, Saline, Virus, Derivative Assay, In Vitro, Two Tailed Test