Journal: Cell Reports

Article Title: The HIF transcription network exerts innate antiviral activity in neurons and limits brain inflammation

doi: 10.1016/j.celrep.2024.113792

Figure Lengend Snippet: HIF deficiency in neurons renders mice susceptible to HSV-2 infection in the CNS (A) Illustration of experimental setup for HSV-2 infection of mice. Image was generated with BioRender. (B) C57BL/6 mice were infected in the cornea with 400 PFU/mL HSV-2. After 5 days, mice were injected with pimonidazole 2 h before they were sacrificed, and brain stem sections were monitored for staining. Scale bars: 100 μm (top row) and 20 μm (bottom row). (C) nHif1a/Hif2a ff/ff and nHif1a/Hif2a ΔΔ/ΔΔ mice were infected in the cornea with 400 PFU/mL HSV-2. Brain stems were harvested 5 days later for measurement of Eno1 mRNA by RT-qPCR. Data were normalized to 18S rRNA and are shown as means ±SD. n = 4–5 mice per group. (D–F) Weight change, clinical scores, and survival were monitored measured from the day of infection. n = 9 mice per group. (G–K) Brain stems were isolated 5 days post-infection, and homogenates were analyzed for (G) levels of infectious HSV-2 by plaque assay, (H) phosphorylation of TBK1 at S172 by immunoblotting (lysates were pooled in groups, 4 mice per group), (I and J) Cxcl10 and Tnfa mRNA levels by RT-qPCR, and (K) CXCL10 protein by ELISA. For data in (D), (I), and (J), n = 20 mice per group. For data in (K), n = 20 mice per group. Data are shown as means ±SD. p values were calculated using two-way ANOVA with Bonferroni’s post hoc test. See also Figure S8 .

Article Snippet: Taqman primers and probe CXCL10 , Thermo Fisher , #Mm00445235_m1.

Techniques: Infection, Generated, Injection, Staining, Quantitative RT-PCR, Isolation, Plaque Assay, Phospho-proteomics, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports

Article Title: The HIF transcription network exerts innate antiviral activity in neurons and limits brain inflammation

doi: 10.1016/j.celrep.2024.113792

Figure Lengend Snippet: Activation of the HIF pathway induces antiviral activity in human neurons (A) Image of hESC-derived neurons. Scale bar, 100 μm. (B) Neurons were infected with HSV-1 (MOI 1) for the 24 and 48 h, and lysates were isolated and immunoblotted for the indicate antigens. We used PMA-differentiated THP1 macrophage-like cells stimulated with cGAMP (4 μg/mL) for 2 h as positive control for the STING pathway. VCN, vinculin. (C and D) HSV transcripts (gB) were measured by RT-qPCR analysis of total RNA from hESC-derived neurons isolated 48 h after infection with HSV-1 and -2 (MOI 1) in the presence and absence of treatment with ML228 (1.4 μM). Transcript levels were normalized against 18S rRNA and are shown as NRs. n = 2 biological replicates. (E) Levels of HSV-1 and -2 VP5 protein, LC3B, and VCL in hESC-derived neurons 48 h after infection with HSV-1 and -2 (MOI 1) cultures in the presence or absence of treatment with ML228 (1.4 μM). (F) Illustration of experimental setup for testing of effect of neuronal hypoxia on microglia activation. Image was generated with BioRender. (G) HSV-2 gB mRNA transcripts measured by RT-qPCR following infection in ESC-derived neurons during normoxia and hypoxia (1% O 2 ). n = 5 biological replicates. (H) Immunoblot for signaling proteins in microglia lysates treated for 24 h with supernatants (1:5) from neurons infected for 48 h (MOI 1) with HSV-2 under normoxic or hypoxic (1% O 2 ) conditions. (I and J) Total RNA was isolated from microglia treated as described in (H) and examined for levels of CXCL10 and TNFA . n = 4 biological replicates. Transcript levels were normalized against 18S rRNA and shown as NRs. The data in (C), (D), (G), (I), and (J) are shown as means ± SD. Individual values are represented by dots. For (C) and (D), Student’s t test, two-tailed, parametric distribution was used to identify statistical significance. For (G), (I), and (J), Mann-Whitney was applied to identify statistical significance. See also Figure S9 .

Article Snippet: Taqman primers and probe CXCL10 , Thermo Fisher , #Mm00445235_m1.

Techniques: Activation Assay, Activity Assay, Derivative Assay, Infection, Isolation, Positive Control, Quantitative RT-PCR, Generated, Western Blot, Two Tailed Test, MANN-WHITNEY

Journal: Cell Reports

Article Title: The HIF transcription network exerts innate antiviral activity in neurons and limits brain inflammation

doi: 10.1016/j.celrep.2024.113792

Figure Lengend Snippet:

Article Snippet: Taqman primers and probe CXCL10 , Thermo Fisher , #Mm00445235_m1.

Techniques: Virus, Variant Assay, Recombinant, Isolation, Extraction, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Sample Prep, Expressing, Software, Imaging, Modification, Knock-Out