(A–H) iPSC derived microglia (A–D) and neurons (E–H) were transfected with IKBKE or control siRNA. (A and E) The efficiency of transfection was examined by measuring IKBKE mRNA levels. Cells were infected with HSV-2 for 6 hours, and IFNA2, IFNB1, and TNFA mRNA levels were quantified. (I–L) iPSC-derived microglia were transfected with siRNA targeting cGAS (I and J) or TLR3 (K and L). Microglia were infected with HSV-2, and IFNB1 mRNA was quantified (J and L). Unpaired t test was used for statistical analysis. (M) Graphical illustration of experimental setup for microglia-neuron crosstalk experiments. Created with BioRender.com. (N) iPSC-derived neurons were treated with Human Type 1 IFN Neutralizing Antibody Mixture or control IgG (both 1:100) 30 minutes before addition of supernatants from HSV-2–infected microglia or treatment with IFN-β (10 ng/mL). Six hours later, the medium was removed, and the neurons were infected with HSV-2. Supernatants were collected after 16 hours for plaque assay (O). Microglia subjected to IKBKE or control knockdown with siRNA were infected with HSV-2. The cells were washed, and the medium was replaced after 1 hour. Supernatants were collected from the microglia after 24 hours and added to the neurons. Six hours later, the medium was removed, and the neurons were infected with HSV-2. Supernatants were collected 16 hours later for plaque assay. Data presented are pooled from 2 independently performed experiments. (P) Cells were treated as in panel N, and culture supernatants were analyzed for cell viability. Data presented are from 1 of 2 experiments performed. (N–P) Groups were compared with Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test. Error bars represent SEM (A–H) and SD (N–P), and *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.