An IKBKE variant conferring functional cGAS/STING pathway deficiency and susceptibility to recurrent HSV-2 meningitis
Azadeh Reyahi, Marie Studahl, Morten K. Skouboe, Stefanie Fruhwürth, Ryo Narita, Fanghui Ren, Moa Bjerhem Viklund, Marie B. Iversen, Mette Christiansen, Alexandra Svensson, Trine H. Mogensen, Kristina Eriksson, Søren R. Paludan
Azadeh Reyahi, Marie Studahl, Morten K. Skouboe, Stefanie Fruhwürth, Ryo Narita, Fanghui Ren, Moa Bjerhem Viklund, Marie B. Iversen, Mette Christiansen, Alexandra Svensson, Trine H. Mogensen, Kristina Eriksson, Søren R. Paludan
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Research Article Immunology Infectious disease

An IKBKE variant conferring functional cGAS/STING pathway deficiency and susceptibility to recurrent HSV-2 meningitis

Abstract

The mechanisms underlying susceptibility to recurrent herpes simplex virus type 2 (HSV-2) meningitis remain incompletely understood. In a patient experiencing multiple episodes of HSV-2 meningitis, we identified a monoallelic variant in the IKBKE gene, which encodes the IKKε kinase involved in induction of antiviral IFN genes. Patient cells displayed impaired induction of IFN-β1 (IFNB1) expression upon infection with HSV-2 or stimulation with double-stranded DNA (dsDNA) and failed to induce phosphorylation of STING, an activation marker of the DNA-sensing cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) pathway. The patient allele encoded a truncated IKKε protein with loss of kinase activity and also capable of exerting dominant-negative activity. In stem cell–derived microglia, HSV-2–induced expression of IFNB1 was dependent on cGAS, TANK binding kinase 1 (TBK1), and IKBKE, but not TLR3, and supernatants from HSV-2–treated microglia exerted IKBKE-dependent type I IFN–mediated antiviral activity upon neurons. Reintroducing wild-type IKBKE into patient cells rescued IFNB1 induction following treatment with HSV-2 or dsDNA and restored antiviral activity. Collectively, we identify IKKε to be important for protection against HSV-2 meningitis and suggest a nonredundant role for the cGAS/STING pathway in human antiviral immunity.

Authors

Azadeh Reyahi, Marie Studahl, Morten K. Skouboe, Stefanie Fruhwürth, Ryo Narita, Fanghui Ren, Moa Bjerhem Viklund, Marie B. Iversen, Mette Christiansen, Alexandra Svensson, Trine H. Mogensen, Kristina Eriksson, Søren R. Paludan

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Figure 2

Impaired IFNB1 response and STING activation following HSV-2 infection in PBMCs from P1.

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Impaired IFNB1 response and STING activation following HSV-2 infection i...
(A–D) PBMCs were infected with HSV-2 with MOI 9. Total RNA was isolated after 6 hours for measurement of IFNA2 (A), IFNB1 (B), ISG54 (C), and TNFA (D) mRNA by reverse transcription quantitative PCR (RT-qPCR). (E and F) Whole-cell lysates from PBMCs treated for 18 hours with HSV-2, as indicated, were examined for expression of IKKε (N- and C-terminal–targeting antibody), p-IKKε (S172), TBK1, p-TBK1 (S172), IRF3, p-IRF3 (S386), STING, and p-STING (S366). Data shown are representative of 4 (E) and 2 (F) independently performed experiments. (G and H) Fibroblasts were transfected with cGAMP or dsDNA (4 μg/mL) for 4 hours, and lysates were immunoblotted for p-STING (S366), STING, and Vinculin. (I–K) PBMCs were stimulated with dsDNA (4 μg/mL) or poly(IC) (4 μg/mL intracellular, 50 μg/mL extracellular). (L–P) PBMCs were infected for 6 hours with Sendai virus (SeV), influenza A virus (IAV) (MOI 0.1), encephalomyocarditis virus (EMCV) (MOI 0.1), HSV-1 (MOI 3), and HSV-2 (MOI 3), and IFNB1 mRNA was quantified by qPCR. All measurements were done in triplicates, relative to housekeeping genes GAPDH or BACTIN and normalized to the pooled uninfected PBMCs from 10 (A–D), 9 (I, J, and L–N), 4 (K), 2 young (control 1), and 4 around 50 years old (control 2) (O and P) healthy controls. Results were obtained from 3 (A–D) (except for mother and son PBMCs, which were examined once) and (I–N) or 2 (O and P) independent experiments. The nonparametric Mann-Whitney (A–D) and unpaired t test (I–P) were used to evaluate statistical significance between groups. Error bars represent standard error of mean (SEM). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤0.0001.