NEMO is a key component of NF-κB- and IRF-3-dependent TLR3-mediated immunity to herpes simplex virus

Abstract

Background: Children with germline mutations in Toll-like receptor 3 (TLR3), UNC93B1, TNF receptor-associated factor 3, and signal transducer and activator of transcription 1 are prone to herpes simplex virus-1 encephalitis, owing to impaired TLR3-triggered, UNC-93B-dependent, IFN-α/β, and/or IFN-λ-mediated signal transducer and activator of transcription 1-dependent immunity.

Objective: We explore here the molecular basis of the pathogenesis of herpes simplex encephalitis in a child with a hypomorphic mutation in nuclear factor-κB (NF-κB) essential modulator, which encodes the regulatory subunit of the inhibitor of the Iκβ kinase complex.

Methods: The TLR3 signaling pathway was investigated in the patient's fibroblasts by analyses of IFN-β, IFN-λ, and IL-6 mRNA and protein levels, by quantitative PCR and ELISA, respectively, upon TLR3 stimulation (TLR3 agonists or TLR3-dependent viruses). NF-κB activation was assessed by electrophoretic mobility shift assay and interferon regulatory factor 3 dimerization on native gels after stimulation with a TLR3 agonist.

Results: The patient's fibroblasts displayed impaired responses to TLR3 stimulation in terms of IFN-β, IFN-λ, and IL-6 production, owing to impaired activation of both NF-κB and IRF-3. Moreover, vesicular stomatitis virus, a potent IFN-inducer in human fibroblasts, and herpes simplex virus-1, induced only low levels of IFN-β and IFN-λ in the patient's fibroblasts, resulting in enhanced viral replication and cell death, as reported for UNC-93B-deficient fibroblasts.

Conclusion: Herpes simplex encephalitis may occur in patients carrying NF-κB essential modulator mutations, due to the impairment of NF-κB- and interferon regulatory factor 3-dependent-TLR3-mediated antiviral IFN production.

Figure 1. Induction of IFN-β, IFN-λ and IL-6 in response to TLR3 agonists

(A) Production of IFN-β, IFN-λ and IL-6 by SV40-fibroblasts from a healthy control (C), the patient (P), a patient with complete NEMO deficiency (NEMO IP) and a patient with UNC-93B deficiency (UNC-93B^−/−), unstimulated or stimulated with poly(I:C) and IPH31, without or with (poly(I:C)+L and IPH31+L) Lipofectamine (L), assessed by ELISA. (B) Production of IFN-β, IFN-λ and IL-6 by SV40-fibroblasts in response to various doses of poly(I:C) for 24 hours, assessed by ELISA. For A and B, the panels show mean values ± SD of three independent experiments. The reported p values for differences between the responses of patients and controls are for unpaired Student's t tests for each experiment (*: p<0.05; #: p<0.07). (C) IFN-β, IFN-λ and IL-6 mRNA levels in SV40-fibroblasts left unstimulated or stimulated for 2, 4, 6 or 24 hours with poly(I:C). The panels illustrate results from two independent experiments performed in duplicate.

Figure 2. Induction of NF-κB, IRF-3 and IRF-7 in response to poly(I:C)

(A) IRF-3 monomer and dimer formation in total cell extracts from healthy control (C), the patient (P), NEMO IP and UNC-93B ^−/− SV40-fibroblasts, upon stimulation with poly(I:C) for 30 to 180 minutes, as assessed by western blotting. Fold-induction of IRF3 dimers, presented below as a histogram, was determined from the intensity of the signal on immunoblots, normalized with respect to monomer levels. (B) NF-κB translocation, assessed by EMSA, in SV40-fibroblasts from a control C, the patient P, NEMO IP and UNC-93B ^−/− cells, following stimulation with poly(I:C) for 1 hour and with IL1-β for 20 minutes. The panels are representative of two independent experiments. (C) IRF-7 induction in SV40-fibroblasts after 16 hours of poly(I:C) stimulation, analyzed by western blotting and normalized with respect to tubulin levels. This panel is representative of three independent experiments.

Figure 3. IFN production and cell mortality in response to viral stimulation

(A) SV40-fibroblasts from a healthy control (C), the patient (P), a NEMO IP patient and an UNC-93B ^−/− patient were left uninfected or infected with VSV (MOI from 0.001 to 100) for 24 hours (upper panels) or with HSV-1 (MOI from 0.0001 to 1) (lower panels) for 72 hours. The production of IFN-β (left panels) and IFN-λ (right panels) was assessed by ELISA. (B) Cell mortality was estimated by LDH assay, 24 hours after infection with VSV at various MOI, without (left panel) or with (right panel) prior treatment with IFN-α2b. (C) The percentage of live cells was estimated by resazurin oxidation/reduction, 72 hours after infection with HSV-1, at various MOI, without (left panel) or with (right panel) prior treatment with IFN-α2b.