Human TRAF3 adaptor molecule deficiency leads to impaired Toll-like receptor 3 response and susceptibility to herpes simplex encephalitis

Abstract

Tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3) functions downstream of multiple TNF receptors and receptors that induce interferon-α (IFN-α), IFN-β, and IFN-λ production, including Toll-like receptor 3 (TLR3), which is deficient in some patients with herpes simplex virus-1 encephalitis (HSE). Mice lacking TRAF3 die in the neonatal period, preventing direct investigation of the role of TRAF3 in immune responses and host defenses in vivo. Here, we report autosomal dominant, human TRAF3 deficiency in a young adult with a history of HSE in childhood. The TRAF3 mutant allele is loss-of-expression, loss-of-function, dominant-negative and associated with impaired, but not abolished, TRAF3-dependent responses upon stimulation of both TNF receptors and receptors that induce IFN production. TRAF3 deficiency is associated with a clinical phenotype limited to HSE resulting from the impairment of TLR3-dependent induction of IFN. Thus, TLR3-mediated immunity against primary infection by HSV-1 in the central nervous system is critically dependent on TRAF3.

Fig. 1. Heterozygous TRAF3 mutation in a child with HSE (P1)

(A) Family pedigree, with allele segregation. The patient with HSE, in black, is heterozygous for the mutation. (B) Heterozygous c.705C>T mutation in patient 1 (P1). The sequence of the polymerase chain reaction products of genomic DNA from leukocytes of a control (C) and P1 is shown. (C) Schematic representation of TRAF3 protein structure. Human TRAF3 has eleven exons, encoding a protein composed of a ring finger and five zinc-finger domains in the N-terminal region, followed by an isoleucine zipper and a TRAF domain in the C-terminal region. P1 carries the c.705C>T mutation, which results in an arginine (R) to tryptophan (W) substitution at amino-acid position 118 (R118W) in the first zinc-finger domain.

Fig. 2. TRAF3 expression levels and TLR3 response of a child with HSE (P1)

(A) (Top) TRAF3 mRNA expression in SV40-fibroblasts from a control (C) and P1. β-glucuronidase (GUS) was used for normalization. (Bottom) Representative immunoblot analysis of TRAF3 in SV40-fibroblasts from a control (C) and P1. TRAF3 (%) indicates densitometry results, normalized with respect to GADPH and expressed as a percentage of control TRAF3. The control is a representative example of the five controls analyzed. (B) (Top) TRAF3 mRNA levels normalized with respect to 18S rRNA and (Bottom) immunoblot analysis of TRAF3 in RAW cells and RAW Traf3^−/− cells not transfected and stably transfected with human wild-type (WT) (pWPI-TRAF3) or R118W mutant TRAF3 (pBABE-TRAF3-R118W) constructs. As a negative control, we transfected cells with a mock WT vector (Mock-1, pWPI) or with a mock R118W mutant vector (Mock-2, pBABE). TRAF3 (%) indicates densitometry results normalized with respect to GADPH amounts and expressed as a percentage of TRAF3 amounts in RAW cells. (C) Production of IFN-β (measured in structural units, IU/ml), IFN-λ and IL-6 by SV40-fibroblasts after poly(I:C) stimulation (25 μg/ml), at various time points (left panels) and for various doses of poly(I:C), for 24 hours (right panels), as assessed by ELISA. C is the positive control and P1 is patient 1; UNC93B^−/− is the UNC-93B-deficient patient. (D) NF-κB ELISA of nuclear extracts from SV40-fibroblasts from a control (C), P1 and a NEMO-deficient patient (negative control), after stimulation with TNF-α and IL-1β for 30 minutes, or stimulation with poly(I:C) for 120 minutes. (E) IRF-3 monomers and dimers in total cell extracts of SV40-fibroblasts from a control (C) and P1, after stimulation with poly(I:C) for 1 and 2 hours, as assessed by immunoblot. TLR3- and UNC-93B-deficient patients were used as negative controls.

Fig. 3. Complementation of the patient’s cells and dominant negative effect

(A) Immunoblot analysis of TRAF3 amounts in SV40-fibroblasts from a control (C) and P1, not transfected (P1) and stably transfected with human WT TRAF3 (P1 WT) or mock vector (P1-mock), with and without stimulation with poly(I:C) for 24 h. TRAF3 (%) indicates densitometry results normalized with respect to GADPH amounts and expressed as a percentage of control TRAF3 amounts. (B) Production of IFN-β and IL-6, as assessed by ELISA, in SV40-fibroblasts from a control (C), P1, an UNC-93B-deficient patient (UNC93B^−/−), P1 cells transfected with human WT TRAF3 (P1-WT) or mock vector (P1-mock). (C) Immunoblot analysis of TRAF3 amounts in RAW cells, non transfected RAW Traf3^−/− cells, and RAW cells stably transfected with R118W mutant TRAF3 (R118W), WT TRAF3 (WT) or mock vector (Mock). (D) IFN-β production, as assessed by ELISA, in RAW cells, not transfected or transfected with R118W mutant TRAF3 or mock vector. All transfections generated stable cell lines. Glyceraldehyde phosphate dehydrogenase (GADPH) was used as an internal expression control for WB. The panels illustrate results from a single experiment, representative of three. Mean values ± SD were calculated from three independent experiments.

Fig. 4. SV40-fibroblast studies: production of type I IFN and cytokines

(A) Production of IFN-β, IFN-λ and IL-6 after stimulation with VSV, as assessed by ELISA, in SV40-fibroblasts from a control (C), P1 and an UNC-93B-deficient patient (UNC93B^−/−). (B) Viable cell percentages, estimated by resazurin oxidation/reduction, for SV40-fibroblasts from a healthy control (C), P1, TLR3^+/−, UNC93B^−/− and STAT1^−/− patients, 24 h after infection with VSV, at various multiplicities of infection. The cells were either not treated (left panel), or were subjected to prior treatment (right panel) with recombinant IFN-α for 18 hours. (C) VSV titers, estimated on Vero cells, in SV40-fibroblasts from a healthy control (C), P1, STAT1 ^−/−, UNC93B ^−/− and TLR3 ^+/− patients, at various times after VSV infection without (left panel) or with (right panel) 18 hours of pretreatment with IFN-α. (D) The production of IFN-β and IL-6, assessed by ELISA, in SV40-fibroblasts from a control (C), P1 (UNC93B ^−/−), P1 SV40-fibroblasts transfected with human WT TRAF3 (P1-WT) or mock vector (P1-mock). (E) Viable cell percentages, estimated by resazurin oxidation and reduction, for SV40-fibroblasts from a healthy control (C), P1, P1 cells transfected with WT TRAF3 (P1-WT) and P1 cells transfected with mock vector (P1-mock), 24 hours after infection with VSV, at various multiplicities of infection. The cells were either not treated (left panel), or subjected to prior treatment (right panel) with recombinant IFN-α for 18 hours. (F) VSV titers, estimated on Vero cells, in SV40-fibroblasts from a healthy control (C), P1, P1 cells transfected with WT TRAF3 (P1-WT) or mock vector (P1-mock), at various times after VSV infection, without (left panel) or with (right panel) 18 hours of pretreatment with IFN-α. Mean values ± SD were calculated from three independent experiments.

Fig. 5. Production of type I IFN and cytokines in immune system cells

(A) Production of IFN-α and IL-12p40 after stimulation with LPS and R-848, as assessed by ELISA, in MDDCs. C is the positive control (one of the eight controls tested) and P1 is patient 1. (B) Production of IFN-λ and IL-12p40 after stimulation with LPS and R-848, as assessed by ELISA, in MDMs. C is the positive control (one of the eight controls tested) and P1 is patient 1. (C) Production of IFN-β, IFN-λ and IL-6, as assessed by ELISA, in SV40-fibroblasts after stimulation with poly(I:C)+Lipofectamine or 7sk-as. C is the positive control and P1 is patient 1; UNC93B ^−/− is an UNC-93B-deficient patient. (D) IFN-α production after stimulation with poly(I:C) and various viruses, as assessed by ELISA, in PBMCs. C is the positive control and P1 is patient 1. The panel illustrates results from a single experiment, representative of two. (E) Production of IFN-β and IFN-λ, as assessed by ELISA, in SV40-fibroblasts after stimulation with HSV-1. C is the positive control and P1 is patient 1; UNC93B ^−/− is an UNC-93B-deficient patient. Mean values ± SD were calculated from three independent experiments. (F) Production of IFN-β and IFN-λ, as assessed by ELISA, in SV40-fibroblasts, after stimulation with various viruses. C is the positive control and P1 is patient 1; UNC93B ^−/−, TLR3 ^+/− and MyD88 ^−/− are patients with the corresponding genotypes.

Fig. 6. TRAF3 in TNFR pathways

The production of IL-6 (A) and IL-12p40 (B) was assessed by ELISA in MDDC, after 24 h of incubation with L-cells transfected with human CD40L (CD40L) and non transfected L-cells (Neg control), for a healthy control (C) and P1. (C) Cell death assay, based on resazurin oxidation/reduction, for SV40-fibroblasts from a healthy control (C) and P1 treated with various doses of LTα1β2, with or without IFN-γ (80 IU/ml) for 72 h. (D) IL-8 production, assessed by ELISA, in SV40-fibroblasts from control (C), P1, P1 cells transfected with human WT TRAF3 (P1-WT) or mock vector (P1-mock), after activation with LTα1β2 at the concentration indicated. (E) WB analysis of the p52 subunit in EBV-B cells not stimulated (NS) or stimulated with BAFF at a concentration of 100 ng/ml (BAFF). C is the positive control, P1 is patient 1 and BAFFR ^−/− is EBV-B cells from a BAFFR-deficient patient. “Fold induction” indicates densitometry results normalized with respect to GADPH levels, expressed as a fold induction over non stimulated control cells. GADPH was used as an internal control for WB. The panel illustrates results from a single experiment, representative of three. (F) IL-10 production, assessed by ELISA, in EBV-B cells, after BAFF activation (100 ng/ml). C is the positive control; P1 is patient 1 and BAFFR ^−/− is a BAFFR-deficient patient. Mean values ± SD were calculated from three independent experiments.