Inborn errors of TLR3- or MDA5-dependent type I IFN immunity in children with enterovirus rhombencephalitis

Figures

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  Figure 1.. Biallelic TLR3 or IFIH1 mutations in two patients with EVE. (A) Family pedigrees, showing segregation of the TLR3 and IFIH1 mutations. The black symbols indicate patients. (B) Brain imaging showing EVE lesions in P1 and P2. In P1, post-contrast T1-FLAIR imaging showed pathological tegmental hypersignal (yellow arrows) associated with cervical myelitis, highly suggestive of rhombencephalitis. In P2, post-contrast T1-FLAIR imaging showed pathological tegmental hypersignal with evidence of diffusion restriction in the superior cerebellar peduncles and the ponto-bulbar junction (yellow arrows), suggestive of rhombencephalitis. (C) Confirmation of the mutations by Sanger sequencing. The single nucleotide substitutions for each patient relative to the sequence for a healthy control are indicated by red squares. (D) Schematic diagram of the structure of the human TLR3 gene and protein, featuring the leader sequence (L), leucine-rich repeats (LRRs), transmembrane domain (TM), linker region (LR), and Toll/IL-1 receptor (TIR) domain. Roman numerals indicate the coding exons. Mutations previously identified in patients with HSE (E110K, L297V, L360P, P554S, G743D, E746*, R811I, R867Q), IAV…

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  Figure S1.. Biallelic TLR3 or IFIH1 mutations in two patients with EVE. (A) Brain imaging showing EVE lesions in P1 and P2. In P1, post-contrast T1-FLAIR imaging showed pathological tegmental hypersignal (yellow arrows) associated with cervical myelitis, highly suggestive of rhombencephalitis. In P2, post-contrast T1-FLAIR imaging showed pathological tegmental hypersignal, with evidence of diffusion restriction in the superior cerebellar peduncles and the ponto-bulbar junction (yellow arrows), suggestive of rhombencephalitis. (B) Tests for IgG antibodies against various viruses were performed at the age of 8 yr for P1, and at the age of 5 yr for P2. The threshold value for each viral serological test is indicated. N, negative; NA, not applicable. (C) Alignment of TLR3 protein sequences around the D280 residue, across species.

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  Figure 2.. Production and function of the mutant forms of TLR3 of P1 in vitro. (A) TLR3 mRNA levels were determined by RT-qPCR in P2.1 TLR3-deficient fibrosarcoma cells, without transfection, or after transfection with an empty vector (Vec), HA-tagged TLR3 WT, D280N, or F322fs2*. TLR3 W769* and R867Q were used as experimentally confirmed loss-of-function or hypomorphic control mutants. (B) TLR3 was detected by immunoblotting. P2.1 cells, either untransfected (NT) or stably transfected with empty vector (Vec), HA-tagged WT or mutant TLR3 were immunoblotted with a C-terminal (C) HA-specific antibody or N-terminal (N) TLR3-specific antibody. GAPDH was used as a loading control. (C and D) RT-qPCR for IFNL1 (C) or IFNB (D) mRNA induction without stimulation (NS) or after 4 h of stimulation with 25 µg/ml poly(I:C) (PIC-4h) in P2.1 cells not transfected (NT) or stably transfected with empty vector (Vec), HA-tagged WT or mutant TLR3. (A, C, and D) Mean values ± SD were calculated from four independent experiments. P values were obtained by one-way ANOVA and subsequent Tamhane’s T2’s multiple comparison tests, and the respective P values are indicated. ns, P > 0.05; *, P < 0.05; ***, P < 0.001.

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  Figure S2.. Function of P1’s TLR3 mutants in vitro. RT-qPCR for IFNB and IFNL1 mRNA levels, without stimulation (NS) or after 2 and 4 h of stimulation with 25 µg/ml poly(I:C) (PIC), in P2.1 cells not transfected (NT) or stably transfected with empty vector (Vec), HA-tagged TLR3 WT, D280N, or F322fs2*. VSV M51R, at an MOI of 1, was used as a positive control for IFN induction by TLR3-independent pathways.

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  Figure 3.. Impairment of TLR3-dependent extracellular poly(I:C) responsiveness in P1’s SV40-fibroblasts and rescue by WT TLR3. (A) TOPO-TA cloning of TLR3 cDNA from P1’s SV40-fibroblasts and primary fibroblasts, and comparison with that from a healthy control. The percentage of the TLR3 sequences in cDNA clones from P1, 101 clones from SV40-fibroblasts, and 101 clones from primary fibroblasts that contained the D280N and F322fs2* mutations is shown, and compared with that for clones isolated in a similar manner from healthy control cells. (B) Production of IFN-β, IFN-λ, and IL-6 by SV40-fibroblasts from three healthy controls (C1, C2, C3), P1, TLR3 P554S/WT, TLR3 G743D+R811I/WT, or TLR3−/− patients, 24 h after stimulation with 1, 5, or 25 µg/ml poly(I:C) (PIC), and 25 µg/ml poly(I:C) in the presence of Lipofectamine (Lipo; PIC25+Lipo), or Lipo alone, as assessed by ELISA. (C) IFNB and IFNL1 mRNA levels in SV40-fibroblasts from two controls (C1, C2), P1, TLR3 P554S/WT, TLR3 G743D+R811I/WT, or TLR3−/− patients, not stimulated (NS), stimulated for 2 and 4 h with 25 µg/ml poly(I:C), or infected with VSV M51R at an MOI of 1 for 18 h. GUS was included for normalization. (D) IFNB and IFNL1 mRNA…

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  Figure 4.. Impairment of MDA5-dependent intracellular poly(I:C) responsiveness in P2’s fibroblasts and rescue by WT MDA5. (A) IFIH1 PCR products from fibroblast cDNA, from P2 and two healthy controls (C1, C2), amplified with a forward primer binding to part of exon 6 and a reverse primer binding to part of exon 10. The result shown is representative of two independent experiments. (B) Sanger sequencing results for IFIH1 from P2’s fibroblast cDNA. (C) cDNA TOPO cloning and sequencing results demonstrating completely aberrant splicing of IFIH1 in fibroblasts from P2. At least 50 transcripts were sequenced for the patient and the control. The result shown is the sum of two independent experiments. (D) IFIH1 mRNA levels in SV40-fibroblasts from two healthy controls (C1, C2), P2, and MDA5 KO SV40-fibroblast, with primers spanning exons 5–6, exons 8–9, and exons 9–10, respectively; GUS was used as an expression control. Mean values and SD from three independent experiments, each with technical duplicates, are shown. (E) Western blot for MDA5 in SV40-fibroblasts from two healthy controls (C1, C2), P2, and MDA5 KO SV40-fibroblasts. One antibody recognizing aa 78–555 of MDA5 (M) and another…

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  Figure 5.. Impaired basal or virus-induced IFN production in TLR3- or MDA5-deficient fibroblasts. (A and B) mRNA levels for IFNB (A) and IFNL1 (B) in unstimulated SV40-fibroblasts from three healthy controls (Ctrls), P1, P2, a TLR3−/− patient, and MDA5 KO SV40-fibroblasts, as quantified by RT-qPCR. (C and D) mRNA levels for IFNB and IFNL1, as assessed by RT-qPCR, in SV40-fibroblasts from three healthy controls (Ctrls), P1, P2, a TLR3−/− patient, and MDA5 KO SV40-fibroblasts, without viral infection, or following 24 h of infection with EV30 (C) or EV71 (D) at an MOI of 5. Data shown are from five independent experiments, with two biological replicates from each experiment (A and B), or three biological replicates from each experiment for the controls and one biological sample from each experiment for the patient-specific cell lines (C and D). Mean values ± SD were calculated from five independent experiments. P values were obtained by one-way ANOVA and subsequent least significant difference multiple comparison tests (A and B) or paired samples t test (C and D). ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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  Figure S3.. ISG induction in TLR3- or MDA5-deficient fibroblasts following EV30 or EV71 infection. (A and B) mRNA levels for CXCL10, MX1, OAS1, and ISG15 in SV40-fibroblasts from three healthy controls (Ctrls), P1, P2, a TLR3−/− patient, and MDA5 KO SV40-fibroblasts, as quantified by RT-qPCR following 24 h of infection with EV30 (A) or EV71 (B) at an MOI of 5. Mean values ± SD were calculated from three independent experiments. P values were obtained by paired samples t test. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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  Figure 6.. Enhanced susceptibility to EV30 and EV71 in fibroblasts with TLR3 and MDA5 mutations, and rescue by WT TLR3 or MDA5 or by IFN-α2b treatment. (A–D) Replication of EV30 (A and C) and EV71 (B and D), quantified by viral titration, in SV40-fibroblasts from three healthy controls (Ctrls), P1, P2, a TLR3−/− patient, MDA5 KO SV40-fibroblasts, and SV40-fibroblasts from an IFNAR1−/− patient, following infection with EV30 or EV71 at an MOI of 0.1, at various time points. IFN-α2b treatment was initiated 18 h before infection, then removed upon or continued during infection (A and B), or was started 4 h or 9 h after infection (C and D). (E) EV30 replication was assessed by viral titration, in SV40-fibroblasts from P1 that were left untransfected, or were transfected with an empty vector (Vec), TLR3 WT, or TLR3 D280N or F322fs2*, at various time points after infection, at an MOI of 0.1. (F) EV71 replication was evaluated by viral titration in SV40-fibroblasts from three healthy controls (Ctrls), or from P2 after stable transduction with WT MDA5 or empty vector (Vec), at various time points after infection at an MOI of 0.1. (A–F) Mean values ± SD from at least two independent experiments are…

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  Figure S4.. Measurement of HSV-1 susceptibility and EV-induced IFN production in human SV40-fibroblasts. (A and B) Levels of HSV-1 replication (A) and cell viability (B) were assessed for SV40-fibroblasts from three healthy controls (Ctrls), P1, P2, a TLR3−/− patient, MDA5 KO SV40-fibroblasts, and an IFNAR1−/− patient, at various time points after infection with HSV-1 at an MOI of 0.001. NI, not infected. (C) Levels of IFNB and IFNL1 mRNA, in SV40-fibroblasts from one healthy control, at various time points after infection with EV30 or EV71 at an MOI of 5. NS, not stimulated. Mean values ± SD were calculated from three independent experiments (A–C).