Impaired intrinsic immunity to HSV-1 in human iPSC-derived TLR3-deficient CNS cells

In the course of primary infection with herpes simplex virus 1 (HSV-1), children with inborn errors of TLR3 immunity are prone to HSV-1 encephalitis (HSE) 1–3. We tested the hypothesis that the pathogenesis of HSE involves non hematopoietic central nervous system (CNS)-resident cells. We derived induced pluripotent stem cells (iPSCs) from the dermal fibroblasts of TLR3- and UNC-93B-deficient patients and from controls. These iPSCs were differentiated into highly purified populations of neural stem cells (NSCs), neurons, astrocytes and oligodendrocytes. The induction of IFN-β and/or IFN-γ1 in response to poly(I:C) stimulation was dependent on TLR3 and UNC-93B in all cells tested. However, the induction of IFN-β and IFN-γ1 in response to HSV-1 infection was impaired selectively in UNC-93B-deficient neurons and oligodendrocytes. These cells were also much more susceptible to HSV-1 infection than control cells, whereas UNC-93B-deficient NSCs and astrocytes were not. TLR3-deficient neurons were also found to be susceptible to HSV-1 infection. The rescue of UNC-93B- and TLR3-deficient cells with the corresponding wild-type allele demonstrated that the genetic defect was the cause of the poly(I:C) and HSV-1 phenotypes. The viral infection phenotype was further rescued by treatment with exogenous IFN-α/β, but not IFN-γ1.Thus, impaired TLR3- and UNC-93B-dependent IFN-α/β intrinsic immunity to HSV-1 in the CNS, in neurons and oligodendrocytes in particular, may underlie the pathogenesis of HSE in children with TLR3 pathway deficiencies.

study. e-Immunocytochemical analysis of three-month-old cells two days after PDGFRα sorting. PDGFR + cell populations contained fewer O4 + OPCs than PDGFR-negative cells.
Suppl. Figure 5 Suppl. Figure 5. UNC-93Bdeficiency does not affect global gene expression profile in neurons and astrocytes, as shown by comparison with control cells differentiated from hESCs Clustering based on all the genes displaying significant differential expression with respect to hESCs. The gene expression profile in astrocytes and neurons differentiated from UNC-93B-deficient iPSCs line C (UNC-93B -/-) or control hESCs line H9 (H9) and that of control hESCs are shown. UNC-93B-deficiency did not significantly alter the overall pattern of gene expression in neurons and astrocytes derived from UNC-93B-deficient iPSCs, as shown by comparison with hESC-derived cells. Results for biological duplicates (for neurons and astrocytes) or triplicates (for undifferentiated H9 hESCs) are shown.
Student's t tests showed a significant induction of IFN-β, IFN-λ1, NFKB1 or MX1 in all cells from all the controls tested (p<0.05), but not in UNC-93B-deficient cells. One healthy control line 'C + iPSC 3' was used in a-, d-, eand h-. One healthy control line (C + iPSC 2) is used in b-, d-, f-, gand m-. Two healthy controls lines (C + iPSC 3 and C + iPSC 4) were used in k-and l-. Mean values + SEM calculated from the two iPSC controls are presented. The UNC-93B-deficient iPSC line 'UNC-93B -/-C' was used in experiments athrough n-.
Suppl. Figure 7 Suppl. Figure 7. HSV-1-GFP infection in central nervous system cells Representative images showing GFP expression in HSV-1-GFP infected healthy control central nervous system (CNS) cells. After 2 or 24 hours of infection with HSV-1-GFP at a MOI of 1, GFP expression in neurons, oligodendrocytes, astrocytes and NSCs differentiated from hESCs from a healthy control was visualized under a fluorescence microscope and shown. Phase-contrast photomicrographs of the NSCs, neurons, oligodendrocytes and astrocytes from the same field are also shown.
Suppl. Figure 8 Suppl. Figure 8. HSV-1-GFP infection in UNC-93B-deficient and control NSCs a-Representative images showing GFP expression in HSV-1-GFP infected NSCs from healthy controls or UNC-93B-deficient patient. After 2 and 24 hours of infection with HSV-1-GFP at a MOI of 1, GFP expression in NSCs differentiated from hESCs from a healthy control (C + ) (left panel) or from UNC-93B-deficient (UNC-93B -/-) iPSCs (right panel), with or without IFN-α2b, IFN-β or IFN-λ1 treatment, was visualized by fluorescence microcopy. Phase-contrast photomicrographs of the NSCs from the same field are also shown. The UNC-93B-deficient iPSC line 'UNC-93B -/-C' was used here for NSC differentiation for this experiment. b-After 24 hours of infection with various MOI of HSV-1-GFP, the GFP expression in NSCs differentiated from two lines of UNC-93B-deficient iPSCs (UNC-93B -/-C, UNC-93B -/-H), from iPSCs from two healthy controls, one line each (C + iPSC 3, C + iPSC 4), or from hESCs from a healthy control (C + (hESC)),without IFN treatment, was assessed with a fluorescence plate reader. The difference in GFP fluorescence intensity between HSV-1-GFP-infected cells and uninfected cells is shown. ANOVA and subsequent Dunnett t tests showed that GFP levels in UNC-93B-deficient NSCs were not significantly higher than those of the healthy controls tested. c-, d-, e-, f-After 24 hours of infection with various MOI of HSV-1-GFP, the GFP expression in NSCs differentiated from UNC-93B-deficient iPSCs, or from hESCs from a healthy control, without IFN treatment (c-) or upon treatment with IFN-α2b (d-), IFN-β (e-) or IFN-λ1 (f-), was assessed with a fluorescence plate reader. The difference in GFP fluorescence intensity between HSV-1-GFP-infected cells and uninfected cells is shown. In cto f-, the UNC-93B-deficient iPSC line 'UNC-93B -/-C' was used for NSC differentiation. For the data shown in b-, mean values + SEM were calculated from a single experiment, carried out in triplicate. For the data shown in c-, d-, eand f-, mean values + SEM were calculated from two independent experiments, each carried out in triplicate. Figure 9 Suppl. Figure 9. HSV-1-GFP infection in UNC-93B-deficient and control astrocytes a-Representative images showing GFP expression in HSV-1-GFP infected astrocytes from healthy controls or UNC-93B-deficient patient. After 2 and 24 hours of infection with HSV-1-GFP at a MOI of 1, GFP expression in astrocytes differentiated from hESCs from a healthy control (C + ) (left panel) or from UNC-93B-deficient iPSCs (UNC-93B -/-) (right panel), with or without IFN-α2b, IFN-β or IFN-λ1 treatment, was visualized by fluorescence microcopy. Phase-contrast photomicrographs of the astrocytes from the same field are also shown. Suppl. Figure 10 Suppl. Figure 10. Protective effect of poly(I:C) treatment against HSV-1-GFP infection in control but not UNC-93B-deficient astrocytes a-After 24 hours of infection with various MOI of HSV-1-GFP, GFP expression in astrocytes differentiated from UNC-93B-deficient iPSCs (UNC-93B -/-) or from hESCs from a healthy control (C + ), without IFN treatment, treated with IFN-α2b, poly(I:C), CpG-A or CpG-C, was measured with a fluorescence plate reader.

Suppl.
The difference in GFP fluorescence intensity between HSV-1-GFP-infected cells and uninfected cells is shown.
Mean values + SEM were calculated from two independent experiments, each carried out in triplicate. ANOVA was performed for data shown in a-, from UNC-93B -/or from C + astrocytes, without IFN treatment, treated with IFN-α2b or poly(I:C). When significant, Dunnett t tests were performed for 2X2 comparisons, and Suppl. Figure 12 Suppl. Figure 12. Responses to HSV-1 infection in neurons and glial cells a-, band c-IL-6 production, as measured by ELISA, in astrocytes (a-), neurons (b-) and oligodendrocytes (c-) differentiated from UNC-93B-deficient iPSCs (UNC-93B -/-), hESCs from a healthy control (C + (hESC)) and iPSCs from a healthy control (C + (iPSC)), after 24 hours of infection with HSV-1. Mean values + SEM were calculated from two independent experiments, each carried out in duplicate. d-, e-, IFN-λ1 mRNA induction, as measured by RT-qPCR, after 24 hours of HSV-1 infection, in NSCs (d-) and astrocytes (e-) differentiated from UNC-93B-deficient iPSCs (UNC-93B -/-), hESCs from a healthy control (C + hESC), or iPSCs from a healthy control (C + iPSC). f-IFN-β mRNA induction, as measured by RT-qPCR, after 24 hours of HSV-1 infection, in neurons differentiated from UNC-93B-deficient iPSCs (UNC-93B -/-), hESCs from a healthy control (C + hESC), or iPSCs from a healthy control (C + iPSC). g-IFN-λ1 mRNA induction, as measured by RT-qPCR, after 24 hours of HSV-1 infection, in oligodendrocytes differentiated from UNC-93B-deficient iPSCs (UNC-93B -/-), hESCs from a healthy control (C + hESC), or iPSCs from a healthy control (C + iPSC). For the data shown in dto g-, mean values + SEM were calculated from three independent experiments, each carried out in duplicate. h-MX1 mRNA induction, after 24 hours of stimulation with HSV-1, in oligodendrocytes differentiated from UNC-93B-deficient iPSCs line C (UNC-93B -/-), from hESCs from a healthy control (C + hESC), or from iPSCs from a healthy control (C + iPSC). Mean values + SEM were calculated from two independent experiments, each carried out in duplicate. In ato h-, UNC-93B-deficient iPSC line 'UNC-93B -/-C' and one hESC healthy control line were used for the differentiation of various types of CNS cells, the healthy control iPSC line 'C + iPSC 2' was used for the differentiation of neurons and oligodendrocytes, the healthy control iPSC line 'C + iPSC 3' was used for the differentiation of NSCs and astrocytes. ANOVA was performed for data shown in ato h-. When significant, Dunnett t tests were performed for 2X2 comparisons, and significant results are indicated in corresponding panels (* p<0.05, ** p<0.01). 4A-C, S10, S11 Rescue of HSV-1-GFP replication with IFN-α2b or IFN-β in NSCs and astrocytes line C iPSC 3, 4 and hESC H9 S10A, S10C-E, S11A, S11C-E, Rescue of HSV-1-GFP replication in control, but not in UNC93B -/astrocytes, with poly(I:C) line C iPSC 3,4 and hESC H9 S11F No rescue of HSV-1-GFP replication with IFN-λ1 in NSCs and astrocytes line C hESC H9 S10F, S11G CpG fails to reduce HSV-1 replication in astrocytes line C hESC H9 S12A-D 4H-K, S15 Note: Suppl. Note: * Total number of variant = total number of single nucleotide variations and indels.
* Intersection = number of variant calls common to the two call sets being compared.
* Concordance = percent genotype concordance of the intersection call set.
* Differences between the number of variants and concordance were checked by manual inspection of the alignments and result from: 1. Ambiguous alignments in repeat-rich and duplicated regions, leading to false positive variant calls.
2. Technical limitations in indel calling resulting in a higher error rate than for single-nucleotide variant calls.
3. Insufficient read coverage to make confident genotype calls Suppl. Table 3. List of genes carrying non reported exonic variants revealed by whole-exome sequencing in control or patient iPSC lines, or their parental fibroblasts. Only variants not common to iPSC lines and their respective parental fibroblasts are taken into account here.