Impaired intrinsic immunity to HSV-1 in human iPSC-derived TLR3-deficient CNS cells - PubMed (original) (raw)

. 2012 Nov 29;491(7426):769-73.

doi: 10.1038/nature11583. Epub 2012 Oct 28.

Itai M Pessach, Shen-Ying Zhang, Michael J Ciancanelli, Melina Herman, Avinash Abhyankar, Shui-Wang Ying, Sotirios Keros, Peter A Goldstein, Gustavo Mostoslavsky, Jose Ordovas-Montanes, Emmanuelle Jouanguy, Sabine Plancoulaine, Edmund Tu, Yechiel Elkabetz, Saleh Al-Muhsen, Marc Tardieu, Thorsten M Schlaeger, George Q Daley, Laurent Abel, Jean-Laurent Casanova, Lorenz Studer, Luigi D Notarangelo

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

Fabien G Lafaille et al. Nature. 2012.

Abstract

In the course of primary infection with herpes simplex virus 1 (HSV-1), children with inborn errors of toll-like receptor 3 (TLR3) immunity are prone to HSV-1 encephalitis (HSE). We tested the hypothesis that the pathogenesis of HSE involves non-haematopoietic 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 interferon-β (IFN-β) and/or IFN-λ1 in response to stimulation by the dsRNA analogue polyinosinic:polycytidylic acid (poly(I:C)) was dependent on TLR3 and UNC-93B in all cells tested. However, the induction of IFN-β and IFN-λ1 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 showed that the genetic defect was the cause of the poly(I:C) and HSV-1 phenotypes. The viral infection phenotype was rescued further by treatment with exogenous IFN-α or IFN-β ( IFN-α/β) but not IFN-λ1. 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.

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Figures

Figure 1

Figure 1. Derivation and purification of CNS cells

a- Schematic diagram of the differentiation and purification protocols used. b- Representative phase-contrast images showing the morphology of the hPSCs, neural rosettes and NPC clusters, from healthy control hESCs, healthy control iPSCs, and UNC-93B−/− iPSCs. Immunocytochemistry analysis revealed the expression of rosette markers (PLZF/ZO1) and a forebrain marker FOXG1. c- Characterization of UNC-93B−/− iPSC-derived CNS cell types:

Upper panels

: phase-contrast images illustrating the characteristic morphology of each type of neural cell;

Lower panels

: immunofluorescence analysis for markers of neural stem cells (nestin), neurons (Tuj1), oligodendrocyte progenitor cells (O4) and astrocytes (GFAP). **d-**Quantification of marker expression for each neural cell type derived from control hESCs, UNC-93B−/− iPSCs, and control iPSCs (error bars = SEM). * Scale bars in b- correspond to 100 μm; those in c- correspond to 50 μm, except for O4 staining (100 μm).

Figure 2

Figure 2. UNC-93B-dependent IFN responses to TLR3 in neurons and glial cells

a- UNC93B1 mRNA levels in CNS cells differentiated from hESCs from a healthy control (C+ (hESC)) and iPSCs from a healthy control (C+ (iPSC)) and an UNC-93B-deficient patient (UNC-93B−/−). b-, c- IFN-β (b-) or IFN-γ1 (c-) mRNA induction, after six hours of poly(I:C) stimulation, in neurons (b-) or oligodendrocytes (c-) differentiated from one healthy control hESC line, one healthy control iPSC line, and in UNC-93B−/− neurons (b-) or oligodendrocytes (c-), without lentiviral infection, or after infection with a lentivirus containing human WT UNC93B1 (UNC-93B−/− + UNC93B1) or a mock construct (UNC-93B−/− + mock). d- IFN-β (upper panel) or IFN-γ1 (lower panel) mRNA induction, after four (upper panel) or six (lower panel) hours of poly(I:C) stimulation, in astrocytes differentiated from hESCs from a healthy control, in UNC-93B−/− astrocytes, without lentiviral infection, or after infection with a lentivirus containing human WT UNC93B1 (UNC-93B−/− + UNC93B1) or a mock construct (UNC-93B−/− + mock). In a- to d-, Mean values ± SEM were calculated from three (a-) or two (b–d) independent experiments, each carried out in duplicate. ANOVA was performed for data shown in a-. When significant, Dunnett t tests were performed for 2X2 comparisons, significant results are indicated in corresponding panels (* p<0.05, ** p<0.01).

Figure 3

Figure 3. High HSV-1 susceptibility in UNC-93B-deficient neurons and oligodendrocytes

24 hours of infection with HSV-1-GFP, at a multiplicity of infection of 1, was performed in experiments shown. a- GFP expression in CNS cells differentiated from UNC-93B−/− iPSCs or from hESCs from a healthy control (C+) was visualized by fluorescence microscopy. Phase-contrast photomicrographs from the same view are also shown. b-, c-, d- GFP expression in NSCs (b-), astrocytes (c-) and neurons (d-) differentiated from UNC-93B−/− iPSCs, from hESCs from a healthy control (C+ (hESC)) and one to two iPSC lines each from up to three healthy controls (C_+_ (iPSC)), was assessed with a fluorescence plate reader. The difference in GFP intensity between HSV-1-GFP-infected cells and uninfected cells is shown. e- GFP expression in neurons differentiated from two lines of UNC-93B−/− iPSCs, one line of TLR3−/− iPSCs, one or two iPSC lines each from three healthy controls, or from one C+ hESCs line. f- GFP expression, in neurons from one C+ iPSC line, in UNC-93B−/− neurons, without lentiviral infection, or after infection with a lentivirus containing human WT UNC93B1 (UNC-93B−/− + UNC93B1) or a mock construct (UNC-93B−/− + Mock). g- GFP expression in oligodendrocytes differentiated from UNC-93B−/− iPSCs, from a C+ hESC line and a C+ iPSC line. Error bars = SEM, calculated from two to three experiments, from the C+ hESC, from the C+ iPSC lines and from the UNC-93B−/− lines, each studied in triplicate (b-, c-, d-, e- and g-); or from one experiment carried out in triplicate, representative of two independent experiments (f-). ANOVA was performed for the data shown in b- to g-. When significant, Dunnett t tests were performed for 2X2 comparisons, significant results are indicated in corresponding panels (* p<0.05, ** p<0.01, *** p<0.001).

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