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

This article has been updated


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)1,2,3. 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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Derivation and purification of CNS cells.
Figure 2: UNC-93B-dependent IFN responses to TLR3 in neurons and glial cells.
Figure 3: High HSV-1 susceptibility in UNC-93B-deficient neurons and oligodendrocytes.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

The transcriptome data have been deposited in the Gene Expression Omnibus database under accession number GSE40593.

Change history

  • 28 November 2012

    Minor typographical corrections were made to Figs 1a and 3e.


  1. 1

    Casrouge, A. et al. Herpes simplex virus encephalitis in human UNC-93B deficiency. Science 314, 308–312 (2006)

    ADS  CAS  Article  Google Scholar 

  2. 2

    Zhang, S. Y. et al. TLR3 deficiency in patients with herpes simplex encephalitis. Science 317, 1522–1527 (2007)

    ADS  CAS  Article  Google Scholar 

  3. 3

    Guo, Y. et al. Herpes simplex virus encephalitis in a patient with complete TLR3 deficiency: TLR3 is otherwise redundant in protective immunity. J. Exp. Med. 208, 2083–2098 (2011)

    CAS  Article  Google Scholar 

  4. 4

    Whitley, R. J. Herpes simplex encephalitis: adolescents and adults. Antiviral Res. 71, 141–148 (2006)

    ADS  CAS  Article  Google Scholar 

  5. 5

    Abel, L. et al. Age-dependent Mendelian predisposition to herpes simplex virus type 1 encephalitis in childhood. J. Pediatr. 157, 623–629 (2010)

    Article  Google Scholar 

  6. 6

    Kim, Y. M., Brinkmann, M. M., Paquet, M. E. & Ploegh, H. L. UNC93B1 delivers nucleotide-sensing toll-like receptors to endolysosomes. Nature 452, 234–238 (2008)

    ADS  CAS  Article  Google Scholar 

  7. 7

    Pérez de Diego, R. et al. Human TRAF3 adaptor molecule deficiency leads to impaired Toll-like receptor 3 response and susceptibility to herpes simplex encephalitis. Immunity 33, 400–411 (2010)

    Article  Google Scholar 

  8. 8

    Sancho-Shimizu, V. et al. Herpes simplex encephalitis in children with autosomal recessive and dominant TRIF deficiency. J. Clin. Invest. 121, 4889–4902 (2011)

    CAS  Article  Google Scholar 

  9. 9

    Herman, M. et al. Heterozygous TBK1 mutations impair TLR3 immunity and underlie herpes simplex encephalitis of childhood. J. Exp. Med. 209, 1567–1582 (2012)

    CAS  Article  Google Scholar 

  10. 10

    Jacquemont, B. & Roizman, B. RNA synthesis in cells infected with herpes simplex virus. X. Properties of viral symmetric transcripts and of double-stranded RNA prepared from them. J. Virol. 15, 707–713 (1975)

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11

    Weber, F., Wagner, V., Rasmussen, S. B., Hartmann, R. & Paludan, S. R. Double-stranded RNA is produced by positive-strand RNA viruses and DNA viruses but not in detectable amounts by negative-strand RNA viruses. J. Virol. 80, 5059–5064 (2006)

    CAS  Article  Google Scholar 

  12. 12

    Bsibsi, M., Ravid, R., Gveric, D. & van Noort, J. M. Broad expression of Toll-like receptors in the human central nervous system. J. Neuropathol. Exp. Neurol. 61, 1013–1021 (2002)

    CAS  Article  Google Scholar 

  13. 13

    Préhaud, C., Megret, F., Lafage, M. & Lafon, M. Virus infection switches TLR-3-positive human neurons to become strong producers of beta interferon. J. Virol. 79, 12893–12904 (2005)

    Article  Google Scholar 

  14. 14

    Jack, C. S. et al. TLR signaling tailors innate immune responses in human microglia and astrocytes. J. Immunol. 175, 4320–4330 (2005)

    CAS  Article  Google Scholar 

  15. 15

    Zhou, L. et al. Activation of Toll-like receptor-3 induces interferon-lambda expression in human neuronal cells. Neuroscience 159, 629–637 (2009)

    CAS  Article  Google Scholar 

  16. 16

    Mitchell, B. M., Bloom, D. C., Cohrs, R. J., Gilden, D. H. & Kennedy, P. G. Herpes simplex virus-1 and varicella-zoster virus latency in ganglia. J. Neurovirol. 9, 194–204 (2003)

    CAS  Article  Google Scholar 

  17. 17

    Lokensgard, J. R. et al. Robust expression of TNF-alpha, IL-1beta, RANTES, and IP-10 by human microglial cells during nonproductive infection with herpes simplex virus. J. Neurovirol. 7, 208–219 (2001)

    CAS  Article  Google Scholar 

  18. 18

    Bello-Morales, R., Fedetz, M., Alcina, A., Tabares, E. & Lopez-Guerrero, J. A. High susceptibility of a human oligodendroglial cell line to herpes simplex type 1 infection. J. Neurovirol. 11, 190–198 (2005)

    CAS  Article  Google Scholar 

  19. 19

    Marques, C. P., Hu, S., Sheng, W. & Lokensgard, J. R. Microglial cells initiate vigorous yet non-protective immune responses during HSV-1 brain infection. Virus Res. 121, 1–10 (2006)

    CAS  Article  Google Scholar 

  20. 20

    Pessach, I. M. et al. Induced pluripotent stem cells: a novel frontier in the study of human primary immunodeficiencies. J. Allergy Clin. Immunol. 127, 1400–1407 (2011)

    CAS  Article  Google Scholar 

  21. 21

    Chambers, S. M. et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nature Biotechnol. 27, 275–280 (2009)

    CAS  Article  Google Scholar 

  22. 22

    Kriks, S. et al. Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease. Nature 480, 547–551 (2011) 10.1038/nature10648

    ADS  CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. 23

    Elkabetz, Y. et al. Human ES cell-derived neural rosettes reveal a functionally distinct early neural stem cell stage. Genes Dev. 22, 152–165 (2008)

    CAS  Article  Google Scholar 

  24. 24

    Tabar, V. et al. Migration and differentiation of neural precursors derived from human embryonic stem cells in the rat brain. Nature Biotechnol. 23, 601–606 (2005)

    CAS  Article  Google Scholar 

  25. 25

    Jackson, A. C., Rossiter, J. P. & Lafon, M. Expression of Toll-like receptor 3 in the human cerebellar cortex in rabies, herpes simplex encephalitis, and other neurological diseases. J. Neurovirol. 12, 229–234 (2006)

    CAS  Article  Google Scholar 

  26. 26

    Farina, C. et al. Preferential expression and function of Toll-like receptor 3 in human astrocytes. J. Neuroimmunol. 159, 12–19 (2005)

    CAS  Article  Google Scholar 

  27. 27

    Taupin, P. & Gage, F. H. Adult neurogenesis and neural stem cells of the central nervous system in mammals. J. Neurosci. Res. 69, 745–749 (2002)

    CAS  Article  Google Scholar 

  28. 28

    Desai, P. & Person, S. Incorporation of the green fluorescent protein into the herpes simplex virus type 1 capsid. J. Virol. 72, 7563–7568 (1998)

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Reinert, L. S. et al. TLR3 deficiency renders astrocytes permissive to herpes simplex virus infection and facilitates establishment of CNS infection in mice. J. Clin. Invest. 122, 1368–1376 (2012)

    CAS  Article  Google Scholar 

  30. 30

    Bieniasz, P. D. Intrinsic immunity: a front-line defense against viral attack. Nature Immunol. 5, 1109–1115 (2004)

    CAS  Article  Google Scholar 

  31. 31

    Park, I. H. et al. Disease-specific induced pluripotent stem cells. Cell 134, 877–886 (2008)

    CAS  Article  Google Scholar 

  32. 32

    Barberi, T. et al. Neural subtype specification of fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice. Nature Biotechnol. 21, 1200–1207 (2003)

    CAS  Article  Google Scholar 

  33. 33

    Perrier, A. L. et al. Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc. Natl Acad. Sci. USA 101, 12543–12548 (2004)

    ADS  CAS  Article  Google Scholar 

  34. 34

    Somers, A. et al. Generation of transgene-free lung-disease specific human iPS cells using a single excisable lentiviral stem cell cassette. Stem Cells 28, 1728–1740 (2010)

    CAS  Article  Google Scholar 

  35. 35

    Mostoslavsky, G., Fabian, A. J., Rooney, S., Alt, F. W. & Mulligan, R. C. Complete correction of murine Artemis immunodeficiency by lentiviral vector-mediated gene transfer. Proc. Natl Acad. Sci. USA 103, 16406–16411 (2006)

    ADS  CAS  Article  Google Scholar 

  36. 36

    Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009)

    CAS  Article  Google Scholar 

  37. 37

    McKenna, A. et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20, 1297–1303 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009)

    Article  Google Scholar 

  39. 39

    Ying, S. W. & Goldstein, P. A. Propofol-block of SK channels in reticular thalamic neurons enhances GABAergic inhibition in relay neurons. J. Neurophysiol. 93, 1935–1948 (2005)

    CAS  Article  Google Scholar 

  40. 40

    Ying, S. W. et al. Dendritic HCN2 channels constrain glutamate-driven excitability in reticular thalamic neurons. J. Neurosci. 27, 8719–8732 (2007)

    CAS  Article  Google Scholar 

  41. 41

    Park, I. H. et al. Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451, 141–146 (2008)

    ADS  CAS  Article  Google Scholar 

  42. 42

    Yang, K. et al. Human TLR-7-, -8-, and -9-mediated induction of IFN-alpha/beta and -lambda Is IRAK-4 dependent and redundant for protective immunity to viruses. Immunity 23, 465–478 (2005)

    CAS  Article  Google Scholar 

Download references


We thank our patients, their families and physicians; and the members of the three laboratories for helpful discussions and critical reading of this manuscript. The work was funded by grant number 8UL1TR000043 from the National Center for Translational Sciences (NCATS), the National Institutes of Health (NIH), the Rockefeller University, the St. Giles Foundation, the ANR, INSERM, Paris Descartes University, the March of Dimes, NIH grant 5R01NS072381-02 (to J.-L.C., L.S. and L.D.N.), NIH grant 1R03AI0883502-01 (to L.D.N.), NIH grant 1R01NS066390 and the Manton Foundation, the Israeli Centers of Research Excellence (I-CORE), and Gene Regulation in Complex Human Disease, Center No 41/11 (to I.M.P.). F.G.L. is supported by the New York Stem Cell Foundation.

Author information




F.G.L., I.M.P., S.-Y.Z., J.-L.C., L.S. and L.D.N. designed the experiments. F.G.L., I.M.P., S.-Y.Z., M.J.C., M.H., A. A., G.M., S.-W.Y., S.K., P.A.G, J.O.-M., E.J., E.T., Y.E. and T.M.S. carried out the experiments. S.A. and M.T. helped to obtain materials from patients and interpret the findings. G.Q.D. and L.A. helped to analyse and describe the data. S.-Y.Z. and J.-L.C. wrote the manuscript with the aid of F.G.L., I.M.P., L.S. and L.D.N. F.G.L., I.M.P. and S.-Y. Zhang are equal first authors. J.L.C., L.S. and L.D.N. are co-senior authors.

Corresponding authors

Correspondence to Shen-Ying Zhang or Jean-Laurent Casanova.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-12, Supplementary Tables 1-4 and Supplementary References. (PDF 5803 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Lafaille, F., Pessach, I., Zhang, S. et al. Impaired intrinsic immunity to HSV-1 in human iPSC-derived TLR3-deficient CNS cells. Nature 491, 769–773 (2012).

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing