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T cells promote microglia-mediated synaptic elimination and cognitive dysfunction during recovery from neuropathogenic flaviviruses

Nature Neuroscience volume 22, pages 1276–1288 (2019)Cite this article

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Abstract

T cells clear virus from the CNS and dynamically regulate brain functions, including spatial learning, through cytokine signaling. Here we determined whether hippocampal T cells that persist after recovery from infection with West Nile virus (WNV) or Zika virus (ZIKV) impact hippocampal-dependent learning and memory. Using newly established models of viral encephalitis recovery in adult animals, we show that in mice that have recovered from WNV or ZIKV infection, T cell-derived interferon-γ (IFN-γ) signaling in microglia underlies spatial-learning defects via virus-target-specific mechanisms. Following recovery from WNV infection, mice showed presynaptic termini elimination with lack of repair, while for ZIKV, mice showed extensive neuronal apoptosis with loss of postsynaptic termini. Accordingly, animals deficient in CD8+ T cells or IFN-γ signaling in microglia demonstrated protection against synapse elimination following WNV infection and decreased neuronal apoptosis with synapse recovery following ZIKV infection. Thus, T cell signaling to microglia drives post-infectious cognitive sequelae that are associated with emerging neurotropic flaviviruses.

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Fig. 1: ZIKV targets the hippocampus.
Fig. 2: ZIKV induces neuroinflammation and neuronal loss in the hippocampus.
Fig. 3: T cells express IFN-γ in the post-infected hippocampus.
Fig. 4: Loss of IFN-γ signaling protects animals against spatial-learning deficits.
Fig. 5: IFN-γ promotes neuronal apoptosis in ZIKV-infected animals post recovery.
Fig. 6: IFNGR is required for repair of presynaptic and postsynaptic termini in flavivirus-infected animals post recovery.
Fig. 7: IFN-γ potentiates microglial engulfment of postsynaptic termini and neurons in ZIKV-infected animals post recovery.
Fig. 8: Loss of IFN-γ signaling in microglia during recovery protects animals against spatial-learning deficits.

Data availability

The data from this study are tabulated in the main paper and supplementary materials. All reagents are available from R.S.K. under a material transfer agreement with Washington University. The data that support the findings of this study are also available from the corresponding author upon request.

References

  1. Murray, K. O. et al. Survival analysis, long-term outcomes, and percentage of recovery up to 8 years post-infection among the Houston West Nile virus cohort. PLoS One 9, e102953 (2014).

    Article  Google Scholar 

  2. Mehta, R. et al. The spectrum of neurological disease associated with Zika and chikungunya viruses in adults in Rio de Janeiro, Brazil: a case series. PLoS Negl. Trop. Dis. 12, e0006212 (2018).

    Article  Google Scholar 

  3. Shrestha, B., Gottlieb, D. & Diamond, M. S. Infection and injury of neurons by West Nile encephalitis virus. J. Virol. 77, 13203–13213 (2003).

    Article  CAS  Google Scholar 

  4. Ogata, A. et al. Japanese encephalitis virus neurotropism is dependent on the degree of neuronal maturity. J. Virol. 65, 880–886 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Lum, F. M. et al. Zika virus infects human fetal brain microglia and induces inflammation. Clin. Infect. Dis. 64, 914–920 (2017).

    Article  CAS  Google Scholar 

  6. Garcez, P. P. et al. Zika virus impairs growth in human neurospheres and brain organoids. Science 352, 816–818 (2016).

    Article  CAS  Google Scholar 

  7. Neunuebel, J. P. & Knierim, J. J. CA3 retrieves coherent representations from degraded input: direct evidence for CA3 pattern completion and dentate gyrus pattern separation. Neuron 81, 416–427 (2014).

    Article  CAS  Google Scholar 

  8. Hong, S. et al. Complement and microglia mediate early synapse loss in Alzheimer mouse models. Science 352, 712–716 (2016).

    Article  CAS  Google Scholar 

  9. Lui, H. et al. Progranulin deficiency promotes circuit-specific synaptic pruning by microglia via complement activation. Cell 165, 921–935 (2016).

    Article  CAS  Google Scholar 

  10. Vasek, M. J. et al. A complement–microglial axis drives synapse loss during virus-induced memory impairment. Nature 534, 538–543 (2016).

    Article  CAS  Google Scholar 

  11. Parkhurst, C. N. et al. Microglia promote learning-dependent synapse formation through brain-derived neurotrophic factor. Cell 155, 1596–1609 (2013).

    Article  CAS  Google Scholar 

  12. Sierra, A. et al. Microglia shape adult hippocampal neurogenesis through apoptosis-coupled phagocytosis. Cell Stem Cell 7, 483–495 (2010).

    Article  CAS  Google Scholar 

  13. Garber, C. et al. Astrocytes decrease adult neurogenesis during virus-induced memory dysfunction via IL-1. Nat. Immunol. 19, 151–161 (2018).

    Article  CAS  Google Scholar 

  14. Brochard, V. et al. Infiltration of CD4+ lymphocytes into the brain contributes to neurodegeneration in a mouse model of Parkinson disease. J. Clin. Invest 119, 182–192 (2009).

    CAS  PubMed  Google Scholar 

  15. Grebing, M. et al. Myelin-specific T cells induce interleukin-1beta expression in lesion-reactive microglial-like cells in zones of axonal degeneration. Glia 64, 407–424 (2016).

    Article  Google Scholar 

  16. Togo, T. et al. Occurrence of T cells in the brain of Alzheimer’s disease and other neurological diseases. J. Neuroimmunol. 124, 83–92 (2002).

    Article  CAS  Google Scholar 

  17. Paz, J., Yao, H., Lim, H. S., Lu, X. Y. & Zhang, W. The neuroprotective role of attractin in neurodegeneration. Neurobiol. Aging 28, 1446–1456 (2007).

    Article  CAS  Google Scholar 

  18. Filiano, A. J. et al. Unexpected role of interferon-gamma in regulating neuronal connectivity and social behaviour. Nature 535, 425–429 (2016).

    Article  CAS  Google Scholar 

  19. Kim, I. J., Beck, H. N., Lein, P. J. & Higgins, D. Interferon gamma induces retrograde dendritic retraction and inhibits synapse formation. J. Neurosci. 22, 4530–4539 (2002).

    Article  CAS  Google Scholar 

  20. Li, L., Walker, T. L., Zhang, Y., Mackay, E. W. & Bartlett, P. F. Endogenous interferon gamma directly regulates neural precursors in the non-inflammatory brain. J. Neurosci. 30, 9038–9050 (2010).

    Article  CAS  Google Scholar 

  21. Monteiro, S. et al. Absence of IFNgamma promotes hippocampal plasticity and enhances cognitive performance. Transl. Psychiatry 6, e707 (2016).

    Article  CAS  Google Scholar 

  22. Schenkel, J. M. et al. T cell memory. Resident memory CD8 T cells trigger protective innate and adaptive immune responses. Science 346, 98–101 (2014).

    Article  CAS  Google Scholar 

  23. Tripathi, S. et al. A novel Zika virus mouse model reveals strain specific differences in virus pathogenesis and host inflammatory immune responses. PLoS Pathog. 13, e1006258 (2017).

    Article  Google Scholar 

  24. Daffis, S. et al. 2′-O methylation of the viral mRNA cap evades host restriction by IFIT family members. Nature 468, 452–456 (2010).

    Article  CAS  Google Scholar 

  25. Durrant, D. M., Robinette, M. L. & Klein, R. S. IL-1R1 is required for dendritic cell-mediated T cell reactivation within the CNS during West Nile virus encephalitis. J. Exp. Med. 210, 503–516 (2013).

    Article  CAS  Google Scholar 

  26. Wakim, L. M., Woodward-Davis, A. & Bevan, M. J. Memory T cells persisting within the brain after local infection show functional adaptations to their tissue of residence. Proc. Natl Acad. Sci. USA 107, 17872–17879 (2010).

    Article  CAS  Google Scholar 

  27. Kaabinejadian, S. et al. Immunodominant West Nile virus T cell epitopes are fewer in number and fashionably late. J. Immunol. 196, 4263–4273 (2016).

    Article  CAS  Google Scholar 

  28. Mahad, D. et al. Modulating CCR2 and CCL2 at the blood–brain barrier: relevance for multiple sclerosis pathogenesis. Brain 129, 212–223 (2006).

    Article  Google Scholar 

  29. Eigenbrod, T., Bode, K. A. & Dalpke, A. H. Early inhibition of IL-1beta expression by IFN-gamma is mediated by impaired binding of NF-kappaB to the IL-1beta promoter but is independent of nitric oxide. J. Immunol. 190, 6533–6541 (2013).

    Article  CAS  Google Scholar 

  30. Ebner, F. et al. Microglial activation milieu controls regulatory T cell responses. J. Immunol. 191, 5594–5602 (2013).

    Article  CAS  Google Scholar 

  31. Goldmann, T. et al. Origin, fate and dynamics of macrophages at central nervous system interfaces. Nat. Immunol. 17, 797–805 (2016).

    Article  CAS  Google Scholar 

  32. Kreutzfeldt, M. et al. Neuroprotective intervention by interferon-gamma blockade prevents CD8+ T cell-mediated dendrite and synapse loss. J. Exp. Med 210, 2087–2103 (2013).

    Article  CAS  Google Scholar 

  33. Nowakowski, T. J. et al. Expression analysis highlights AXL as a candidate Zika virus entry receptor in neural stem cells. Cell Stem Cell 18, 591–596 (2016).

    Article  CAS  Google Scholar 

  34. Hastings, A. K. et al. TAM receptors are not required for Zika virus infection in mice. Cell Rep. 19, 558–568 (2017).

    Article  CAS  Google Scholar 

  35. Prieto, A. L., O’Dell, S., Varnum, B. & Lai, C. Localization and signaling of the receptor protein tyrosine kinase Tyro3 in cortical and hippocampal neurons. Neuroscience 150, 319–334 (2007).

    Article  CAS  Google Scholar 

  36. Ottum, P. A., Arellano, G., Reyes, L. I., Iruretagoyena, M. & Naves, R. Opposing roles of interferon-gamma on cells of the central nervous system in autoimmune neuroinflammation. Front. Immunol. 6, 539 (2015).

    Article  Google Scholar 

  37. Neher, J. J. et al. Phagocytosis executes delayed neuronal death after focal brain ischemia. Proc. Natl Acad. Sci. USA 110, E4098–E4107 (2013).

    Article  CAS  Google Scholar 

  38. Nakatomi, H. et al. Regeneration of hippocampal pyramidal neurons after ischemic brain injury by recruitment of endogenous neural progenitors. Cell 110, 429–441 (2002).

    Article  CAS  Google Scholar 

  39. Wang, X., Haroon, F., Karray, S., Martina, D. & Schluter, D. Astrocytic Fas ligand expression is required to induce T-cell apoptosis and recovery from experimental autoimmune encephalomyelitis. Eur. J. Immunol. 43, 115–124 (2013).

    Article  CAS  Google Scholar 

  40. Canty, A. J. et al. Synaptic elimination and protection after minimal injury depend on cell type and their prelesion structural dynamics in the adult cerebral cortex. J. Neurosci. 33, 10374–10383 (2013).

    Article  CAS  Google Scholar 

  41. Vilalta, A. & Brown, G. C. Neurophagy, the phagocytosis of live neurons and synapses by glia, contributes to brain development and disease. FEBS J 285, 3566–3575 (2018).

    Article  CAS  Google Scholar 

  42. Lian, H. et al. Astrocyte–microglia cross talk through complement activation modulates amyloid pathology in mouse models of Alzheimer’s disease. J. Neurosci. 36, 577–589 (2016).

    Article  CAS  Google Scholar 

  43. Savarin, C. et al. Astrocyte response to IFN-gamma limits IL-6-mediated microglia activation and progressive autoimmune encephalomyelitis. J. Neuroinflamm. 12, 79 (2015).

    Article  Google Scholar 

  44. Liddelow, S. A. et al. Neurotoxic reactive astrocytes are induced by activated microglia. Nature 541, 481–487 (2017).

    Article  CAS  Google Scholar 

  45. Nicastri, E., Castilletti, C., Balestra, P., Galgani, S. & Ippolito, G. Zika virus infection in the central nervous system and female genital tract. Emerg. Infect. Dis. 22, 2228–2230 (2016).

    Article  Google Scholar 

  46. Zucker, J. et al. Zika virus-associated cognitive impairment in adolescent, 2016. Emerg. Infect. Dis. 23, 1047–1048 (2017).

    Article  Google Scholar 

  47. Sejvar, J. J. The long-term outcomes of human West Nile virus infection. Clin. Infect. Dis. 44, 1617–1624 (2007).

    Article  Google Scholar 

  48. Borchardt, S. M., Feist, M. A., Miller, T. & Lo, T. S. Epidemiology of West Nile virus in the highly epidemic state of North Dakota, 2002–2007. Public Health Rep. 125, 246–249 (2010).

    Article  Google Scholar 

  49. Readhead, B. et al. Multiscale analysis of independent Alzheimer’s cohorts finds disruption of molecular, genetic, and clinical networks by human herpesvirus. Neuron 99, 64–82 e67 (2018).

    Article  CAS  Google Scholar 

  50. Unger, M. S. et al. Doublecortin expression in CD8+ T-cells and microglia at sites of amyloid-beta plaques: a potential role in shaping plaque pathology? Alzheimers Dement. 14, 1022–1037 (2018).

    Article  Google Scholar 

Download references

Acknowledgements

The authors acknowledge M. Diamond, J. Miner, A. Smith, L. Thackray, and K. Funk for helpful discussions and technical support. They also thank W. Beatty at the Molecular Microbiology Imaging facility at Washington University School of Medicine. This work was supported by NIH grants U19 AI083019, R01 NS052632, and R01 AI101400 to R.S.K.

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Author notes

  1. These authors contributed equally: Charise Garber and Allison Soung.

Authors and Affiliations

  1. Department of Internal Medicine, Washington University School of Medicine, St Louis, MO, USA

    Charise Garber, Allison Soung, Lauren L. Vollmer, Marlene Kanmogne, Aisling Last, Jasmine Brown & Robyn S. Klein

  2. Department of Pathology and Immunology, Washington University School of Medicine, St Louis, MO, USA

    Robyn S. Klein

  3. Department of Neuroscience, Washington University School of Medicine, St Louis, MO, USA

    Robyn S. Klein

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  1. Charise Garber
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Contributions

C.G., A.S., and R.S.K. designed the experiments. C.G., A.S., L.L.V., M.K., A.L., and J.B. performed experiments. C.G., A.S., L.L.V., M.K., and R.S.K analyzed the data. R.S.K., C.G., and A.S. wrote the paper. All authors read and edited the manuscript.

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Correspondence to Robyn S. Klein.

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The authors declare no competing interests.

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Journal peer review information: Nature Neuroscience thanks Ryuta Koyama, Susanna Rosi, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Integrated supplementary information

Supplementary Figure 1 ZIKV targets the hippocampus.

(a, b) ZIKV preferentially targets the hippocampus (white box). Additional representative images of in situ hybridization for viral RNA (RNA-ISH) at 7 dpi as in Fig 1e, (n=4 mice per group). (c, d) Neither WNV nor ZIKV targets astrocytes (GFAP+) or microglia (IBA1+) in the CA3 region of the hippocampus. (e, f) Neither WNV nor ZIKV targets neural progenitor cells (DCX+) but does infect NeuN+ cells in the dentate gyrus of the hippocampus. Data from one independent experiment. Scale bar 20 µm.

Supplementary Figure 2 Kinetics of immune cell infiltration to the hippocampus.

Cx3CR1GFPCCR2RFP reporter animals were infected with WNV or ZIKV at 8 weeks old. Cells were isolated from the hippocampus at indicated dpi. (a) Gating strategy to identify putative microglia (P1: CD45midCD11b+), macrophage (P2: CD45hiCD11b+), and lymphocyte (P3: CD45hiCD11b-) populations. (b, c) Quantification of indicated populations at 0, 7, and 25 dpi revealed infiltration of putative macrophage and lymphocyte populations by 7 dpi, the latter of which persisted in the hippocampus at 25 dpi. Data are representative of two independent experiments (n=3 mice per group, mean ± SD, b: p<0.0001, =0.0006, <0.0001 c: p<0.0001, =0.0020, 0.0140, <0.0001). (d) Additional gates were applied to P1 and P3 populations to assess MHC-II expression in CD45midCD11b+Cx3CR1+CCR2- microglia populations and CD103 expression in CD45hiCD11b- CD8+ T cells (quantification in main Figure 2). (e, f) CD45hiCD11b- CD8+ T cells (identified by gating strategy in a,d) enter the hippocampus by 7 dpi and persist to 25 dpi (n=3 mice per group, mean ± SD, e: p=0.0018, <0.0001, =0.0001, 0.0017 f: p=0.0012, 0.0043). Data was analyzed by two-way (b,c) or one-way (e,f) ANOVA, and corrected for multiple comparisons. *,P<0.05, **, P<0.005, ***, P<0.001, ****, P<0.0001.

Supplementary Figure 3 IFNγ+ T cells persist in the hippocampus.

Most of the IFNg+ cells in the hippocampus during recovery are CD8+ T cells, and these persist in the hippocampus for at least a month after viral clearance. IFNγ-Thy1.1 reporter mice were infected with ZIKV or WNV and cells were isolated from the hippocampus at 25 or 52 dpi for flow cytometric analysis. (a) Representative gating strategy to identify CD45+IFNg+ cells, which were further analyzed according to the gating strategy shown in main Fig. 3. (b, c) Data from main Fig. 3f, g represented as number of cells (n=4 mice per group, mean ± SD, b: p<0.0001, =0.0289, 0.0038, 0.0015 c: p=0.0106, 0.0052, 0.0056). (d, e) Analysis of CD45+IFNγ+ cells at 52 dpi showed that CD45hiCD11b- CD8+ T cells persist in the hippocampus at least one month after viral clearance. Data are representative of two independent experiments (d: n=4 mice per group; e: n=3 mice per group, mean ± SD, d: p=0.0001, 0.0107, <0.0001 e: p<0.0001, =0.0001, 0.0108). Data was analyzed by one-way ANOVA, and corrected for multiple comparisons. *,P<0.05, **, P<0.005, ***, P<0.001, ****, P<0.0001.

Supplementary Figure 4 Ifngr1-/- mice display similar clinical course and viral burden as WT mice.

(a-f) Loss of IFNγ signaling does not affect survival, weight loss, or clinical score. (a,d) Survival analysis (n indicated in the figure). (b,e) No difference in weight loss following infection, expressed as % of day 0 body weight (b: n=4 (WT Mock), 10 (WT WNV), 5 (Ifngr1-/- WNV) mice per group; e: n=20 (WT mock), 15 (WT ZIKV), 16 (Ifngr1-/- ZIKV) mice per group, mean ± SD, b: p=0.0001, 0.0002 e: p=0.0002, 0.0001). (c,f) No difference in clinical score following infection (n as in b,e, mean ± SD). (g-i) Loss of IFNγ signaling does not influence replication or clearance of infectious virus, with the exception of slight delay in the cerebellum. Viral burden was quantified via standard plaque assays. Data are pooled from two independent experiments (n=10 mice per group; mean ± SD, i: p=0.0040). Data was analyzed by log rank (Mantel-Cox) test (a,d) or two-way ANOVA (b,c,e-i) and corrected for multiple comparisons. **, P<0.005.

Supplementary Figure 5 Ifngr1-/- mice have slight decrease in CD103+CD8+ T cells, which express high levels of CCR2.

(a, b) Loss of IFNγ signaling led to a small but significant decrease in CD103 expression of CD45hiCD11b-CD8+ T cells isolated from the hippocampus at 25 dpi. Flow cytometric analysis of cells isolated from the hippocampus of Cx3CR1GFPCCR2RFP or Ifngr1-/-Cx3CR1GFPCCR2RFP animals as in Fig. 2e, f (gating strategy in Supplementary Fig 2). Data are pooled from two independent experiments (a: n=8 mice per group; b: n=3 (WT Mock, Ifngr1-/- Mock), 8 (WT WNV, Ifngr1-/- WNV) mice per group, mean ± SD, a: p<0.0001, =0.0060 b: p<0.0001, =0.0004). (c, d) Loss of IFNγ signaling did not influence CCR2 expression, which was high increased in CD45hiCD11b-CD8+ T cells isolated from the hippocampus at 25 dpi. Flow cytometric analysis of cells isolated from the hippocampus of Cx3CR1GFPCCR2RFP or Ifngr1-/-Cx3CR1GFPCCR2RFP animals as in Fig. 2e, f (gating strategy in Supplementary Fig 2). Data are pooled from two independent experiments (n=3 mice per group, mean ± SD, c: p<0.0001, d: p<0.0001). Data was analyzed by two-way ANOVA and corrected for multiple comparisons (a-d). **, P<0.005, ***, P<0.001, ****, P<0.0001.

Supplementary Figure 6 Decreases in post-synaptic terminals and increase in apoptotic neurons are observed during ZIKV acute infection and long-term recovery.

(a, c) ZIKV infection does not target glutamatergic termini (Vglut1) or GABAergic (GAD67) neurons at 25 dpi. Data are pooled from two independent experiments (n=7 mice per group, mean ± SD). (b) No difference in new neurons within the DG during recovery. Representative images of recovered hippocampus at 52 dpi, assessed after BrdU labeling during acute infection; BrdU (red), NeuN (green), DAPI (blue). Data from one experiment (n=4 mice, mean ± SD). Scale bar 50 µm. (d) ZIKV infection leads to decreased number of neurons at 52 dpi. More neurons were found in Ifngr1-/- recovered mice. Representative images of the recovered hippocampus, NeuN (green). Data are pooled from two independent experiments (n=4 (WT Mock, WT ZIKV, Ifngr1-/- ZIKV), 5 (Ifngr1-/- Mock) mice per group, mean ± SD, p<0.0001, =0.0094). Scale bar 20 µm. (e) WNV infection leads to the acute loss of synaptophysin+ pre-synaptic terminals. Data are pooled from 2 independent experiments (n=4 mice per group, mean ± SD). Scale bar 20 µm. (f-g) ZIKV infection leads to acute loss of homer1+ post-synaptic terminals, but preservation of synaptophysin+ pre-synaptic terminals (f) Representative immunostaining in the hippocampus at 7 dpi; Synaptophysin (red), DAPI (blue). Data are pooled from 2 independent experiments (n=4 mice per group, mean ± SD). Scale bar 10 µm. (g) Representative immunostaining in the hippocampus at 7 dpi; Homer1 (green), DAPI (blue). Data are pooled from 2 independent experiments (n=4 mice per group, mean ± SD, p=0.0199, 0.0367). Scale bar 10 µm. Data was analyzed by two-way ANOVA and corrected for multiple comparisons (a-g). *,P<0.05, **, P<0.005, ***, P<0.001.

Supplementary Figure 7 Significant differences in numbers of microglia in the hippocampus of WT and Ifngr1-/- WNV and ZIKV-recovered mice.

(a) Astrocyte expression of IL-1β was similarly elevated in WT and Ifngr1-/- mice. Representative immunostaining in the hippocampus; GFAP (green), IL-1β (red), DAPI (blue). Data are pooled from 3 independent experiments (n=9 (WT Mock), 4 (WT ZIKV, Ifngr1-/- Mock, Ifngr1-/- ZIKV) mice per group, mean ± SD, p<0.0001). Scale bar 50 µm. (b-e) ZIKV- and WNV-recovered animals demonstrate similar numbers of microglia in the hippocampus at 25 dpi. (b,c) No differences in the number of CD45midCD11b+Cx3CR1+CCR2- microglia were observed in cells isolated from the ZIKA- or WNV-recovered hippocampus of reporter mice at 25 dpi. Representative plots of CD45midCD11b+ cells. Data are representative of two independent experiments (n=3 mice per group, mean ± SD). (d,e) No differences in the number of Tmem119+ microglia were observed in the hippocampus of WT and Ifngr1-/- infected animals at 25 dpi. Representative immunostaining of Tmem119 (green) and DAPI (blue) in the hippocampus. Data are pooled from two independent experiments (d: n=5 (WT), 4 (Ifngr1-/-) mice per group; e: n=6 (WT), 8 (Ifngr1-/-) mice per group, mean ± SD). Data was analyzed by unpaired two-sided t test (a-d). (f,g) WNV or ZIKV infection induces increased expression of MHC-II in hippocampal microglia at 25 dpi. Representative plots from flow cytometric analysis of MHC-II expression in CD45midCD11b+Cx3CR1+CCR2- microglia isolated from the recovered hippocampus of infected WT and Ifngr1-/- animals. Data are pooled from two independent experiments (n=8 mice per group, mean ± SD, p<0.0001). Data was analyzed by two-way ANOVA and corrected for multiple comparisons (f,g). *,P<0.05, ***, P<0.001, ****, P<0.0001.

Supplementary Figure 8 Conditional deletion of IFNgR in Cx3CR1+ microglia.

Specific deletion of IFNgR in microglia was achieved as in Fig. 8a–g. (a, b) Flow cytometric analysis of CD45midCD11b+ microglia isolated from the hippocampus of Cx3CR1CreERIfngrfl/fl (Cre+) mice and Cx3CR1Cre-Ifngrfl/fl (Cre-) littermate controls after behavioral testing demonstrated significant deletion of IFNgR in Cre+ mice. Data are pooled from two independent experiments (a: n=4 (Cre- Mock, Cre+ ZIKV), 5 (Cre- ZIKV), 7 (Cre+ Mock) mice per group; b: n=5 (Cre- Mock, Cre+ Mock), 6 (Cre- WNV, Cre+ WNV) mice per group, mean ± SD, p<0.0001). (c) Analysis of peripheral myeloid cells isolated from the blood (PBMCs) and spleen revealed no difference in IFNgR expression between Cre+ and Cre- mice. Data are representative of two independent experiments (n=4 mice per group, mean ± SD). (d) Tamoxifen treatment did not alter IFNgR expression in CD45hiCD11b-CD8+ or CD45hiCD11b-CD4+ T cells isolated from the hippocampus of Cre+ and Cre- mice. Data are representative of two independent experiments (n=4 mice per group, mean ± SD). (e, f) Conditional deletion of IFNgR in microglia during recovery led to significant attenuation of MHC-II expression in microglia isolated from the hippocampus of Cre+ but not Cre- mice after behavioral testing. Data are pooled from two independent experiments (e: n=4 (Cre- Mock, Cre+ ZIKV), 5 (Cre- ZIKV, Cre+ Mock) mice per group; f: n=5 (Cre- Mock, Cre+ Mock, Cre+ WNV), 6 (Cre- WNV) mice per group, mean ± SD, e: p=0.0060, 0.0367 f: p=0.0182, 0.0369). Data was analyzed by two-way ANOVA and corrected for multiple comparisons (a,b,e,f) or by unpaired two-sided t test (c,d). *,P<0.05, **, P<0.005, ****, P<0.0001.

Supplementary Figure 9 T cell-derived IFNγ targets microglia to mediate neurocognitive dysfunction during recovery from WNV and ZIKV infection.

(a) WNV infects neurons throughout the CNS. Anti-viral T cells that persist in the CNS after viral recovery produce IFNγ. IFNγ targeting of microglia leads to the elimination of presynaptic termini, most prominently in the CA3 region of the hippocampus, and the development of spatial learning and memory deficits. (b) ZIKV preferentially targets neurons in the hippocampus. Like WNV, IFNγ producing T cells persist in the CNS after ZIKV recovery. Targeting microglia, IFNγ induced apoptosis of neurons and the elimination of post-synaptic termini throughout the hippocampus. ZIKV inflicted mice subsequently develop spatial learning and memory deficits.

Supplementary information

Supplementary Figures 1–9.

Reporting Summary

Supplementary Video 1

3D reconstruction from confocal Z-stack images of immunostaining of microglia engulfment of homer+ postsynaptic termini at 25 dpi: This video shows a 3D reconstruction from confocal Z-stack images of immunostaining of the postsynaptic marker, homer, and the microglia marker, IBA1, in hippocampus of ZIKV-infected mouse at 25 dpi.

Supplementary Video 2

3D reconstruction from confocal Z-stack images of immunostaining of microglia engulfment of NeuN+ perikarya at 25 dpi: This video shows a 3D reconstruction from confocal Z-stack images of immunostaining of the neural cell body marker, NeuN, and the microglia marker, IBA1, in hippocampus of ZIKV-infected mouse at 25 dpi.

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Garber, C., Soung, A., Vollmer, L.L. et al. T cells promote microglia-mediated synaptic elimination and cognitive dysfunction during recovery from neuropathogenic flaviviruses. Nat Neurosci 22, 1276–1288 (2019). https://doi.org/10.1038/s41593-019-0427-y

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