Inhibition of innate immune response ameliorates Zika virus-induced neurogenesis deficit in human neural stem cells

Abstract

Global Zika virus (ZIKV) outbreaks and their strong link to microcephaly have raised major public health concerns. ZIKV has been reported to affect the innate immune responses in neural stem/progenitor cells (NS/PCs). However, it is unclear how these immune factors affect neurogenesis. In this study, we used Asian-American lineage ZIKV strain PRVABC59 to infect primary human NS/PCs originally derived from fetal brains. We found that ZIKV overactivated key molecules in the innate immune pathways to impair neurogenesis in a cell stage-dependent manner. Inhibiting the overactivated innate immune responses ameliorated ZIKV-induced neurogenesis reduction. This study thus suggests that orchestrating the host innate immune responses in NS/PCs after ZIKV infection could be promising therapeutic approach to attenuate ZIKV-associated neuropathology.

Fig 1. An in vitro hNS/PCs culture system to study ZIKV infection.

(A-C) hNS/PCs are derived from human fetal brains. They proliferate to form neurospheres. After a 4-day priming and 9-day differentiation, they can generate neurons (Tuj1, red) and astrocytes (GFAP, green). Blue, nuclear counterstain. Scale bars: (B) 100 μm, (C) 20 μm. (D) ZIKV replicates rapidly in the proliferating hNS/PC lines. Culture medium was collected on Days 1, 3, 5, 7, 9 and 12 for plaque assay. Representative images of plaques (10⁻¹ dilution of the medium) are shown on the left. Data are presented as mean ± SD (n = 2).

Fig 2. ZIKV reduced neurogenesis in hNS/PCs during early stage infection.

(A) The in vitro system to study ZIKV infection at the different developmental stages of hNS/PCs. (B) Newly generated neurons were stained by Tuj1 (red). Blue, nuclear counterstain. Scale bars: 20 μm. (C) Quantification data are presented as mean ± SD (n = 4), * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, one-way ANOVA with a Tukey post-hoc test.

Fig 3. ZIKV infection in hNS/PC-generated astrocytes and neurons.

(A) Newly generated astrocytes were stained by GFAP (red). ZIKV was stained by anti-ZIKV E protein antibodies (green). Scale bar: 40 μm. (B) Newly generated neurons were stained by Tuj1 (red). ZIKV was stained by anti-ZIKV E protein antibodies (green). Blue, nuclear counterstain. Scale bar: 40 μm. (C) Quantification data were presented as mean ± SD (n = 4), ** p<0.01, *** p<0.001, t-test.

Fig 4. ZIKV-induced innate immune responses in the hNS/PC lines during the early stage of infection.

(A) The in vitro system to study ZIKV infection at different developmental stages in hNS/PCs. (B-C) Transcription levels of innate immune genes were measured by RT-qPCR. Data are presented as mean ± SD (n = 4), * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, two-way ANOVA with a Dunnett’s multiple comparisons test. (D) Protein expression and phosphorylation levels were assessed by Western blot in G010. Data are presented as mean ± SD (n = 3) * p<0.05, *** p<0.001, **** p<0.0001, t-test.

Fig 5. FAMP-reduced neurogenesis deficits caused by ZIKV infection.

(A) The in vitro system to study the effects of FAMP on K048? hNS/PCs after ZIKV infection. (B) Gene transcription levels were measured by RT-qPCR. Data are presented as mean ± SD (n = 4), * p<0.05, ** p<0.01, **** p<0.0001, two-way ANOVA with a Dunnett’s multiple comparisons test. (C) Newly generated neurons were stained by Tuj1 (red). Blue, nuclear counterstain. Scale bars: 40 μm. (D) Quantification data are presented as mean ± SD (n = 4), ** p<0.01, **** p<0.0001, one-way ANOVA with a Tukey post hoc test. (E) ZIKV RNA loads in the culture medium collected on Days 1, 2, 6, 9 and 12 were measured by RT-qPCR. Data are presented as mean ± SD (n = 3).

Fig 6. Lack of rescue of ZIKV-induced neurogenesis deficits with FAMP treatment at the late stage.

(A) The in vitro system to study the effects of FAMP on hNS/PCs after ZIKV infection. ZIKV was inoculated at the early proliferating stage. FAMP was treated at the early proliferating or the late differentiating stage. (B) Gene transcription levels were measured by RT-qPCR. Data are presented as mean ± SD (n = 2), **** p<0.0001, two-way ANOVA with a Dunnett’s multiple comparisons test. (C) Newly generated neurons were stained by Tuj1 (red). Blue, nuclear counterstain. Scale bar: 40 μm. (D) Quantification data are presented as mean ± SD (n = 2), * p<0.05, **** p<0.0001, one-way ANOVA with a Tukey post-hoc test.

Fig 7. A hypothetical model of modulating the ZIKV-induced overactivation of innate immune responses in NS/PCs to attenuate neurogenesis deficits.

The entry of ZIKV is mediated by unknown cell surface receptors. The virus membrane then fuses with the endosomal membrane and the ssRNA genome of the virus is released. TLR3 recognizes nucleic acids that are inside the endosome. TRIF is the sole adapter of TLR3. TRIF recruits a signaling complex that is consists of TRAF3 and 5 and the downstream mediator TBK-1. Activation of TBK-1 phosphorylates and activates the transcription factor IRF7 to drive a robust transcriptional activation of the type-I IFN responses. IFN-α and IFN-β bind to the IFNAR to activate STAT1/2 heterodimers. Phosphorylation and translocation of STAT1 leads to activation of transcriptional activity in several STAT1-associated genes, including IRF1 and NLRC5. Both of them can act as transcriptional activators of MHC class I genes, including B2M and TAP1. B2M is a potential pro-aging factor associated with decreased neurogenesis. Targeting the ZIKV-induced overactivated innate immune responses in NS/PCs (e.g., STAT1 inhibitor FAMP) could be a promising therapeutic strategy to attenuate ZIKV-related neuropathology. TRIF, TIR domain containing adaptor protein inducing IFN-β; TRAF, TNF receptor associated factor; TBK-1, TANK Binding Kinase 1; IRF, interferon regulatory transcription factor; IFNAR, IFN-α receptor; STAT, signal transducer and activator of transcription; NLRC5, NLR Family CARD Domain Containing 5; B2M, beta-2-microglobulin; TAP1, Transporter 1, ATP Binding Cassette Subfamily B Member; FAMP, fludarabine.