Loss of the TAM Receptor Axl Ameliorates Severe Zika Virus Pathogenesis and Reduces Apoptosis in Microglia

Abstract

The TAM receptor, Axl, has been implicated as a candidate entry receptor for Zika virus (ZIKV) infection but has been shown as inessential for virus infection in mice. To probe the role of Axl in murine ZIKV infection, we developed a mouse model lacking the Axl receptor and the interferon alpha/beta receptor (Ifnar^-/-Axl^-/-), conferring susceptibility to ZIKV. This model validated that Axl is not required for murine ZIKV infection and that mice lacking Axl are resistant to ZIKV pathogenesis. This resistance correlates to lower pro-interleukin-1β production and less apoptosis in microglia of ZIKV-infected mice. This apoptosis occurs through both intrinsic (caspase 9) and extrinsic (caspase 8) manners, and is age dependent, as younger Axl-deficient mice are susceptible to ZIKV pathogenesis. These findings suggest that Axl plays an important role in pathogenesis in the brain during ZIKV infection and indicates a potential role for Axl inhibitors as therapeutics during viral infection.

Keywords: Biological Sciences; Neurotoxicology; Virology.

Graphical abstract

Figure 1

The TAM Receptor Axl Is Not Required for Replication of ZIKV In Vivo but Is Involved in Viral Pathogenesis (A–H) (A–F) Six-week old or (G and H) three-week old Ifnar1^−/− or Ifnar1^−/−Axl^−/− mice were subcutaneously infected via footpad injection with 10⁵ plaque-forming unit Cambodian strain of ZIKV. (A) Diagram of Axl knockout mouse model describing the purpose of gene ablation. (B) Whole blood was collected at days 2, 4, and 6 post-ZIKV infection from both groups and analyzed by qRT-PCR. (C and D) At day 6, when mice begin to show viral pathogenesis, mice were sacrificed, brains were collected, and ZIKV levels were analyzed using (C) qRT-PCR and (D) plaque assay. (E and G) Both groups were weighed daily for two weeks or until sacrificed due to severe pathogenesis, and data were graphed as a percentage of original body. (F and H) Mice were also monitored for survival for two weeks after ZIKV infection. ZIKV RNA levels were normalized to mouse β-actin (ACTB) RNA levels. (n = 8–10/group for each genotype). Significance was tested by (B) two-way ANOVA with a post-hoc Tukey test, (C and D) Student's t test, or (F and H) log rank (Mantel-Cox) test. Error bars represent SEM.

Figure 2

Axl Expression Is Important for IL-1β Expression during ZIKV Infection Six-week old Ifnar1^−/− or Ifnar1^−/−Axl^−/− mice were subcutaneously infected via footpad injection with 10⁵ plaque-forming unit Cambodian strain of ZIKV. (A–E) At day 6, when mice begin to show viral pathogenesis, they were sacrificed, brains were collected and (A) pro-IL-1β, (B) TGF-β, (C) TNF-α, (D) IFN-γ, and (E) IL-6 levels were analyzed using qRT-PCR. ZIKV RNA levels were normalized to mouse β-actin (ACTB) RNA levels. Data are expressed as a percentage of the average expression in the Ifnar1−/− group. Significance was tested using Student's t test (n = 8–10/group for each genotype).

Figure 3

Axl Expression Does Not Drive Differences in Immune Cell Infiltration during ZIKV Infection Six-week old Ifnar1^−/− or Ifnar1^−/−Axl^−/− mice were subcutaneously infected via footpad injection with 10⁵ plaque-forming unit Cambodian strain of ZIKV. (A–H) At day 6, when mice begin to show viral pathogenesis, mice were sacrificed, brains were collected, and (A) monocytes, (B) neutrophils, (C) macrophages, (D) dendritic cells, (E) microglia, (F) activated microglia, (G) CD4⁺ T cells, and (H) CD8⁺ T cells were analyzed using flow cytometry. Data are expressed as total number of cells present in the brain. Significance was tested by two-way ANOVA with a post-hoc Tukey test. No significant differences were detected among any groups. Error bars represent SEM (n = 8/group for each infected group and n = 2/group for each uninfected group).

Figure 4

Axl Drives Apoptosis in the Brain of ZIKV-Infected Mice Six-week old Ifnar1^−/− or Ifnar1^−/−Axl^−/− mice were subcutaneously infected via footpad injection with 10⁵ plaque-forming unit Cambodian strain of ZIKV. (A–C) At day 6, when mice begin to show viral pathogenesis, mice were sacrificed, brains were collected, fixed in buffered formalin, and prepared for histology. Slides were stained using the TUNEL assay, and the entire brain was imaged at 20×. Shown are (A) the ventral striatum, (B) the cerebellum, and (C) the hippocampus (n = 4/group for each genotype). Scale bar, 500 μM in main image and 100 μM in inset image.

Figure 5

ZIKV Induces Apoptosis in Microglia Six-week old Ifnar1^−/− or Ifnar1^−/−Axl^−/− mice were subcutaneously infected via footpad injection with 10⁵ plaque-forming unit Cambodian strain of ZIKV. (A–C) At day 6, when mice begin to show viral pathogenesis, mice were sacrificed, brains were collected, fixed in buffered formalin, and prepared for histology. Slides were stained using the TUNEL assay and antibodies toward (A) NeuN (neurons), (B) GFAP (astrocytes), or (C) Iba1 (microglia) and imaged at 20×. Insets are digitally enlarged by 3× to show individual cells. Scale bar is 100 μM in main image. Shown is representative image from Ifnar1^−/− mice. (D) Percent microglia that were TUNEL+ were quantified and graphed from two to three random fields in the brains of each genotype. Significance was tested by two-way ANOVA. Error bars represent SEM (n = 4/group for each genotype).

Figure 6

ZIKV Induces Apoptosis by a PARP-Dependent Caspase-Dependent Mechanism Six-week old Ifnar1^−/− or Ifnar1^−/−Axl^−/− mice were subcutaneously infected via footpad injection with 10⁵ plaque-forming unit Cambodian strain of ZIKV. (A–J) At day 6, when mice begin to show viral pathogenesis, they were sacrificed, brains were collected in radioimmunoprecipitation assay (RIPA) buffer with protease inhibitor, and a western blot was performed. Membrane was probed with antibodies toward proteins involved in apoptosis. Protein expressions of (A) PARP, (B) cleaved PARP, (C) caspase 3, (D) cleaved caspase 3, (E) caspase 8, (F) caspase 9, (G) cleaved caspase 9, (H) caspase 12, and (I) cleaved caspase 12 were quantified using ImageJ. Data shown are representative of at least two independent experiments. Significance was tested with two-tailed Mann-Whitney U test. Error bars represent SEM (n = 4/group for each genotype).