Immune cells promote paralytic disease in mice infected with enterovirus D68

Figures

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  Figure 1. EV-D68 US/IL/14-18952 infects and causes paralysis in immunocompetent neonatal mice. Three-day-old WT mice were inoculated i.c. with PBS (mock), 105 PFU EV-D68 USA/Fermon (Fermon), US/MO/14-18949 (MO49), or US/IL/14-18952 (IL52). (A) Brain and spinal tissue were resected at 3 dpi from EV-D68–inoculated mice, and viral titers were determined by plaque assay. (B) Brain and spinal tissue were resected from IL52-inoculated mice at 1, 3, or 5 dpi, and viral titers were determined by plaque assay. (C) Spinal columns were resected at 5 dpi from IL52-inoculated paralyzed mice or day-matched mock-inoculated controls. Samples were processed for immunohistochemistry staining. DAPI, blue; NeuN, green; EV-D68 VP1, red. Scale bar: 100 μM. (D) Inoculated mice were monitored daily and euthanized upon signs of paralysis. n = 12–27 mice per group. (E) Neutralizing antibody titers in sera collected at 5 or 14 dpi from IL52-inoculated mice. Dotted lines indicate the limit of detection. Data are representative of 2–3 independent experiments. Each symbol represents an individual mouse. Error bars indicate mean ± SD. Mann-Whitney U test (A, B, and E) or log-rank test (D): ***P ≤ 0.001; ****P ≤ 0.0001.

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  Figure 2. Cytokines in spinal tissue of EV-D68–inoculated mice. Three-day-old WT mice were inoculated i.c. with MO49, IL52, or PBS (mock). Spinal tissue was resected 3 dpi and analyzed by Luminex protein assay. (A) Cytokine concentrations are presented as mean values in pg/mL from 7–12 mice and shown as a heatmap normalized to concentrations in mock-inoculated mice. (B) CCL2, CCL7, and CCL12 concentrations normalized to those in mock-inoculated mice are shown. Data are representative of 2–3 independent experiments. Each symbol represents an individual mouse. Error bars indicate mean ± SD. Mann-Whitney U test (B): *P ≤ 0.05.

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  Figure 3. Neurovirulent EV-D68 efficiently infects hSCOs. Pools of 8–12 hSCOs at 14 days after differentiation were either mock inoculated (PBS) or inoculated with 105 PFU EV-D68 MO49 or IL52. (A) At 3 dpi, whole organoids were processed for immunofluorescence. DAPI, blue; NeuN, green; EV-D68 VP1, red. (B) hSCO supernatants were collected at 3 dpi and analyzed by Luminex protein assay. Protein levels are presented as mean values in pg/mL from 2–3 independent samples and shown as a heatmap normalized to levels in mock-infected organoids.

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  Figure 4. Mice inoculated with neurovirulent EV-D68 have altered populations of immune cells in the spinal cord. Three-day-old WT mice were inoculated i.c. with PBS (mock), EV-D68 MO49, or IL52. Spinal cords were resected from paralyzed IL52-inoculated mice or day-matched mice inoculated with MO49 or PBS (mock). Single-cell suspensions were prepared, stained, and analyzed by flow cytometry. (A) Numbers and (B) percentages of the indicated cell types are shown. Data are representative of 2–4 independent experiments. Each symbol represents an individual mouse. Error bars indicate mean ± SD. Kruskal-Wallis test: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

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  Figure 5. Mice lacking CCR2 develop less paralysis relative to WT mice following inoculation with neurovirulent EV-D68. Three-day-old WT or Ccr2–/– mice were inoculated i.c. with PBS (mock) or EV-D68 IL52. (A) Brain and spinal tissue of virus-inoculated mice were resected at 1, 3, or 5 dpi, and viral titers were determined by plaque assay. Dotted lines indicate the limit of detection. (B) Virus-inoculated mice were monitored daily and euthanized upon signs of paralysis. n = 21–28 mice per group. (C) Spinal tissue was resected 3 dpi and analyzed by Luminex protein assay. Data from WT mice are the same as those from the IL52-inoculated mice represented in Figure 2A, as these experiments were conducted concurrently. Cytokine concentrations are presented as mean values in pg/mL from 3–12 mice and shown as a heatmap normalized to mock-inoculated mice. The CCL2 outlier in the Ccr2–/– cohort was excluded from statistical analysis but left on graph for transparency. (D) Levels of CCL2, CCL7, and CCL12 are shown. Data are representative of 2–3 independent experiments. Each symbol represents an individual mouse. Error bars indicate mean ± SD. Mann-Whitney U test (A and D) or log-rank test (B): *P ≤…

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  Figure 6. Loss of CCR2 results in altered immune cell recruitment following neurovirulent EV-D68 inoculation. Three-day-old WT or Ccr2–/– mice were inoculated i.c. with EV-D68 IL52. Spinal cords were resected at 5–6 dpi from paralyzed WT mice or day-matched paralyzed and nonparalyzed Ccr2–/– mice. Single-cell suspensions were prepared, stained, and analyzed by flow cytometry. (A) Numbers and (B) percentages of selected cell types are shown. Each symbol represents an individual mouse. Error bars indicate mean ± SD. Mann-Whitney U test: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

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  Figure 7. Mice lacking mature B and T cells have diminished EV-D68–induced paralysis relative to WT mice. Three-day-old WT or Rag1–/– mice were inoculated i.c. with PBS (mock) or EV-D68 IL52. (A) Brain and spinal tissue were resected at 3 dpi, and viral titers were determined by plaque assay. Data from WT mice are the same as those presented in Figure 5A, as these experiments were conducted concurrently. Dotted lines indicate the limit of detection. (B) Virus-inoculated mice were monitored daily and euthanized upon signs of paralysis. n = 16–21 mice per group. Data from WT mice are the same as those presented in Figure 5C, as these experiments were conducted concurrently. (C) Spinal tissue was resected 3 dpi and analyzed by Luminex protein assay. Data from WT mice are the same as those presented in Figure 2A, as these experiments were conducted concurrently. Cytokine concentrations are presented as mean values in pg/mL from 7–12 mice and shown as a heatmap normalized to concentrations in mock-inoculated mice. (D) Levels of CCL2, CCL7, and CCL12 are shown. Data are representative of 2–3 independent experiments. Each symbol represents an individual mouse. Error bars indicate mean ± SD.…

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  Figure 8. Antibody-mediated depletion of T cells protects mice from EV-D68–mediated paralysis. Three-day-old WT mice were inoculated i.c. with EV-D68 IL52 and subsequently inoculated i.p. with anti-CD4, anti-CD8, or isotype control antibody. Mice received a second dose of antibody at 3 dpi. Mice were monitored daily for signs of disease and euthanized upon detection of paralysis. (A) Experimental workflow. (B) Percent survival (to day of paralysis onset) following treatment with either isotype control antibody or with anti-CD4 or anti-CD8 antibodies. n = 14–18 mice per group. Data are representative of 2 independent experiments. Log-rank test: *P ≤ 0.05; ****P ≤ 0.0001.