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. 2016 Sep 2;12(9):e1005875.
doi: 10.1371/journal.ppat.1005875. eCollection 2016 Sep.

Non-invasive Imaging of Sendai Virus Infection in Pharmacologically Immunocompromised Mice: NK and T Cells, but Not Neutrophils, Promote Viral Clearance After Therapy With Cyclophosphamide and Dexamethasone

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Free PMC article

Non-invasive Imaging of Sendai Virus Infection in Pharmacologically Immunocompromised Mice: NK and T Cells, but Not Neutrophils, Promote Viral Clearance After Therapy With Cyclophosphamide and Dexamethasone

Heba H Mostafa et al. PLoS Pathog. .
Free PMC article

Abstract

In immunocompromised patients, parainfluenza virus (PIV) infections have an increased potential to spread to the lower respiratory tract (LRT), resulting in increased morbidity and mortality. Understanding the immunologic defects that facilitate viral spread to the LRT will help in developing better management protocols. In this study, we immunosuppressed mice with dexamethasone and/or cyclophosphamide then monitored the spread of viral infection into the LRT by using a noninvasive bioluminescence imaging system and a reporter Sendai virus (murine PIV type 1). Our results show that immunosuppression led to delayed viral clearance and increased viral loads in the lungs. After cessation of cyclophosphamide treatment, viral clearance occurred before the generation of Sendai-specific antibody responses and coincided with rebounds in neutrophils, T lymphocytes, and natural killer (NK) cells. Neutrophil suppression using anti-Ly6G antibody had no effect on infection clearance, NK-cell suppression using anti-NK antibody delayed clearance, and T-cell suppression using anti-CD3 antibody resulted in no clearance (chronic infection). Therapeutic use of hematopoietic growth factors G-CSF and GM-CSF had no effect on clearance of infection. In contrast, treatment with Sendai virus-specific polysera or a monoclonal antibody limited viral spread into the lungs and accelerated clearance. Overall, noninvasive bioluminescence was shown to be a useful tool to study respiratory viral progression, revealing roles for NK and T cells, but not neutrophils, in Sendai virus clearance after treatment with dexamethasone and cyclophosphamide. Virus-specific antibodies appear to have therapeutic potential.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. SeV infection measured by bioluminescence imaging in living mice after treatment with Dexa and/or Cy.
(A) Drug treatment regimen and timing of infection. Arrows denote days on which drug injections were performed or 7,000 PFU Sendai virus (SeV) was intranasally inoculated in 5 μL PBS. Bioluminescence was measured after i.p. injection of 150 mg/kg D-luciferin and imaging with a Xenogen machine. (B–D) Bioluminescence of the nasal cavity (B), trachea (C), and lungs (D) in mice inoculated 1 day before starting drug treatment. Data shown are averages of 2 independent experiments with 10 mice at each time point. (E–G) Bioluminescence of the nasal cavity (E), trachea (F), and lungs (G) in mice inoculated 4 days after starting drug treatment. The data shown are averages of 3 independent experiments with 15 mice at each time point. For panels B-G, all groups were inoculated with SeV and symbols denote the following treatment groups: PBS (black circles), Dexa (green diamonds), Cy (orange squares), and Dexa + Cy (blue triangles). Lighter symbols in panels B-D correspond to groups inoculated with SeV 1 day before drug treatment, and darker symbols in panels E-G correspond to groups inoculated with SeV 4 days after drug treatment started. Error bars represent standard deviation. d.p.i., days postinfection; d.a.d.s., days after drug started.
Fig 2
Fig 2. Viral load in the respiratory tracts of mice inoculated with SeV 4 days after treatment with Dexa and/or Cy.
(A–C) Viral loads in the nasopharynx (A), trachea (B), and lungs (C). The data shown are averages of 3 animals at each time point. Bar colors for the groups are as follows: PBS (black), Dexa (green), Cy (orange), and Dexa + Cy (blue). Dashed lines represent the limit of detection. Error bars represent standard deviation. d.p.i., days postinfection; d.a.d.s., days after drug started. Statistics: 2-Way ANOVA; *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 3
Fig 3. Histopathologic changes 13 d.a.d.s. (9 d.p.i.) in the respiratory tracts of mice inoculated with SeV 4 days after treatment with Dexa and /or Cy.
Sections of the nasal cavity (A) and lungs (B) were stained with hematoxylin and eosin (H&E) (left panels), with a mAb to CD3 to show T-cell infiltration (middle panels), or with a mAb to SeV (right panels). Sections from Dexa + Cy-treated mice (bottom panels) were compared to sections from untreated controls (upper panels). The data are representative of the 4 different animals in each group.
Fig 4
Fig 4. Immunological responses in immunosuppressed mice inoculated with SeV at 4 d.a.d.s.
(A) Percent change in starting weight. (B) Peripheral blood neutrophil counts. (C) Peripheral blood lymphocyte counts. (D) Ratio of spleen weight to total body weight. For panels A-D, groups are shown as follows: PBS (black circles), Dexa (green diamonds), Cy (orange squares), and Dexa + Cy (blue triangles). (E) Splenic neutrophil counts. (F) Splenic lymphocyte counts. (G) BALF neutrophil counts. (H) BALF lymphocyte counts. (I and J) SeV-specific IgG levels in the BALF (I) and serum (J) of mice infected with SeV 4 days after treatment with Dexa and/or Cy. (K) Chemokines/cytokines in the BALF of SeV-infected mice at 12 d.p.i. (16 d.a.d.s). For panels E-K, bars are colored as follows: PBS (black), Dexa (green), Cy (orange), and Dexa + Cy (blue). The data shown are a representative of 2 independent experiments with 3 to 5 mice in each group. Error bars represent standard deviation. d.p.i., days postinfection; d.a.d.s., days after drug started. Statistics: 2-way ANOVA; *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 5
Fig 5. Effect of anti-neutrophil antibody Ly6G on viral clearance.
(A) Drug treatment regimen with anti-neutrophil antibody (anti-Ly6G). Arrows denote days on which anti-Ly6G was i.p. injected, drug injections were performed, and 7,000 PFU Sendai virus (SeV) was intranasally inoculated in 5 μL PBS. (B) Peripheral blood neutrophil counts. (C) Peripheral blood lymphocyte counts. (D) Percent change in starting weight. (E-G) Bioluminescence in the nasopharynx (E), trachea (F), and lungs (G). Groups include PBS (black circles), Dexa + Cy (blue triangles), and Dexa + Cy + anti-Ly6G (red circles). The data shown are averages of 5 mice from each group. In all graphs, error bars represent the standard deviation. d.p.i., days postinfection; d.a.d.s., days after drug started.
Fig 6
Fig 6. Effect of granulocyte-colony stimulating factor (G-CSF) on viral clearance.
(A) Drug treatment regimen with G-CSF. Arrows denote days on which G-CSF was i.p. injected, drug injections were performed, and 7,000 PFU Sendai virus (SeV) was intranasally inoculated in 5 μL PBS. (B) Peripheral blood neutrophil counts. (C) Peripheral blood lymphocyte counts. (D) Bioluminescence in the nasopharynx (N), trachea (T), and lungs (L). Groups include PBS (black circles), Dexa + Cy (blue triangles), and Dexa + Cy + G-CSF (purple circles). The data shown are a representative of 2 independent experiments with 5 mice per group. In all graphs, error bars represent the standard deviation. d.p.i., days postinfection; d.a.d.s., days after drug started.
Fig 7
Fig 7. Effect of drug treatment on peripheral lymphocyte populations.
(A) B cell, (B) CD4+ T cell, (C) CD8+ T cell, and (D) NK cell percentages were determined for peripheral blood collected at the indicated time points. Dexa and Cy injections were performed as described previously, and 7,000 PFU SeV was intranasally inoculated in 5 μL PBS at 4 d.a.d.s. Groups include PBS (black bars) and Dexa + Cy (blue bars). The data shown are averages of 5 mice per group. In all graphs, error bars represent the standard deviation. d.p.i., days postinfection; d.a.d.s., days after drug started. * P < 0.05, *** P < 0.001.
Fig 8
Fig 8. Effect of anti-NK (anti-asialo GM1) antibody on viral clearance.
(A) Drug treatment regimen with anti-NK antibody. Arrows denote days on which anti-NK or normal rabbit serum (NRS) were i.p. injected, drug injections were performed, and 7,000 PFU SeV was intranasally inoculated in 5 μL PBS. (B) Peripheral NK cell absolute numbers at 16 d.a.d.s. (C-E) Bioluminescence in the nasopharynx (C), trachea (D), and lungs (E). (F) Percent change in starting weight. Groups include PBS (black bars and circles), Dexa + Cy (light blue bars and triangles), Dexa + Cy + anti-NK (orange bars and octagons), and Dexa + Cy + NRS (green bars and stars). The data shown are averages of 5 mice per group. In all graphs, error bars represent the standard deviation. d.p.i., days postinfection; d.a.d.s., days after drug started. *** P < 0.001.
Fig 9
Fig 9. Effect of anti-CD3 (anti-T cell) antibody on viral clearance.
(A) Drug treatment regimen with anti-CD3 antibody. Arrows denote days on which anti-CD3 or isotype control IgG were i.p. injected, drug injections were performed, and 7,000 PFU SeV was intranasally inoculated in 5 μL PBS. (B) Percent change in starting weight. (C-E) Percentages of (C) CD4+ T cells (D) CD8+ T cells, and (E) NK cells in peripheral blood. (F-H) Bioluminescence in the nasopharynx (F), trachea (G), and lungs (H). Groups include PBS (black bars and circles), Dexa + Cy (light blue bars and triangles), Dexa + Cy + anti-CD3 (burnt orange bars and rectangles), control IgG (orange bars and diamonds). The data shown are averages of 5 mice per group. In all graphs, error bars represent the standard deviation. d.p.i., days postinfection; d.a.d.s., days after drug started. * p < 0.05, *** p < 0.001.
Fig 10
Fig 10. Effects of SeV-specific hyperimmune pooled serum and monoclonal antibodies on controlling SeV infection in immunosuppressed mice.
(A) Drug treatment regimen with SeV-specific hyperimmune serum or mAbs. Arrows denote days on which serum or monoclonal antibodies were i.p. injected, drug injections were performed, and 7,000 PFU Sendai virus (SeV) was intranasally inoculated in 5 μL PBS. (B–D) Bioluminescence in the nasopharynx (B), trachea (C), and lungs (D) after infection with SeV and the following conditions: no immunosuppression and no hyperimmune serum (black circles), Dexa + Cy without hyperimmune serum (blue triangles), Dexa + Cy with hyperimmune serum (brown diamonds), and Dexa + Cy with nonspecific control serum (dark purple triangles). (E-G) Bioluminescence in the nasopharynx (E), trachea (F), and lungs (G) after infection with SeV and the following conditions: no immunosuppression and no monoclonal antibodies (black circles), Dexa + Cy without monoclonal antibodies (blue triangles), Dexa + Cy + M16 [anti-F protein monoclonal antibody] (orange circles), Dexa + Cy + S2 [anti-HN protein monoclonal antibody] (green diamonds), and Dexa + Cy + isotype control mouse IgG (small purple squares). The data shown are averages of 5 mice per group. In all graphs, error bars represent the standard deviation. d.p.i., days postinfection; d.a.d.s., days after drug started.
Fig 11
Fig 11. Mice infected with SeV after treatment with Dexa + Cy are protected from a secondary lethal challenge.
(A) Serum SeV-specific IgG levels measured 1 day before challenge. The data are representative of 2 independent experiments with 3 mice per group. (B–D) Bioluminescence in the nasopharynx (B), trachea (C), and lungs (D) after challenge with SeV. The data are averages of 2 independent experiments with 8 mice per group. Error bars represent standard deviation. Statistics: 2-Way ANOVA; ***P < 0.001.

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