Redundant Function of Plasmacytoid and Conventional Dendritic Cells Is Required To Survive a Natural Virus Infection

Abstract

Viruses that spread systemically from a peripheral site of infection cause morbidity and mortality in the human population. Innate myeloid cells, including monocytes, macrophages, monocyte-derived dendritic cells (mo-DC), and dendritic cells (DC), respond early during viral infection to control viral replication, reducing virus spread from the peripheral site. Ectromelia virus (ECTV), an orthopoxvirus that naturally infects the mouse, spreads systemically from the peripheral site of infection and results in death of susceptible mice. While phagocytic cells have a requisite role in the response to ECTV, the requirement for individual myeloid cell populations during acute immune responses to peripheral viral infection is unclear. In this study, a variety of myeloid-specific depletion methods were used to dissect the roles of individual myeloid cell subsets in the survival of ECTV infection. We showed that DC are the primary producers of type I interferons (T1-IFN), requisite cytokines for survival, following ECTV infection. DC, but not macrophages, monocytes, or granulocytes, were required for control of the virus and survival of mice following ECTV infection. Depletion of either plasmacytoid DC (pDC) alone or the lymphoid-resident DC subset (CD8α(+) DC) alone did not confer lethal susceptibility to ECTV. However, the function of at least one of the pDC or CD8α(+) DC subsets is required for survival of ECTV infection, as mice depleted of both populations were susceptible to ECTV challenge. The presence of at least one of these DC subsets is sufficient for cytokine production that reduces ECTV replication and virus spread, facilitating survival following infection.

Importance: Prior to the eradication of variola virus, the orthopoxvirus that causes smallpox, one-third of infected people succumbed to the disease. Following successful eradication of smallpox, vaccination rates with the smallpox vaccine have significantly dropped. There is now an increasing incidence of zoonotic orthopoxvirus infections for which there are no effective treatments. Moreover, the safety of the smallpox vaccine is of great concern, as complications may arise, resulting in morbidity. Like many viruses that cause significant human diseases, orthopoxviruses spread from a peripheral site of infection to become systemic. This study elucidates the early requirement for innate immune cells in controlling a peripheral infection with ECTV, the causative agent of mousepox. We report that there is redundancy in the function of two innate immune cell subsets in controlling virus spread early during infection. The viral control mediated by these cell subsets presents a potential target for therapies and rational vaccine design.

FIG 1

Survival of ECTV infection does not require inflammatory monocytes or mo-DC, granulocytes, or macrophages. (A) Representative gating of myeloid cell populations from ECTV-infected mice. All gates were defined by fluorescence minus one controls, and the numbers represent percentages of cells. Large arrows indicate the flow of the analyses, with each population being a subset of the previous one. (B) Quantification of myeloid cell populations from WT mice infected with ECTV and injected i.v. with either CLL or PBS at 1 day p.i. Spleens were harvested at 2 days p.i. (C) WT or CCR2KO mice were infected with ECTV and monitored for survival until 21 days p.i. (D and E) WT mice were injected i.v. with 0.5 ml of 1.4 mg/ml anti-Ly6G depleting antibody (1A8) or with an IgG2a control antibody (2A3) in PBS on day −1 and then every third day and infected with ECTV on day 0. Depletion of Ly6C⁺ Ly6G⁺ cells in the spleen at 21 days p.i. (D) and survival (E) were measured. (F and G) WT mice were injected i.p. with 3 mg/mouse anti-CD115 depleting antibody or vehicle control every other day starting at day −5 and infected with ECTV. Depletion of splenic macrophage populations (F) and survival (G) were monitored until 22 days p.i. (B to G) Data were pooled from two independent experiments (means ± SEM). ***, P < 0.001; Student's unpaired t test.

FIG 2

CD11c⁺ cells control viral replication to mediate survival of ECTV infection. (A) Mice were infected in the footpad with 1 × 10⁶ PFU ECTV expressing NP-S-EGFP (green). Twelve hours postinfection, D-LN were harvested and then stained with anti-CD11c-Alexa 647 (red). The data are representative of the results of 7 independent experiments. (B and C) Mice were infected with 3 × 10³ PFU ECTV in the footpad, and at 2 days p.i., D-LN were harvested and stained for flow cytometry. The cells were gated on live cells, singlets, and NK cell- and T cell-negative status and then on CD11b⁺ CD11c⁺ (conventional DC), CD11b⁺ CD11c⁻ (macrophages), and CD11b⁻ CD11c⁺ (pDC and CD8α⁺ DC). Representative histograms of IFN-α expression are shown (B), and the percentages of IFN-α⁺ cells were quantified (C). “Uninfected” represents IFN-α production by CD11b⁻ CD11c⁺ cells in the absence of ECTV infection. The data are representative of two independent experiments. The error bars indicate SEM. (D) CD11c:cre × iDTR mice were either (i) injected i.p. with DT on day 1 and mock infected in the left hind footpad on day 0; (ii) injected i.p. with PBS on day 1 and then infected with ECTV on day 0; or (iii) infected with ECTV on day 0 and then injected i.p. with DT on day −1 or day 1, 3, or 5 postinfection. Survival was monitored until 15 days p.i. The data from four independent experiments were combined. (E to H) WT or CD11c:cre × iDTR mice were injected i.p. with DT or PBS on day 1 p.i. All the mice were infected with ECTV as for panel D, and infected footpads (E), D-LN (F), livers (G), and spleens (H) were harvested at 5 days p.i. The tissues were processed and plated in serial dilutions on cell monolayers for viral titer analysis. The data from two independent experiments were combined. (I) CD11c:cre × iDTR mice were infected with ECTV as for panel D and injected i.p. with DT on day 1 p.i. The mice received 100 mg/kg cidofovir or PBS i.p. at 3 days p.i. and 6 days p.i. Survival was monitored to 21 days p.i. The data from two individual experiments were combined. *, P < 0.05; Student's unpaired t test.

FIG 3

CD8α⁺ DC and pDC are depleted in susceptible DT-injected CD11c:cre × iDTR mice and produce IFN-α in response to live ECTV. (A and B) WT or CD11c:cre × iDTR mice were infected with ECTV on day 0 and injected i.p. with DT or PBS at 1 day p.i. D-LN (A) or spleens (B) were harvested at 2 days p.i., and the cells were stained for flow cytometry for CD11b⁺ DCs, pDCs, and CD8α⁺ DCs as in Fig. 1A. The data are representative of two independent experiments (means ± SEM). (C) Spleens from WT mice were harvested 10 to 14 days after administration of Flt3L. Purified DC were left untreated (naive) or treated with live WT ECTV (WT ECTV) or UVC-inactivated WT ECTV (UVC ECTV) at an MOI of 10 for 6 h. The cells were stained for flow cytometry and gated on pDC, CD8α⁺ DC, or CD11b⁺ DC producing IFN-α. The data are representative of four independent experiments with experimental triplicates (means ± SEM). *, P < 0.05; **, P < 0.01; ***, P < 0.001; Student's unpaired t test.

FIG 4

CD8α⁺ DC or pDC are required for survival of mice following ECTV infection. (A) WT or Batf3KO mice were mock infected or infected with ECTV, and survival was monitored to 21 days p.i. The data from three independent experiments were pooled. (B) WT mice were injected i.p. with pDC-depleting or isotype control antibody and infected with ECTV. Survival was monitored to 14 days p.i. The data from three independent experiments were combined. (C) Batf3KO mice were injected i.p. with pDC-depleting antibody or control antibody, and survival was monitored to 14 days p.i. The data from three independent experiments were combined. (D) WT or Batf3KO mice were injected i.p. with pDC-depleting or control antibody and infected with ECTV. Spleens were harvested at 3 days p.i., and cells were stained for flow cytometry for pDC and CD8α⁺ DC as for Fig. 1A. The data are representative of two independent experiments (means ± SEM). *, P < 0.05; **, P < 0.01; ***, P < 0.001; Student's unpaired t test.

FIG 5

CD8α⁺ DC or pDC are required to control ECTV infection. (A, B, and C) WT or Batf3KO mice were injected i.p. with pDC-depleting or control antibody and infected with ECTV. Infected footpads (A), D-LN (B), and spleens (C) were harvested at 5 days p.i. The tissues were processed, and the titer of virus was determined. The data from two independent experiments were combined. (D) Batf3KO mice were injected i.p. with pDC-depleting antibody and infected with ECTV as for panel A. The mice received 100 mg/kg cidofovir or PBS i.p. at 3 and 6 days p.i. Survival was monitored to 17 days p.i. The data from two independent experiments were combined. *, P < 0.05; **, P < 0.01; ***, P < 0.001; Student's unpaired t test.

FIG 6

The absence of both CD8α⁺ DC and pDC does not result in reduced numbers of NK cells, T_CD4+, or T_CD8+. WT or Batf3KO mice were injected i.p. with pDC-depleting or control antibody and infected with ECTV. Spleens were harvested at 4 days p.i., processed, and stained for NK cell subsets (A), T_CD4+ subsets (B), or T_CD8+ subsets (C). The data are representative of two independent experiments (n = 2 or 3). Student's unpaired t test was used to compare the pDC-depleted Batf3KO group to either the pDC-depleted WT group or the control antibody Batf3KO group. Statistics would have been shown only if both of these comparisons had a significant P value (P < 0.05).