Induction of natural killer cell responses by ectromelia virus controls infection

Abstract

Natural killer (NK) cells play a pivotal role in the innate immune response to viral infections, particularly murine cytomegalovirus (MCMV) and human herpesviruses. In poxvirus infections, the role of NK cells is less clear. We examined disease progression in C57BL/6 mice after the removal of NK cells by both antibody depletion and genetic means. We found that NK cells were crucial for survival and the early control of virus replication in spleen and to a lesser extent in liver in C57BL/6 mice. Studies of various knockout mice suggested that gammadelta T cells and NKT cells are not important in the C57BL/6 mousepox model and CD4+ and CD8+ T cells do not exhibit antiviral activity at 6 days postinfection, when the absence of NK cells has a profound effect on virus titers in spleen and liver. NK cell cytotoxicity and/or gamma interferon (IFN-gamma) secretion likely mediated the antiviral effect needed to control virus infectivity in target organs. Studies of the effects of ectromelia virus (ECTV) infection on NK cells demonstrated that NK cells proliferate within target tissues (spleen and liver) and become activated following a low-dose footpad infection, although the mechanism of activation appears distinct from the ligand-dependent activation observed with MCMV. NK cell IFN-gamma secretion was detected by intracellular cytokine staining transiently at 32 to 72 h postinfection in the lymph node, suggesting a role in establishing a Th1 response. These results confirm a crucial role for NK cells in controlling an ECTV infection.

FIG. 1.

Lack of NK cells increases susceptibility of resistant C57BL/6 mice to lethal disease. (a) C57BL/6 mice were treated with either anti-NK1.1 or an isotype control anti-MAR. Anti-NK1.1 MAb-treated and control-treated C57BL/6 mice were infected with various doses of ECTV in the footpad two days following MAb treatment, and mortality after infection was recorded. Six days following infection with 50 PFU of ECTV, spleens (b) and livers (c) from anti-NK1.1 MAb-treated and control-treated mice were harvested and virus titers were determined by a virus plaque assay. Circles represent individual animals and lines represent means. (n = 4 to 5 mice/group; representative of two experiments; *, P = 0.03). (d) Transgenic NKD mice and C57BL/6 controls were infected with various doses of ECTV in the footpad, and mortality was recorded. Six days following infection with 50 PFU of ECTV, spleens (e) and livers (f) from infected NKD and control mice were harvested and virus titers were determined by plaque assay (n = 6 to 7; representative of two experiments).

FIG. 2.

Lack of NKT cells does not increase susceptibility to ECTV infection. CD1 knockout, Jα18 knockout, and intact mice were infected with 50 PFU of ECTV in the footpad. Mortality was recorded, and virus titers in spleens and livers were determined by plaque assays.

FIG. 3.

Lack of NK cells in SCID mice increases severity of ECTV infection. (a) SCID and C57BL/6 mice were treated with either anti-NK1.1 MAb or anti-MAR isotype control 2 days prior to intravenous infection with 300 PFU of ECTV, and mortality was recorded (n = 8; representative of two experiments; P < 0.001). Infected mice were sacrificed at 6 days p.i., and virus titers in spleens (b) and livers (c) were determined by plaque assays (n = 8; representative of two experiments).

FIG. 4.

γδ T cells (^gdT), CD4⁺ T cells, and CD8⁺ T cells do not appear important in early control of ECTV infection despite the critical need for IFN-γ and perforin. (a) C57BL/6 controls and strains lacking CD8⁺, CD4⁺, and γδ T cells were infected with 50 PFU of ECTV by the footpad route, and mortality was recorded (n = 8 to 12 mice). Infected mice were sacrificed at 6 days p.i., and virus titers in spleens (b) and livers (c) were determined by plaque assays (n = 5 to 6 mice). (d) C57BL/6 controls and strains lacking IFN-γ and perforin were infected via the footpad route with 50 PFU of ECTV, and mortality was recorded (n = 8 to 12 mice). Infected mice were sacrificed at 6 days p.i., and virus titers in spleens (e) and livers (f) were determined by plaque assays (n = 10 to 12 mice; *, indicates a P value of 0.03 for spleen IFN-γ, 0.003 for spleen perforin, and 0.01 for liver perforin).

FIG. 5.

NK cell populations expand in response to ECTV infection. C57BL/6 mice were infected with 50 PFU of ECTV in the footpad and sacrificed at various times p.i. Spleens, livers, and popliteal lymph nodes were harvested, and NK cell populations were analyzed by flow cytometry (a to d) (n = 5 to 14 mice/time point; representative of two experiments). Flow cytometric analysis of BrdU incorporation into splenic (e) and liver (f) NK and T cells was performed on tissues isolated at 2 and 6 days p.i. (n = 5 mice/time point; representative of two experiments).

FIG. 6.

Association of hepatic NK cells with ECTV lesions. C57BL/6 mice were infected or mock infected with 50 PFU of ECTV in the footpad and sacrificed at 6 days p.i. Livers were embedded in embedding media, sectioned at 5 μm, and stained for orthopoxvirus antigens (red) and NK, CD4, or CD8 antigens (brown). (a) Uninfected; (b and d) anti-NK1.1, infected (*, P = 0.0001); (c and e) anti-CD4, infected; (f) anti-CD8, infected (*, P = 0.002).

FIG. 7.

NK cells demonstrate increased IFN-γ production and cytolytic activity in response to ECTV infection. C57BL/6 mice were infected with 50 PFU of ECTV in the footpad and sacrificed at various times p.i. Spleens and popliteal lymph nodes were harvested, and NK cell IFN-γ secretion and lytic activity were measured. (a) NK cell IFN-γ production at 56 h p.i. (n = 4 to 5 mice/time point; representative of two experiments). (b) Kinetics of NK cell-associated IFN-γ production in draining lymph nodes (n = 4 to 5 mice/time point; representative of two experiments). (c) Splenocyte lytic activity at 6 days p.i. (n = 3 to 4 mice/time point).

FIG. 8.

IL-12 does not appear to play a role in controlling ECTV infection. IL-12 knockout and C57BL/6 mice were infected with 50 PFU of ECTV by the footpad route. Mortality was recorded, and virus titers in spleen and liver were determined by plaque assays.