TLR3 increases disease morbidity and mortality from vaccinia infection

Abstract

Innate immunity is required for effective control of poxvirus infections, but cellular receptors that initiate the host response to these DNA viruses remain poorly defined. Given this information and the fact that functions of TLRs in immunity to DNA viruses remain controversial, we investigated effects of TLR3 on pathogenesis of vaccinia virus, a prototype poxvirus. We used a recombinant strain Western Reserve vaccinia virus that expresses firefly luciferase to infect wild-type C57BL/6 and TLR3-/- mice through intranasal inoculation. Bioluminescence imaging showed that TLR3-/- mice had substantially lower viral replication in the respiratory tract and diminished dissemination of virus to abdominal organs. Mice lacking TLR3 had reduced disease morbidity, as measured by decreased weight loss and hypothermia after infection. Importantly, TLR3-/- mice also had improved survival relative to wild-type mice. Infected TLR3-/- mice had significantly reduced lung inflammation and recruitment of leukocytes to the lung. Mice lacking TLR3 also had lower levels of inflammatory cytokines, including IL-6, MCP-1, and TNF-alpha in serum and/or bronchoalveolar lavage fluid, but levels of IFN-beta did not differ between genotypes of mice. To our knowledge, our findings show for the first time that interactions between TLR3 and vaccinia increase viral replication and contribute to detrimental effects of the host immune response to poxviruses.

Figure 1. TLR3^−/− C57BL/6 mice are resistant to vaccinia infection

WT (filled symbols) and TLR3^−/− (open symbols) mice (n = 5 mice/genotype) were infected i.n. with 1×10⁵ PFU luminescent reporter vaccinia virus. A and B, Chest (A) and abdominal (B) luminescence. Whole body bioluminescence imaging was performed each day postinfection, and photon flux in defined regions of interest was quantified by Living Image software. Data are expressed as mean values for photon flux. Error bars, SEM. C, Weight loss. Baseline weight was measured the day of infection, and weights were taken each day thereafter. Values are given as mean percentage of initial weight. D, Body temperature. On days 5 and 6 postinfection, core body temperatures of infected mice and an uninfected control were measured with a rectal probe.

Figure 2

A. TLR3^−/− mice are resistant to vaccinia at a dose that is approximately the LD50 for WT. Representative bioluminescence images of TLR3^−/− (left) and WT (right) mice on day 4 postinfection. Mice were infected i.n. with 1×10⁴ PFU luminescent reporter vaccinia virus (n = 10 WT and n = 11 TLR3^−/−). Photon flux is depicted on a pseudocolor scale with red representing the highest and blue the lowest values. B–D. TLR3^−/− mice are resistant to vaccinia at a dose that is approximately the LD50 for WT. Quantitative bioluminescence imaging data for head (B), chest (C), and abdomen (D) regions of interest. Mice were infected i.n. with 1×10⁴ PFU luminescent reporter vaccinia virus (n = 10 WT and n = 11 TLR3^−/−). WT are indicated by filled symbols, and TLR3^−/− are denoted with open symbols. Whole body bioluminescence imaging was performed each day postinfection, and photon flux in defined regions of interest was quantified by Living Image software. Data are expressed as mean values for photon flux ± SEM. E–H. TLR3^−/− mice are resistant to vaccinia at a dose that is approximately the LD50 for WT. Plaque assays and illness. Mice were infected i.n. with 1×10⁴ PFU luminescent reporter vaccinia virus (n = 10 WT and n = 11 TLR3^−/−). WT are indicated by filled symbols, and TLR3^−/− are denoted with open symbols. E, Lung viral titers in WT (filled bars) and TLR3^−/− (open bars) on day 4 or 7 postinfection (n = 7 WT and n = 6 TLR3^−/−). Plaque data are combined results of two experiments. F, Brain viral titers in WT (filled bars) and TLR3^−/− (open bars) on day 4 or 7 postinfection (n = 7 WT day 4 and n = 6 TLR3^−/− and WT day 7). G, Weight loss. Data are expressed as mean percent of initial weight (n mean percent weight loss = 10 WT or 11 TLR3^−/− on day 1, decreasing to n = 2 WT and n = 7 TLR3^−/− on day 13). *, Significant differences between genotypes (p < 0.05). H, Survival curve. A subset of mice (n = 5 WT and n = 7 TLR3^−/−) were monitored throughout the experiment until they recovered or were euthanized for 30% weight loss. Five WT and four TLR3^−/− mice were euthanized at an intermediate time points.

Figure 3. TLR3^−/− mice recruit fewer leukocytes to the lung in response to vaccinia

Mice (n = 4/genotype at each time point) were infected i.n. with 1×10⁴ PFU Vac-FL, and leukocytes in the lung were quantified and characterized by flow cytometry. A, Number of CD45+ cells at indicated times divided by number of CD45+ cells in uninfected mouse of same genotype. B, Mac-3+ cells in uninfected mice (day 0) and in TLR3^−/− and WT mice on days 3 and 7 postinfection. C, CD19+ cells; D, Ly6G+ cells. Values are expressed as a percent of total cells. Error bars denote SEM. *, Significant differences between genotypes (p < 0.05). **, p < 0.01.

Figure 4

TLR3^−/− and WT mice produce similar quantities of activated CTLs in response to vaccinia infection. Mice (n = 3/genotype) were infected i.n. with 1×10⁴ PFU Vac-FL, and CD8 T cells in the lung were analyzed by flow cytometry 7 days postinfection. A, CD8+ cells, expressed as a percent of total cells. B, Granzyme-positive cells, expressed as a percent of CD8+ cells. C, Granzyme and perforin double-positive cells, expressed as a percent of CD8+ cells. Error bars denote SEM. *, Significant differences between genotypes (p < 0.05). Positive control, Splenocytes stimulated in vitro for 4 days with IL-2.

Figure 5. Histology of vaccinia-infected lungs

Lungs were harvested on day 3 or 7 postinfection. (A), Photomicrograph of representative section of TLR3^−/− lung, day 7 postinfection, ×12 magnification. Inset, ×23. Arrows denote foci of inflammation. B, bronchus; V, blood vessel. (B), Representative WT lung section, day 7 postinfection, ×12 magnification. Inset, ×23. Arrows denote foci of inflammation. B, bronchus; V, blood vessel; N, region of necrosis. C, Foci of inflammation per lung section. Foci were counted on a lung section from each mouse. Data are expressed as mean number of foci per section (n = 6). Error bars, SEM.

Figure 6. WT mice have significantly higher cytokine levels in BAL fluid and plasma

Mice were infected i.n. with 1×10⁴ PFU Vac-GFL. On days 3 and 7 postinfection, lungs were lavaged with 1 ml of PBS. Blood samples were collected on the same days, centrifuged, and plasma were separated from cells. Cytokine levels were measured by ELISA. Single points indicate individual mice (WT filled, TLR3^−/− open), and horizontal bars are mean values. A, BAL TNF-α, (*, p < 0.05). B, Plasma IL-6 (*, p < 0.01). C, Plasma MCP-1 (*, p < 0.01).