Alternate mechanisms of initial pattern recognition drive differential immune responses to related poxviruses

Abstract

Vaccinia immunization was pivotal to successful smallpox eradication. However, the early immune responses that distinguish poxvirus immunization from pathogenic infection remain unknown. To address this, we developed a strategy to map the activation of key signaling networks in vivo and applied this approach to define and compare the earliest signaling events elicited by immunizing (vaccinia) and lethal (ectromelia) poxvirus infections in mice. Vaccinia induced rapid TLR2-dependent responses, leading to IL-6 production, which then initiated STAT3 signaling in dendritic and T cells. In contrast, ectromelia did not induce TLR2 activation, and profound mouse strain-dependent responses were observed. In resistant C57BL/6 mice, the STAT1 and STAT3 pathways were rapidly activated, whereas in susceptible BALB/c mice, IL-6-dependent STAT3 activation did not occur. These data link early immune signaling events to infection outcome and suggest that activation of different pattern-recognition receptors early after infection may be important in determining vaccine efficacy.

Figure 1

Early in vivo response to TLR ligands is characterized by ligand-specific Type I IFN and IL-6 dependent pSTAT1 and pSTAT3 activation in multiple cell types. (A) Relative response to various TLR ligands in different cells as determined by levels of STAT phosphorylation. The indicated natural and synthetic microbial ligands specific for TLR4 (LPS), TLR9 (CpG), TLR7 (R848), TLR3 (poly I:C), TLR1/2 (PAM3CSK4), TLR2/6 (FSL1), and phosphate-buffered saline (PBS) controls were intravenously injected into C57BL/6 mice (20 μg/mouse). One hour after injection, spleens were harvested and immediately fixed and prepared for surface marker and pSTAT 1, 3, 4, 5, and 6 staining. Cell populations were identified as CD11c⁺ B220⁻ (cDCs); CD4⁺ T cells; CD8⁺ T cells; B220⁺ CD11c⁻ (B cell), or CD11b^hi (granulocytes). The percentage of cells showing greater than basal STAT phosphorylation is shown as a heat map (black, 0%; bright yellow, 100%). (B) STAT1 and 3 phosphorylation is TLR dependent. Wild-type (green) or TLR2^−/−, TLR3^−/−, TLR4 mutant, and TLR9^−/− mice (all in blue) were challenged with PAM3CSK4, poly (I:C), LPS, and CpG, respectively. Control PBS treatment is shown in red. Spleens were prepared for intracellular staining after 1 hour. Levels of pSTAT1 and pSTAT3 in CD11c+ cells are shown. (C) STAT1 and STAT3 phosphorylation are type I IFN and IL-6 dependent respectively. IFNAR^−/− and IL6^−/− mice were injected with 20 μg LPS; 1 hour later cells were harvested and prepared as before. All experiments were performed in at least three separate mice; data from one representative experiment is shown.

Figure 2

Systemic vaccinia infection elicits TLR2 and IL-6-dependent responses that promote viral clearance and neutralizing antibody production. (A) Early in vivo STAT response to vaccinia virus infection resembles the PAM3CSK4 (TLR1/2 ligand) response. pSTAT1 and pSTAT3 activation in CD11c⁺ cells at 1 hour after intravenous (i.v.) injection with PBS control, CpG, poly (I:C), or PAM3CSK4, compared to vaccinia virus infection (1 × 10⁷ pfu; Western Reserve strain). (B) Vaccinia induction of pSTAT3 is TLR2 and IL-6 dependent. Wild-type (green), TLR2^−/−, or IL6^−/− (both in blue) mice were infected with vaccinia virus (VV) as before, and, after one hour, spleens were excised, dissociated, and prepared for intracellular analysis. pSTAT3 levels were determined in CD11c⁺, CD4⁺, and CD8⁺ cells. (C) IL-6 production in response to vaccinia infection is TLR2 dependent. Wild-type and TLR2^−/− mice were infected i.v. with 1 × 10⁷ pfu of vaccinia virus; after 1 hour serum was harvested for detection of secreted IL-6 by ELISA. Serum from uninfected mice was used as control. (D) Vaccinia is recognized by TLR2 in vitro. HEK-293 cells transfected with mouse TLR2 and an NFκ B-driven luciferase reporter and untransfected control cells with the NFκ B-driven luciferase reporter alone were exposed to UV-inactivated vaccinia virus (5 viral particles per cell). NFkB-driven luciferase expression was evaluated by bioluminescence signal (after addition of luciferase) 24 hours later (data represented as mean ± SD).

Figure 3

Robust long-term immune response to vaccinia is dependent on early TLR2 and IL-6 signaling. (A) TLR2^−/− or IL6^−/− mice display delayed clearance of vaccinia virus. Mice (wild-type, TLR2^−/−, or IL6^−/− of the C57BL/6 background) were infected i.v. with 1 × 10⁷ pfu of Western Reserve strain vaccinia expressing luciferase. Overall viral load was determined at different times by bioluminescence imaging (BLI) after luciferin substrate delivery by intraperitoneal injection in an IVIS200 (Xenogen). No differences were seen in initial overall viral gene expression (correlating with viral replication) or biodistribution (except that an early spleen signal seen in C57/BL6 mice was absent from the gene knock-out strains). Significant differences were not seen until 6 days after infection, when the gene knock-out strains displayed delayed viral clearance (data represented as mean ± SD, with n = at least 3 mice per group). (B) Addition of exogenous IL-6 can substitute for loss of TLR2. C57BL/6, TLR2^−/−, and IL6^−/− mice were infected with vaccinia expressing luciferase as before; in one group, TLR2^−/− mice were additionally treated with 500ng IL-6 delivered intraperitoneally. Viral luciferase gene expression is shown for whole animals as determined 7 days post infection (data represented as mean ± SD, with n = at least 3 mice per group) (C) Loss of TLR2-IL6-pSTAT3 early signaling pathway leads to reduced levels of anti-vaccinia neutralizing antibody. Mice treated as above were sacrificed 21 days after initial viral infection, and levels of vaccinia specific antibody in the serum were determined with a viral neutralization assay. The TLR2^−/− and IL6^−/− strains both displayed reduced levels of neutralizing antibody.

Figure 4

Resistance to poxvirus infection correlates with early and potent STAT network activation in dendritic cells and T cells. (A) Ectromelia virus infection produces delayed and reduced pSTAT1 and pSTAT3 induction, especially in sensitive BALB/c mice. In vivo time course of the early immune responses in C57BL/6 and BALB/c mice infected intravenously with 1 × 10⁷ pfu of either vaccinia virus or ectromelia virus (Moscow strain) was determined as in Figure 1. Percentage of cells demonstrating pSTAT1 or pSTAT3 activation at different times after infection is plotted for conventional dendritic cells (CD11c⁺), T cells (CD4⁺ or CD8⁺), B cells (B220⁺), and granulocytes (CD11b^hi) (data represented as mean ± SD). (B) Ectromelia virus induction of pSTAT3 is IL-6- but not TLR2-dependent. Wild-type (C57BL/6), TLR2^−/−, and IL6^−/− mice were infected with 1 × 10⁷ pfu ectromelia virus. After 1 hour spleens were excised, dissociated, and prepared for intracellular analysis. Levels of pSTAT3 in CD11c⁺ cells are shown (data represented as mean +/− SD; two-tailed, unpaired t-test: * p<0.02 for IL6^−/− compared to WT and TLR2^−/−, WT compared to TLR2^−/− was not significant). (C) IL-6 induction is necessary for resistance to ectromelia virus infection in C57BL/6 mice. Wild-type and IL6^−/− mice of the C57BL/6 background were subcutaneously infected with 1 × 10³ pfu of ectromelia virus. Survival was monitored for 20 days following infection. Livers were extracted from separate groups of infected mice on day 7 and a plaque-forming assay was used to monitor viral burden. (D) TLR2 activation prior to ectromelia infection reduces viral burden. PBS, 1 × 10⁷ pfu of MVA (modified vaccinia Ankara), or 20 μg of PAM3CSK4 was intravenously injected into C57BL/6 mice one hour before systemic infection with 1 × 10⁷ pfu of ectromelia. Spleens were excised 72 hours later and ectromelia burden was quantified using a plaque-forming assay (data represented as mean ± SD; two-tailed, unpaired t-test: * p< 0.01 MVA compared to PBS, p< 0.007 PAM3CSK4 compared to PBS).