The recognition of pathogen-associated molecular patterns (PAMPs) occurs during the earliest steps of an innate immune response. Pattern recognition causes the release of numerous cytokines capable of initiating gene regulatory programs through the activation of transcription factor networks. Until now it has been impossible to examine how this transcription factor network is activated in response to a microbial infection in the context of an intact immune system, within a living host. Direct measurement of serum cytokines in vivo
demonstrates the presence of specific interleukins but does not reveal which immune cells actually respond to a particular cytokine. Because of this, our current understanding of cytokine responsiveness is based largely on in vitro
experiments in which bone marrow-derived or sorted primary cells are treated with recombinant cytokines and then subjected to western blot analysis. In vivo
cytokine responsiveness may differ drastically from the in vitro
responsiveness of cellular monocultures due to competition between different cell types for available cytokines (Pandiyan et al., 2007
), as well as biophysical constraints imposed by the stromal microarchitecture of secondary lymphoid organs (Gretz et al., 2000
). Here we have developed a novel strategy to study the kinetics and levels of activation of different signaling pathways within diverse immune cell populations at times after exposure to a pathogen. This approach allowed us to define the earliest STAT signaling pathways induced by different (immunizing or pathogenic) poxviruses in the context of a living organism, and to correlate network activation with different immune outcomes.
Vaccinia was key to what is possibly mankind's greatest medical achievement, the eradication of smallpox. Attenuated and wild-type vaccinia strains continue to be evaluated clinically as recombinant vaccine vehicles for a number of infectious agents, including HIV (Manrique et al., 2009
; Rodriguez et al., 2009
). Deeper comprehension of the immune response to vaccinia may produce strategies to enhance vaccine efficacy and predict potentially harmful side effects, while an understanding of how the early immune response differs in vaccinating and pathogenic infections may lead to alternative therapeutic approaches or means to predict the outcome of infection.
A number of innate and adaptive cell populations have been linked to protective responses against orthopoxviruses (Xu et al., 2004
). Innate immunity is thought to be highly dependent upon type I IFN-activated natural killer cells (Martinez et al., 2008
) and γ δ T cells (Selin et al., 2001
). Adaptive CD8+
T cell responses are often critical for destruction of virally infected cells; however, only a minor role for CD8+
T cell mediated cellular immunity has been described for resistance against vaccinia infection (Spriggs et al., 1992
; Xu et al., 2004
). In contrast, humoral immune responses are known to be important in controlling vaccinia in both humans (Lane et al., 1969
) and mice (Galmiche et al., 1999
). In mice depleted of either CD4+
T cells or B cells, severe defects in neutralizing antibody production are associated with enhanced viral replication. These studies revealed the CD4+ T cell dependence of a large portion of the anti-vaccinia neutralizing antibody response.
Here, we demonstrate how vaccinia infection induces immune network signals that promote humoral immunity. Early IL-6 production, preferential activation of the STAT3 pathway, and potent neutralizing antibody responses all depended on viral recognition via TLR2. We found that IL-6 was necessary for robust activation of the STAT3 pathway, although numerous cytokines can potentially activate STAT3 in T cells (including IL-10 and IL-21). IL-6 is thus a non-redundant regulator of STAT3 programming with reported effects ranging from increasing T cell viability (Takeda et al., 1998
), to the deactivation of regulatory T cells (Korn et al., 2007
), and, prominently, the promotion of antibody responses.
Surprisingly, although IL-6 was first discovered as a B cell hybridoma factor (Hirano et al., 1985
), in our experiments, naïve B cells were only minimally sensitive to IL-6 in vivo
at times early after exposure to vaccinia (). Our observation is in agreement with a recent study that suggests B cells are indirectly affected by IL-6 through the IL-6-dependent generation of IL-21 producing T follicular-helper (TFH
) cells (Dienz et al., 2009
). Alternatively, B cell receptor stimulation increases IL-6Rα expression (Burdin et al., 1996
), thus B cells most likely achieve robust STAT3 activation indirectly through IL-21 produced by TFH
cells or via IL-6 directly only after antigen recognition.
In the ectromelia model, at least two lines of evidence indicate that the early induction of IL-6-dependent pSTAT3 can significantly impact on the outcome of infection. First, pSTAT3 is induced rapidly in resistant C57BL/6 mice whereas in the susceptible strain it is not. Second, C57BL/6 mice deficient in IL-6 are highly susceptible to infection. Although the precise molecular mechanism(s) through which IL-6-dependent pSTAT3 induction restricts ectromelia replication is not yet known, our data suggests it acts early and may likely have a role in the innate and inflammatory responses. This is because the absence of IL-6 does not affect generation of a robust antiviral cytotoxic T cell response (data not shown) and IL6−/−
mice succumb to mousepox significantly earlier than B cell-deficient mice (Chaudhri et al., 2006
). Nevertheless, it is likely that IL-6 plays an important role early during the innate response and later in generation of anti-ectromelia antibody responses.
A recent study has shown that TLR9 recognition of ectromelia is necessary for IL-6 production by dendritic cells in vitro
(Samuelsson et al., 2008
). It is therefore probable that TLR9 sensing is also responsible for IL-6 production and STAT activation in our in vivo
model. Additionally, cytosolic recognition of vaccinia DNA is most likely responsible for the IL-6 production observed later in infection in the absence of TLR2 (Figure S2
). These results suggest that a fundamental difference exists in the initial immune response to these closely related poxviruses: vaccinia immediately triggers TLR2 and eventually activates microbial nucleic acid sensing receptors, whereas ectromelia evades TLR2 recognition and is only recognized later by nucleic acid sensors such as TLR9. If this initial IL-6 response to ectromelia in vivo
is purely TLR9-dependent, then the mechanism for TLR9 evasion by ectromelia in BALB/c mice demands further investigation. This fundamental difference in the early host response to vaccinia versus ectromelia may be a key determinant in the maintenance of host-species specificity and could underlie the unique and potent immunogenicity of vaccinia virus.
The single-cell approach to determining the status of signaling pathways utilized in this study facilitated the in vivo deconstruction of the antiviral cytokine response network subsequent to poxvirus infection. Furthermore, we found early and robust STAT1 and STAT3 pathway activation was directly correlated with positive long-term immune outcomes. As single-cell biochemical analysis advances, it will be possible to simultaneously interrogate the activation states of all critical immune signaling pathways across the entire spectrum of hematopoietic cell populations. Definitive in vivo network maps revealing how cytokines program the immune system during homeostasis and disease are the likely byproduct of evaluating the immune system cell-by-cell and may provide means to develop novel therapeutic approaches, to closely assess an ongoing immune response, and to predict immune outcome.