In the work presented above, the interplay between cytokines and intracellular signaling molecules in the regulation of NK cell activation and function is defined at the earliest site of viral infection in immunocompetent mice. Production of IFN-α, IFN-β, and IFN-γ in the peritoneal cavity was shown to peak at 30 h after LCMV infection. NK cells were the major producers of IFN-γ and uniquely expressed high levels of STAT4. The pathway for induction of NK cell IFN-γ production was dependent on responsiveness to type 1 IFNs and on the STAT4 signaling molecule. The elicited IFN-γ enhanced defense because loss of responsiveness to the factor resulted in increased viral burdens. Thus, type 1 IFN induction of IFN-γ production by NK cells, dependent on STAT4, is conclusively proven to occur and to lead to downstream effects under immunocompetent conditions.
Previous work from this laboratory has established that under basal conditions, splenic NK cells express high STAT4 and low STAT1 levels and preferentially activate STAT4 over STAT1 after ex vivo
exposure to type 1 IFNs (9
). Nevertheless, it has only been possible to identify type 1 IFN activation of STAT4 and IFN-γ in the spleens of STAT1-deficient mice during LCMV infection because the type 1 IFNs and IFN-γ induce STAT1 levels to block the pathway to STAT4 (9
). The results reported here answer the question of why a pathway from type 1 IFN to STAT4 for IFN-γ expression would be maintained when it is rapidly turned off by concurrent STAT1 induction; it is used to locally access a short burst of IFN-γ at very early times after infection. In this case, “locally” is the peritoneal cavity. In addition to the detectable NK cell IFN-γ response, innate responses to LCMV at this site differed from the well-characterized responses in spleen and serum (7
) with regard to the kinetics and magnitude of type 1 IFN production and the kinetics of STAT1 induction in NK cells (9
). Type 1 IFN production in the peritoneal cavity was very early and of short duration—detectable at 24 h, peaking at 30 h, and resolving by 36 h after infection ()—and although total lymphocytes and T cells had dramatically elevated STAT1 levels early, induction was delayed by 8 to 16 h in the NK cells (). Thus, in contrast to the spleen, the environment in the peritoneal cavity allows type 1 IFN to access STAT4 prior to STAT1 induction in NK cells ().
FIG 7 Compartmental differences in innate immune response to LCMV infection. Shown is a schematic representation of the differences in the immune response to LCMV in the peritoneal cavity compared to the spleen. The results presented here studying the peritoneal (more ...)
Why was STAT1 elevation delayed in the NK cells compared to the other PEC populations examined? Promoter sequences in the STAT1 gene specific for STAT1 complexes may act to enhance STAT1 induction whenever STAT1 is activated (26
). Thus, any cytokine signaling through STAT1 would be expected to induce elevated STAT1 levels. In addition to type 1 IFN receptors, receptors for IFN-γ also activate STAT1 (10
). The results presented here suggest that NK cells might first respond to type 1 IFN exposure with IFN-γ because of their high STAT4 levels, but once IFN-γ is induced, it acts back on the cells to induce STAT1. This would explain the delay in STAT1 induction in NK cells compared to other subsets. Alternatively or additionally, it might just take longer for type 1 IFN to induce STAT1 in populations with high STAT4 levels. The NK cells in both the spleen (9
) and peritoneum () consistently have lower levels of STAT1. Because the spleen is a secondary compartment of infection, most of the NK cells may be experiencing cytokines produced earlier at other sites and/or not be synchronized to make a detectable short burst of IFN-γ in this compartment. In any case, it is remarkable that there is a window of opportunity for IFN-γ production under the conditions in the peritoneum given the link between this response and shutting it off. The pathway suggests that there will be a very early source of NK cell IFN-γ whenever type 1 IFNs are induced.
Our studies are helping to resolve conflicting reports in the literature on potential type 1 IFN consequences for IFN-γ expression (5
), adding information to the growing literature reporting cellular differences in type 1 IFN responses (27
) and advancing the mechanistic understanding of how the biological effects of STAT cytokines are regulated to access diverse and paradoxical effects as needed. Type 1 IFNs have long been known to exert antiviral effects and enhance NK cell cytotoxicity through the activation of STAT1 (4
). The NK cells, however, are positioned to respond to type 1 IFNs with STAT4 activation because high basal levels of STAT4 are associated with the type 1 IFN receptor (9
). Increasing the endogenous levels of total STAT1 protein negatively regulates STAT4 access in part by displacing STAT4 from the receptor, and this induction of STAT1, as observed in the spleen, is beneficial because it protects from dysregulated systemic IFN-γ production and cytokine-mediated disease (9
). In contrast, the results reported here show that the type 1 IFNs have early access to STAT4 and the NK cell IFN-γ pathway prior to STAT1 induction and that this pathway promotes resistance to infection. Recent studies have shown that similar STAT4-STAT1 regulation is in place in humans; type 1 IFNs induce STAT4 activation in NK cells isolated from healthy individuals, but the response is diminished in NK cells from individuals chronically infected with hepatitis C virus. The activation of STAT4 negatively correlates with STAT1 levels in these populations (29
). Thus, modulation of STAT4 and STAT1 concentrations in different cell types under different conditions of infections in mice and humans helps explain how type 1 IFNs can enhance or antagonize IFN-γ stimulation, and the studies with mice provide explanations for how the balance, in both directions, works to the benefit of the host. A change in relative STAT ratios might also play a role in conditioning NK cells to modify type 1 IFN responses from promoting cytokine production to enhancing cytotoxic function.
In addition to our earlier studies with STAT1-deficient mice, there have been a few other reports suggesting a role of type 1 IFN in NK cell IFN-γ expression. One examined a number of activation responses, including intracellular IFN-γ, of type 1 IFN receptor-deficient compared to WT NK cells following in vitro
exposure to vaccinia virus and dendritic cells (30
). Early in vivo
IFN-γ induction has been reported in the mouse peritoneum during different viral infections (31
), and intracellular IFN-γ expression by splenic NK cells has been observed following treatments with the chemical analogue of viral nucleic acids, polyinosinic-polycytidylic acid [poly(I
). These in vivo
studies, however, have been done under conditions where the type 1 IFN effects were not delineated from those that might have been mediated by other cytokines induced under the experimental conditions used, including reported IL-12. In one study with poly(I
), the intracellular NK cell IFN-γ expression was shown to be blocked by treatments with antibodies neutralizing type 1 IFN function, but the consequences of the treatments on other cytokine responses were not evaluated. Here, in vivo
responses under which the type 1 IFN receptor was blocked were evaluated by different approaches, including adoptive transfer of peritoneal populations from WT or type 1 IFN receptor-deficient mice into WT-infected mice (). This method allows direct comparison of donor and recipient cells in the context of the complete endogenous immune responses. Thus, to our knowledge, this is the first demonstration of type 1 IFNs inducing IFN-γ production from NK cells under immunocompetent conditions of viral infection.
The studies do not exclude possible roles for accessory cytokines in enhancing type 1 IFN induction of IFN-γ. Certainly, IL-12 is an activator of STAT4 and a potent inducer of IFN-γ (13
). The cytokine IL-18 can enhance the stimulation of IFN-γ by either type 1 IFNs or IL-12 (7
), and new studies deciphering pathways from different sensors have demonstrated synergism between type 1 IFNs and IL-12 for IFN-γ induction in human cells (38
). The results presented here, however, suggest that there may be a short burst of IFN-γ induced by type 1 IFNs independent of IL-12, and our previous work examining murine cytomegalovirus infections of mice has shown that induced type 1 IFNs promote NK cell cytotoxicity but IL-12 is required for splenic and systemic NK cell IFN-γ (3
). The experiments carried out here neutralizing IL-12 in vivo
show that IL-12 does not play a role in the LCMV-induced NK cell IFN-γ response (). Interestingly, the positive controls for these studies (i.e., effects on LPS induction of IFN-γ in the peritoneal cavity) () demonstrate that when it is elicited, IL-12 is important in eliciting a peritoneal NK cell IFN-γ, but also that there is residual NK cell IFN-γ after neutralization. This is consistent with early reports of incomplete IFN-γ blockade by IL-12 neutralization following LPS treatment in other settings (21
) and the known LPS induction of IFN-β (39
). Taken together, the studies suggest that IL-12 is an important inducer of NK cell IFN-γ if elicited and the critical cytokine for IFN-γ induction once STAT1 levels are increased. There are likely to be additional regulatory factors in play, however, as the presence of STAT1 eventually results in reduced IFN-γ induction by either type 1 IFNs or IL-12 (6
). Thus, much remains to be done to map the complex interactions between type 1 IFN and IL-12 for shaping NK cell responses, but eliciting different type 1 IFN and IL-12 responses may be part of directing the magnitude of, and continued access to, the NK cell IFN-γ response.
The peritoneal responses reported here demonstrate that by being receptive to early type 1 IFN signaling with IFN-γ production, NK cells can serve as sensors for and responders to the presence of viral pathogens. The studies surprisingly reveal a previously elusive role for NK cells in early defense against LCMV. In comparison to other viral infections in compartments outside the peritoneal cavity, LCMV has been shown to be relatively insensitive to the direct antiviral effects of NK cells (40
) or IFN-γ (43
). Our previous attempts to access NK cell IFN-γ in defense against LCMV have shown that administration of IL-12 prior to infection can induce the response to result in under a 1-log decrease in splenic viral titers (44
). In this report, infection-induced IFN-γ, dependent on NK cells, was shown to proportionally limit viral burden at this first site of infection. Thus, though modest, there is a documented effect of the NK cells and IFN-γ during LCMV infection. The mechanism for the antiviral effect mediated by IFN-γ is not known. Given the rapid kinetics, it is likely to be a result of the activation of direct antiviral effects in infected cells or macrophages rather than enhancement of adaptive immune functions resulting from promoting major histocompatibility complex (MHC) class 1 expression or CD8 T cell effector functions (45
). It remains to be determined whether or not the NK cell IFN-γ elicited by type 1 IFNs will be a more important first response in defense against other more sensitive viral infections and/or for promoting immunoregulatory functions.
In summary, these studies discover a novel pathway for NK cell stimulation at an initial site of infection. Moreover, they advance understanding of the regulation of type 1 IFN responses and provide critical insights into how the immune response is finely regulated to deliver maximal protection without being detrimental to the host.