Sepsis is a highly lethal disorder that induces multiple defects in cells of both the innate and adaptive immune system (14
). Sepsis targets T cells, B cells, DCs, NK cells, and monocytes for apoptotic destruction (17
). Sepsis also induces an immunosuppressive phenotype consisting of decreased responsiveness of immune effector cells (13
). The net result of these sepsis-induced alterations is a severe impairment in immunity such that patients are often unable to eradicate their primary infection and are vulnerable to secondary nosocomial infections, often with organisms that are not pathogenic in patients with competent immune systems. Perhaps the strongest evidence for the profound immunosuppression in patients with sepsis is their loss of the delayed type hypersensitivity response to positive controls and their reactivation of latent viral pathogens (32
). Two recent studies of critically ill immuno-competent patients (many of whom had sepsis) requiring prolonged intensive care unit stays showed a high incidence of reactivation of CMV (33%) and HSV (21%) (32
). Reactivation of these viral pathogens is presumably due to loss in T cell immunity, thereby allowing reactivation of latent virus.
Given the compelling evidence of immunosuppression as a major pathologic sequela of sepsis, immunostimulatory cytokines are rational agents to study in animal models of sepsis as potential therapeutics. In the current study, we evaluated the efficacy of therapeutic IL-15 in the disorder and identified cellular mechanisms for its salutary effects.
IL-15 can activate both innate and adaptive immunity (1
) and is required for the differentiation of activated CD8 T cells into effector CD8 T cells, a requirement for an effective immune response to invading pathogens. IL-15 is also necessary for DC priming of NK cells (5
) that may be beneficial in sepsis by production of IFN- γ, a key macrophage-activating cytokine. Eradicating pathogens in sepsis requires coordinated action of cells of both the innate and adaptive immune system and IL-15 is advantageous in this regard because of its broad effects on cells in both systems.
As an IL-2 family member, IL-15 also has potent antiapoptotic activity that we hypothesized would override the widespread apoptosis of immune cells in sepsis. Previous studies support this assertion; IL-15was found to protect against lethal apoptosis in vivo and prevent death of lymphocytes and hepatocytes against multiple apoptotic stimuli (11
). In the current study, IL-15 prevented the sepsis-induced apoptotic depletion of DCs, NK cells, and CD8 T cells. The potential significance of this antiapoptotic effect of IL-15 should be viewed in the context of numerous animal studies showing that prevention of sepsis-induced apoptosis by a variety of different means improves survival in the disorder (17
Attesting to its potent and diverse antiapoptotic effects, IL-15 also prevented sepsis-induced death of intestinal epithelial cells that have been shown to play a key role in sepsis (31
).Work from our group has shown that transgenic mice that overexpress Bcl-2 in the intestinal epithelium had markedly decreased gut apoptosis and conferred a >3-fold improvement in survival (35
). Additional work from our group has shown that administration of epidermal growth factor ameliorated the increase in proapoptotic proteins, reduced gut apoptosis, and decreased mortality from 60% to 30% in the mouse CLP model (36
). One theory of the role of the gut in sepsis relates to its barrier function. It is postulated that the loss of bowel integrity in sepsis results in translocation of bacteria or bacterial products, for example, endotoxin, into the circulation, leading to the theory that the gut represents the “motor” of the systemic inflammatory response.
Survival of hematopoietic cells is due to the antagonistic balance between pro- and antiapoptotic Bcl-2 family members (37
). One of the antiapoptotic mechanisms of action of IL-15 in CD8 T cells in sepsis is due to its effects to increase Bcl-2 as demonstrated in the current study (). CLP mice treated with IL-15 had an increase in intracellular Bcl-2 staining in CD4 T, CD8 T, NK, and DC cells compared with non-IL-15–treated CLP mice (). In addition to increasing antiapoptotic Bcl-2, IL-15 also prevented the sepsis-induced increase in proapoptotic Bim and PUMA protein (). Bim and PUMA protein abundance were unchanged in IL-15– treated septic mice compared with sham-operated mice. Previous work from our laboratory has shown that Bim null and PUMA null mice have a marked decrease in sepsis-induced apoptosis; Bim null mice also had an improved survival in sepsis (30
). The current results in CD8 T cells are consistent with work by Huntington et al. who showed that IL-15–mediated survival of NK cells was due to the effect of IL-15 to decrease Bim (38
). These investigators showed that IL-15 decreased Bim via its effects on phosphorylation of Erk1 and Erk2 kinases (38
). To the best of our knowledge, the current work demonstrating that IL-15 decreases PUMA in CD8 T cells is the first report to note this particular mechanism of action of IL-15. These findings, showing the effects of IL-15 on Bim and PUMA protein in sepsis, are consistent with recent work from our laboratory in which IL-15 prevented the sepsis-induced increase in mRNA for both Bim and PUMA in CD8 T cells (data not shown). In short, antiapoptotic effects of IL-15 are due in part to increasing anti-apoptotic Bcl-2 family member expression while preventing the injury-induced increases in proapoptotic Bcl-2 family members.
The proper host reaction to invading pathogens is a vigorous but controlled proinflammatory immune response (13
). A failure of the host to mount an initial robust immune response results in further pathogen multiplication. Alternatively, an excessive unbridled proinflammatory response may result in “cytokine storm”-mediated organ injury and mortality. The effect of sepsis to increase CD4 T cell activation markers and to increase circulating proin-flammatory cytokines is indicative of the establishment of a heightened host immune response to the severe infection. Because of the potent effects of IL-15 on CD8 T cells, DCs, and NK cells, the authors were concerned that IL-15 might exacerbate the proin-flammatory response in sepsis and lead to worsened survival in the current study. The results reported in this study indicate that IL-15 does not cause excessive immune stimulation and “cytokine storm” using this dosing regimen. In this regard, IL-15 did not further increase CD4 or CD8 T cell activation markers in the septic animals (Supplemental Fig. 2
). Furthermore, although IL-15 did increase circulating IFN-γ, it modestly decreased circulating IL-6 and TNF-α, two prototypical proinflammatory cytokines in sepsis (). One possible explanation for the decreased IL-6 and TNF-α in IL-15–treated mice with sepsis could be that IL-15 resulted in a more rapid containment of the infectious process and thereby a less robust immune response.
As noted, IL-15 caused an increase in circulating IFN-γ, a key macrophage-activating cytokine. In addition, IL-15 more than doubled the percentage of IFN-γ–positive NK cells in both sham and septic mice as well as increased IFN-γ production in stimulated splenocytes (). The effect of IL-15 on IFN-γ production may be particularly important. Sepsis impairs the production of IFN-γ by immune cells and although there is some controversy regarding exogenous administration of IFN-γ, studies have shown that restoration of production of endogenous IFN-γ can improve survival (39
To date there are few studies examining the role of IL-15 treatment in animal models of bacterial infection. Hiromatsu and associates demonstrated that mice treated with IL-15 immediately after injection of live E. coli
had improved survival and reduced apoptosis in the peritoneal cavity, liver, spleen, and lung (10
). Although the study by Hiromatsu et al. provides important data, the injection of live E. coli
is not considered to be a clinically relevant animal model of sepsis (41
). There is also an earlier report examining the role of IL-15 in the more widely accepted CLP model of sepsis using knockout mice. In that study Orinska and colleagues found that IL-15 null mice had improved survival in the CLP model of sepsis, and they attributed this improvement to an inhibitory role of intracellular IL-15 in mast cell cytokine maturation (21
). The confounding nature of the findings between Orinska et al. and Hiromatsu et al. motivated the current study.
The current results are consistent with the report from Hiromatsu et al., suggesting that there is a predominant immunosupportive and immunostimulatory effect of therapeutic IL-15. In contrast, Orinska and associates showed a key role for mast cell-specific intracellular
). Their work established that mast cell-specific IL-15, that is, IL-15 that was confined intracellularly
within the mast cells, inhibited mast cell chymase activity. Mast cell chymase activity is essential for mast cell antibacterial effects by regulating activation of several biological mediators (especially chemokines and cytokines) that assist in host defenses. Deletion of IL-15 resulted in enhanced mast cell chymase activity, increased chemokine/cytokine processing, and more efficient attraction of polymorphonuclear cells into the peritoneal cavity after CLP. In the current study, there were no differences in the recruitment of cells to the peritoneum in septic mice between IL-15 and vehicle treatment, suggesting that exogenous IL-15 does not alter mast cell-mediated recruitment of myeloid cells to the site of infection (Supplemental Fig. 3
There is no clear explanation for the differences in the findings of Orinska et al. versus the findings in the current study. One possibility is that Orinska et al. used IL-15 null mice and it is known that the host immune system compensates for genetic deletion of various key components. Thus, the IL-15 null mice may have had other unknown compensatory mechanisms that were operative in sepsis. To further address this issue, we conducted studies testing an anti–IL-15 Ab in sepsis. Mice treated with anti–IL-15 Ab had a significantly worsened mortality compared with septic mice that did not receive the Ab (data not shown).
In conclusion, IL-15 had broad antiapoptotic effects and protected CD8 T, NK, DC, and intestinal epithelial cells from sepsis-induced apoptosis and augmented IFN-γ production in septic animals. These effects were associated with improved survival in two widely used models of sepsis. These attributes make IL-15 an attractive target for further development as a potential therapy for sepsis.