In this study, we tested the hypothesis that IL-7, a potent T-cell antiapoptotic cytokine, would mediate the early innate immune response to sepsis. First, we demonstrated the necessity of αβ and/or γδ T cells for the IL-7-induced increase in survival of septic wild-type mice. Additionally, we show the upregulation of Bcl-2 expression on CD4 T cells as early as 3 h following CLP in mice treated with rhIL-7. Here, mice treated with rhIL-7 were shown to have increased neutrophil recruitment to the peritoneum without increased end-organ tissue damage compared to that of WT controls. Elevated neutrophil recruitment at the site of infection was correlated with elevated local and systemic production of CXCL1/KC and IL-17. We determined that following rhIL-7 treatment, septic mice had increased numbers of γδ T cells in the peritoneum, producing elevated amounts of IL-17. Additionally, γδ T cells were necessary for an elevated peritoneal IL-17 concentration following IL-7 treatment. Previously, we demonstrated that T cells isolated from IL-7-treated mice had a reversal of the sepsis-induced defective production of IFN-γ (55
). Here, we built upon these findings by demonstrating an IL-7-induced increase in peritoneal IFN-γ levels. Increased IFN-γ production has been shown to promote macrophage or endothelial CXCL10/IP-10 expression (3
), a ligand for the CXCR3 receptor. We show that this receptor is expressed upon γδ T cells. These results suggest that IL-7 treatment improves the host response to sepsis through the rapid recruitment of γδ T cells to the site of infection, coupled with accelerated γδ T cell-mediated recruitment of neutrophils to the site of infection. All together, these data are summarized in Fig. .
FIG. 8. Model for IL-7's role in modulating neutrophil recruitment in sepsis. This figure represents a schematic hypothesis, demonstrating how rhIL-7 treatment, through maintenance of appropriate T-cell immunocompetence, results in enhanced neutrophil recruitment (more ...)
The work detailing that rhIL-7 treatment improves the murine host response to sepsis has recently been reported (55
), although its effects on lymphocyte apoptosis and proliferation in human diseases such as hepatitis C and cancer stem from ongoing clinical trials (46
). At 24 h following CLP, IL-7 treatment significantly increased Bcl-2 expression in splenic CD4 and CD8 T-cell subsets compared to that in wild-type animals and produced a relative change of mRNA abundance in CD4 T cells (55
). Additionally, rhIL-7 blunted both splenic and mesenteric lymph node T-cell apoptosis at 24 h following CLP, resulting in significantly elevated numbers of CD4 and CD8 T cells in treated versus WT mice at this time point (55
). Clinical findings of decreased Bcl-2 gene expression in septic human patients at 12 and 24 h following diagnosis were previously noted to correlate highly with decreased absolute numbers of CD4 lymphocytes (4
). Furthermore, prevention of apoptosis with overexpression of Bcl-2 is known to prevent T-cell apoptosis, providing protection from sepsis-related mortality (26
). Here, we show that naive CD4, CD8, and effector memory CD4 cells obtained from rhIL-7-treated animals have significantly elevated Bcl-2 protein expression at 3 h following CLP, suggesting that apoptotic signaling in T cells takes place soon after induction of sepsis. One key effect of early T-cell apoptosis in sepsis is reduced immunocompetence through decreased IFN-γ production. The production of IFN-γ by CD4 T cells during sepsis is associated with improved survival, decreased bacterial load, and increased neutrophil functionality (37
). Interestingly, we show that IL-7 treatment significantly increases the peritoneal concentration of IFN-γ as early as 3 h following CLP. These data suggest that rhIL-7 treatment upregulates Bcl-2 expression very soon after treatment, such that T-cell apoptosis can be reduced and IFN-γ production can be preserved.
The enhanced production of IFN-γ in rhIL-7-treated mice relative to WT controls exerts downstream effects on both adaptive and innate cells. Gamma interferon-inducible protein 10 (CXCL10/IP-10) is a chemokine secreted by endothelial cells, macrophages, and neutrophils upon stimulation with IFN-γ and serves as a chemoattractant for T lymphocytes (7
). It has been suggested that CXCL10/IP-10 plays a distinct role in the recruitment of T cells expressing CXCR3 to sites of inflammation (7
), consistent with findings that TCR γδ T cells express CXCR3 and transmigrate upon stimulation with IP-10 (11
). Here, we demonstrate that a large percentage of γδ T cells and neutrophils present at the site of infection soon following CLP express CXCR3. Further, septic mice treated with IL-7 had significantly larger amounts of CXCL10/IP-10 at the peritoneum. Consistent with these findings, we observed that rhIL-7 treatment resulted in an elevated number of γδ T cells at the site of infection. All together, we postulate that IL-7 treatment enhanced T-cell IFN-γ production such that CXCL10/IP-10 was increased and γδ T cells entered the site of infection.
After exposure to CXCL10/IP-10, γδ T cells can transmigrate and mediate inflammatory processes in tissue compartments such as the lung and peritoneum. The inflammatory effects of γδ T cells result, in part, from production of the cytokine IL-17 in response to early and sustained stimulation by IL-1β, TLR-2, and IL-23 (1
). IL-17 acts upon mesothelial cells to produce CXCL1/KC and CXCL2/MIP-2 (15
) and has been shown to be necessary for the promotion of granulopoiesis and neutrophil recruitment in peripheral tissues (14
). Here, we show that local and systemic levels of IL-17 are elevated with rhIL-7 treatment following CLP, with a subsequent and complementary increase in CXCL1/KC. Further, elevated IL-17 following rhIL-7 treatment in sepsis is dependent upon the presence of γδ T cells. This demonstrates a sequential and mechanistic pathway through which rhIL-7-mediated attenuation of T-cell dysfunction results in increased neutrophil recruitment to the site of infection following CLP. The elevated number of neutrophils early in the septic course is likely responsible for the decreased bacterial load in rhIL-7-treated mice, even in the absence of neutrophil functionality changes. The rapid return of neutrophil numbers to WT levels at 24 h may explain the comparable levels of end-organ tissue damage seen in these groups, consistent with previous findings in this model assessing the role of neutrophils in sepsis (22
). The lack of a functional effect on neutrophils following IL-7 treatment may further explain the consonant end-organ results in these studies. All together, we speculated that the ability of rhIL-7 to maintain numbers and functionality of CD4 T cells following sepsis resulted in accelerated γδ T-cell recruitment and functionality at the site of infection, ultimately aiding bacterial clearance without worsening tissue damage.
Our data reported here demonstrate that rhIL-7 treatment increased peritoneal IFN-γ levels, IP-10 levels, and CXCR3-expressing γδ T-cell accumulation. However, one limitation to this report is a lack of data verifying that IP-10, through CXCR3, recruits γδ T cells to the peritoneum. While trying to address this, we subjected CXCR3-deficient mice to CLP and found that these mice had significantly increased peritoneal bacterial loads compared to those of WT mice. In addition, neutrophils isolated from these mice demonstrated a decreased phagocytic capacity. Finally, the CXCR3 knockout (KO) had increased numbers of neutrophils and macrophages at the site of infection compared to those of controls. We speculate that the CXCR3-mediated defect in phagocytosis resulted in sustained leukocyte chemotaxis that ultimately confounded the experimental results. Thus, the use of more specific reagents will be needed to fully address this potentially important mechanism.
Sepsis involves a complex interplay between the innate and adaptive immune systems that occurs very early after the onset of disease and not in a delayed fashion, as previously accepted. γδ T cells, through production of IL-17, appear to play a central role in immune response cross talk between the two arms of the immune response. In addition, the existence of cross talk between these two responses suggests that multiple targets for future therapeutic intervention may be available. One such therapeutic target is the cytokine IL-7, shown to play a direct role in prevention of T-cell apoptosis, with subsequent downstream effects resulting in an enhanced innate response to sepsis. Going forward, these insights may play a role in the ultimate production of a viable sepsis therapeutic target that reduces the health care burden imposed by this disease process.