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The Journal of Infectious Diseases logoLink to The Journal of Infectious Diseases
. 2012 Jun 12;206(4):606–616. doi: 10.1093/infdis/jis383

Interleukin-7 Ameliorates Immune Dysfunction and Improves Survival in a 2-Hit Model of Fungal Sepsis

Jacqueline Unsinger 1, Carey-Ann D Burnham 2, Jacquelyn McDonough 1, Michel Morre 3, Priya S Prakash 4, Charles C Caldwell 4, W Michael Dunne Jr 2, Richard S Hotchkiss 1,5,6
PMCID: PMC3491749  PMID: 22693226

Abstract

Background. Secondary hospital-acquired fungal infections are common in critically-ill patients and mortality remains high despite antimicrobial therapy. Interleukin-7 (IL-7) is a potent immunotherapeutic agent that improves host immunity and has shown efficacy in bacterial and viral models of infection. This study examined the ability of IL-7, which is currently in multiple clinical trials (including hepatitis and human immunodeficiency virus), to improve survival in a clinically relevant 2-hit model of fungal sepsis.

Methods. Mice underwent cecal ligation and puncture to induce peritonitis. Four days later, surviving mice had intravenous injection with Candida albicans. Following Candida infection, mice were treated with IL-7 or saline control. The effect of IL-7 on host immunity and survival was recorded.

Results. IL-7 ameliorated the loss of immune effector cells and increased lymphocyte functions, including activation, proliferation, expression of adhesion molecules, and interferon-γ production. These beneficial effects of IL-7 were associated with an increase in global immunity as reflected by an enhanced delayed type hypersensitivity response and a 1.7-fold improvement in survival.

Conclusions. The present findings showing that IL-7 improves survival in fungal sepsis, together with its previously reported efficacy in bacterial and viral infectious models, further supports its use as a novel immunotherapeutic in sepsis.

Sepsis is a leading cause of death in most intensive care units (ICUs) [1, 2]. In many cases of protracted sepsis, the early hyperinflammatory phase of sepsis progresses to a state of relative immunosuppression characterized by severe defects in immunity thereby inhibiting their ability to control their primary infection and predisposing the patient to secondary nosocomial infections [3–6]. In addition to apoptosis-induced depletion of critical cellular components of the immune system, the function of surviving immune cells is profoundly depressed [5–8]. Increased T-regulatory cells and myeloid-derived suppressor cells also contribute to sepsis-induced immunosuppression [7, 8]. Although the immune defect in sepsis may manifest itself in many ways, it is often recognized by reactivation of latent cytomegalovirus and herpes simplex virus and by acquisition of secondary infections [6, 9, 10].

One of the most common secondary infections in septic patients is due to Candida albicans, which induces an opportunistic infection termed candidiasis [11–16]. Candida infections are now the third most common cause of bloodstream infections in many ICUs [11, 13]. Despite excellent antimicrobial therapy against most Candida infections, mortality remains >30%–40% [11–15]. Candida albicans resides as a commensal of the human mucosal and gastrointestinal tract [15, 16]. In immunocompromised hosts, saprophytic colonization often leads to opportunistic mucosal or life-threatening deep organ infections [11, 13, 14]. A wide range of factors that compromise host immunity or alter host bacterial flora predispose patients to infections by Candida [11–16].

Interleukin-7 (IL-7) is a pluripotent cytokine produced by bone marrow and thymic stromal cells that is required for T-cell survival [17–22]. IL-7 induces proliferation of naive and memory T cells [17, 20, 23], potentially supporting replenishment of the peripheral T-cell pool, which is severely depleted during sepsis [3, 6]. IL-7 also improves activation of T cells and increases expression of cell adhesion molecules, which improve the ability of T cells to traffic to sites of infection [24]. IL-7 is currently undergoing numerous clinical trials, including in patients with cancer, human immunodeficiency virus 1 (HIV-1), and viral hepatitis [25]. Unlike other closely related common γ-chain cytokines such as IL-2, IL-7 does not induce a hyperinflammatory “cytokine storm” response and is generally well tolerated [26–29].

IL-7 improves survival in a polymicrobial peritonitis model of sepsis [24]. Additionally, IL-7 restored immunity and decreased mortality in a viral model of lymphocytic choriomeningitis [30]. Thus, IL-7 has shown efficacy in combating diverse pathogens. The present study was designed to test IL-7 in a 2-hit model of sepsis in which a sublethal peritonitis infection was followed by secondary C. albicans infection. This 2-hit model of sepsis was used because it induces an immunosuppressed state in which the host is more vulnerable to secondary pathogens. This situation reflects the clinical condition that occurs in patients in ICUs who develop hospital-acquired fungal infections. Survival in Candida infections is critically dependent on collaboration between neutrophils, macrophages, and lymphocytes with the latter being closely regulated by IL-7 [31, 32]. We also investigated potential mechanisms for its putative beneficial effect, including actions to reverse the sepsis-induced loss in the delayed type hypersensitivity response, prevent cell death, and improve cell function.

METHODS

(See “Supplement” online for complete methods.)

Mice

Eight- to ten-week-old male C57BL6 or CD1 mice were used in studies. Procedures were approved by the Animal Studies Committee.

Antibodies and Reagents

Information on antibodies, reagents, and cytokine kits are provided in “Supplement” online.

Cecal Ligation and Puncture Model of Sepsis

The cecal ligation and puncture (CLP) model was used to induce peritonitis [24]. Mice were anesthetized and a midline abdominal incision performed. The cecum was ligated and punctured twice. Imipenem (25 mg/kg) was given subcutaneously 4 hours post-CLP.

Determination of Immune Status Prior to Infection With Candida

Four days after surgery, splenocytes were harvested, tested for viability, and stained for cell-surface markers [33]. Cells were stimulated for 18 hours with CD3/CD28 and stained for intracellular interferon-γ (IFN-γ) [34].

Second-Hit C. albicans Model

Candida albicans (ATCC MYA-2430) was grown overnight in Difco Sabouraud dextrose broth medium, harvested, washed, and suspended in saline to obtain an optical density of 0.5A600. Four days post-CLP, surviving mice received 50 μL Candida suspension intravenously. Previous studies demonstrated that mice had increased susceptibility to Candida (consistent with impaired immunity) at this time point [24, 35]. This dose of Candida caused <10% mortality in naive mice; therefore, the much higher mortality in mice that had undergone CLP prior to Candida challenge highlights their compromised immunity. Mice received a single dose of fluconazole (50 μg intraperitoneally) 4 days after Candida injection. For survival studies, animals received 2.5 µg IL-7 subcutaneously, starting at day 6 and continuing for 5 days. Animals were observed for 14 days. Because of the high lethality of Candida at 14 days and because IL-7-treated mice began to show survival benefit at 11 days (see “Results”), mice were killed at day 11 (7 days post–Candida injection) for those studies evaluating immunologic/cellular effects of Candida and IL-7 treatment.

Recombinant Human IL-7

Recombinant human IL-7 was provided by Cytheris Corporation as described [24]. Human IL-7 can bind and signal via the murine IL-7 receptor [36].

Determination of Cell Proliferation-Ki-67 Staining

Mice were treated with 2.5 μg IL-7 or saline subcutaneous for 5 days, starting at day 5 post-CLP (1 day post–Candida injection). At day 11, cells were harvested and cell proliferation quantitated [35].

Quantification of Absolute Cell Counts and CD4 and CD8 T-Cell Subsets

Total cell counts per spleen, naive, effector memory, and central memory CD4 and CD8 T cells were determined [35].

Quantitation of Cytokines

Cytokines were quantified via enzyme-linked immunosorbent assay [35].

Determination of Delayed-Type Hypersensitivity

At day 4 post-surgery, mice received 50 μL Candida suspension. One group received 2.5 μg IL-7 subcutaneously 1 day post–Candida infection for 5 consecutive days, while the control group receiving saline. Seven days post-CLP, mice were immunized with 100 μL of 10 mM 2,4,6-trinitrobenzenesulfonic acid (TNBS) subcutaneously. At day 11, mice had antigenic challenge with 30 μL of 10 mM TNBS in the right footpad. Phosphate-buffered saline was injected in the left footpad as a control. Measurements (micrometers) of footpad swelling represent the difference between the right and left footpad.

Determination of Intracellular IFN-γ Production

IFN-γ plays a central role in control of Candida infection [37]. Thus, we also examined the impact of IL-7 on IFN-γ production post–Candida infection. Twenty-four hours post–Candida infection, 1 group of mice started treatment with 2.5 µg IL-7 for 5 consecutive days. At day 11, splenocyte suspensions were stimulated overnight with CD3/CD28 [24, 35]. Supernatants were harvested and cytokines analyzed [35, 38]. For the detection of intracellular cytokine production, Brefeldin A was added for an additional 4 hours of incubation. Cells were fixed, permeabilized, and stained for IFN-γ.

Determination of Tissue Candida Colony Forming-Units

To determine if IL-7 had an effect to decrease tissue Candida concentrations, kidney and liver were harvested at days 3 and 7 post–fungal infection and Candida colony-forming units (CFUs) quantitated.

Statistical Analysis

Data are reported as mean ± SEM. Data were analyzed using the statistical program Prism (GraphPad, San Diego, CA). Data were analyzed using 1-way analysis of variance with Dunnett's post-test or Student's t test. Survival data were analyzed utilizing log-rank or χ2. Significance was accepted at P < .05.

RESULTS

Characterization of the Host Immune Status Prior to Candida Challenge

Prior to testing IL-7, we performed a series of studies to characterize host immune status at day 4 post-CLP, the time point when Candida was injected. Studies have shown that mice are much more susceptible to pathogen challenge 4 days after CLP [38], yet few studies have examined the phenotypic and functional changes in host innate and adaptive immunity at this time point. To determine which cell subsets were impacted by sepsis, we enumerated and characterized T-cell and macrophage subsets (Figure 1). As compared to healthy mice, septic mice had decreased absolute numbers of naive and central memory CD4 and CD8 T cells (Figure 1A). In addition, T cells from septic animals had decreased CD44 expression, and upon T-cell activation, decreased IFN-γ production and CD69 expression (Figure 1B–D). Interestingly, we observed a significant increase in F4/80−/CD68+ macrophages (Figure 1E). Finally, both macrophage subsets demonstrated reduced major histocompatibility complex class II and CD80 expression, key molecules involved in antigen presentation (Figure 1F). Collectively, the data demonstrate reduced T-cell numbers and functionality, and decreased potential of macrophages to act as potent antigen-presenting cells.

Figure 1.

Figure 1.

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Immune status of splenic leukocytes prior to secondary Candida challenge. Ex vivo splenocytes were isolated from septic or healthy mice 4 days after surgery, and T cells were (A) enumerated and (B) CD44 expression determined as described in the methods. In addition, splenocytes were stimulated with anti-CD3 and CD28 for 18 hours and analyzed for (C) intensity of IFN-γ production and (D) expression of the T-cell activation marker, CD69, as described in the methods. In a parallel set of experiments, splenic macrophages were (E) enumerated and (F) analyzed for major histocompatibility complex class II (MHC II) and CD80 expression. The intensity of expression is measured by the mean fluorescence intensity, which is a measure of the number of molecules of interest (ie, CD44, IFN-γ, MHC II, or CD80 on a per-cell-basis). The sample size for each group was 6. Data were analyzed by Student's t test. *P < .05, septic versus wild type.

IL-7 Improves Survival in Candida Infections

Following Candida infection, mice were divided into IL-7 versus saline control groups. Both dose and timing of administration of IL-7 were important in improving survival. The treatment regimen for IL-7 that improved survival consisted of the following: at day 6 post-CLP (48 hours after Candida), mice had a subcutaneous injection of 2.5 µg IL-7 daily for 5 days (5 doses). Control mice received saline. As demonstrated (Figure 2A), mice treated with IL-7 had a 1.7-fold improvement in survival versus control (P < .002). Additional studies were performed in which mice received a larger inoculum of Candida to determine if IL-7 would improve survival in a more lethal model. In the more lethal model, no control mice survived at day 14, while 40% of IL-7–treated mice survived (Figure 2B; P < .05).

Figure 2.

Figure 2.

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IL-7 improves survival in 2-hit Candida sepsis. Survival studies were conducted in the age-matched outbred strain CD1. Mice received cecal ligation and puncture surgery at day 0. At day 4, mice were injected with C. albicans intravenously. At day 6, IL-7 treatment was started with 1 daily dose of 2.5 μg subcutaneously for 5 days. Survival of mice was followed for 14 days. (A) Results are a combination of 2 independent studies. Data were analyzed by utilizing a log-rank test. Mice treated with IL-7 had an improved survival compared to control mice; *P < .002, log-rank. (B) Candida survival studies were conducted in a more lethal model in which an increased inoculum of Candida suspension (60 µL) was employed. At day 14, no control mice survived, while 40% of IL-7–treated mice survived. *At day 14, there was a statistically different improvement in survival in IL-7–treated mice by χ2, P < .05.

IL-7 Treatment Improves the Delayed Type Hypersensitivity Response

Clinical studies of septic patients have shown that the intensity of the delayed-type hypersensitivity (DTH) response correlates with survival [39]. B6 mice that underwent CLP surgery followed by Candida infection failed to mount a DTH response to antigenic challenge, consistent with the prevailing concept that sepsis leads to severe immunosuppression (Figure 3). Although the immune response of CLP-Candida–infected mice that received IL-7 was reduced compared to naive control group, the DTH response was improved by 39% compared to saline-treated mice (P < .03). Therefore, IL-7 improved immunity even when administered after onset of primary and secondary infections.

Figure 3.

Figure 3.

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IL-7 improves the delayed-type hypersensitivity response. C57BL6 mice underwent cecal ligation and puncture (CLP) surgery. On day 4 post-surgery, mice were injected with C. albicans. IL-7 treatment was started at day 5 with a single dose of 2.5 μg IL-7 and continued for 5 consecutive days. At day 7 post–primary surgery, all mice were immunized with injections of 100 μL of 10 mM 2,4,6-trinitrobenzenesulfonic acid (TNBS) subcutaneously. At day 11, all mice had antigenic challenge with 30 μL of 10 mM TNBS in the right footpad. Phosphate-buffered saline was injected in the left footpad as a control. Twenty-four hours post–antigen challenge, the individual immune responses were determined by measuring footpad swelling. Three groups were tested: (i) immune control (unmanipulated mice that had received primary immunization followed by antigenic footpad challenge), (ii) CLP + Candida (mice that had CLP followed by secondary Candida infection), and (iii) CLP + Candida + IL-7 (mice that had CLP followed by secondary Candida infection and treatment with IL-7). Each value represents the mean of 5 to 9 mice. Data were analyzed by Student's t test. Septic mice treated with IL-7 had an improved delayed-type hypersensitivity response compared to non-IL-7–treated septic mice; *P < .03.

IL-7 Increases Absolute Splenic CD4 and CD8 T-Cell Counts

Twenty-four hours following Candida injection, B6 mice received 2.5 μg IL-7 daily for 5 days and were sacrificed on day 11 post-CLP, a time point at which the IL-7–treated septic mice showed improvement in survival (Figure 2). Whereas control septic mice showed an approximately 50% loss in splenic CD4 T cells compared to naive mice, CD4 T cells in the IL-7–treated group was reduced by only approximately 30% compared to naive. This represents an approximately 38% increase relative to control septic mice (Figure 4A). The effect of IL-7 on various subsets of CD4 T cells was also examined. IL-7 caused significant increases in naive and effector memory CD4 T cells compared to control but not in central memory CD4 T cells (Figure 4A). A more dramatic effect of IL-7 was seen in CD8 T cells. The control septic mice had a reduction in CD8 T-cell counts by 32% compared to naive mice, while CD8 T cells in septic mice treated with IL-7 were actually increased by approximately 126% and 52% compared to control septic mice and naive mice, respectively; (Figure 4B; P < .01). Evaluation of CD8 T-cell subsets showed that IL-7 markedly increased absolute numbers of naive, effector memory, and central memory CD8 T cells compared to control septic mice.

Figure 4.

Figure 4.

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IL-7 improves splenic naive and memory CD4 and CD8 T-cell counts during sepsis. Mice underwent cecal ligation and puncture (CLP) surgery at day 0 and received a Candida inoculum at day 4 post-CLP. Treatment with 2.5 μg IL-7 subcutaneously was initiated at day 5 and continued for 4 consecutive days. Splenocytes were harvested and analyzed by flow cytometry at day 11, a time point when the separation of the survival of the IL-7–treated group versus the non-IL-7–treated groups generally occurred. The absolute numbers of CD4 and CD8 T-cell subsets in the spleen were enumerated. Naive, effector memory, and central memory CD4 and CD8 T cells were identified via expression of CD44 and CD62L as detailed in the “Methods” section. Values are expressed as total counts per spleen. Each value represents the mean of 9 mice for the naive (ie, unmanipulated) group, 14 mice for the septic non-IL-7–treated group, and 18 mice for the IL-7–treated experimental group. Values result from 2 independent experiments. Data were analyzed by 1-way analysis of variance with Dunnett's post-test. A, IL-7 caused significant increases in total CD4 T cells as well as in naive and effector memory CD4 T cells. B, IL-7 caused significant increases in total CD8 T cells as well as in naive, effector memory, and central memory CD T cells.

IL-7 Improves Proliferation and Activation of CD4 and CD8 T Cells

Previous work has shown that sepsis inhibits activation and proliferation of T cells and that IL-7 can partially reverse these defects [24, 35, 38]. In the present study, we evaluated the effect of IL-7 in a more protracted case of sepsis due to a different class of pathogens. Whereas the number of proliferating CD4 T cells in septic spleens was reduced by 44% compared to naive, IL-7 restored CD4 T-cell proliferation to normal (Figure 5A). CD8 T cells responded with an even more robust proliferative response to IL-7. Whereas CD8 T-cell proliferation was reduced by 30% in the untreated group compared to naive, CD8 T-cell proliferation in septic mice treated with IL-7 increased by approximately 170% compared to untreated septic mice, and even exceeded CD8 T-cell proliferation in naive mice by 87% (Figure 5A).

Figure 5.

Figure 5.

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IL-7 improves CD4 and CD8 T-cell proliferation and activation. Mice underwent cecal ligation and puncture (CLP) surgery at day 0 and received a Candida inoculum at day 4 post-CLP. Treatment with 2.5 μg IL-7 subcutaneously was initiated at day 5 and continued for 4 consecutive days. Splenocytes were harvested at day 11, a time point when the separation of the survival of the IL-7–treated septic mice versus the non-IL-7–treated septic mice generally occurred, and stained for CD4 and CD8 T-cell markers. Cell proliferation was quantitated via expression of Ki67. Cell activation was quantitated via expression of the activation marker CD69. Each value is expressed as total counts per spleen and represents a mean of 5 to 9 mice from 2 independent experiments. Data were analyzed by 1-way analysis of variance with Dunnett's post-test. Note that CD4 and CD8 T cells from septic mice that had received IL-7 therapy had increased proliferation and activation compared to non-IL-7–treated septic mice.

We also tested the ability of IL-7 to enhance cell activation via examining expression of CD69 and CD25 on CD4 and CD8 T cells. Sepsis caused a decrease in expression of CD69 on CD4 and CD8 T cells, although this was not statistically significant (Figure 5B). IL-7 reversed the sepsis-induced suppression in CD4 T-cell activation (P < .01) and actually increased CD69 expression in CD8 T cells by approximately 33% relative to naive (P < .05). IL-7 also reduced the sepsis-induced decrease in CD25 expression in CD4 and CD8 T cells, but this difference did not reach significance (data not shown).

IL-7 Increases Expression of the Adhesion Molecule Leukocyte Function–Associated Antigen-1

Integrins are essential for recruiting cells to sites of infection and for optimal activation of CD4 and CD8 T cells [40]. Previous work from our laboratory showed that IL-7 increased integrin expression in lymphocytes during bacterial sepsis. To investigate if adhesion molecule expression is also increased with IL-7 treatment in fungal sepsis, mice were sacrificed at day 11 and splenic CD4 and CD8 T cells were stained for leukocyte function–associated antigen-1 (LFA-1) and very-late antigen-4 (VLA-4). LFA-1 expression was measured as mean fluorescence intensity (MFI), a measure of the number of molecules of interest expressed on a per-cell basis, and was significantly increased in both CD4 and CD8 T cells in IL-7 versus saline-treated control mice (Figure 6) (P < .01). There was no change in MFI of VLA-4 in the different groups (data not shown).

Figure 6.

Figure 6.

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IL-7 upregulates expression of the leukocyte function–associated antigen-1 (LFA-1) marker (CD11α) in sepsis. Mice underwent cecal ligation and puncture (CLP) surgery at day 0 and received a Candida inoculum at day 4 post-CLP. Treatment with 2.5 μg IL-7 subcutaneously was initiated at day 5 and continued for 4 consecutive days. Splenocytes were harvested and analyzed by flow cytometry for the cell adhesion marker LFA-1 (CD11α) at day 11, a time point when the separation of the survival of the treated versus the untreated groups generally occurred. The mean fluorescent intensity (a measure of the number of cell surface receptors) of LFA-1 expression of the naive unmanipulated mice was set at a value of 1.0, and values of the experimental groups were expressed as relative to this value. Values are expressed as fold-increase compared to the naive group. Each value represents the mean of 9 to 18 mice from 2 separate experiments. Data were analyzed by 1-way analysis of variance with Dunnett's post-test. Note that IL-7 treatment increased expression of LFA-1 on both CD4 and CD8 T cells; P < .01.

IL-7 Treatment Reverses the T-Cell Defect in Cytokine Production

Studies show either globally decreased pro- and anti-inflammatory cytokines or a shift from T-helper 1 (Th1) to Th2 phenotype during sepsis [41–43]. This sepsis-induced decrease in cytokines, which are essential in host defense, is thought to be central to sepsis pathology. We determined the effect of IL-7 on T-cell cytokine production induced by stimulation with CD3/CD28. At day 11, splenocytes were harvested and 1 × 107 cells stimulated with CD3/CD28 for 18 hours [24, 35, 36]. Splenocytes from non-IL-7–treated septic mice showed marked decreases in proinflammatory cytokines IFN-γ, IL-2, and TNF-α (91%, 65%, and 82% decrease, respectively) compared to unmanipulated controls (Figure 7A). In contrast, septic mice treated with IL-7 showed increased cytokine production compared to untreated controls. Of special interest is the recovery of INF-γ production in IL-7–treated mice. Previous work has demonstrated the vital role of IFN-γ in surviving sepsis [6]. IL-7 treatment increased IFN-γ production approximately 7.5-fold compared to untreated septic mice (P < .01). IL-7 also increased IL-6 production relative to both naive and non-IL-7–treated septic mice (P < .05).

Figure 7.

Figure 7.

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IL-7 improves splenocyte cytokine production and decreases fungal burden. Mice underwent cecal ligation and puncture (CLP) surgery at day 0 and received a Candida inoculum at day 4 post-CLP. Treatment with 2.5 μg IL-7 subcutaneously was initiated at day 5 and continued for 4 consecutive days. At day 11, splenocytes were harvested and cell suspensions were prepared. A, Cytokine supernatants: 10 million splenocytes were plated in 24 wells and stimulated with CD3 and CD28 overnight. Supernatant were harvested at 18 hours and the production of the different cytokines were analyzed using enzyme-linked immunosorbent assay. Note that splenocytes from septic mice treated with IL-7 had increased IL-2, IFN-γ, TNF-α, and IL-6 production compared to septic mice that did not receive IL-7. B and C, Intracellular IFN-γ: splenocytes were harvested and cultivated overnight as mentioned above. Next, Brefeldin A was added to the overnight cultures and incubated for an additional 4 hours. Cells were harvested, stained for CD8 T-cell marker, fixed, permeabilized, and finally stained for intracellular IFN-γ. B, Representative flow cytometry dot plot for IFN-γ–producing CD8 T cells for a naive, control CLP plus Candida mouse, and CLP plus Candida mouse treated with IL-7. The percentage of CD8 T cells that are IFN-γ producers (in parentheses) is demonstrated in the right upper quadrant of each graph. C, Bar graphs demonstrating the mean for CD8-producing T cells. Each value represents the mean of 5 to 9 mice over 2 separate experiments. Data were analyzed by 1-way analysis of variance with Dunnett's post-test. The percentage of CD8 T cells that produced IFN-γ was significantly increased in septic mice treated with IL-7 compared to non-IL-7–treated septic mice; P < .01. D, Tissue Candida colony-forming units (CFUs): kidney and liver were homogenized 3 days post–Candida injection, serially diluted, and plated on blood agar plates with chloramphenicol. At 24 hours, the number of CFUs in liver was less in mice treated with IL-7; P < .007.

The effect of IL-7 on intracellular IFN-γ in CD4 and CD8 T cells was also quantitated. Splenocytes were stained for CD4 and CD8 and stimulated overnight with CD3/CD28. Brefeldin A was added to the cell culture for 4 hours to block cytokine secretion. Cells were harvested and stained for IFN-γ. Only a small percentage (<5%) of CD4 T cells from either control or septic mice were positive for IFN-γ, and there were no group differences (data not shown). In contrast, a large percentage of CD8 T cells from sham-operated mice stained positive for IFN-γ (Figure 7B). Sepsis caused a 45% decrease in the percentage of CD8 T cells positive for IFN-γ. The percentage of CD8 T cells positive for IFN-γ was increased by 49% in IL-7–treated mice (P < .01) and this was comparable to naive mice (Figure 7C).

IL-7 Decreased Tissue Fungal Colony Counts in Liver

Kidney and liver were harvested from mice at days 3 and 7 post–Candida infection and CFUs were determined. There was a decrease in CFUs of Candida in liver in IL-7–treated mice at day 3 post–Candida infection (P < .01; Figure 7D). There was a trend toward a decrease in Candida CFUs in kidney, but it did not reach statistical significance (Figure 7D). At 7 days after Candida infection, kidneys of control mice had 2.9 × 105 CFUs, while IL-7–treated mice had only 8.4 × 104 CFUs (N = 4 mice per group; data not shown). This did not reach statistical significance.

DISCUSSION

Increasing evidence suggests that sepsis can induce a state of prolonged immunosuppression that follows the initial proinflammatory period [44]. This sepsis-induced immunosuppression is manifested by decreased whole blood–stimulated cytokine production, blunted response to antigens, and increased infections with opportunistic pathogens [3, 4, 9, 10, 39, 45–47]. Candidiasis, cytomegalovirus reactivation, and secondary infections by relatively avirulent bacterial pathogens (eg, Stenotrophomonas maltophilia, Acinetobacter calcoaceticus-baumannii complex, and Enterococcus spp.) are commonplace in patients during prolonged ICU stays. In this regard, in the 2-hit model employed in the current study, neither CLP nor Candida infection is lethal by itself; however, the combination of CLP followed by Candida leads to severe infection and death from systemic candidiasis because of the weakened immune state following CLP [35, 38]. Thus, the present model mimics the compromised immune state that occurs in the ICU setting.

The incidence of fungal infections is increasing, and candidemia currently represents the third to fourth most common healthcare-associated bloodstream infection in the ICU [12–15]. The ability of the host to sense and contain invading Candida requires a coordinated response by many elements of the innate and adaptive immune system, both of which are impaired in sepsis [4, 6, 44]. One measure of global immune integrity is the DTH response to immunizing antigens. The effect of IL-7 to improve the impaired DTH response in this 2-hit sepsis model (Figure 3) is consistent with earlier work from our group in the CLP model of sepsis, and strongly supports the wide-ranging beneficial effects of IL-7 on host immunity. IL-7 not only ameliorated sepsis-induced death of CD4 and CD8 T cells but also caused significant proliferation. IL-7 also increased lymphocyte activation, thus enabling them to more effectively respond to Candida. In addition, IL-7 increased expression of the integrin LFA-1 which improves lymphocyte migration to infected sites.

Another salutary effect of IL-7, which is likely a major cause of its beneficial properties in candidiasis, is its action to increase IFN-γ. Prophylactic IFN-γ reduces the risk of invasive fungal infections in patients with chronic granulomatous disease [16]. IFN-γ enhances various phagocytic cell functions (eg, increased production of superoxide, increased expression of major histocompatibility complex, and reduction of phagocytic vacuole pH), which will improve their ability to engulf and kill Candida. IL-7 treatment increased both production of IFN-γ and the percentage of IFN-γ–producing CD8 T cells (Figure 7).

The current study adds to the increasing evidence supporting the potential use of IL-7 in the immunotherapy of sepsis. Previous work by our group demonstrated that IL-7 was efficacious in an acute polymicrobial CLP sepsis model [24]. The current results show that IL-7 is also beneficial in a fungal model of sepsis that reproduces the delayed secondary infections typical of patients in the ICU. Additional studies indicate that IL-7 improves survival in a primary Pseudomonas aeruginosa pneumonia model (data not shown). Recently, Pellegrini et al. [30] administered IL-7 to mice that were chronically infected with lymphocytic choriomeningitis. IL-7 increased viral clearance, increased T-cell numbers, and improved survival. Thus, IL-7 has been remarkably effective in the laboratory against a range of diverse pathogens, including bacteria, fungi, and viruses. A major potential benefit of IL-7 is that it represents an adjuvant therapy that could be combined with current antifungal agents. Many of the current antifungal agents have excellent in vitro antifungal activity against C. albicans, but patients are dying despite appropriate antifungal therapy. IL-7 offers the potential to act in a synergistic fashion to improve fungal killing by improving host immunity. IL-7 is currently being tested as a positive immunostimulatory molecule in 4 multinational clinical trials, including patients with HIV-1, cancer, and hepatitis C [25–29]. In a dose-escalation drug safety study at the National Cancer Institute, 16 patients with refractory cancer who received IL-7 had a greater than 2-fold increase in circulating CD4 and CD8 T cells and a >60% increase in size of spleen and lymph nodes [26]. Additionally, a recent trial in HIV-1–infected patients who had persistently low CD4 T cells despite effective viral suppression showed that IL-7 was associated with robust and sustained increases in circulating CD4 and CD8 T cells [29]. A recent case report on the use of IL-7 in a patient with idiopathic low CD4 T cells suffering from progressive multifocal leukoencephalopathy showed that IL-7 therapy led to a rapid increase in lymphocytes, decreased circulating levels of John Cunningham (JC) virus, and resolution of disease [48]. Thus, as demonstrated in these clinical studies, a key property of IL-7 is its ability to reverse loss of critical immune effector cells, a sepsis hallmark. Another significant feature of IL-7 is its low incidence of side effects [26–29]. IL-7 is well tolerated in patients and unlike IL-2 (a closely related cytokine), IL-7 rarely induces fever, capillary leak syndrome, or other clinical abnormalities associated with excessive proinflammatory cytokines. Collectively, these studies, together with the present findings, suggest that IL-7 offers promise as a means to reverse the sepsis-induced immune dysfunction and improve survival in candidiasis.

In conclusion, IL-7 acts at multiple levels to improve host immunity during sepsis. The beneficial effects of IL-7 on host immunity resulted in improved survival in a secondary infection model of C. albicans, an increasingly problematic pathogen in patients in the ICU. Given the reported beneficial clinical safety profile of IL-7 to date, as well as its demonstrated efficacy against a number of diverse pathogens, IL-7 should move rapidly into clinical trials in sepsis.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online (http://www.oxfordjournals.org/our_journals/jid/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

Supplementary Data
supp_206_4_606__index.html (1KB, html)

Notes

Financial support. This work was supported by funding from the National Institute of General Medical Sciences (NIGMS; GM 44118 and GM 55194).

Potential conflicts of interest. M. M. is CEO of Cytheris, which holds the patent on use of IL-7 in sepsis and other diseases, including cancer and viral infections. R. S. H. has research funding from Pfizer for the study of anti-CTLA4 in vitro in sepsis. All other authors report no potential conflicts.

All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

References

  • 1.Angus DC, Linde-Zwirble WT, Lidicker J, et al. Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Crit Care Med. 2001;29:1303–10. doi: 10.1097/00003246-200107000-00002. [DOI] [PubMed] [Google Scholar]
  • 2.Murphy SL. Deaths: final data for 1998. Natl Vital Stat Rep. 1998;48:1–105. [PubMed] [Google Scholar]
  • 3.Monnert G, Venet F. Immunomodulatory cell therapy in sepsis: have we learnt lessons from the past? Expert Rev Anti Infect Ther. 2010;8:1109–12. doi: 10.1586/eri.10.107. [DOI] [PubMed] [Google Scholar]
  • 4.Munford RS, Pugin J. Normal responses to injury prevent systemic inflammation and can be immunosuppressive. Am J Respir Crit Care Med. 2001;163:316–21. doi: 10.1164/ajrccm.163.2.2007102. [DOI] [PubMed] [Google Scholar]
  • 5.Weighardt H, Heidecke CD, Emmanuilidis K, et al. Sepsis after major visceral surgery is associated with sustained and interferon-gamma-resistant defects of monocyte cytokine production. Surgery. 2000;127:309–15. doi: 10.1067/msy.2000.104118. [DOI] [PubMed] [Google Scholar]
  • 6.Hotchkiss RS, Coopersmith CM, McDunn JE, Ferguson TA. The sepsis seesaw: tilting toward immunosuppression. Nat Med. 2009;15:496–7. doi: 10.1038/nm0509-496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Venet F, Chung CS, Monneret G, et al. Regulatory T cell populations in sepsis and trauma. J Leukoc Biol. 2008;83:523–35. doi: 10.1189/jlb.0607371. [DOI] [PubMed] [Google Scholar]
  • 8.Delano MJ, Scumpia PO, Weinstein JS, et al. MyD88-dependent expansion of an immature GR-1+CD11b+ population induces T cell suppression and Th2 polarization in sepsis. J Exp Med. 2007;204:1463–74. doi: 10.1084/jem.20062602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Luyt CE, Combes A, Deback C, et al. Herpes simplex virus lung infection in patients undergoing prolonged mechanical ventilation. Am J Respir Crit Care Med. 2007;175:935–42. doi: 10.1164/rccm.200609-1322OC. [DOI] [PubMed] [Google Scholar]
  • 10.Limaye AP, Kirby KA, Rubenfeld GD, et al. Cytomegalovirus reactivation in critically ill immunocompetent patients. JAMA. 2008;300:413–22. doi: 10.1001/jama.300.4.413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Lipsett PA. Surgical critical care: fungal infections in surgical patients. Crit Care Med. 2006;34(suppl):S215–24. doi: 10.1097/01.CCM.0000231883.93001.E0. [DOI] [PubMed] [Google Scholar]
  • 12.Miceli MH, Dâiaz JA, Lee SA. Emerging opportunistic yeast infections. Lancet Infect Dis. 2011;11:142–51. doi: 10.1016/S1473-3099(10)70218-8. [DOI] [PubMed] [Google Scholar]
  • 13.Arendrup MC. Epidemiology of invasive candidiasis. Curr Opin Crit Care. 2010;16:445–52. doi: 10.1097/MCC.0b013e32833e84d2. [DOI] [PubMed] [Google Scholar]
  • 14.Lepak A, Andes D. Fungal sepsis: optimizing antifungal therapy in the critical care setting. Crit Care Clin. 2011;27:123–47. doi: 10.1016/j.ccc.2010.11.001. [DOI] [PubMed] [Google Scholar]
  • 15.Romani L. The T cell response against fungal infections. Curr Opin Immunol. 1997;9:484–90. doi: 10.1016/s0952-7915(97)80099-4. [DOI] [PubMed] [Google Scholar]
  • 16.Antachopoulos C. Invasive fungal infections in congenital immunodeficiencies. Clin Microbiol Infect. 2010;16:1335–42. doi: 10.1111/j.1469-0691.2010.03289.x. [DOI] [PubMed] [Google Scholar]
  • 17.Mazzucchelli R, Durum SK. Interleukin-7 receptor expression: intelligent design. Nat Rev Immunol. 2007;7:144–54. doi: 10.1038/nri2023. [DOI] [PubMed] [Google Scholar]
  • 18.Fry TJ, Mackall CL. The many faces of IL-7: from lymphopoiesis to peripheral T cell maintenance. J Immunol. 2005;174:6571–6. doi: 10.4049/jimmunol.174.11.6571. [DOI] [PubMed] [Google Scholar]
  • 19.Ma A, Koka R, Burkett P. Diverse functions of IL-2, IL-15, and IL-7 in lymphoid homeostasis. Annu Rev Immunol. 2006;24:657–79. doi: 10.1146/annurev.immunol.24.021605.090727. [DOI] [PubMed] [Google Scholar]
  • 20.Boyman O, Ramsey C, Kim DM, Sprent J, Surh CD. IL-7/anti-IL-7 mAb complexes restore T cell development and induce homeostatic T cell expansion without lymphopenia. J Immunol. 2008;180:7265–75. doi: 10.4049/jimmunol.180.11.7265. [DOI] [PubMed] [Google Scholar]
  • 21.Alpdogan O, van den Brink MR. IL-7 and IL-15: therapeutic cytokines for immunodeficiency. Trends Immunol. 2005;26:56–64. doi: 10.1016/j.it.2004.11.002. [DOI] [PubMed] [Google Scholar]
  • 22.Fry TJ, Christensen BL, Komschlies KL, Gress RE, Mackall CL. Interleukin-7 restores immunity in athymic T-cell–depleted hosts. Blood. 2001;97:1525–33. doi: 10.1182/blood.v97.6.1525. [DOI] [PubMed] [Google Scholar]
  • 23.Schluns KS, Kieper WC, Jameson SC, Lefranðcois L. Interleukin-7 mediates the homeostasis of naive and memory CD8 T cells in vivo. Nat Immunol. 2000;1:426–32. doi: 10.1038/80868. [DOI] [PubMed] [Google Scholar]
  • 24.Unsinger J, McGlynn M, Kasten KR, et al. IL-7 promotes T cell viability, trafficking, and functionality and improves survival in sepsis. J Immunol. 2010;184:3768–79. doi: 10.4049/jimmunol.0903151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Mackall CL, Fry TJ, Gress RE. Harnessing the biology of IL-7 for therapeutic application. Nat Rev Immunol. 2011;11:330–42. doi: 10.1038/nri2970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Rosenberg SA, Sportes C, Ahmadzadeh M, et al. IL-7 administration to humans leads to expansion of CD8+ and CD4+ cells but a relative decrease of CD4+ T-regulatory cells. J Immunother. 2006;29:313–9. doi: 10.1097/01.cji.0000210386.55951.c2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Sportes C, Hakim FT, Memon SA, et al. Administration of rhIL-7 in humans increases in vivo TCR repertoire diversity by preferential expansion of naive T cell subsets. J Exp Med. 2008;205:1701–14. doi: 10.1084/jem.20071681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Levy Y, Lacabaratz C, Weiss L, et al. Enhanced T cell recovery in HIV-1-infected adults through IL-7 treatment. J Clin Invest. 2009;119:997–1007. doi: 10.1172/JCI38052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Sereti I, Dunham RM, Spritzler J, et al. IL-7 administration drives T cell-cycle entry and expansion in HIV-1 infection. Blood. 2009;113:6304–14. doi: 10.1182/blood-2008-10-186601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Pellegrini M, Calzascia T, Toe JG, et al. IL-7 engages multiple mechanisms to overcome chronic viral infection and limit organ pathology. Cell. 2011;144:601–13. doi: 10.1016/j.cell.2011.01.011. [DOI] [PubMed] [Google Scholar]
  • 31.Glocker E, Grimbacher B. Chronic mucocutaneous candidiasis and congenital susceptibility to Candida. Curr Opin Allergy Clin Immunol. 2010;10:542–50. doi: 10.1097/ACI.0b013e32833fd74f. [DOI] [PubMed] [Google Scholar]
  • 32.Romani L, Mencacci A, Cenci E, et al. An immunoregulatory role for neutrophils in CD4+ T helper subset selection in mice with candidiasis. J Immunol. 1997;158:2356–62. [PubMed] [Google Scholar]
  • 33.Tschop J, Martignoni A, Reid MD, et al. Differential immunological phenotypes are exhibited following scald and flame burns. Shock. 2008;31:157–63. doi: 10.1097/SHK.0b013e31817fbf4d. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Kasten KR, Prakash PS, Unsinger J, et al. Interleukin-7 (IL-7) treatment accelerates neutrophil recruitment through gamma delta T-cell IL-17 production in a murine model of sepsis. Infect Immun. 2010;78:4714–22. doi: 10.1128/IAI.00456-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Unsinger J, Herndon JM, Davis CG, Muenzer JT, Hotchkiss RS, Ferguson TA. The role of TCR engagement and activation-induced cell death in sepsis-induced T cell apoptosis. J Immunol. 2006;177:7968–73. doi: 10.4049/jimmunol.177.11.7968. [DOI] [PubMed] [Google Scholar]
  • 36.Capitini CM, Chisti AA, Mackall CL. Modulating T-cell homeostasis with IL-7: preclinical and clinical studies. J Inter Med. 2009;266:141–153. doi: 10.1111/j.1365-2796.2009.02085.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Haraguchi N, Ishii Y, Morishima Y, et al. Impairment of host defense against disseminated candidiasis in mice overexpressing GATA-3. Infect Immun. 2010;78:2302–11. doi: 10.1128/IAI.01398-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Unsinger J, Kazama H, McDonough JS, Hotchkiss RS, Ferguson TA. Differential lymphopenia-induced homeostatic proliferation for CD4+ and CD8+ T cells following septic injury. J Leukoc Biol. 2009;85:382–90. doi: 10.1189/jlb.0808491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Christou NV, Meakins JL, Gordon J, et al. The delayed hypersensitivity response and host resistance in surgical patients. 20 years later. Ann Surg. 1995;222:534–46. doi: 10.1097/00000658-199522240-00011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.von Andrian UH, Mempel TR. Homing and cellular traffic in lymph nodes. Nat Rev Immunol. 2003;3:867–78. doi: 10.1038/nri1222. [DOI] [PubMed] [Google Scholar]
  • 41.Ayala A, Deol ZK, Lehman DL, Herdon CD, Chaudry IH. Polymicrobial sepsis but not low-dose endotoxin infusion causes decreased splenocyte IL-2/IFN-gamma release while increasing IL-4/IL-10 production. J Surg Res. 1994;56:579–85. doi: 10.1006/jsre.1994.1092. [DOI] [PubMed] [Google Scholar]
  • 42.Adib-Conquy M, Cavaillon JM. Compensatory anti-inflammatory response syndrome. Thromb Haemost. 2009;101:36–47. [PubMed] [Google Scholar]
  • 43.Remick DG. Pathophysiology of sepsis. Am J Pathol. 2007;170:1435–44. doi: 10.2353/ajpath.2007.060872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Hotchkiss RS, Karl IE. The pathophysiology and treatment of sepsis. N Engl J Med. 2003;348:138–50. doi: 10.1056/NEJMra021333. [DOI] [PubMed] [Google Scholar]
  • 45.Hotchkiss RS, Tinsley KW, Swanson PE, et al. Sepsis-induced apoptosis causes progressive profound depletion of B and CD4+ T lymphocytes in humans. J Immunol. 2001;166:6952–63. doi: 10.4049/jimmunol.166.11.6952. [DOI] [PubMed] [Google Scholar]
  • 46.Hotchkiss RS, Tinsley KW, Swanson PE, et al. Depletion of dendritic cells, but not macrophages, in patients with sepsis. J Immunol. 2002;168:2493–500. doi: 10.4049/jimmunol.168.5.2493. [DOI] [PubMed] [Google Scholar]
  • 47.Felmet KA, Hall MW, Clark RS, Jaffe R, Carcillo JA. Prolonged lymphopenia, lymphoid depletion, and hypoprolactinemia in children with nosocomial sepsis and multiple organ failure. J Immunol. 2005;174:3765–72. doi: 10.4049/jimmunol.174.6.3765. [DOI] [PubMed] [Google Scholar]
  • 48.Patel A, Patel J, Ikwuagwu J. A case of progressive multifocal leukoencephalopathy and idiopathic CD4+ lymphocytopenia. J Antimicrob Chemother. 2010;65:2697–8. doi: 10.1093/jac/dkq359. [DOI] [PubMed] [Google Scholar]
  • 49.Gaffen SL, Hernandez-Santos N, Peterson AC. IL-17 signaling in host defense against Candida albicans. Immunol Res. 2011;50:181–187. doi: 10.1007/s12026-011-8226-x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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