Interleukin-7 ameliorates immune dysfunction and improves survival in a 2-hit model of fungal sepsis

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.

Figures

Figure 1.

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.

Figure 2.

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.

Figure 3.

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.

Figure 4.

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.

Figure 5.

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.

Figure 6.

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.

Figure 7.

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.