Viral Delivery of IL-7 Is a Potent Immunotherapy Stimulating Innate and Adaptive Immunity and Confers Survival in Sepsis Models.

Persistence of an immunosuppressive state plays a role in septic patient morbidity and late mortality. Both innate and adaptive pathways are impaired, pointing toward the need for immune interventions targeting both arms of the immune system. We developed a virotherapy using the nonpropagative modified vaccinia virus Ankara (MVA), which harbors the intrinsic capacity to stimulate innate immunity, to deliver IL-7, a potent activator of adaptive immunity. The rMVA-human IL-7 (hIL-7)-Fc encoding the hIL-7 fused to the human IgG2-Fc was engineered and shown to express a dimeric, glycosylated, and biologically active cytokine. Following a single i.v. injection in naive mice, the MVA-hIL-7-Fc increased the number of total and activated B, T, and NK cells but also myeloid subpopulations (Ly6Chigh, Ly6Cint, and Ly6Cneg cells) in both lung and spleen. It triggered differentiation of T cells in central memory, effector memory, and acute effector phenotypes and enhanced polyfunctionality of T cells, notably the number of IFN-γ-producing cells. The MVA vector contributed significantly to immune cell activation, particularly of NK cells. The MVA-hIL-7-Fc conferred a significant survival advantage in the cecal ligation and puncture (CLP) and Candida albicans sepsis models. It significantly increased cell numbers and activation in both spleen and lung of CLP mice. Comparatively, in naive and CLP mice, the rhIL-7-Fc soluble counterpart overall induced less vigorous, shorter lasting, and narrower immune activities than did the MVA-hIL-7-Fc and favored TNF-α-producing cells. The MVA-hIL-7-Fc represents a novel class of immunotherapeutic with clinical potential for treatment of septic patients.

Temporal Changes in Innate and Adaptive Immunity During Sepsis as Determined by ELISpot.

BACKGROUND: The inability to evaluate host immunity in a rapid quantitative manner in patients with sepsis has severely hampered development of novel immune therapies. The ELISpot assay is a functional bioassay that measures the number of cytokine-secreting cells and the relative amount of cytokine produced at the single-cell level. A key advantage of ELISpot is its excellent dynamic range enabling a more precise quantifiable assessment of host immunity. Herein, we tested the hypothesis that the ELISpot assay can detect dynamic changes in both innate and adaptive immunity as they often occur during sepsis. We also tested whether ELISpot could detect the effect of immune drug therapies to modulate innate and adaptive immunity. METHODS: Mice were made septic using sublethal cecal ligation and puncture (CLP). Blood and spleens were harvested serially and ex vivo IFN-γ and TNF-α production were compared by ELISpot and ELISA. The capability of ELISpot to detect changes in innate and adaptive immunity due to in vivo immune therapy with dexamethasone, IL-7, and arginine was also evaluated. RESULTS: ELISpot confirmed a decreased innate and adaptive immunity responsiveness during sepsis progression. More importantly, ELISpot was also able to detect changes in adaptive and innate immunity in response to immune-modulatory reagents, for example dexamethasone, arginine, and IL-7 in a readily quantifiable manner, as predicted by the reagents known mechanisms of action. ELISpot and ELISA results tended to parallel one another although some differences were noted. CONCLUSION: ELISpot offers a unique capability to assess the functional status of both adaptive and innate immunity over time. The results presented herein demonstrate that ELISpot can also be used to detect and follow the in vivo effects of drugs to ameliorate sepsis-induced immune dysfunction. This capability would be a major advance in guiding new immune therapies in sepsis.

Blockade of the negative co-stimulatory molecules PD-1 and CTLA-4 improves survival in primary and secondary fungal sepsis.

INTRODUCTION: Fungal sepsis is an increasingly common problem in intensive care unit patients.Mortality from fungal sepsis remains high despite antimicrobial therapy that is highly active against most fungal pathogens, a finding consistent with defective host immunity that is present in many patients with disseminated fungemia.One recently recognized immunologic defect that occurs in patients with sepsis is T cell "exhaustion" due to increased expression of programmed cell death -1 (PD-1).This study tested the ability of anti-PD-1 and anti-programmed cell death ligand -1 (anti-PD-L1) antagonistic antibodies to improve survival and reverse sepsis-induced immunosuppression in two mouse models of fungal sepsis. METHODS: Fungal sepsis was induced in mice using two different models of infection, that is, primary fungal sepsis and secondary fungal sepsis occurring after sub-lethal cecal ligation and puncture (CLP).Anti-PD-1 and anti-PD-L1 were administered 24 to 48 h after fungal infection and effects on survival, interferon gamma production, and MHC II expression were examined. RESULTS: Anti-PD-1 and anti-PD-L1 antibodies were highly effective at improving survival in primary and secondary fungal sepsis.Both antibodies reversed sepsis-induced suppression of interferon gamma and increased expression of MHC II on antigen presenting cells.Blockade of cytotoxic T-lymphocyte antigen-4 (CTLA-4), a second negative co-stimulatory molecule that is up-regulated in sepsis and acts like PD-1 to suppress T cell function, also improved survival in fungal sepsis. CONCLUSIONS: Immuno-adjuvant therapy with anti-PD-1, anti-PD-L1 and anti-CTLA-4 antibodies reverse sepsis-induced immunosuppression and improve survival in fungal sepsis.The present results are consistent with previous studies showing that blockade of PD-1 and CTLA-4 improves survival in bacterial sepsis.Thus, immuno-adjuvant therapy represents a novel approach to sepsis and may have broad applicability in the disorder.Given the relative safety of anti-PD-1 antibody in cancer clinical trials to date, therapy with anti-PD-1 in patients with life-threatening sepsis who have demonstrable immunosuppression should be strongly considered.

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

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.

Intravenously administered interleukin-7 to reverse lymphopenia in patients with septic shock: a double-blind, randomized, placebo-controlled trial.

BACKGROUND: Profound lymphopenia is an independent predictor of adverse clinical outcomes in sepsis. Interleukin-7 (IL-7) is essential for lymphocyte proliferation and survival. A previous phase II study showed that CYT107, a glycosylated recombinant human IL-7, administered intramuscularly reversed sepsis-induced lymphopenia and improved lymphocyte function. Thepresent study evaluated intravenous administration of CYT107. This prospective, double-blinded, placebo-controlled trial was designed to enroll 40 sepsis patients, randomized 3:1 to CYT107 (10 µg/kg) or placebo, for up to 90 days. RESULTS: Twenty-one patients were enrolled (fifteen CYT107 group, six placebo group) at eight French and two US sites. The study was halted early because three of fifteen patients receiving intravenous CYT107 developed fever and respiratory distress approximately 5-8 h after drug administration. Intravenous administration of CYT107 resulted in a two-threefold increase in absolute lymphocyte counts (including in both CD4+ and CD8+ T cells (all p < 0.05)) compared to placebo. This increase was similar to that seen with intramuscular administration of CYT107, was maintained throughout follow-up, reversed severe lymphopenia and was associated with increase in organ support free days (OSFD). However, intravenous CYT107 produced an approximately 100-fold increase in CYT107 blood concentration compared with intramuscular CYT107. No cytokine storm and no formation of antibodies to CYT107 were observed. CONCLUSION: Intravenous CYT107 reversed sepsis-induced lymphopenia. However, compared to intramuscular CYT107 administration, it was associated with transient respiratory distress without long-term sequelae. Because of equivalent positive laboratory and clinical responses, more favorable pharmacokinetics, and better patient tolerability, intramuscular administration of CYT107 is preferable. TRIAL REGISTRATION: Clinicaltrials.gov, NCT03821038. Registered 29 January 2019, https://clinicaltrials.gov/ct2/show/NCT03821038?term=NCT03821038&draw=2&rank=1 .

Sepsis-induced apoptosis leads to active suppression of delayed-type hypersensitivity by CD8+ regulatory T cells through a TRAIL-dependent mechanism.

Patients who survive severe sepsis often display severely compromised immune function. One hallmark of such immune suppression in septic patients is an impaired delayed-type hypersensitivity (DTH) response, manifested by a loss of skin testing to recall Ags. Because sepsis induces significant apoptosis in lymphoid and myeloid cells, and apoptotic cells are themselves tolerogenic, we tested the hypothesis that suppression of DTH is mediated by tolerogenic properties of the apoptotic cells generated during sepsis. Mice subjected to cecal ligation and puncture demonstrated a loss of DTH for the 7 d following cecal ligation and puncture; however, the immune response returned to normal by day 10. Blocking sepsis-induced apoptosis via Bcl-2 overexpression or Bim deficiency prevented the loss of DTH. Importantly, injection of apoptotic cells into Bim-/- mice prevented an effective DTH response, thereby suggesting a causal link between apoptotic cells and immune suppression. Surprisingly, when TRAIL null mice were examined, we found that these animals had significant apoptosis but retained their DTH responses. Further studies revealed that apoptotic cells generated during sepsis induced a CD8+ regulatory T cell that suppressed DTH by TRAIL production. These results establish a link between apoptotic cells and immune suppression during sepsis and suggest TRAIL may be a viable therapeutic target for boosting the adaptive immune response following sepsis.

IL-15 prevents apoptosis, reverses innate and adaptive immune dysfunction, and improves survival in sepsis.

IL-15 is a pluripotent antiapoptotic cytokine that signals to cells of both the innate and adaptive immune system and is regarded as a highly promising immunomodulatory agent in cancer therapy. Sepsis is a lethal condition in which apoptosis-induced depletion of immune cells and subsequent immunosuppression are thought to contribute to morbidity and mortality. This study tested the ability of IL-15 to block apoptosis, prevent immunosuppression, and improve survival in sepsis. Mice were made septic using cecal ligation and puncture or Pseudomonas aeruginosa pneumonia. The experiments comprised a 2 x 2 full factorial design with surgical sepsis versus sham and IL-15 versus vehicle. In addition to survival studies, splenic cellularity, canonical markers of activation and proliferation, intracellular pro- and antiapoptotic Bcl-2 family protein expression, and markers of immune cell apoptosis were evaluated by flow cytometry. Cytokine production was examined both in plasma of treated mice and splenocytes that were stimulated ex vivo. IL-15 blocked sepsis-induced apoptosis of NK cells, dendritic cells, and CD8 T cells. IL-15 also decreased sepsis-induced gut epithelial apoptosis. IL-15 therapy increased the abundance of antiapoptotic Bcl-2 while decreasing proapoptotic Bim and PUMA. IL-15 increased both circulating IFN-gamma, as well as the percentage of NK cells that produced IFN-gamma. Finally, IL-15 increased survival in both cecal ligation and puncture and P. aeruginosa pneumonia. In conclusion, IL-15 prevents two immunopathologic hallmarks of sepsis, namely, apoptosis and immunosuppression, and improves survival in two different models of sepsis. IL-15 represents a potentially novel therapy of this highly lethal disorder.

IL-7 promotes T cell viability, trafficking, and functionality and improves survival in sepsis.

Sepsis is a highly lethal disorder characterized by widespread apoptosis-induced depletion of immune cells and the development of a profound immunosuppressive state. IL-7 is a potent antiapoptotic cytokine that enhances immune effector cell function and is essential for lymphocyte survival. In this study, recombinant human IL-7 (rhIL-7) efficacy and potential mechanisms of action were tested in a murine peritonitis model. Studies at two independent laboratories showed that rhIL-7 markedly improved host survival, blocked apoptosis of CD4 and CD8 T cells, restored IFN-gamma production, and improved immune effector cell recruitment to the infected site. Importantly, rhIL-7 also prevented a hallmark of sepsis (i.e., the loss of delayed-type hypersensitivity), which is an IFN-gamma- and T cell-dependent response. Mechanistically, rhIL-7 significantly increased the expression of the leukocyte adhesion markers LFA-1 and VLA-4, consistent with its ability to improve leukocyte function and trafficking to the infectious focus. rhIL-7 also increased the expression of CD8. The potent antiapoptotic effect of rhIL-7 was due to increased Bcl-2, as well as to a dramatic decrease in sepsis-induced PUMA, a heretofore unreported effect of IL-7. If additional animal studies support its efficacy in sepsis and if current clinical trials continue to confirm its safety in diverse settings, rhIL-7 should be strongly considered for clinical trials in sepsis.

Low-dose interleukin-2 reverses chronic migraine-related sensitizations through peripheral interleukin-10 and transforming growth factor beta-1 signaling.

Low-dose interleukin-2 (LD-IL-2) treatment has been shown to effectively reverse chronic migraine-related behaviors and the sensitization of trigeminal ganglion (TG) neurons through expansion and activation of peripheral regulatory T cells (Tregs) in mice. In this study, we investigated the molecular mechanisms underlying the effects of LD-IL-2 and Treg cells. LD-IL-2 treatment increases the production of cytokines interleukin-10 (IL-10) and transforming growth factor beta-1 (TGFß1) in T cells, especially Treg cells, suggesting that they may mediate the therapeutic effect of LD-IL-2. Indeed, neutralizing antibodies against either IL-10 or TGFß completely blocked the effects of LD-IL-2 on the facial mechanical hypersensitivity as well as the sensitization of TG neurons resulting from repeated nitroglycerin (NTG, a reliable trigger of migraine in patients) administration in mice, indicating that LD-IL-2 and Treg cells engage both peripheral IL-10 and TGFß signaling pathways to reverse chronic-migraine related sensitizations. In an in vitro assay, incubation of TG culture with exogenous IL-10 or TGFß1 fully reversed NTG-induced sensitization of TG neurons, suggesting that the IL-10 and TGFß1 signaling in TG neurons contribute to LD-IL-2's therapeutic effects. Collectively, these results not only elucidate the molecular mechanisms through which LD-IL-2 and Treg cells reverse chronic-migraine related sensitizations, but also suggest that the IL-10 and TGFß1 signaling pathways in TG neurons are potential targets for chronic migraine therapy.

Frontline Science: OX40 agonistic antibody reverses immune suppression and improves survival in sepsis.

A defining feature of protracted sepsis is development of immunosuppression that is thought to be a major driving force in the morbidity and mortality associated with the syndrome. The immunosuppression that occurs in sepsis is characterized by profound apoptosis-induced depletion of CD4 and CD8 T cells and severely impaired T cell function. OX40, a member of the TNF receptor superfamily, is a positive co-stimulatory molecule expressed on activated T cells. When engaged by OX40 ligand, OX40 stimulates T cell proliferation and shifts the cellular immune phenotype toward TH1 with increased production of cytokines that are essential for control of invading pathogens. The purpose of the present study was to determine if administration of agonistic Ab to OX40 could reverse sepsis-induced immunosuppression, restore T cell function, and improve survival in a clinically relevant animal model of sepsis. The present study demonstrates that OX40 agonistic Ab reversed sepsis-induced impairment of T cell function, increased T cell IFN-γ production, increased the number of immune effector cells, and improved survival in the mouse cecal ligation and puncture model of sepsis. Importantly, OX40 agonistic Ab was not only effective in murine sepsis but also improved T effector cell function in PBMCs from patients with sepsis. The present results provide support for the use of immune adjuvants that target T cell depletion and T cell dysfunction in the therapy of sepsis-induced immunosuppression. In addition to the checkpoint inhibitors anti-PD-1 and anti-PD-L1, OX40 agonistic Ab may be a new therapeutic approach to the treatment of this highly lethal disorder.

A Peptide-Based Checkpoint Immunomodulator Alleviates Immune Dysfunction in Murine Polymicrobial Sepsis.

ABSTRACT: Sepsis-induced immunosuppression involves both innate and adaptive immunity and is associated with the increased expression of checkpoint inhibitors, such as programmed cell-death protein 1 (PD-1). The expression of PD-1 is associated with poor outcomes in septic patients, and in models of sepsis, blocking PD-1 or its ligands with antibodies increased survival and alleviated immune suppression. While inhibitory antibodies are effective, they can lead to immune-related adverse events (irAEs), in part due to continual blockade of the PD-1 pathway, resulting in hyperactivation of the immune response. Peptide-based therapeutics are an alternative drug modality that provide a rapid pharmacokinetic profile, reducing the incidence of precipitating irAEs. We recently reported that the potent, peptide-based PD-1 checkpoint antagonist, LD01, improves T-cell responses. The goal of the current study was to determine whether LD01 treatment improved survival, bacterial clearance, and host immunity in the cecal-ligation and puncture (CLP)-induced murine polymicrobial sepsis model. LD01 treatment of CLP-induced sepsis significantly enhanced survival and decreased bacterial burden. Altered survival was associated with improved macrophage phagocytic activity and T-cell production of interferon-γ. Further, myeloperoxidase levels and esterase-positive cells were significantly reduced in LD01-treated mice. Taken together, these data establish that LD01 modulates host immunity and is a viable therapeutic candidate for alleviating immunosuppression that characterizes sepsis and other infectious diseases.

Interleukin-7 (IL-7) treatment accelerates neutrophil recruitment through gamma delta T-cell IL-17 production in a murine model of sepsis.

The sepsis syndrome represents an improper immune response to infection and is associated with unacceptably high rates of mortality and morbidity. The interactions between T cells and the innate immune system while combating sepsis are poorly understood. In this report, we observed that treatment with the potent, antiapoptotic cytokine interleukin-7 (IL-7) accelerated neutrophil recruitment and improved bacterial clearance. We first determined that T cells were necessary for the previously observed IL-7-mediated enhanced survival. Next, IL-7 increased Bcl-2 expression in T cells isolated from septic mice as early as 3 h following treatment. This treatment resulted in increased gamma interferon (IFN-γ) and IP-10 production within the septic peritoneum together with local and systemic increases of IL-17 in IL-7-treated mice. We further demonstrate that the increase in IL-17 was largely due to increased recruitment and production by γδ T cells, which express CXCR3. Consistent with increased IL-17 production, IL-7 treatment increased CXCL1/KC production, neutrophil recruitment, and bacterial clearance. Significantly, end-organ tissue injury was not significantly different between vehicle- and IL-7-treated mice. Collectively, these data illustrate that IL-7 can mediate the cross talk between Th1 and Th17 lymphocytes during sepsis such that neutrophil recruitment and bacterial clearance is improved while early tissue injury is not increased. All together, these observations may underlay novel potential therapeutic targets to improve the host immune response to sepsis.

Low-dose interleukin-2 reverses behavioral sensitization in multiple mouse models of headache disorders.

Headache disorders are highly prevalent and debilitating, with limited treatment options. Previous studies indicate that many proinflammatory immune cells contribute to headache pathophysiology. Given the well-recognized role of regulatory T (Treg) cells in maintaining immune homeostasis, we hypothesized that enhancing Treg function may be effective to treat multiple headache disorders. In a mouse model of chronic migraine, we observed that repeated nitroglycerin (NTG, a reliable trigger of migraine in patients) administration doubled the number of CD3 T cells in the trigeminal ganglia without altering the number of Treg cells, suggesting a deficiency in Treg-mediated immune homeostasis. We treated mice with low-dose interleukin-2 (ld-IL2) to preferentially expand and activate endogenous Treg cells. This not only prevented the development of NTG-induced persistent sensitization but also completely reversed the established facial skin hypersensitivity resulting from repeated NTG administration. The effect of ld-IL2 was independent of mouse sex and/or strain. Importantly, ld-IL2 treatment did not alter basal nociceptive responses, and repeated usage did not induce tolerance. The therapeutic effect of ld-IL2 was abolished by Treg depletion and was recapitulated by Treg adoptive transfer. Furthermore, treating mice with ld-IL2 1 to 7 days after mild traumatic brain injury effectively prevented as well as reversed the development of behaviors related to acute and chronic post-traumatic headache. In a model of medication overuse headache, Ld-IL2 completely reversed the cutaneous hypersensitivity induced by repeated administration of sumatriptan. Collectively, this study identifies ld-IL2 as a promising prophylactic for multiple headache disorders with a mechanism distinct from the existing treatment options.

Severe immunosuppression and not a cytokine storm characterizes COVID-19 infections.

COVID-19-associated morbidity and mortality have been attributed to a pathologic host response. Two divergent hypotheses have been proposed: hyperinflammatory cytokine storm; and failure of host protective immunity that results in unrestrained viral dissemination and organ injury. A key explanation for the inability to address this controversy has been the lack of diagnostic tools to evaluate immune function in COVID-19 infections. ELISpot, a highly sensitive, functional immunoassay, was employed in 27 patients with COVID-19, 51 patients with sepsis, 18 critically ill nonseptic (CINS) patients, and 27 healthy control volunteers to evaluate adaptive and innate immune status by quantitating T cell IFN-É£ and monocyte TFN-α production. Circulating T cell subsets were profoundly reduced in COVID-19 patients. Additionally, stimulated blood mononuclear cells produced less than 40%-50% of the IFN-É£ and TNF-α observed in septic and CINS patients, consistent with markedly impaired immune effector cell function. Approximately 25% of COVID-19 patients had increased IL-6 levels that were not associated with elevations in other canonical proinflammatory cytokines. Collectively, these findings support the hypothesis that COVID-19 suppresses host functional adaptive and innate immunity. Importantly, IL-7 administered ex vivo restored T cell IFN-É£ production in COVID-19 patients. Thus, ELISpot may functionally characterize host immunity in COVID-19 and inform prospective therapies.

Deletion of MyD88 markedly attenuates sepsis-induced T and B lymphocyte apoptosis but worsens survival.

Sepsis induces widespread lymphocyte apoptosis, resulting in impaired immune defenses and increased morbidity and mortality. There are multiple potential triggers or signaling molecules involved in mediating death signals. Elucidating the specific signaling pathways that are involved in mediating lymphocyte apoptosis may lead to improved therapies of this lethal disorder. We investigated a number of key cellular receptors and intracellular signaling pathways that may be responsible for apoptotic cell death. Specifically, we investigated the role of pathogen-associated molecular patterns (TLR2, TLR4, and IL-1R), intracellular signaling proteins (MyD88 and TRIF), cytoplasmic transcription factors (STAT1 and STAT4), and the MAPK pathway (JNK1) in sepsis-induced lymphocyte apoptosis. Studies were performed in the cecal ligation and puncture (CLP) model of sepsis using specific gene-targeted deletions. CLP-induced lymphocyte apoptosis was evaluated 20 h post-operation by active caspase-3 and TUNEL staining. Surprisingly, the only genetic construct that ameliorated T and B lymphocyte sepsis-induced apoptosis ( approximately 80% and 85%, respectively) occurred in MyD88(-/-) mice. Despite the marked decrease in sepsis-induced apoptosis, MyD88(-/-) mice had a worsened survival. In conclusion, lymphocyte death in sepsis likely involves multiple pathogen-sensing receptors and redundant signaling pathways. MyD88 was effective in blocking apoptosis, as it is essential in mediating most pathogen recognition pathways; however, MyD88 is also critical for host survival in a model of severe peritonitis.

Bim siRNA decreases lymphocyte apoptosis and improves survival in sepsis.

To assess the degree of lymphocyte apoptosis and survival in mice treated with small interfering RNA (siRNA) targeted to Bim, a proapoptotic molecule from the Bcl-2 family, within a clinically relevant model of sepsis. C57BL/6 mice were treated with a single dose of Bim siRNA complexed in cationic liposomes via tail vein injection. Approximately 24 h later, mice were subjected to either cecal ligation and puncture (CLP) or sham surgery. Animals were killed at 20 h postsurgery, and spleens were harvested for fluorescence-activated cell sorting analysis using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling as a marker for apoptosis. A second cohort of mice was followed for survival for 7 days. The degree of lymphocyte apoptosis in Bim siRNA-treated mice was markedly decreased compared with controls. Fluorescent activated cell sorter analysis demonstrated 13.1% +/- 1.2% B-cell apoptosis and 11.5% +/- 1.5% T-cell apoptosis in control mice compared with 2.7% +/- 0.4% B-cell apoptosis and 3.9% +/- 0.3% T-cell apoptosis in Bim siRNA-treated mice after CLP (P < 0.001 and P < 0.01, respectively). This striking difference in lymphocyte apoptosis correlated with a significant survival advantage in Bim siRNA-treated mice. At 7 days, there was 90% overall survival in Bim siRNA-treated septic mice compared with 50% overall survival in control septic mice (P < 0.05). Treatment with Bim siRNA in vivo has the potential to be an effective therapy in the treatment of sepsis.

Multiple triggers of cell death in sepsis: death receptor and mitochondrial-mediated apoptosis.

Lymphocyte apoptosis plays a central role in the pathophysiology of sepsis. Lymphocyte apoptosis was examined in mice with defective death receptor pathways due to transgenic expression of a dominant negative mutant of Fas-associated death domain (FADD-DN) or Bid-/- and in mice with defective mitochondrial-mediated pathways due to loss of Bim-/-, Puma-/-, or Noxa-/-. FADD-DN transgenic and Bid-/- mice had significant albeit incomplete protection, and this protection was associated with increased survival. Surprisingly, splenic B cells were also protected in FADD-DN mice although transgene expression was confined to T cells, providing evidence for an indirect protective mechanism. Bim-/- provided virtually complete protection against lymphocyte apoptosis whereas Puma-/- and Noxa-/- mice had modest or no protection, respectively. Bim-/- mice had improved survival, and adoptive transfer of splenocytes from Bim-/- mice into Rag 1-/- mice demonstrated that this was a lymphocyte intrinsic effect. The improved survival was associated with decreased interleukin (IL) -10 and IL-6 cytokines. Collectively, these data indicate that numerous death stimuli are generated during sepsis, and it therefore appears unlikely that blocking a single "trigger" can inhibit apoptosis. If siRNA becomes practical therapeutically, proapoptotic proteins would be potential targets.

Interleukin-7 restores lymphocytes in septic shock: the IRIS-7 randomized clinical trial.

BACKGROUND: A defining pathophysiologic feature of sepsis is profound apoptosis-induced death and depletion of CD4+ and CD8+ T cells. Interleukin-7 (IL-7) is an antiapoptotic common γ-chain cytokine that is essential for lymphocyte proliferation and survival. Clinical trials of IL-7 in over 390 oncologic and lymphopenic patients showed that IL-7 was safe, invariably increased CD4+ and CD8+ lymphocyte counts, and improved immunity. METHODS: We conducted a prospective, randomized, double-blind, placebo-controlled trial of recombinant human IL-7 (CYT107) in patients with septic shock and severe lymphopenia. Twenty-seven patients at academic sites in France and the United States received CYT107 or placebo for 4 weeks. Primary aims were to determine the safety of CYT107 in sepsis and its ability to reverse lymphopenia. RESULTS: CYT107 was well tolerated without evidence of inducing cytokine storm or worsening inflammation or organ dysfunction. CYT107 caused a 3- to 4-fold increase in absolute lymphocyte counts and in circulating CD4+ and CD8+ T cells that persisted for weeks after drug administration. CYT107 also increased T cell proliferation and activation. CONCLUSIONS: This is the first trial of an immunoadjuvant therapy targeting defects in adaptive immunity in patients with sepsis. CYT107 reversed the marked loss of CD4+ and CD8+ immune effector cells, a hallmark of sepsis and a likely key mechanism in its morbidity and mortality. CYT107 represents a potential new way forward in the treatment of patients with sepsis by restoring adaptive immunity. Such immune-based therapy should be broadly protective against diverse pathogens including multidrug resistant bacteria that preferentially target patients with impaired immunity. TRIAL REGISTRATION: Trials registered at clinicaltrials.gov: NCT02640807 and NCT02797431. FUNDING: Revimmune, NIH National Institute of General Medical Sciences GM44118.

Interleukin 7 immunotherapy improves host immunity and survival in a two-hit model of Pseudomonas aeruginosa pneumonia.

Patients with protracted sepsis develop impaired immunity, which predisposes them to acquiring secondary infections. One of the most common and lethal secondary infections is Pseudomonas aeruginosa pneumonia. Immunoadjuvant therapy is a promising approach to reverse sepsis-induced immunosuppression and improve morbidity and mortality from secondary infections. Interleukin-7 is an immunoadjuvant that improves survival in clinically relevant animal models of polymicrobial peritonitis and in fungal sepsis. This study investigated the effect of recombinant human interleukin-7 (rhIL-7) on survival in a 2-hit model of sublethal cecal ligation and puncture followed by P. aeruginosa pneumonia. Potential immunologic mechanisms responsible for the rhIL-7 putative beneficial effect were also examined, focusing on IL-17, IL-22, IFN-γ, and TNF-α, cytokines that are critical in the control of sepsis and pulmonary Pseudomonas infections. Results showed that rhIL-7 was highly effective in preventing P. aeruginosa-induced death, i.e., 92% survival in rhIL-7-treated mice versus 56% survival in control mice. rhIL-7 increased absolute numbers of immune effector cells in lung and spleen and ameliorated the sepsis-induced loss of lung innate lymphoid cells (ILCs). rhIL-7 also significantly increased IL-17-, IFN-γ-, and TNF-α-producing lung ILCs and CD8 T cells as well as IFN-γ- and TNF-α-producing splenic T cell subsets and ILCs. Furthermore, rhIL-7 enhanced NF-κB and STAT3 signaling in lungs during sepsis and pneumonia. Given the high mortality associated with secondary P. aeruginosa pneumonia, the ability of rhIL-7 to improve immunity and increase survival in multiple animal models of sepsis, and the remarkable safety profile of rhIL-7, clinical trials with rhIL-7 should be considered.

Pneumonia after cecal ligation and puncture: a clinically relevant "two-hit" model of sepsis.

Sepsis continues to be the primary cause of death among patients in surgical intensive care units. In many cases, death does not result from the initial septic event but rather from subsequent nosocomial infection with pneumonia being the most common etiology. In addition, most deaths in patients with sepsis occur after the first 72 h. By contrast, in most animal models of sepsis, most deaths occur within the first 72 h. The purpose of this study was to develop a clinically relevant "two-hit" model of sepsis that would reflect delayed mortality because of secondary nosocomial infection. The well-accepted and widely used cecal ligation and puncture (CLP) model was used as the "first hit". Pseudomonas aeruginosa or Streptococcus pneumoniae was used to induce pneumonia in mice 72 h after CLP as a "second hit." In this study, mortality in mice undergoing CLP followed by pneumonia was significantly higher than in mice receiving pneumonia or CLP alone. S. pneumoniae pneumonia after CLP resulted in a 95% mortality compared with a 20% mortality for pneumonia alone, P < 0.0001. Similarly, mortality of P. aeruginosa pneumonia after CLP (85%) was significantly higher than P. aeruginosa alone (20%), P < 0.0001. Mice undergoing CLP followed by P. aeruginosa pneumonia also had significantly higher levels of B- and T-cell apoptotic death. Finally, mice undergoing CLP followed by P. aeruginosa or S. pneumoniae pneumonia had significantly decreased concentrations of proinflammatory mediators monocyte chemoattractant protein-1 and interleukin (IL)-6 compared with mice undergoing CLP or pneumonia alone. In conclusion, a primary sublethal infection impairs the immune system thus rendering the host more susceptible to secondary infection and death. Double injury, that is, CLP followed by pneumonia, provides a useful tool in the study of sepsis, creating a prolonged period of infection as opposed to CLP alone. The extended duration of infection may lead to a better understanding of the mechanism of the immune dysregulation seen in clinical sepsis and therefore provides for evaluation of potential therapies that target specific stages of the immune response.