Naeem K. Patil

Immunotherapy: A promising approach to reverse sepsis-induced immunosuppression.

Sepsis is defined as life-threatening organ dysfunction caused by dysregulated host responses to infection (Third International Consensus definition for Sepsis and septic shock). Despite decades of research, sepsis remains the leading cause of death in intensive care units. More than 40 clinical trials, most of which have targeted the sepsis-associated pro-inflammatory response, have failed. Thus, antibiotics and fluid resuscitation remain the mainstays of supportive care and there is intense need to discover and develop novel, targeted therapies to treat sepsis. Both pre-clinical and clinical studies over the past decade demonstrate unequivocally that sepsis not only causes hyper-inflammation, but also leads to simultaneous adaptive immune system dysfunction and impaired antimicrobial immunity. Evidences for immunosuppression include immune cell depletion (T cells most affected), compromised T cell effector functions, T cell exhaustion, impaired antigen presentation, increased susceptibility to opportunistic nosocomial infections, dysregulated cytokine secretion, and reactivation of latent viruses. Therefore, targeting immunosuppression provides a logical approach to treat protracted sepsis. Numerous pre-clinical studies using immunomodulatory agents such as interleukin-7, anti-programmed cell death 1 antibody (anti-PD-1), anti-programmed cell death 1 ligand antibody (anti-PD-L1), and others have demonstrated reversal of T cell dysfunction and improved survival. Therefore, identifying immunosuppressed patients with the help of specific biomarkers and administering specific immunomodulators holds significant potential for sepsis therapy in the future. This review focusses on T cell dysfunction during sepsis and discusses the potential immunotherapeutic agents to boost T cell function during sepsis and improve host resistance to infection.

Role of G-CSF in monophosphoryl lipid A-mediated augmentation of neutrophil functions after burn injury

Infection is the leading cause of death in severely burned patients that survive the acute phase of injury. Neutrophils are the first line of defense against infections, but hospitalized burn patients frequently cannot mount an appropriate innate response to infection. Thus, immune therapeutic approaches aimed at improving neutrophil functions after burn injury may be beneficial. Prophylactic treatment with the TLR4 agonist monophosphoryl lipid A is known to augment resistance to infection by enhancing neutrophil recruitment and facilitating bacterial clearance. This study aimed to define mechanisms by which monophosphoryl lipid A treatment improves bacterial clearance and survival in a model of burn‐wound sepsis. Burn‐injured mice were treated with monophosphoryl lipid A or vehicle, and neutrophil mobilization was evaluated in the presence or absence of Pseudomonas aeruginosa infection. Monophosphoryl lipid A treatment induced significant mobilization of neutrophils from the bone marrow into the blood and sites of infection. Neutrophil mobilization was associated with decreased bone marrow neutrophil CXCR4 expression and increased plasma G‐CSF concentrations. Neutralization of G‐CSF before monophosphoryl lipid A administration blocked monophosphoryl lipid A‐induced expansion of bone marrow myeloid progenitors and mobilization of neutrophils into the blood and their recruitment to the site of infection. G‐CSF neutralization ablated the enhanced bacterial clearance and survival benefit endowed by monophosphoryl lipid A in burn‐wound‐infected mice. Our findings provide convincing evidence that monophosphoryl lipid A‐induced G‐CSF facilitates early expansion, mobilization, and recruitment of neutrophils to the site of infection after burn injury, allowing for a robust immune response to infection.

The role of MyD88- and TRIF-dependent signaling in monophosphoryl lipid A-induced expansion and recruitment of innate immunocytes

Treatment with the TLR4 agonist MPLA augments innate resistance to common bacterial pathogens. However, the cellular and molecular mechanisms by which MPLA augments innate immunocyte functions are not well characterized. This study examined the importance of MyD88‐ and TRIF‐dependent signaling for leukocyte mobilization, recruitment, and activation following administration of MPLA. MPLA potently induced MyD88‐ and TRIF‐dependent signaling. A single injection of MPLA caused rapid mobilization and recruitment of neutrophils, a response that was largely mediated by the chemokines CXCL1 and ‐2 and the hemopoietic factor G‐CSF. Rapid neutrophil recruitment and chemokine production were regulated by both pathways although the MyD88‐dependent pathway showed some predominance. In further studies, multiple injections of MPLA potently induced mobilization and recruitment of neutrophils and monocytes. Neutrophil recruitment after multiple injections of MPLA was reliant on MyD88‐dependent signaling, but effective monocyte recruitment required activation of both pathways. MPLA treatment induced expansion of myeloid progenitors in bone marrow and upregulation of CD11b and shedding of L‐selectin by neutrophils, all of which were attenuated in MyD88‐ and TRIF‐deficient mice. These results show that MPLA‐induced neutrophil and monocyte recruitment, expansion of bone marrow progenitors and augmentation of neutrophil adhesion molecule expression are regulated by both the MyD88‐ and TRIF‐dependent pathways.

IL-15 Superagonist Expands mCD8+ T, NK and NKT Cells after Burn Injury but Fails to Improve Outcome during Burn Wound Infection

Background Severely burned patients are highly susceptible to opportunistic infections and sepsis, owing to the loss of the protective skin barrier and immunological dysfunction. Interleukin-15 (IL-15) belongs to the IL-2 family of common gamma chain cytokines and stimulates the proliferation and activation of T (specifically memory CD8), NK and NKT cells. It has been shown to preserve T cell function and improve survival during cecal ligation and puncture (CLP)-induced sepsis in mice. However, the therapeutic efficacy of IL-15 or IL-15 superagonist (SA) during infection after burn injury has not been evaluated. Moreover, very few, if any, studies have examined, in detail, the effect of burn injury and infection on the adaptive immune system. Thus, we examined the effect of burn and sepsis on adaptive immune cell populations and the effect of IL-15 SA treatment on the host response to infection. Methods Mice were subjected to a 35% total body surface area burn, followed by wound infection with Pseudomonas aeruginosa. In some experiments, IL-15 SA was administered after burn injury, but before infection. Leukocytes in spleen, liver and peritoneal cavity were characterized using flow cytometry. Bacterial clearance, organ injury and survival were also assessed. Results Burn wound infection led to a significant decline in total white blood cell and lymphocyte counts and induced organ injury and sepsis. Burn injury caused decline in CD4+ and CD8+ T cells in the spleen, which was worsened by infection. IL-15 treatment inhibited this decline and significantly increased cell numbers and activation, as determined by CD69 expression, of CD4+, CD8+, B, NK and NKT cells in the spleen and liver after burn injury. However, IL-15 SA treatment failed to prevent burn wound sepsis-induced loss of CD4+, CD8+, B, NK and NKT cells and failed to improve bacterial clearance and survival. Conclusion Cutaneous burn injury and infection cause significant adaptive immune dysfunction. IL-15 SA does not augment host resistance to burn wound sepsis in mice despite inducing proliferation and activation of lymphocyte subsets.

Flt3 Ligand Treatment Attenuates T Cell Dysfunction and Improves Survival in a Murine Model of Burn Wound Sepsis.

Introduction: Sepsis is a leading cause of death among severely burned patients. Burn injury disrupts the protective skin barrier and causes immunological dysfunction. In our previous studies, we found that burn injury and wound infection causes a significant decline in lymphocyte populations, implying adaptive immune system dysfunction. In the present study, we examined the effect of treatment with Fms-like tyrosine kinase-3 Ligand (Flt3L) on T cell phenotype and function in a model of burn wound sepsis. FLt3L is an essential cytokine required for hematopoietic progenitor cell development and expansion of both myeloid and lymphoid lineages. Flt3L has been shown to potentiate innate immune functions of dendritic cells and neutrophils during burn wound sepsis. However, the ability of Flt3L to improve T cell function during burn wound sepsis has not been previously evaluated. Methods: Mice underwent 35% total body surface area scald burn and were treated with Flt3L (10 &mgr;g) or vehicle daily via the intraperitoneal route starting 1 day after burn injury. On day 4 after burn injury, Pseudomonas aeruginosa was used to induce wound infection. Leukocytes in spleen and wound draining lymph nodes were characterized using flow cytometry. Bacterial clearance, organ injury, and survival were also assessed. Results: Flt3L treatment prevented the decline in splenic CD4+ and CD8+ T cells caused by burn injury and infection. Flt3L treatment also attenuated the decline in CD28 expression on CD4+ and CD8+ T cells and IFN&ggr; production by CD8+ T cells in the spleen and wound draining lymph nodes. Furthermore, Flt3L decreased the levels of programmed death ligand 1 expression on splenic dendritic cells and macrophages. Flt3 treatment improved systemic bacterial clearance, decreased liver and kidney injury, and significantly improved survival in mice with burn wound sepsis. Conclusion: Burn injury and associated sepsis causes significant loss of T cells and evidence of T cell dysfunction. Flt3L attenuates T cell dysfunction and improves host resistance to burn wound sepsis in mice.

Immunobiology of the IL-15/IL-15Rα complex as an antitumor and antiviral agent

Interleukin (IL)-15 is essential for natural killer (NK), NKT and memory (m) CD8+ T cell development and function, and is currently under investigation as an immunotherapeutic agent for the treatment of cancer. Recently, the creation of IL-15 superagonist by complexing IL-15 and its high affinity receptor alpha (IL-15 Rα) in solution, inspired by the natural trans-presentation of IL-15, advances the potential of IL-15-based tumor immunotherapy. IL-15 superagonist shows promising advantages over monomeric IL-15 such as sustaining high circulating concentrations due to prolonged half-life and more potently stimulating NK and CD8+ T effector lymphocytes. So far, there are three different forms of recombinant IL-15 superagonist fusion protein based on configurational modifications. Gene therapy using engineered cells co-expressing IL-15/IL-15 Rα complex for cancer treatment is also emerging. All forms have demonstrated efficacy in causing tumor regression in animal studies, which provides strong rationale for advancing IL-15 superagonist through clinical trials. To date, there are fourteen phase I/II IL-15 superagonist trials in cancer patients and one phase I trial in HIV patients. Information generated by ongoing trials regarding the toxicity and efficacy of IL-15 superagonist is awaited. Finally, we elaborate on immunotoxicity caused by IL-15 superagonist in preclinical studies and discuss important safety considerations.

IL-15 Enables Septic Shock by Maintaining NK Cell Integrity and Function

Interleukin 15 is essential for the development and differentiation of NK and memory CD8+ (mCD8+) T cells. Our laboratory previously showed that NK and CD8+ T lymphocytes facilitate the pathobiology of septic shock. However, factors that regulate NK and CD8+ T lymphocyte functions during sepsis are not well characterized. We hypothesized that IL-15 promotes the pathogenesis of sepsis by maintaining NK and mCD8+ T cell integrity. To test our hypothesis, the pathogenesis of sepsis was assessed in IL-15–deficient (IL-15 knockout, KO) mice. IL-15 KO mice showed improved survival, attenuated hypothermia, and less proinflammatory cytokine production during septic shock caused by cecal ligation and puncture or endotoxin-induced shock. Treatment with IL-15 superagonist (IL-15 SA, IL-15/IL-15Rα complex) regenerated NK and mCD8+ T cells and re-established mortality of IL-15 KO mice during septic shock. Preventing NK cell regeneration attenuated the restoration of mortality caused by IL-15 SA. If given immediately prior to septic challenge, IL-15–neutralizing IgG M96 failed to protect against septic shock. However, M96 caused NK cell depletion if given 4 d prior to septic challenge and conferred protection. IL-15 SA treatment amplified endotoxin shock, which was prevented by NK cell or IFN-γ depletion. IL-15 SA treatment also exacerbated septic shock caused by cecal ligation and puncture when given after the onset of sepsis. In conclusion, endogenous IL-15 does not directly augment the pathogenesis of sepsis but enables the development of septic shock by maintaining NK cell numbers and integrity. Exogenous IL-15 exacerbates the severity of sepsis by activating NK cells and facilitating IFN-γ production.

Targeting Immune Cell Checkpoints during Sepsis

Immunosuppression is increasingly being recognized as one of the causes of increased morbidity and mortality during sepsis. Both innate and adaptive immune system dysfunction have been shown to cause an impaired ability to eradicate the primary infection and also lead to frequent occurrence of secondary opportunistic infections. Pre-clinical and clinical studies have shown that inhibitory immune checkpoint molecules, including programmed death-1 (PD-1), programmed death ligand-1 (PD-L1), cytotoxic T lymphocyte antigen-4 (CTLA-4), T cell membrane protein-3 (TIM-3), Lymphocyte activation-gene-3 (LAG-3) and 2B4, are upregulated during the course of sepsis. Engagement of these inhibitory molecules on various immune cells has been consistently shown to inhibit innate immune cell functions (e.g., phagocytosis, cytokine production and pathogen clearance) and also lead to impaired T cell competence. In numerous pre-clinical models of sepsis, therapeutic agents aimed at blocking engagement of inhibitory immune checkpoints on immune cells have been shown to improve innate and adaptive immune cell functions, increase host resistance to infection and significantly improve survival. Therefore, immunotherapy with immune cell checkpoint inhibitors holds significant potential for the future of sepsis therapy and merits further investigation.

The biology of natural killer cells during sepsis

Natural killer (NK) cells are large granular lymphocytes largely recognized for their importance in tumour surveillance and the host response to viral infections. However, as the major innate lymphocyte population, NK cells also coordinate early responses to bacterial infections by amplifying the antimicrobial functions of myeloid cells, especially macrophages, by production of interferon‐γ (IFN‐γ). Alternatively, excessive NK cell activation and IFN‐γ production can amplify the systemic inflammatory response during sepsis resulting in increased physiological dysfunction and organ injury. Our understanding of NK cell biology during bacterial infections and sepsis is mostly derived from studies performed in mice. Human studies have demonstrated a correlation between altered NK cell functions and outcomes during sepsis. However, mechanistic understanding of NK cell function during human sepsis is limited. In this review, we will review the current understanding of NK cell biology during sepsis and discuss the challenges associated with modulating NK cell function during sepsis for therapeutic benefit.

Comparative Transcriptome Profiles of Human Blood in Response to the Toll-like Receptor 4 Ligands Lipopolysaccharide and Monophosphoryl Lipid A.

Monophosphoryl lipid A (MPLA), a less toxic derivative of lipopolysaccharide (LPS), is employed as a vaccine adjuvant and is under investigation as a non-specific immunomodulator. However, the differential response of human leukocytes to MPLA and LPS has not been well characterized. The goal of this study was to compare the differential transcriptomic response of human blood to LPS and MPLA. Venous blood from human volunteers was stimulated with LPS, MPLA or vehicle. Gene expression was determined using microarray analysis. Among 21,103 probes profiled, 136 and 130 genes were differentially regulated by LPS or MPLA, respectively. Seventy four genes were up-regulated and 9 were down-regulated by both ligands. The remaining genes were differentially induced by either agent. Ingenuity Pathway Analysis predicted that LPS and MPLA share similar upstream regulators and have comparable effects on canonical pathways and cellular functions. However, some pro-inflammatory cytokine and inflammasome-associated transcripts were more strongly induced by LPS. In contrast, only the macrophage-regulating chemokine CCL7 was preferentially up-regulated by MPLA. In conclusion, LPS and MPLA induce similar transcriptional profiles. However, LPS more potently induces pro-inflammatory cytokine and inflammasome-linked transcripts. Thus, MPLA is a less potent activator of the pro-inflammatory response but retains effective immunomodulatory activity.