Benjamin A. Fensterheim

IL-15 Superagonist–Mediated Immunotoxicity: Role of NK Cells and IFN-γ

IL-15 is currently undergoing clinical trials to assess its efficacy for treatment of advanced cancers. The combination of IL-15 with soluble IL-15Rα generates a complex termed IL-15 superagonist (IL-15 SA) that possesses greater biological activity than IL-15 alone. IL-15 SA is considered an attractive antitumor and antiviral agent because of its ability to selectively expand NK and memory CD8+ T (mCD8+ T) lymphocytes. However, the adverse consequences of IL-15 SA treatment have not been defined. In this study, the effect of IL-15 SA on physiologic and immunologic functions of mice was evaluated. IL-15 SA caused dose- and time-dependent hypothermia, weight loss, liver injury, and mortality. NK (especially the proinflammatory NK subset), NKT, and mCD8+ T cells were preferentially expanded in spleen and liver upon IL-15 SA treatment. IL-15 SA caused NK cell activation as indicated by increased CD69 expression and IFN-γ, perforin, and granzyme B production, whereas NKT and mCD8+ T cells showed minimal, if any, activation. Cell depletion and adoptive transfer studies showed that the systemic toxicity of IL-15 SA was mediated by hyperproliferation of activated NK cells. Production of the proinflammatory cytokine IFN-γ, but not TNF-α or perforin, was essential to IL-15 SA–induced immunotoxicity. The toxicity and immunological alterations shown in this study are comparable to those reported in recent clinical trials of IL-15 in patients with refractory cancers and advance current knowledge by providing mechanistic insights into IL-15 SA–mediated immunotoxicity.

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.

The Cytokine Response to Lipopolysaccharide Does Not Predict the Host Response to Infection

The magnitude of the LPS-elicited cytokine response is commonly used to assess immune function in critically ill patients. A suppressed response, known as endotoxin tolerance, is associated with worse outcomes, yet endotoxin tolerance-inducing TLR4 ligands are known to protect animals from infection. Thus, it remains unknown whether the magnitude of the LPS-elicited cytokine response provides an accurate assessment of antimicrobial immunity. To address this, the ability of diverse TLR ligands to modify the LPS-elicited cytokine response and resistance to infection were assessed. Priming of mice with LPS, monophosphoryl lipid A (MPLA), or poly(I:C) significantly reduced plasma LPS–elicited proinflammatory cytokines, reflecting endotoxin tolerance, whereas CpG-ODN–primed mice showed augmented cytokine production. In contrast, LPS, MPLA, and CpG-ODN, but not poly(I:C), improved the host response to a Pseudomonas aeruginosa infection. Mice primed with protective TLR ligands, including CpG-ODN, showed reduced plasma cytokines during P. aeruginosa infection. The protection imparted by TLR ligands persisted for up to 15 d yet was independent of the adaptive immune system. In bone marrow–derived macrophages, protective TLR ligands induced a persistent metabolic phenotype characterized by elevated glycolysis and oxidative metabolism as well as augmented size, granularity, phagocytosis, and respiratory burst. Sustained augmentation of glycolysis in TLR-primed cells was dependent, in part, on hypoxia-inducible factor 1-α and was essential for increased phagocytosis. In conclusion, the magnitude of LPS-elicited cytokine production is not indicative of antimicrobial immunity after exposure to TLR ligands. Additionally, protective TLR ligands induce sustained augmentation of phagocyte metabolism and antimicrobial function.

Bruton's Tyrosine Kinase Deficiency Inhibits Autoimmune Arthritis in Mice but Fails to Block Immune Complex-Mediated Inflammatory Arthritis.

Brutons tyrosine kinase (BTK) is a B cell signaling protein that also contributes to innate immunity. BTK inhibitors prevent autoimmune arthritis but have off‐target effects, and the mechanisms of protection remain unknown. We undertook these studies using genetic deletion to investigate the role of BTK in adaptive and innate immune responses that drive inflammatory arthritis.

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.

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.

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.

The TLR4 Agonist Monophosphoryl Lipid A Drives Broad Resistance to Infection via Dynamic Reprogramming of Macrophage Metabolism

Monophosphoryl lipid A (MPLA) is a clinically used TLR4 agonist that has been found to drive nonspecific resistance to infection for up to 2 wk. However, the molecular mechanisms conferring protection are not well understood. In this study, we found that MPLA prompts resistance to infection, in part, by inducing a sustained and dynamic metabolic program in macrophages that supports improved pathogen clearance. Mice treated with MPLA had enhanced resistance to infection with Staphylococcus aureus and Candida albicans that was associated with augmented microbial clearance and organ protection. Tissue macrophages, which exhibited augmented phagocytosis and respiratory burst after MPLA treatment, were required for the beneficial effects of MPLA. Further analysis of the macrophage phenotype revealed that early TLR4-driven aerobic glycolysis was later coupled with mitochondrial biogenesis, enhanced malate shuttling, and increased mitochondrial ATP production. This metabolic program was initiated by overlapping and redundant contributions of MyD88- and TRIF-dependent signaling pathways as well as downstream mTOR activation. Blockade of mTOR signaling inhibited the development of the metabolic and functional macrophage phenotype and ablated MPLA-induced resistance to infection in vivo. Our findings reveal that MPLA drives macrophage metabolic reprogramming that evolves over a period of days to support a macrophage phenotype highly effective at mediating microbe clearance and that this results in nonspecific resistance to infection.