Julia K. Bohannon

The immunobiology of toll-like receptor 4 agonists: from endotoxin tolerance to immunoadjuvants.

ABSTRACT Lipopolysaccharide (LPS, endotoxin) is a structural component of the gram-negative outer membrane. The lipid A moiety of LPS binds to the LPS receptor complex expressed by leukocytes, endothelial cells, and parenchymal cells and is the primary component of gram-negative bacteria that is recognized by the immune system. Activation of the LPS receptor complex by native lipid A induces robust cytokine production, leukocyte activation, and inflammation, which is beneficial for clearing bacterial infections at the local level but can cause severe systemic inflammation and shock at higher challenge doses. Interestingly, prior exposure to LPS renders the host resistant to shock caused by subsequent LPS challenge, a phenomenon known as endotoxin tolerance. Treatment with lipid A has also been shown to augment the host response to infection and to serve as a potent vaccine adjuvant. However, the adverse effects associated with the pronounced inflammatory response limit the use of native lipid A as a clinical immunomodulator. More recently, analogs of lipid A have been developed that possess attenuated proinflammatory activity but retain attractive immunomodulatory properties. The lipid A analog monophosphoryl lipid A exhibits approximately 1/1,000th of the toxicity of native lipid A but retains potent immunoadjuvant activity. As such, monophosphoryl lipid A is currently used as an adjuvant in several human vaccine preparations. Because of the potency of lipid A analogs as immunoadjuvants, numerous laboratories are actively working to identify and develop new lipid A mimetics and to optimize their efficacy and safety. Based on those characteristics, lipid A analogs represent an attractive family of immunomodulators.

Prophylactic Treatment with Fms-Like Tyrosine Kinase-3 Ligand after Burn Injury Enhances Global Immune Responses to Infection

Severely burned patients are susceptible to infections with opportunistic organisms due to altered immune responses and frequent wound contamination. Immunomodulation to enhance systemic and local responses to wound infections may be protective after burn injury. We previously demonstrated that pretreatments with fms-like tyrosine kinase-3 (Flt3) ligand (Flt3L), a dendritic cell growth factor, increase the resistance of mice to a subsequent burn injury and wound infection by a dendritic cell-dependent mechanism. This study was designed to test the hypothesis that Flt3L administration after burn injury decreases susceptibility to wound infections by enhancing global immune cell activation. Mice were treated with Flt3L after burn injury and examined for survival, wound and systemic bacterial clearance, and immune cell activation after wound inoculation with Pseudomonas aeruginosa. To gain insight into the local effects of Flt3L at the burn wound, localization of Langerhans cells was examined. Mice treated with Flt3L had significantly greater numbers of CD25-expressing T cells and CD69-expressing T and B cells, neutrophils, and macrophages after, but not before, infection. Overall leukocyte apoptosis in response to infection was decreased with Flt3L treatment. Survival and local and systemic bacterial clearance were enhanced by Flt3L. Langerhans cells appeared in the dermis of skin bordering the burn wound, and further increased in response to wound infection. Flt3L augmented the appearance of Langerhans cells in response to both injury and infection. These data suggest that dendritic cell enhancement by Flt3L treatments after burn injury protects against opportunistic infections through promotion of local and systemic immune responses to infection.

Dendritic Cell Modification of Neutrophil Responses to Infection after Burn Injury

Burn patients are highly susceptible to infections due to increased exposure through wounds and impairments in a number of immune functions. Dendritic cells (DCs) are important in activation of numerous immune responses that are essential for the clearance of infections. We have found that prophylactic treatment of burn-injured mice with the DC growth factor FLT3 ligand (FL) significantly increases resistance to burn wound infections in a DC-dependent manner that is correlated closely with enhanced bacterial clearance. However, as DCs are not typically microbicidal, the mechanisms by which DC modulation enhances bacterial clearance are not known. Due to the rapid response of neutrophils to cutaneous wounds, and the reported interactions between DCs and neutrophils, we investigated the role of neutrophils in FL-mediated resistance to burn wound infection. This was examined both in vivo and in vitro through neutrophil depletion, supplementation of neutrophils, and assessment of neutrophil chemotaxis following FL treatment. To test the involvement of DCs, CD11c-diphtheria toxin receptor transgenic mice were used to deplete DCs during FL treatment. Studies revealed that neutrophils do play a critical role in FL-mediated resistance to a burn wound infection. Additionally, treatment with FL after a burn injury enhances neutrophil-mediated control of bacterial spread, neutrophil migratory capacity, and myeloperoxidase production in a DC-dependent manner. The results of this study provide new insight into immunological mechanisms that can offer protection against infection after burn injury.

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.

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.

STAT1-deficient mice are resistant to cecal ligation and puncture-induced septic shock.

ABSTRACT STAT1 (signal transducer and activator of transcription 1) is a member of the JAK-STAT signaling family and plays a key role in facilitating gene transcription in response to activation of the types I and II interferon (IFN) receptors. TYK2 is essential for type I, but not type II, IFN-induced STAT1 activation. Previous studies show that STAT1-deficient mice are resistant to endotoxin-induced shock. The goal of the present study was to assess the response of STAT1- and TYK2-deficient mice to septic shock caused by cecal ligation and puncture (CLP). End points included survival, core temperature, organ injury, systemic cytokine production, and bacterial clearance. Results showed that survival rates were significantly higher in STAT1 knockout (STAT1KO) mice compared with wild-type controls (80% vs. 10%). The improved survival of STAT1KO mice was associated with less hypothermia, metabolic acidosis, hypoglycemia, and hepatocellular injury. Plasma interleukin 6, MIP-2, CXCL10, and IFN-&agr; concentrations were significantly lower in STAT1KO mice than in wild-type mice. In the absence of antibiotic treatment, blood and lung bacterial counts were significantly lower in STAT1KO mice than in controls. However, treatment with antibiotics ablated that difference. A survival advantage was not observed in TYK2-deficient mice compared with control. However, CLP-induced hypothermia and systemic interleukin 6 and CXCL10 production were significantly attenuated in TYK2-deficient mice. These results indicate that STAT1 activation is an important factor in the pathogenesis of CLP-induced septic shock and is associated with the development of systemic inflammation and organ injury. TYK2 activation also appears to contribute to CLP-induced inflammation, but to a lesser extent than STAT1.

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.