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Pretreatment with 3-deacyl 6-acyl phosphorylated hexaacyl disaccharide; a novel synthetic Toll-like receptor 4 agonist, preserves kidney function during ischemia-reperfusion injury-induced acute kidney injury.

Objectives: Nosocomial pneumonia is a common complication in critically ill patients. The goal of this study was to examine the efficacy of the Toll-like receptor 4 agonist 3-deacyl phosphorylated hexacyl disaccharide (3D PHAD), in a clinically relevant murine model of pneumonia, and assess the cellular mechanisms that mediate the protective response. Design: Mice received intrapulmonary 3D PHAD (20 μg) or vehicle for 2 consecutive days before challenge with intrapulmonary Klebsiella pneumoniae (2.3 × 10 3 colony-forming units). Mice were followed for 14-day survival, pulmonary K. pneumoniae burden, lung leukocyte profile, leukocyte phagocytic capacity, and cytokine production. Pneumonia severity and leukocyte recruitment were further assessed by histological evaluation. Setting: Research laboratory. Subjects: Wild-type, male C57BL/6 J mice. Interventions: Intrapulmonary treatment with 20 μg 3D PHAD for 2 consecutive days. Measurements and main results: Intrapulmonary treatment with 3D PHAD decreased lung K. pneumoniae colony-forming units and pneumonia severity with an associated improvement in survival compared with mice treated with vehicle. The numbers of neutrophils, monocytes, and macrophages in the lungs of 3D PHAD-treated mice were higher than those in vehicle-treated mice before infection but were not significantly different from vehicle-treated mice at 48 h after K. pneumoniae challenge. Lung innate leukocytes from 3D PHAD-treated mice had increased phagocytic capacity. Treatment with 3D PHAD alone increased cytokines in the lungs but decreased cytokines in plasma during K. pneumoniae pneumonia as compared with control. Conclusions: Intrapulmonary treatment with 3D PHAD augments innate immunity in the lung and facilitates resistance to K. pneumoniae pneumonia.

Unlike the adaptive immune system, the innate immune system has classically been characterized as being devoid of memory functions. However, recent research shows that innate myeloid and lymphoid cells have the ability to retain memory of prior pathogen exposure and become primed to elicit a robust, broad-spectrum response to subsequent infection. This phenomenon has been termed innate immune memory or trained immunity. Innate immune memory is induced via activation of pattern recognition receptors and the actions of cytokines on hematopoietic progenitors and stem cells in bone marrow and innate leukocytes in the periphery. The trained phenotype is induced and sustained via epigenetic modifications that reprogram transcriptional patterns and metabolism. These modifications augment antimicrobial functions, such as leukocyte expansion, chemotaxis, phagocytosis, and microbial killing, to facilitate an augmented host response to infection. Alternatively, innate immune memory may contribute to the pathogenesis of chronic diseases, such as atherosclerosis and Alzheimer’s disease.

Bacterial infections are a common and deadly threat to vulnerable patients. Alternative strategies to fight infection are needed. β-Glucan, an immunomodulator derived from the fungal cell wall, provokes resistance to infection by inducing trained immunity, a phenomenon that persists for weeks to months. Given the durability of trained immunity, it is unclear which leukocyte populations sustain this effect. Macrophages have a life span that surpasses the duration of trained immunity. Thus, we sought to define the contribution of differentiated macrophages to trained immunity. Our results show that β-glucan protects mice from Pseudomonas aeruginosa infection by augmenting recruitment of innate leukocytes to the site of infection and facilitating local clearance of bacteria, an effect that persists for more than 7 d. Adoptive transfer of macrophages, trained using β-glucan, into naive mice conferred a comparable level of protection. Trained mouse bone marrow–derived macrophages assumed an antimicrobial phenotype characterized by enhanced phagocytosis and reactive oxygen species production in parallel with sustained enhancements in glycolytic and oxidative metabolism, increased mitochondrial mass, and membrane potential. β-Glucan induced broad transcriptomic changes in macrophages consistent with early activation of the inflammatory response, followed by sustained alterations in transcripts associated with metabolism, cellular differentiation, and antimicrobial function. Trained macrophages constitutively secreted CCL chemokines and robustly produced proinflammatory cytokines and chemokines in response to LPS challenge. Induction of the trained phenotype was independent of the classic β-glucan receptors Dectin-1 and TLR-2. These findings provide evidence that β-glucan induces enhanced protection from infection by driving trained immunity in macrophages.

BACKGROUND: Monophosphoryl lipid A (MPLA) is a TLR4 agonist that has potent immunomodulatory properties and modulates innate immune function to improve host resistance to infection with common nosocomial pathogens in mice. The goal of this study was to assess the safety and efficacy of MPLA in a sheep model of burn injury and Pseudomonas aeruginosa pneumonia. The sheep provides a favorable model for pre-clinical testing as their response to TLR4 agonists closely mimics that of humans. METHODS: Twelve chronically instrumented adult female Merino sheep received 20% total body surface area, third-degree cutaneous burn under anesthesia and analgesia. At 24 hours after burn, sheep were randomly allocated to receive; 1) MPLA (2.5 μg/kg IV, n=6), or 2) vehicle (IV, n=6). At 24 hours after MPLA or vehicle treatment, Pseudomonas aeruginosa pneumonia was induced. Sheep were mechanically ventilated, fluid resuscitated and cardiopulmonary variables were monitored for 24 hours after induction of pneumonia. Cytokine production, vascular barrier function, and lung bacterial burden were also measured. RESULTS: MPLA infusion induced small and transient alterations in core body temperature, heart rate, pulmonary artery pressure, and pulmonary vascular resistance. Pulmonary mechanics were not altered. Vehicle-treated sheep developed severe acute lung injury during Pseudomonas aeruginosa pneumonia, which was attenuated by MPLA as indicated by improved PaO(2)/FiO(2) ratio, oxygenation index, and shunt fraction. Sheep treated with MPLA also exhibited less vascular leak, lower blood lactate levels, and lower modified organ injury score. MPLA treatment attenuated systemic cytokine production and decreased lung bacterial burden. CONCLUSIONS: MPLA was well tolerated in burned sheep and attenuated development of acute lung injury, lactatemia, cytokinemia, vascular leak, and hemodynamic changes caused by Pseudomonas aeruginosa pneumonia.

Objectives To determine whether synthetic phosphorylated hexa-acyl disaccharides provide antimicrobial protection in clinically relevant models of bacterial infection. Design Laboratory study. Setting University laboratory. Subjects BALB/c, C57BL/10J, and C57BL/10ScNJ mice. Interventions Mice were treated with lactated Ringer's (vehicle) solution, monophosphoryl lipid A, or phosphorylated hexa-acyl disaccharides at 48 and 24 hours prior to intraperitoneal Pseudomonas aeruginosa or IV Staphylococcus aureus infection. Leukocyte recruitment, cytokine production, and bacterial clearance were measured 6 hours after P. aeruginosa infection. In the systemic S. aureus infection model, one group of mice was monitored for 14-day survival and another for S. aureus tissue burden at 3 days postinfection. Duration of action for 3-deacyl 6-Acyl phosphorylated hexa-acyl disaccharide was determined at 3, 10, and 14 days using a model of intraperitoneal P. aeruginosa infection. Effect of 3-deacyl 6-Acyl phosphorylated hexa-acyl disaccharide on in vivo leukocyte phagocytosis and respiratory burst was examined. Leukocyte recruitment, cytokine production, and bacterial clearance were measured after P. aeruginosa infection in wild-type and toll-like receptor 4 knockout mice treated with 3-deacyl 6-Acyl phosphorylated hexa-acyl disaccharide or vehicle to assess receptor specificity. Measurements and main results During intraperitoneal P. aeruginosa infection, phosphorylated hexa-acyl disaccharides significantly attenuated infection-induced hypothermia, augmented leukocyte recruitment and bacterial clearance, and decreased cytokine production. At 3 days post S. aureus infection, bacterial burden in lungs, spleen, and kidneys was significantly decreased in mice treated with monophosphoryl lipid A or phosphorylated hexa-acyl disaccharides, which was associated with improved survival. Leukocyte phagocytosis and respiratory burst functions were enhanced after treatment with monophosphoryl lipid A or phosphorylated hexa-acyl disaccharides. A time course study showed that monophosphoryl lipid A- and 3-deacyl 6-Acyl phosphorylated hexa-acyl disaccharide-mediated protection against P. aeruginosa lasts for up to 10 days. Partial loss of augmented innate antimicrobial responses was observed in toll-like receptor 4 knockout mice treated with 3-deacyl 6-Acyl phosphorylated hexa-acyl disaccharide. Conclusions Phosphorylated hexa-acyl disaccharides significantly augment resistance against clinically relevant Gram-negative and Gram-positive infections via enhanced leukocyte recruitment, phagocytosis, and respiratory burst functions of innate leukocytes. Improved antimicrobial protection persists for up to 10 days and is partially mediated through toll-like receptor 4.

Infection is the leading cause of death and prolonged hospitalization in severely burned patients that survive the acute phase of injury. Here we describe a murine model of severe burn injury followed by subsequent postburn infection, both local and systemic, that leads to sepsis. A detailed description of the full-thickness scald burn procedure is provided, followed by description of infection with two common burn-associated nosocomial pathogens, Pseudomonas aeruginosa and Staphylococcus aureus.

Cellular metabolism is a means of generating ATP to provide energy for key cellular functions. However, recent research shows that citric acid cycle intermediates target vital cellular functions of the innate immune system. Succinate, itaconate, citrate, and fumarate have been shown to mediate or regulate important myeloid cell functions during infection and inflammation. This review covers the regulatory functions of citric acid cycle intermediates in myeloid cells and discusses potential translational applications, key mechanistic questions, and future research directions.

Hematopoietic myeloblasts give rise to macrophages, dendritic cells, and neutrophils. Circulating myeloid cells detect invading microbes using pattern recognition receptors and subsequently orchestrate an innate immune response to contain and kill the pathogens. This innate immune response establishes an inflammatory niche characterized by hypoxia due to host and pathogen factors. Hypoxia-inducible factor (HIF) transcription factors are the primary regulators of the myeloid response to hypoxia. In particular, HIF-1α is a critical hub that integrates hypoxic and immunogenic signals during infection or inflammation. Hypoxia induces HIF-1α stabilization, which drives metabolic and phenotypic reprogramming of myeloid cells to maximize antimicrobial potential. HIF-1α activity in myeloid-derived cells enhances the host response to infection, but may also play a role in pathogenic inflammatory processes, such as atherosclerosis. In this review, we summarize recent advances that have elucidated the mechanism by which myeloid cells regulate HIF-1α activity and, in turn, how HIF-1α shapes myeloid cell function.

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.

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.

Discussion on leukocyte metabolism and the subsequent changes in intracellular metabolite concentrations.

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.

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.

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.

Nipah virus (NiV) infection can lead to severe respiratory or neurological disease in humans. Transmission of NiV has been shown to occur through contact with virus contaminated fomites or consumption of contaminated food. Previous results using the African green monkey (AGM) model of NiV infection identified aspects of infection that, while similar to humans, don’t fully recapitulate disease. Previous studies also demonstrate near uniform lethality that is not consistent with human NiV infection. In these studies, aerosol exposure using an intermediate particle size (7μm) was used to mimic potential human exposure by facilitating virus deposition in the upper respiratory tract. Computed tomography evaluation found some animals developed pulmonary parenchymal disease including consolidations, ground-glass opacities, and reactive adenopathy. Despite the lack of neurological signs, magnetic resonance imaging identified distinct brain lesions in three animals, similar to those previously reported in NiV-infected patients. Immunological characterization of tissues collected at necropsy suggested a local pulmonary inflammatory response with increased levels of macrophages in the lung, but a limited neurologic response. These data provide the first clear evidence of neurological involvement in the AGM that recapitulates human disease. With the development of a disease model that is more representative of human disease, these data suggest that NiV infection in the AGM may be appropriate for evaluating therapeutic countermeasures directed at virus-induced neuropathogenesis., Author summary The development of effective therapeutic approaches to the treatment of human diseases requires an understanding of the disease process induced by an infectious agent. Historically the development of medical countermeasures for highly pathogenic viruses required the use of a uniformly lethal animal model. While this approach is useful in some regards, it frequently does not provide a true indication of the disease process. In the work presented here, the approach was to use a virus exposure method that mimicked a potential route of human exposure and used a dose that might be more representative of one a human would receive. Using this method and advanced medical imaging techniques, we were able to demonstrate an extended disease course with mixed respiratory and neurological disease like that seen in humans. This study also found that the response to infection in the lungs was inflammatory and that the disease in the brain was limited despite clear evidence of lesions. These data support the development of animal models that mimic human disease and allow for the identification of potential therapeutic approaches that target the disease process rather than only the virus.

Cowpox virus (CPXV) inoculation of nonhuman primates (NHPs) has been suggested as an alternate model for smallpox (Kramski et al., 2010, PLoS One, 5, e10412). Previously, we have demonstrated that intrabronchial inoculation of CPXV-Brighton Red (CPXV-BR) into cynomolgus monkeys resulted in a disease that shared many similarities to smallpox; however, severe respiratory tract disease was observed (Smith et al., 2011, J. Gen. Virol.). Here we describe the course of disease after small particle aerosol exposure of rhesus monkeys using computed tomography (CT) to monitor respiratory disease progression. Subjects developed a severe respiratory disease that was uniformly lethal at 5.7 log10 PFU of CPXV-BR. CT indicated changes in lung architecture that correlated with changes in peripheral blood monocytes and peripheral oxygen saturation. While the small particle aerosol inoculation route does not accurately mimic human smallpox, the data suggest that CT can be used as a tool to monitor real-time disease progression for evaluation of animal models for human diseases.

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.

Aerosol aerodynamic particle size is known to affect deposition patterns of inhaled aerosol particles, as well as the virulence of inhaled bioaerosol particles. While a significant amount of work has been performed to describe the deposition of aerosol particles in the human respiratory tract, only a limited amount of work has been performed to describe the deposition of aerosol particles in the respiratory tract of nonhuman primates, an animal model commonly utilized in pharmacological and toxicological studies, especially in the biodefense field. In this study, anesthetized rhesus macaques inhaled radiolabeled aerosols with MMADs of 1.7, 3.6, 7.4 and 11.8 µm to characterize regional deposition patterns. The results demonstrate that the regional deposition pattern shifts as particle size increases, with greater deposition in more proximal regions of the respiratory tract and decreased deposition in the pulmonary region. The results of this study extend the findings of previous studies which demonstrated a similar shift in the deposition pattern as a function of particle size by providing greater resolution of deposition patterns. These data on regional deposition patterns provide a starting point to begin to explore potential mechanisms responsible for the differences in virulence of infectious bioaerosols as a function of particle size and deposition pattern reported in previous studies. Additionally, the data are useful to assess the performance of various deposition models that have been published in the literature.

Burn patients are susceptible to infections due, in part, to immune dysfunction. Upregulation of programmed death-1 (PD-1) receptor on T cells and programmed cell death ligand-1 (PD-L1) on myeloid cells contribute to immune dysfunction in nonburn-related sepsis. We hypothesized that PD-1/PDL1 interactions contribute to immune dysfunction after burn injury. To determine the impact of burn injury and infection on PD-L1, PD-1 and costimulatory receptor expression by leukocytes and its relationship to T cell functions. The efficacy of anti-PD-L1 antibody was evaluated in a clinically relevant mouse model of burn injury and bacterial infection. Mice underwent 35% scald burn followed by Pseudomonas aeruginosa or Staphylococcus aureus infection on day 4 postburn. Anti-PD-L1 was administered on day 3 postburn. Numbers and phenotype of leukocytes, plasma cytokine concentrations, bacterial clearance, organ injury, and survival were assessed. Burn injury and infection with P. aeruginosa caused a significant upregulation of PD-L1 on myeloid cells, along with a decrease in T cell numbers and function, significant multiorgan injury, and decreased survival. Treatment with anti-PD-L1 antibody improved bacterial clearance, reduced organ injury, and enhanced survival during Pseudomonas burn wound infection. Furthermore, anti-PD-L1 effectively protected against multiorgan injury, and improved bacterial clearance and survival following systemic S. aureus infection after burn injury. Blockade of PD-1/PD-L1 interactions might represent a viable treatment to improve outcomes among critically ill burn-injured subjects and increased leukocyte PD-L1 expression could serve as a valuable biomarker to select appropriate patients for such treatment.

Infectious diseases remain a threat to critically ill patients, particularly with the rise of antibiotic-resistant bacteria. Septic shock carries a mortality of up to ∼40% with no compelling evidence of promising therapy to reduce morbidity or mortality. Septic shock survivors are also prone to nosocomial infections. Treatment with toll-like receptor 4 (TLR4) agonists have demonstrated significant protection against common nosocomial pathogens in various clinically relevant models of infection and septic shock. TLR4 agonists are derived from a bacteria cell wall or synthesized de novo, and more recently novel small molecule TLR4 agonists have also been developed. TLR4 agonists augment innate immune functions including expansion and recruitment of innate leukocytes to the site of infection. Recent studies demonstrate TLR4-induced leukocyte metabolic reprogramming of cellular metabolism to improve antimicrobial function. Metabolic changes include sustained augmentation of macrophage glycolysis, mitochondrial function, and tricarboxylic acid cycle flux. These findings set the stage for the use of TLR4 agonists as standalone therapeutic agents or antimicrobial adjuncts in patient populations vulnerable to nosocomial infections.

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.

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.

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.Mice underwent 35% total body surface area scald burn and were treated with Flt3L (10 μg) 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.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 IFNγ 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.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.

We previously demonstrated that small-particle (0.5–3.0 µm) aerosol infection of rhesus monkeys (Macaca mulatta) with cowpox virus (CPXV)-Brighton Red (BR) results in fulminant respiratory tract disease characterized by severe lung parenchymal pathology but only limited systemic virus dissemination and limited classic epidermal pox-like lesion development ( Johnson et al., 2015 ). Based on these results, and to further develop CPXV as an improved model of human smallpox, we evaluated a novel large-particle aerosol (7.0–9.0 µm) exposure of rhesus monkeys to CPXV-BR and monitored for respiratory tract disease by serial computed tomography (CT). As expected, the upper respiratory tract and large airways were the major sites of virus-induced pathology following large-particle aerosol exposure. Large-particle aerosol CPXV exposure of rhesus macaques resulted in severe upper airway and large airway pathology with limited systemic dissemination.

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-α 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.

Burn patients are susceptible to opportunistic infections partly because of decreased immune functions, especially TH1-driven antigen-specific responses, which are regulated by dendritic cells. The dendritic cell growth factor, fms-like tyrosine kinase-3 ligand (FL), has been shown to increase resistance to Pseudomonas aeruginosa, in a dendritic cell-dependent manner, in a mouse model of burn wound infection. The specific mechanisms of protection are not known. This study tested the hypothesis that FL can enhance production of P. aeruginosa-specific antibodies after burn wound infection. Mice that had been previously exposed to P. aeruginosa were infected after burn injury by wound inoculation or challenged by intraperitoneal injection of heat-killed P. aeruginosa. In response to wound infection, FL treatments enhanced bacterial clearance and induced a shift from immunoglobulin (Ig) M toward IgG and IgA. However, serum levels of neither P. aeruginosa-specific antibodies nor interferon gamma (IFN-gamma) were significantly increased by FL, possibly because of decreased systemic exposure to bacteria. After challenge with heat-killed bacteria, which ensured equal exposures, FL-treated mice produced significantly greater levels of P. aeruginosa-specific IgG2a, which correlated with an increase in serum levels of interferon gamma and enhanced opsonization capacity. IL-12, IL-10, and transforming growth factor beta were significantly increased in FL-treated mice, regardless of the type of challenge. These findings indicate that FL treatments after burn injury enhance cytokine responses to recall antigens and increase bacterial clearance. In addition, through its ability to promote TH1-associated antigen-specific responses, FL may have potential as an immunotherapy to enhance adaptive immunity after severe 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.

Burn patients with concomitant pulmonary Pseudomonas aeruginosa (PA) infection have mortality rates as high as 50%, despite antibiotic therapy. Sphingosine is generated from ceramide via ceramidase and has been reported to have antimicrobial properties. We observed a reduction in sphingosine and a concurrent increase in ceramide in bronchial epithelial cells after burn injury. After PA inoculation, these mice had a significant decrease in survival compared to noninjured mice. However, when injured mice were pretreated with sphingosine or neutral ceramidase and subsequently infected, mortality and bacterial levels were robustly reduced. We further observed that sphingosine directly kills PA. Together, these results demonstrate that reduction in sphingosine is associated with an increased susceptibility to pulmonary infection after burn injury. Restoration of sphingosine levels through direct sphingosine administration or conversion of the increased ceramide to sphingosine by neutral ceramidase reduces mortality and mitigates pulmonary infection after burn injury.

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.

Infection is the leading cause of death in severely burned patients. Prophylactic treatment with the TLR4 agonist monophosphoryl lipid A (MPLA) induces resistance to subsequent bacterial challenge. Treatment of mice with MPLA enhances bacterial clearance, leading to improved survival in a model of P. aeruginosa burn wound infection. The current study was aimed to define the mechanisms responsible for improved bacterial clearance and survival in MPLA-treated burn-infected mice, and to determine if MPLA could protect against clinically relevant Gram positive and fungal pathogens. Mice underwent severe burn injury, followed by systemic treatment with MPLA or vehicle control for 2 days. Mice were then inoculated with P. aeruginosa topically or intraperitoneally, and responding neutrophils were measured in bone marrow, blood, burn wound and peritoneal cavity. In later experiments, burned mice were challenged systemically with S. aureus or C. albicans. MPLA treatment induced G-CSF production, decreased bone marrow neutrophil numbers and increased neutrophil numbers in the blood, peritoneal cavity and burn wound site. G-CSF was essential for MPLA-mediated survival, bacterial clearance and neutrophil trafficking. MPLA also improved survival against S. aureus and C. albicans systemic infections after burn. This suggests that G-CSF facilitates MPLA-induced trafficking of neutrophils, allowing for a more rapid response to sites of infection. The ability of MPLA to enhance antimicrobial responses and its ability to protect against a variety of clinically relevant pathogens make it an attractive therapeutic candidate for use in burn patients for the prevention of post-burn infections.

BackgroundFms-like tyrosine kinase-3 ligand (Flt3L) is a hemopoietic cytokine and dendritic cell (DC) growth factor that promotes the proliferation and differentiation of progenitor cells into DCs. We have previously found that treatment of severely burned mice with recombinant Flt3L significantly enhances DC production and bacterial clearance from infected burn wounds, and increases global immune cell activation and survival in response to a burn wound infection. These significant benefits of Flt3L treatment after burn injury have prompted the question of whether or not severe burn injury induces deficits in endogenous Flt3L levels that could affect DCs and subsequent responses to infection.ResultsTo address this, male BALB/c mice received a 30% total body surface area scald burn. Blood, spleens, and wound-draining lymph nodes were harvested at various time-points after injury. Some mice received a wound inoculation withP. aeruginosa. Murine Flt3L and G-CSF levels were measured by ELISA. Burn injury had no significant effect on Flt3L levels at any post-burn time-point examined compared to normal Flt3L levels in the sera, spleen, or lymph nodes. Additionally, Flt3L levels in the sera, spleen, and lymph nodes were not significantly altered when wounds were inoculated on the day of burn injury or at post-burn time points examined. Alternatively, levels of G-CSF were increased in response to burn injury and burn wound infection. Additionally, DC numbers and functions were not altered following burn injury alone. There was no significant difference between the number of DCs in the spleens of sham-injured mice and mice at 5 days after burn injury. When naïve T cells from sham-injured mice were co-cultured with DCs from either sham- or burn-injured mice, IFN-γ production was similar, however, IFN-γ levels produced by T cells harvested from burn-injured mice were significantly lower than those produced by T cells from sham mice, regardless of which DC group, sham or burn, was used in the coculture.ConclusionThese data suggest that the beneficial effects of Flt3L treatments after burn injury are not due to correction of a burn-associated Flt3L deficiency but rather, are likely due to supplementary stimulation of DC production and 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.

Introduction The chemokine CXCL10 is produced during infection and inflammation to activate the chemokine receptor CXCR3, an important regulator of lymphocyte trafficking and activation. The goal of this study was to assess the contributions of CXCL10 to the pathogenesis of experimental septic shock in mice. Methods Septic shock was induced by cecal ligation and puncture (CLP) in mice resuscitated with lactated Ringer’s solution and, in some cases, the broad spectrum antibiotic Primaxin. Studies were performed in CXCL10 knockout mice and mice treated with anti-CXCL10 immunoglobulin G (IgG). Endpoints included leukocyte trafficking and activation, core body temperature, plasma cytokine concentrations, bacterial clearance and survival. Results CXCL10 was present at high concentrations in plasma and peritoneal cavity during CLP-induced septic shock. Survival was significantly improved in CXCL10 knockout (CXCL10KO) mice and mice treated with anti-CXCL10 IgG compared to controls. CXCL10KO mice and mice treated with anti-CXCL10 IgG showed attenuated hypothermia, lower concentrations of interleukin-6 (IL-6) and macrophage inhibitory protein-2 (MIP-2) in plasma and lessened natural killer (NK) cell activation compared to control mice. Compared to control mice, bacterial burden in blood and lungs was lower in CXCL10-deficient mice but not in mice treated with anti-CXCL10 IgG. Treatment of mice with anti-CXCL10 IgG plus fluids and Primaxin at 2 or 6 hours after CLP significantly improved survival compared to mice treated with non-specific IgG under the same conditions. Conclusions CXCL10 plays a role in the pathogenesis of CLP-induced septic shock and could serve as a therapeutic target during the acute phase of septic shock.

During the course of infection, natural killer (NK) cells contribute to innate immunity by producing cytokines, particularly interferon-gamma (IFN-γ). In addition to their beneficial effects against infection, NK cells may play a detrimental role during systemic inflammation, causing lethality during sepsis. Little is known on the immune status of NK cells in patients with systemic inflammatory response syndrome (SIRS) or sepsis in terms of cell surface markers expression and IFN-γ production.We investigated 27 sepsis patients and 11 patients with non-infectious SIRS. CD56bright and CD56dim NK cell subsets were identified by flow cytometry and Toll-like receptor (TLR)2, TLR4, TLR9, CX3CR1, CD16 and CD69 expression were analyzed, as well as ex vivo IFN-γ production by NK cells in whole blood samples.We first showed that in NK cells from healthy controls, TLR2 and TLR4 expression is mainly intracellular, similarly to TLR9. Intracellular levels of TLR2 and TLR4, in both CD56bright and CD56dim NK cell subsets from sepsis patients, were increased compared to healthy subjects. In addition, the percentage of CD69+ cells was higher among NK cells of sepsis patients. No difference was observed for TLR9, CX3CR1, and CD16 expression. The ex vivo stimulation by TLR4 or TLR9 agonists, or whole bacteria in synergy with accessory cytokines (IL-15+IL-18), resulted in significant production of IFN-γ by NK cells of healthy controls. In contrast, for SIRS and sepsis patients this response was dramatically reduced.This study reports for the first time an intracellular expression of TLR2 and TLR4 in human NK cells. Surface TLR4 expression allows discriminating sepsis and SIRS. Furthermore, during these pathologies, NK cells undergo an alteration of their immune status characterized by a profound reduction of their capacity to release IFN-γ.

Background Influenza results in up to 500,000 deaths annually. Seasonal influenza vaccines have an estimated 60% effectiveness, but provide little or no protection against novel subtypes, and may be less protective in high-risk groups. Neuraminidase inhibitors are recommended for the treatment of severe influenza infection, but are not proven to reduce mortality in severe disease. Preclinical models of severe influenza infection that closely correlate to human disease are needed to assess efficacy of new vaccines and therapeutics. Methods We developed a nonhuman primate model of influenza and bacterial co-infection that recapitulates severe pneumonia in humans. Animals were infected with influenza A virus via intra-bronchial or small-particle aerosol inoculation, methicillin-resistant Staphylococcus aureus , or co-infected with influenza and methicillin-resistant S. aureus combined. We assessed the severity of disease in animals over the course of our study using tools available to evaluate critically ill human patients including high-resolution computed tomography imaging of the lungs, arterial blood gas analyses, and bronchoalveolar lavage. Results Using an intra-bronchial route of inoculation we successfully induced severe pneumonia following influenza infection alone and following influenza and bacterial co-infection. Peak illness was observed at day 6 post-influenza infection, manifested by bilateral pulmonary infiltrates and hypoxemia. The timing of radiographic and physiologic manifestations of disease in our model closely match those observed in severe human influenza infection. Discussion This was the first nonhuman primate study of influenza and bacterial co-infection where high-resolution computed tomography scanning of the lungs was used to quantitatively assess pneumonia over the course of illness and where hypoxemia was correlated with pneumonia severity. With additional validation this model may serve as a pathway for regulatory approval of vaccines and therapeutics for the prevention and treatment of severe influenza pneumonia.

Background Patients experiencing large thermal injuries are susceptible to opportunistic infections that can delay recovery and lead to sepsis. Dendritic cells (DC) are important for the detection of pathogens and activation of the innate and acquired immune responses. DCs are significantly decreased in burn patients early after injury, and the development of sepsis is associated with persistent DC depletion. In a murine model of burn wound infection, the enhancement of DCs after injury by treatment with the DC growth factor Fms-like tyrosine kinase-3 ligand (FL) enhances neutrophil migration to infection, improves bacterial clearance, and increases survival in a DC-dependent manner. FL expands the production of both conventional DCs (cDC) and plasmacytoid DCs (pDC). It has been established that cDCs are required for some of the protective effects of FL after burn injury. This study was designed to determine the contribution of the pDC subset. Methods Mice were subjected to full-thickness scald burns under deep anesthesia and were provided analgesia. pDCs were depleted by injection of anti-plasmacytoid dendritic cell antigen-1 antibodies. Survival, bacterial clearance, and neutrophil responses in vivo and in vitro were measured. Results Depletion of preexisting pDCs, but not FL-expanded pDCs, abrogated the beneficial effects of FL on survival, bacterial clearance, and neutrophil migration in response to burn wound infection. This requisite role of pDCs for FL-mediated enhancement of neutrophil migratory capacity is not due to direct effects of pDCs on neutrophils. cDCs, but not pDCs, directly increased neutrophil migratory capacity after co-culture. Conclusions The protective effects of FL treatment after burn injury are mediated by both pDCs and cDCs. Pharmacological enhancement of both DC subtypes by FL is a potential therapeutic intervention to enhance immune responses to infection and improve outcome after burn injury.