Innate Immune Memory and the Host Response to Infection.

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.

Pretreatment with a novel Toll-like receptor 4 agonist attenuates renal ischemia-reperfusion injury.

Acute kidney injury (AKI) is common in surgical and critically ill patients. This study examined whether pretreatment with a novel Toll-like receptor 4 agonist attenuated ischemia-reperfusion injury (IRI)-induced AKI (IRI-AKI). We performed a blinded, randomized-controlled study in mice pretreated with 3-deacyl 6-acyl phosphorylated hexaacyl disaccharide (PHAD), a synthetic Toll-like receptor 4 agonist. Two cohorts of male BALB/c mice received intravenous vehicle or PHAD (2, 20, or 200 µg) at 48 and 24 h before unilateral renal pedicle clamping and simultaneous contralateral nephrectomy. A separate cohort of mice received intravenous vehicle or 200 µg PHAD followed by bilateral IRI-AKI. Mice were monitored for evidence of kidney injury for 3 days postreperfusion. Kidney function was assessed by serum blood urea nitrogen and creatinine measurements. Kidney tubular injury was assessed by semiquantitative analysis of tubular morphology on periodic acid-Schiff (PAS)-stained kidney sections and by kidney mRNA quantification of injury [neutrophil gelatinase-associated lipocalin (Ngal), kidney injury molecule-1 (Kim-1), and heme oxygenase-1 (Ho-1)] and inflammation [interleukin-6 (IL-6), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (Tnf-α)] using quantitative RT-PCR. Immunohistochemistry was used to quantify proximal tubular cell injury and renal macrophages by quantifying the areas stained with Kim-1 and F4/80 antibodies, respectively, and TUNEL staining to detect the apoptotic nuclei. PHAD pretreatment yielded dose-dependent kidney function preservation after unilateral IRI-AKI. Histological injury, apoptosis, Kim-1 staining, and Ngal mRNA were lower in PHAD-treated mice and IL-1ß mRNA was higher in PHAD-treated mice. Similar pretreatment protection was noted with 200 mg PHAD after bilateral IRI-AKI, with significantly reduced Kim-1 immunostaining in the outer medulla of mice treated with PHAD after bilateral IRI-AKI. In conclusion, PHAD pretreatment leads to dose-dependent protection from renal injury after unilateral and bilateral IRI-AKI in mice.NEW & NOTEWORTHY 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.

ß-Glucan Induces Distinct and Protective Innate Immune Memory in Differentiated Macrophages.

Bacterial infections are a common and deadly threat to vulnerable patients. Alternative strategies to fight infection are needed. ß-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 ß-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 ß-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. ß-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 ß-glucan receptors Dectin-1 and TLR-2. These findings provide evidence that ß-glucan induces enhanced protection from infection by driving trained immunity in macrophages.

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.

Phosphorylated Hexa-Acyl Disaccharides Augment Host Resistance Against Common Nosocomial Pathogens.

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.

Immunobiology and application of toll-like receptor 4 agonists to augment host resistance to infection.

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.

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.

The biology of natural killer cells during sepsis.

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

Targeting Immune Cell Checkpoints during Sepsis.

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

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.

Pharmacologic targeting of sphingosine-1-phosphate receptor 1 improves the renal microcirculation during sepsis in the mouse.

Microvascular failure is hallmark of sepsis in humans and is recognized as a strong predictor of mortality. In the mouse subjected to cecal ligation and puncture (CLP) to induce a clinically relevant sepsis, renal microvascular permeability increases and peritubular capillary perfusion declines rapidly in the kidney leading to acute kidney injury (AKI). Sphingosine-1-phosphate (S1P) is a key regulator of microvascular endothelial function. To investigate the role of S1P in the development of microvascular permeability and peritubular capillary hypoperfusion in the kidney during CLP-induced AKI, we used a pharmacologic approach and a clinically relevant delayed dosing paradigm. Evans blue dye was used to measure renal microvascular permeability and intravital video microscopy was used to quantitate renal cortical capillary perfusion. The S1P receptor 1 (S1P1) agonist SEW2871 [5-[4-phenyl-5-(trifluoromethyl)-2-thienyl]-3-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazole] and S1P2 antagonist JTE-013 [N-(2,6-dichloro-4-pyridinyl)-2-[1,3-dimethyl-4-(1-methylethyl)-1H-pyrazolo[3,4-b]pyridin-6-yl]-hydrazinecarboxamide] were administered at the time of CLP and produced a dose-dependent but partial reduction in renal microvascular permeability at 6 hours after CLP. However, neither agent improved capillary perfusion at 6 hours. With delayed administration at 6 hours after CLP, only SEW2871 reversed microvascular permeability when measured at 18 hours. Importantly, SEW2871 also restored capillary perfusion and improved renal function. These data suggest that S1P1 and S1P2 do not regulate the early decline in renal capillary perfusion. However, later in the course of sepsis, pharmacologic stimulation of S1P1, even when delaying therapy until after injury has occurred, improves capillary and renal function, suggesting this approach should be evaluated as an adjunct therapy during sepsis.

Inactivation of renal mitochondrial respiratory complexes and manganese superoxide dismutase during sepsis: mitochondria-targeted antioxidant mitigates injury.

Acute kidney injury (AKI) is a complication of sepsis and leads to a high mortality rate. Human and animal studies suggest that mitochondrial dysfunction plays an important role in sepsis-induced multi-organ failure; however, the specific mitochondrial targets damaged during sepsis remain elusive. We used a clinically relevant cecal ligation and puncture (CLP) murine model of sepsis and assessed renal mitochondrial function using high-resolution respirometry, renal microcirculation using intravital microscopy, and renal function. CLP caused a time-dependent decrease in mitochondrial complex I and II/III respiration and reduced ATP. By 4 h after CLP, activity of manganese superoxide dismutase (MnSOD) was decreased by 50% and inhibition was sustained through 36 h. These events were associated with increased mitochondrial superoxide generation. We then evaluated whether the mitochondria-targeted antioxidant Mito-TEMPO could reverse renal mitochondrial dysfunction and attenuate sepsis-induced AKI. Mito-TEMPO (10 mg/kg) given at 6 h post-CLP decreased mitochondrial superoxide levels, protected complex I and II/III respiration, and restored MnSOD activity by 18 h. Mito-TEMPO also improved renal microcirculation and glomerular filtration rate. Importantly, even delayed therapy with a single dose of Mito-TEMPO significantly increased 96-h survival rate from 40% in untreated septic mice to 80%. Thus, sepsis causes sustained inactivation of three mitochondrial targets that can lead to increased mitochondrial superoxide. Importantly, even delayed therapy with Mito-TEMPO alleviated kidney injury, suggesting that it may be a promising approach to treat septic AKI.

Bacteria- and fungus-derived PAMPs induce innate immune memory via similar functional, metabolic, and transcriptional adaptations.

Exposure to pathogen-associated molecular patterns (PAMPs) induces an augmented, broad-spectrum antimicrobial response to subsequent infection, a phenomenon termed innate immune memory. This study examined the effects of treatment with ß-glucan, a fungus-derived dectin-1 ligand, or monophosphoryl lipid A (MPLA), a bacteria-derived Toll-like receptor 4 ligand, on innate immune memory with a focus on identifying common cellular and molecular pathways activated by these diverse PAMPs. Treatment with either PAMP prepared the innate immune system to respond more robustly to Pseudomonas aeruginosa infection in vivo by facilitating mobilization of innate leukocytes into blood, recruitment of leukocytes to the site of infection, augmentation of microbial clearance, and attenuation of cytokine production. Examination of macrophages ex vivo showed amplification of metabolism, phagocytosis, and respiratory burst after treatment with either agent, although MPLA more robustly augmented these activities and more effectively facilitated killing of bacteria. Both agents activated gene expression pathways in macrophages that control inflammation, antimicrobial functions, and protein synthesis and suppressed pathways regulating cell division. ß-glucan treatment minimally altered macrophage differential gene expression in response to lipopolysaccharide (LPS) challenge, whereas MPLA attenuated the magnitude of the LPS-induced transcriptional response, especially cytokine gene expression. These results show that ß-glucan and MPLA similarly augment the innate response to infection in vivo. Yet, MPLA more potently induces alterations in macrophage metabolism, antimicrobial functions, gene transcription and the response to LPS.

Rolipram improves renal perfusion and function during sepsis in the mouse.

Microcirculatory dysfunction is correlated with increased mortality among septic patients and is believed to be a major contributor to the development of acute kidney injury (AKI). Rolipram, a selective phosphodiesterase 4 (PDE4) inhibitor, has been shown to reduce microvascular permeability and in the kidney, increase renal blood flow (RBF). This led us to investigate its potential to improve the renal microcirculation and preserve renal function during sepsis using a murine cecal ligation and puncture (CLP) model to induce sepsis. Rolipram, tested at doses of 0.3-10 mg/kg i.p., acutely restored capillary perfusion in a bell-shaped dose-response effect with 1 mg/kg being the lowest most efficacious dose. This dose also acutely increased RBF despite transiently decreasing mean arterial pressure. Rolipram also reduced renal microvascular permeability. It is noteworthy that delayed treatment with rolipram at 6 hours after CLP restored the renal microcirculation, reduced blood urea nitrogen and serum creatinine, and increased glomerular filtration rate at 18 hours. However, delayed treatment with rolipram did not reduce serum nitrate/nitrite levels, a marker of nitric oxide production, nor reactive nitrogen species generation in renal tubules. These data show that restoring the microcirculation with rolipram, even with delayed treatment, is enough to improve renal function during sepsis despite the generation of oxidants and suggest that PDE4 inhibitors should be evaluated further for their ability to treat septic-induced AKI.

INTRAPULMONARY TREATMENT WITH A NOVEL TLR4 AGONIST CONFERS PROTECTION AGAINST KLEBSIELLA PNEUMONIA.

ABSTRACT: 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.

MyD88-dependent signaling drives toll-like receptor-induced trained immunity in macrophages.

Immunocompromised populations are highly vulnerable to developing life-threatening infections. Strategies to protect patients with weak immune responses are urgently needed. Employing trained immunity, whereby innate leukocytes undergo reprogramming upon exposure to a microbial product and respond more robustly to subsequent infection, is a promising approach. Previously, we demonstrated that the TLR4 agonist monophosphoryl lipid A (MPLA) induces trained immunity and confers broad resistance to infection. TLR4 signals through both MyD88- and TRIF-dependent cascades, but the relative contribution of each pathway to induction of trained immunity is unknown. Here, we show that MPLA-induced resistance to Staphylococcus aureus infection is lost in MyD88-KO, but not TRIF-KO, mice. The MyD88-activating agonist CpG (TLR9 agonist), but not TRIF-activating Poly I:C (TLR3 agonist), protects against infection in a macrophage-dependent manner. MPLA- and CpG-induced augmentation of macrophage metabolism and antimicrobial functions is blunted in MyD88-, but not TRIF-KO, macrophages. Augmentation of antimicrobial functions occurs in parallel to metabolic reprogramming and is dependent, in part, on mTOR activation. Splenic macrophages from CpG-treated mice confirmed that TLR/MyD88-induced reprogramming occurs in vivo. TLR/MyD88-triggered metabolic and functional reprogramming was reproduced in human monocyte-derived macrophages. These data show that MyD88-dependent signaling is critical in TLR-mediated trained immunity.

A Murine Model of Full-Thickness Scald Burn Injury with Subsequent Wound and Systemic Bacterial Infection.

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.

TLR Agonists as Mediators of Trained Immunity: Mechanistic Insight and Immunotherapeutic Potential to Combat Infection.

Despite advances in critical care medicine, infection remains a significant problem that continues to be complicated with the challenge of antibiotic resistance. Immunocompromised patients are highly susceptible to development of severe infection which often progresses to the life-threatening condition of sepsis. Thus, immunotherapies aimed at boosting host immune defenses are highly attractive strategies to ward off infection and protect patients. Recently there has been mounting evidence that activation of the innate immune system can confer long-term functional reprogramming whereby innate leukocytes mount more robust responses upon secondary exposure to a pathogen for more efficient clearance and host protection, termed trained immunity. Toll-like receptor (TLR) agonists are a class of agents which have been shown to trigger the phenomenon of trained immunity through metabolic reprogramming and epigenetic modifications which drive profound augmentation of antimicrobial functions. Immunomodulatory TLR agonists are also highly beneficial as vaccine adjuvants. This review provides an overview on TLR signaling and our current understanding of TLR agonists which show promise as immunotherapeutic agents for combating infection. A brief discussion on our current understanding of underlying mechanisms is also provided. Although an evolving field, TLR agonists hold strong therapeutic potential as immunomodulators and merit further investigation for clinical translation.

Immune Checkpoints: Novel Therapeutic Targets to Attenuate Sepsis-Induced Immunosuppression.

Sepsis is a leading cause of death in intensive care units and survivors develop prolonged immunosuppression and a high incidence of recurrent infections. No definitive therapy exists to treat sepsis and physicians rely on supportive care including antibiotics, intravenous fluids, and vasopressors. With the rising incidence of antibiotic resistant microbes, it is becoming increasingly critical to discover novel therapeutics. Sepsis-induced leukocyte dysfunction and immunosuppression is recognized as an important contributor towards increased morbidity and mortality. Pre-clinical and clinical studies show that specific cell surface inhibitory immune checkpoint receptors and ligands including PD-1, PD-L1, CTLA4, BTLA, TIM3, OX40, and 2B4 play important roles in the pathophysiology of sepsis by mediating a fine balance between host immune competency and immunosuppression. Pre-clinical studies targeting the inhibitory effects of these immune checkpoints have demonstrated reversal of leukocyte dysfunction and improved host resistance of infection. Measurement of immune checkpoint expression on peripheral blood leukocytes may serve as a means of stratifying patients to direct individualized therapy. This review focuses on advances in our understanding of the role of immune checkpoints in the host response to infections, and the potential clinical application of therapeutics targeting the inhibitory immune checkpoint pathways for the management of septic patients.