Cell-free hemoglobin triggers macrophage cytokine production via TLR4 and MyD88.

Cell-free hemoglobin (CFH) is elevated in the airspace of patients with acute respiratory distress syndrome (ARDS) and is sufficient to cause acute lung injury in a murine model. However, the pathways through which CFH causes lung injury are not well understood. Toll-like receptor 4 (TLR4) is a mediator of inflammation after detection of damage- and pathogen-associated molecular patterns. We hypothesized that TLR4 signaling mediates the proinflammatory effects of CFH in the airspace. After intratracheal CFH, BALBc mice deficient in TLR4 had reduced inflammatory cell influx into the airspace [bronchoalveolar lavage (BAL) cell counts, median TLR4 knockout (KO): 0.8 × 104/mL [IQR 0.4-1.2 × 104/mL], wild-type (WT): 3.0 × 104/mL [2.2-4.0 × 104/mL], P < 0.001] and attenuated lung permeability (BAL protein, TLR4KO: 289 µg/mL [236-320], WT: 488 µg/mL [422-536], P < 0.001). These mice also had attenuated production of interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α in the airspace. C57Bl/6 mice lacking TLR4 on myeloid cells only (LysM.Cre+/-TLR4fl/fl) had reduced cytokine production in the airspace after CFH, without attenuation of lung permeability. In vitro studies confirm that WT primary murine alveolar macrophages exposed to CFH (0.01-1 mg/mL) had dose-dependent increases in IL-6, IL-1 ß, CXC motif chemokine ligand 1 (CXCL-1), TNF-α, and IL-10 (P < 0.001). Murine MH-S alveolar-like macrophages show TLR4-dependent expression of IL-1ß, IL-6, and CXCL-1 in response to CFH. Primary alveolar macrophages from mice lacking TLR4 adaptor proteins myeloid differentiation primary response 88 (MyD88) or TIR-domain-containing adapter-inducing interferon-ß (TRIF) revealed that MyD88KO macrophages had 71-96% reduction in CFH-dependent proinflammatory cytokine production (P < 0.001), whereas macrophages from TRIFKO mice had variable changes in cytokine responses. These data demonstrate that myeloid TLR4 signaling through MyD88 is a key regulator of airspace inflammation in response to CFH.NEW & NOTEWORTHY Cell-free hemoglobin (CFH) is elevated in the airspace of most patients with acute respiratory distress syndrome and causes severe inflammation. Here, we identify that CFH contributes to macrophage-induced cytokine production via Toll-like receptor 4 (TLR4) and myeloid differentiation primary response 88 (MyD88) signaling. These data increase our knowledge of the mechanisms through which CFH contributes to lung injury and may inform development of targeted therapeutics to attenuate inflammation.

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.

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.

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.

Fms-like tyrosine kinase-3 ligand increases resistance to burn wound infection through effects on plasmacytoid dendritic cells.

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.

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.

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.

The role of CXCL10 in the pathogenesis of experimental septic shock.

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.

TGFß macrophage reprogramming: a new dimension of macrophage plasticity.

The August 2023 article in Science Signaling, "TGF-ß uncouples glycolysis and inflammation in macrophages and controls survival during sepsis," challenges the traditional M1/M2 macrophage classification by investigating the impact of transforming growth factor ß on macrophage metabolism and function. Despite its conventional anti-inflammatory role, transforming growth factor ß-treated macrophages exhibit a distinct phenotype marked by heightened glycolysis, suppressed proinflammatory cytokines, and increased coagulation factor expression. The study identifies phosphofructokinase, liver type as a crucial glycolytic enzyme regulated by transforming growth factor ß via the mTOR-c-MYC pathway. Epigenetic changes induced by transforming growth factor ß, such as increased Smad3 activation and reduced proinflammatory transcription factor motif enrichment, contribute to the anti-inflammatory profile. The research extends its implications to sepsis, revealing the role of transforming growth factor ß in exacerbating coagulation and reducing survival in mouse models. This effect involves upregulation of coagulation factor F13A1, dependent on phosphofructokinase, liver type activity and glycolysis in macrophages. Connections to COVID-19 pathology are drawn, as transforming growth factor ß-treated macrophages and SARS-CoV-2 E protein-exposed cells display similar metabolic profiles. Bioinformatic analysis of COVID-19 patient data suggests correlations between myeloid expression of TGFßR1, PFKL, and F13A1 with disease severity. The study challenges the M1/M2 classification, emphasizing the complexity of macrophage responses influenced by transforming growth factor ß, proposing transforming growth factor ß as a potential therapeutic target for conditions like sepsis and COVID-19 where dysregulated coagulation is significant. Overall, the research provides valuable insights into transforming growth factor ß-mediated immunometabolic regulation, paving the way for future investigations and potential therapeutic interventions.

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.

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.

Advantages and Disadvantages of Using Small and Large Animals in Burn Research: Proceedings of the 2021 Research Special Interest Group.

Multiple animal species and approaches have been used for modeling different aspects of burn care, with some strategies considered more appropriate or translatable than others. On April 15, 2021, the Research Special Interest Group of the American Burn Association held a virtual session as part of the agenda for the annual meeting. The session was set up as a pro/con debate on the use of small versus large animals for application to four important aspects of burn pathophysiology: burn healing/conversion, scarring, inhalation injury, and sepsis. For each of these topics, two experienced investigators (one each for small and large animal models) described the advantages and disadvantages of using these preclinical models. The use of swine as a large animal model was a common theme due to anatomic similarities with human skin. The exception to this was a well-defined ovine model of inhalation injury; both of these species have larger airways which allow for incorporation of clinical tools such as bronchoscopes. However, these models are expensive and demanding from labor and resource standpoints. Various strategies have been implemented to make the more inexpensive rodent models appropriate for answering specific questions of interest in burns. Moreover, modeling burn-sepsis in large animals has proven difficult. It was agreed that the use of both small and large animal models has merit for answering basic questions about the responses to burn injury. Expert opinion and the ensuing lively conversations are summarized herein, which we hope will help inform experimental design of future research.

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.