Improving Neuromuscular Monitoring Through Education-Based Interventions and Studying Its Association With Adverse Postoperative Outcomes: A Retrospective Observational Study.

BACKGROUND: We assessed the association between education-based interventions, the frequency of train-of-four (TOF) monitoring, and postoperative outcomes. METHODS: We studied adults undergoing noncardiac surgery from February 1, 2020 through October 31, 2021. Our education-based interventions consisted of 3 phases. An interrupted time-series analysis, adjusting for patient- and procedure-related characteristics and secular trends over time, was used to assess the associations between education-based interventions and the frequency of TOF monitoring, postoperative pulmonary complications (PPCs), 90-day mortality, and sugammadex dosage. For each outcome and intervention phase, we tested whether the intervention at that phase was associated with an immediate change in the outcome or its trend (weekly rate of change) over time. In a sensitivity analysis, the association between education-based interventions and postoperative outcomes was adjusted for TOF monitoring. RESULTS: Of 19,422 cases, 11,636 (59.9%) had documented TOF monitoring. Monitoring frequency increased from 44.2% in the first week of preintervention stage to 83.4% in the final week of the postintervention phase. During the preintervention phase, the odds of TOF monitoring trended upward by 0.5% per week (odds ratio [OR], 1.005; 95% confidence interval [CI], 1.002-1.007). Phase 1 saw an immediate 54% increase (OR, 1.54; 95% CI, 1.33-1.79) in the odds, and the trend OR increased by 3% (OR, 1.03; 95% CI, 1.01-1.05) to 1.035, or 3.5% per week (joint Wald test, P < .001). Phase 2 was associated with a further immediate 29% increase (OR, 1.29; 95% CI, 1.02-1.64) but no significant association with trend (OR, 0.96; 95% CI, 0.93-1.01) of TOF monitoring (joint test, P = .04). Phase 3 and postintervention phase were not significantly associated with the frequency of TOF monitoring (joint test, P = .16 and P = .61). The study phases were not significantly associated with PPCs or sugammadex administration. The trend OR for 90-day mortality was larger by 24% (OR, 1.24; 95% CI, 1.06-1.45; joint test, P = .03) in phase 2 versus phase 1, from a weekly decrease of 8% to a weekly increase of 14%. However, this trend reversed again at the transition from phase 3 to the postintervention phase (OR, 0.82; 95% CI, 0.68-0.99; joint test, P = .05), from a 14% weekly increase to a 6.2% weekly decrease in the odds of 90-day mortality. In sensitivity analyses, adjusting for TOF monitoring, we found similar associations between study initiatives and postoperative outcomes. TOF monitoring was associated with lower odds of PPCs (OR, 0.69; 95% CI, 0.55-0.86) and 90-day mortality (OR, 0.79; 95% CI, 0.63-0.98), but not sugammadex dosing (mean difference, -0.02; 95% CI, -0.04 to 0.01). CONCLUSIONS: Our education-based interventions were associated with both TOF utilization and 90-day mortality but were not associated with either the odds of PPCs or sugammadex dosing. TOF monitoring was associated with reduced odds of PPCs and 90-day mortality.

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.

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.

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.

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.

Trained Immunity Enhances Human Monocyte Function in Aging and Sepsis.

Aging plays a critical role in the incidence and severity of infection, with age emerging as an independent predictor of mortality in sepsis. Trained immunity reprograms immunocytes to respond more rapidly and effectively to pathogens and serves as a potential approach to improve immune function in aging and/or sepsis. However, there is very little data on trained immunity in the aging immune system or in the presence of sepsis. We examined the impact of ß-glucan induced innate immune training on monocytes from aging healthy humans (>60 years old) as well as sepsis patients. We observed increased metabolic capacity, upregulated cytokine secretion, increased H3K27 acetylation, and upregulation of crucial intracellular signaling pathways in trained monocytes from healthy aging subjects. The response to trained immunity in healthy aging monocytes was equivalent to the response of monocytes from younger, i.e., 18 - 59 years, individuals. Additionally, we found that trained immunity induced a unique expression pattern of cell surface markers in monocytes that was consistent across age groups. Trained monocytes from sepsis patients also displayed enhanced metabolic capacity and increased cytokine production. These results indicate that immune training can be induced in aging monocytes as well as monocytes from critically ill sepsis patients.

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.

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.

Reversal of sepsis-induced T cell dysfunction: OX-40 to the rescue?

The study of Unsinger and colleagues provide important insights into OX40 mediated immunotherapy as a potential approach for the treatment of sepsis induced immune suppression.

The Metabolic Basis of Immune Dysfunction Following Sepsis and Trauma.

Critically ill, severely injured and high-risk surgical patients are vulnerable to secondary infections during hospitalization and after hospital discharge. Studies show that the mitochondrial function and oxidative metabolism of monocytes and macrophages are impaired during sepsis. Alternatively, treatment with microbe-derived ligands, such as monophosphoryl lipid A (MPLA), peptidoglycan, or ß-glucan, that interact with toll-like receptors and other pattern recognition receptors on leukocytes induces a state of innate immune memory that confers broad-spectrum resistance to infection with common hospital-acquired pathogens. Priming of macrophages with MPLA, CPG oligodeoxynucleotides (CpG ODN), or ß-glucan induces a macrophage metabolic phenotype characterized by mitochondrial biogenesis and increased oxidative metabolism in parallel with increased glycolysis, cell size and granularity, augmented phagocytosis, heightened respiratory burst functions, and more effective killing of microbes. The mitochondrion is a bioenergetic organelle that not only contributes to energy supply, biosynthesis, and cellular redox functions but serves as a platform for regulating innate immunological functions such as production of reactive oxygen species (ROS) and regulatory intermediates. This review will define current knowledge of leukocyte metabolic dysfunction during and after sepsis and trauma. We will further discuss therapeutic strategies that target leukocyte mitochondrial function and might have value in preventing or reversing sepsis- and trauma-induced immune dysfunction.

Monophosphoryl lipid A pretreatment suppresses sepsis- and LPS-induced proinflammatory cytokine production in the medullary thick ascending limb.

Sepsis is the leading cause of acute kidney injury in critically ill patients. Tumor necrosis factor-α (TNF-α) has been implicated in the pathogenesis of septic kidney injury; however, the sites and mechanisms of renal TNF-α production during sepsis remain to be defined. In the present study, we showed that TNF-α expression is increased in medullary thick ascending limbs (MTALs) of mice with sepsis induced by cecal ligation and puncture. Treatment with lipopolysaccharide (LPS) for 3 h in vitro also increased MTAL TNF-α production. Sepsis and LPS increased MTAL TNF-α expression through activation of the myeloid differentiation factor 88 (MyD88)-IL-1 receptor-associated kinase 1-ERK signaling pathway. Pretreatment with monophosphoryl lipid A (MPLA), a nontoxic immunomodulator that protects against bacterial infection, eliminated the sepsis- and LPS-induced increases in MTAL TNF-α production. The suppressive effect of MPLA on TNF-α was mediated through activation of a phosphatidylinositol 3-kinase-dependent pathway that inhibits MyD88-dependent ERK activation. This likely involves MPLA-phosphatidylinositol 3-kinase-mediated induction of Tollip, which negatively regulates the MyD88-ERK pathway by inhibiting activation of IL-1 receptor-associated kinase 1. These regulatory mechanisms are similar to those previously shown to mediate the effect of MPLA to prevent sepsis-induced inhibition of MTAL [Formula: see text] absorption. These results identify the MTAL as a site of local TNF-α production in the kidney during sepsis and identify molecular mechanisms that can be targeted to attenuate renal TNF-α expression. The ability of MPLA pretreatment to suppress MyD88-dependent ERK signaling in the MTAL during sepsis has the dual beneficial effects of protecting tubule transport functions and attenuating harmful proinflammatory responses.

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.

Monophosphoryl Lipid a Attenuates Multiorgan Dysfunction During Post-Burn Pseudomonas Aeruginosa Pneumonia in Sheep.

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 preclinical 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 h after burn, sheep were randomly allocated to receive: MPLA (2.5 µg/kg i.v., n = 6), or vehicle (i.v., n = 6). At 24 h after MPLA or vehicle treatment, Pseudomonas aeruginosa pneumonia was induced. Sheep were mechanically ventilated, fluid resuscitated and cardiopulmonary variables were monitored for 24 h 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 PaO2/FiO2 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.

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.

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.

Monophosphoryl lipid A induces protection against LPS in medullary thick ascending limb through induction of Tollip and negative regulation of IRAK-1.

LPS inhibits HCO3- absorption in the medullary thick ascending limb (MTAL) through a Toll-like receptor 4 (TLR4)-myeloid differentiation factor 88 (MyD88)-extracellular signal-regulated kinase (ERK) pathway that is upregulated by sepsis. Pretreatment with the nontoxic immunomodulator monophosphoryl lipid A (MPLA) prevents inhibition by LPS through activation of a TLR4-TIR-domain-containing adaptor-inducing interferon-ß (TRIF)-phosphatidylinositol 3-kinase (PI3K) pathway that prevents LPS-induced ERK activation. Here, we identified the molecular mechanisms that underlie the protective inhibitory interaction between the MPLA-PI3K and LPS-ERK pathways. Treatment of mouse MTALs with LPS in vitro increased phosphorylation of IL-1 receptor-associated kinase (IRAK)-1, a critical mediator of LPS signaling downstream of TLR4-MyD88. Activation of ERK by LPS was eliminated by a selective IRAK-1 inhibitor, establishing IRAK-1 as the upstream mediator of ERK activation. Pretreatment of MTALs with MPLA in vitro prevented LPS-induced IRAK-1 activation; this effect was dependent on PI3K. Treatment of MTALs with MPLA increased expression of Toll-interacting protein (Tollip), an inducible protein that negatively regulates LPS signaling by inhibiting IRAK-1. The MPLA-induced increase in Tollip protein level was prevented by PI3K inhibitors. In coimmunoprecipitation experiments, MPLA increased the amount of Tollip stably bound to IRAK-1, an interaction that inhibits IRAK-1 activation. These results support a mechanism whereby MPLA increases Tollip expression in the MTAL through a PI3K-dependent pathway. Tollip, in turn, inhibits LPS-induced TLR4 signaling by suppressing activation of IRAK-1, thereby preventing activation of ERK that inhibits HCO3- absorption. These studies show that MPLA induces reprogramming of MTAL cells that protects against LPS stimulation and identify IRAK-1 and Tollip as new therapeutic targets to prevent renal tubule dysfunction in response to infectious and inflammatory stimuli.