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Anemia and renal failure are independent risk factors for perioperative stroke, prompting us to assess the combined impact of acute hemodilutional anemia and bilateral nephrectomy (2Nx) on microvascular brain P o 2 (PBr o 2) in a rat model. Changes in PBr o 2 (phosphorescence quenching) and cardiac output (CO, echocardiography) were measured in different groups of anesthetized Sprague-Dawley rats (1.5% isoflurane, n = 5–8/group) randomized to Sham 2Nx or 2Nx and subsequently exposed to acute hemodilutional anemia (50% estimated blood volume exchange with 6% hydroxyethyl starch) or time-based controls (no hemodilution). Outcomes were assessed by ANOVA with significance assigned at P < 0.05. At baseline, 2Nx rats demonstrated reduced CO (49.9 ± 9.4 vs. 66.3 ± 19.3 mL/min; P = 0.014) and PBr o 2 (21.1 ± 2.9 vs. 32.4 ± 3.1 mmHg; P < 0.001) relative to Sham 2Nx rats. Following hemodilution, 2Nx rats demonstrated a further decrease in PBr o 2 (15.0 ± 6.3 mmHg, P = 0.022). Hemodiluted 2Nx rats did not demonstrate a comparable increase in CO after hemodilution compared with Sham 2Nx (74.8 ± 22.4 vs. 108.9 ± 18.8 mL/min, P = 0.003) that likely contributed to the observed reduction in PBr o 2. This impaired CO response was associated with reduced fractional shortening (33 ± 9 vs. 51 ± 5%) and increased left ventricular end-systolic volume (156 ± 51 vs. 72 ± 15 µL, P < 0.001) suggestive of systolic dysfunction. By contrast, hemodiluted Sham 2Nx animals demonstrated a robust increase in CO and preserved PBr o 2. These data support the hypothesis that the kidney plays a central role in maintaining cerebral perfusion and initiating the adaptive increase in CO required to optimize PBr o 2 during acute anemia. NEW & NOTEWORTHY: This study has demonstrated that bilateral nephrectomy acutely impaired cardiac output (CO) and microvascular brain P o 2 (PBr o 2), at baseline. Following acute hemodilution, nephrectomy prevented the adaptive increase in CO associated with acute hemodilution leading to a further reduction in PBr o 2, accentuating the degree of cerebral tissue hypoxia. These data support a role for the kidney in maintaining PBr o 2 and initiating the increase in CO that optimized brain perfusion during acute anemia. [ABSTRACT FROM AUTHOR]

Background: Liver transplantation is the life‐saving treatment for many end‐stage pediatric liver diseases. The perioperative course, including surgical and anesthetic factors, have an important influence on the trajectory of this high‐risk population. Given the complexity and variability of the immediate postoperative course, there would be utility in identifying risk factors that allow prediction of adverse outcomes and intensive care unit trajectories. Aims: The aim of this study was to develop and validate a risk prediction model of prolonged intensive care unit length of stay in the pediatric liver transplant population. Methods: This is a retrospective analysis of consecutive pediatric isolated liver transplant recipients at a single institution between April 1, 2013 and April 30, 2020. All patients under the age of 18 years receiving a liver transplant were included in the study (n = 186). The primary outcome was intensive care unit length of stay greater than 7 days. Results: Recipient and donor characteristics were used to develop a multivariable logistic regression model. A total of 186 patients were included in the study. Using multivariable logistic regression, we found that age < 12 months (odds ratio 4.02, 95% confidence interval 1.20–13.51, p =.024), metabolic or cholestatic disease (odds ratio 2.66, 95% confidence interval 1.01–7.07, p =.049), 30‐day pretransplant hospital admission (odds ratio 8.59, 95% confidence interval 2.27–32.54, p =.002), intraoperative red blood cells transfusion >40 mL/kg (odds ratio 3.32, 95% confidence interval 1.12–9.81, p =.030), posttransplant return to the operating room (odds ratio 11.45, 95% confidence interval 3.04–43.16, p =.004), and major postoperative respiratory event (odds ratio 32.14, 95% confidence interval 3.00–343.90, p <.001) were associated with prolonged intensive care unit length of stay. The model demonstrates a good discriminative ability with an area under the receiver operative curve of 0.888 (95% confidence interval, 0.824–0.951). Conclusions: We develop and validate a model to predict prolonged intensive care unit length of stay in pediatric liver transplant patients using risk factors from all phases of the perioperative period. [ABSTRACT FROM AUTHOR]

Phagocytosis is a key component of the innate immune response and of the clearance of apoptotic bodies. Phagosome formation and subsequent maturation require extensive cytoskeletal rearrangement and precisely choreographed vesicular fusion and fission events. The objectives of this review are to highlight the functional importance of lipids in the phagocytic process, to discuss how pathogenic microorganisms can in some cases manipulate host lipid metabolism to either co-opt or disrupt phagosome maturation and promote their own survival, and to describe how defective phagosomal lipid metabolism can result in disease., Introduction Specialized cells of the immune system, such as macrophages and neutrophils, engage and actively engulf invading microorganisms into an intracellular vacuole by a process termed phagocytosis (1), (2). Inside [...]

Author(s): Cheryl L. Birmingham [1, 2]; Veronica Canadien [1]; Natalia A. Kaniuk [1]; Benjamin E. Steinberg [1, 3]; Darren E. Higgins [4]; John H. Brumell (corresponding author) [1, 2, 3] [...]

Ingestion of foreign particles by macrophages and neutrophils and the fate of the vacuole that contains the ingested material are generally monitored by optical microscopy. Invasion of host cells by pathogenic bacteria and their intracellular proliferation are similarly studied by microscopy or by plating assays. These labor-intensive and time-consuming methods limit the number of assays that can be performed. The effort required to test multiple reagents or conditions can be prohibitive. We describe high-throughput assays of phagocytosis and of phagosomal maturation. An automated fluorescence microscope-based platform and associated analysis software were used to study Fc[gamma]/receptor-mediated phagocytosis of IgG-opsonized particles by cultured murine macrophages. Phagosomal acidification was measured as an index of maturation. The same platform was similarly used to implement high-throughput assays of invasion of mammalian cells by pathogenic bacteria. The invasion of HeLa cells by Salmonella and the subsequent intracellular proliferation of the bacteria were measured rapidly and reliably in large populations of cells. These high-throughput methods are ideally suited for the efficient screening of chemical libraries to select potential drugs and of small interference RNA libraries to identify essential molecules involved in critical steps of the immune response. Salmonella; phosphatidylinositol 3-kinase; actin; vacuolar pH

T lymphocytes are increasingly implicated in pain signaling. A subset of T lymphocytes, termed TChAT, express the rate-limiting enzyme for acetylcholine (ACh) production, choline acetyltransferase (ChAT), and mediate numerous physiological functions. Given that cholinergic signaling has long been known to modulate pain processing and is the basis for several analgesics used clinically, we asked whether TChAT could be the intersection between T lymphocyte and cholinergic mediation of pain signaling. In this study, we used a mouse gene knockout strategy to ablate ChAT specifically from T lymphocytes and examined the development and expression of mechanical and thermal hypersensitivity in a spared nerve injury (SNI) mouse model of neuropathic pain. We found that mice with ChAT knockout in T cells (floxed Chat plus CD4-Cre recombinase) did not differ from control mice with intact ChAT (floxed Chat, but no Cre recombinase) in their expression of mechanical sensitivity before or after injury. Similarly, thermal sensitivity was unaffected after injury, with control mice expressing similar patterns of thermal preference to mice whose T cells do not express ChAT. Our experiments demonstrate that cholinergic signaling initiated by T lymphocytes neither dampens nor exacerbates the expression of mechanical or thermal sensitivity in neuropathic mice. Thus, while both cholinergic signaling and T lymphocytes have established roles in modulating pain phenotypes, it is not cholinergic signaling initiated by T lymphocytes that drive this. Our findings will help to narrow in on which aspects of T-cell modulation may prove useful as therapies. [ABSTRACT FROM AUTHOR]

Inflammasomes are multiprotein complexes tasked with sensing endogenous or exogenous inflammatory signals and integrating this signal into a downstream response. Inflammasome activation has been implicated in a variety of pulmonary diseases, including pulmonary hypertension, bacterial pneumonia, COPD, and asthma. Of increasing interest is the contribution of inflammasome activation in the context of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Inflammasome activation in both the lung parenchyma and resident immune cells generates intereukin-1β (IL-1β) and IL-18, both of which drive the cascade of lung inflammation forward. Blockade of these responses has been shown to be beneficial in animal models and is a focus of translational research in the field. In this review, we will discuss the assembly and regulation of inflammasomes during lung inflammation, highlighting therapeutically viable effector steps. We will examine the importance of IL-1β and IL-18, two key products of inflammasome activation, in ALI, as well as the contribution of the pulmonary endothelial cell to this process. Finally, we will explore translational research moving toward anti-inflammasome therapies for ALI/ARDS and speculate toward future directions for the field. [ABSTRACT FROM AUTHOR]

Background: The COVID-19 pandemic has yielded an unprecedented quantity of new publications, contributing to an overwhelming quantity of information and leading to the rapid dissemination of less stringently validated information. Yet, a formal analysis of how the medical literature has changed during the pandemic is lacking. In this analysis, we aimed to quantify how scientific publications changed at the outset of the COVID-19 pandemic. Methods: We performed a cross-sectional bibliometric study of published studies in four high-impact medical journals to identify differences in the characteristics of COVID-19 related publications compared to non-pandemic studies. Original investigations related to SARS-CoV-2 and COVID-19 published in March and April 2020 were identified and compared to non-COVID-19 research publications over the same two-month period in 2019 and 2020. Extracted data included publication characteristics, study characteristics, author characteristics, and impact metrics. Our primary measure was principal component analysis (PCA) of publication characteristics and impact metrics across groups. Results: We identified 402 publications that met inclusion criteria: 76 were related to COVID-19; 154 and 172 were non-COVID publications over the same period in 2020 and 2019, respectively. PCA utilizing the collected bibliometric data revealed segregation of the COVID-19 literature subset from both groups of non-COVID literature (2019 and 2020). COVID-19 publications were more likely to describe prospective observational (31.6%) or case series (41.8%) studies without industry funding as compared with non-COVID articles, which were represented primarily by randomized controlled trials (32.5% and 36.6% in the non-COVID literature from 2020 and 2019, respectively). Conclusions: In this cross-sectional study of publications in four general medical journals, COVID-related articles were significantly different from non-COVID articles based on article characteristics and impact metrics. COVID-related studies were generally shorter articles reporting observational studies with less literature cited and fewer study sites, suggestive of more limited scientific support. They nevertheless had much higher dissemination. [ABSTRACT FROM AUTHOR]

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Blood pressure regulation is known to be maintained by a neuro-endocrine circuit, but whether immune cells contribute to blood pressure homeostasis has not been determined. We previously showed that CD4+ T lymphocytes that express choline acetyltransferase (ChAT), which catalyzes the synthesis of the vasorelaxant acetylcholine, relay neural signals. Here we show that these CD4+CD44hiCD62Llo T helper cells by gene expression are a distinct T-cell population defined by ChAT (CD4 TChAT). Mice lacking ChAT expression in CD4+ cells have elevated arterial blood pressure, compared to littermate controls. Jurkat T cells overexpressing ChAT (JTChAT) decreased blood pressure when infused into mice. Co-incubation of JTChAT and endothelial cells increased endothelial cell levels of phosphorylated endothelial nitric oxide synthase, and of nitrates and nitrites in conditioned media, indicating increased release of the potent vasorelaxant nitric oxide. The isolation and characterization of CD4 TChAT cells will enable analysis of the role of these cells in hypotension and hypertension, and may suggest novel therapeutic strategies by targeting cell-mediated vasorelaxation. [ABSTRACT FROM AUTHOR]

Background: Physicians have traditionally been at the forefront of medical research, bringing clinical questions to the laboratory and returning with ideas for treatment. However, we have anecdotally observed a decline in the popularity of basic science research among trainees. We hypothesized that fewer resident physicians have been pursuing basic science research training over time. Methods: We examined records from residents in the Surgeon–Scientist and Clinician–Investigator programs at the University of Toronto (1987–2016). Research by residents was categorized independently by 2 raters as basic science, clinical epidemiology or education-related based on the title of the project, the name of the supervisor and Pubmed searches. The study population was divided into quintiles of time, and the proportion pursuing basic science training in each quintile was calculated. Results: Agreement between the raters was 100%; the categorization of the research topic remained unclear in 9 cases. The proportion of trainees pursuing basic science training dropped by 60% from 1987 to 2016 (p = 0.005). Interpretation: Significantly fewer residents in the Surgeon–Scientist and Clinician–Investigator Programs at the University of Toronto are pursuing training in the basic sciences as compared with previous years. [ABSTRACT FROM AUTHOR]

Inflammation and immunity are regulated by neural reflexes. Recent basic science research has demonstrated that a neural reflex, termed the inflammatory reflex, modulates systemic and regional inflammation in a multiplicity of clinical conditions encountered in perioperative medicine and critical care. In this review, the authors describe the anatomic and physiologic basis of the inflammatory reflex and review the evidence implicating this pathway in the modulation of sepsis, ventilator-induced lung injury, postoperative cognitive dysfunction, myocardial ischemia-reperfusion injury, and traumatic hemorrhage. The authors conclude with a discussion of how these new insights might spawn novel therapeutic strategies for the treatment of inflammatory diseases in the context of perioperative and critical care medicine. [ABSTRACT FROM AUTHOR]

Explosive growth in our understanding of genomics and molecular biology have fueled calls for the pursuit of personalized medicine, the notion of harnessing biologic variability to provide patient-specific care. This vision will necessitate a deep understanding of the underlying pathophysiology in each patient. Medical journals play a pivotal role in the education of trainees and clinicians, yet we suspected that the amount of basic science in the top medical journals has been in decline. We conducted an automated search strategy in PubMed to identify basic science articles and calculated the proportion of articles dealing with basic science in the highest impact journals for 8 different medical specialties from 1994 to 2013. We observed a steep decline (40-60%) in such articles over time in almost all of the journals examined. This rapid decline in basic science from medical journals is likely to affect practitioners' understanding of and interest in the basic mechanisms of disease and therapy. In this Life Sciences Forum, we discuss why this decline may be occurring and what it means for the future of science and medicine. [ABSTRACT FROM AUTHOR]

R3460 --> 555.16 --> Hypothesis: We hypothesize that renal sensing of hypoxia is essential for activation of cardiovascular responses needed to maintain cerebral oxygen delivery during acute anemia and that bilateral nephrectomy will negatively affect brain oxygen delivery during acute hemodilutional anemia. Methods: All experiments were approved by the Animal Care and Use Committee at St. Michael's Hospital and conducted in accordance with ARRIVE‐2 guidelines. Sham or bilateral nephrectomy (n=5) were performed on isoflurane anesthetized Sprague‐Dawley rats prior to performing acute hemodilution (50% estimated blood volume) 1:1 with 6% hydroxyethyl starch. Heart rate (HR), mean arterial pressure (MAP) and rectal temperature were monitored continuously. Arterial blood gases and cooximetry samples were measured before and after hemodilution. Continuous measurements of brain microvascular oxygen tension (PbrO2) were measured via a phosphorescence quenching method using an intravascular oxygen probe, Oxyphor PdG4, and an OxyLED phosphorometer. Data were tested for normality and analysis performed by two‐way ANOVA with p<0.05 taken to be significant. Results: Bilateral nephrectomy resulted in a reduction in baseline HR (p<0.004). Hemodilution resulted in a comparable reduction in hemoglobin concentration in both groups (Sham: 5.1±0.3 g/dL; Nephrectomy: 4.2±0.4 g/dL) with no differences in arterial PaO2 or PaCO2. After hemodilution, HR increased significantly in both groups (p<0.05). MAP tended to be lower in the bilateral nephrectomy group and decreased transiently in both groups following hemodilution (p<0.01). Brain microvascular pO2 (PbrO2)was not significantly different between nephrectomy and sham at baseline. Post‐hemodilution the PbrO2 was significantly lower in nephrectomised rats (11.5±5.1 mmHg) compared to sham operated rats (25.4±11.3 mmHg) (p=0.031). Conclusions: We observed that bilateral nephrectomy resulted in changes in baseline physiology, including a lower HR. Hemodilution resulted in a comparable reduction in hemoglobin concentration, without difference in PaO2 or PaCO2. MAP decreased more profoundly immediately after hemodilution in nephrectomised animals, and recovered with time. Nephrectomy resulted in a significant drop in brain microvascular pO2 (PbrO2) immediately after hemodilution. This was not observed in sham controls. These data suggest that the kidney serves an important sensing role during acute anemia which supports cardiovascular responses and preserves the adequacy of brain tissue oxygen delivery. Hypoxia sensing by the kidney, and other integrative systemic mechanisms, may be central to these findings. [ABSTRACT FROM AUTHOR]

The article focuses on the conflicting literature surrounding the identity of the putative lysosomal chloride counter ion pathway. It indicates that the discrepant conclusions within the literature can be accounted for by differences in the methodologies used to assay organellar potential of hydrogen (pH). Furthermore, it compares the techniques used to measure organellar pH, highlighting their respective strengths and limitations.

Autophagy plays an important role in immunity to microbial pathogens. The autophagy system can target bacteria in phagosomes. promoting phagosome maturation and preventing pathogen escape into the cytosol. Recently, Toll-like receptor (TLR) signaling from phagosomes was found to initiate their targeting by the autophagy system, but the mechanism by which TLR signaling activates autophagy is unclear. Here we show that autophagy targeting of phagosomes is not exclusive to those containing TLR ligands. Engagement of either TLRs or the Fcy receptors (FcγRs) during phagocytosis induced recruitment of the autophagy protein LC3 to phagosomes with similar kinetics. Both receptors are known to activate the NOX2 NADPH oxidase, which plays a central role in microbial killing by phagocytes through the generation of reactive oxygen species (ROS). We found that NOX2generated ROS are necessary for LC3 recruitment to phagosomes. Antibacterial autophagy in human epithelial cells, which do not express NOX2, was also dependent on ROS generation. These data reveal a coupling of oxidative and nonoxidative killing activities of the NOX2 NADPH oxidase in phagocytes through autophagy. Furthermore, our results suggest a general role for members of the NOX family in regulating autophagy. [ABSTRACT FROM AUTHOR]

Here we have identified 'triple D' (3d), a recessive N-ethyl-N-nitrosourea-induced mutation and phenotype in which no signaling occurs via the intracellular Toll-like receptors 3, 7 and 9 (sensors for double-stranded RNA, single-stranded RNA and unmethylated DNA, respectively). The 3d mutation also prevented cross-presentation and diminished major histocompatibility complex class II presentation of exogenous antigen; it also caused hypersusceptibility to infection by mouse cytomegalovirus and other microbes. By positional identification, we found 3d to be a missense allele of Unc93b1, which encodes the 12-membrane-spanning protein UNC-93B, a highly conserved molecule found in the endoplasmic reticulum with multiple paralogs in mammals. Innate responses to nucleic acids and exogenous antigen presentation, which both initiate in endosomes, thus seem to depend on an endoplasmic reticulum–resident protein, which suggests communication between these organellar systems. [ABSTRACT FROM AUTHOR]

Chronic pain has been widely recognized as a major public health problem that impacts multiple aspects of patient quality of life. Unfortunately, chronic pain is often resistant to conventional analgesics, which are further limited by their various side effects. New therapeutic strategies and targets are needed to better serve the millions of people suffering from this devastating disease. To this end, recent clinical and preclinical studies have implicated the epidermal growth factor receptor signaling pathway in chronic pain states. EGFR is one of four members of the ErbB family of receptor tyrosine kinases that have key roles in development and the progression of many cancers. EGFR functions by activating many intracellular signaling pathways following binding of various ligands to the receptor. Several of these signaling pathways, such as phosphatidylinositol 3-kinase, are known mediators of pain. EGFR inhibitors are known for their use as cancer therapeutics but given recent evidence in pilot clinical and preclinical investigations, may have clinical use for treating chronic pain. Here, we review the clinical and preclinical evidence implicating EGFR in pathological pain states and provide an overview of EGFR signaling highlighting how EGFR and its ligands drive pain hypersensitivity and interact with important pain pathways such as the opioid system. [ABSTRACT FROM AUTHOR]

ClC-4 is closely related to ClC-5, a member of the ClC family of transporters and channels. Unlike ClC-5, for which a role in the regulation of endosomal function was well established, the cellular function of ClC-4 was uncertain. In the present study, we tested for a specific role for ClC-4 in recycling endosomes by comparing transferrin (Tfn) receptor function in primary cell lines generated from ClC-4-null mice and their wild-type siblings. We found that endosomal pH is relatively alkaline and receptor-mediated uptake of Tfn is reduced in ClC-4-null fibroblasts. Surprisingly, this reduction in Tfn uptake occurs, despite a minor increase in the total surface expression of the Tfn receptor in ClC-4-null fibroblasts. As impaired Tfn uptake by ClC-4-null fibroblasts could be rescued to wild-type levels by addition of the iron chelator: desoxiferramine, the primary defect in these cells is related to the failure of iron to dissociate from Tfn, a pH-dependent event in endosomes that precedes the dissociation of Tfn from its receptor at the cell surface. Interestingly, ClC-4 depletion had no effect on epidermal growth factor receptor (EGFR) trafficking to lysosomes for degradation pointing to its specific role in recycling endosomes. These observations provide direct evidence supporting an essential role for ClC-4 in the modulation of Tfn receptor accessibility at the cell surface through its role in endosomal acidification. [ABSTRACT FROM AUTHOR]

Summary: Autophagy is known to be important in presentation of cytosolic antigens on MHC class II (MHC II). However, the role of autophagic process in antigen presentation in vivo is unclear. Mice with dendritic cell (DC)-conditional deletion in Atg5, a key autophagy gene, showed impaired CD4+ T cell priming after herpes simplex virus infection and succumbed to rapid disease. The most pronounced defect of Atg5 −/− DCs was the processing and presentation of phagocytosed antigens containing Toll-like receptor stimuli for MHC class II. In contrast, cross-presentation of peptides on MHC I was intact in the absence of Atg5. Although induction of metabolic autophagy did not enhance MHC II presentation, autophagic machinery was required for optimal phagosome-to-lysosome fusion and subsequent processing of antigen for MHC II loading. Thus, our study revealed that DCs utilize autophagic machinery to optimally process and present extracellular microbial antigens for MHC II presentation. [Copyright &y& Elsevier]

The ongoing metabolic and microbicidal pathways that support and protect cellular life generate potentially damaging reactive oxygen species (ROS). To counteract damage, cells express peroxidases, which are antioxidant enzymes that catalyze the reduction of oxidized biomolecules. Glutathione peroxidase 4 (GPX4) is the major hydroperoxidase specifically responsible for reducing lipid peroxides; this homeostatic mechanism is essential, and its inhibition causes a unique type of lytic cell death, ferroptosis. The mechanism(s) that lead to cell lysis in ferroptosis, however, are unclear. We report that the lipid peroxides formed during ferroptosis accumulate preferentially at the plasma membrane. Oxidation of surface membrane lipids increased tension on the plasma membrane and led to the activation of Piezo1 and TRP channels. Oxidized membranes thus became permeable to cations, ultimately leading to the gain of cellular Na+ and Ca2+ concomitant with loss of K+. These effects were reduced by deletion of Piezo1 and completely inhibited by blocking cation channel conductance with ruthenium red or 2-aminoethoxydiphenyl borate (2-APB). We also found that the oxidation of lipids depressed the activity of the Na+/K+-ATPase, exacerbating the dissipation of monovalent cation gradients. Preventing the changes in cation content attenuated ferroptosis. Altogether, our study establishes that increased membrane permeability to cations is a critical step in the execution of ferroptosis and identifies Piezo1, TRP channels, and the Na+/K+-ATPase as targets/effectors of this type of cell death. [Display omitted] • Lipid peroxidation increases plasma membrane tension and opens Piezo1 • Piezo1 and TRP channels cooperatively promote ferroptosis by facilitating cation flux • Collapse of transmembrane cation gradients is magnified by Na+/K+-ATPase inhibition • Blocking sodium influx and/or potassium efflux suppresses ferroptosis Hirata et al. report that peroxidation of lipids increases plasma membrane tension and leads to the activation of mechanosensitive cation channels including Piezo1. Gating of these channels induces loss of K+ and gain of Na+ that promote ferroptosis. These findings demonstrate key roles of mechanosensing channels in the execution of ferroptosis. [ABSTRACT FROM AUTHOR]

It is commonly assumed that all phagosomes have identical molecular composition. This assumption has remained largely unchallenged due to a paucity of methods to distinguish individual phagosomes. We devised an assay that extends the utility of nitro blue tetrazolium for detection and quantification of NAPDH oxidase (NOX) activity in individual phagosomes. Implementation of this assay revealed that in murine macrophages there is heterogeneity in the ability of individual phagosomes to generate superoxide, both between and within cells. To elucidate the molecular basis of the variability in NOX activation, we employed genetically encoded fluorescent biosensors to evaluate the uniformity in the distribution of phospholipid mediators of the oxidative response. Despite variability in superoxide generation, the distribution of phosphatidylinositol 3,4,5-trisphosphate, phosphatidylinositol 3-phosphate, and phosphatidic acid was nearly identical in all phagosomes. In contrast, diacylglycerol (DAG) was not generated uniformly across the phagosomal population, varying in a manner that directly mirrored superoxide production. Modulation of DAG levels suggested that NOX activation is precluded when phagosomes fail to reach a critical DAG concentration. In particular, forced expression of diacylglycerol kinase abrogated DAG accumulation at the phagosome, leading to impaired respiratory burst. Conversely, pharmacological inhibition of DAG kinases or expression of an inactive diacylglycerol kinase mutant increased the proportion of DAG-positive phagosomes, concomitantly potentiating phagosomal NOX activity. Our data suggest that diacylglycerol kinases limit the extent of NADPH oxidase activation, curtailing the production of potentially harmful reactive oxygen species. The resulting heterogeneity in phagosome responsiveness could enable the survival of a fraction of invading microorganisms. [ABSTRACT FROM AUTHOR]