Enrichment of Bioactive Lipids in Urinary Extracellular Vesicles and Evidence of Apoptosis in Kidneys of Hypertensive Diabetic Cathepsin B Knockout Mice after Streptozotocin Treatment.

Cathepsin B (CtsB) is a ubiquitously expressed cysteine protease that plays important roles in health and disease. Urinary extracellular vesicles (uEVs) are released from cells associated with urinary organs. The antibiotic streptozotocin (STZ) is known to induce pancreatic islet beta cell destruction, diabetic nephropathy, and hypertension. We hypothesized that streptozotocin-induced diabetic kidney disease and hypertension result in the release of bioactive lipids from kidney cells that induce oxidative stress and renal cell death. Lipidomics was performed on uEVs isolated from CtsB knockout mice treated with or without STZ, and their kidneys were used to investigate changes in proteins associated with cell death. Lysophosphatidylethanolamine (LPE) (18:1), lysophosphatidylserine (LPS) (22:6), and lysophosphatidylglycerol (LPG) (22:5) were among the bioactive lipids enriched in uEVs from CtsB knockout mice treated with STZ compared to untreated CtsB mice (n = 3 uEV preparations per group). Anti-oxidant programming was activated in the kidneys of the CtsB knockout mice treated with STZ, as indicated by increased expression of glutathione peroxidase 4 (GPX4) and the cystine/glutamate antiporter SLC7A11 (XCT) (n = 4 mice per group), which was supported by a higher reactivity to 4-hydroxy-2-nonenal (4-HNE), a marker for oxidative stress (n = 3 mice per group). Apoptosis but not ferroptosis was the ongoing form of cell death in these kidneys as cleaved caspase-3 levels were significantly elevated in the STZ-treated CtsB knockout mice (n = 4 mice per group). There were no appreciable differences in the pro-ferroptosis enzyme acyl-CoA synthetase long-chain family member 4 (ACSL4) or the inflammatory marker CD93 in the kidneys (n = 3 mice per group), which further supports apoptosis as the prevalent mechanism of pathology. These data suggest that STZ treatment leads to oxidative stress, inducing apoptotic injury in the kidneys during the development of diabetic kidney disease and hypertension.

Functional consequence of Iron dyshomeostasis and ferroptosis in systemic lupus erythematosus and lupus nephritis.

Systemic lupus erythematosus (SLE) and its renal manifestation Lupus nephritis (LN) are characterized by a dysregulated immune system, autoantibodies, and injury to the renal parenchyma. Iron accumulation and ferroptosis in the immune effectors and renal tubules are recently identified pathological features in SLE and LN. Ferroptosis is an iron dependent non-apoptotic form of regulated cell death and ferroptosis inhibitors have improved disease outcomes in murine models of SLE, identifying it as a novel druggable target. In this review, we discuss novel mechanisms by which iron accumulation and ferroptosis perpetuate immune cell mediated pathology in SLE/LN. We highlight intra-renal dysregulation of iron metabolism and ferroptosis as an underlying pathogenic mechanism of renal tubular injury. The basic concepts of iron biology and ferroptosis are also discussed to expose the links between iron, cell metabolism and ferroptosis, that identify intracellular pro-ferroptotic enzymes and their protein conjugates as potential targets to improve SLE/LN outcomes.

Animal models of lupus nephritis: the past, present and a future outlook.

Lupus nephritis (LN) is the most severe end-organ pathology in Systemic Lupus Erythematosus (SLE). Research has enhanced our understanding of immune effectors and inflammatory pathways in LN. However, even with the best available therapy, the rate of complete remission for proliferative LN remains below 50%. A deeper understanding of the resistance or susceptibility of renal cells to injury during the progression of SLE is critical for identifying new targets and developing effective long-term therapies. The complex and heterogeneous nature of LN, combined with the limitations of clinical research, make it challenging to investigate the aetiology of this disease directly in patients. Hence, multiple murine models resembling SLE-driven nephritis are utilised to dissect LN's cellular and genetic mechanisms, identify therapeutic targets, and screen novel compounds. This review discusses commonly used spontaneous and inducible mouse models that have provided insights into pathogenic mechanisms and long-term maintenance therapies in LN.

Male kidney-specific BMAL1 knockout mice are protected from K+-deficient, high-salt diet-induced blood pressure increases.

The circadian clock protein basic helix-loop-helix aryl hydrocarbon receptor nuclear translocator-like protein 1 (BMAL1) is a transcription factor that impacts kidney function, including blood pressure (BP) control. Previously, we have shown that male, but not female, kidney-specific cadherin Cre-positive BMAL1 knockout (KS-BMAL1 KO) mice exhibit lower BP compared with littermate controls. The goal of this study was to determine the BP phenotype and immune response in male KS-BMAL1 KO mice in response to a low-K+ high-salt (LKHS) diet. BP, renal inflammatory markers, and immune cells were measured in male mice following an LKHS diet. Male KS-BMAL1 KO mice had lower BP following the LKHS diet compared with control mice, yet their circadian rhythm in pressure remained unchanged. Additionally, KS-BMAL1 KO mice exhibited lower levels of renal proinflammatory cytokines and immune cells following the LKHS diet compared with control mice. KS-BMAL1 KO mice were protected from the salt-sensitive hypertension observed in control mice and displayed an attenuated immune response following the LKHS diet. These data suggest that BMAL1 plays a role in driving the BP increase and proinflammatory environment that occurs in response to an LKHS diet.NEW & NOTEWORTHY We show here, for the first time, that kidney-specific BMAL1 knockout mice are protected from blood pressure (BP) increases and immune responses to a salt-sensitive diet. Other kidney-specific BMAL1 knockout models exhibit lower BP phenotypes under basal conditions. A salt-sensitive diet exacerbates this genotype-specific BP response, leading to fewer proinflammatory cytokines and immune cells in knockout mice. These data demonstrate the importance of distal segment BMAL1 in BP and immune responses to a salt-sensitive environment.

COVID-19-associated pulmonary aspergillosis in immunocompetent patients: a virtual patient cohort study.

The opportunistic fungus Aspergillus fumigatus infects the lungs of immunocompromised hosts, including patients undergoing chemotherapy or organ transplantation. More recently however, immunocompetent patients with severe SARS-CoV2 have been reported to be affected by COVID-19 Associated Pulmonary Aspergillosis (CAPA), in the absence of the conventional risk factors for invasive aspergillosis. This paper explores the hypothesis that contributing causes are the destruction of the lung epithelium permitting colonization by opportunistic pathogens. At the same time, the exhaustion of the immune system, characterized by cytokine storms, apoptosis, and depletion of leukocytes may hinder the response to A. fumigatus infection. The combination of these factors may explain the onset of invasive aspergillosis in immunocompetent patients. We used a previously published computational model of the innate immune response to infection with Aspergillus fumigatus. Variation of model parameters was used to create a virtual patient population. A simulation study of this virtual patient population to test potential causes for co-infection in immunocompetent patients. The two most important factors determining the likelihood of CAPA were the inherent virulence of the fungus and the effectiveness of the neutrophil population, as measured by granule half-life and ability to kill fungal cells. Varying these parameters across the virtual patient population generated a realistic distribution of CAPA phenotypes observed in the literature. Computational models are an effective tool for hypothesis generation. Varying model parameters can be used to create a virtual patient population for identifying candidate mechanisms for phenomena observed in actual patient populations.

Metformin Alleviates Diabetes-Associated Hypertension by Attenuating the Renal Epithelial Sodium Channel.

Diabetic nephropathy is the primary cause of morbidity in type 2 diabetes mellitus (T2DM) patients. New data indicate that hypertension, a common comorbidity in T2DM, can worsen outcomes of diabetic nephropathy. While metformin is a commonly prescribed drug for treating type 2 diabetes, its blood pressure regulating ability is not well documented. The aim of this study was to investigate the effect of metformin on normalizing blood pressure in salt-loaded hypertensive diabetic db/db mice. Sixteen-week-old male and female diabetic db/db mice were individually placed in metabolic cages and then randomized to a control vehicle (saline) or metformin treatment group. We evaluated the blood pressure reducing ability of metformin in salt-induced hypertension and progression of nephropathy in db/db mice. We observed that metformin- normalized systolic blood pressure in hypertensive diabetic mice. Mechanistically, metformin treatment reduced renal cathepsin B expression. Low cathepsin B expression was associated with reduced expression and activity of the epithelial sodium channel (ENaC), sodium retention, and thus control of hypertension. In addition, we identified that urinary extracellular vesicles (EVs) from the diabetic mice are enriched in cathepsin B. Compared to treatment with urinary EVs of vehicle-treated hypertensive diabetic mice, the amiloride-sensitive transepithelial current was significantly attenuated upon exposure of renal collecting duct cells to urinary EVs isolated from metformin-treated db/db mice or cathepsin B knockout mice. Collectively, our study identifies a novel blood pressure reducing role of metformin in diabetic nephropathy by regulating the cathepsin B-ENaC axis.

Kidney tubular epithelial cell ferroptosis links glomerular injury to tubulointerstitial pathology in lupus nephritis.

Ferroptosis is a druggable, iron-dependent form of cell death that is characterized by lipid peroxidation but has received little attention in lupus nephritis. Kidneys of lupus nephritis patients and mice showed increased lipid peroxidation mainly in the tubular segments and an increase in Acyl-CoA synthetase long-chain family member 4, a pro-ferroptosis enzyme. Nephritic mice had an attenuated expression of SLC7A11, a cystine importer, an impaired glutathione synthesis pathway, and low expression of glutathione peroxidase 4, a ferroptosis inhibitor. Lipidomics of nephritic kidneys confirmed ferroptosis. Using nephrotoxic serum, we induced immune complex glomerulonephritis in congenic mice and demonstrate that impaired iron sequestration within the proximal tubules exacerbates ferroptosis. Lupus nephritis patient serum rendered human proximal tubular cells susceptibility to ferroptosis which was inhibited by Liproxstatin-2, a novel ferroptosis inhibitor. Collectively, our findings identify intra-renal ferroptosis as a pathological feature and contributor to tubular injury in human and murine lupus nephritis.

COVID-19-associated pulmonary aspergillosis in immunocompetent patients: A virtual patient cohort study.

Purpose: The opportunistic fungus Aspergillus fumigatus infects the lungs of immunocompromised hosts, including patients undergoing chemotherapy or organ transplantation. More recently however, immunocompetent patients with severe SARS-CoV2 have been reported to be affected by COVID-19 Associated Pulmonary Aspergillosis (CAPA), in the absence of the conventional risk factors for invasive aspergillosis. This paper explores the hypothesis that contributing causes are the destruction of the lung epithelium permitting colonization by opportunistic pathogens. At the same time, the exhaustion of the immune system, characterized by cytokine storms, apoptosis, and depletion of leukocytes may hinder the response to A. fumigatus infection. The combination of these factors may explain the onset of invasive aspergillosis in immunocompetent patients. Methods: We used a previously published computational model of the innate immune response to infection with Aspergillus fumigatus . Variation of model parameters was used to create a virtual patient population. A simulation study of this virtual patient population to test potential causes for co-infection in immunocompetent patients. Results: The two most important factors determining the likelihood of CAPA were the inherent virulence of the fungus and the effectiveness of the neutrophil population, as measured by granule half-life and ability to kill fungal cells. Varying these parameters across the virtual patient population generated a realistic distribution of CAPA phenotypes observed in the literature. Conclusions: Computational models are an effective tool for hypothesis generation. Varying model parameters can be used to create a virtual patient population for identifying candidate mechanisms for phenomena observed in actual patient populations.

Correlation of alpha-1 antitrypsin levels and exosome associated neutrophil elastase endothelial injury in subjects with SARS-CoV2 infection.

BACKGROUND: Severe acute respiratory syndrome caused by a novel coronavirus 2 (SARS-CoV-2) has infected more than 18 million people worldwide. The activation of endothelial cells is a hallmark of signs of SARS-CoV-2 infection that includes altered integrity of vessel barrier and endothelial inflammation. OBJECTIVES: Pulmonary endothelial activation is suggested to be related to the profound neutrophil elastase (NE) activity, which is necessary for sterilization of phagocytosed bacterial pathogens. However, unopposed activity of NE increases alveolocapillary permeability and extracellular matrix degradation. The uncontrolled protease activity of NE during the inflammatory phase of lung diseases might be due to the resistance of exosome associated NE to inhibition by alpha-1 antitrypsin. METHOD: 31 subjects with a diagnosis of SARS-CoV2 infection were recruited in the disease group and samples from 30 voluntaries matched for age and sex were also collected for control. RESULTS: We measured the plasma levels of exosome-associated NE in SARS-CoV-2 patients which, were positively correlated with sign of endothelial damage in those patients as determined by plasma levels of LDH. Notably, we also found strong correlation with plasma levels of alpha-1 antitrypsin and exosome-associated NE in SARS-CoV-2 patients. Using macrovascular endothelial cells, we also observed that purified NE activity is inhibited by purified alpha-1 antitrypsin while, NE associated with exosomes are resistant to inhibition and show less sensitivity to alpha-1 antitrypsin inhibitory activity, in vitro. CONCLUSIONS: Our results point out the role of exosome-associated NE in exacerbation of endothelial injury in SARS-CoV-2 infection. We have demonstrated that exosome-associated NE could be served as a new potential therapeutic target of severe systemic manifestations of SARS-CoV-2 infection.

Computational Modeling of Macrophage Iron Sequestration during Host Defense against Aspergillus.

Iron is essential to the virulence of Aspergillus species, and restricting iron availability is a critical mechanism of antimicrobial host defense. Macrophages recruited to the site of infection are at the crux of this process, employing multiple intersecting mechanisms to orchestrate iron sequestration from pathogens. To gain an integrated understanding of how this is achieved in aspergillosis, we generated a transcriptomic time series of the response of human monocyte-derived macrophages to Aspergillus and used this and the available literature to construct a mechanistic computational model of iron handling of macrophages during this infection. We found an overwhelming macrophage response beginning 2 to 4 h after exposure to the fungus, which included upregulated transcription of iron import proteins transferrin receptor-1, divalent metal transporter-1, and ZIP family transporters, and downregulated transcription of the iron exporter ferroportin. The computational model, based on a discrete dynamical systems framework, consisted of 21 3-state nodes, and was validated with additional experimental data that were not used in model generation. The model accurately captures the steady state and the trajectories of most of the quantitatively measured nodes. In the experimental data, we surprisingly found that transferrin receptor-1 upregulation preceded the induction of inflammatory cytokines, a feature that deviated from model predictions. Model simulations suggested that direct induction of transferrin receptor-1 (TfR1) after fungal recognition, independent of the iron regulatory protein-labile iron pool (IRP-LIP) system, explains this finding. We anticipate that this model will contribute to a quantitative understanding of iron regulation as a fundamental host defense mechanism during aspergillosis. IMPORTANCE Invasive pulmonary aspergillosis is a major cause of death among immunosuppressed individuals despite the best available therapy. Depriving the pathogen of iron is an essential component of host defense in this infection, but the mechanisms by which the host achieves this are complex. To understand how recruited macrophages mediate iron deprivation during the infection, we developed and validated a mechanistic computational model that integrates the available information in the field. The insights provided by this approach can help in designing iron modulation therapies as anti-fungal treatments.

Metabolic regulation of follicular helper T cell differentiation in a mouse model of lupus.

Follicular helper T (TFH) cells are expanded in systemic lupus erythematosus (SLE), where they are required for production of high affinity autoantibodies. A better understanding of the mechanisms that regulate the differentiation of TFH cells is critical. Naïve T cells from lupus-prone B6.NZM2410.Sle1.Sle2.Sle3 (TC) mice showed an intrinsic higher capacity to differentiate into TFH cells. Metabolic reprogramming is a vital regulatory mechanism for T cell differentiation, but how metabolic pathways contribute to TFH cell expansion in SLE remains elusive. Here we show that glycolysis, mTOR signaling, FAO, and the activity of complex V of the electron transport chain support TFH lineage commitment. Blocking complex I uniquely decreased the expansion of TFH cells from lupus-prone mice, and inhibition of some pathways had a greater effect in lupus-prone than control TFH cells. However, blocking glutaminolysis, complex III and ADP/ATP translocase did not affect TFH cell expansion. Together, our results identified novel intrinsic metabolic requirements for TFH cell differentiation, and further defined the differential metabolic pathways that support the expansion of TFH cells in lupus-prone mice. Together, our data indicates the crucial but distinct roles for metabolic pathways in TFH cell differentiation and provide a comprehensive experimental basis for fully understanding the precise roles of distant metabolic signaling in regulating the TFH cell differentiation.

Labile iron accumulation augments T follicular helper cell differentiation.

T follicular helper (Tfh) cells are a subset of CD4+ T cells that are essential in the pathogenesis of systemic lupus erythematosus (SLE). Notably, iron is required for activated CD4+ T lymphocytes to sustain high proliferation and metabolism. In this issue of the JCI, Gao et al. showed that CD4+ T cells from patients with SLE accumulated iron, augmenting their differentiation into Tfh cells and correlating with disease activity. Using human cells and murine models, the authors demonstrated that miR-21 was overexpressed in lupus T cells and inhibited 3-hydroxybutyrate dehydrogenase-2 (BDH2). The subsequent loss of BDH2 drove labile iron to accumulate in the cytoplasm and promoted TET enzyme activity, BCL6 gene demethylation, and Tfh cell differentiation. This work identifies a role for iron in CD4+ T cell biology and the development of pathogenic effectors in SLE. We await future investigations that could determine whether modulating iron levels could regulate Tfh cells in human health and disease.

Multi-scale mechanistic modelling of the host defence in invasive aspergillosis reveals leucocyte activation and iron acquisition as drivers of infection outcome.

Aspergillus species are ubiquitous environmental moulds, with spores inhaled daily by most humans. Immunocompromised hosts can develop an invasive infection resulting in high mortality. There is, therefore, a pressing need for host-centric therapeutics for this infection. To address it, we created a multi-scale computational model of the infection, focused on its interaction with the innate immune system and iron, a critical nutrient for the pathogen. The model, parameterized using published data, was found to recapitulate a wide range of biological features and was experimentally validated in vivo. Conidial swelling was identified as critical in fungal strains with high growth, whereas the siderophore secretion rate seems to be an essential prerequisite for the establishment of the infection in low-growth strains. In immunocompetent hosts, high growth, high swelling probability and impaired leucocyte activation lead to a high conidial germination rate. Similarly, in neutropenic hosts, high fungal growth was achieved through synergy between high growth rate, high swelling probability, slow leucocyte activation and high siderophore secretion. In summary, the model reveals a small set of parameters related to fungal growth, iron acquisition and leucocyte activation as critical determinants of the fate of the infection.

Aspergillus Utilizes Extracellular Heme as an Iron Source During Invasive Pneumonia, Driving Infection Severity.

BACKGROUND: Depriving microbes of iron is critical to host defense. Hemeproteins, the largest source of iron within vertebrates, are abundant in infected tissues in aspergillosis due to hemorrhage, but Aspergillus species have been thought to lack heme import mechanisms. We hypothesized that heme provides iron to Aspergillus during invasive pneumonia, thereby worsening the outcomes of the infection. METHODS: We assessed the effect of heme on fungal phenotype in various in vitro conditions and in a neutropenic mouse model of invasive pulmonary aspergillosis. RESULTS: In mice with neutropenic invasive aspergillosis, we found a progressive and compartmentalized increase in lung heme iron. Fungal cells cultured under low iron conditions took up heme, resulting in increased fungal iron content, resolution of iron starvation, increased conidiation, and enhanced resistance to oxidative stress. Intrapulmonary administration of heme to mice with neutropenic invasive aspergillosis resulted in markedly increased lung fungal burden, lung injury, and mortality, whereas administration of heme analogs or heme with killed Aspergillus did not. Finally, infection caused by fungal germlings cultured in the presence of heme resulted in a more severe infection. CONCLUSIONS: Invasive aspergillosis induces local hemolysis in infected tissues, thereby supplying heme iron to the fungus, leading to lethal infection.

Circadian rhythms and renal pathophysiology.

The reality of life in modern times is that our internal circadian rhythms are often out of alignment with the light/dark cycle of the external environment. This is known as circadian disruption, and a wealth of epidemiological evidence shows that it is associated with an increased risk for cardiovascular disease. Cardiovascular disease remains the top cause of death in the United States, and kidney disease in particular is a tremendous public health burden that contributes to cardiovascular deaths. There is an urgent need for new treatments for kidney disease; circadian rhythm-based therapies may be of potential benefit. The goal of this Review is to summarize the existing data that demonstrate a connection between circadian rhythm disruption and renal impairment in humans. Specifically, we will focus on chronic kidney disease, lupus nephritis, hypertension, and aging. Importantly, the relationship between circadian dysfunction and pathophysiology is thought to be bidirectional. Here we discuss the gaps in our knowledge of the mechanisms underlying circadian dysfunction in diseases of the kidney. Finally, we provide a brief overview of potential circadian rhythm-based interventions that could provide benefit in renal disease.

Iron Inhibits the Translation and Activity of the Renal Epithelial Sodium Channel.

Hypertension is associated with an increased renal expression and activity of the epithelial sodium channel (ENaC) and iron deficiency. Distal tubules absorb iron, causing perturbations that may influence local responses. In this observational study, we investigated the relationship between iron content and ENaC expression and activity using two cell lines and hepcidin knockout mice (a murine model of iron overload). We found that iron did not transcriptionally regulate ENaC in hepcidin knockout mice or in vitro in collecting duct cells. However, the renal tubules of hepcidin knockout mice have a lower expression of ENaC protein. ENaC activity in cultured Xenopus 2F3 cells and mpkCCD cells was inhibited by iron, which could be reversed by iron chelation. Thus, our novel findings implicate iron as a regulator of ENaC protein and its activity.

The Mechanism of Mitochondrial Injury in Alpha-1 Antitrypsin Deficiency Mediated Liver Disease.

Alpha-1 antitrypsin deficiency (AATD) is caused by a single mutation in the SERPINA1 gene, which culminates in the accumulation of misfolded alpha-1 antitrypsin (ZAAT) within the endoplasmic reticulum (ER) of hepatocytes. AATD is associated with liver disease resulting from hepatocyte injury due to ZAAT-mediated toxic gain-of-function and ER stress. There is evidence of mitochondrial damage in AATD-mediated liver disease; however, the mechanism by which hepatocyte retention of aggregated ZAAT leads to mitochondrial injury is unknown. Previous studies have shown that ER stress is associated with both high concentrations of fatty acids and mitochondrial dysfunction in hepatocytes. Using a human AAT transgenic mouse model and hepatocyte cell lines, we show abnormal mitochondrial morphology and function, and dysregulated lipid metabolism, which are associated with hepatic expression and accumulation of ZAAT. We also describe a novel mechanism of ZAAT-mediated mitochondrial dysfunction. We provide evidence that misfolded ZAAT translocates to the mitochondria for degradation. Furthermore, inhibition of ZAAT expression restores the mitochondrial function in ZAAT-expressing hepatocytes. Altogether, our results show that ZAAT aggregation in hepatocytes leads to mitochondrial dysfunction. Our findings suggest a plausible model for AATD liver injury and the possibility of mechanism-based therapeutic interventions for AATD liver disease.

Iron Metabolism: An Under Investigated Driver of Renal Pathology in Lupus Nephritis.

Nephritis is a common manifestation of systemic lupus erythematosus, a condition associated with inflammation and iron imbalance. Renal tubules are the work horse of the nephron. They contain a large number of mitochondria that require iron for oxidative phosphorylation, and a tight control of intracellular iron prevents excessive generation of reactive oxygen species. Iron supply to the kidney is dependent on systemic iron availability, which is regulated by the hepcidin-ferroportin axis. Most of the filtered plasma iron is reabsorbed in proximal tubules, a process that is controlled in part by iron regulatory proteins. This review summarizes tubulointerstitial injury in lupus nephritis and current understanding of how renal tubular cells regulate intracellular iron levels, highlighting the role of iron imbalance in the proximal tubules as a driver of tubulointerstitial injury in lupus nephritis. We propose a model based on the dynamic ability of iron to catalyze reactive oxygen species, which can lead to an accumulation of lipid hydroperoxides in proximal tubular epithelial cells. These iron-catalyzed oxidative species can also accentuate protein and autoantibody-induced inflammatory transcription factors leading to matrix, cytokine/chemokine production and immune cell infiltration. This could potentially explain the interplay between increased glomerular permeability and the ensuing tubular injury, tubulointerstitial inflammation and progression to renal failure in LN, and open new avenues of research to develop novel therapies targeting iron metabolism.

Neutrophil-Derived Tumor Necrosis Factor Drives Fungal Acute Lung Injury in Chronic Granulomatous Disease.

Chronic granulomatous disease (CGD) results from deficiency of nicotinamide adenine dinucleotide phosphate(NADPH) oxidase and impaired reactive oxygen species (ROS) generation. This leads to impaired killing of Aspergillus and, independently, a pathologic hyperinflammatory response to the organism. We hypothesized that neutrophil-derived ROS inhibit the inflammatory response to Aspergillus and that acute lung injury in CGD is due to failure of this regulation. Mice with gp91phox deficiency, the most common CGD mutation, had more severe lung injury, increased neutrophilinfiltration, and increased lung tumor necrosis factor (TNF) after Aspergillus challenge compared with wild-types. Neutrophils were surprisingly the predominant source of TNF in gp91phox-deficient lungs. TNF neutralization inhibited neutrophil recruitment in gp91phox-deficient mice and protected from lung injury. We propose that, in normal hosts, Aspergillus stimulates TNF-dependent neutrophil recruitment to the lungs and neutrophil-derived ROS limit inflammation. In CGD, in contrast, recruited neutrophils are the dominant source of TNF, promoting further neutrophil recruitment in a pathologic positive-feedback cycle, resulting in progressive lung injury.