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1.  BMPR2 Preserves Mitochondrial Function and DNA Integrity During Reoxygenation to Promote Endothelial Survival and Reverse Pulmonary Hypertension 
Cell metabolism  2015;21(4):596-608.
Mitochondrial dysfunction, inflammation and mutant bone morphogenetic protein receptor (BMPR)2 are associated with pulmonary arterial hypertension (PAH), an incurable disease characterized by pulmonary arterial (PA) endothelial cell (EC) apoptosis, decreased microvessels and occlusive vascular remodeling. We hypothesized that reduced BMPR2 induces PAEC mitochondrial dysfunction, promoting a pro-inflammatory or pro-apoptotic state. Mice with EC-deletion of BMPR2 develop hypoxia-induced pulmonary hypertension that, in contrast to non-transgenic littermates, does not reverse upon reoxygenation and is associated with reduced PA microvessels and lung EC p53, PGC1α and TFAM, regulators of mitochondrial biogenesis and mitochondrial DNA. Decreasing PAEC BMPR2 by siRNA during reoxygenation represses p53, PGC1α, NRF2, TFAM, mitochondrial membrane potential and ATP and induces mitochondrial DNA deletion and apoptosis. Reducing PAEC BMPR2 in normoxia increases p53, PGC1α, TFAM, mitochondrial membrane potential, ATP production and glycolysis, induces mitochondrial fission, and a pro-inflammatory state. These features are recapitulated in PAEC from PAH patients with mutant BMPR2.
PMCID: PMC4394191  PMID: 25863249
2.  Fatty acid synthesis and NLRP3-inflammasome 
Oncotarget  2015;6(26):21765-21766.
PMCID: PMC4673109  PMID: 26314848
Immunology and Microbiology Section; Immune response; Immunity; fatty acid synthesis; NLRP3 inflammasome
3.  UCP2-induced fatty acid synthase promotes NLRP3 inflammasome activation during sepsis 
Cellular lipid metabolism has been linked to immune responses; however, the precise mechanisms by which de novo fatty acid synthesis can regulate inflammatory responses remain unclear. The NLRP3 inflammasome serves as a platform for caspase-1–dependent maturation and secretion of proinflammatory cytokines. Here, we demonstrated that the mitochondrial uncoupling protein-2 (UCP2) regulates NLRP3-mediated caspase-1 activation through the stimulation of lipid synthesis in macrophages. UCP2-deficient mice displayed improved survival in a mouse model of polymicrobial sepsis. Moreover, UCP2 expression was increased in human sepsis. Consistently, UCP2-deficient mice displayed impaired lipid synthesis and decreased production of IL-1β and IL-18 in response to LPS challenge. In macrophages, UCP2 deficiency suppressed NLRP3-mediated caspase-1 activation and NLRP3 expression associated with inhibition of lipid synthesis. In UCP2-deficient macrophages, inhibition of lipid synthesis resulted from the downregulation of fatty acid synthase (FASN), a key regulator of fatty acid synthesis. FASN inhibition by shRNA and treatment with the chemical inhibitors C75 and cerulenin suppressed NLRP3-mediated caspase-1 activation and inhibited NLRP3 and pro–IL-1β gene expression in macrophages. In conclusion, our results suggest that UCP2 regulates the NLRP3 inflammasome by inducing the lipid synthesis pathway in macrophages. These results identify UCP2 as a potential therapeutic target in inflammatory diseases such as sepsis.
PMCID: PMC4319445  PMID: 25574840
4.  Autophagy: A Crucial Moderator of Redox Balance, Inflammation, and Apoptosis in Lung Disease 
Antioxidants & Redox Signaling  2014;20(3):474-494.
Significance: Autophagy is a fundamental cellular process that functions in the turnover of subcellular organelles and protein. Activation of autophagy may represent a cellular defense against oxidative stress, or related conditions that cause accumulation of damaged proteins or organelles. Selective forms of autophagy can maintain organelle populations or remove aggregated proteins. Autophagy can increase survival during nutrient deficiency and play a multifunctional role in host defense, by promoting pathogen clearance and modulating innate and adaptive immune responses. Recent Advances: Autophagy has been described as an inducible response to oxidative stress. Once believed to represent a random process, recent studies have defined selective mechanisms for cargo assimilation into autophagosomes. Such mechanisms may provide for protein aggregate detoxification and mitochondrial homeostasis during oxidative stress. Although long studied as a cellular phenomenon, recent advances implicate autophagy as a component of human diseases. Altered autophagy phenotypes have been observed in various human diseases, including lung diseases such as chronic obstructive lung disease, cystic fibrosis, pulmonary hypertension, and idiopathic pulmonary fibrosis. Critical Issues: Although autophagy can represent a pro-survival process, in particular, during nutrient starvation, its role in disease pathogenesis may be multifunctional and complex. The relationship of autophagy to programmed cell death pathways is incompletely defined and varies with model system. Future Directions: Activation or inhibition of autophagy may be used to alter the progression of human diseases. Further resolution of the mechanisms by which autophagy impacts the initiation and progression of diseases may lead to the development of therapeutics specifically targeting this pathway. Antioxid. Redox Signal. 20, 474–494.
PMCID: PMC3894710  PMID: 23879400
5.  Carbon Monoxide Confers Protection in Sepsis by Enhancing Beclin 1-Dependent Autophagy and Phagocytosis 
Antioxidants & Redox Signaling  2014;20(3):432-442.
Aims: Sepsis, a systemic inflammatory response to infection, represents the leading cause of death in critically ill patients. However, the pathogenesis of sepsis remains incompletely understood. Carbon monoxide (CO), when administered at low physiologic doses, can modulate cell proliferation, apoptosis, and inflammation in pre-clinical tissue injury models, though its mechanism of action in sepsis remains unclear. Results: CO (250 ppm) inhalation increased the survival of C57BL/6J mice injured by cecal ligation and puncture (CLP) through the induction of autophagy, the down-regulation of pro-inflammatory cytokines, and by decreasing the levels of bacteria in blood and vital organs, such as the lung and liver. Mice deficient in the autophagic protein, Beclin 1 (Becn1+/−) were more susceptible to CLP-induced sepsis, and unresponsive to CO therapy, relative to their corresponding wild-type (Becn1+/+) littermate mice. In contrast, mice deficient in autophagic protein microtubule-associated protein-1 light chain 3B (LC3B) (Map1lc3b−/−) and their corresponding wild-type (Map1lc3b+/+) mice showed no differences in survival or response to CO, during CLP-induced sepsis. CO enhanced bacterial phagocytosis in Becn1+/+ but not Becn1+/− mice in vivo and in corresponding cultured macrophages. CO also enhanced Beclin 1-dependent induction of macrophage protein signaling lymphocyte-activation molecule, a regulator of phagocytosis. Innovation: Our findings demonstrate a novel protective effect of CO in sepsis, dependent on autophagy protein Beclin 1, in a murine model of CLP-induced polymicrobial sepsis. Conclusion: CO increases the survival of mice injured by CLP through systemic enhancement of autophagy and phagocytosis. Taken together, we suggest that CO gas may represent a novel therapy for patients with sepsis. Antioxid. Redox Signal. 20, 432–442.
PMCID: PMC3894711  PMID: 23971531
6.  Mitophagy-dependent necroptosis contributes to the pathogenesis of COPD 
The Journal of Clinical Investigation  2014;124(9):3987-4003.
The pathogenesis of chronic obstructive pulmonary disease (COPD) remains unclear, but involves loss of alveolar surface area (emphysema) and airway inflammation (bronchitis) as the consequence of cigarette smoke (CS) exposure. Previously, we demonstrated that autophagy proteins promote lung epithelial cell death, airway dysfunction, and emphysema in response to CS; however, the underlying mechanisms have yet to be elucidated. Here, using cultured pulmonary epithelial cells and murine models, we demonstrated that CS causes mitochondrial dysfunction that is associated with a reduction of mitochondrial membrane potential. CS induced mitophagy, the autophagy-dependent elimination of mitochondria, through stabilization of the mitophagy regulator PINK1. CS caused cell death, which was reduced by administration of necrosis or necroptosis inhibitors. Genetic deficiency of PINK1 and the mitochondrial division/mitophagy inhibitor Mdivi-1 protected against CS-induced cell death and mitochondrial dysfunction in vitro and reduced the phosphorylation of MLKL, a substrate for RIP3 in the necroptosis pathway. Moreover, Pink1–/– mice were protected against mitochondrial dysfunction, airspace enlargement, and mucociliary clearance (MCC) disruption during CS exposure. Mdivi-1 treatment also ameliorated CS-induced MCC disruption in CS-exposed mice. In human COPD, lung epithelial cells displayed increased expression of PINK1 and RIP3. These findings implicate mitophagy-dependent necroptosis in lung emphysematous changes in response to CS exposure, suggesting that this pathway is a therapeutic target for COPD.
PMCID: PMC4151233  PMID: 25083992
7.  Metabolomic Derangements Are Associated with Mortality in Critically Ill Adult Patients 
PLoS ONE  2014;9(1):e87538.
To identify metabolomic biomarkers predictive of Intensive Care Unit (ICU) mortality in adults.
Comprehensive metabolomic profiling of plasma at ICU admission to identify biomarkers associated with mortality has recently become feasible.
We performed metabolomic profiling of plasma from 90 ICU subjects enrolled in the BWH Registry of Critical Illness (RoCI). We tested individual metabolites and a Bayesian Network of metabolites for association with 28-day mortality, using logistic regression in R, and the CGBayesNets Package in MATLAB. Both individual metabolites and the network were tested for replication in an independent cohort of 149 adults enrolled in the Community Acquired Pneumonia and Sepsis Outcome Diagnostics (CAPSOD) study.
We tested variable metabolites for association with 28-day mortality. In RoCI, nearly one third of metabolites differed among ICU survivors versus those who died by day 28 (N = 57 metabolites, p<.05). Associations with 28-day mortality replicated for 31 of these metabolites (with p<.05) in the CAPSOD population. Replicating metabolites included lipids (N = 14), amino acids or amino acid breakdown products (N = 12), carbohydrates (N = 1), nucleotides (N = 3), and 1 peptide. Among 31 replicated metabolites, 25 were higher in subjects who progressed to die; all 6 metabolites that are lower in those who die are lipids. We used Bayesian modeling to form a metabolomic network of 7 metabolites associated with death (gamma-glutamylphenylalanine, gamma-glutamyltyrosine, 1-arachidonoylGPC(20:4), taurochenodeoxycholate, 3-(4-hydroxyphenyl) lactate, sucrose, kynurenine). This network achieved a 91% AUC predicting 28-day mortality in RoCI, and 74% of the AUC in CAPSOD (p<.001 in both populations).
Both individual metabolites and a metabolomic network were associated with 28-day mortality in two independent cohorts. Metabolomic profiling represents a valuable new approach for identifying novel biomarkers in critically ill patients.
PMCID: PMC3907548  PMID: 24498130
8.  Circulating Mitochondrial DNA in Patients in the ICU as a Marker of Mortality: Derivation and Validation 
PLoS Medicine  2013;10(12):e1001577.
In this paper, Choi and colleagues analyzed levels of mitochondrial DNA in two prospective observational cohort studies and found that increased mtDNA levels are associated with ICU mortality, and improve risk prediction in medical ICU patients. The data suggests that mtDNA could serve as a viable plasma biomarker in MICU patients.
Mitochondrial DNA (mtDNA) is a critical activator of inflammation and the innate immune system. However, mtDNA level has not been tested for its role as a biomarker in the intensive care unit (ICU). We hypothesized that circulating cell-free mtDNA levels would be associated with mortality and improve risk prediction in ICU patients.
Methods and Findings
Analyses of mtDNA levels were performed on blood samples obtained from two prospective observational cohort studies of ICU patients (the Brigham and Women's Hospital Registry of Critical Illness [BWH RoCI, n = 200] and Molecular Epidemiology of Acute Respiratory Distress Syndrome [ME ARDS, n = 243]). mtDNA levels in plasma were assessed by measuring the copy number of the NADH dehydrogenase 1 gene using quantitative real-time PCR. Medical ICU patients with an elevated mtDNA level (≥3,200 copies/µl plasma) had increased odds of dying within 28 d of ICU admission in both the BWH RoCI (odds ratio [OR] 7.5, 95% CI 3.6–15.8, p = 1×10−7) and ME ARDS (OR 8.4, 95% CI 2.9–24.2, p = 9×10−5) cohorts, while no evidence for association was noted in non-medical ICU patients. The addition of an elevated mtDNA level improved the net reclassification index (NRI) of 28-d mortality among medical ICU patients when added to clinical models in both the BWH RoCI (NRI 79%, standard error 14%, p<1×10−4) and ME ARDS (NRI 55%, standard error 20%, p = 0.007) cohorts. In the BWH RoCI cohort, those with an elevated mtDNA level had an increased risk of death, even in analyses limited to patients with sepsis or acute respiratory distress syndrome. Study limitations include the lack of data elucidating the concise pathological roles of mtDNA in the patients, and the limited numbers of measurements for some of biomarkers.
Increased mtDNA levels are associated with ICU mortality, and inclusion of mtDNA level improves risk prediction in medical ICU patients. Our data suggest that mtDNA could serve as a viable plasma biomarker in medical ICU patients.
Please see later in the article for the Editors' Summary
Editors' Summary
Intensive care units (ICUs, also known as critical care units) are specialist hospital wards that provide care for people with life-threatening injuries and illnesses. In the US alone, more than 5 million people are admitted to ICUs every year. Different types of ICUs treat different types of problems. Medical ICUs treat patients who, for example, have been poisoned or who have a serious infection such as sepsis (blood poisoning) or severe pneumonia (inflammation of the lungs); trauma ICUs treat patients who have sustained a major injury; cardiac ICUs treat patients who have heart problems; and surgical ICUs treat complications arising from operations. Patients admitted to ICUs require constant medical attention and support from a team of specially trained nurses and physicians to prevent organ injury and to keep their bodies functioning. Monitors, intravenous tubes (to supply essential fluids, nutrients, and drugs), breathing machines, catheters (to drain urine), and other equipment also help to keep ICU patients alive.
Why Was This Study Done?
Although many patients admitted to ICUs recover, others do not. ICU specialists use scoring systems (algorithms) based on clinical signs and physiological measurements to predict their patients' likely outcomes. For example, the APACHE II scoring system uses information on heart and breathing rates, temperature, levels of salts in the blood, and other signs and physiological measurements collected during the first 24 hours in the ICU to predict the patient's risk of death. Existing scoring systems are not perfect, however, and “biomarkers” (molecules in bodily fluids that provide information about a disease state) are needed to improve risk prediction for ICU patients. Here, the researchers investigate whether levels of circulating cell-free mitochondrial DNA (mtDNA) are associated with ICU deaths and whether these levels can be used as a biomarker to improve risk prediction in ICU patients. Mitochondria are cellular structures that produce energy. Levels of mtDNA in the plasma (the liquid part of blood) increase in response to trauma and infection. Moreover, mtDNA activates molecular processes that lead to inflammation and organ injury.
What Did the Researchers Do and Find?
The researchers measured mtDNA levels in the plasma of patients enrolled in two prospective observational cohort studies that monitored the outcomes of ICU patients. In the Brigham and Women's Hospital Registry of Critical Illness study, blood was taken from 200 patients within 24 hours of admission into the hospital's medical ICU. In the Molecular Epidemiology of Acute Respiratory Distress Syndrome study (acute respiratory distress syndrome is a life-threatening inflammatory reaction to lung damage or infection), blood was taken from 243 patients within 48 hours of admission into medical and non-medical ICUs at two other US hospitals. Patients admitted to medical ICUs with a raised mtDNA level (3,200 or more copies of a specific mitochondrial gene per microliter of plasma) had a 7- to 8-fold increased risk of dying within 28 days of admission compared to patients with mtDNA levels of less than 3,200 copies/µl plasma. There was no evidence of an association between raised mtDNA levels and death among patients admitted to non-medical ICUs. The addition of an elevated mtDNA level to a clinical model for risk prediction that included the APACHE II score and biomarkers that are already used to predict ICU outcomes improved the net reclassification index (an indicator of the improvement in risk prediction algorithms offered by new biomarkers) of 28-day mortality among medical ICU patients in both studies.
What Do These Findings Mean?
These findings indicate that raised mtDNA plasma levels are associated with death in medical ICUs and show that, among patients in medical ICUs, measurement of mtDNA plasma levels can improve the prediction of the risk of death from the APACHE II scoring system, even when commonly measured biomarkers are taken into account. These findings do not indicate whether circulating cell-free mtDNA increased because of the underlying severity of illness or whether mtDNA actively contributes to the disease process in medical ICU patients. Moreover, they do not provide any evidence that raised mtDNA levels are associated with an increased risk of death among non-medical (mainly surgical) ICU patients. These findings need to be confirmed in additional patients, but given the relative ease and rapidity of mtDNA measurement, the determination of circulating cell-free mtDNA levels could be a valuable addition to the assessment of patients admitted to medical ICUs.
Additional Information
Please access these websites via the online version of this summary at
The UK National Health Service Choices website provides information about intensive care
The Society of Critical Care Medicine provides information for professionals, families, and patients about all aspects of intensive care
MedlinePlus provides links to other resources about intensive care (in English and Spanish)
The UK charity ICUsteps supports patients and their families through recovery from critical illness; its booklet Intensive Care: A Guide for Patients and Families is available in English and ten other languages; its website includes patient experiences and relative experiences of treatment in ICUs
Wikipedia has a page on ICU scoring systems (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
PMCID: PMC3876981  PMID: 24391478
9.  Inflammasome-regulated Cytokines Are Critical Mediators of Acute Lung Injury 
Rationale: Despite advances in clinical management, there are currently no reliable diagnostic and therapeutic targets for acute respiratory distress syndrome (ARDS). The inflammasome/caspase-1 pathway regulates the maturation and secretion of proinflammatory cytokines (e.g., IL-18). IL-18 is associated with injury in animal models of systemic inflammation.
Objectives: We sought to determine the contribution of the inflammasome pathway in experimental acute lung injury and human ARDS.
Methods: We performed comprehensive gene expression profiling on peripheral blood from patients with critical illness. Gene expression changes were assessed using real-time polymerase chain reaction, and IL-18 levels were measured in the plasma of the critically ill patients. Wild-type mice or mice genetically deficient in IL-18 or caspase-1 were mechanically ventilated using moderate tidal volume (12 ml/kg). Lung injury parameters were assessed in lung tissue, serum, and bronchoalveolar lavage fluid.
Measurements and Main Results: In mice, mechanical ventilation enhanced IL-18 levels in the lung, serum, and bronchoalveolar lavage fluid. IL-18–neutralizing antibody treatment, or genetic deletion of IL-18 or caspase-1, reduced lung injury in response to mechanical ventilation. In human patients with ARDS, inflammasome-related mRNA transcripts (CASP1, IL1B, and IL18) were increased in peripheral blood. In samples from four clinical centers, IL-18 was elevated in the plasma of patients with ARDS (sepsis or trauma-induced ARDS) and served as a novel biomarker of intensive care unit morbidity and mortality.
Conclusions: The inflammasome pathway and its downstream cytokines play critical roles in ARDS development.
PMCID: PMC3373064  PMID: 22461369
acute respiratory distress syndrome; inflammasome; interleukin-18; mechanical ventilation
10.  Statins and Pulmonary Fibrosis 
Rationale: The role of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) in the development or progression of interstitial lung disease (ILD) is controversial.
Objectives: To evaluate the association between statin use and ILD.
Methods: We used regression analyses to evaluate the association between statin use and interstitial lung abnormalities (ILA) in a large cohort of smokers from COPDGene. Next, we evaluated the effect of statin pretreatment on bleomycin-induced fibrosis in mice and explored the mechanism behind these observations in vitro.
Measurements and Main Results: In COPDGene, 38% of subjects with ILA were taking statins compared with 27% of subjects without ILA. Statin use was positively associated in ILA (odds ratio, 1.60; 95% confidence interval, 1.03–2.50; P = 0.04) after adjustment for covariates including a history of high cholesterol or coronary artery disease. This association was modified by the hydrophilicity of statin and the age of the subject. Next, we demonstrate that statin administration aggravates lung injury and fibrosis in bleomycin-treated mice. Statin pretreatment enhances caspase-1–mediated immune responses in vivo and in vitro; the latter responses were abolished in bone marrow–derived macrophages isolated from Nlrp3−/− and Casp1−/− mice. Finally, we provide further insights by demonstrating that statins enhance NLRP3-inflammasome activation by increasing mitochondrial reactive oxygen species generation in macrophages.
Conclusions: Statin use is associated with ILA among smokers in the COPDGene study and enhances bleomycin-induced lung inflammation and fibrosis in the mouse through a mechanism involving enhanced NLRP3-inflammasome activation. Our findings suggest that statins may influence the susceptibility to, or progression of, ILD.
Clinical trial registered with (NCT 00608764).
PMCID: PMC3297101  PMID: 22246178
statins; interstitial lung disease; pulmonary fibrosis; inflammasome; mitochondrial reactive oxygen species
11.  Carbon Monoxide Activates Autophagy via Mitochondrial Reactive Oxygen Species Formation 
Autophagy, an autodigestive process that degrades cellular organelles and protein, plays an important role in maintaining cellular homeostasis during environmental stress. Carbon monoxide (CO), a toxic gas and candidate therapeutic molecule, confers cytoprotection in animal models of acute lung injury. The mechanisms underlying CO-dependent lung cell protection and the role of autophagy in this process remain unclear. Here, we demonstrate that CO exposure time-dependently increased the expression and activation of the autophagic protein, microtubule-associated protein–1 light chain-3B (LC3B) in mouse lung, and in cultured human alveolar (A549) or human bronchial epithelial cells. Furthermore, CO increased autophagosome formation in epithelial cells by electron microscopy and green fluorescent protein (GFP)-LC3 puncta assays. Recent studies indicate that reactive oxygen species (ROS) play an important role in the activation of autophagy. CO up-regulated mitochondria-dependent generation of ROS in epithelial cells, as assayed by MitoSOX fluorescence. Furthermore, CO-dependent induction of LC3B expression was inhibited by N-acetyl-L-cysteine and the mitochondria-targeting antioxidant, Mito-TEMPO. These data suggest that CO promotes the autophagic process through mitochondrial ROS generation. We investigated the relationships between autophagic proteins and CO-dependent cytoprotection using a model of hyperoxic stress. CO protected against hyperoxia-induced cell death, and inhibited hyperoxia-associated ROS production. The ability of CO to protect against hyperoxia-induced cell death and caspase-3 activation was compromised in epithelial cells infected with LC3B-small interfering (si)RNA, indicating a role for autophagic proteins. These studies uncover a new mechanism for the protective action of CO, in support of potential therapeutic application of this gas.
PMCID: PMC3208612  PMID: 21441382
apoptosis; autophagy; carbon monoxide; epithelial cells; hyperoxia
12.  Characterization of macroautophagic flux in vivo using a leupeptin-based assay 
Autophagy  2011;7(6):629-642.
Macroautophagy is a highly conserved catabolic process that is crucial for organ homeostasis in mammals. However, methods to directly measure macroautophagic activity (or flux) in vivo are limited. In this study we developed a quantitative macroautophagic flux assay based on measuring LC3b protein turnover in vivo after administering the protease inhibitor leupeptin. Using this assay we then characterized basal macroautophagic flux in different mouse organs. We found that the rate of LC3b accumulation after leupeptin treatment was greatest in the liver and lowest in spleen. Interestingly we found that LC3a, an ATG8/LC3b homologue and the LC3b-interacting protein p62 were degraded with similar kinetics to LC3b. However, the LC3b-related proteins GABARAP and GATE-16 were not rapidly turned over in mouse liver, implying that different LC3b homologues may contribute to macroautophagy via distinct mechanisms. Nutrient starvation augmented macroautophagic flux as measured by our assay, while refeeding the animals after a period of starvation significantly suppressed flux. We also confirmed that beclin 1 heterozygous mice had reduced basal macroautophagic flux compared to wild-type littermates. These results illustrate the usefulness of our leupeptin-based assay for studying the dynamics of macroautophagy in mice.
PMCID: PMC3127049  PMID: 21460622
macroautophagy; autophagy; flux; mice; in vivo; LC3; GABARAP; GATE-16; leupeptin; cycloheximide
13.  Autophagy proteins regulate innate immune response by inhibiting NALP3 inflammasome-mediated mitochondrial DNA release 
Nature immunology  2010;12(3):222-230.
Autophagy, a cellular process for organelle and protein turnover, regulates innate immune responses. We demonstrate that depletion of autophagic proteins microtubule associated protein-1 light chain 3B (LC3B) and Beclin 1 enhances caspase-1 activation and secretion of interleukin-1β and interleukin-18. Autophagic protein depletion promoted accumulation of dysfunctional mitochondria and cytosolic translocation of mitochondrial DNA (mtDNA) in response to lipopolysaccharide (LPS) and ATP in macrophages. Release of mtDNA into the cytosol depended on the NALP3 inflammasome and mitochondrial ROS. Cytosolic mtDNA contributed to IL-1β and IL-18 secretion in response to LPS and ATP. LC3B-deficient mice produced more caspase-1-dependent cytokines in two sepsis models and were susceptible to LPS-induced mortality. Our study suggests that autophagic proteins regulate NALP3-dependent inflammation by preserving mitochondrial integrity.
PMCID: PMC3079381  PMID: 21151103
14.  Egr-1 Regulates Autophagy in Cigarette Smoke-Induced Chronic Obstructive Pulmonary Disease 
PLoS ONE  2008;3(10):e3316.
Chronic obstructive pulmonary disease (COPD) is a progressive lung disease characterized by abnormal cellular responses to cigarette smoke, resulting in tissue destruction and airflow limitation. Autophagy is a degradative process involving lysosomal turnover of cellular components, though its role in human diseases remains unclear.
Methodology and Principal Findings
Increased autophagy was observed in lung tissue from COPD patients, as indicated by electron microscopic analysis, as well as by increased activation of autophagic proteins (microtubule-associated protein-1 light chain-3B, LC3B, Atg4, Atg5/12, Atg7). Cigarette smoke extract (CSE) is an established model for studying the effects of cigarette smoke exposure in vitro. In human pulmonary epithelial cells, exposure to CSE or histone deacetylase (HDAC) inhibitor rapidly induced autophagy. CSE decreased HDAC activity, resulting in increased binding of early growth response-1 (Egr-1) and E2F factors to the autophagy gene LC3B promoter, and increased LC3B expression. Knockdown of E2F-4 or Egr-1 inhibited CSE-induced LC3B expression. Knockdown of Egr-1 also inhibited the expression of Atg4B, a critical factor for LC3B conversion. Inhibition of autophagy by LC3B-knockdown protected epithelial cells from CSE-induced apoptosis. Egr-1−/− mice, which displayed basal airspace enlargement, resisted cigarette-smoke induced autophagy, apoptosis, and emphysema.
We demonstrate a critical role for Egr-1 in promoting autophagy and apoptosis in response to cigarette smoke exposure in vitro and in vivo. The induction of autophagy at early stages of COPD progression suggests novel therapeutic targets for the treatment of cigarette smoke induced lung injury.
PMCID: PMC2552992  PMID: 18830406
15.  Carbon monoxide differentially inhibits TLR signaling pathways by regulating ROS-induced trafficking of TLRs to lipid rafts 
The Journal of Experimental Medicine  2006;203(10):2377-2389.
Carbon monoxide (CO), a byproduct of heme catabolism by heme oxygenase (HO), confers potent antiinflammatory effects. Here we demonstrate that CO derived from HO-1 inhibited Toll-like receptor (TLR) 2, 4, 5, and 9 signaling, but not TLR3-dependent signaling, in macrophages. Ligand-mediated receptor trafficking to lipid rafts represents an early event in signal initiation of immune cells. Trafficking of TLR4 to lipid rafts in response to LPS was reactive oxygen species (ROS) dependent because it was inhibited by diphenylene iodonium, an inhibitor of NADPH oxidase, and in gp91phox-deficient macrophages. CO selectively inhibited ligand-induced recruitment of TLR4 to lipid rafts, which was also associated with the inhibition of ligand-induced ROS production in macrophages. TLR3 did not translocate to lipid rafts by polyinosine-polycytidylic acid (poly(I:C)). CO had no effect on poly(I:C)-induced ROS production and TLR3 signaling. The inhibitory effect of CO on TLR-induced cytokine production was abolished in gp91phox-deficient macrophages, also indicating a role for NADPH oxidase. CO attenuated LPS-induced NADPH oxidase activity in vitro, potentially by binding to gp91phox. Thus, CO negatively controlled TLR signaling pathways by inhibiting translocation of TLR to lipid rafts through suppression of NADPH oxidase–dependent ROS generation.
PMCID: PMC2118097  PMID: 17000866

Results 1-15 (15)