Kelley M. Bradley

Pore-Forming Toxins Induce Macrophage Necroptosis during Acute Bacterial Pneumonia.

Necroptosis is a highly pro-inflammatory mode of cell death regulated by RIP (or RIPK)1 and RIP3 kinases and mediated by the effector MLKL. We report that diverse bacterial pathogens that produce a pore-forming toxin (PFT) induce necroptosis of macrophages and this can be blocked for protection against Serratia marcescens hemorrhagic pneumonia. Following challenge with S. marcescens, Staphylococcus aureus, Streptococcus pneumoniae, Listeria monocytogenes, uropathogenic Escherichia coli (UPEC), and purified recombinant pneumolysin, macrophages pretreated with inhibitors of RIP1, RIP3, and MLKL were protected against death. Alveolar macrophages in MLKL KO mice were also protected during S. marcescens pneumonia. Inhibition of caspases had no impact on macrophage death and caspase-1 and -3/7 were determined to be inactive following challenge despite the detection of IL-1β in supernatants. Bone marrow-derived macrophages from RIP3 KO, but not caspase-1/11 KO or caspase-3 KO mice, were resistant to PFT-induced death. We explored the mechanisms for PFT-induced necroptosis and determined that loss of ion homeostasis at the plasma membrane, mitochondrial damage, ATP depletion, and the generation of reactive oxygen species were together responsible. Treatment of mice with necrostatin-5, an inhibitor of RIP1; GW806742X, an inhibitor of MLKL; and necrostatin-5 along with co-enzyme Q10 (N5/C10), which enhances ATP production; reduced the severity of S. marcescens pneumonia in a mouse intratracheal challenge model. N5/C10 protected alveolar macrophages, reduced bacterial burden, and lessened hemorrhage in the lungs. We conclude that necroptosis is the major cell death pathway evoked by PFTs in macrophages and the necroptosis pathway can be targeted for disease intervention.

Chlorine Gas Exposure Causes Systemic Endothelial Dysfunction by Inhibiting Endothelial Nitric Oxide Synthase–Dependent Signaling

Chlorine gas (Cl(2)) exposure during accidents or in the military setting results primarily in injury to the lungs. However, the potential for Cl(2) exposure to promote injury to the systemic vasculature leading to compromised vascular function has not been studied. We hypothesized that Cl(2) promotes extrapulmonary endothelial dysfunction characterized by a loss of endothelial nitric oxide synthase (eNOS)-derived signaling. Male Sprague Dawley rats were exposed to Cl(2) for 30 minutes, and eNOS-dependent vasodilation of aorta as a function of Cl(2) dose (0-400 ppm) and time after exposure (0-48 h) were determined. Exposure to Cl(2) (250-400 ppm) significantly inhibited eNOS-dependent vasodilation (stimulated by acetycholine) at 24 to 48 hours after exposure without affecting constriction responses to phenylephrine or vasodilation responses to an NO donor, suggesting decreased NO formation. Consistent with this hypothesis, eNOS protein expression was significantly decreased (∼ 60%) in aorta isolated from Cl(2)-exposed versus air-exposed rats. Moreover, inducible nitric oxide synthase (iNOS) mRNA was up-regulated in circulating leukocytes and aorta isolated 24 hours after Cl(2) exposure, suggesting stimulation of inflammation in the systemic vasculature. Despite decreased eNOS expression and activity, no changes in mean arterial blood pressure were observed. However, injection of 1400W, a selective inhibitor of iNOS, increased mean arterial blood pressure only in Cl(2)-exposed animals, suggesting that iNOS-derived NO compensates for decreased eNOS-derived NO. These results highlight the potential for Cl(2) exposure to promote postexposure systemic endothelial dysfunction via disruption of vascular NO homeostasis mechanisms.

Administration of nitrite after chlorine gas exposure prevents lung injury: effect of administration modality.

Cl(2) gas toxicity is complex and occurs during and after exposure, leading to acute lung injury (ALI) and reactive airway syndrome (RAS). Moreover, Cl(2) exposure can occur in diverse situations encompassing mass casualty scenarios, highlighting the need for postexposure therapies that are efficacious and amenable to rapid and easy administration. In this study, we assessed the efficacy of a single dose of nitrite (1 mg/kg) to decrease ALI when administered to rats via intraperitoneal (ip) or intramuscular (im) injection 30 min after Cl(2) exposure. Exposure of rats to Cl(2) gas (400 ppm, 30 min) significantly increased ALI and caused RAS 6-24h postexposure as indexed by BAL sampling of lung surface protein and polymorphonucleocytes (PMNs) and increased airway resistance and elastance before and after methacholine challenge. Intraperitoneal nitrite decreased Cl(2)-dependent increases in BAL protein but not PMNs. In contrast im nitrite decreased BAL PMN levels without decreasing BAL protein in a xanthine oxidoreductase-dependent manner. Histological evaluation of airways 6h postexposure showed significant bronchial epithelium exfoliation and inflammatory injury in Cl(2)-exposed rats. Both ip and im nitrite improved airway histology compared to Cl(2) gas alone, but more coverage of the airway by cuboidal or columnar epithelium was observed with im compared to ip nitrite. Airways were rendered more sensitive to methacholine-induced resistance and elastance after Cl(2) gas exposure. Interestingly, im nitrite, but not ip nitrite, significantly decreased airway sensitivity to methacholine challenge. Further evaluation and comparison of im and ip therapy showed a twofold increase in circulating nitrite levels with the former, which was associated with reversal of post-Cl(2) exposure-dependent increases in circulating leukocytes. Halving the im nitrite dose resulted in no effect in PMN accumulation but significant reduction of BAL protein levels, indicating a distinct nitrite dose dependence for inhibition of Cl(2)-dependent lung permeability and inflammation. These data highlight the potential for nitrite as a postexposure therapeutic for Cl(2) gas-induced lung injury and also suggest that administration modality is a key consideration in nitrite therapeutics.

Sodium nitrite protects against kidney injury induced by brain death and improves post-transplant function

Renal injury induced by brain death is characterized by ischemia and inflammation and limiting it is a therapeutic goal that could improve outcomes in kidney transplantation. Brain death resulted in decreased circulating nitrite levels and increased infiltrating inflammatory cell infiltration into the kidney. Since nitrite stimulates nitric oxide signaling in ischemic tissues, we tested whether nitrite therapy was beneficial in a rat model of brain death followed by kidney transplantation. Nitrite, administered over 2 hours of brain death, blunted the increased inflammation without affecting brain death-induced alterations in hemodynamics. Kidneys were transplanted after 2 hours of brain death and renal function followed over 7 days. Allografts collected from nitrite-treated brain dead rats showed significant improvement in function over the first 2 to 4 days post transplantation compared to untreated brain dead animals. Gene microarray analysis after 2 hours of brain death without or with nitrite therapy showed the latter significantly altered the expression of about 400 genes. Ingenuity Pathway analysis indicated multiple signaling pathways were affected by nitrite, including those related to hypoxia, transcription and genes related to humoral immune responses. Thus, nitrite-therapy attenuates brain death-induced renal injury by regulating responses to ischemia and inflammation, ultimately leading to better post-transplant kidney function.

A Randomized Clinical Trial Testing the Anti-Inflammatory Effects of Preemptive Inhaled Nitric Oxide in Human Liver Transplantation

Decreases in endothelial nitric oxide synthase derived nitric oxide (NO) production during liver transplantation promotes injury. We hypothesized that preemptive inhaled NO (iNO) would improve allograft function (primary) and reduce complications post-transplantation (secondary). Patients at two university centers (Center A and B) were randomized to receive placebo (n = 20/center) or iNO (80 ppm, n = 20/center) during the operative phase of liver transplantation. Data were analyzed at set intervals for up to 9-months post-transplantation and compared between groups. Patient characteristics and outcomes were examined with the Mann-Whitney U test, Student t-test, logistic regression, repeated measures ANOVA, and Cox proportional hazards models. Combined and site stratified analyses were performed. MELD scores were significantly higher at Center B (22.5 vs. 19.5, p<0.0001), surgical times were greater at Center B (7.7 vs. 4.5 hrs, p<0.001) and warm ischemia times were greater at Center B (95.4 vs. 69.7 min, p<0.0001). No adverse metabolic or hematologic effects from iNO occurred. iNO enhanced allograft function indexed by liver function tests (Center B, p<0.05; and p<0.03 for ALT with center data combined) and reduced complications at 9-months (Center A and B, p = 0.0062, OR = 0.15, 95% CI (0.04, 0.59)). ICU (p = 0.47) and hospital length of stay (p = 0.49) were not decreased. iNO increased concentrations of nitrate (p<0.001), nitrite (p<0.001) and nitrosylhemoglobin (p<0.001), with nitrite being postulated as a protective mechanism. Mean costs of iNO were

Original ArticleSodium nitrite protects against kidney injury induced by brain death and improves post-transplant function

1,020 per transplant. iNO was safe and improved allograft function at one center and trended toward improving allograft function at the other. ClinicalTrials.gov with registry number 00582010 and the following URL:http://clinicaltrials.gov/show/NCT00582010.

Quercetin induced tissue-type plasminogen activator expression is mediated through Sp1 and p38 mitogen-activated protein kinase in human endothelial cells

Renal injury induced by brain death is characterized by ischemia and inflammation and limiting it is a therapeutic goal that could improve outcomes in kidney transplantation. Brain death resulted in decreased circulating nitrite levels and increased infiltrating inflammatory cell infiltration into the kidney. Since nitrite stimulates nitric oxide signaling in ischemic tissues, we tested whether nitrite therapy was beneficial in a rat model of brain death followed by kidney transplantation. Nitrite, administered over 2 hours of brain death, blunted the increased inflammation without affecting brain death-induced alterations in hemodynamics. Kidneys were transplanted after 2 hours of brain death and renal function followed over 7 days. Allografts collected from nitrite-treated brain dead rats showed significant improvement in function over the first 2 to 4 days post transplantation compared to untreated brain dead animals. Gene microarray analysis after 2 hours of brain death without or with nitrite therapy showed the latter significantly altered the expression of about 400 genes. Ingenuity Pathway analysis indicated multiple signaling pathways were affected by nitrite, including those related to hypoxia, transcription and genes related to humoral immune responses. Thus, nitrite-therapy attenuates brain death-induced renal injury by regulating responses to ischemia and inflammation, ultimately leading to better post-transplant kidney function.

Chlorine gas exposure disrupts nitric oxide homeostasis in the pulmonary vasculature

Summary.  Background: Wine polyphenol quercetin upregulates tissue‐type plasminogen activator (t‐PA) transcription in cultured human umbilical cord vein endothelial cells (HUVECs). However, the regulatory elements and signaling pathways involved in this regulation are unknown. Objectives: We aimed to localize quercetin‐responsive t‐PA promoter elements, identify the proteins that bind these elements, and decipher signaling pathways involved in the regulation of t‐PA. Methods: To localize quercetin‐responsive elements, HUVECs were transiently transfected with various t‐PA promoter–reporter constructs. Element functionality was evaluated by mutational analysis. Nuclear protein–t‐PA element interactions were evaluated by electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation (ChIP) analysis. Mitogen‐activated protein kinase (MAPK) inhibitors were used to determine the signaling pathways involved in t‐PA regulation. MAPK inhibition effects were evaluated by real‐time PCR, immunoblotting analysis, and transfections. Coimmunoprecipitation was used to evaluate MAPK and transcription factor interaction. Results: Deletion of the t‐PA promoter region − 288 to − 250 resulted in loss of quercetin responsiveness. This region contains putative Sp1‐binding elements, which we termed Sp1a and Sp1b. Sp1b mutation abolished the quercetin‐inducible response, whereas Sp1a mutation had no effect. EMSA and ChIP analysis demonstrated quercetin‐enhanced Sp1 binding to Sp1b. Inhibition of p38 MAPK abrogated basal and quercetin‐induced t‐PA expression and promoter activity, as well as quercetin‐induced Sp1 binding to Sp1b. Quercetin enhanced p38 MAPK and Sp1 physical association, which was similarly diminished by p38 MAPK inhibition. Conclusions: We showed, for the first time, the presence of a functional Sp1‐binding element in the t‐PA promoter controlling quercetin induction via the p38 MAPK pathway. Understanding these mechanisms may provide new insights into polyphenol cardioprotective effects.

Pore-forming toxin-mediated ion dysregulation leads to death receptor-independent necroptosis of lung epithelial cells during bacterial pneumonia

Exposure to chlorine (Cl2) gas during industrial accidents or chemical warfare leads to significant airway and distal lung epithelial injury that continues post exposure. While lung epithelial injury is prevalent, relatively little is known about whether Cl2 gas also promotes injury to the pulmonary vasculature. To determine this, rats were subjected to a sub-lethal Cl2 gas exposure (400 ppm, 30 min) and then brought back to room air. Pulmonary arteries (PA) were isolated from rats at various times post-exposure and contractile (phenylephrine) and nitric oxide (NO)-dependent vasodilation (acetylcholine and mahmanonoate) responses measured ex vivo. PA contractility did not change, however significant inhibition of NO-dependent vasodilation was observed that was maximal at 24-48 h post exposure. Superoxide dismutase restored NO-dependent vasodilation suggesting a role for increased superoxide formation. This was supported by ∼2-fold increase in superoxide formation (measured using 2-hydroethidine oxidation to 2-OH-E+) from PA isolated from Cl2 exposed rats. We next measured PA pressures in anesthetized rats. Surprisingly, PA pressures were significantly (∼4 mmHg) lower in rats that had been exposed to Cl2 gas 24 h earlier suggesting that deficit in NO-signaling observed in isolated PA experiments did not manifest as increased PA pressures in vivo. Administration of the iNOS selective inhibitor 1400W, restored PA pressures to normal in Cl2 exposed, but not control rats suggesting that any deficit in NO-signaling due to increased superoxide formation in the PA, is offset by increased NO-formation from iNOS. These data indicate that disruption of endogenous NO-signaling mechanisms that maintain PA tone is an important aspect of post-Cl2 gas exposure toxicity.

Bacterial Pore-Forming Toxins Promote the Activation of Caspases in Parallel to Necroptosis to Enhance Alarmin Release and Inflammation During Pneumonia

We report that pore-forming toxins (PFTs) induce respiratory epithelial cell necroptosis independently of death receptor signaling during bacterial pneumonia. Instead, necroptosis was activated as a result of ion dysregulation arising from membrane permeabilization. PFT-induced necroptosis required RIP1, RIP3 and MLKL, and could be induced in the absence or inhibition of TNFR1, TNFR2 and TLR4 signaling. We detected activated MLKL in the lungs from mice and nonhuman primates experiencing Serratia marcescens and Streptococcus pneumoniae pneumonia, respectively. We subsequently identified calcium influx and potassium efflux as the key initiating signals responsible for necroptosis; also that mitochondrial damage was not required for necroptosis activation but was exacerbated by MLKL activation. PFT-induced necroptosis in respiratory epithelial cells did not involve CamKII or reactive oxygen species. KO mice deficient in MLKL or RIP3 had increased survival and reduced pulmonary injury during S. marcescens pneumonia. Our results establish necroptosis as a major cell death pathway active during bacterial pneumonia and that necroptosis can occur without death receptor signaling.