Disruption of cytochrome P4501A2 in mice leads to increased susceptibility to hyperoxic lung injury

Hyperoxia contributes to acute lung injury in diseases such as acute respiratory distress syndrome. Cytochrome P450 (CYP) 1A enzymes have been implicated in hyperoxic lung injury, but the mechanistic role of CYP1A2 in pulmonary injury is not known. We hypothesized that mice lacking the gene Cyp1a2 (which is predominantly expressed in the liver) will be more sensitive to lung injury and inflammation mediated by hyperoxia and that CYP1A2 will play a protective role by attenuating lipid peroxidation and oxidative stress in the lung. Eight- to ten-week-old WT (C57BL/6) or Cyp1a2^−/− mice were exposed to hyperoxia (>95% O₂) or maintained in room air for 24–72 h. Lung injury was assessed by determining the ratio of lung weight/body weight (LW/BW) and by histology. Extent of inflammation was determined by measuring the number of neutrophils in the lung as well as cytokine expression. The Cyp1a2^−/− mice under hyperoxic conditions showed increased LW/BW ratios, lung injury, neutrophil infiltration, and IL-6 and TNF-α levels and augmented lipid peroxidation, as evidenced by increased formation of malondialdehyde– and 4-hydroxynonenal–protein adducts and pulmonary isofurans compared to WT mice. In vitro experiments showed that the F₂-isoprostane PGF₂-α is metabolized by CYP1A2 to a dinor metabolite, providing evidence for a catalytic role for CYP1A2 in the metabolism of F₂-isoprostanes. In summary, our results support the hypothesis that hepatic CYP1A2 plays a critical role in the attenuation of hyperoxic lung injury by decreasing lipid peroxidation and oxidative stress in vivo.     

Loss of parietal cell superoxide dismutase leads to gastric oxidative stress and increased injury susceptibility in mice

Mitochondrial superoxide dismutase (SOD2) prevents accumulation of the superoxide that arises as a consequence of oxidative phosphorylation. However, SOD2 is a target of oxidative/nitrosative inactivation, and reduced SOD2 activity has been demonstrated to contribute to portal hypertensive gastropathy. We investigated the consequences of gastric parietal cell-specific SOD2 deficiency on mitochondrial function and gastric injury susceptibility. Mice expressing Cre recombinase under control of the parietal cell Atpase4b gene promoter were crossed with mice harboring loxP sequences flanking the sod2 gene (SOD2 floxed mice). Cre-positive mice and Cre-negative littermates (controls) were used in studies of SOD2 expression, parietal cell function (ATP synthesis, acid secretion, and mitochondrial enzymatic activity), increased oxidative/nitrosative stress, and gastric susceptibility to acute injury. Parietal cell SOD2 deficiency was accompanied by a 20% (P < 0.05) reduction in total gastric SOD activity and a 93% (P < 0.001) reduction in gastric SOD2 activity. In SOD2-deficient mice, mitochondrial aconitase and ATP synthase activities were impaired by 36% (P < 0.0001) and 44% (P < 0.005), respectively. Gastric tissue ATP content was reduced by 34% (P < 0.002). Basal acid secretion and peak secretagogue (histamine)-induced acid secretion were reduced by 43% (P < 0.0001) and 40% (P < 0.0005), respectively. There was a fourfold (P < 0.02) increase in gastric mucosal apoptosis and 41% (P < 0.001) greater alcohol-induced gastric damage in the parietal cell SOD2-deficient mice. Our findings indicate that loss of parietal cell SOD2 leads to mitochondrial dysfunction, resulting in perturbed energy metabolism, impaired parietal cell function, and increased gastric mucosal oxidative stress. These alterations render the gastric mucosa significantly more susceptible to acute injury.     

Lung injury and mortality with hyperoxia are increased in peroxiredoxin 6 gene-targeted mice

Overexpression of peroxiredoxin 6 (Prdx6) has been shown to protect lungs of mice against hyperoxia-mediated injury. In this study, we evaluated whether genetic inactivation of Prdx6 in mice increases sensitivity to oxygen toxicity. We evaluated mouse survival, lung histopathology, total protein and nucleated cells in bronchoalveolar lavage fluid (BALF), and oxidation of lung protein and lipids by measurement of protein carbonyls and thiobarbituric reactive substances (TBARS), respectively. The duration of survival for Prdx6 -/- mice was significantly shorter than that observed in wild-type mice on exposure to 85 or 100% O(2); survival of Prdx6 +/- mice was intermediate. After 72-h exposure to 100% O(2), lungs of Prdx6-/- mice showed more severe injury than wild-type with increased wet/dry weight, epithelial cell necrosis and alveolar edema on microscopic examination, increased protein and nucleated cells in BALF, and higher content of TBARS and protein carbonyls in lung homogenate. These findings show that Prdx6 -/- mice have increased sensitivity to hyperoxia and provide in vivo evidence that Prdx6 is an important lung antioxidant enzyme.     

Syndecan-4 deficiency leads to high mortality of lipopolysaccharide-injected mice

Syndecan-4 is a transmembrane heparan sulfate proteoglycan belonging to the syndecan family. Following intraperitoneal injection of lipopolysaccharide (LPS), syndecan-4-deficient mice exhibited high mortality compared with wild-type controls. Severe endotoxin shock was observed in the deficient mice: systolic blood pressure and left ventricular fractional shortening were lower in the deficient mice than in the wild-type controls 9 h after LPS injection. Although histological examinations revealed no apparent differences between two groups, the plasma level of interleukin (IL)-1beta was higher in the deficient mice than in the wild-type controls 9 h after LPS injection. Consistent with the regulatory roles of syndecan-4, its expression in monocytes and endothelial cells of microvasculature increased in the wild-type mice after LPS administration. Although IL-1beta was produced to the same extent by macrophages from syndecan-4-deficient and wild-type mice after LPS stimulation, inhibition of its production by transforming growth factor-beta1 was impaired in the syndecan-4-deficient macrophages. These results indicate that syndecan-4 could be involved in prevention of endotoxin shock, at least partly through the inhibitory action of transforming growth factor-beta1 on IL-1beta production.     

Cyclooxygenase-2 deficiency leads to intestinal barrier dysfunction and increased mortality during polymicrobial sepsis

Sepsis remains the leading cause of death in critically ill patients, despite modern advances in critical care. Intestinal barrier dysfunction may lead to secondary bacterial translocation and the development of the multiple organ dysfunction syndrome during sepsis. Cyclooxygenase (COX)-2 is highly upregulated in the intestine during sepsis, and we hypothesized that it may be critical in the maintenance of intestinal epithelial barrier function during peritonitis-induced polymicrobial sepsis. COX-2(-/-) and COX-2(+/+) BALB/c mice underwent cecal ligation and puncture (CLP) or sham surgery. Mice chimeric for COX-2 were derived by bone marrow transplantation and underwent CLP. C2BBe1 cells, an intestinal epithelial cell line, were treated with the COX-2 inhibitor NS-398, PGD(2), or vehicle and stimulated with cytokines. COX-2(-/-) mice developed exaggerated bacteremia and increased mortality compared with COX-2(+/+) mice following CLP. Mice chimeric for COX-2 exhibited the recipient phenotype, suggesting that epithelial COX-2 expression in the ileum attenuates bacteremia following CLP. Absence of COX-2 significantly increased epithelial permeability of the ileum and reduced expression of the tight junction proteins zonula occludens-1, occludin, and claudin-1 in the ileum following CLP. Furthermore, PGD(2) attenuated cytokine-induced hyperpermeability and zonula occludens-1 downregulation in NS-398-treated C2BBe1 cells. Our findings reveal that absence of COX-2 is associated with enhanced intestinal epithelial permeability and leads to exaggerated bacterial translocation and increased mortality during peritonitis-induced sepsis. Taken together, our results suggest that epithelial expression of COX-2 in the ileum is a critical modulator of tight junction protein expression and intestinal barrier function during sepsis.     

Inactivation of PKCtheta leads to increased susceptibility to obesity and dietary insulin resistance in mice

In this study, we investigated the metabolic phenotype of PKCtheta knockout mice (C57BL/6J) on chow diet and high-fat diet (HFD). The knockout (KO) mice are normal in growth and reproduction. On the chow diet, body weight and food intake were not changed in the KO mice; however, body fat content was increased with a corresponding decrease in body lean mass. Energy expenditure and spontaneous physical activity were decreased in the KO mice. On HFD, energy expenditure and physical activity remained low in the KO mice. The body weight and fat content were increased rapidly in the KO mice. At 8 wk on HFD, severe insulin resistance was detected in the KO mice with hyperinsulinemic euglycemic clamp and insulin tolerance test. Insulin action in both hepatic and peripheral tissues was reduced in the KO mice. Plamsa free fatty acid was increased, and expression of adiponectin in the adipose tissue was decreased, in the KO mice on HFD. This study suggests that loss of PKCtheta reduces energy expenditure and increases the risk of dietary obesity and insulin resistance in mice.     

Trypanosoma cruzi: host selenium deficiency leads to higher mortality but similar parasitemia in mice

Selenium is an essential trace element and its deficiency was implicated in heart diseases. We recently showed low Se levels in chronic chagasic patients with cardiomyopathy. Herein, mice were depleted in Se by feeding the mothers with chow containing only 0.005 mg Se/kg and maintaining this diet for offspring, that were further infected with Trypanosoma cruzi. Survival rate was significantly lower in Se deficient than in control mice. Parasitemia was similar in all groups. Necrotic heart lesions were found after infection (high CK-MB levels). No outbreaks of parasite growth were detected in chronic survivors submitted or not to a second Se depletion. The present results confirm our hypothesis that a nutritional deficiency in Se is associated to a higher mortality during T. cruzi infection. The potential beneficial effect of Se supplementation is a perspective. Hypothesis to explain the higher susceptibility of Se-depleted mice to T. cruzi infection are discussed.     

IgA immunodeficiency leads to inadequate Th cell priming and increased susceptibility to influenza virus infection

IgA is considered to be the principal Ab involved in defense against pathogens in the mucosal compartment. Using mice with a targeted disruption in IgA gene expression (IgA(-/-) mice), we have examined the precise role of IgA in protective anti-influenza responses after intranasal vaccination. IgA(-/-) mice immunized intranasally with soluble hemagglutinin (hemagglutinin subtype 1) and neuraminidase (neuraminidase subtype 1) vaccine in the absence of adjuvant were found to be more susceptible to influenza virus infection than IgA(+/+) mice (13 vs 75% survival after virus challenge). Inclusion of IL-12 during immunization restored the protective efficacy of the vaccine to that seen in IgA(+/+) animals. IgA(-/-) mice had no detectable IgA expression, but displayed enhanced serum and pulmonary IgM and IgG Ab levels after IL-12 treatment. Assessment of T cell function revealed markedly depressed splenic lymphoproliferative responses to PHA in IgA(-/-) animals compared with IgA(+/+) mice. Furthermore, IgA(-/-) animals displayed impaired T cell priming to the H1N1 subunit vaccine, with concomitant reduction in recall memory responses due to a defect in APC function. Collectively, these results provide evidence that a major role of IgA is to facilitate presentation of Ag to mucosal T cells. IL-12 treatment can overcome IgA deficiency by providing adequate T cell priming during vaccination.     

Mycoplasma pulmonis infection augments natural killer cell activity in mice

The goal of this study was to determine if experimental Mycoplasma pulmonis infection augmented splenic natural killer (NK) cell activity in mice. A 4 hour 51Cr-release in vitro assay using YAC-1 tumor target cells was employed to measure splenic NK cell activity in C57BL/6J mice infected intraperitoneally with M. pulmonis and in uninfected controls. Transient augmentation of the NK cells was observed, peaking at day 3 postinoculation (PI) and gradually returning to normal levels by day 10 PI. Selective depletion studies showed that the cells responsible for killing target cells were NK cells. They were nonadherent to nylon wool, not susceptible to Thy-1.2 antibody and susceptible to asialo GM1 ganglioside antibody. Inadvertent augmentation of the NK cell system due to M. pulmonis infection may complicate the interpretation of research data, especially in immunology and cancer studies.     

细菌耐受噬菌体感染的分子机制研究进展

噬菌体是能感染细菌的病毒。为了抵抗噬菌体的感染,细菌进化出多种抵抗噬菌体感染的机制,这些机制的阐析极大地促进了基因编辑领域的发展,同时也为噬菌体治疗的开展奠定了基础。本文就细菌针对噬菌体感染的各个环节所进行的抵抗及其分子机制进行了简要综述,同时讨论了这些防御系统的存在对细菌自身的影响,分析了当前细菌耐受噬菌体机制研究存在的局限性,并对未来研究进行了展望。     

Molecular mechanisms for changes in hepatic protein synthesis induced by schistosomiasis infection in mice

Mice infected with Schistosoma mansoni and littermate controls were evaluated serially for 12 weeks. Infected mice gained weight at the same rate as controls, but starting with the sixth week their livers became enlarged with granulomas and fibrous tissue, and they developed hypoalbuminemia. To evaluate the regulation of the albumin and type I collagen gene expression, total RNA was isolated from infected and control mice and translated in an mRNA-dependent rabbit reticulocyte lysate system. Protein synthesis was decreased 1.5-3-fold with RNA from infected vs. control liver. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cell-free products showed a reduction in albumin but an increase in type I procollagen synthesis in infected mice. Immunoprecipitation of the cell-free product confirmed that albumin synthesis was reduced in greater proportion than other liver proteins in schistosome-infected mice. Hybridization of RNA from infected liver with cloned mouse albumin cDNA (pmalb-2) demonstrated a reduction in albumin mRNA to 37% of control, while hybridization with a chick type I pro alpha 2 collagen cDNA probe (pCg-45) revealed increased procollagen mRNA in infected liver beginning at 6 weeks postinfection. These results suggest that in murine schistosomiasis a reduction in biologically active albumin mRNA results in decreased albumin synthesis and may be responsible in part for hypoalbuminemia. In addition, increased collagen mRNA is associated with increased collagen synthesis during hepatic fibrosis.     

Reduced adrenal response and increased mortality after systemic Klebsiella pneumoniae infection in interleukin-6-deficient mice.

During bacterial infections, both the immune system and the hypothalamus-pituitary-adrenal (HPA) axis are activated. The role of IL-6 in the activation of the HPA axis during bacterial sepsis is not fully understood. The aim of the present study was to investigate the role of endogenous IL-6 in a potentially lethal infection with Klebsiella pneumoniae and the concomitant activation of the HPA axis. We examined the mortality of IL-6-/- and IL-6+/+ mice after intravenous (i.v.) infection with K. pneumoniae as well as the bacterial outgrowth in several organs. Subsequently, the influence of endogenous IL-6 on the effect of i.v. administration of K. pneumoniae on the plasma levels of corticosterone and the pro-inflammatory cytokines TNF-alpha and IL-1alpha was investigated in these mice. The present study demonstrates that IL-6-/- mice are more susceptible than IL-6+/+ mice to a systemic Gram-negative infection with K. pneumoniae, leading to increased outgrowth of microorganisms in the organs of the mice. Moreover, this infection is associated with a reduced adrenal response in IL-6-/- mice. We conclude that IL-6-/- mice are more susceptible to Gram-negative bacterial infections, which is mainly due to an impaired recruitment of granulocytes to the site of infection in the absence of IL-6. Furthermore, the reduced adrenal response may be an explanation for the strong inflammatory response with higher TNF-alpha plasma levels in IL-6-/- mice.     

COX-2 disruption leads to increased central vasopressin stores and impaired urine concentrating ability in mice

It was hypothesized that cyclooxygenase-2 (COX-2) activity promotes urine concentrating ability through stimulation of vasopressin (AVP) release after water deprivation (WD). COX-2-deficient (COX-2(-/-), C57BL/6) and wild-type (WT) mice were water deprived for 24 h, and water balance, central AVP mRNA and peptide level, AVP plasma concentration, and AVP-regulated renal transport protein abundances were measured. In male COX-2(-/-), basal urine output and water intake were elevated while urine osmolality was decreased compared with WT. Water deprivation resulted in lower urine osmolality, higher plasma osmolality in COX-2(-/-) mice irrespective of gender. Hypothalamic AVP mRNA level increased and was unchanged between COX-2(-/-) and WT after WD. AVP peptide content was higher in COX-2(-/-) compared with WT. At baseline, plasma AVP concentration was elevated in conscious chronically catheterized COX-2(-/-) mice, but after WD plasma AVP was unchanged between COX-2(-/-) and WT mice (43 ± 11 vs. 70 ± 16 pg/ml). Renal V2 receptor abundance was downregulated in COX-2(-/-) mice. Medullary interstitial osmolality increased and did not differ between COX-2(-/-) and WT after WD. Aquaporin-2 (AQP2; cortex-outer medulla), AQP3 (all regions), and UT-A1 (inner medulla) protein abundances were elevated in COX-2(-/-) at baseline and further increased after WD. COX-2(-/-) mice had elevated plasma urea and creatinine and accumulation of small subcapsular glomeruli. In conclusion, hypothalamic COX-2 activity is not necessary for enhanced AVP expression and secretion in response to water deprivation. Renal medullary COX-2 activity negatively regulates AQP2 and -3. The urine concentrating defect in COX-2(-/-) is likely caused by developmental glomerular injury and not dysregulation of AVP or collecting duct aquaporins.     

Augmented inducible nitric oxide synthase expression and increased NO production reduce sepsis-induced lung injury and mortality in myeloperoxidase-null mice

The myeloperoxidase (MPO)-hydrogen peroxide-halide system is an efficient oxygen-dependent antimicrobial component of polymorphonuclear leukocyte (PMN)-mediated host defense. However, MPO deficiency results in few clinical consequences indicating the activation of compensatory mechanisms. Here, we determined possible mechanisms protecting the host using MPO(-/-) mice challenged with live gram-negative bacterium Escherichia coli. We observed that MPO(-/-) mice unexpectedly had improved survival compared with wild-type (WT) mice within 5-12 h after intraperitoneal E. coli challenge. Lungs of MPO(-/-) mice also demonstrated lower bacterial colonization and markedly attenuated increases in microvascular permeability and edema formation after E. coli challenge compared with WT. However, PMN sequestration in lungs of both groups was similar. Basal inducible nitric oxide synthase (iNOS) expression was significantly elevated in lungs and PMNs of MPO(-/-) mice, and NO production was increased two- to sixfold compared with WT. Nitrotyrosine levels doubled in lungs of WT mice within 1 h after E. coli challenge but did not change in MPO(-/-) mice. Inhibition of iNOS in MPO(-/-) mice significantly increased lung edema and reduced their survival after E. coli challenge, but iNOS inhibitor had the opposite effect in WT mice. Thus augmented iNOS expression and NO production in MPO(-/-) mice compensate for the lack of HOCl-mediated bacterial killing, and the absence of MPO-derived oxidants mitigates E. coli sepsis-induced lung inflammation and injury.     

Ischemia-reperfusion injury leads to distinct temporal cardiac remodeling in normal versus diabetic mice

Diabetes is associated with higher incidence of myocardial infarction (MI) and increased propensity for subsequent events post-MI. Here we conducted a temporal analysis of the influence of diabetes on cardiac dysfunction and remodeling after ischemia reperfusion (IR) injury in mice. Diabetes was induced using streptozotocin and IR performed by ligating the left anterior descending coronary artery for 30 min followed by reperfusion for up to 42 days. We first evaluated changes in cardiac function using echocardiography after 24 hours reperfusion and observed IR injury significantly decreased the systolic function, such as ejection fraction, fractional shortening and end systolic left ventricular volume (LVESV) in both control and diabetic mice. The longitudinal systolic and diastolic strain rate were altered after IR, but there were no significant differences between diabetic mice and controls. However, a reduced ability to metabolize glucose was observed in the diabetic animals as determined by PET-CT scanning using 2-deoxy-2-((18)F)fluoro-D-glucose. Interestingly, after 24 hours reperfusion diabetic mice showed a reduced infarct size and less apoptosis indicated by TUNEL analysis in heart sections. This may be explained by increased levels of autophagy detected in diabetic mice hearts. Similar increases in IR-induced macrophage infiltration detected by CD68 staining indicated no change in inflammation between control and diabetic mice. Over time, control mice subjected to IR developed mild left ventricular dilation whereas diabetic mice exhibited a decrease in both end diastolic left ventricular volume and LVESV with a decreased intraventricular space and thicker left ventricular wall, indicating concentric hypertrophy. This was associated with marked increases in fibrosis, indicted by Masson trichrome staining, of heart sections in diabetic IR group. In summary, we demonstrate that diabetes principally influences distinct IR-induced chronic changes in cardiac function and remodeling, while a smaller infarct size and elevated levels of autophagy with similar cardiac function are observed in acute phase.     

Molecular mechanisms of hepatic ischemia-reperfusion injury and preconditioning

Ischemia-reperfusion injury is, at least in part, responsible for the morbidity associated with liver surgery under total vascular exclusion or after liver transplantation. The pathophysiology of hepatic ischemia-reperfusion includes a number of mechanisms that contribute to various degrees in the overall injury. Some of the topics discussed in this review include cellular mechanisms of injury, formation of pro- and anti-inflammatory mediators, expression of adhesion molecules, and the role of oxidant stress during the inflammatory response. Furthermore, the roles of nitric oxide in preventing microcirculatory disturbances and as a substrate for peroxynitrite formation are reviewed. In addition, emerging mechanisms of protection by ischemic preconditioning are discussed. On the basis of current knowledge, preconditioning or pharmacological interventions that mimic these effects have the greatest potential to improve clinical outcome in liver surgery involving ischemic stress and reperfusion.