Hyperbaric oxygen alleviates selective domains of cognitive and motor deficits in female 5xFAD mice.

Treatment of Alzheimer's disease (AD) has been limited to managing of symptoms or anti-amyloid therapy with limited results and uncertainty. Seeking out new therapies that can reverse the effects of this devastating disease is important. Hyperbaric oxygen (HBO) therapy could be such a candidate as it has been shown to improve brain function in certain neurological conditions. Furthermore, the role sex plays in the vulnerability/resilience to AD remains equivocal. An understanding of what makes one sex more vulnerable to AD could unveil new pathways for therapy development. In this study, we investigated the effects of HBO on cognitive, motor, and affective function in a mouse model of AD (5xFAD) and assessed protein oxidation in peripheral tissues as a safety indicator. The motor and cognitive abilities of 5xFAD mice were significantly impaired. HBO therapy improved cognitive flexibility and associative learning of 5xFAD females but not males, but HBO had no effect other aspects of cognition. HBO also reversed AD-related declines in balance but had no impact on gait and anxiety-like behavior. HBO did not affect body weights or oxidative stress in peripheral tissues. Our study provides further support for HBO therapy as a potential treatment for AD and emphasizes the importance of considering sex as a biological variable in therapeutic development. Further investigations into the underlying mechanisms of HBO's sex-specific responses are warranted, as well as optimizing treatment protocols for maximum benefits.

Extracellular Superoxide Dismutase Enhances Recruitment of Immature Neutrophils to the Liver.

Listeria monocytogenes is a Gram-positive intracellular pathogen that causes spontaneous abortion in pregnant women, as well as septicemia, meningitis, and gastroenteritis, primarily in immunocompromised individuals. Although L. monocytogenes can usually be effectively treated with antibiotics, there is still around a 25% mortality rate with individuals who develop clinical listeriosis. Neutrophils are innate immune cells required for the clearance of pathogenic organisms, including L. monocytogenes The diverse roles of neutrophils during both infectious and noninfectious inflammation have recently gained much attention. However, the impact of reactive oxygen species, and the enzymes that control their production, on neutrophil recruitment and function is not well understood. Using congenic mice with varying levels of extracellular superoxide dismutase (ecSOD) activity, we have recently shown that the presence of ecSOD decreases clearance of L. monocytogenes while increasing the recruitment of neutrophils that are not protective in the liver. The data presented here show that ecSOD activity does not lead to a cell-intrinsic increase in neutrophil-homing potential or a decrease in protection against L. monocytogenes Instead, ecSOD activity enhances the production of neutrophil-attracting factors and protects hyaluronic acid (HA) from damage. Furthermore, neutrophils from the livers of ecSOD-expressing mice have decreased intracellular and surface-bound myeloperoxidase, are less capable of killing phagocytosed L. monocytogenes, and have decreased oxidative burst. Collectively, our data reveal that ecSOD activity modulates neutrophil recruitment and function in a cell-extrinsic fashion, highlighting the importance of the enzyme in protecting tissues from oxidative damage.

Extracellular superoxide dismutase inhibits innate immune responses and clearance of an intracellular bacterial infection.

Reactive oxygen species and reactive nitrogen species play important roles during immune responses to bacterial pathogens. Extracellular superoxide dismutase (ecSOD) regulates extracellular concentrations of reactive oxygen species and reactive nitrogen species and contributes to tissue protection during inflammatory insults. The participation of ecSOD in immune responses seems therefore intuitive, yet is poorly understood. In the current study, we used mice with varying levels of ecSOD activity to investigate the involvement of this enzyme in immune responses against Listeria monocytogenes. Surprisingly, our data demonstrate that despite enhanced neutrophil recruitment to the liver, ecSOD activity negatively affected host survival and bacterial clearance. Increased ecSOD activity was accompanied by decreased colocalization of neutrophils with bacteria, as well as increased neutrophil apoptosis, which reduced overall and neutrophil-specific TNF-α production. Liver leukocytes from mice lacking ecSOD produced equivalent NO· compared with liver leukocytes from mice expressing ecSOD. However, during infection, there were higher levels of peroxynitrite (NO(3)·(-)) in livers from mice lacking ecSOD compared with livers from mice expressing ecSOD. Neutrophil depletion studies revealed that high levels of ecSOD activity resulted in neutrophils with limited protective capacity, whereas neutrophils from mice lacking ecSOD provided superior protection compared with neutrophils from wild-type mice. Taken together, our data demonstrate that ecSOD activity reduces innate immune responses during bacterial infection and provides a potential target for therapeutic intervention.

Allele-specific effects of ecSOD on asbestos-induced fibroproliferative lung disease in mice.

Previous work by others suggests that there is a strain-dependent variation in the susceptibility to inflammatory lung injury in mice. Specifically, the 129/J mice appear to be more resistant to asbestos-induced pulmonary fibrosis than the C57BL/6 strain. A separate line of evidence suggests that extracellular superoxide dismutase (ecSOD) may play an important role in protecting the lung from such injuries. We have recently reported that the 129/J strain of mice has an ecSOD genotype and phenotype distinctly different from those of the C57BL/6 mice. In order to identify ecSOD as a potential "asbestos-injury resistance" gene, we bred congenic mice, on the C57BL/6 background, carrying the wild type (sod3wt) or the 129/J (sod3129) allele for ecSOD. This allowed us to examine the role of ecSOD polymorphism in susceptibility to lung injury in an otherwise identical genetic background. Interestingly, asbestos treatment induces a significant (~40%) increase in plasma ecSOD activity in the sod3129 mice, but not in the sod3wt mice. Asbestos administration results in a loss of ecSOD activity and protein from lung tissue of both congenic strains, but the lung ecSOD activity remains significantly higher in sod3129 mice. As expected, asbestos treatment results in a significant recovery of ecSOD protein in bronchoalveolar lavage fluid (BALF). The BALF of sod3129 mice also have significantly lower levels of proteins and inflammatory cells, especially neutrophils, accompanied by a significantly lower extent of lung injury, as measured by a pathology index score or hydroxyproline content. Immunohistochemistry reveals a significant loss of ecSOD from the tips of the respiratory epithelial cells in response to asbestos treatment and that the loss of immunodetectable ecSOD is compensated for by enzyme expression by infiltrating cells, especially in the sod3wt mice. Our studies thus identify ecSOD as an important anti-inflammatory gene, responsible for most, if not all of the resistance to asbestos-induced lung injury reported for the 129/J strain of mice. The data further suggest allele-specific differences in the regulation of ecSOD expression. These congenic mice therefore represent a very useful model to study the role of this enzyme in all inflammatory diseases. Polymorphisms in human ecSOD have also been reported and it appears logical to assume that such variations may have a profound effect on disease susceptibility.

Extracellular superoxide dismutase polymorphism in mice: Allele-specific effects on phenotype.

Extracellular superoxide dismutase (ecSOD) protects the extracellular matrix from oxidative stress. We previously reported a new allele for ecSOD, expressed in 129P3/J mice (129), which differs from the wild type (wt), expressed in C57BL/6J and other strains, by two amino acid substitutions and a 10-bp deletion in the 3' UTR of the mRNA (A. Pierce et al., 2003, Arterioscler. Thromb. Vasc. Biol.23:1820-1825). The newly discovered allele is associated with a phenotype of significantly increased circulating and heparin-releasable enzyme activities and levels. To examine the properties of the two forms of ecSOD in an identical environment we generated, by extensive backcrossing of ecSOD heterozygous progeny to C57BL/6J females, a congenic C57 strain with the 129 (or wt) allele of ecSOD. These mice are homozygous for nearly 5000 SNPs across all chromosomes, as determined by the Affymetrix Parallele Mouse 5K SNP panel. This study describes the generation of the congenic mice (genetically >99.8% identical) and their ecSOD phenotype. The congenic mouse plasma ecSOD activity before and after heparin administration recapitulates the differences reported in the founder mice. Tissue enzyme distribution is similar in both congenic groups, although the 129 allele is associated with higher levels of enzyme expression despite lower levels of enzyme mRNA. In these characteristics the phenotype is allele driven, with little impact from the rest of the genome. The congenic mice carrying the 129 allele have mRNA levels that are in between those in the founder 129P3/J and C57BL/6J strains. We conclude that the ecSOD phenotype in most aspects of enzyme expression is allele driven, with the exception of tissue mRNA levels, for which a significant contribution by the surrounding (host) genome is observed. These results also suggest potential allele-specific differences in the regulation of ecSOD synthesis and intracellular processing/secretion of ecSOD, independent of the genotype context. Most importantly, the congenic mice offer an excellent model to examine the regulatory mechanisms of ecSOD expression and the role of ecSOD in various diseases involving oxidative stress.

Hyperbaric oxygen treatment attenuates the pro-inflammatory and immune responses in apolipoprotein E knockout mice.

Chronic hyperbaric oxygen (HBO) therapy significantly attenuates atherosclerosis in New Zealand white rabbits as well as the apoE knockout (KO) mice, independent of plasma lipid concentrations and lipoprotein profiles. Because atherosclerosis has many features of a chronic inflammatory disease, in which both cell-mediated and humoral immune responses participate, we examined the effect of HBO treatment on various aspects of the immune response. We now demonstrate that in apoE KO mice, HBO treatment significantly reduces the circulating levels of antibodies to (MDA)LDL, both in the IgG and IgM class, as well as the delayed-type hypersensitivity (DTH) response to oxLDL challenge. Furthermore, HBO treatment results in a profound attenuation in the production of pro-inflammatory cytokines in response to an inflammatory stimulus (LPS), which is accompanied by a marked increase in the constitutive production of the anti-inflammatory cytokine IL-10 by spleen cells, independent of antigen specificity, as indicated by polyclonal activation of T cells. Our results demonstrate that HBO treatment results in the dampening of T and B cell-mediated responses to oxLDL or inflammatory stimuli.

Chronic hyperbaric oxygen treatment elicits an anti-oxidant response and attenuates atherosclerosis in apoE knockout mice.

We previously demonstrated that hyperbaric oxygen (HBO) treatment inhibits diet-induced atherosclerosis in New Zealand White rabbits. In the present study we investigate the mechanisms that might be involved in the athero-protective effect of HBO treatment in a well-accepted model of atherosclerosis, the apoE knockout (KO) mouse. We examine the effects of daily HBO treatment (for 5 and 10 weeks) on the components of the anti-oxidant defense mechanism and the redox state in blood, liver and aortic tissues and compare them to those of untreated apoE KO mice. HBO treatment results in a significant reduction of aortic cholesterol content and decreased fatty streak formation. These changes are accompanied by a significant reduction of autoantibodies against oxidatively modified LDL and profound changes in the redox state of the liver and aortic tissues. A 10-week treatment significantly reduces hepatic levels of TBARS and oxidized glutathione, while significantly increases the levels of reduced glutathione, glutathione reductase (GR), transferase, Se-dependent glutathione peroxidase and catalase (CAT). The effects of HBO treatment are similar in the aortic tissues. These observations provide evidence that HBO treatment has a powerful effect on the redox state of relevant tissues and produces an environment that inhibits oxidation. The anti-oxidant response may be the key to the anti-atherogenic effect of HBO treatment.

Cloning and characterization of two alleles of the murine extracellular superoxide dismutase gene.

We have recently documented the existence of a second allele of ecSOD in mice. Thus far, this allele was only found in the 129P3/J strain. It is characterized by two point mutations leading to amino acid changes as well as a 10 bp deletion from the 3' UTR. We have also shown that the phenotype is profoundly affected by the genotype. In order to obtain a tool to investigate the differences in the properties as well as the posttranscriptional regulation of expression of the two alleles we now describe the creation and characterization of stably transfected CHO-K1 cell lines expressing either of these alleles. CHO-K1 cells were chosen because they do not express endogenous ecSOD and are easy to transfect. We demonstrate that the transfected cells secrete substantial amounts of glycosylated ecSOD, detected by Western blot analyses, ConA-Sepharose affinity chromatography and activity measurements.

Extracellular superoxide dismutase polymorphism in mice.

OBJECTIVE: In this study, we describe a previously unrecognized murine extracellular superoxide dismutase (ecSOD) allele and examine its distribution among various strains and its effect on the ecSOD phenotype. METHODS AND RESULTS: Polymerase chain reaction analysis of genomic and cDNA from apolipoprotein E/LDLR-/- mice indicates the presence of 2 distinct transcripts for this enzyme independent of the extent of atherosclerosis or age. Sequencing and genotyping analyses reveal the presence of 2 alleles for ecSOD. One is a short variant with a 10-base pair deletion in the 3'UTR, accompanied by a single nucleotide substitution (position 61) found in the 129P3/J strain of mice. By contrast, all other strains examined carry the long form. Both free and heparin-releasable ecSOD activities in the 129P3/J strain are more than 3-fold higher than those in the C57Bl/6 mice. Corresponding differences in plasma enzyme mass are observed by immunoblotting. A clear allele dose effect can be observed in F2 hybrids of these 2 strains; free and total ecSOD activities in mice homozygous for the short allele are twice those of mice homozygous for the long allele, with the heterozygote values in between. CONCLUSIONS: These data clearly demonstrate the allele-specific effects on the ecSOD phenotype independent of other strain-specific factors and underline the need for backcrossing of genetically modified mice.

Cellular apoptosis is associated with increased caveolin-1 expression in macrophages.

Macrophage apoptosis is an important factor in determining the efficiency of the immune response, atherosclerotic lesion stability, and clearance of aged cells by phagocytosis. The involvement of caveolin-1 in the regulation of apoptosis has been previously suggested in fibroblasts and epithelial cells. Here we show that treatment of thioglycollate-elicited mouse peritoneal macrophages with various unrelated apoptotic agents, including simvastatin, camptothecin, or glucose deprivation, is associated with a specific and large increase in caveolin-1 expression. In contrast, caveolin-2 levels remain unaffected. Induction of apoptosis was measured by changes in cell morphology, annexin V-labeling, and DNA fragmentation. We demonstrate that caveolin-1 in macrophages is present in lipid rafts and colocalizes with phosphatidylserine (PS) at the cell surface of apoptotic macrophages. Our data suggest that caveolin-1 increase is an early event, closely accompanied by PS externalization and independent of caspase activation and nuclear DNA fragmentation. The increase in caveolin-1 levels does not require new protein synthesis, as cycloheximide does not prevent the apoptosis-mediated increase in caveolin-1 levels. We propose that increased levels of caveolin-1 characterize the apoptotic phenotype of macrophages. Caveolin-1 may be involved in the efficient externalization of PS at the surface of the apoptotic cells.

Caveolins and macrophage lipid metabolism.

The identification of caveolin-1 more than a decade ago initiated active research into its role in the formation of caveolae, membrane trafficking, signal transduction pathways, and lipid homeostasis. Although caveolins are ubiquitously expressed, the majority of the available information comes from differentiated cells rich in caveolins, such as fibroblasts, adipocytes, and endothelial cells. During the development of atherosclerosis, macrophages play a pivotal role in the formation of the fatty streak lesions. They take up large amounts of lipids and accumulate in the subendothelial space, forming foam cells that fill up the lesion area. Since caveolins have been implicated in the regulation of cellular cholesterol metabolism in several cell types, it is of interest to examine their potential function in macrophages. In this review, we attempt to summarize current knowledge and views on the role of caveolins in cholesterol metabolism with emphasis on macrophages.