Inhalation of ozone induces DNA strand breaks and inflammation in mice

Ozone (O₃) is a well-known oxidant pollutant present in photochemical smog. Although ozone is suspected to be a respiratory carcinogen it is not regulated as a carcinogen in most countries.The genotoxic and inflammatory effects of ozone were investigated in female mice exposed to ozone for 90 min. The tail moment in bronchoalveolar lavage (BAL) cells from BALB/c mice was determined by the comet assay as a measure of DNA strand breaks. Within the first 200 min after exposure, the BAL cells from the mice exposed to 1 or 2 ppm ozone had 1.6- and 2.6-fold greater tail moments than unexposed mice. After 200 min there was no effect. It could be ruled out that the effect during the first 200 min was due to major infiltration of lymphocytes or neutrophils. Unexpectedly, ozone had no effect on the content of 8-oxo-deoxyguanosine (8-oxo-dG) in nuclear DNA or on oxidised amino acids in the lung tissue. The mRNA level of the repair enzyme ERCC1 was not increased in the lung tissue. Inflammation was measured by the cytokine mRNA level in lung homogenates. An up to 150-fold induction of interleukin-6 (IL-6) mRNA was detected in the animals exposed to 2 ppm ozone compared to the air-exposed control mice. Also at 1 ppm ozone, the IL-6 mRNA was induced. The large induction of IL-6 mRNA in the lung took place after DNA strand breaks were induced in BAL. This does not support the notion that inflammatory reactions are the cause of DNA damage. To determine whether these exposures were mutagenic, Muta™Mice were exposed to 2 ppm ozone, 90 min per day for 5 days. No treatment-related mutations could be detected in the cII transgene.These results indicate that a short episode of ozone exposure at five times the threshold limit value (TLV) in US induces lung inflammatory mediators and DNA damage in the cells in the lumen of the lung. This was not reflected by an induction of mutations in the lung of Muta™Mice.     

Inhalation of ozone induces DNA strand breaks and inflammation in mice

Ozone (O3) is a well-known oxidant pollutant present in photochemical smog. Although ozone is suspected to be a respiratory carcinogen it is not regulated as a carcinogen in most countries.The genotoxic and inflammatory effects of ozone were investigated in female mice exposed to ozone for 90 min. The tail moment in bronchoalveolar lavage (BAL) cells from BALB/c mice was determined by the comet assay as a measure of DNA strand breaks. Within the first 200 min after exposure, the BAL cells from the mice exposed to 1 or 2 ppm ozone had 1.6- and 2.6-fold greater tail moments than unexposed mice. After 200 min there was no effect. It could be ruled out that the effect during the first 200 min was due to major infiltration of lymphocytes or neutrophils. Unexpectedly, ozone had no effect on the content of 8-oxo-deoxyguanosine (8-oxo-dG) in nuclear DNA or on oxidised amino acids in the lung tissue. The mRNA level of the repair enzyme ERCC1 was not increased in the lung tissue. Inflammation was measured by the cytokine mRNA level in lung homogenates. An up to 150-fold induction of interleukin-6 (IL-6) mRNA was detected in the animals exposed to 2 ppm ozone compared to the air-exposed control mice. Also at 1 ppm ozone, the IL-6 mRNA was induced. The large induction of IL-6 mRNA in the lung took place after DNA strand breaks were induced in BAL. This does not support the notion that inflammatory reactions are the cause of DNA damage. To determine whether these exposures were mutagenic, Muta Mice were exposed to 2 ppm ozone, 90 min per day for 5 days. No treatment-related mutations could be detected in the cII transgene.These results indicate that a short episode of ozone exposure at five times the threshold limit value (TLV) in US induces lung inflammatory mediators and DNA damage in the cells in the lumen of the lung. This was not reflected by an induction of mutations in the lung of Muta Mice.     

Chlamydophila pneumoniae induces a sustained airway hyperresponsiveness and inflammation in mice

Background

It has been reported that Chlamydophila (C.) pneumoniae is involved in the initiation and promotion of asthma and chronic obstructive pulmonary diseases (COPD). Surprisingly, the effect of C. pneumoniae on airway function has never been investigated.

Methods

In this study, mice were inoculated intranasally with C. pneumoniae (strain AR39) on day 0 and experiments were performed on day 2, 7, 14 and 21.

Results

We found that from day 7, C. pneumoniae infection causes both a sustained airway hyperresponsiveness and an inflammation. Interferon-γ (IFN-γ) and macrophage inflammatory chemokine-2 (MIP-2) levels in bronchoalveolar lavage (BAL)-fluid were increased on all experimental days with exception of day 7 where MIP-2 concentrations dropped to control levels. In contrast, tumor necrosis factor-α (TNF-α) levels were only increased on day 7. From day 7 to 21 epithelial damage and secretory cell hypertrophy was observed. It is suggested that, the inflammatory cells/mediators, the epithelial damage and secretory cell hypertrophy contribute to initiation of airway hyperresponsiveness.

Conclusion

Our study demonstrates for the first time that C. pneumoniae infection can modify bronchial responsiveness. This has clinical implications, since additional changes in airway responsiveness and inflammation-status induced by this bacterium may worsen and/or provoke breathlessness in asthma and COPD.     

Differential response of a(2)-macroglobulin-deficient mice in models of lethal TNF-induced inflammation

Tumor necrosis factor (TNF) is an essential mediator in the pathogenesis of Gram-negative septic shock. Injection of TNF into normal mice leads to systemic, lethal inflammation, which is indistinguishable from lipopolysaccharide (LPS)-induced lethal inflammation. alpha(2)-macroglobulin (A2M) is a major positive acute phase protein with broad-spectrum protease-inhibitory activity. Mouse A2M-deficient (MAM-/-) mice were significantly protected against lethal systemic inflammation induced by TNF. The protection is not due to faster clearance of the injected TNF. The induction of tolerance to TNF-induced lethality by repetitive administration of small doses of human TNF for five consecutive days was equally efficient in both mutant mice compared to wild-type mice. In D-(+)-galactosamine (GalN)-sensitized mice, TNF induces lethal inflammatory hepatitis. MAM(-/-) mice are equally sensitive to the lethal combination of TNF/GalN. Furthermore, interleukin-1-induced desensitization to TNF/GalN was not impaired in MAM(-/-) mice. We conclude that MAM plays a mediating role in TNF-induced lethal shock and that MAM deficiency does not reduce changes in efficiency of tolerance and desensitization to TNF and TNF/GalN-induced lethality, respectively.     

LysGH15 reduces the inflammation caused by lethal methicillin-resistant Staphylococcus aureus infection in mice

The endolysin LysGH15, derived from staphylococcal phage GH15, has a wide lytic spectrum and strong lytic activity against Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), in vitro and in vivo. Here, the ability of lethal MRSA to induce mRNA levels of interleukin-6 (IL-6), interleukin-4 (IL-4), and interferon-γ (IFN-γ) in spleen tissues of mice was studied. A large number of bacteria were detected in spleens. The bacteria caused elevated expression levels of these three cytokines. Administration of LysGH15 significantly reduced the number of bacteria and the levels of IL-6, IL-4, and IFN-γ mRNA in spleen cells compared with those in untreated mice at 24 h (P < 0.05). LysGH15 can eliminate a large number of bacteria and effectively alleviate inflammation induced by infection with lethal MRSA.     

Dietary citrate acutely induces insulin resistance and markers of liver inflammation in mice

Citrate is widely used as a food additive being part of virtually all processed foods. Although considered inert by most of the regulatory agencies in the world, plasma citrate has been proposed to play immunometabolic functions in multiple tissues through altering a plethora of cellular pathways. Here, we used a short-term alimentary intervention (24 hours) with standard chow supplemented with citrate in amount corresponding to that found in processed foods to evaluate its effects on glucose homeostasis and liver physiology in C57BL/6J mice. Animals supplemented with dietary citrate showed glucose intolerance and insulin resistance as revealed by glucose and insulin tolerance tests. Moreover, animals supplemented with citrate in their food displayed fed and fasted hyperinsulinemia and enhanced insulin secretion during an oral glucose tolerance test. Citrate treatment also amplified glucose-induced insulin secretion in vitro in INS1-E cells. Citrate supplemented animals had increased liver PKCα activity and altered phosphorylation at serine or threonine residues of components of insulin signaling including IRS-1, Akt, GSK-3 and FoxO1. Furthermore, citrate supplementation enhanced the hepatic expression of lipogenic genes suggesting increased de novo lipogenesis, a finding that was reproduced after citrate treatment of hepatic FAO cells. Finally, liver inflammation markers were higher in citrate supplemented animals. Overall, the results demonstrate that dietary citrate supplementation in mice causes hyperinsulinemia and insulin resistance both in vivo and in vitro, and therefore call for a note of caution on the use of citrate as a food additive given its potential role in metabolic dysregulation.     

Prior burn insult induces lethal acute lung injury in endotoxemic mice: effects of cytokine inhibition

Many patients who experience surgical stress, including burn injury, become susceptible to severe sepsis and septic organ dysfunction, which remains the primary contributor to morbidity and mortality in burn patients. In the present study, we developed a murine model of burn-primed sublethal endotoxemia by producing a 15% BSA full-thickness burn on the dorsum of BALB/c mice under ether anesthesia and administering 10 mg/kg of LPS intravenously on day 11 to model endotoxemia. The prior burn injury in this model induced two-peaked production of cytokines, TNF-alpha, and macrophage inflammatory protein-2 at 2 and 12 h after LPS administration, and it was associated with increased mortality. We also assessed the effect of pharmacological modulation with cytokine synthesis inhibitors prednisolone and JTE-607 and found that pretreatment with them attenuated later cytokine production and decreased mortality after LPS administration. We speculate that the prior burn injury primed the mice for the secondary insult via cytokine production. These results also suggested that an anticytokine strategy might serve as a novel prophylactic therapy for septic organ dysfunction in burn-primed patients.     

Molecular cloning and partial characterization of rat procarboxypeptidase R and carboxypeptidase N

Carboxypeptidase R (EC 3.4.17.20) (CPR) and carboxypeptidase N (EC 3.4.17.3) (CPN) cleave carboxy-terminal arginine or lysine residues from biologically active peptides such as kinins or anaphylatoxins in the circulation thereby regulating their activities. Although CPN is present in a stable active form in plasma, CPR is generated from proCPR, a plasma zymogen, by proteolytic enzymes such as thrombin, thrombin-thrombomodulin complex and plasmin. We have isolated rat proCPR and CPN cDNA clones which can induce enzymatic activities in culture supernatants of the transfected cells. mRNA of proCPR was detected only in rat liver by Northern hybridization and showed hepatocyte-specific expression. Expression of proCPR mRNA was enhanced following LPS injection, indicating that proCPR production is increased under inflammatory conditions.     

Baculovirus induces an innate immune response and confers protection from lethal influenza virus infection in mice

A recombinant baculovirus expressing the hemagglutinin gene of the influenza virus, A/PR/8/34 (H1N1), under the control of the chicken beta-actin promoter, was constructed. To determine the induction of protective immunity in vivo, mice were inoculated with the recombinant baculovirus by intramuscular, intradermal, i.p., and intranasal routes and then were challenged with a lethal dose of the influenza virus. Intramuscular or i.p. immunization with the recombinant baculovirus elicited higher titers of antihemagglutinin Ab than intradermal or intranasal administration. However, protection from a lethal challenge of the influenza virus was only achieved by intranasal immunization of the recombinant baculovirus. Surprisingly, sufficient protection from the lethal influenza challenge was also observed in mice inoculated intranasally with a wild-type baculovirus, as evaluated by reductions in the virus titer, inflammatory cytokine production, and pulmonary consolidations. These results indicate that intranasal inoculation with a wild-type baculovirus induces a strong innate immune response, which protects mice from a lethal challenge of influenza virus.     

Systemic maternal inflammation and neonatal hyperoxia induces remodeling and left ventricular dysfunction in mice

Aims

The impact of the neonatal environment on the development of adult cardiovascular disease is poorly understood. Systemic maternal inflammation is linked to growth retardation, preterm birth, and maturation deficits in the developing fetus. Often preterm or small-for-gestational age infants require medical interventions such as oxygen therapy. The long-term pathological consequences of medical interventions on an immature physiology remain unknown. In the present study, we hypothesized that systemic maternal inflammation and neonatal hyperoxia exposure compromise cardiac structure, resulting in LV dysfunction during adulthood.

Methods and Results

Pregnant C3H/HeN mice were injected on embryonic day 16 (E16) with LPS (80 µg/kg; i.p.) or saline. Offspring were placed in room air (RA) or 85% O₂ for 14 days and subsequently maintained in RA. Cardiac echocardiography, cardiomyocyte contractility, and molecular analyses were performed. Echocardiography revealed persistent lower left ventricular fractional shortening with greater left ventricular end systolic diameter at 8 weeks in LPS/O₂ than in saline/RA mice. Isolated cardiomyocytes from LPS/O₂ mice had slower rates of contraction and relaxation, and a slower return to baseline length than cardiomyocytes isolated from saline/RA controls. α-/β-MHC ratio was increased and Connexin-43 levels decreased in LPS/O₂ mice at 8 weeks. Nox4 was reduced between day 3 and 14 and capillary density was lower at 8 weeks of life in LPS/O₂ mice.

Conclusion

These results demonstrate that systemic maternal inflammation combined with neonatal hyperoxia exposure induces alterations in cardiac structure and function leading to cardiac failure in adulthood and supports the importance of the intrauterine and neonatal milieu on adult health.     

IL-33 induces Th17-mediated airway inflammation via mast cells in ovalbumin-challenged mice

Allergic asthma is characterized by infiltration of eosinophils, elevated Th2 cytokine levels, airway hyperresponsiveness, and IgE. In addition to eosinophils, mast cells, and basophils, a variety of cytokines are also involved in the development of allergic asthma. The pivotal role of eosinophils in the progression of the disease has been a subject of controversy. To determine the role of eosinophils in the progression of airway inflammation, we sensitized and challenged BALB/c wild-type (WT) mice and eosinophil-deficient ΔdblGATA mice with ovalbumin (OVA) and analyzed different aspects of inflammation. We observed increased eosinophil levels and a Th2-dominant response in OVA-challenged WT mice. In contrast, eosinophil-deficient ΔdblGATA mice displayed an increased proportion of mast cells and a Th17-biased response following OVA inhalation. Notably, the levels of IL-33, an important cytokine responsible for Th2 immune deviation, were not different between WT and eosinophil-deficient mice. We also demonstrated that mast cells induced Th17-differentiation via IL-33/ST2 stimulation in vitro. These results indicate that eosinophils are not essential for the development of allergic asthma and that mast cells can skew the immune reaction predominantly toward Th17 responses via IL-33 stimulation.     

Modulation of procarboxypeptidase R (ProCPR) activation by complementary peptides to thrombomodulin

We designed complementary peptides (C-peptides) using a novel computer program (MIMETIC), which generates a series of peptides designed to interact with a target peptide sequence. Carboxypeptidase R (CPR) is an unstable basic carboxypeptidase found in fresh serum in addition to carboxypeptidase N (CPN) which is stable. CPR is generated from its precursor form (proCPR) by trypsin-like enzymes, and its activation is mediated by thrombin generated in the coagulation cascade. The efficiency of activation is enhanced approximately 1,200-fold when thrombin (T) is bound to thrombomodulin (TM). We attempted to generate C-peptides which recognize the T-binding site within TM assuming that some of these might interfere with the generation of T and TM complexes (T-TM). Among three peptides designed, two inhibited the enhancement in activation of proCPR by T in the presence of TM. One of the peptides at 16 microM reduced the activation of proCPR to the level obtained by T alone.     

Female-specific dominant lethal effects in mice

For some chemicals, induction of presumed dominant lethal mutations has been observed only in female mice and not in males. In those cases, questions arise as to (1) whether the increased embryonic mortality is due to genetic effects of the chemicals in the oocyte or, (2) is caused indirectly through maternal toxicity, and, if genetic, (3) the basis for the sex difference. These questions were studied using the compounds adriamycin and platinol. Neither compound induces dominant lethals in male germ cells, but both increased early embryonic mortality when females were treated prior to mating (treatment of maturing oocytes). Reciprocal zygote transfer experiments rules out, either entirely or for the large part, maternal toxicit as the cause, and cytogenetic analysis of first-cleavage metaphases revealed high incidences of chromosomal aberrations. The results of both of these experiments thus provide evidence that the early embryonic mortality resulted from genetic effects induced in oocytes. Most interestingly, each compound produced unexpected types of chromosomal aberrations. Adriamycin produced deletions, rings, and presumed chromosome-type rearrangements. Platinol, on the other hand, produced a few chromatid-type aberrations, but the bulk of aberrations were characterized by disorganization of the chromatin, minute fragments, and thread-lik chromatin bridges between fragments and chromosomes or between two or more chromosomes. The latter type of cytogenetic damage was observed primarily in the compounds are associated with the diffused state of the maturing oocyte chromosomes.     

Dobrava, but not Saaremaa, hantavirus is lethal and induces nitric oxide production in suckling mice

Hantaviruses are the causative agents of HFRS and HCPS (hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome), two severe, and often fatal human diseases. Mortality from HFRS varies between hantaviruses; Hantaan and Dobrava show the highest, Seoul intermediate, and Puumala low mortality. Saaremaa, genetically closely related to Dobrava, is also known to induce HFRS, with low or no mortality. In this study, mice were inoculated with Dobrava and Saaremaa viruses to test for infectibility, lethality, viremia, nitric oxide production and antibody responses. Out of suckling mice intracerebrally inoculated with 50, 500 and 5,000 focus-forming units of Dobrava virus, respectively, 1/8, 2/8 and 7/8 died within 18-26 days. In all but one of the lethally infected mice high levels of replicating virus were detected, and most were positive for neutralizing antibodies and showed elevated levels of nitric oxide production. All suckling mice intracerebrally inoculated with 50, 500, or 5,000 focus-forming units of Saaremaa virus survived and all seroconverted. Clearly lower viral titers were observed for the Saaremaa virus-inoculated mice, also when sacrificed at day 18 after infection, compared to those in mice that died following Dobrava virus infection. Dobrava, Saaremaa, Puumala and Hantaan virus infections of adult mice were asymptomatic, and the anti-nucleocapsid protein IgG2a/IgG1-titer ratio was higher in mice inoculated with Dobrava virus than in those inoculated with Saaremaa virus. Elevated nitric oxide production was not detected in asymptomatically infected mice, and iNOS-/- mice, like normal mice, cleared viremia. In conclusion, we show that Dobrava virus and Saaremaa virus induce distinct differences in terms of survival, viremia, nitric oxide production and antibody responses in mice.     

Maternal obesity induces gut inflammation and impairs gut epithelial barrier function in nonobese diabetic mice

Impairment of gut epithelial barrier function is a key predisposing factor for inflammatory bowel disease, type 1 diabetes (T1D) and related autoimmune diseases. We hypothesized that maternal obesity induces gut inflammation and impairs epithelial barrier function in the offspring of nonobese diabetic (NOD) mice. Four-week-old female NOD/ShiLtJ mice were fed with a control diet (CON; 10% energy from fat) or a high-fat diet (HFD; 60% energy from fat) for 8 weeks to induce obesity and then mated. During pregnancy and lactation, mice were maintained in their respective diets. After weaning, all offspring were fed the CON diet. At 16 weeks of age, female offspring were subjected to in vivo intestinal permeability test, and then ileum was sampled for biochemical analyses. Inflammasome mediators, activated caspase-1 and mature forms of interleukin (IL)-1β and IL-18 were enhanced in offspring of obese mothers, which was associated with elevated serum tumor necrosis factor α level and inflammatory mediators. Consistently, abundance of oxidative stress markers including catalase, peroxiredoxin-4 and superoxide dismutase 1 was heightened in offspring ileum (P<.05). Furthermore, offspring from obese mothers had a higher intestinal permeability. Morphologically, maternal obesity reduced villi/crypt ratio in the ileum of offspring gut. In conclusion, maternal obesity induced inflammation and impaired gut barrier function in offspring of NOD mice. The enhanced gut permeability in HFD offspring might predispose them to the development of T1D and other gut permeability-associated diseases.     

Chronic sleep disorder induced by psychophysiological stress induces glucose intolerance without adipose inflammation in mice

Sleep disturbances are associated with various metabolic diseases such as hypertension and diabetes. We had previously established a mouse model of a psychophysiological stress-induced chronic sleep disorder (CSD) characterized by disrupted circadian rhythms of wheel-running activity, core body temperature, and sleep-wake cycles. To evaluate the underlying mechanisms of metabolic disorders induced by CSD, we created mice with CSD for six weeks and fed them with a high-fat diet. Glucose intolerance with hyperglycemia resulted, although plasma insulin levels and body weight increases were identical between control and CSD mice. Gluconeogenesis and glycolysis were enhanced and suppressed, respectively, in the livers of CSD mice, because the mRNA expression of Pck1 was significantly increased, whereas that of Gck and Pklr were significantly decreased in the CSD mice. Adipose inflammation induced by the high-fat diet seemed suppressed by the CSD, because the mRNA expression levels of Adgre1, Ccl2, and Tnf were significantly downregulated in the adipose tissues of CSD mice. These findings suggest that CSD impair glucose tolerance by inducing gluconeogenesis and suppressing glycolysis. Hyperphasia with hypoleptinemia, hypercorticosteronemia, and increased plasma free fatty acids might be involved in the impaired glucose metabolism under a CSD. Further studies are needed to elucidate the endocrine and molecular mechanisms underlying the associations between sleep disorders and impaired glucose homeostasis that consequently causes diabetes.