Modulation of murine macrophage function by N-acetylcysteine in a model of endotoxic shock

In previous studies we have observed changes in several functions of peritoneal macrophages from BALB/c mice with irreversible endotoxic shock caused by intraperitoneal injection of E. coli lipopolysaccharide (LPS) (100 mg/kg), which were associated with a high production of superoxide anion. Since antioxidants, such as N-acetylcysteine (NAC), are free radical scavengers that improve the immune response, in the present work we have studied different functions of peritoneal macrophages from BALB/c mice suffering the endotoxic shock above indicated and administered N-acetylcysteine (150 mg/kg i.p.) at 30 minutes after LPS injection. In the peritoneal macrophages obtained at 2, 4, 12 and 24 h after LPS injection, the following functions were studied: adherence to substrate, mobility, ingestion of particles, and production of superoxide anion and tumour necrosis factor (TNF alpha). The increase in adherence, ingestion and superoxide anion and TNF alpha production shown by macrophages from animals with endotoxic shock was counteracted by NAC injection. Moreover, the survival time of mice with endotoxic shock was increased in the presence of NAC. These data suggest that NAC, administered intraperitoneally, may be useful for the treatment of irreversible endotoxic shock by modulation of the function of macrophages with decreased superoxide anion and TNF alpha production and concomitant increase of survival time.     

Altered macrophage function is associated with severe Burkholderia pseudomallei infection in a murine model of type 2 diabetes

This study used a murine model of type 2 diabetes (BKS.Cg-Dock7(m) +/+Lepr(db)/J mice) to investigate the inflammatory and cellular mechanisms predisposing to Burkholderia pseudomallei infection and co-morbid diabetes. Homozygous db/db (diabetic) mice developed extreme obesity, dyslipidaemia and glucose intolerance leading to hyperglycaemia and overt type 2 diabetes. Compared to their heterozygous db/+ (non-diabetic) littermates, diabetic mice rapidly succumbed to subcutaneous B. pseudomallei infection, paralleled by severe hypoglycaemia and increased expression of the proinflammatory cytokines, tumour necrosis factor (TNF)-α and interleukin (IL)-1β, in the spleen, despite comparable bacterial loads in the spleen of non-diabetic mice. Neutrophil oxidative burst and dendritic cell uptake and killing of B. pseudomallei were similar between diabetic and non-diabetic mice. Compared to peritoneal macrophages from non-diabetic mice, macrophages from diabetic mice were unable to contain and kill B. pseudomallei. Functional differences between macrophages of diabetic and non-diabetic mice toward B. pseudomallei may contribute to rapid dissemination and more severe disease progression in hosts with co-morbid type 2 diabetes.     

Effect of ageing on the murine venous circulation

The effect of ageing on the morphology of veins, venous valves and arteries was investigated in male wild-type mice using an adapted procedure with injection of a silicone polymer Microfil^? that preserves morphology of the vasculature. Throughout the hind limb the arterial, but not the venous, lumen area and wall thickness were significantly greater in 24-month as compared to 10-week-old C57BL/6 mice. Venous valves were most frequently located at the sapheno-femoral vein junction in the lower extremities, and appeared thicker at the base supported by structurally intact collagen fibers, and thinner towards the proximal end of the valve leaflet, with less organized collagen. Overall, valves were less supported by structurally intact collagen at 24 months as compared to 10 weeks. Endothelial expression of CD31, endothelial protein C receptor or von Willebrand factor (VWF) was not affected by age, while thrombomodulin expression was lower in aged versus young arteries. At both ages, expression of VWF was lower at venous valves versus veins. Evaluation of the blood coagulation profile revealed that aged mice had shortened prothrombin time, elevated plasma levels of factor (F)VII, FVIII and VWF and increased neutrophil and platelet counts. Thus, our data indicate that in mice with ageing, venous valves become more fragile, in association with a procoagulant and inflammatory blood phenotype. Taken together, we found that the procoagulant state in ageing, is accompanied by mild vascular changes.     

Effect of a low-protein diet supplemented with keto-acids on autophagy and inflammation in 5/6 nephrectomized rats

Ketoacids (KA) are known to preserve muscle mass among patients with chronic kidney disease (CKD) on a low-protein diet (LPD). The present study was to compare the effects of KA supplemented diet therapy in autophagy and inflammation in CKD rats'' skeletal muscle. Rats with 5/6 nephrectomy were randomly divided into three groups and fed with either 11 g/kg/day protein [normal-protein diet (NPD)], 3 g/kg/day protein (LPD) or 3 g/kg/day protein which including 5% protein plus 1% KA (LPD + KA) for 24 weeks. Sham-operated rats with NPD intake were used as control. LPD could improve body weight, gastrocnemius muscle mass, as well as gastrocnemius muscle cross-sectional area, with the effect being more obvious in the LPD + KA group. The autophagy marker LC3 (microtubule-associated protein 1 light chain 3), p62, Parkin and PTEN induced putative kinase 1 (PINK1) were significantly attenuate in LPD + KA group than LPD group. LPD + KA group had the lower total mtDNA (mitochondiral DNA) and cytosol mtDNA, NACHT-PYD-containing protein 3 (NALP3) inflammasome than LPD group, but its reactive oxygen species (ROS), caspase-1 and apoptosis-associated speck-like protein containing a CARD (ASC) level was higher. Immunoblotting showed IL-1β (interleukin-1-beta) was lower in LPD and LPD + KA group than the NPD group, but IL-18 showed no significant difference among control and CKD group; toll-like receptor signalling-dependent IL-6 was higher in LPD + KA group than LPD group, but tumor necrosis factor-α (TNF-α) was not significantly changed between LPD + KA and LPD group. Systematic changes of the four cytokines were different from that of the tissue. Although LPD + KA could further ameliorate-activated autophagy than LPD, its effect on the activated inflammation state in CKD was not distinctly. Further study is still required to explore the method of ameliorating inflammation to provide new therapeutic approaches for CKD protein energy wasting (PEW).     

Administration of a murine diet supplemented with conjugated linoleic acid increases the expression and activity of hepatic uncoupling proteins

Daily intake of conjugated linoleic acid (CLA) has been shown to reduce body fat accumulation and to increase body metabolism; this latter effect has been often associated with the up-regulation of uncoupling proteins (UCPs). Here we addressed the effects of a CLA-supplemented murine diet (~2?% CLA mixture, cis-9, trans-10 and trans-10, cis-12 isomers; 45?% of each isomer on alternating days) on mitochondrial energetics, UCP2 expression/activity in the liver and other associated morphological and functional parameters, in C57BL/6 mice. Diet supplementation with CLA reduced both lipid accumulation in adipose tissues and triacylglycerol plasma levels, but did not augment hepatic lipid storage. Livers of mice fed a diet supplemented with CLA showed high UCP2 mRNA levels and the isolated hepatic mitochondria showed indications of UCP activity: in the presence of guanosine diphosphate, the higher stimulation of respiration promoted by linoleic acid in mitochondria from the CLA mice was almost completely reduced to the level of the stimulation from the control mice. Despite the increased generation of reactive oxygen species through oxi-reduction reactions involving NAD(+)/NADH in the Krebs cycle, no oxidative stress was observed in the liver. In addition, in the absence of free fatty acids, basal respiration rates and the phosphorylating efficiency of mitochondria were preserved. These results indicate a beneficial and secure dose of CLA for diet supplementation in mice, which induces UCP2 overexpression and UCP activity in mitochondria while preserving the lipid composition and redox state of the liver.     

A mitochondrial model for premature ageing of somatically cloned mammals

Cloned sheep have recently been discovered to have an unexpectedly advanced biological age. We propose that the explanation is a simple consequence of inheritance of acquired, free radical-induced cellular damage with somatic mitochondria that contribute to the mitochondrial population of cloned cells but not to zygotes produced by fertilization in normal sexual reproduction. Each increment of ageing in cloning experiments is therefore predicted to be maternally inherited. The hypothesis suggests practical ways of decreasing the effect. The hypothesis is itself a prediction of the recent proposal that mitochondria of the female germ line function primarily as genetic templates.     

Ameliorative effects of l-carnitine on rats raised on a diet supplemented with lead acetate

Lead intoxication has been a major health hazard in humans. It affects people at all ages. Its toxicity is associated with various organs of the body and affects different metabolic pathways. Based on histological data, l-carnitine reduced the severity of tissue damage produced as a result of exposure of rats to lead acetate. The main objective of this study was to evaluate the underlying mechanism of protection offered by l-carnitine against lead acetate intoxication using male Sprague–Dawley rats.Forty male Sprague–Dawley rats were randomly divided into four groups with ten rats in each. The first group (G1) served as the control group and animals received standard diet only. The second group (G2) received lead acetate in their diet. The third group (G3) was the l-carnitine treated group and received the normal standard diet supplemented with l-carnitine. While the fourth group (G4) had a diet supplemented with both lead acetate and l-carnitine. At the end of each experiment, blood (serum and whole blood) were collected from each animal and analyzed for the following parameters: serum testosterone levels, serum nitric oxide and serum malondialdehyde. This is in addition to looking at the enzymatic activities of two important enzymes (superoxide dismutase and catalase) and on (glutathione reductase) which are indicative of the antioxidant activities in the whole blood. The results indicated that l-carnitine will counteract the undesirable effects of lead intoxication. It exerted its antioxidant potential by reducing the production of ROS and scavenging free radicals by maintaining and protecting the level of the of antioxidant enzymes SOD, CAT and glutathione peroxidase.Conclusion:l-Carnitine may play an important role in reversing the undesirable effects of lead intoxication. Future studies should be conducted to see whether such an effect is applicable in humans exposed to lead poising.     

Effect of iron and phagocytosis on murine macrophage activation in vitro

Iron-exposed murine macrophages have a modified bactericidal activity as shown by previous observations. In order to assess the role of iron in macrophage activation, as measured by free radical production and by intracellular bacterial killing, murine peritoneal macrophages were cultivated in the presence of various sources of iron, human iron-saturated transferrin and ammonium ferric citrate, or iron chelators, Desferal, and human Apo-transferrin, and were infected with an enteropathogenic strain ofE. coli. The release of nitrite (NO₂ ^?), and the production of superoxide anion (O₂ ^?) and hydrogen peroxide (H₂O₂) by the phagocytes were measured and compared to the production by uninfected macrophages. The synergistic action with murine r.IFN-γ was also studied in the radical production reaction and for the bactericidal activity of macrophages. Our results show that in vitro phagocytosis ofE. coli induced elevated production of NO₂ ^? and H₂O₂ by macrophages, and that oxygen derivatives were released independently of the presence of added iron or chelator. Despite a phagocytosis-related enhancement of NO₂ ^? release, reactive nitrogen intermediates (RNI) are not directly involved in the bactericidal mechanism, as revealed by increased intracellular killing owing to RNI inhibitors. Moreover, bacterial killing may depend on oxygen derivatives, as suggested by the effect of the antioxidant sodium ascorbate leading to both a diminished H₂O₂ production and a decreased bactericidal activity of macrophages.     

Effect of fish-fat or beef-fat supplemented diet on immune complex-induced entheropathy in the rat

In contrast to animals on a beef fat-supplemented diet (BFD), animals maitained on a fish fat-supplemented diet (FFD) incorporate increased amounts of eicosapentaenoic acid (EPA) into membrane phospholipids. Generation of lipid mediators from such tissues favors the formation of compounds with less pro-inflammatory activity than are derived from tissues poor in EPA. Nevertheless, the FFD has not had a uniformly beneficial effect on animal models of inflammatory diseases. We previously showed that intravenous injection of rat anti-BSA-BSA complexes (IC) prepared in 5x antigen excess rapidly induced a striate pattern of serosal (to mucosal) hemorrhage and vascular congestion throughout the small intestine. In this study, we tested the effect of a BFD and FFD on immune complex-induced enteropathy. After six (Expt. 1) or eight weeks (Expt. 2) on the diet, rats were injected with IC and the severity of serosal hyperemia in the small intestine was scored. In some FFD, no lesions were seen under conditions which elicited moderate to severe lesions in BFD rats. In Expt. 1 involving 22 rats and in Expt. 2 involving 28 rats, those on the FFD had a significantly lower composite lesional score compared to those on the BFD, p < 0.005 and p < 0.005, respectively. These results indicate that the FFD had a beneficial effect on IC-induced enteropathy. It is suggested that this effect of the FFD may be mediated primarily by a reduction in availability of platelet-activating factor.     

Effect of fish-fat or beef-fat supplemented diet on immune complex-induced enteropathy in the rat

In contrast to animals on a beef fat-supplemented diet (BFD), animals maintained on a fish fat-supplemented diet (FFD) incorporate increased amounts of eicosapentaenoic acid (EPA) into membrane phospholipids. Generation of lipid mediators from such tissues favors the formation of compounds with less pro-inflammatory activity than are derived from tissues poor in EPA. Nevertheless, the FFD has not had a uniformly beneficial effect on animal models of inflammatory diseases. We previously showed that intravenous injection of rat anti-BSA-BSA complexes (IC) prepared in 5x antigen excess rapidly induced a striate pattern of serosal (to mucosal) hemorrhage and vascular congestion throughout the small intestine. In this study, we tested the effect of a BFD and FFD on immune complex-induced enteropathy. After six (Expt. 1) or eight weeks (Expt. 2) on the diet, rats were injected with IC and the severity of serosal hyperemia in the small intestine was scored. In some FFD, no lesions were seen under conditions which elicited moderate to severe lesions in BFD rats. In Expt. 1 involving 22 rats and in Expt. 2 involving 28 rats, those on the FFD had a significantly lower composite lesional score compared to those on the BFD, p less than 0.005 and p less than 0.005, respectively. These results indicate that the FFD had a beneficial effect on IC-induced enteropathy. It is suggested that this effect of the FFD may be mediated primarily by a reduction in availability of platelet-activating factor.     

Effect of anti-mouse macrophage serum on murine cytomegalovirus infection.

Rabbit anti-mouse macrophage serum (AMS) was used to study the role played by macrophages against murine cytomegalovirus infection. Treatment of mice with AMS enhanced morbidity and mortality following virus infection. These results are discussed in relation to the role of macrophages against virus infections.     

Effect of hyperbaric oxygenation on macrophage function in mice

Hyperbaric oxygenation (HBO) has an immunosuppressive effect. The possible mechanisms of this immunosuppressive effect were assessed by determining the production of interleukin-1 (IL-1), interleukin-6 (IL-6), and prostaglandin E2 (PGE2), as well as phagocytosis by splenic macrophages from HBO-treated mice. Although HBO treatment did not have any significant effect on IL-6 production and phagocytotic activity, a marked decrease in IL-1 production and a significant decrease in PGE2 production were observed. These results suggest that the reduction of IL-1 production may play an important role in the immunosuppressive effect of HBO.     

Influence of aging on murine neutrophil and macrophage function against Candida albicans

Previous work by our group showed that aged C57BL/6 mice develop an altered innate and adaptive immune response to Candida albicans and are more susceptible to systemic primary candidiasis. In this work, we used young (2-3 months old) and aged (18-20 months old) C57BL/6 mice to study in vitro the influence of aging on (1) the fungicidal activity of neutrophils and macrophages, (2) the production of cytokines by resident peritoneal macrophages in response to C. albicans, and (3) cell surface Toll-like receptor (TLR) 2 expression on resident peritoneal macrophages. Our results indicate that murine phagocytes have a fungicidal activity well preserved with aging. In vitro production of proinflammatory cytokines (IL-6, IL-1beta, and tumor necrosis factor-alpha and chemokines (MIP-2) by purified (CD11b(+)) peritoneal macrophages in response to yeasts and hyphae of C. albicans was significantly lower in aged mice as compared with young mice. However, the production of IL-10 by macrophages, in response to C. albicans, was similar in both young and aged animals. Moreover, baseline TLR2 surface expression level was lower on aged macrophages than on control macrophages. Taken together, these data indicate that the increased susceptibility to C. albicans disseminated infections in aged mice is correlated with defects in TLR2 expression and in cytokine production, but not with an impaired fungicidal activity.     

Decreased alveolar macrophage apoptosis is associated with increased pulmonary inflammation in a murine model of pneumococcal pneumonia

Regulation of the inflammatory infiltrate is critical to the successful outcome of pneumonia. Alveolar macrophage apoptosis is a feature of pneumococcal infection and aids disease resolution. The host benefits of macrophage apoptosis during the innate response to bacterial infection are incompletely defined. Because NO is required for optimal macrophage apoptosis during pneumococcal infection, we have explored the role of macrophage apoptosis in regulating inflammatory responses during pneumococcal pneumonia, using inducible NO synthase (iNOS)-deficient mice. iNOS(-/-) mice demonstrated decreased numbers of apoptotic macrophages as compared with wild-type C57BL/6 mice following pneumococcal challenge, greater recruitment of neutrophils to the lung and enhanced expression of TNF-alpha. Pharmacologic inhibition of iNOS produced similar results. Greater pulmonary inflammation was associated with greater levels of early bacteremia, IL-6 production, lung inflammation, and mortality within the first 48 h in iNOS(-/-) mice. Labeled apoptotic alveolar macrophages were phagocytosed by resident macrophages in the lung and intratracheal instillation of exogenous apoptotic macrophages decreased neutrophil recruitment in iNOS(-/-) mice and decreased TNF-alpha mRNA in lungs and protein in bronchial alveolar lavage, as well as chemokines and cytokines including IL-6. These changes were associated with a lower probability of mice becoming bacteremic. This demonstrates the potential of apoptotic macrophages to down-regulate the inflammatory response and for the first time in vivo demonstrates that clearance of apoptotic macrophages decreases neutrophil recruitment and invasive bacterial disease during pneumonia.     

An abundant tissue macrophage population in the adult murine heart with a distinct alternatively-activated macrophage profile

Cardiac tissue macrophages (cTMs) are a previously uncharacterised cell type that we have identified and characterise here as an abundant GFP(+) population within the adult Cx(3)cr1(GFP/+) knock-in mouse heart. They comprise the predominant myeloid cell population in the myocardium, and are found throughout myocardial interstitial spaces interacting directly with capillary endothelial cells and cardiomyocytes. Flow cytometry-based immunophenotyping shows that cTMs exhibit canonical macrophage markers. Gene expression analysis shows that cTMs (CD45(+)CD11b(+)GFP(+)) are distinct from mononuclear CD45(+)CD11b(+)GFP(+) cells sorted from the spleen and brain of adult Cx(3)cr1(GFP/+) mice. Gene expression profiling reveals that cTMs closely resemble alternatively-activated anti-inflammatory M2 macrophages, expressing a number of M2 markers, including Mrc1, CD163, and Lyve-1. While cTMs perform normal tissue macrophage homeostatic functions, they also exhibit a distinct phenotype, involving secretion of salutary factors (including IGF-1) and immune modulation. In summary, the characterisation of cTMs at the cellular and molecular level defines a potentially important role for these cells in cardiac homeostasis.     

Inflammation-mediated memory dysfunction and effects of a ketogenic diet in a murine model of multiple sclerosis

A prominent clinical symptom in multiple sclerosis (MS), a progressive disorder of the central nervous system (CNS) due to heightened neuro-inflammation, is learning and memory dysfunction. Here, we investigated the effects of a ketogenic diet (KD) on memory impairment and CNS-inflammation in a murine model of experimental autoimmune encephalomyelitis (EAE), using electrophysiological, behavioral, biochemical and in vivo imaging approaches. Behavioral spatial learning deficits were associated with motor disability in EAE mice, and were observed concurrently with brain inflammation. The KD improved motor disability in the EAE model, as well as CA1 hippocampal synaptic plasticity (long-term potentiation) and spatial learning and memory (assessed with the Morris Water Maze). Moreover, hippocampal atrophy and periventricular lesions in EAE mice were reversed in KD-treated EAE mice. Finally, we found that the increased expression of inflammatory cytokines and chemokines, as well as the production of reactive oxygen species (ROS), in our EAE model were both suppressed by the KD. Collectively, our findings indicate that brain inflammation in EAE mice is associated with impaired spatial learning and memory function, and that KD treatment can exert protective effects, likely via attenuation of the robust immune response and increased oxidative stress seen in these animals.