Genetic Background and Kinetics Define Wound Bed Extracellular Vesicles in a Mouse Model of Cutaneous Injury.

Extracellular vesicles (EVs) have an important role in mediating intercellular signaling in inflammation and affect the kinetics of wound healing, however, an understanding of the mechanisms regulating these responses remains limited. Therefore, we have focused on the use of cutaneous injury models in which to study the biology of EVs on the inflammatory phase of wound healing. For this, the foreign body response using sterile subcutaneous polyvinylalcohol (PVA) sponges is ideally suited for the parallel analysis of immune cells and EVs without the need for tissue dissociation, which would introduce additional variables. We have previously used this model to identify mediators of EV biogenesis, establishing that control of how EVs are made affects their payload and biological activity. These studies in normal mice led us to consider how conditions such as immunodeficiency and obsesity affect the profile of immune cells and EVs in this model using genetically defined mutant mice. Since EVs are intrinsically heterogenous in biological fluids, we have focused our studies on a novel technology, vesicle flow cytometry (vFC) to quantify changes in EVs in mouse models. Here, we show that myeloid-derived immune cells and EVs express proteins relevant in antigen presentation in PVA sponge implants that have distinct profiles in wildtype, immune-deficient (NOD scid) vs. diabetic (Leprdb) mice. Together, these results establish a foundation for the parallel analysis of both immune cells and EVs with technologies that begin to address the heterogeneity of intercellular communication in the wound bed.

Diet-induced obese mice exhibit altered immune responses to early Salmonella Typhimurium oral infection.

Obesity is a chronic disease associated with different metabolic diseases as well as alterations in immune cell function. It is characterized by a chronic systemic low grade inflammation. There are several studies demonstrating the influence of obesity on the impaired immune response to infection. However, it is not completely clear whether the obese environment influences the development or maintenance of the immune response against infections. The aim of this study was to determine how obesity induced by a high-fat diet affects the immune response to an early oral Salmonella infection. Four groups of mice were kept in separate cages. Two of these designated as controls, fed with a normal diet; whereas other two groups were fed with a high fat diet for 10 weeks. Some mice were used for Salmonella oral infection. After 7 days of oral infection with S. Thypimurium the proportions of spleen cell subsets expressing activation markers in normal diet and HFD obese mice were stained with monoclonal antibodies and analyzed by flow cytometry. Also, mRNA levels of different cytokines were quantified by RT-PCR. It was found that obesity affects the function of the immune system against an early oral Salmonella infection, decreasing NK cells, altering the expression of activation molecules as well as cytokines mRNA levels. Interestingly, the expression some activation molecules on T lymphocytes was reestablished after Salmonella infection, but not the CD25 expression. Immune alterations could lead to immunosuppression or increased susceptibility to infections in HFD obese mice.

Immunomodulatory glycan LNFPIII alleviates hepatosteatosis and insulin resistance through direct and indirect control of metabolic pathways.

Parasitic worms express host-like glycans to attenuate the immune response of human hosts. The therapeutic potential of this immunomodulatory mechanism in controlling the metabolic dysfunction that is associated with chronic inflammation remains unexplored. We demonstrate here that administration of lacto-N-fucopentaose III (LNFPIII), a Lewis(X)-containing immunomodulatory glycan found in human milk and on parasitic helminths, improves glucose tolerance and insulin sensitivity in diet-induced obese mice. This effect is mediated partly through increased interleukin-10 (Il-10) production by LNFPIII-activated macrophages and dendritic cells, which reduces white adipose tissue inflammation and sensitizes the insulin response of adipocytes. Concurrently, LNFPIII treatment upregulates nuclear receptor subfamily 1, group H, member 4 (Fxr-α, also known as Nr1h4) to suppress lipogenesis in the liver, conferring protection against hepatosteatosis. At the signaling level, the extracellular signal-regulated kinase (Erk)-activator protein 1 (Ap1) pathway seems to mediate the effects of LNFPIII on both inflammatory and metabolic pathways. Our results suggest that LNFPIII may provide new therapeutic approaches to treat metabolic diseases.

Malaria and obesity: obese mice are resistant to cerebral malaria.

BACKGROUND: The relationship between malaria and obesity are largely unknown. This is partly due to the fact that malaria occurs mainly in tropical areas where, until recently, obesity was not prevalent. It now appears, however, that obesity is emerging as a problem in developing countries. To investigate the possible role of obesity on the host-parasite response to malarial infection, this study applied a murine model, which uses the existence of genetically well characterized obese mice. METHODS: The receptivity of obese homozygous ob/ob mice was compared to the receptivity of control heterozygous ob/+ lean mice after a single injection of Plasmodium berghei ANKA sporozoites. Both parasitaemia and mortality in response to infection were recorded. RESULTS: The control mice developed the expected rapid neurological syndromes associated with the ANKA strain, leading to death after six days, in absence of high parasitaemia. The obese mice, on the other hand, did not develop cerebral malaria and responded with increasing parasitaemia, which produced severe anemia leading to death 18-25 days after injection. CONCLUSION: The observed major differences in outward symptoms for malarial infection in obese versus control mice indicate a link between obesity and resistance to the infection which could be addressed by malariologists studying human malaria.

Detection of nuclear protein antigens to antinuclear antibodies in serum of NOD mouse.

Nuclear protein antigens to the antinuclear antibodies in serum of non-obese diabetic (NOD) mice were investigated. In the serum of diabetic NOD female mice (20 weeks old), the antinuclear antibodies were detected by indirect immunofluorescence assay using frozen sections of liver of C 57 BL/6 J or NOD mice as antigen. Nuclei were separated from the liver of C 57 BL/6 J mice and solubilized. Solubilized nuclear antigens were analyzed by SDS PAGE-Western immunoblotting techniques. Nuclear protein antigens with molecular weights of 26,000, 32,000 and 65,000 showed strongly positive reactions with the antinuclear antibodies in the serum of the NOD mouse.

T-lymphopenia and T-cell imbalance in diabetic db/db mice.

The diabetic db/db mice of the C57 BL/KsJ strain display anti-islet immunity, thymic dysfunction, and lymphopenia. In the present work, lymphocytes, T-cells, and T-cell subsets were enumerated in thymus and spleen from diabetic db/db mice and their db/ + heterozygote littermates from the 10th day to the 10th month of life. A significant lymphopenia was detected in thymus and spleen from the second month on, involving specifically the T-cell compartment, as assessed by use of a monoclonal anti-Thy1 antibody in indirect fluorescence. The study of T-cell subsets by monoclonal anti-Lyt1 and anti-Lyt2 antibodies revealed a significant increase in Lyt1+ cells and a decrease in Lyt2+ cells, with a corresponding increase of the Lyt1+/Lyt2+ ratio. These anomalies appeared early in life, and were apparently linked neither with the degree of hyperglycemia nor with weight loss or infection. The T-cell depletion in thymus was more pronounced in young male (less than 3 mo) than in young female db/db mice. These alterations may correspond to an increase in the helper/suppressor-cytotoxic ratio and could be linked with the thymic anomalies present in these mice, contributing to the development of anti-islet autoimmunity.

Islet cell surface antibodies in genetically obese hyperglycaemic (ob/ob) mice.

A quantitative method for circulating islet cell surface antibodies (ICSA), based on the binding of 125I-protein A to insulin-producing RINm5F cells, was used to evaluate ICSA in plasma of 4- to 40-week-old Aston obese hyperglycaemic (ob/ob) mice and normal control (+/+) mice. RINm5F cells bound 2502 +/- 1196 c.p.m. 125I-protein A per 10(5) cells (mean +/- S.D., n = 54) after incubation with +/+ plasma. ICSA positive plasma (defined as 125I-protein A binding, mean +/- 2 S.D. of +/+ plasma) was detected in 3 out of 54 +/+ mice and 3 out of 54 ob/ob mice. ICSA were not observed in ob/ob mice before the onset of diabetes (7 weeks of age), but were detected at 9, 20 and 40 weeks. At 20 weeks 125I-protein A binding produced by ob/ob plasma was 35% greater than +/+ plasma (P less than 0.05). The low occurrence of ICSA in ob/ob mice (6%) suggests that factors other than ICSA are responsible for B-cell dysfunction and eventual islet degeneration observed in Aston ob/ob mice.

Intracisternal Type A particles in murine pancreatic B cells. Immunocytochemical demonstration of increased antigen (p73) in genetically diabetic mice.

Intracisternal Type A particles (IAPs) are retroviruslike structures identified by a core protein antigen (p73) and found in mouse embryos, in many mouse tumor cells, and in pancreatic B cells of some strains of genetically diabetic mice. Using both peroxidase-antiperoxidase and protein A-gold immunocytochemical techniques to localize p73, the authors have observed differences in intracellular antigen distribution between MOPC-104E, a mouse tumor cell line rich in IAP, and B cells from genetically diabetic (db/db) mice of the CBA/LtJ and C57BL/KsJ strain. In MOPC-104E cells studied by electron microscopy, localization of protein A-gold complex label was almost exclusively limited to IAP and their sites of assembly on the rough endoplasmic reticulum. In contrast, p73 appeared widely distributed throughout the cytoplasm of B cells from hyperglycemic db/db mice but not normal littermate controls. In addition to distribution over budding IAP, label was also found dispersed through other cytoplasmic organelles involved in secretion, including Golgi complexes and secretory granules. Patch labeling of B cell surfaces was sometimes observed. An ultrastructural survey of islets isolated from normal mice of 7 inbred genetic backgrounds on which the "diabetes" (db) gene has been studied showed that constitutive ability to produce IAP was associated with strain susceptibility to severe diabetes (eg, C57BL/KsJ, DBA/2J, CBA/LtJ, and C3HeB/FeJ). Strains whose B cells failed to show constitutive expression in situ or glucose-inducible expression in cell culture were resistant to the diabetogenic action of db genes. The possibility is discussed that p73 may represent a "neoantigen" which sensitizes the diabetic mouse to reject, by autoimmune mechanisms, the B cells expressing it.

Arginine stimulates thymic immune function and ameliorates the obesity and the hyperglycemia of genetically obese mice.

The effect of 6-day dietary arginine supplementation on the weight gain, blood glucose, thymus weight, thymic lymphocyte content, and in vitro thymic lymphocyte immune reactivity was studied in obese (C57BL/6J-OB/)B) and heterozygous lean mice. Control mice were fed a commercial laboratory chow (1.8% arginine content) and drank tap water, while supplemented mice were given 0.5% arginine in the chow and 0.5% arginine solution for drinking. All mice ate and drank ad libitum. Supplemental arginine significantly decreased the weight gain (1.2 g vs. 2.2 g, p less than 0.01) and blood glucose levels (303 mg% vs 236 mg%, p less than 0.02) of the OB/OB mice; no such effects were noted in the lean heterozygotes, all of which had normal blood glucose levels. OB/OB mice had thymus glands which weighed less and contained significantly fewer lymphocytes than their lean littermates. In vitro mitogen-stimulated thymic lymphocyte protein synthetic rates were equal in chow-fed lean and OB/OB mice. In both groups, supplemental arginine significantly increased thymus weight, the number of thymic lymphocytes per gland, and thymic lymphocyte immunoreactivity in vitro. The hormonal secretagogue activity of arginine on the pituitary may explain its beneficial effects on the rate of weight gain, hyperglycemia, and depressed thymic immune function of OB/OB mice.

Cell-mediated immunity in genetically obese C57BL/6J ob/ob) mice.

The thymus and spleen of genetically obese (C57BL/6J ob/ob) mice weighed less and contained fewer mononuclear cells and Thy 1.2-positive lymphocytes compared with findings in lean controls. The number of splenic antibody forming cells, particularly IgG-producing lymphocytes, was reduced. There was a marked increase in natural killer cell activity and a slight increase in antibody-dependent cell-mediated cytotoxicity in the obese animals. Cytotoxic response of spleen cells of obese mice immunized in vivo was markedly lower than that of lean controls, whereas the generation of T killer cells against alloantigens after in vitro immunization was unimpaired, which points to a deleterious "microenvironment" in obesity. It is suggested that metabolic, nutritional, and endocrine changes in obesity may contribute to altered immunocompetence.