Increased Recruitment but Impaired Function of Leukocytes during Inflammation in Mouse Models of Type 1 and Type 2 Diabetes

BACKGROUND: Patients suffering from diabetes show defective bacterial clearance. This study investigates the effects of elevated plasma glucose levels during diabetes on leukocyte recruitment and function in established models of inflammation. METHODOLOGY/PRINCIPAL FINDINGS: Diabetes was induced in C57Bl/6 mice by intravenous alloxan (causing severe hyperglycemia), or by high fat diet (moderate hyperglycemia). Leukocyte recruitment was studied in anaesthetized mice using intravital microscopy of exposed cremaster muscles, where numbers of rolling, adherent and emigrated leukocytes were quantified before and during exposure to the inflammatory chemokine MIP-2 (0.5 nM). During basal conditions, prior to addition of chemokine, the adherent and emigrated leukocytes were increased in both alloxan- (62±18% and 85±21%, respectively) and high fat diet-induced (77±25% and 86±17%, respectively) diabetes compared to control mice. MIP-2 induced leukocyte emigration in all groups, albeit significantly more cells emigrated in alloxan-treated mice (15.3±1.0) compared to control (8.0±1.1) mice. Bacterial clearance was followed for 10 days after subcutaneous injection of bioluminescent S. aureus using non-invasive IVIS imaging, and the inflammatory response was assessed by Myeloperoxidase-ELISA and confocal imaging. The phagocytic ability of leukocytes was assessed using LPS-coated fluorescent beads and flow cytometry. Despite efficient leukocyte recruitment, alloxan-treated mice demonstrated an impaired ability to clear bacterial infection, which we found correlated to a 50% decreased phagocytic ability of leukocytes in diabetic mice. CONCLUSIONS/SIGNIFICANCE: These results indicate that reduced ability to clear bacterial infections observed during experimentally induced diabetes is not due to reduced leukocyte recruitment since sustained hyperglycemia results in increased levels of adherent and emigrated leukocytes in mouse models of type 1 and type 2 diabetes. Instead, decreased phagocytic ability observed for leukocytes isolated from diabetic mice might account for the impaired bacterial clearance

Helicobacter pylori and Gastric Protection Mechanisms : An in vivo Study in Mice and Rats

The stomach is frequently exposed to hazardous agents and to resist this harsh environment, several protective mechanisms exist. Of special interest is the gastric pathogen Helicobacter pylori which causes gastritis, ulcers and cancer but the mechanism leading to these diseases are still unclear. However it is very likely that H. pylori negatively influence the protection mechanisms that exist in the stomach. The aims of the present investigation were first to develop an in vivo mouse model in which different protection mechanisms could be studied, and second to investigate the influence of H. pylori on these mechanisms. An in vivo preparation of the gastric mucosa in mice was developed. This preparation allows studies of different gastric mucosal variables and can also be applied for studies in other gastro-intestinal organs. Mice chronically infected with H. pylori, were shown to have a reduced ability of the mucosa to maintain a neutral pH at the epithelial cell surface. This could be due to the thinner inner, firmly adherent mucus gel layer, and/or to defective bicarbonate transport across the epithelium. The Cl-/HCO3- exchanger SLC26A9 was inhibited by NH4+, which also is produced by H. pylori. The mRNA levels of SLC26A9 were upregulated in infected mice, suggesting a way to overcome the inhibition of the transporter. Furthermore, the hyperemic response to acid pH 2 and 1.5 was abolished in these mice. The mechanisms by which the bacteria could alter the blood flow response might involve inhibition of the epithelial iNOS. Water extracts of H. pylori (HPE) reduces the blood flow acutely through an iNOS and nerve-mediated pathway, possibly through the endogenous iNOS inhibitor ADMA. Furthermore, HPE alters the blood flow response to acid as the hyperemic response to acid pH 0.8 is accentuated in mice treated with HPE

Acute effects of Helicobacter pylori extracts on gastric mucosal blood flow in the mouse

AIM: To investigate the mechanisms underlying the reduction in gastric blood flow induced by a luminal water extract of Helicobacter pylori (HPE)

Changes in energy metabolism due to acute rotenone-induced mitochondrial complex I dysfunction – An in vivo large animal model

Metabolic crisis is a clinical condition primarily affecting patients with inherent mitochondrial dysfunction in situations of augmented energy demand. To model this, ten pigs received an infusion of rotenone, a mitochondrial complex I inhibitor, or vehicle. Clinical parameters, blood gases, continuous indirect calorimetry, in vivo muscle oxygen tension, ex vivo mitochondrial respiration and metabolomics were assessed. Rotenone induced a progressive increase in blood lactate which was paralleled by an increase in oxygen tension in venous blood and skeletal muscle. There was an initial decrease in whole body oxygen utilization, and there was a trend towards inhibited mitochondrial respiration in platelets. While levels of succinate were decreased, other intermediates of glycolysis and the TCA cycle were increased. This model may be suited for evaluating pharmaceutical interventions aimed at counteracting metabolic changes due to complex I dysfunction

Increased weight, blood glucose and insulin, and impaired glucose tolerance in high fat diet (HFD) mice.

<p><b>A</b>, Weight gain in control mice (white, n = 7) and mice fed a HFD (black, n = 5) and <b>B</b>, plasma glucose concentrations in mice on control (white) or high fat diet (black) over time. <b>C</b>, Serum insulin concentrations in mice after 15 weeks of control or high fat diet. <b>D</b>, Glucose tolerance test in control or high fat fed mice presented as area under curve (AUC) of plasma glucose levels×time, 15 weeks after diet start. *<i>p</i><0.05. Data are means ± SEM.</p

Decreased phagocytic ability of leukocytes in diabetic mice.

<p>Percentage of phagocytic leukocytes phagocytizing LPS-coated fluorescent beads in the peritoneal cavity in control (n = 5) and alloxan-treated diabetic (n = 4) mice. *<i>p</i><0.05. Data are means ± SEM.</p

Increased leukocyte recruitment to MIP-2 in diabetic mice.

<p>The number of adherent (<b>A</b>) and emigrated cells (<b>B</b>) in the cremaster muscle of control (white, n = 6), alloxan-treated (dark grey, n = 6), high fat diet-induced diabetic (light grey, n = 5) and acute glucose-infused (black, n = 4) mice before (time 0) and after MIP-2 addition to the superfusate (0.5 nmol/L). *<i>p</i><0.05 compared to other groups at the same time point. †p<0.05 compared to time 0 in the same group. Data are means ± SEM.</p