Increased Neutrophils presence at the site of infection as a compensatory mechanism to deficiencies in neutrophils-bactericidal functions in a STZ-induced diabetic murine model

RODRIGUEZ-RODRIGUES N; MARTIRE-GRECO D; LANDONI V.I; BALBOA L; FERNÁNDEZ G C

Medellin

Congreso; Immunocolombia; 2015

ALAI

Diabetes mellitus can be considered one of the main problems of global health. From the clinical perspective, diabetes comprises a heterogeneous group of processes whose common characteristic is hyperglycemia resulting from defects in insulin secretion, usually due to autoimmune destruction of pancreatic beta cells in diabetes mellitus type 1 or a progressive resistance to the peripheral action of insulin in diabetes mellitus type 2. In both cases, the development of the disease is attributed to a combination of genetic and a number of environmental factors. Diabetic patients have a higher risk for serious chronic complications including blindness, kidney failure, amputations and cardiovascular disorders. In addition, these patients are prone to infections that can result not only recurrent but also severe. Since neutrophils (PMN) are the first line of defense against infections, and their function is altered in patients with diabetes, our aim was to study the PMN response against a peritoneal infection in a murine model of diabetes. To reproduce in mouse the state of hyperglycemia observed in diabetic patients we used the drug Streptozotocin (STZ). STZ is a naturally occurring chemical that is particularly toxic to the insulin-producing beta cells of the pancreas in mammals. Multiple low doses of STZ are largely used in medical research to produce an animal model for Type 2 diabetes. Taking this into account to develop this project mice were fasted for four hours prior to injection. 50 mg/kg of STZ per mouse were inoculated i.p. for five consecutive days. With this protocol, the animals generated a maximum hyperglycemia 30 days after the last dose of STZ inoculation (STZ group). At the same time a group of mice was inoculated with PBS (Control group). After 30 days of the last injection of STZ blood samples were collected to determine the basal functional state of PMN. Then a polymicrobial suspension containing 5x107 CFU was inoculated i.p. to mice. Four hours after bacterial challenge, blood samples were collected, and peritoneal lavages were performed after mice were sacrificed. We first decided to study PMN function under basal condition, before conducting bacterial challenge. Both the absolute number and percentage of PMN present in whole blood, measured using an hematology analyzer were increased in the STZ group (Absolute PMN x109/L, Ctrl: 0.78 ± 0.29 vs. STZ: 3.57 ± 0.25 p=0.0011, n=6; % PMN Ctrl: 6.21 ± 1.53 vs. STZ: 16.75 ± 3.76, p=0.026, n=6). Regarding activation status of PMN, no differences were observed when expression of the activation marker CD11b was contemplated, measuring the mean fluorescence intensity (MFI) by flow cytometry. Additionally, reactive oxygen species (ROS) generation was tested in whole blood of both groups, using the fluorescent dye dihydrorhodamine (DHR) by flow cytometry. We found that the percentage of PMN producing ROS (DHR+Gr-1+ CD11b+) from the STZ group was increased (% Gr-1+ CD11b+ DHR+, Ctrl: 26.9 ± 4.72 vs. STZ: 45.9 ± 4.05, p=0.016, n=6). Finally, considering that lipopolysaccharide (LPS) present in the outer membrane of gram negative bacteria is a proinflammatory stimulus, we assess the ex vivo response of PMN to LPS. For this PMN were stimulated with 1 µg/ml LPS. The expression of CD11b and ROS generation were measured in whole blood in Ctrol and STZ mice by flow cytometry after one hour. The increase in CD11b expression and ROS generation caused by the incubation with LPS were similar for both groups. Then we directed the experiments in order to study PMN function after bacterial challenge. The absolute number of PMN recovered in the peritoneal fluid was broadly higher in the STZ group (Absolute Gr-1+ CD11b+ x106, Ctrl: 0.93 ± 0.13 vs STZ: 2.9 ± 0.3, p less than 0.0001, n=22). Again, no differences were observed in the degree of activation level of peritoneal PMN measured by the MFI of CD11b. The numbers of remaining bacteria in peritoneal lavages were analyzed counting the CFU grown in McConkey agar plates. No differences in the remaining CFU were observed between groups (CFU/ml x106 Ctrl: 1.19 ± 0.11 vs STZ: 1.35 ± 0.15, n=22). This result and the fact that STZ mice needed a higher number of PMN to control the infection indicated that some mechanism of bacterial elimination may be affected. With this in mind we decided to analyze one of the most important bactericidal mechanisms of PMN, the neutrophil extracellular traps (NETs) formation, by confocal microscopy. For this, 1x105 peritoneal PMN recovered from peritoneal lavages after bacterial challenge were gently seeded onto a slide and labeled using propidium iodide to stain the DNA and anti-Elastase present in NETs. We observed that, qualitatively, the NETs in the STZ group were smaller and were present in lower proportion compared to the control group. Our results indicate that the resolution of an infection was preserved in STZ mice by increasing the number of PMN at the site of bacterial challenge. This reveals a compensatory mechanism to counteract deficiencies in PMN-mediated bactericidal functions, such NETs formation described herein.