?Increased PMN presence at the site of infection as a compensatory mechanism todeficiencies in PMN-bactericidal functions in a STZ-induced diabetic murine model

RODRIGUEZ RODRIGUES, NAHUEL; MARTIRE GRECO D; LANDONI V.I; BALBOA L; FERNANDEZ G.C

Medellin

Congreso; Inmunocolombia; 2015

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 by 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 turn, these patients are more likely to have urinary tract infections, respiratory soft tissue infections and periodontal diseases.Since 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 the mouse the state of hyperglycemia observed in diabetic patients we used the Streptozotocin (STZ) drug. 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. 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, mice were sacrifice, blood samples were collected and peritoneal lavages were performed. Both the absolute number and percentage of PMN present in whole blood before bacterial challenge, measured using a 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). No differences were observed when the expression of the activation marker CD11b was contemplated, measuring the mean fluorescence intensity by flow cytometry.Additionally, reactive oxygen species (ROS) generation in whole blood of both groups was tested prior to bacterial challenge, using the fluorescent dye dihydrorhodamine (DHR) by flow cytometry. We found that in basal conditions, the % of DHR+ PMN (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). In order to assess the response of PMN to ex vivo stimulation with LPS (1 µg/ml), the expression of CD11b and ROS generation were measured in whole blood in Ctrol and STZ mice. The increase in CD11b expression and ROS generation caused by the incubation with LPS were similar for both groups.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