Androgens promote a higher infiltration of neutrophils to the prostate in response to bacterial stimuli

SCALERANDI MV; GARCÍA LN; LEIMGRUBER C; PEINETTI N; MALDONADO CA; QUINTAR AA

Congreso; "IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología"; 2015

It has been widely established that androgens have suppressive effects on adaptive immunity. However, there is little evidence about their role on innate immunity and the early inflammatory response. We previously reported that testosterone maintains a reduced expression of key elements for innate immunity and diminishes the antibacterial ability of the prostate. In the present study, we focused on the neutrophils, which are the main cells of innate immune system in clearing bacterial infection. We characterized the neutrophil recruitment to the prostate gland in two complimentary acute models of protatitis under different androgen status. Methodology. Male Wistar rats (3-month-old, 250 ? 350 g) were orchiectomized via the scrotal route under ketamine (80 mg⁄ kg) ⁄ xylazine (8 mg⁄ kg) anaesthesia and divided into two groups containing eight animals each. One group received s.c. testosterone (Sustanon, Organon; 2.5 mg/rat/day, group T), while the other was treated with the vehicle (group OX). These groups were further divided into 2 groups (n = 4 per group) and assigned to one of two experimental protocols detailed below. Protocol 1: the animals were anesthetized and subjected to laparotomy to expose the ventral prostate. Bacterial prostatitis (BP) was induced on the day 1 post-castration by direct inoculation of 200 ul of a solution containing uropathogenic E. coli (108 UFC/ml) (OX+BP and T+BP groups). The animals were sacrificed at 5 days after bacterial inoculation and the ventral prostates were harvested and processed for morphological analysis at both photonic and ultrastructural levels. The progression of the infection was assessed by immunocytochemistry by using an anti-E. coli antibody, and expression of the antimicrobial peptide βdefensin1. Protocol 2: the animals were inoculated with 50 ul of a solution of lipopolysaccharide (LPS, 20 mg/ml) using the same surgical procedure described above at the day 2 post-castration (OX+LPS and T+LPS groups). At 24 hours after LPS inoculation, the animals were sacrificed and the ventral prostates were harvested and processed for morphological analysis and flow cytometry. To determine to amount of neutrophils infiltrating the gland, the cells obtained after a mechanical and enzymatic digestion of the prostate were incubated with anti-granulocyte-FITC and anti-defensin1 antibodies and analysed with a Cytoron Absolute Flow Cytometer followed by data analysis with the Flowjob software. The values were expressed as mean + standard error of the mean and processed statistically using SPSS 17.0 (SPSS Inc., Chicago, Ill., USA). Student?s t test was used to compare OX+LPS versus T+LPS groups. Significant differences were considered at p < 0.05. As controls, rats subjected to the same surgical procedures were used, replacing bacterial or LPS solution by PBS. Blood sample from animals of all experimental groups were obtained by cardiac puncture to confirm the androgen status.Results. The direct inoculation of bacteria into the prostate gland caused an acute inflammatory response independently on the androgen status. However, this process was significantly influenced by the testosterone levels. At 5 days after bacterial infection, animals with physiological levels of androgens showed a significantly higher neutrophil infiltration and invasion into the prostatic acini compared to animals with low testosterone levels. However, the OX+BP group exhibited a bacterial reduction, revealing by the weak immunostaining for E. coli in the prostatic tissues. The ultrastructural analysis confirmed the presence of phagocytosed bacteria in active intracinar neutrophils in T+BP group, in line with an intense immunostaining for defensin1. On the other hand, the scarce intracinar neutrophils found in the OX+BP group evidenced apoptotic features and were free of bacteria. To determine whether an active infection is required for this differential neutrophil recruitment, E. coli LPS was used instead of the live bacterium as stimulus. In agreement with the findings observed in the bacterial model, the number of infiltrating neutrophils per gland after LPS was three times higher in animals with physiological levels of testosterone compare to those with a low androgenic status (26x104 + 2x104 vs. 68x104 + 11x104; p= 0.019). These results correlated with the largest inflammatory infiltrate observed at the photonic level in T+LPS group. No differences in the frequency of defensin1 positive cells were observed (64 + 1 vs. 67 + 3 %; p= 0.514). Conclusion. Androgens favor the recruitment of neutrophils to the site of injury regardless of whether the stimulus comes from an active bacterial infection or from a soluble specific bacterial component. However, under a normal androgenic status the higher recruitment of neutrophils was not accompanied by an efficient resolution of the infection. These findings suggest that testosterone modulates the neutrophil behavior and their ability to resolve bacterial prostatitis, which provides the basis for future research to develop alternative therapies for inflammatory and immune-related prostatic diseases based on homeostatic androgen functions.ACKNOWLEDGMENTS. We gratefully acknowledge the excellent technical assistance of Phd. Carolina Leimgruber.The authors have declared no conflict of interest.