Stress response during the development of DSS-induced colitis worsens intestinal inflammation

V Ferraz-de-Paula1,2, A Ribeiro2, JR Santin1, IM Daufenback1, ML Pinheiro2, C Costola-de-Souza2, J Palermo-Neto2, SHP Farsky1. 1University of Sao Paulo, Department of Clinical and Toxicological Analyses - School of Pharmaceutical Sciences, Brazil, 2University of Sao Paulo, Neuroimmunomodulation Research Group - School of Veterinary Medicine, Brazil

Introduction: The inflammatory bowel diseases are defined as chronic inflammatory diseases causing long-term and sometimes irreversible damage to the gastrointestinal structure and function. The etiology of these diseases are not completely understood, however it has been shown that multiple factors, such as genetic, environmental, and immune response to the intestinal bacterial flora are involved. The role of environmental factors in the pathogenesis of IBD have been neglected in the past years, however it is well established a positive correlation between environmental factors, such as physical and/or psychological stressors, and the relapse of the disease. Therefore, the aim of our work was investigate the role of stress on the neutrophil migration and activity during the development of a murine model of DSS-induced intestinal inflammation.

Methods: C57Bl/6 male mice (n=6/group), weighing 20-25 g and approximately 60 days old, were divided in 4 groups: Control (C), DSS, Stress (ST) and Stress+DSS (ST+DSS). Colitis was induced by the addition of DSS [40KDa, 3.5% (wt/vol)] to the autoclaved drinking water. Mice were treated with DSS or regular water for 5 days. Mice of group C and DSS were maintained in their home cage during all protocol and mice of groups ST and ST+DSS were submitted to restraint stress protocol for 2 hours on the experimental days 1, 3 and 5 of the DSS treatment. Body weight was assessed daily during the DSS treatment period. Blood, colon and bone marrow were collected 24h after the experimental day 5. Total and differential leukocytes counts were performed in the blood and bone marrow. The length and weigh t of the colon were measured, and histological assessment of colitis was performed by HE staining. Neutrophil infiltration into colon was quantified by measuring myeloperoxidase activity (MPO). All experiments were approved by “Ethics Committee in the use of Animals” of the School of Veterinary Medicine and Animal Science of University of Sao Paulo (Protocol nº2403). Data (mean±sem) sets were examined by one-way ANOVA, a P value <0.05 was considered significant.

Results: No changes were observed in food and water intake between the groups. We observed a decrease of the % of body weight (C:-0.9446±0.6559, ST:-2.686±0.9469, DSS:-5,250±1,865, ST+DSS:-5,467±1,501) and colon length (C:8.5±0.3, ST:7.6±0.2, DSS:5.8±0.2, ST+DSS:5.1±0.1), associated to an increase in the colon length/weight ratio (C:0.025±0.001, ST:0.026±0.001, DSS:0.035±0.001, ST+DSS:0.046±0.003) in the groups DSS and ST+DSS when compared to groups C and ST (P < 0.05). Additionally, we observed that stress was able to enhance these effects in the group ST+DSS when compared to group DSS (P < 0.05). No difference was found in the total cellularity of blood and bone marrow, but we observed an increase of neutrophil blood count in the experimental groups when compared to C group (C:1.6±0.1, ST:2.2±0.2, DSS:2.8±0.1, ST+DSS:2.9±0.3) and a decrease of granulocytes from the bone marrow in the groups DSS and ST+DSS (P < 0.05) (C:1.7±0.1, ST:2.3±0.1, DSS:1.2±0.1, ST+DSS:1.2±0.1) compared to ST group (P < 0.05). Stress augmented neutrophil infiltration in the colon in the group ST+DSS when we compared to group DSS (P < 0.05), which was demonstrated by the increase of MPO activity (C:2.2±0.7, ST:0.6±0.4, DSS:93±30, ST+DSS:191±23) and histological analyses.

Conclusion: Altogether these results show that stress during the development of DSS-induced colitis is able to worsen the acute colitis by augmenting neutrophil migration. Currently more studies are in progress in order to understand this phenomenon.