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Hyperbaric oxygen treatment ameliorates the course of DSS-induced colitis in BALB/c mice and reduces the Foxp3+ regulatory T cell expansion

Several studies in both animals and men showed beneficial effects of hyperbaric oxygenation (HBO2) on the course of experimentally induced colitis and inflammatory bowel disease. However, underling mechanisms of its action remain elusive. It was found that adoptive transfer or induction of CD4+CD25+FoxP3+ regulatory T cells (Treg) can prevent disease and reverse the course of established colitis (1). In addition, recent findings suggest that the mesenteric lymph nodes (mLN) are an important site of Treg induction (2). In the present study, we have assessed the effects of HBO2 on the frequencies of Treg in mLN of mice with DSS-induced colitis. BALB/c mice at the age of 10-12 weeks were randomized into 4 groups (n=9 mice): control mice, control mice undergoing HBO2, mice receiving dextran sodium sulphate (DSS) and DSS treated mice undergoing HBO2. Mice were fed 5% w/v of DSS (Mr 36.000-50.000, MPBomedicals, France) in drinking water ad libidum for 7 consecutive days. HBO2 was initiated at day 1 and was administered until the end of experiment, twice a day 100% O2 for 60 minutes at 2.3 bars with addition of 15 minutes for gradual compression and decompression. Disease activity (DAI) was assessed by daily recording of animal body weight, stool consistency and the presence of occult or gross blood per rectum. On the day 9 mice were scarified by cervical dislocation and their mLN collected and further processed for flow cytometric assessment of Treg. The data are presented as mean±SEM, and were compared by student t test and one-way ANOVA. The study was approved by the Ethical Committee of Faculty of Medicine Osijek. Mice treated with DSS exhibited severe symptoms of colitis, including diarrhea and gross rectal bleeding starting from day 6. DSS treated mice that underwent HBO2 presented with occult bleeding at day 8 and only a loose stool starting at day 7. DSS treated mice have lost 14.8±1.9% of body weight, whereas the mice which additionally underwent HBO2 lost 5.9±3.4% body weight. There was a significant difference in disease severity between those two groups, as evidenced by DAI on days 6-8 (p<0.001, Figure 1). Frequency of Treg in mLN increased significantly in DSS treated mice (p=0.026), and was reduced to control values after HBO2 (p=0.03, Figure 2). DSS induced inflammation of the colon resulted in increased frequencies of Treg cells in the mLN. Consistent to previous studies HBO2 significantly reduced symptoms of disease and severity of colitis, which resulted in reversed frequencies of Treg cells, at comparable levels with those found in control mice. Thus our results suggest that the improved course of DSS-induced colitis in BALB/c mice does not involve Treg expansion.

Foxp3+ regulatory T cell; hyperbaric oxygen; colitis

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