A depleção de células TREGS agrava a mucosite intestinal experimental induzida por irinotecano

Abstract

Intestinal mucositis (IM) is a common side effect related to anti-cancer chemotherapy based on irinotecan. MI is characterized by severe diarrhea and significantly affects patients' quality of life, increasing morbidity and health costs. Up to 80% of irinotecan-treated patients develop some degree of MI. The pathophysiology of this side effect is not fully understood and, unfortunately, there is no specific and effective treatment. The investigation of pathophysiological mechanisms may potentially guide the discovery of biomarkers or new therapeutic strategies. Previous studies point to the involvement of inflammatory molecules and cells, such as nitric oxide, COX-2, KC (analogous to human IL-8), TNF-α, IL-1, IL-18 and IL-33, toll-like receptors and neutrophils in the pathogenesis of MI in murine models. However, the participation of other innate and adaptive immune system cells, regulated in part by these cytokines, has not been investigated yet. Aim: To identify the expression profile of Tregs, Th7, Th (non-reg and non-Th17 cells) and type 3 innate lymphoid cells along the development of irinotecan-induced IM and to evaluate the role of Tregs in the pathogenesis. Methods: C57BL/6 (20-25g) received saline or irinotecan (75 mg/kg, i.p.), once daily/4 days, for induction of MI. Euthanasia occurred on days 1 (D1), D3, D5 or D7. For depletion of the Tregs the mice were pretreated with cyclophosphamide (100 mg/kg), 2h before the first dose of irinotecan, and euthanized on D7. Lymphocytes from intestinal lamina propria were isolated by enzymatic digestion and percoll gradient and splenic cells by mechanical digestion. The frequency of different lymphocyte subpopulations was identified by flow cytometry. On the day of euthanasia, a peripheral blood sample was collected for blood leukocyte count (x 103/l) and ileum samples for MPO, cytokine and histopathological assay. Results: The mice with MI had weight loss (P<0.05), leucopenia (P<0.05), diarrhea (P <0.05), intestinal damage (P<0.05) and reduction of villus/crypt ratio (P<0.05). In addition, irinotecan injection induced the increase of the % of Tregs (P<0.05) and % of Th17 cells (P<0.05), intestinal and splenic. The ratio of intestinal %Treg / Th17 correlated positively with severity parameters, such as diarrhea (P<0.05) and histopathological (P<0.05) scores of mice. There was a negative correlation between % Treg / Th17 splenic and these same parameters (P<0.05). Tregs depletion aggravated MI, increasing weight loss (P<0.05), diarrhea scores (P<0.05), mortality (100%) and reducing villus/crypt ratio (P<0, 05). The number of intestinal neutrophils increased after irinotecan injection, with the peak on D5 (P<0.05). In D7, there was a reduction in the number of these cells vs D5 (P<0.05). In addition, there was a negative correlation between the number of neutrophils and the % of Tregs (P<0.05). The frequency of type 3 innate lymphoid cells (P<0.05) and other Th cells, non-Treg and non-Th17 cells (P<0.05), decreased after irinotecan injection and were positively modulated after Tregs depletion. In addition, irinotecan injection elevated TNF-, IL-1, KC and IL-17A cytokines in the gut (P<0.05). With Tregs depletion, the levels of KC (P <0.05) and TNF- (P<0.05) were potentialized, and there was a reduction of IL-10 (P<0.05). Conclusion: Tregs cells are important to counterbalancing inflammation in irinotecan induced-IM.