Interleukin-22 Regulates Intestinal Epithelial Cell Stemness And Differentiation

Background and Objective:As a part of the digestive system,the small intestine is an important organ for food digestion and nutrients absorption.The inner wall of small intestine is folded.Each villus is surrounded by some concave crypts where the intestinal stem cells are located at the basement of crypts.Under normal physiologic condition,intestinal stem cells produce the transit amplifying(TA)cells that differentiate into enterocytes,goblet cells and enteroendocrine cells along the crypt-villus axis.Under pathological condition,constant self-renewal and repair of intestinal epithelium depends on the vitality of intestinal stem cells.Once the small intestinal epithelium gets damaged,quiescent stem cell will be activated,and the differentiation of intestinal stem cells will be accelerated to help repair the wounds.Interleukin-22(IL-22)is a cytokine secreted by Thl7,Th22,NK cells and some other immune cells.It functions in the digestive tract,respiratory and skin cells,especially in epithelial cells.IL-22 promotes the inflammatory response on one hand,and helps to repair tissue damage on the other hand.However,it's still unclear whether IL-22 aggravates or relieves inflammatory in DSS mouse model.In vitro culture of enteroids is used as experimental object in this study.We aim to explore the influence of IL-22 on small intestinal stem cell sternness and differentiation and find out the underlying mechanism as far as possible.Method:Recombinant IL-22 was added into the medium during the procedure of 3D culture of intestinal stem cells in vitro,and further testing experiment was carried out after sample collection.Lgr5-EGFP-IRES-CreERT2 mice were injected intraperitoneally with interleukin-22 to observe whether in vivo influence would be caused by interleukin-22.The small intestine tissues were embedded in paraffin and O.C.T compound,and mice were injected intraperitoneally with EdU in advance for proliferative detection.Hematoxylin-eosin(HE)staining and EdU staining were carried on after tissue slides being prepared.The condition of cell death was observed by staining with propidium Iodide(PI).CellEventTM Caspase-3/7 staining was used to identify whether cell apoptosis was induced by interleukin-22.The enteroids were collected for RNA extraction,and qRT-PCR analysis was used for detecting the expression of related genes at the transcriptional level later.Chemical inhibitors of JAK-STAT3 and Notch signaling were used to confirm the roles of related signaling pathways.Results:Less small intestinal organoids were formed in vitro after IL-22 treatment,and morphological abnormalities were caused by interleukin-22 during the procedure of in vitro 3D culture of small intestinal stem cells.Although EdU staining showed an increased number of proliferating cells in enteroids treated with IL-22,results of PI staining showed a significant increase in necrosis cells of enteroids after prolonged treatment of IL-22.CellEvent TM Caspase-3/7 staining furtherly revealed that IL-22 accelerated the proliferation of organoid cells and increased the production of apoptotic cells.The results of Real-Time qPCR showed that the expression of Lgr5,Ascl2 and Oflm4 in IL-22 treated group was significantly decreased.The expression of Alpi and Villin in the epithelial cells were also significantly decreased.JAK-STAT3 pathway inhibitors can alleviate IL-22-induced apoptosis of enteroids.Notch signaling pathways,including Jagl,Dlll,Notchl,Hesl and some other related gene expression were also significantly reduced.There was no significant change in the length of small intestine of IL-22 mice.Paraffin section with HE staining showed that the small intestinal epithelium was damaged and the number of small intestine crypts was significantly reduced.The fluorescent density and number of Lgr5-EGFP+ stem cells isolated from Lgr5-EGFP-IRES-CreERT2 mice injected with IL-22 were both weakened.Conclusions:In the enteroids model,transient IL-22 treatment can promote the proliferation of small intestinal epithelial cells,but long-term IL-22 treatment can lead to the number reduction and sternness loss of Lgr5+ small intestinal stem cells.IL-22 can cause small intestinal epithelial structure damage,and lead to intestinal epithelial cell proliferation activity weakened in vivo.Small intestine crypts morphological changes and crypts number reduction are also induced by IL-22.IL-22 may reduce the sternness of Lgr5+ small intestinal stem cells,and inhibit the differentiation of progenitor cells into mature absorption epithelial cells by suppressing Notch signaling by JAK-STAT3.Therefore,the transient expression of IL-22 may promote the repair of small intestinal epithelial injury,but long-term high expression of IL-22 can lead to Lgr5+ stem cell stemness loss and differentiation abnormality,which will be detrimental to small intestinal epithelium self-renewal and injury repair.