Interleukin-28A Attenuates Intestinal Ischemia Reperfusion Injury By Maintaining The Intestinal Barrier Function

BackgroundIntestinal ischemia reperfusion(I/R)occurs frequently with numerous pathological processes,such as trauma,hemorrhagic shock,obstruction of the intestine,sever burns,small bowel transplantation and other major surgeries.Acute small intestinal I/R induces severe epithelial damage and increases intestinal permeability.Loss of intestinal epithelial barrier function leads to bacterial translocation,resulting in systemic inflammation and multiple organ failure with high mortality.The underlying mechanisms of intestinal I/R injury are very complicated,and accumulating studies show that intestinal barrier dysfunction plays a critical role in intestinal I/R.The intestinal epithelial barrier(IEB)is an important defense barrier between the lumen and the intestinal mucosa.The barrier comprises an intact single-cell layer of intestinal epithelial cells(IECs)and tight junctions(TJs)between the IECs.TJs,a group of transmembrane proteins such as claudins,occludin,and zonula occludens(ZO),are located at the apical membrane and regulate the passage of ions,nutrients and water.Evidence has suggested that the destruction of expression or distribution of TJs is responsible for the increase in intestinal permeability and dysfunction of the intestinal epithelial barrier caused by intestinal I/R.Interleukin-28A(IL-28 A or interferon-λ2),belonging to type III IFNs,is reported to be involved in the maintenance of intestinal mucosal homeostasis.However,the mechanisms are still not entirely clear.A recent study showed that IL-28 A regulates the proliferation of IECs in mice with colitis and accelerates intestinal mucosal healing.Additionally,the administration of exogenous IL-28 can maintain the endothelial junctions in the blood-brain barrier.However,little is known about the role of IL-28 A in intestinal epithelial barrier function.In this study,we established a mouse model of intestinal I/R and a hypoxic Caco-2 cell culture model to explore the role and mechanism of IL-28 A in intestinal I/R and intestinal epithelial barrier function,using Western blot,immunohistochemistry,immunofluorescence staining,transepithelial electrical resistance(TER)and so on.Methods1.Adult C57BL/6 mice were randomly divided into three groups: Sham,I/R and I/R +IL-28 A.The mice in I/R + IL-28 A group were given injections of recombinant mouse IL-28 A for 12 h before the operation.Mice were sacrificed 6 h after reperfusion.The expression of IL-28 A in the Sham and I/R groups was assessed by Western blot and immunofluorescence staining.The mucosal permeability was investigated by the permeability tracer fluorescein isothiocyanate(FITC)-dextran 4000.The small intestines were collected and photographed.Histology analyses were performed by hematoxylin-eosin(H&E)staining and Chiu’s score.2.The expression and distribution of TJ proteins(ZO-1、occludin and claudin-1)were examined by Western blot and immunohistochemical staining.The expression of signal transducer and activator of transcription 1(STAT1)and p STAT1 was assessed by Western blot and immunofluorescence staining.3.In vitro,a hypoxic Caco-2 cell culture model was established with or without IL-28 A treatment.Epithelial permeability was measured by transepithelial electrical resistance(TER).The expression of TJ proteins and STAT1/p STAT1 was assessed by Western blot.The expression and distribution of claudin-1 was examined by immunofluorescence staining.4.Fludarabine was used to inhibit p STAT1 with or without IL-28 A treatment in vitro.The expression of TJ proteins and STAT1/p STAT1 was assessed by western blot.The expression and distribution of claudin-1 was examined by immunofluorescence staining.Results1.The cells expressing IL-28 A were mainly in the lamina propria,and I/R significantly decreased the number of IL-28A-positive cells.The expression of IL-28 A protein in the small bowel after intestinal I/R was obviously decreased compared with that of the sham group.2.Administration of IL-28 A significantly attenuated the I/R-induced edema and hyperemia of the intestinal tract.The histological examination showed that I/R caused short villi,shedding of the epithelial surface,loss of entire crypts and immune cell infiltration,while IL-28 A pretreatment attenuated these effects.3.I/R led to an increased FITC-dextran level in the serum compared with that of the sham group,while IL-28 A pretreatment partially rescued this effect.IL-28 A treatment significantly attenuated intestinal I/R-induced disruption of TJ proteins,including zonula occludens-1(ZO-1),occludin and claudin-1.Western blot showed that IL-28 A prevented the reduction in claudin-1,although it had no effect on the expression of ZO-1 and occludin.4.In vitro,IL-28 A treatment reversed the decrease in TER of Caco-2 monolayers exposed to hypoxic environments.Western blot analysis showed that hypoxia obviously decreased the expression of ZO-1,occludin,and claudin-1.Although it did not affect the expression of ZO-1 and occludin,IL-28 A treatment up-regulated the expression of claudin-1both under normoxia and hypoxia conditions.5.IL-28 A led to the activation of STAT1 both in vivo and in vitro.Fludarabine successfully inhibited STAT1 phosphorylation with or without IL-28 A treatment.Blocking STAT1 phosphorylation decreases the expression of claudin-1 and counteracts the IL-28A-mediated upregulation of claudin-1 expression,although the expression of ZO-1 and occludin was not altered.Conclusions1.IL-28 A was mainly expressed in intestinal lamina propria and I/R caused a downregulation of IL-28 A in the small intestines of mice.IL-28 A maintained intestinal barrier function and relieved intestinal damage caused by intestinal I/R.2.IL-28 A maintained the distribution of intestinal epithelial tight junctions and upregulated the expression of claudin-1 both in an intestinal I/R injury model and a hypoxia-challenged Caco-2 cell model.3.Inhibiting phosphorylation of STAT1 reversed the effects of IL-28 A on the expression and distribution of claudin-1 in Caco-2 cells.The protective roles of IL-28 A in the intestinal barrier are through or at least partly through the JAK-STAT1 signaling pathway.