Effects And Mechanisms Of Antimicrobial Peptide CWA On Intestinal Inflammation And Intestinal Barrier Functions In Weaned Piglets

Weaning stress causes intestinal disorders and low immunity, and leads to diarrhea, decreased growth performance, and death in weaned piglets, seriously affecting the healthy development of pig farming. Although many factors affect diarrhea, intestinal inflammation and barrier function damages are leading causes of diarrhea in weaned piglets. A lot of antibiotics have been used to treat or prevent diarrhea in weaned piglets, leading to bacterial resistance, drug residues and environmental pollution. In addition, antibiotics disrupt gut homeostasis and damage intestinal barrier function. Antimicrobial peptides (AMPs) are important molecules in innate immunity. Because of broad-spectrum antimicrobial activity, membrane-disrupting mechanism and not easy to develop bacterial resistance, AMPs attract many attention. New findings show that AMPs not only regulate immune function, but also improve intestinal epithelial barrier function. However, whether AMPs could be applied for the prevention and treatment of diarrhea and what effects of AMPs on intestinal inflammation and epithelial barrier function in weaned piglets are still unknown. In our previous studies, the antimicrobial peptide Cathelicidin-WA (CWA) has showed high antimicrobial activity and low hemolysis. Thus, in this study, we will reveal effects of CWA on the prevention and treatment of diarrhea in weaned piglets, and then investigate immunoregulatory and epithelial barrier protective properties of CWA in the intestine of weaned piglets, and further examine effects of CWA on intestinal inflammation and impaired barrier function in weaned mice infected by Enterhemorrhagic Escherichia coli (EHEC) O157:H7, and at last explore mechanisms of inhibiting intestinal inflammation and protecting intestinal barrier function by CWA in vitro. The main findings are as follows:1 Effects of CWA on intestinal inflammation and barrier function in weaned pigelts(1) Effects of CWA on intestinal inflammtion in piglets during weaningA total of 18 healthy piglets were weaned at 21 d and then treated with saline (control) and CWA by intraperitoneal injection. Piglets were fed for 7 d after injection. We found that CWA treatment significantly increased growth performance and reduced diarrheal index in weanling piglets. Serum IgG, IL-6, IL-22 and TNF-α production were decreased by CWA treatment. Compared with the control, CWA significantly increased the villus height and the villus height:crypt depth ratio in the jejunum (P＜0.05), and decreased the crypt depth (P＜0.05) and increased the villus height:crypt depth ratio in the ileum (P＜0.05). We demonstrated that CWA inhibited the expression of the inflammatory cytokines TNF-α, IL-6 and IL-8 but increased the expression of the anti-inflammatory cytokine IL-10 in the intestine, especially in the jejunum. We found that CWA reduced gene expression levels of TLR4 and NF-κB in the jejunum (P＜0.05). We also demonstrated that CWA suppressed inflammation by down-regulating the nuclear factor-κB (NF-κB) signaling pathway in the jejunum during weaning.(2) Effects of CWA on intestinal inflammation and barrier function in weaned piglets with diarrheaA total of 108 piglets with clinical diarrhea were selected and treated with saline (control), enrofloxacin (Enro) or CWA for 4 days. The objective of this study was to investigate whether the CWA could be an effective therapy for diarrhea during weaning and to explore the immunoregulatory and epithelial barrier protective properties of CWA in the intestine of weaned piglets with clinic diarrhea. Both CWA and Enro effectively attenuated diarrhea and improved the growth performance in weaned piglets with diarrhea. Compared with the control, CWA decreased IL-6, IL-8 and IL-22 levels and reduced neutrophil infiltration into the jejunum. CWA inhibited inflammation by down-regulating the TLR4-, MyD88- and NF-κB-dependent pathways. Additionally, CWA improved intestinal morphology by increasing villus and microvillus heights and enhancing intestinal barrier function by increasing tight junction (TJ) protein expression. CWA also improved microbiota composition and increased short-chain fatty acid (SCFA) levels in feces. By contrast, Enro not only disrupted the intestinal barrier but also negatively affected microbiota composition and SCFA levels in the intestine. In conclusion, CWA effectively attenuated inflammation, enhanced intestinal barrier function, and improved microbiota composition in the intestines of weaned piglets with diarrhea.2 Effects of CWA on intestinal inflammation and barrier function in E.coli-infected miceA total of 36 weaned mice were divided into control group, E.coli group, E.coli+Enro group, E.coli+CWA group. Mice infected with EHEC 0157:H7 were treated by intraperitoneal injection with saline, Enro or CWA for 4 times. We found that CWA and Enro improved the survival rate and the body weight of mice infected by E.coli. CWA and Enro relieved villous atrophy and crypt hyperplasia in the small intestine and colonic epithelial damages caused by E.coli infection. CWA and Enro decreased levels of TNF-a, IL-6, and IL-10 in serum and colon, and decreased the level of IL-6 in the jejunum in E.coli-infected mice. In terms of antimicrobial proteins in intestines, CWA and Enro decreased REG3y expression in the jejunum and Remlβ expression in the colon (P<0.05), but CWA increased TFF3 expression in the jejunum and colon (P＜0.05), indicating that CWA could improve the repair function of the intestinal epithelium. CWA and Enro increased goblet cells and the mucus layer in the jejunum while decreased the goblet cells in the colon in E.coli-infected mice. Meanwhile, the results of Western blot and immunofluorescence demonstrated that CWA and Enro alleviated the reduction of MUC-2 expression in the jejunum and the increases of MUC-2 expression in the colon caused by E.coli infection. Furthermore, CWA and Enro increased the ZO-1 expression in the jejunum in E.coli-infected mice, and CWA also alleviated the reduction of occludin expression in the jejunum caused by E.coli infection. In intestinal microflora and SCFAs concentrations, although there are no differences on total bacteria, bifidobacterium, or bacillus in cecal contents among groups, CWA and Enro alleviated the increases of Escherichia and the decreases of Lactobacillus in cecal contents caused by E.coli infection. CWA also alleviated the reduction of Lactobacillus/Escherichia coli caused by E.coli infection. Further, CWA and Enro showed improvement effects on SCFAs concentrations in cecal contents of E.coli-infected mice, but the effects were not significant. These data further demonstrated that CWA attenuated inflammation and enhanced barrier function in the intestine.3 Mechanisms of CWA on the regulation of intestinal inflammation and epithelial barrier function(1) Mechanisms of CWA on the regulation of intestinal inflammationWith LPS-induced inflammation in porcine macrophage, we found that Enro had no effects on LPS-induced expression of the pro-inflammatory cytokine IL-6 in porcine macrophages, but CWA inhibited IL-6 production in a concentration-dependent manner. Meanwhile, CWA alleviated the pro-inflammatory cytokines IL-6, IL-8, and IL-22 expressions and the levels of the phosphorylation of NF-κB and IκB-α in LPS-induced macrophages. Furthermore, using siRNA-mediated knockdown, we found that CWA had no significant effects on LPS-induced IL-6 expression in MyD88-silenced or TLR4-silenced macrophages. Interestingly, CWA dramatically activated the phosphorylation of STAT-1, which partly coincided with the expression sites of Mac2 in the jejunum of weaned piglets, indicating that CWA could affect functions of intestinal macrophages. Using FITC-labeled dextran and flow cytometry, we demonstrated that CWA could enhance the phagocytosis of macrophages. With a selective STAT-1 inhibitor (Fludarabine), we demonstrated that CWA enhanced macrophage via STAT-1-dependent manner. These results suggest that mechanisms of CWA alleviating intestinal inflammation may be through inhibiting inflammation by down-regulating TLR4-MyD88-NF-KB signaling pathway and enhancing macrophage phagocytosis by STAT-1 pathway.(2) Mechanisms of intestinal epithelial barrier function improved by CWAUsing Caco-2 monolayer cells, we studied effects of CWA on the repair capabilities in damaged intestinal epithelial cells. The results showed that CWA increased the wound healing and significantly reduced the wound width of Caco-2 monolayer cells at 48 h, indicating that CWA improve the repair capabilities of damaged intestinal epithelial cells. In addition, using porcine jejunal epithelial cells (IPEC-J2), we found that CWA alleviated the decline of TER values in IPEC-J2 monolayer cells caused by LPS. Meanwhile, CWA alleviate the decline of occludin and ZO-1 expression in epithelial cells induced by LPS. Furthermore, with Racl selective inhibitor NSC 23766, we studied whether the regulation of TJ proteins by CWA mediated by Racl. The results showed that CWA improved ZO-1 and occludin expression in IPEC-J2 cells, but the enhanced ZO-1 and occludin expression and reduced EHEC 0157:H7 adhesion by CWA were inhibited by NSC 23766 in IPEC-J2 cells. These results suggested that CWA can improve the repair function of damaged intestinal epithelial cells, and enhance the intestinal epithelial barrier function by up-regulating tight junction protein via Racl activation.This study not only provides basic data and guidance for applying antimicrobial peptide in the prevention and treatment for weaning diarrhea and reducing the use of antibiotics in pigs, but also provides new therapeutic strategies for other intestinal diseases caused by intestinal inflammation and intestinal barrier function damages in human or animals.