Reduced Expression of Mucosal Antimicrobial Peptides in the Small Intestine During Acute Rejection after Orthotopic Small Bowel Transplantation

Abstract

Background: After small bowel transplantation, episodes of acute rejection are frequent and often associated with severe systemic inflammatory response and sepsis. One potential mechanism is bacterial overgrowth and subsequent translocation of microorganisms through the mucosal barrier. Expression of antimicrobial peptides constitutes a well established defense mechanism of intact intestinal mucosa against such microbial assault, alteration of these antimicrobial peptides could facilitate translocation and prove deleterious to graft and host. Methods: We analyzed the impact of small bowel transplantation and acute rejection on the expression of antimicrobial peptides in intestinal iso- and allografts of rats. For allogenic transplantation, Brown Norway intestinal grafts were transplanted into Lewis hosts (n=9) and for isogenic transplantation Lewis rats served likewise as donor and host (n=9). Graft and host mucosa was sampled at 7 d posttransplant for RT-PCR expression analysis of known antimicrobial peptides. Only laparotomy and mucosa sampling was performed in both BN and Lew rats for sham groups (n=4, respectively). Statistical significance of mRNA expression levels was determined by non-parametric testing (Kruskal-Wallis test with Bonferroni correction and Dunn's multiple comparison test on p< 0,05 level). For histologic grading of acute rejection, the Wu rejection score was applied on HE stained graft sections by two independent reviewers. Results: Histology at 7d posttransplant displayed acute rejection in allogenic grafts and residual inflammatory changes in isogenic grafts as observed by two independent reviewers (interrater reliability analysis showed substantial agreement; Cohen's κ = 0,66, p< 0,001). In RT-PCR expression analysis of host and graft mucosa normalized to β-Actin, Cryptdin 7 and neutrophil defensin 3 (NP 3) were significantly reduced in allogenic (BN to LEW) graft mucosa versus isogenic (Lew to Lew) graft mucosa (Cryptdin7: 11fold, p< 0,05/NP3: 14fold, p< 0,05 reduction respectively). Compared to sham control (laparotomy only), the Cryptdins 5+7 and NP3 were significantly reduced in allogenic graft mucosa (Cryptdin5: 18fold, p< 0,001/Cryptdin7: 17 fold, p< 0,001/NP3: 21fold, p< 0,001 reduction respectively), but not in isogenic graft mucosa. Of the other antimicrobial peptides tested (BD1+2, HIP/PAP3, CRAMP, RELM, Lysozyme), some showed a tendency towards reduced expression in allogenic grafts which did not reach statistical significance. Conclusion: A variety of important antimicrobial peptides, both of Paneth cell- (Cryptdin 5+7), and non-Paneth cell origin (NP3) were found to be significantly reduced in the setting of acute rejection after allogenic intestinal transplantation. The operative trauma as induced by isogenic intestinal transplantation failed to negatively affect the expression of antimicrobial peptides in a similar fashion. If the reduced expression and secretion of antimicrobial peptides is a causative mechanism of bacterial translocation and sepsis during acute rejection of intestinal grafts remains to be determined.