Characterization of acute cellular rejection in the different layers of rat transplanted intestines.

STRINGA, P; ROMANIN, D; LAUSADA, N; GOBI, RODRIGO PAPA; VECCHIO, L; GONDOLESI, G; RUMBO, M

Madrid

Congreso; 27th international congress of the transplantation society; 2018

The transplantation Society

NTRODUCCIONIntestinal transplantation (IT) faces many challenges, among them, the necessity to understand and early detect rejection processes. Rodent models of IT are used to provide evidence for intervention strategies as well as improve knowledge of small bowel transplant biology. Theaim of our study was to determine the kinetics of small bowel rejection using a Heterotopic IT (HIT) model, with emphasis in the characterization of acute cellular rejection (ACR) phenomenon in the different layers of the transplanted intestine, since most of the information coming from the clinics is on mucosal layer, the only one that is accesible by endoscopic biopsies.METHODSAllogeneic (ALLO) HIT was performed following standard procedure and rejection was monitored by clinical scoring and a descriptive evaluation of hematoxylin-eosin staining intestinal grafts. Also, real time PCR from microdissected samples (epithelial, muscular and serosa layers) or whole intestinal graft to determine gene expresion was performed at 5 and 10-12 post operative (POD) after HIT. In addition, isogenic HIT group was performed as a control group.RESULTSGraft lesions compatible with ACR were observed since 5 POD in ALLOgroup. Mild rejection was the most characteristic grade. The remaining samples showed indeterminate ACR and no signs of ACR. On the other hand, severe ACR was diagnosed in all ALLO samples taken at 10-12 POD. Considering the descriptive analysis, samples showed a well preserved architecture at 5 POD. Also, confluent and loose apoptotic cells in the intestinal epithelium and perivascular infiltrate in mesenteric vessels and muscular and serosa layers were evident. Lesions along the transplanted intestines were seen heterogeneously at the late phase of ACR. Significant cellular infiltrate, epithelial damage, absence of enterocytes in some areas of the graft villi, ulcerated areas and necrosis zones, and increase of apoptotic cells in the entire graft were observed. The muscular layer showed inflammatory cell infiltrate and increased intercellular edema. Histological changes at the serosa layer were like those reported at 5 POD, with the addition of histological signs compatible with fibrosis.At 5 POD, some markers were consistently increased in ALLO group when compared with ISO controls such as CXCL10 that showed a 120 ± 80-fold increase compared with nontransplanted tissue. Furthermore, IFNg and IDO, which are known to be dependent on IFNg stimulation,showed a trend to be increased at these timepoints. Remarkably, other inflammation-related genes, such as CXCL1 and IL6 showed consistent increase in ALLO groups at 10 to 12 POD, when severe rejection process was established. Interestingly, when a principal component analysis of the overall gene expression markers was performed, ALLO group at 10 to 12 POD clearly separated from the other conditions (ALLO group at 5 POD and ISO group at bothearly and late time points), which were group together, indicating that major changes in gene expression of the immune related genes evaluated was altered mainly in subsequent stages of ACR. We wanted to determine if there were differences at the gene expression level among different graft tissue layers during ACR. To this aim, we recovered serosa layer, muscular layer, and epithelial layer using laser dissection microscopy measured gene expression by qPCR. The overall picture coming from this analysis was coincident with whole tissue biopsies, with several interesting features: higher levels of IL6 and CXCL1 were observed in ALLO groups at 10 to 12 POD with important activation of this response in serosa and muscular layer. Interestingly, IL22 expression was only measurable in epithelial layer samples in ALLO groups at 10 to 12 POD, indicating ACR-induced expresion in this compartment. Serosa layer showed some of the highest relative increases in proinflammatory gene expression also in ALLO groups at 10 to 12 POD.CONCLUSIONAlthough in the clinics mucosal rejection has been extensively characterized, in our animal model we could document that all graft layers are affected by ACR since the initial stages. Serosa and muscular layer show high expression of proinflammatory markers, withdifferential expression of IL22 in the epithelial compartment. This information could be useful in the search of early biomarkers of rejection.