Paneth and Stem Cells Are Not Affected During Initial Stages of Acute Cellular Rejection in Small Bowel Transplantation in Humans

Introduction: In spite of the last developments and improvements in long term outcome after intestinal transplantation, acute cellular rejection (ACR) remains as the most common cause of graft loss[1]. The diagnosis of ACR is still based on pathological findings[1][2], but it is unclear, which cell type and which crypt zone (bottom (BZ) or transit-amplifying zone (TZ)) are the main target cells. Therefore, we wanted to elucidate which crypt zone is mainly disturbed by apoptosis and whether the early target cells of ACR are Paneth cells (PC) or stem cells (SC) as has been shown in other pathologies[3]. Methods: Intestinal biopsies from ITx-patients classified by an experimented pathologist as normal (N, n=8) or with mild (MR, n=8) or moderate (MoR, n=8) ACR were used. Immunostainings were performed with antibodies against αdefensin-5 (DEFA5), interleukin-22 receptor (IL22RA1), cleaved-caspase-3 (CASP3) to identify PC, SC and apoptotic events. The expression of DEFA5, lysozyme (LYZ, PC marker), Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) and IL22RA1 genes (SC markers) was assayed by qPCR in N (n=5) and MoR (n=6). Results: Our results show that there was an increasing number of apoptotic bodies in BZ as the diagnosis worsens from N to MoR. Apoptosis per crypt in TZ during MoR exhibited statistical differences compared with the TZ in N group (N: 0.13±0.17, MoR: 0.64±0.25; P<0.05). According to this, the number of PC remained constant in N and MR groups and was not significantly lower in MoR (N: 3.57±1.0, MR: 3.56±0.85, MoR: 2.54±0.97; P:NS). Moreover, the expression of antimicrobial peptides had the same level or even higher in MoR than that in normal group (DEFA5 N: 1.23±0.94, MoR: 2.02±2.04; LYZ N: 1.20±1.08, MoR: 1.35±1.13; P:NS). Regarding SC, there were no differences in the expression level of LGR5 between groups (N: 0.89±1.10, MoR: 1.08±1.16; P:NS), whereas IL22RA1 had two-fold increase in rejection group (N: 0.46±0.33, MoR: 1.05±0.80; P:NS). In addition, no variations in the number of IL22RA1+ comparing groups were found (N: 1.74±0.32, MR: 1.55±0.44, MoR: 1.25±0.58, P:NS). Conclusion: We were able to show that cells are primarily affected in the TZ during ACR, and that PC and SC remain undamaged during MR and MoR. Our results highlight the relevance of future research in the IL22-IL22R+SC axis as a future therapeutic target to promote epithelial regeneration and avoid intestinal barrier disruption during rejection processes. References: 1. Bharadwaj S. Current status of intestinal and multivisceral transplantation. Gastroenterology Report. 2017; 1–9. 2. Ruiz P. Histological Criteria for the Identification of Acute Cellular Rejection in Human Small Bowel Allografts: Results of the Pathology Workshop at the VIII International Small Bowel Transplant Symposium. Transplantation Proceedings. 2004; 36: 335–337. 3. Levine J. Low Paneth cell numbers at onset of gastrointestinal graft-versus-host disease identify patients at high risk for nonrelapse mortality. Blood. 2016; 122 (8): 1505–1509.