Normothermic Machine Perfusion Mitigates Allograft Inflammation in Intestinal Transplantation

The only life-saving therapy for intestinal failure patients who no longer tolerate parenteral nutrition is transplantation. Intestinal transplantation (IT) following the traditionally employed allograft static cold storage (CS) method results in frequent rejection. Improved outcomes of other solid organ transplantation have been achieved using normothermic machine perfusion (NMP)-based storage, in part through allograft immune modulation. The exploration of NMP in IT is nascent. Preliminary assays demonstrate that NMP stabilizes CD3 intraepithelial lymphocytes in the jejunum and ileum while decreasing lamina propria natural killer cell populations at the end of storage. The objective of this study was to further evaluate the impact of NMP on intestinal inflammation using targeted genomic and shotgun proteomic assessments. We hypothesized that NMP would reduce allograft inflammatory signaling and improve IT outcomes. Porcine small intestine was procured (T0), stored for 6H at either 4°C (CS_T6) or perfused at 34°C (NMP_T6), and transplanted. Samples were collected at T0, T6, 1-hour post-transplant and reperfusion (T1RP), and euthanasia (T48). Mucosal scrapings of the jejunum and ileum were collected for proteomic and transcriptomic evaluations. Proteins from NMP and CS-stored intestine were extracted, separated, and ionized prior to identification with mass spectrometry (n=4-5). RNA from NMP-stored intestine was extracted, hybridized to a custom codeset, and analyzed using nanoString nCounter (n=3). Significance was set at p<0.05. Proteomic assessments comparing CS and NMP identified elevated calprotectin in CS-ileum, a clinical marker of intestinal inflammation and rejection, though our conclusions were limited by low survival in CS transplant recipients. Because of the low survival of CS recipients, transcriptomic analyses were used to profile allograft changes over the duration of NMP-storage. Proinflammatory TNFA, CD69, IFNG, IL21, IL17, IL12, TBET, and TGFB increased following reperfusion, however, all except TNFA decreased by T48, with many returning to T0 levels. Cytokines associated with reduced inflammatory injury following intestinal transplantation, such as IL6, IL5, and IL21, significantly increased in jejunum over time. Additionally, the transcription of anti-inflammatory IL13 and GATA3 and markers of successful intestinal transplantation such as CCR10, IL22, CTLA4, and FOXP3 were significantly upregulated in T1RP and T48 jejunum. NMP increased key anti-inflammatory and epithelial recovery transcripts while reducing protein markers of inflammation compared to CS. While many of the cytokines elevated during NMP have been historically associated with inflammation, current literature has demonstrated dual roles that are anti-inflammatory ( IL6, 2, 5 ). Furthermore, transient inflammation in transplantation is inevitable given the unavoidable manipulation and ischemia that occurs during bowel procurement. Overall, NMP supported resolution of procurement-associated inflammation and a favorable immune environment which may improve small intestinal transplantation outcomes. NIH 5T32OD011130-15; U.S. Department of Defense PR181265; NIH K01OD010199 SERCA,This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Duke Scholars

Author Andrew Serghios Barbas Surgery, Abdominal Transplant Surgery