Graduate school of medicine, Kyoto University
Pancreatic islet culture induces tolerogenic effects as well as stimulates immune cell trafficking by regulating inflammatory cytokines
Takayuki Anazawa1,2, Junichiro Haga1, Naoya Sato1, Akira Kenjo1, Shigeru Marubashi1, Takashi Kimura1, Tsutomu Mori3, Hideaki Okajima2, Shinji Uemoto2, Mitsukazu Gotoh1.
1Regenerative Surgery, Fukushima Medical University, Fukushima, Japan; 2Surgery, Kyoto University, Kyoto, Japan; 3Human Lifesciences, Fukushima Medical University School of Nursing, Fukushima, Japan
Objective: The utility of cultured pancreatic islet cells for clinical transplantation remains controversial. Pretransplant cultured islets provide the initiation of time-dependent immunosuppression and sufficient opportunity for additional quality controls; however, isolated islets are frequently lost during the culture period. This drawback may be overcome if the host inflammatory response to implanted cultured islets could be modulated or if cultured cells could be made less immunogenic. A comprehensive understanding of biological responses of islets during isolation and culture is required for the establishment of efficient culture conditions. The aim of the present study is to identify the genome-wide effects of islet culture using a suite of modern bioinformatics tools allowing the establishment of more effective pretransplantation islet treatment.
Methods: Fresh isolates from Wistar rats and 1- or 3-day cultures were used to assess islet recovery and viability. Freshly isolated or cultured islets were transplanted into the renal subcapsular space of STZ-induced diabetic C57BL/6 mice to evaluate graft survival. Total RNA was purified from cultured islet cells, and RNA expression was analyzed using microarray. Normalization, filtering, and gene ontology analysis were performed. Resultant data were then analyzed using Ingenuity Pathway Analysis (IPA).
Results: After in vitro culture for 1 and 3 days, marked islet loss was observed. Although culture was found to have minimal effect on islet viability, statistically significant prolongation of graft survival was demonstrated following transplantation of islets cultured for 1 day (15.5 ± 3.3) or 3 days (14.2 ± 2.6) compared to freshly isolated islets (10.3 ± 2.3). Transplant studies suggested that cultured islets are less immunogenic; however, culture conditions were unable to achieve graft unresponsiveness. Microarray analysis revealed marked upregulation of HMOX1 and IL-6 in cultured islets. Increased levels of IL-6 in culture supernatants and increased HO-1 expression, which was reported to increase graft survival, in cultured islets were confirmed by in vitro validation analyses. The top bioprocess identified by IPA following islet culture involved “immune cell trafficking.” IL-1β, TNF, and TGFβ1 were identified as the top upstream regulators. We also observed the upregulation of multiple chemotaxis factors involved in immune cell trafficking, including monocyte chemotactic protein-3, matrix metalloprotease-2, and IL-33, in cultured islets. These findings indicate harmonized downregulation of chemotaxis factors is required for clinically relevant prolongation of graft survival.
Conclusion: Our findings indicate that islet culture induces HO-1 expression parallel with increased IL-6 levels and may provide HO-1-mediated tolerogenic effects. However, islet isolation and subsequent culture was also found to induce immune cell trafficking through the regulation of inflammatory cytokine expression. The use of novel pharmacological agents during islet culture to inhibit immune cell trafficking are required to establish effective pretransplant islet culture treatment.
15:30 - 17:00
|Modifying Islet Immunity - Adaptive, Innate, and Xenoimmunity||Pancreatic islet culture induces tolerogenic effects as well as stimulates immune cell trafficking by regulating inflammatory cytokines||Plenary Room 1|