The effect of rewarming ischemia on tissue transcriptome signatures: a clinical observational study in lung transplantation

BACKGROUND: In lung transplantation (LuTx), various ischemic phases exist, yet the rewarming ischemia time (RIT) during implantation has often been overlooked. During RIT, lungs are deflated and exposed to the body temperature in the recipient's chest cavity. Our prior clinical findings demonstrated that prolonged RIT increases the risk of primary graft dysfunction. However, the molecular mechanisms of rewarming ischemic injury in this context remain unexplored. We aimed to characterize the rewarming ischemia phase during LuTx by measuring organ temperature and comparing transcriptome and metabolome profiles in tissue obtained at the end versus the start of implantation. METHODS: In a clinical observational study, 34 double-LuTx with ice preservation were analyzed. Lung core and surface temperature (n=65 and 55 lungs) was measured during implantation. Biopsies (n=59 lungs) were wedged from right middle lobe and left lingula at start and end of implantation. Tissue transcriptomic and metabolomic profiling were performed. RESULTS: Temperature increased rapidly during implantation, reaching core/surface temperatures of 21.5°C/25.4°C within 30min. Transcriptomics showed increased pro-inflammatory signaling and oxidative stress at the end of implantation. Upregulation of NLRP3 and NFKB1 correlated with RIT. Metabolomics indicated elevated levels of amino acids, hypoxanthine, uric acid, cysteineglutathione disulfide alongside decreased levels of glucose and carnitines. Arginine, tyrosine, and 1-carboxyethylleucine showed correlation with incremental RIT. CONCLUSIONS: The final rewarming ischemia phase in LuTx involves rapid organ rewarming, accompanied by transcriptomic and metabolomic changes indicating pro-inflammatory signaling and disturbed cell metabolism. Limiting implantation time and lung cooling represent potential interventions to alleviate rewarming ischemic injury. Lung tissue biopsy pieces preserved at -80°C were homogenized in Total RNA Lysis Solution (Bio-Rad, Cat#7326820, US) with a 3mm tungsten carbide bead (Qiagen, Cat#69997, Netherlands) using the TissueLyser II (Qiagen, Netherlands). The homogenate underwent RNA extraction with TRIzol (Invitrogen, Cat#15596026, US) and RNA purification with the Aurum Total RNA mini Kit (Bio-Rad, Cat#7326820, US). RNA quality was verified using the NanoPhotometer NP80 Touch (Implen, Germany). We did nCounter (NanoString Technologies, US) digital gene expression analysis with the Immunology V2 panel targeting 579 immune system-associated genes. The workflow was carried out as established before in critical COVID-191, fatal COVID-19 nursing home outbreaks2, and respiratory infections at the emergency department3. In short, transcripts -including 15 housekeeping genes - were quantified by hybridization with specific fluorescent barcodes linked to a 50 bp reporter probe and an adjacent 50 bp capture probe. Data were normalized for background (negative control probes), internal positive control probes and housekeeping genes using nSolver software version 4.0 (NanoString Technologies, US). Differentially expressed genes and predefined biological pathway scores were determined using nSolver, with correction for multiple testing by the Benjamini-Hochberg method at a 5% false discovery rate for multiple comparisons cut-off. Comparison of baseline RNA levels for lung side, mechanical ventilation time, donation type and cold ischemia time was done according to the negative binomial distribution with generalized linear models. References 1. Menezes SM, Braz M, Llorens-Rico V, Wauters J, Van Weyenbergh J. Endogenous IFNβ expression predicts outcome in critical patients with COVID-19. Lancet Microbe. Jun 2021;2(6):e235-e236. doi:10.1016/s2666-5247(21)00063-x 2. Cuypers L, Keyaerts E, Hong SL, et al. Immunovirological and environmental screening reveals actionable risk factors for fatal COVID-19 during post-vaccination nursing home outbreaks. Nat Aging. Jun 2023;3(6):722-733. doi:10.1038/s43587-023-00421-1 3. Fukutani KF, Nascimento-Carvalho CM, Bouzas ML, et al. In situ Immune Signatures and Microbial Load at the Nasopharyngeal Interface in Children With Acute Respiratory Infection. Front Microbiol. 2018;9:2475. doi:10.3389/fmicb.2018.02475