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. 2021 Mar 30;204(9):1060–1074. doi: 10.1164/rccm.202012-4306OC

Figure 5.

Figure 5.

Two murine models of primary graft dysfunction. (A, B, E, and F) RAG1−/− mice (n = 12) received PBS or 0.5 × 106 T-regulatory cells (Tregs) intravenously; 18 hours later, lung ischemia–reperfusion was performed, which consisted of 1 hour of left lung ischemia and 2 hours of reperfusion; and then lungs were collected for quantitative PCR (qPCR) analysis (B) and histologic evaluation (E [left] and F). (C–F) RAG1−/− mice (n = 17) received intravenous 0.7 × 106 Tregs, which were preliminarily treated with 200 ng/ml IL-18 or PBS for 3 hours and then washed and cryopreserved. Eighteen hours later, lung ischemia–reperfusion was performed, which consisted of 1 hour of left lung ischemia and 2 hours of reperfusion; then both lungs were collected for (C) qPCR analysis and (H [right] and F) histologic evaluation. The median number (interquartile range) of samples evaluated for each marker is as follows: 7 (7–8) in B; 13.5 (12–14.75) in D. (F) Representative examples and (E) scoring from histology of left lungs (hematoxylin and eosin staining; scale bars, 100 μm; n = 12). (G–I) Wild-type B6 donor lungs were kept for 24 hours at 4°C to induce prolonged cold ischemia. On the next day, those lungs were transplanted into three different recipients: normal (N)-weight male mice receiving a control “inflammatory” high-fat diet (iHFD), obese male mice receiving an iHFD, and obese mice receiving an iHFD and IL-18 binding peptide (IL-18BP) treatment. For the last group, we injected 7 mg/kg IL-18BP intraperitoneally at 1 hour before operation. On the next day, both lungs were collected for dry/wet evaluation (see Figure E4H in the online supplement) before undergoing (H) qPCR and (I and J) histologic evaluation (scale bars, 100 μm). Ten mice in total were evaluated for qPCR analysis and histologic scoring: four with N weight, three untreated iHFD mice, and three IL-18BP–treated iHFD mice. More data are presented in Figures E4D–E4J. A Mann-Whitney U test was used in B and D, and two-way ANOVA with a Sidak’s test was used in E, H, and J. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. PMN = polymorphonuclear neutrophils.