Hepatic CEACAM1 expression indicates donor liver quality and prevents early transplantation injury (original) (raw)
Hepatic CC1 null mutation exacerbates IRI in mouse OLT. We first aimed to determine the influence of graft-specific disruption of CEACAM1 signaling on the severity of hepatic IRI in a clinically relevant mouse OLT model with extended ex vivo cold storage (4°C/18 hours), which mimics the marginal human liver graft scenario. At 6 hours after transplantation into WT recipients, _CC1_-deficient (CC1–/–; KO) liver grafts (n = 6) exhibited increased sinusoidal congestion, edema vacuolization, and hepatocellular necrosis (Figure 1A); enhanced Suzuki’s histological IRI grading (WT → WT = 3.5 ± 1.0 vs. CC1-KO → WT = 6.0 ± 1.3, P = 0.0005, Figure 1B); higher serum levels of alanine aminotransferase (sALT) and aspartate aminotransferase (sAST) (sAST: WT → WT = 3053 ± 501 vs. CC1-KO → WT = 6097 ± 1324 IU/L, P < 0.0001; sALT: WT → WT = 6616 ± 1065 vs. CC1-KO → WT = 9807 ± 2655, P = 0.0087; Figure 1C); and elevated frequency of TUNEL-positive necrotic/apoptotic cells (WT → WT = 46.6 ± 4.9 vs. CC1-KO → WT = 83.7 ± 14.7/HPF, P < 0.0001; Figure 1, D and E) as compared with CC1 proficient (WT → WT) grafts (n = 6). Thus, disruption of CEACAM1 signaling in the donor liver augmented IRI and enhanced hepatocellular death in murine OLT.
Hepatic _CC1_-null mutation exacerbates hepatocellular damage and inflammatory response in IR-stressed mouse OLT. Livers from groups of WT and CC1-KO C57BL/6 donor mice, stored in UW solution (4°C/18 hours), were transplanted to WT C57BL/6 recipient mice. OLT and serum samples were analyzed 6 hours after reperfusion. The sham group underwent the same procedures except for OLT. (A) Representative H&E staining (original magnification ×100). (B) Suzuki’s histological grading of liver IRI. (C) sAST and sALT levels (IU/L). (D) Representative TUNEL staining and immunohistochemical staining of OLT-infiltrating CD11b+ and Ly6G+ cells (original magnification ×200). (E) Quantification of TUNEL-positive cells/HPF. (F) Serum HMGB1 (ng/mL) and MCP1 (pg/mL) levels measured by ELISA. (G) Quantification of hepatic CD11b+ and Ly6G+ cells/HPF. (H) Real-time reverse transcription PCR–assisted (qRT-PCR–assisted) detection of mRNA coding for MCP1, CXCL1, CXCL2, and CXCL10 in OLT. Data were normalized to HPRT gene expression. Data are mean ± SD. *P < 0.05, 1-way ANOVA followed by Tukey’s HSD test (B, C, and E–G) or Student’s t test (H), n = 5–6/group.
Hepatic CC1 ablation enhances IR-inflammatory phenotype in mouse OLT. Since the release of DAMPs, such as HMGB1, from damaged cells triggers a cascade of inflammatory cytokine/chemokine events, which further aggravate organ damage (17), we aimed to evaluate the impact of graft CC1 deficiency on the release of HMGB1 and accompanied innate-immune response in our model. At 6 hours after reperfusion, CC1-KO liver grafts (CC1-KO → WT) showed higher serum HMGB1 levels (Figure 1F) and increased frequency of intragraft infiltration by CD11b-positive (macrophage)/Ly6G-positive (neutrophil) cells (Figure 1, D and G), along with elevated serum MCP1 (Figure 1F) and hepatic mRNA levels coding for MCP1, CXCL1, CXCL2, and CXCL10 (Figure 1H), as compared with controls (WT → WT). These data indicate the importance of graft CC1 signaling to suppress secretion of DAMPs, mitigate innate immune activation, and alleviate hepatocellular damage in IR-stressed OLT.
Hepatic CC1 deletion augments cell damage by enhancing reactive oxygen species (ROS) and HMGB1 translocation during liver cold storage. Although restoration of blood flow at reperfusion is the principal cause of liver IRI (17), cold storage itself can also trigger hepatocellular damage (8). Having demonstrated the importance of graft CC1 expression on HMGB1 release in OLT (Figure 1F), we next asked whether CEACAM1 may affect graft injury and HMGB1 signaling during ex vivo cold storage (before revascularization). Herein, we focused on the liver effluent obtained by flushing the liver with physiological saline (2 mL) via a cuff placed at portal vein immediately after 18 hours of cold stimulation (Figure 2A). Indeed, the flush from CC1-deficient livers contained increased HMGB1 and histone H3 levels as compared with CC1-proficient (WT) livers (Figure 2B), suggesting higher susceptibility of CC1-KO grafts to peritransplant cold stress. Since the generation of ROS is one of the principal factors leading to cell death in cold-stored tissue (8, 18), we then evaluated 4-Hydroxynonenal (4HNE, a ROS metabolite) levels in our model. Cold stimulation alone increased 4HNE expression, mainly in hepatic parenchymal cells, whereas CC1 deficiency markedly enhanced 4HNE upregulation (Figure 2C, upper panel, red stains). Moreover, cold stress led to cytoplasmic HMGB1 translocation, while CC1 disruption further augmented extranuclear HMGB1 localization (Figure 2C, middle panel). Thus, disruption of hepatic CC1 signaling increased effluence of damage-associated molecules (HMGB1/histone H3) while enhancing 4HNE expression and extranuclear HMGB1 translocation during cold storage. These data suggest that graft-specific CEACAM1 was essential to inhibit local ROS generation and prevent cell damage in the early hepatic cold storage phase. Noteworthy, cold stimulation alone did not affect the frequency of TUNEL-positive cells in WT or CC1-KO liver grafts (Figure 2C, lower panel), implying hepatic damage leading to DAMPs release (Figure 2B) was likely distinct from apoptotic/necrotic cell death.
CC1 ablation in cold-stored livers enhances ROS and HMGB1 translocation/release to liver flush, which further increases inflammatory gene program in macrophage cultures. (A) Groups of WT and CC1-KO liver grafts stored in UW solution (4°C/18 hours) were perfused with physiological saline (2 mL) via a cuff placed at the portal vein to collect liver flush from inferior vena cava. (B) Liver flush samples (20 μL) from cold-stressed WT or CC1-deficient livers were screened by Western blots for HMGB1/Histone H3 levels (n = 4/group, *P < 0.05, Student’s t test). (C) WT or CC1-KO liver grafts were collected after cold storage (4°C/18 hours). Representative (n = 3/group) immunohistochemical staining of CC1/4HNE (a ROS metabolite), CC1/HMGB1, and TUNEL is shown. Arrowheads indicate extranuclear HMGB1 localization. (D) BMDM cultures (WT) were stimulated (6 hours) with liver flush obtained from WT or CC1 KO cold-stored grafts. qRT-PCR–assisted detection of mRNA coding for MCP1, CXCL2, CXCL10 with β2M normalization (n = 4–6, *P < 0.05, 1-way ANOVA followed by Tukey’s HSD test).
Hepatic flush from CC1-KO cold-stored mouse livers enhances macrophage activation in vitro. Since the leakage of DAMPs from a cold-stored graft is not only a tissue damage indicator, but may also trigger innate inflammatory response, we next asked whether and how liver flush may affect bone marrow–derived macrophage (BMDM) cultures (Figure 2, A and D). Indeed, liver flush from cold-stored WT livers was found to increase mRNA levels coding for MCP1, CXCL2 and CXCL10, whereas liver flush from CC1-KO livers further enhanced the proinflammatory gene expression program as compared with WT cultures. Thus, in the absence of hepatic CC1 signaling, cold stimulation triggered a more pronounced inflammatory response by hepatic flush in vitro, as compared with CC1-proficient (WT) liver flush.
Hepatic CC1 null mutation augments cold stress–triggered upregulation of the ASK1/p-p38 signaling axis. To seek a molecular signaling pathway that underlies cold-related hepatic damage, we contrasted murine WT livers at resting state (naive), after cold storage (4°C/18 hours), and after OLT reperfusion (Figure 3A). Cleavage of caspase-3, an essential step to execute apoptosis, was pronounced at 3 hours after reperfusion, while cold stress alone failed to increase cleaved caspase-3 expression. Consistently, TUNEL-positive (apoptotic/necrotic) cells were increased in OLT (Figure 1D) but not during cold storage alone (Figure 2C). In addition, cold stimulation did not affect RIP3 (a marker of necroptosis) expression (Figure 3B). These findings indicate that the molecular mechanism of cold stress–triggered liver graft damage was likely to be distinct from apoptosis, necrosis, or necroptosis. Since recent reports have demonstrated tissue-specific importance of ASK1/p-p38 signaling in cold-stress cell damage (10, 11), we focused on the ASK1/p-p38 axis in our model. Interestingly, the markedly increased hepatic ASK1 and p-p38 expression seen after cold storage (4°C/18 hours) in WT livers decreased in OLT at 3 hours after reperfusion (Figure 3C). In marked contrast, in the absence of CC1 signaling, liver grafts showed increased ASK1/p-p38 levels not only at resting state (Figure 3D) but also after cold storage (Figure 3E). Thus, CEACAM1 was essential to inhibit a cold stimulation–triggered ASK1/p-p38 signaling pathway.
Donor hepatic CC1 deletion enhances cold stress-triggered ASK1/p-p38 signaling. (A) Groups of WT and CC1-KO livers were stored in UW solution (4°C/18 hours). Liver samples were collected right after cold storage (before OLT) or 3 hours after reperfusion (post-OLT). (B and C) Western blot–assisted detection and relative intensity ratio of cleaved caspase-3, RIP3, CC1, ASK1, p-p38 in naive liver, cold-stored liver or postreperfusion OLT (WT). Vinculin (VCL) expression served as an internal control and was used for normalization (n = 3/group). (D) Western blot–assisted detection and relative intensity ratio of CC1, ASK1, and p-p38 in naive WT or CC1-KO liver. VCL expression served as an internal control and was used for normalization (n = 4/group). (E) Western blot–assisted detection and relative intensity ratio of CC1, ASK1, and p-p38 in cold-stored WT or CC1-KO livers. VCL expression served as an internal control and used for normalization (n = 3/group). Data shown as mean ± SD. *P < 0.05, 1-way ANOVA followed by Tukey’s HSD test (B and C) or Student’s t test (D and E).
Hepatocyte CC1 inhibits p-p38 upregulation and cell death under cold stress via the ASK1 pathway. Having demonstrated the hepatoprotective role of CEACAM1 in cold-stressed liver grafts (Figure 2, B and C) and the inhibitory function of CC1 against ASK1/p-p38 signaling (Figure 3E), we next asked whether the ASK1/p-p38 axis may be essential for CEACAM1-mediated hepatocyte protection. In a refined murine primary hepatocyte culture (WT), 4 hours of cold stimulation (4°C) distinctly increased expression of ASK1 and p-p38 (Figure 4A), upregulated 4HNE expression (Figure 4C, upper panel), induced HMGB1 translocation to cytoplasm (Figure 4C, middle panel), ultimately leading to hepatocyte death (Figure 4C, lower panel, and Figure 4D). Notably, hepatocyte CC1 deficiency further enhanced ASK1/p-p38 (Figure 4B), 4HNE expression, HMGB1 translocation, and cell death (Figure 4, C and D). In contrast, ASK1 silencing using siRNA (ASK1) in cold-stressed _CC1_-deficient hepatocyte cultures inhibited p-p38 overexpression, 4HNE upregulation, HMGB1 translocation, and cell death (Figure 4, B–D). These data indicate the important contribution of ASK1 signaling in p-p38 regulation and CECAM1-mediated hepatocyte protection during cold stimulation.
Hepatocyte CC1 deficiency enhances p-p38 increase, 4HNE overexpression, HMGB1 translocation, and cell death due to cold stress in an ASK1-dependent manner. (A) Primary mouse hepatocytes (WT) with or without cold stimulation (4°C/4 hours) were incubated for the indicated time periods. Western blot–assisted detection and relative intensity ratio of CC1, ASK1, p-p38. VCL expression served as an internal control and used for normalization (n = 2/group). (B–D) Cold-stimulated WT or CC1-KO hepatocytes were pretreated with or without siRNA against ASK1. (B) Western blot–assisted detection and relative intensity ratio of CC1, ASK1, p-p38. VCL expression served as an internal control and used for normalization (n = 3/group). (C) Representative (n = 3/group) immunohistochemical staining of 4HNE (red, upper panels), HMGB1 (red, middle panels), and dead cell detection (red, lower panels). (D) Quantification of dead cells/HPF (n = 4–5/group). *P < 0.05, 1-way ANOVA followed by Tukey’s HSD test.
ASK1 inhibition suppresses p-p38 upregulation, 4HNE overexpression, and HMGB1 translocation in cold-stressed CC1-deficient liver grafts. To verify in vivo relevance of the aforementioned in vitro findings (Figure 4), we next incubated CC1-KO livers with a selective ASK1 inhibitor (19) during 18 hours of cold storage. _CC1_-deficient liver grafts supplemented with ASK1 inhibitor showed suppressed p-p38 upregulation (Figure 5A), neither 4HNE overexpression nor cytoplasmic translocation of HMGB1 (Figure 5B), and also decreased HMGB1 secretion in the liver flush after cold stimulation (Figure 5C). Thus, liver CC1 deficiency increased p-p38/4HNE expression and promoted HMGB1 translocation/tissue damage via ASK1 signaling.
Inhibition of ASK1 in CC1-KO livers prevents cold-triggered p-p38 increase, 4HNE overexpression, and HMGB1 translocation as well as OLT damage and recipient mortality. (A–C) Groups of WT and CC1-KO livers were stored in UW solution (4°C/18 hours) with or without ASK1 inhibitor (10 μg/15 mL). (A) Western blot–assisted detection and relative intensity ratio of CC1 and p-p38. VCL expression served as an internal control and used for normalization (n = 3–4/group). (B) Representative (n = 3/group) immunohistochemical staining of CC1/4HNE and CC1/HMGB1. (C) Liver flush (20 μL) from cold-stressed WT or CC1-KO livers with or without ASK1 inhibitor were analyzed by Western blots for HMGB1 levels (n = 3–4/group). (D–H) Cold-stored (4°C/18 hours) WT or CC1-KO livers were transplanted into recipient mice, and OLT and serum samples were analyzed at 6 hours after reperfusion. Some CC1-KO grafts were preincubated with ASK1 inhibitor (10 μg/15 mL) during cold storage (4°C/18 hours). Separate OLT recipient groups were monitored for 20-day survival. (D) Representative H&E (original magnification ×100) and TUNEL staining. (E) sAST and sALT levels (IU/L; n = 7–8/group). (F) Suzuki’s histological grading of liver IRI (n = 7–8/group). (G) Quantification of TUNEL-positive cells/HPF (n = 7–8/group). Data shown as mean ± SD. *P < 0.05, 1-way ANOVA followed by Tukey’s HSD test. (H) Recipient mice were monitored for 20 days and cumulative survival was analyzed (Kaplan-Meier method). Dotted line: WT → WT; solid line: CC1-KO → WT; bold line: CC1-KO+ASK1 inhibitor → WT (n = 6–9/group; *P < 0.05 vs. CC1-KO → WT, log-rank test).
ASK1 signal inhibition during cold storage alleviates hepatic IRI and improves CC1-KO OLT survival. To investigate the function of ASK1 in aggravated hepatic IR damage, we transplanted CC1-KO livers with or without preincubation with ASK1 inhibitor during cold storage into WT recipients. Addition of ASK1 inhibitor during cold stimulation (4°C/18 hours) attenuated sinusoidal congestion, edema/vacuolization, and hepatocellular necrosis (Figure 5D), decreased sAST/ALT levels (sAST: CC1-KO → WT = 6035 ± 1688 vs. CC1-KO+ASK1 inhibitor → WT = 3552 ± 1083 IU/L, P = 0.0020; sALT: CC1-KO → WT = 10573 ± 2824 vs. CC1-KO+ASK1 inhibitor → WT = 6744 ± 2648, P = 0.0271; Figure 5E), decreased Suzuki’s histological grading of IRI (CC1-KO → WT = 6.3 ± 1.0 vs. CC1-KO+ASK1 inhibitor → WT = 2.0 ± 1.2, P < 0.0001; Figure 5F), and suppressed frequency of TUNEL-positive cell death (CC1-KO → WT = 82.0 ± 11.8 vs. CC1-KO+ASK1 inhibitor → WT = 39.7 ± 5.7/HPF, P < 0.0001; Figure 5, D and G) at 6 hours after reperfusion in otherwise IRI-susceptible CC1-KO grafts. Moreover, unlike recipients of CC1-KO grafts with poor prognosis and outcomes as compared with WT → WT pairs (20-day survival: 0% versus 44%, P = 0.0068), incubation of donor livers with ASK1 inhibitor significantly improved overall survival of CC1-KO → WT hosts (50% vs. 0%, P = 0.0217) (Figure 5H). Thus, preincubation of CC1-KO livers with ASK1 inhibitor during cold storage mitigated hepatic IRI and improved otherwise inferior post-OLT survival.
CEACAM1 levels in cold-stressed human donor livers negatively correlate with ASK1/p-p38 expression in OLT patients. Having demonstrated the regulatory CEACAM1 – ASK1/p-p38 crosstalk in cold-stimulated murine liver grafts, we next aimed to validate its relevance in clinical liver transplant patients. Sixty cold-stored human liver biopsies (Bx), collected at back table before implantation, were screened by Western blots for CEACAM1, ASK1, and p-p38 protein levels with β-actin normalization (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/JCI133142DS1). Hepatic CEACAM1 correlated negatively with ASK1 (r = –0.3424, P = 0.0074, Supplemental Figure 1B) and p-p38 expression (r = –0.2947, P = 0.0222, Supplemental Figure 1C). Based on Western-assisted relative CEACAM1 expression pattern, pretransplant human Bx samples collected from 60 OLT patients were divided into low CEACAM1 (n = 30) and high CEACAM1 expression groups (n = 30), based on the relative CEACAM1/β-actin levels, according to median split method (cutoff = 0.85, Figure 6A). There was no correlation between CEACAM1 classification and recipient/surgical parameters, including age, sexual phenotype, race, BMI, disease etiology, ABO compatibility, model for end-stage liver disease (MELD) score, pretransplant blood tests, preoperative hospital stay, cold ischemia time (CIT), warm ischemia time (WIT), and blood transfusions during surgery (Supplemental Table 1). There was no correlation between CEACAM1 grouping and donor variables, such as age, sexual phenotype, race, BMI, preprocurement blood tests, and donation status (donation after circulatory death [DCD] or donation after brain death [DBD]) (Supplemental Table 2). Consistent with data in Supplemental Figure 1, B and C, the low CEACAM liver transplant clinical cohort showed higher ASK1 (P = 0.0459) and p-p38 (P = 0.0153) levels as compared with high CEACAM1 cases (Figure 6B). Representative Western blots are shown in Figure 6C. Consistent with mouse OLT data (Figure 2C), low CEACAM1 levels in clinical liver Bx samples were associated with increased 4HNE (Figure 6D) and enhanced cytoplasmic HMGB1 translocation (Figure 6E).
Pretransplant CEACAM1 levels are associated with ASK1/p-p38/4HNE expression and HMGB1 translocation in human OLT recipients. Pretransplant (after cold storage) human liver Bx (n = 60) were analyzed by Western blots with β-actin normalization for CEACAM1, ASK1, and p-p38 levels (see Supplemental Figure 1A). (A) Bx samples were divided into low (n = 30) and high (n = 30) CEACAM1 expression groups based on the relative CEACAM1/β-actin levels (cutoff = 0.85, median). (B) Western blot–assisted expression of ASK1 and p-p38. Data shown in dot plots and bars indicate mean ± SEM. #P < 0.05 (Mann-Whitney U test). (C) Four representative Western blots are shown (case 1/2: low CEACAM1, case 3/4: high CEACAM1). (D) Representative (n = 3) CEACAM1/4HNE staining (original magnification ×200). (E) Representative (n = 3) CEACAM1/HMGB1 staining (original magnification ×400).
CEACAM1 levels before liver implantation associate with the hepatocellular function in human OLT recipients. Since disruption of liver CEACAM1 signaling in mouse OLT not only affected stress resistance under cold storage (Figures 2 and 3) but was also critical for postreperfusion injury and recovery (Figure 1), we next aimed to evaluate correlation between pretransplant CEACAM1 levels and postreperfusion hepatic damage in human OLT. Pretransplant CEACAM1 expression correlated negatively with sAST (r = –0.3302, P = 0.0100, Supplemental Figure 1D) and sALT (r = –0.3280, P = 0.0105, Supplemental Figure 1E) levels at postoperative day 1 (POD1). In addition, low CEACAM1 cases (Figure 6A) exhibited significantly higher sAST at POD1–5 (Figure 7A) and sALT at POD1–7 (Figure 7B). We then analyzed liver Bx samples obtained at 2 hours after reperfusion (before the abdominal closure) from the corresponding low vs. high CEACAM1 clinical cases. The TUNEL staining from representative postreperfusion Bx samples is shown (Figure 7C, low CEACAM1: 133.8 ± 26.0/HPF; high CEACAM1: 69.8 ± 24.23/HPF; P = 0.0611, n = 4/group). Pretransplant low CEACAM1 livers exhibited increased mRNA levels coding for TLR4 (P = 0.0065), CD80 (P = 0.0910), CD86 (P = 0.0112), CXCL10 (P = 0.0866), CD68 (P = 0.1004) (macrophage activation markers); Cathepsin G (P = 0.0052) (neutrophil marker); CD28 (P = 0.0189), CD4 (P = 0.0346), IL17 (P = 0.0327) (T cell markers) in OLTs at 2 hours after reperfusion (Figure 8). Thus, pretransplant low CEACAM1 expression was associated with enhanced postreperfusion innate/adaptive immune responses and increased hepatocellular damage in the early post-OLT period. Although low CEACAM1 cases experienced increased frequency of EAD as compared with the high CEACAM1 group (30.0% vs. 16.7%), the differences failed to reach statistical significance (P = 0.3604, Figure 7D). To examine the relationship between pretransplant CEACAM1 expression and OLT outcomes, we analyzed the overall graft survival (median follow-up, 1269 days; range, 3–1892 days) and rejection-free graft survival (median follow-up, 1249 days; range, 3–1892 days). None of the patients underwent secondary liver transplantation. Despite obvious trends, inferior graft survival (P = 0.3293, Figure 7E) and rejection-free graft survival (P = 0.1674, Figure 7F) in the low CEACAM1 group failed to reach statistical significance when compared with the high CEACAM1 group.
Low CEACAM1 levels impair hepatocellular function in human OLT recipients. Pretransplant (after cold storage) human liver Bx samples were divided into low (n = 30) and high (n = 30) CEACAM1 expression groups, based on the relative CEACAM1/β-actin levels (see Figure 6A). (A) Serum AST levels at POD1–7. (B) Serum ALT levels at POD1–7. Data are mean ± SEM. #P < 0.05 (Mann-Whitney U test). (C) Post-OLT Bx were obtained at 2 hours after reperfusion from corresponding clinical cases. Representative (n = 3) TUNEL staining (original magnification ×400). (D) Incidence of EAD (Fisher’s exact test). (E) The cumulative probability of overall graft survival. (F) The cumulative probability of rejection-free graft survival. Solid line indicates low CEACAM1; dotted line indicates high CEACAM1 human OLT patient group (Kaplan-Meier method, log-rank test).
Depressed innate and adaptive gene activation pattern in human OLT is accompanied by high pretransplant hepatic CECAM1 levels. Pretransplant (after cold storage) human liver Bx samples were classified into low (n = 30) and high (n = 30) CEACAM1 (CC1) expression groups (see Figure 6A for details). Post-OLT Bx were obtained at 2 hours after reperfusion from corresponding cases, followed by qRT-PCR–assisted detection of mRNA coding for TLR4, CD80, CD86, CXCL10, CD68, Cathepsin G, CD28, CD4, and IL17. Data normalized to GAPDH gene expression are shown in dot plots and bars indicative of mean ± SEM. #P < 0.05 (Mann-Whitney U test).
CEACAM1 expression before liver implantation dictates the incidence of EAD in human OLT recipients. Having failed to establish a statistical correlation between the EAD rate and pretransplant CEACAM1 expression level when the median level (CEACAM1/β-actin: 0.85) was simply employed as a cutoff (Figure 6A and Figure 7D), we next aimed to seek the optimal cutoff to evaluate predictive ability of pretransplant CEACAM1 for EAD. Based on a ROC curve and Youden index on the basis of best accuracy in relation to an incidence of EAD, the optimal pretransplant CEACAM1/β-actin cutoff values were selected by maximizing sum of sensitivity and specificity (0.71, AUROC = 0.604, sensitivity = 0.761, specificity = 0.643) (Figure 9A and Supplemental Table 3). Based on the cutoff value of CEACAM1/β-actin being 0.71, 60 OLT clinical cases were classified into two groups: CEACAM1/β-actin less than 0.71 (n = 20) and CEACAM1/β-actin greater than 0.71 (n = 40) (Figure 9B). There was no correlation between CEACAM1 classification and recipient age, sexual phenotype, BMI, disease etiology, ABO compatibility, MELD score, pretransplant blood tests, CIT, WIT, and blood transfusion required for OLT operations, whereas we found statistically significant differences in recipient race and preoperative hospital stay (Supplemental Table 4). There was no correlation between CEACAM1 grouping and donor variables, including age, sexual phenotype, race, BMI, preprocurement blood tests, and DCD (Supplemental Table 5). Likewise to the data shown in Figure 7, A and B, the CEACAM1/β-actin less than 0.71 group showed increased sAST levels at POD1, 2, 4, and 5, and higher sALT levels at POD1–7 (Figure 9C). Of note, the CEACAM1/β-actin less than 0.71 cases experienced a significantly increased incidence of EAD as compared with the CEACAM1/β-actin greater than 0.71 cases (45.0% vs. 12.5%, P = 0.0088, Figure 9B). To determine whether a CEACAM1/β-actin level less than 0.71 represents an independent predictor of EAD in our liver transplant patient cohort, we conducted multivariate analysis based on a stepwise logistic regression model by screening the phrase “pre-OLT CEACAM1/β-actin<0.71” simultaneously with recipient age (≥60 years), recipient sexual phenotype (male), recipient race, recipient BMI (≥25 kg/m2), preoperative hospital stay (≥7 days), disease etiology, MELD score (≥35), cold ischemia time (≥420 minutes), warm ischemia time (≥ 50 minutes), donor age (≥40 years), donor sexual phenotype (male), donor BMI (≥ 25 kg/m2), and DCD donor. Remarkably, “pre-OLT CEACAM1/β-actin<0.71” was identified as one of the predictive factors of EAD (OR = 7.209 [95% CI: 1.376–37.755], P = 0.019), along with cold ischemia time (≥420 minutes) (OR = 13.024 [95% CI: 1.873–90.551], P = 0.009) (Figure 9D). Despite obvious trends of inferior graft survival (P = 0.3756, Supplemental Figure 2A) and rejection-free graft survival (P = 0.4093, Supplemental Figure 2B), the patient group with pre-OLT CEACAM1/β-actin less than 0.71 failed to reach statistical significance when compared with the patient group with pre-OLT CEACAM1/β-actin greater than 0.71.
Pretransplant CEACAM1 expression dictates the incidence of EAD in human OLT patients. Pretransplant (after cold storage) human liver Bx samples (n = 60) were analyzed by Western blots with β-actin normalization for CEACAM1 levels. (A) ROC analysis of CEACAM1/β-actin for predicting EAD. Based on a ROC curve and Youden index on the basis of best accuracy in relation to EAD incidence, the CEACAM1/β-actin cutoff value of 0.71 was determined. AUROC, area under the receiver operating characteristic curve. (B) Based on the optimal cutoff value (0.71), 60 human OLTs were classified into CEACAM1/β-actin less than 0.71 (n = 20) and CEACAM1/β-actin greater than 0.71 cases (n = 40), and the incidence of EAD was evaluated. #P < 0.05 (Fisher’s exact test). (C) Serum AST and ALT levels at POD1–7. Data are mean ± SEM. #P < 0.05 (Mann-Whitney U test). (D) Stepwise multivariate logistic regression analysis was performed to identify independent risk factors of EAD.