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Biliary Epithelial Senescence in Cellular Rejection Following Live Donor Liver Transplantation

Published:August 26, 2022DOI:https://doi.org/10.1016/j.jceh.2022.08.004

      Background

      As with the hepatocytes, cholangiocyte senescence can also easily be detected in damaged small bile ducts and bile ductules during liver disease affecting the biliary system and cholangiocytes. Despite cellular senescence being a feature of chronic progressive cholangiopathies in adults, only a few studies have investigated its role in liver transplant rejection.

      Method

      Transplant biopsies displaying features of rejection were reviewed and classified based on the type of rejection and the time since transplantation. An immunohistochemistry panel has been applied for 3 senescent cell markers (p53, p21, p16).

      Results

      Immunohistochemical expression analysis for the biliary senescence markers (53 biopsies) was done in the post-transplantation periods (Group 1–4) for the cases with the histologically proven diagnosis of rejection. In post-transplant group 1 (<3 months), group 2 (3–6 months), group 3 (6–12 months) and group 4 (>12 months), any 2 senescent markers’ positivity was noted in 5/14 (35.7%), 8/13 (61.5%), 16/17 (94.1%) and 9/9 (100%) biopsies respectively and were comparable in all four groups (P = 0.001). A comparison of early biopsies (Group1; 3 months) and late biopsies (Group 2,3&4; >3 months) revealed significantly higher expression in late biopsies (>3 months) (P = 0.001 for any two markers). In ACR, LAR, ECR, and CR/DR any two senescent markers were positive in 14/28 (50%), 12/13 (92.3%) cases, 9/9 (100%), and 3/3cases (100%). Senescent markers (any two) were comparable in all four histological groups (P < 0.001).
      LAR group had increased expression (P = 0.009 for any two markers and 0.001 for all three markers) and has increased progression to CR (P = 0.019) as compared to ACR.

      Conclusion

      This study on a large number of LDLT allograft biopsies demonstrates the role of biliary senescence in rejection and suggests a pathobiological role for senescence in the poor prognosis seen in late acute cellular rejection and chronic rejection.

      Keywords

      Abbreviations:

      ACR (Acute cellular rejection), LAR (Late acute cellular rejection), ECR (Early chronic rejection), CR/DR (Chronic Rejection/Ductopenic Rejection), LDLT (Live donor liver transplantation), RAI (Rejection activity index), MELD (Model for end-stage liver disease), BEC (Biliary epithelial cells)
      Cellular damage, if not repaired, leads to apoptosis or senescence. Senescent cells accumulate as a consequence of cellular senescence, which limits the proliferative potential of damaged cells by causing a stable cell cycle arrest. Senescent cells can be seen in the aging process as well as in certain diseases, such as cirrhosis and fibrosis. Once cellular senescence is established, these cells become apoptosis-resistant.
      • Hampel B.
      • Wagner M.
      • Teis D.
      • Zwerschke W.
      • Huber L.A.
      • Jansen-Durr P.
      Apoptosis resistance of senescent human fibroblasts is correlated with the absence of nuclear IGFBP-3.
      As a consequence, the increased number of senescent cells results in altered physiology and a decline in organ function. The inflammatory nature of senescent cells also results in substantial changes in the surrounding microenvironment, thus disrupting tissue homeostasis.
      • Campisi J.
      • d'Adda di Fagagna F.
      Cellular senescence: when bad things happen to good cells.
      • Collado M.
      • Blasco M.A.
      • Serrano M.
      Cellular senescence in cancer and aging.
      • Herbig U.
      • Ferreira M.
      • Condel L.
      • Carey D.
      • Sedivy J.M.
      Cellular senescence in aging primates.
      • Jeyapalan J.C.
      • Ferreira M.
      • Sedivy J.M.
      • Herbig U.
      Accumulation of senescent cells in mitotic tissue of aging primates.
      The selection of the appropriate marker is one of the most difficult aspects of detecting senescent cells. While expression of senescence-associated -galactosidase enzymatic activity is considered a gold standard, it requires frozen sections, which are not available in most settings. As a result, several markers were used, including morphological changes, the expression of cell cycle inhibitors, changes in the nuclear membrane, and the evaluation of the inflammatory secretome. Most often the senescent cells are classically arrested in the cell cycle at the G1/S transition, so they have DNA content characteristic of the G1 phase and express cells cycles inhibitors such as p21, p16, p27, and p53
      • Ferreira-Gonzalez S.
      • Lu W.Y.
      • Raven A.
      • et al.
      Paracrine cellular senescence exacerbates biliary injury and impairs regeneration.
      • Paradis V.
      • Youssef N.
      • Dargere D.
      • et al.
      Replicative senescence in normal liver, chronic hepatitis C, and hepatocellular carcinomas.
      • Gutierrez-Reyes G.
      • del Carmen Garcia de Leon M.
      • Varela-Fascinetto G.
      • et al.
      Cellular senescence in livers from children with end stage liver disease.
      • Krizhanovsky V.
      • Yon M.
      • Dickins R.A.
      • et al.
      Senescence of activated stellate cells limits liver fibrosis.
      along with an absence of proliferative markers.
      Hepatocyte senescence appears to be a nearly universal phenomenon in the context of chronic liver disease, regardless of etiology.
      • Wiemann S.U.
      • Satyanarayana A.
      • Tsahuridu M.
      • et al.
      Hepatocyte telomere shortening and senescence are general markers of human liver cirrhosis.
      In addition to hepatocytes, the senescence of cholangiocytes is also detected readily within damaged small bile ducts and bile ductules in liver diseases affecting cholangiocytes and the biliary system. Senescence may play a role in biliary atresia, primary sclerosing cholangitis, cellular rejection, and primary biliary cirrhosis.
      • Sasaki M.
      • Ikeda H.
      • Sato Y.
      • Nakanuma Y.
      Decreased expression of Bmi1 is closely associated with cellular senescence in small bile ducts in primary biliary cirrhosis.
      ,
      • Sasaki M.
      • Ikeda H.
      • Yamaguchi J.
      • Nakada S.
      • Nakanuma Y.
      Telomere shortening in the damaged small bile ducts in primary biliary cirrhosis reflects ongoing cellular senescence.
      Although cellular senescence is one of the features of human adult chronic progressive cholangiopathies, only a few studies have looked into its role in liver transplant rejection. Previous research has described the role of cellular senescence in both chronic rejection (CR)
      • Lunz 3rd, J.G.
      • Contrucci S.
      • Ruppert K.
      • et al.
      Replicative senescence of biliary epithelial cells precedes bile duct loss in chronic liver allograft rejection: increased expression of p21(WAF1/Cip1) as a disease marker and the influence of immunosuppressive drugs.
      and acute cellular rejections (ACR) of higher RAI.
      • Brain J.G.
      • Robertson H.
      • Thompson E.
      • et al.
      Biliary epithelial senescence and plasticity in acute cellular rejection.
      In a post-transplant setting, histology frequently differs at different times, with the conspicuous appearance of atypical features at later times. In general, immune cells actively clear senescent cells; however, in a post-transplant scenario with a high dose of immunosuppression, senescent cells may accumulate and impact allograft rejection. Thus, the current study was carried out to assess the accumulation of senescent cells in a series of liver biopsies taken at various time points after transplantation. The study also focuses on the relationship between senescent markers and late acute cellular rejection (LAR) because of the atypical presentations, which often included prominent biliary atypia, poor response to immunosuppressants, and a poor prognosis.

      Method

      This is a single-institutional 10-year study conducted from January 2010 to December 2020. A total of 567 liver transplants were performed at the Institute of Liver and Biliary Sciences, New Delhi, India. Following ethical committee approval, a retrospective analysis was performed on all transplant cases in which liver biopsies indicated rejection (Figure 1) . Gender, age, the etiology of underlying liver disease, and the time of biopsy post-transplantation are among the recipient variables studied. The biopsies having at least 6 portal tracts were assessed for histological variables that included amount and type of portal inflammation, portal vein and hepatic vein endothelialitis, portal eosinophils, interface hepatitis, lobular inflammation, central perivenulitis, bile duct damage, bile duct atypia, and loss). All of the parameters were graded semiquantitatively (Figure 2, Figure 3).
      Figure 1
      Figure 1Consort diagram displaying the study design.
      Figure 2
      Figure 2ACR displaying portal inflammation and bile duct injury (arrow) (A; H&E;400x). LAR portal tracts show less inflammation (B; H&E;200x) and more prominent Zone 3 necroinflammatory lesion (Incet). An abnormal bile duct (arrow) in ECR along with nuclear pleomorphism and loss of polarity produce a ‘dysplastic-like’ appearance (C; H&E;200x). CR/DR shows loss of bile ducts (D; H&E; 200x). ACR, Acute cellular rejection; LAR, Late acute cellular rejection; ECR, Early chronic rejection; CR/DR, Chronic Rejection/Ductopenic Rejection.
      Figure 3
      Figure 3Immunohistochemical stain for the cells senescence markers p16, p21, and p53 in liver allograft biopsy with LAR (A, B, and C, respectively), ECR (D, E, and F, respectively) and in CR/DR (G, H, and I, respectively) show a high level of expression. LAR, Late acute cellular rejection; ECR, Early chronic rejection; CR/DR, Chronic Rejection/Ductopenic Rejection.
      All biopsies were reviewed and classified based on the type of rejection and the time since transplantation. Acute cellular rejection (ACR), late acute cellular rejection (LAR), early chronic rejection (ECR), and chronic rejection or ductopenic rejection were histological diagnostic subcategories (CR). Biopsies displaying evidence of rejection were also subdivided into different time zones: Group 1 (<3 months), Group 2 (3–6 months), Group 3 (6–12 months), and Group 4 (>12months).

      Immunosuppressants used in recipient

      The recipient was given intraoperative methylprednisolone 500 mg followed by 100 mg, 80 mg and 60 mg on day 1,2, and 3 respectively. After 2–3 days if renal function is normal, a low dose of Tacrolimus (0.5 mg BD) was added. According to the biochemical parameters the dose of steroids was tapered down, tacrolimus was increased, and myocphenolate mofetil was sometimes added to make the patient steroid-free at the end of 3 months. Cyclosporine was not prescribed in any of our study group patients.

      Histopathological classification of transplant rejection
      • Demetris A.J.
      • Bellamy C.
      • Hubscher S.G.
      • et al.
      2016 Comprehensive update of the Banff working group on liver allograft pathology: introduction of antibody-mediated rejection.

      For cellular rejection, three components of the diagnostic triad are portal inflammation, bile duct damage, and venular endothelial inflammation. Two of these features are required for the diagnosis of acute rejection. Grading was performed according to the Banff RAI score on a scale of 0–9.
      ACR is characterized by a mixed portal tract inflammation, bile duct damage, and venular endothelial inflammation including portal vein or hepatic vein. Lobular changes are not seen usually.
      LAR is characterized by mononuclear portal inflammation, less inflammation of the bile duct, and portal vein endothelialitis. More prominent interface hepatitis, Zone 3 necroinflammatory lesion, or central perivenulitis, and bile duct degenerative changes are seen in the cases.
      CR is characterized by the presence of ductopenia and arteriopathy. Based on the severity & extent of findings, CR may be considered as early CR & CR or Ductopenic Rejection (DR).
      Early Chronic Rejection (ECR): Two of the following features are mandatory.
      1: Senescence-related changes involving the majority of the ducts or bile duct loss in <50% of the patients. 2: occasional loss of portal tract hepatic arterioles involving <25% of the portal tract. 3: Zone 3 perivenular mononuclear inflammation/necrosis and/or mild perivenular fibrosis. 4: intimal inflammation, focal foam cell deposition without luminal compromise in large perihilar branches, 5: or in large perihilar bile ducts.
      Chronic Rejection/Ductopenic Rejection (CR/DR): Two of the following features are mandatory.
      1: Bile duct loss is seen in >50% of portal tracts, degenerative in the remnant bile duct, and portal tract hepatic arterioles loss involving >25% of portal tracts. 3: variable inflammation in or around terminal hepatic venules, focal obliteration, and moderate changes to severe fibrosis.
      4: Luminal narrowing, fibrointimal hyperplasia in large perihilar hepatic arteriolar branches, 5: mural fibrosis in large perihilar bile ducts.
      Histological features of senescence will include cytoplasmic eosinophilia, cellular and nuclear enlargement, and irregular arrangement with uneven nuclear spacing.

      Immunohistochemistry application and interpretation

      Biopsies displaying 10 or more portal tracts with a minimum of three portal tracts having bile ducts were considered for immunohistochemistry. The IHC has been applied manually for a panel of 3 senescent cell markers (p53, p21, p16) to be assessed in bile ducts.
      Senescent cells were evaluated by applying immunohistochemistry for p16 (clone EP16, mouse monoclonal, Livemore CA-9445, USA, ready to use), p21 (clone WA-1, mouse monoclonal, Meridian Rd, Rockford IL 61101, USA, 1:1000 dilution), and p53 (clone BP-53-12, mouse monoclonal, Livemore CA 94551, USA, ready to use). For the assessment of immunohistochemical expression in small bile ducts, the extent of expression was evaluated blindly by two histopathologists (AR, NN) and scores as 0 (negative), 1 (focal, weakly positive cells in =<1 small bile ducts), and score 2 (moderate to strong positive cells detected in 1–3 & >3 small bile ducts). Staining scores 0 and 1 were considered as Negative and score 2 was taken as Positive immunohistochemistry.

      Statistical analysis

      Statistical analysis was done with the help of SPSS software22.0 (SPSS, Chicago IL). Quantitative variables are indicated as mean ± standard deviation. Categorical variables are represented as frequency (%). Intergroup comparison of categorical variables was done using the Chi–Square test or Fisher Exact test as appropriate. A P-value of <0.05 was considered statistically significant.

      Results

      From the year 2010–2020, 567 liver transplantation surgeries and 366 biopsies from 95 patients (16.7%) with a clinical suspicion of rejection were performed. 53 biopsies were further excluded as they did not contain adequate tissue for diagnosis. We excluded 190 biopsies as they did not show any signs of rejection or other complications/pathologies. We examined the detailed histopathological parameters of 123 biopsies obtained from 68 patients in this study. The observed rejection rate was 12% (68/567). An immunohistochemistry panel for senescent markers was applied to the biopsies (53) having an adequate number of portal tracts (≥10) and these belonged to 38 patients [Figure 1].
      The recipients' average age was 37.3 ± 12.8 years, with a male predominance (60/68; 88.2 percent). Ten patients had acute liver failure, six had Acute on Chronic Failure (ACLF), four had Hepatocellular Carcinoma (HCC), one had polycystic liver disease, one had vascular pathology, and the remaining 46 had chronic liver disease, with a mean MELD Score of 24 ± 7.56. Underlying aetiologies for chronic liver disease patients were alcohol, HBV, HCV, NASH, Cryptogenic, and others with frequencies of 16 (34.8%),4 (8.7%), 2 (4.4%), 14 (30.4%), and 10 (21.7%), respectively.
      The biopsies (n = 123) were classified as ACR (57), LAR (44), ECR,
      • Brain J.G.
      • Robertson H.
      • Thompson E.
      • et al.
      Biliary epithelial senescence and plasticity in acute cellular rejection.
      CR/DR
      • Gutierrez-Reyes G.
      • del Carmen Garcia de Leon M.
      • Varela-Fascinetto G.
      • et al.
      Cellular senescence in livers from children with end stage liver disease.
      Thirty patients (44.1%) have had >2 episodes of rejection.
      Histological parameters revealed that mixed portal inflammation, portal vein endothelialitis, portal eosinophils, and bile duct damage were significantly higher in early biopsies (<3 months) whereas central perivenulitis without hepatic vein (HV) endothelialitis, bile duct atypia, and bile duct loss was more common in the late biopsies (>3 months).
      Bile duct injury may be focal in ACR and showed lymphocytic/mixed inflammatory cells infiltrating the small bile ducts, however, degenerative changes were not prominent. In LAR, inflammation of bile ducts and portal venous endothelium was less obvious but the degenerative changes of the bile ducts are well appreciated.
      Immunohistochemical expression analysis for the biliary senescence markers (53 biopsies) was done in the post-transplantation periods (Group 1–4) for the cases with the histologically proven diagnosis of rejection. In post-transplant group 1 (<3 months), out of 14 biopsies (14/53), positive immunoreactivity for p53, p21, and p16 was noted in 4/14 (28.6%), 6/14 (42.9%), and 3/14 (21.4%) respectively. Any 2 senescent markers’ positivity was noted in 5/14 (35.7%) cases whereas all three markers positivity was noted in only 3/14 (21.4%) cases.
      The post-transplant group 2 (3–6 months) included 13 biopsies (13/53, percent), with 8,9,7 biopsies positive for p53, p21, and p16.8/13 (61.5%) biopsies were positive for any two senescent markers.
      Group 3 (6–12 months) will include 17 biopsies (17/53%) with positive immunoreactivity for p53, p21, and p16 in 16/17 (94.1%), 15/17 (70.6%), and 12/17 (70.6%), respectively. Only 5/17 (29.4%) biopsies were positive for all three senescent cell markers, while 16/17 (94.1%) were positive for any two of the markers.
      Senescent cells were significantly increased in post-transplant group 4 (>12 months). Positive immunoreactivity for p53, p21, and p16 was observed in 9/9 (100 percent), 9/9 (100 percent), and 8/9 (88.9 percent) of the 9 biopsies (9/53, percent). In all 9 cases (100 percent) of biopsies, the positivity rate for any two senescent markers was noted. According to the Fisher exact test, senescent markers (any two) were comparable in all four groups (p 0.001). (Table 1).
      Table 1Expression of an Association of Cell Senescent Markers in Allograft Biopsy of Different Time-Zones Post-LDLT.
      Groupsp53p21p16All 3 markers positiveAny 2 markers positive
      No.%No.%No.%No.%No.%
      Group 1
      • Brain J.G.
      • Robertson H.
      • Thompson E.
      • et al.
      Biliary epithelial senescence and plasticity in acute cellular rejection.
      428.6%642.9%321.4%321.4%535.7%
      Group 2
      • Lunz 3rd, J.G.
      • Contrucci S.
      • Ruppert K.
      • et al.
      Replicative senescence of biliary epithelial cells precedes bile duct loss in chronic liver allograft rejection: increased expression of p21(WAF1/Cip1) as a disease marker and the influence of immunosuppressive drugs.
      861.5%969.2%753.8%215.3%861.5%
      Group 3
      • Gruttadauria S.
      • Vasta F.
      • Mandala L.
      • et al.
      Basiliximab in a triple-drug regimen with tacrolimus and steroids in liver transplantation.
      1694.1%1588.2%1270.6%529.4%1694.1%
      Group 4
      • Krizhanovsky V.
      • Yon M.
      • Dickins R.A.
      • et al.
      Senescence of activated stellate cells limits liver fibrosis.
      9100%9100%888.9%111.1%9100%
      Fisher Exact Test: P-value0.743<0.001
      LDLT, Live donor liver transplantation.
      After observing an increasing trend of senescent cell markers in different time zone groups, a comparison of early biopsies (Group1; 3 months) and late biopsies (Group2,3&4; >3 months) revealed significantly higher expression in late biopsies (>3 months) (P = 0.001 for any two markers). (Table2).
      Table 2Correlation of Cell Senescent Markers Positivity in Early (<3 months) and Late (>3 months) Allograft Biopsies.
      p53p21p16All 3 markers positiveAny 2 markers positive
      No.%No.%No.%No.%No.%
      Early Biopsy: Group 1 [<3mo.]
      • Brain J.G.
      • Robertson H.
      • Thompson E.
      • et al.
      Biliary epithelial senescence and plasticity in acute cellular rejection.
      428.5%642.8%321.4%321.4%535.7%
      Late Biopsy: Group 2,3&4 [>3mo.]3481.7%3384.6%2769.2%820.5%3384.6%
      Chi–Square Test: P-value0.942<0.001
      Similar expression analysis was performed based on the histological diagnosis rendered in the rejection spectrum. Details were provided in Table 3. In ACR, any two senescent markers were positive in 14/28 (50%) cases, whereas all three markers were positive in only 6/28 (21.4%) cases. LAR was positive for any two senescent markers in 12/13 (92.3%) cases, whereas all three markers were positive in 11/13 (84.6%) cases. CR revealed 100 percent positivity for two markers, and 8/9 (88.9 percent) cases had all three markers positive. In the last group with the diagnosis of chronic rejection/ductopenic rejection, any 2 senescent marker positivity was noted in all 3 cases (100%) and all three markers positivity was noted in 2/3 (66.7%). The Fisher exact analysis showed that senescent markers (any two) were comparable in all four histological groups (P < 0.001). (Table 3)
      Table 3Expression and Association of Cell Senescent Markers in Allograft Biopsy With Different Histological Rejection Stages.
      Histological Rejection Groupsp53p21p16All 3 markers positiveAny 2 markers positive
      No.%No.%No.%No.%P < 0.001No.%P = 0.009
      ACR (n = 28)1346.4%1553.6%932.1%621.4%1450%
      LAR (n = 13)1292.3%1292.3%1184.6%1184.6%1292.3%
      ECR (n = 9)9100%9100%888.9%888.9%9100%
      CR (n = 3)3100%3100%266.7%266.6%3100%
      Fisher Exact Test: P-value0.002<0.001
      ACR, Acute cellular rejection; LAR, Late acute cellular rejection; ECR, Early chronic rejection; CR, Chronic Rejection.
      The comparison of the ACR and LAR groups in terms of senescent marker expression revealed that the LAR group had increased expression (P = 0.009 for any two markers and 0.001 for all three markers). When the ACR group was compared to the LAR and CR combined groups, the senescent markers were significantly higher in the latter (P = 0.001) (Table 4).
      Table 4Comparison of Cell Senescent Markers Positivity Between Acute Cellular Rejection and Non-ACR (LAR, ECR & CR/DR).
      Histological Rejection Groups
      • Campisi J.
      • d'Adda di Fagagna F.
      Cellular senescence: when bad things happen to good cells.
      p53p21p16All 3 markers positiveAny 2 markers positive
      No.%No.%No.%No.%No.%
      Group 1: Acute Cellular Rejection [ACR]281346.4%1553.6%932.1%621.4%1450%
      Group 2: Non ACR [LAR &Chronic Rejection] [LAR, ECR, CR]
      • Sebagh M.
      • Rifai K.
      • Feray C.
      • et al.
      All liver recipients benefit from the protocol 10-year liver biopsies.
      2496%2496%2184%2184%2496%


      Chi–Square Test: P-value
      <0.001<0.001
      ACR, Acute cellular rejection; LAR, Late acute cellular rejection; ECR, Early chronic rejection; CR/DR, Chronic Rejection/Ductopenic Rejection.
      All cases were followed up on for at least three months, with a median follow-up period of 24 months. Follow-up data from 38 patients with histologically proven rejection in the first 6 months and a biopsy diagnosis of AR or LAR were analysed for evidence of biopsy-proven progression of immunologic pathology, i.e. chronic rejection. Chronic rejection occurred in nine patients. Progression to chronic rejection is significantly increased (P = 0.019) in LAR cases compared to ACR cases (Table 5).
      Table 5Association of Patients Progressed to Chronic Rejection With a Histological Diagnosis of Rejection.
      No of PatientsProgressed to CRNot Progressed to CRP-value
      ACR
      • Sebagh M.
      • Rifai K.
      • Feray C.
      • et al.
      All liver recipients benefit from the protocol 10-year liver biopsies.
      3/9 (33.3%)22/29 (75.8%)0.019
      LAR
      • Lunz 3rd, J.G.
      • Contrucci S.
      • Ruppert K.
      • et al.
      Replicative senescence of biliary epithelial cells precedes bile duct loss in chronic liver allograft rejection: increased expression of p21(WAF1/Cip1) as a disease marker and the influence of immunosuppressive drugs.
      6/9 (66.7%)7/29 (24.1%)
      ACR, Acute cellular rejection; LAR, Late acute cellular rejection; CR, Chronic Rejection.

      Discussion

      Despite recent advances in liver transplantation, rejection remains a significant problem in the postoperative period.
      • Starzl T.E.
      • Iwatsuki S.
      • Van Thiel D.H.
      • et al.
      Evolution of liver transplantation.
      Acute cellular rejection (ACR) occurs in 15–25% of liver transplant recipients. S Gruttadauria et al. studied 152 liver transplant recipients who had been treated with tacrolimus-based immunosuppressants and found that 15% of the patients had one or multiple episodes of acute cellular rejections.
      • Gruttadauria S.
      • Vasta F.
      • Mandala L.
      • et al.
      Basiliximab in a triple-drug regimen with tacrolimus and steroids in liver transplantation.
      In our study, the biopsy-proven rejection rate was 12% which was similar to the published literature.
      Rejection has been divided into acute and chronic forms based on the time of occurrence (acute: early, chronic: late), and the histological features (ACR, LAR, CR). Diagnosis of rejection biopsies always overlapped in terms of timeline and histological diagnosis.
      • Nacif L.S.
      • Pinheiro R.S.
      • Pecora R.A.
      • et al.
      Late acute rejection in liver transplant: a systematic review.
      The majority of acute cellular rejection episodes occur one to three months after transplantation.
      • Klintmalm G.B.
      • Nery J.R.
      • Husberg B.S.
      • Gonwa T.A.
      • Tillery G.W.
      Rejection in liver transplantation.
      It was found less important because it responded well to the immunosuppressant enhancement and was reversible.
      • Thurairajah P.H.
      • Carbone M.
      • Bridgestock H.
      • et al.
      Late acute liver allograft rejection; a study of its natural history and graft survival in the current era.
      They respond well to immunosuppressant enhancement and are reversible.
      Late acute cellular rejection (LAR) refers to acute cellular rejection that occurs after 3 months. It has different histological features, some in common with typical ACR and some with CR. It was resistant to immunosuppressants, caused causes graft loss, decreased patient survival, progression to chronic rejection, and poses a worse prognosis.
      • Thurairajah P.H.
      • Carbone M.
      • Bridgestock H.
      • et al.
      Late acute liver allograft rejection; a study of its natural history and graft survival in the current era.
      • Ramji A.
      • Yoshida E.M.
      • Bain V.G.
      • et al.
      Late acute rejection after liver transplantation: the Western Canada experience.
      • Uemura T.
      • Ikegami T.
      • Sanchez E.Q.
      • et al.
      Late acute rejection after liver transplantation impacts patient survival.
      A growing body of evidence suggested that cellular senescence plays a role in inflammatory cholangiopathies, including cellular rejection. Histological features are highly subjective; however, the immunohistochemistry could demonstrate the upregulated expression of p53, p21, and p16 in senescent cells.
      • Sasaki M.
      • Ikeda H.
      • Yamaguchi J.
      • Nakada S.
      • Nakanuma Y.
      Telomere shortening in the damaged small bile ducts in primary biliary cirrhosis reflects ongoing cellular senescence.
      ,
      • Sasaki M.
      • Ikeda H.
      • Haga H.
      • Manabe T.
      • Nakanuma Y.
      Frequent cellular senescence in small bile ducts in primary biliary cirrhosis: a possible role in bile duct loss.
      To elucidate its role in inflammatory cholangiopathies, we studied senescence in biliary epithelial cells from late biopsies and LAR & CR combined. We discovered that cellular senescence markers were highly expressed in the biopsies of interest. Previous studies on the role of senescence in a rejection are rare, and none performed in a large series like ours. Sasaki et al. investigated chronic biliary pathologies including 10 chronic liver allograft rejection biopsies in their study of senescence in liver diseases. In comparison to 25 histologically normal liver biopsies, they discovered the extensive-expression of p16 (6/10; 60%) and p21 (8/10; 80%) in small bile ducts in chronic rejection biopsies.
      • Sasaki M.
      • Ikeda H.
      • Haga H.
      • Manabe T.
      • Nakanuma Y.
      Frequent cellular senescence in small bile ducts in primary biliary cirrhosis: a possible role in bile duct loss.
      According to John G et al., the percentage and number of p21+ biliary epithelial cells (BEC) per portal tract were significantly higher in early CR compared to BECs in normal liver allograft biopsies or those with nonspecific changes, chronic hepatitis C, or obstructive cholangiopathy.
      • Lunz 3rd, J.G.
      • Contrucci S.
      • Ruppert K.
      • et al.
      Replicative senescence of biliary epithelial cells precedes bile duct loss in chronic liver allograft rejection: increased expression of p21(WAF1/Cip1) as a disease marker and the influence of immunosuppressive drugs.
      Our study showed concordant results with an 83.3%–100% expression rate (all three markers vs any two markers) in chronic rejection (ECR and CR/DR). We also discovered that senescence markers were strongly expressed in chronic rejection (both early and late) as opposed to acute cellular rejection, where the expression was dismal.
      J G Brain conducted a study to demonstrate the presence of senescent BEC in ACR by assessing the relationship between sen BEC and ACR grade in transplant biopsies. The cases included were 9 ‘time zero’ biopsies as control tissue and 25 transplanted liver biopsies with varying degrees of acute rejection (mild, moderate, and severe). They found a positive correlation (P < 0.0013) between increasing grade T cell-mediated acute rejection and the number of BEC expressing the senescence marker p21. However, the time zone and histological details to ascertain the proportion of LAR were not mentioned in the study.
      • Brain J.G.
      • Robertson H.
      • Thompson E.
      • et al.
      Biliary epithelial senescence and plasticity in acute cellular rejection.
      Our findings suggested that biliary senescence was implicated in the development of late rejection episodes.
      Degenerative changes of the bile ducts are well recognized in LAR. LAR is more frequently associated with the emergence of early CR-related features, such as bile duct atrophy, early duct loss, or centrilobular fibrosis. It was discovered that patients with late acute cellular rejection were more likely to develop chronic rejections (P = 0.019). While these changes were typically associated with AR that does not respond to treatment, cases that presented late may have a more insidious course.
      • Nakazawa Y.
      • Jonsson J.R.
      • Walker N.I.
      • et al.
      Fibrous obliterative lesions of veins contribute to progressive fibrosis in chronic liver allograft rejection.
      ,
      • Sebagh M.
      • Rifai K.
      • Feray C.
      • et al.
      All liver recipients benefit from the protocol 10-year liver biopsies.
      Increased expression of senescent cell markers (p53,p21, and p16) in the small bile ducts of LAR and CR compared to acute cellular rejection may explain the worsening of immunologic damage, therapy resistance, and poor prognosis observed in these cases. There is a need for research to validate the role of senescence in rejection, as well as its relationship to treatment and prognosis.
      This study on a large number of LDLT allograft biopsies demonstrates the role of biliary senescence in rejection and suggests a pathobiological role for senescence in the poor prognosis seen in late acute cellular rejection and chronic rejection.

      Credit authorship contribution statement

      Dr. Archana Rastogi, Dr. Neha Nigam and Dr. Rama Gayatri: Conceptualisation, methodology, design, analysis of data, and writing of the manuscript. Dr. Viniyendra Pamecha and Dr Chhagan Bihari: Reviewing and editing of the paper.

      Conflict of interest

      All authors have none to declare.

      Funding

      None.

      References

        • Hampel B.
        • Wagner M.
        • Teis D.
        • Zwerschke W.
        • Huber L.A.
        • Jansen-Durr P.
        Apoptosis resistance of senescent human fibroblasts is correlated with the absence of nuclear IGFBP-3.
        Aging Cell. 2005; 4: 325-330
        • Campisi J.
        • d'Adda di Fagagna F.
        Cellular senescence: when bad things happen to good cells.
        Nat Rev Mol Cell Biol. 2007; 8: 729-740
        • Collado M.
        • Blasco M.A.
        • Serrano M.
        Cellular senescence in cancer and aging.
        Cell. 2007; 130: 223-233
        • Herbig U.
        • Ferreira M.
        • Condel L.
        • Carey D.
        • Sedivy J.M.
        Cellular senescence in aging primates.
        Science. 2006; 311: 1257
        • Jeyapalan J.C.
        • Ferreira M.
        • Sedivy J.M.
        • Herbig U.
        Accumulation of senescent cells in mitotic tissue of aging primates.
        Mech Ageing Dev. 2007; 128: 36-44
        • Ferreira-Gonzalez S.
        • Lu W.Y.
        • Raven A.
        • et al.
        Paracrine cellular senescence exacerbates biliary injury and impairs regeneration.
        Nat Commun. 2018; 9: 1020
        • Paradis V.
        • Youssef N.
        • Dargere D.
        • et al.
        Replicative senescence in normal liver, chronic hepatitis C, and hepatocellular carcinomas.
        Hum Pathol. 2001; 32: 327-332
        • Gutierrez-Reyes G.
        • del Carmen Garcia de Leon M.
        • Varela-Fascinetto G.
        • et al.
        Cellular senescence in livers from children with end stage liver disease.
        PLoS One. 2010; 5e10231
        • Krizhanovsky V.
        • Yon M.
        • Dickins R.A.
        • et al.
        Senescence of activated stellate cells limits liver fibrosis.
        Cell. 2008; 134: 657-667
        • Wiemann S.U.
        • Satyanarayana A.
        • Tsahuridu M.
        • et al.
        Hepatocyte telomere shortening and senescence are general markers of human liver cirrhosis.
        Faseb J. 2002; 16: 935-942
        • Sasaki M.
        • Ikeda H.
        • Sato Y.
        • Nakanuma Y.
        Decreased expression of Bmi1 is closely associated with cellular senescence in small bile ducts in primary biliary cirrhosis.
        Am J Pathol. 2006; 169: 831-845
        • Sasaki M.
        • Ikeda H.
        • Yamaguchi J.
        • Nakada S.
        • Nakanuma Y.
        Telomere shortening in the damaged small bile ducts in primary biliary cirrhosis reflects ongoing cellular senescence.
        Hepatology. 2008; 48: 186-195
        • Lunz 3rd, J.G.
        • Contrucci S.
        • Ruppert K.
        • et al.
        Replicative senescence of biliary epithelial cells precedes bile duct loss in chronic liver allograft rejection: increased expression of p21(WAF1/Cip1) as a disease marker and the influence of immunosuppressive drugs.
        Am J Pathol. 2001; 158: 1379-1390
        • Brain J.G.
        • Robertson H.
        • Thompson E.
        • et al.
        Biliary epithelial senescence and plasticity in acute cellular rejection.
        Am J Transplant. 2013; 13: 1688-1702
        • Demetris A.J.
        • Bellamy C.
        • Hubscher S.G.
        • et al.
        2016 Comprehensive update of the Banff working group on liver allograft pathology: introduction of antibody-mediated rejection.
        Am J Transplant. 2016; 16: 2816-2835
        • Starzl T.E.
        • Iwatsuki S.
        • Van Thiel D.H.
        • et al.
        Evolution of liver transplantation.
        Hepatology. 1982; 2: 614-636
        • Gruttadauria S.
        • Vasta F.
        • Mandala L.
        • et al.
        Basiliximab in a triple-drug regimen with tacrolimus and steroids in liver transplantation.
        Transplant Proc. 2005; 37: 2611-2613
        • Nacif L.S.
        • Pinheiro R.S.
        • Pecora R.A.
        • et al.
        Late acute rejection in liver transplant: a systematic review.
        Arq Bras Cir Dig. 2015; 28: 212-215
        • Klintmalm G.B.
        • Nery J.R.
        • Husberg B.S.
        • Gonwa T.A.
        • Tillery G.W.
        Rejection in liver transplantation.
        Hepatology. 1989; 10: 978-985
        • Thurairajah P.H.
        • Carbone M.
        • Bridgestock H.
        • et al.
        Late acute liver allograft rejection; a study of its natural history and graft survival in the current era.
        Transplantation. 2013; 95: 955-959
        • Ramji A.
        • Yoshida E.M.
        • Bain V.G.
        • et al.
        Late acute rejection after liver transplantation: the Western Canada experience.
        Liver Transplant. 2002; 8: 945-951
        • Uemura T.
        • Ikegami T.
        • Sanchez E.Q.
        • et al.
        Late acute rejection after liver transplantation impacts patient survival.
        Clin Transplant. 2008; 22: 316-323
        • Sasaki M.
        • Ikeda H.
        • Haga H.
        • Manabe T.
        • Nakanuma Y.
        Frequent cellular senescence in small bile ducts in primary biliary cirrhosis: a possible role in bile duct loss.
        J Pathol. 2005; 205: 451-459
        • Nakazawa Y.
        • Jonsson J.R.
        • Walker N.I.
        • et al.
        Fibrous obliterative lesions of veins contribute to progressive fibrosis in chronic liver allograft rejection.
        Hepatology. 2000; 32: 1240-1247
        • Sebagh M.
        • Rifai K.
        • Feray C.
        • et al.
        All liver recipients benefit from the protocol 10-year liver biopsies.
        Hepatology. 2003; 37: 1293-1301