Digestive and Liver Disease 46 (2014) 363–368
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Digestive and Liver Disease journal homepage: www.elsevier.com/locate/dld
Liver, Pancreas and Biliary Tract
Telomere dysfunction in peripheral blood mononuclear cells from patients with primary biliary cirrhosis Pietro Invernizzi a , Francesca Bernuzzi a , Ana Lleo a , Vanila Pozzoli b , Monica Bignotto b,c , Paola Zermiani b , Andrea Crosignani b , Pier Maria Battezzati b , Massimo Zuin b , Mauro Podda a , Chiara Raggi a,∗ a
Liver Unit and Center for Autoimmune Liver Diseases Humanitas Clinical and Research Center, Rozzano, Italy Gastroenterology and Liver Unit, San Paolo Hospital Medical School, University of Milan, Milan, Italy c Department of Human Morphology and Biomedical Sciences, University of Milan, Italy b
a r t i c l e
i n f o
Article history: Received 29 August 2013 Accepted 19 November 2013 Available online 27 December 2013 Keywords: Autoimmune disease Primary biliary cirrhosis Telomere dysfunction
a b s t r a c t Background: Chromosomal instability in peripheral blood mononuclear cells has a role in the onset of primary biliary cirrhosis. We hypothesized that patients with primary biliary cirrhosis may harbour telomere dysfunction, with consequent chromosomal instability and cellular senescence. Aim: To evaluate the clinical significance of telomerase activity and telomere length in peripheral blood mononuclear cells from patients with primary biliary cirrhosis. Study design: In this population-based case control study, 48 women with primary biliary cirrhosis (25 with cirrhosis), 12 with chronic hepatitis C matched by age and severity of disease, and 55 age-matched healthy women were identified. Mononuclear cells from the peripheral blood of patients and controls were isolated. Telomere length and telomerase activity were measured. Results: Telomere length and telomerase activity did not differ between cases (5.9 ± 1.5 kb) and controls (6.2 ± 1.4 kb, pc = 0.164). Telomere shortening and advanced-stage disease strongly correlated with telomerase activity. Patients with advanced disease retained significantly less telomerase activity than those with early-stage disease (0.6 ± 0.9 OD vs. 1.5 ± 3.7 OD, p = 0.03). Telomere loss correlated with age, suggesting premature cellular ageing in patients with primary biliary cirrhosis. Conclusion: Our data strongly support the telomere hypothesis of human cirrhosis, indicating that telomere shortening and telomerase activity represent a molecular mechanism in the evolution of human cirrhosis in a selected population of patients. © 2013 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.
1. Introduction Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by progressive destruction of intrahepatic bile ducts with cholestasis, portal inflammation, and fibrosis [1,2]. The origin of PBC may be associated with sex chromosome defects mainly based on the observation that X chromosome contains a considerable number of sex- and immune-related genes [3–6]. It has been suggested that environmental factors may trigger immune-mediated aggression against small and medium intrahepatic bile ducts in genetically predisposed individuals [7,8]. The chronic immune characteristic of PBC, similarly to the majority
∗ Corresponding author at: Liver Unit and Center for Autoimmune Liver Diseases, Humanitas Clinical and Research Center, via A. Manzoni 56, 20089 Rozzano, Milan, Italy. Tel.: +39 02 8224 5127; fax: +39 02 8224 5191. E-mail address: [email protected] (C. Raggi).
of autoimmune disorders, can affect the stability, replication and function of chromosomes [5,7,8]. Telomere dysfunction is a hallmark of chromosomal instability [9–11]. Telomeres are chromatin structures formed by kilobases of tandem repeats of the sequences TTAGGG and specific proteins, which cap and protect the end of chromosomes [12]. These telomeric repeats are elongated by telomerase, a ribonucleoprotein enzyme that adds TTAGGG repeats into pre-existent telomeres and extends the 3 end of telomeres. In humans the telomerase is expressed only in germ and stem cells, and prevents critical shortening of their telomeres. Studies in somatic cells have demonstrated that there is a telomere progressive shortening with every cell division and it is believed to be a biological clock, which leads to cellular senescence. Stably capped telomeres contribute greatly to genome stability and a healthy lifespan. Telomerase can counteract replicative senescence by maintaining telomeres and genomic stability. Telomerase also plays a role in ageing and longevity of organisms via its telomere stabilizing function as demonstrated in a cancer-protected mouse model [13].
1590-8658/$36.00 © 2013 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.dld.2013.11.008
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Telomere dysfunction has been described in many human pathologies [12,14] mainly in cancers but also in inflammatory disorders such as autoimmune [15,16] and liver diseases [17]. Indeed, telomerase activity is constantly present in malignant cells which are able to escape senescence and to have unlimited replication potential [10,14]. Accelerated telomere loss (with a reactive, but ineffective, enhanced telomerase activity) has also been observed in different lymphocyte subset from patients with autoimmune diseases, reflecting premature ageing of the immune system [15,16]. To this end, it is important to note that women with PBC and other autoimmune diseases are characterized by an enhanced X monosomy rate in peripheral blood mononuclear cells (PBMCs), particularly T and B lymphocytes [3,18], possibly due to chromosomal instability. Finally, a number of reports showed that telomere dysfunction is a disease-dependent sign of liver cirrhosis thus suggesting that hepatocellular telomere shortening and senescence represent a molecular mechanism in the evolution of human cirrhosis [17]. Similarly, a significant decrease in telomere length was recently found in the biliary epithelial cells of patients with PBC, the target organ of the disease [19]. Remarkably, there are several studies analysing telomeres in liver tissue of patients with liver disorders of different origin but hardly any studies analysing telomere function in PBMC. In this regard, Hoare and colleagues showed the relation between lymphocyte telomere length, age and clinical outcome in non-hereditary liver disorder such as chronic hepatitis C virus (HCV) infection [20]. Since telomere shortening may be a trigger of senescence of immune-related cells and autoimmunity, we here tested the hypothesis that patients with PBC bear telomere dysfunction in PBMCs, by estimating telomere length and telomerase activity.
2. Materials and methods 2.1. Study population Blood samples were obtained from 48 women with PBC. The diagnosis of PBC was based on the internationally accepted criteria, which include the presence of cholestatic liver disease for at least six months, a liver biopsy compatible with the diagnosis, a positive test for anti-mitochondrial antibodies (AMA), serum alkaline phosphatase levels of at least one and a half times more than the upper normal limit, and the absence of biliary obstruction as assessed by ultrasonography, computed tomography or endoscopic cholangiography [2,21]. The characteristics of PBC patients are shown in Table 1. Serum autoantibodies were determined using indirect immunofluorescence [22]. All PBC patients were AMA positive [23]. The following were considered disease-related symptoms: pruritus, jaundice, major complications of portal hypertension, i.e. hepatic encephalopathy, variceal bleeding or ascites requiring diuretic therapy. Disease duration was calculated as the time from the date of the earliest suspected evidence of liver disease to the date of blood collection. Twenty-three patients with no fibrosis on liver biopsy fibrosis (Ludwig’s stage I and II) [24] were considered as having early-stage disease, while the remaining 25 with fibrosis or cirrhosis (stage III or IV) as having advanced disease. At the time of enrolment, 38 (79%) of the PBC patients were receiving ursodeoxycholic acid (UDCA) treatment. The control populations consisted of 12 women with chronic hepatitis C (CHC) who attended our Liver Unit during the same enrolment period as the PBC patients, and 55 age-matched healthy women. The CHC patients were matched with the PBC patients in terms of age (10 five-year age classes ranging from 34 years or younger to 75 years or older) and the severity of liver disease (absence of cirrhosis, presence of compensated cirrhosis, development of major complications of portal hypertension). CHC patients
underwent blood sampling before receiving any anti-viral medication. All subjects provided written consent after being informed about the nature of the study. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki (6th revision, 2008) as reflected in a priori approval by the institution’s human research committee. 2.2. Cell separation PBMCs obtained from a total of 48 female PBC patients were used for this study. PBMCs were isolated from 40 ml of blood with ethylenediaminetetraacetic acid (EDTA) by density-gradient centrifugation using Histopaque-1077 (Sigma). After centrifugation, cells were washed with phosphate-buffered saline (PBS) and the viability of cells was determined using trypan blue. Part of these PBMCs was stored for the study on telomerase activity. 2.3. Telomere length measurement DNA was purified from PBMC using the QIAamp DNA blood Midi Kit (Qiagen, and stored in aliquots at −20 ◦ C. The mean terminal restriction fragment (TRF) length was determined by the use of the TeloTAGGG Telomere Length Assay (Roche). Briefly, 2 g of purified DNA was digested in a final volume of 20 l reaction mixture with HinfI/RsaI enzyme mixture (1 U/l for each enzyme) at 37 ◦ C for 2 h and electrophoresed on 0.8% agarose MP gel in 1× Tris–acetate–EDTA buffer (0.04 M Tris–acetate, 0.001 M EDTA, pH 8) for 2.30 h at 150 V cm. Gels were then denatured, neutralized, and Southern transferred to a positively charged nylon membrane, according to the protocol described by the supplier (Roche). The blotted DNA fragments were hybridized to a digoxigenin (DIG)labelled probe specific for telomeric repeats and incubated with a DIG-specific antibody covalently coupled to alkaline phosphate. To determine the TRF length, the hybridized probe was visualized by chemiluminescent detection which detected TRF DNA on Hyperfilm (Amersham Biosciences). The exposed X-ray film was scanned and analysed densitometrically by Gel Doc with QuantityOne software (Biorad). The amount of telomeric DNA was calculated by integrating the area highest signal intensity of each smear. The TRF length was estimated relative to molecular weight standards that was run in each gel. For quantitative measurement and valid comparison of telomere length in different samples, control-DNA with short telomeres (L) and long telomeres (H) were included in each gel. Fig. 1 shows a representative Southern blotting gel. 2.4. Telomerase activity assay Telomerase activity was measured in PBMCs from PBC patients and controls using the TeloTAGGG Telomerase PCR ELISA kit (Roche), which combines a telomere repeat amplification protocol (TRAP) assay with detection by ELISA. In brief, 2 × 105 PBMCs were added to an elongation/amplification mixture and distilled water in a total volume of 50 l. The resulting elongation products were amplified by polymerase chain reaction (PCR), and PCR products were hybridized to a DIG-labelled telomeric repeat-specific probe bound to a streptavidin-coated 96-well plate. The binding reaction was detected with an anti-DIG-peroxidase antibody, visualized by a colour reaction product, and quantified photometrically. The absorbance of each sample was measured at 450 nm reading against the blank (reference wavelength 690 nm) using an ELISA microplate reader (Labsystems iEMS) within 30 min after addition of the stop reagent. Each sample was measured in duplicates. Each negative sample was obtained by heat treatment (10 min at 80 ◦ C). The samples were regarded as telomerase-positive if the difference
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Table 1 Clinicopathologic characteristics of study population. PBC
No. females Age (years) UDCA treatment Associated diseasesb Disease duration (years)
All
Early diseasea
Advanced diseasea
48 64 ± 11 38 (79%) 15 (31%) 12.7
23 62 ± 11 17 (74%) 5 (22%) 12.3
25 66 ± 11 21 (84%) 10 (40%) 12.8
Controls
CHC
55 55 ± 11 – – –
12 68 ± 8 – – –
Notes: UDCA, ursodeoxycholic acid; CHC, chronic hepatitis C; PBC, primary biliary cirrhosis. a Disease status. b Autoimmune diseases most frequently associated with primary biliary cirrhosis (autoimmune thyroid disease, scleroderma, rheumatoid arthritis and systemic lupus erythematosus).
in absorbance is higher than the twofold background activity. All criteria were fulfilled according to the kit requirements (Roche).
2.5. Statistical analysis The chi-square or Fisher’s exact test were used to compare categorical variables. For the analysis of continuous variables, the Mann–Whitney test was used to compare two groups and the Kruskall–Wallis test, a non-parametric analysis of variance, was used to compare more than two groups. When the analysis of variance, or the overall chi-square test, gave a significant (p < 0.05) result, pairwise comparisons were carried out using Mann–Whitney or Fisher’s exact tests to assess which group differed from the others. In order to control for multiple testing, p-values from such pairwise comparisons were corrected (pc ) by the number of comparisons done, according to the Bonferroni inequality method. Linear regression analysis was used to study the relationship between age, duration of disease and the telomere length. The data in the text and figures are expressed as mean ± SD. The statistical comparisons were made using GraphPad PRISM version 5 (GraphPad). All of the analyses were two-tailed.
3. Results 3.1. Telomere length shortening in advanced stage of primary biliary cirrhosis To test whether premature telomeric loss occurred in PBC patients possibly due to telomere dysfunction, telomere shortening in the PBMCs was analysed on a large selection of PBC patients. The length of telomere restriction fragment (TRF) was analysed using DNA extracted from PBMC samples. The mean TRF length of PBMC did not differ between the PBC (5.9 ± 1.5 kb) and the control group (6.2 ± 1.4 kb, pc = 0.164) or the CHC group (5.8 ± 1.0 kb, pc = 0.095) (Fig. 2A). Interestingly, the mean TRF of PBMC in PBC was significantly shorter in the subgroup with advanced liver disease (n = 25) than that with early-stage disease (n = 23) (5.4 ± 0.8 kb vs. 6.4 ± 1.8 kb, p = 0.03) (Fig. 2B). Furthermore, there was a significant difference between PBC patients with advanced disease and the normal controls (n = 55) (pc = 0.008), while the TRF was similar in the PBC group with early-stage disease compared to the control group (pc = 0.46). To confirm our data we have compared two classes of variables, the first containing the values of the length of telomeres, (collected in two subsets created based on the average of the total), the second containing the stages of the disease (early stage, advanced) with controls. Applying the Fisher test on the two classes of values using a cut off 5.8 kb, we were able to show a dependent relationship between the stages of pathology and the lengths of the telomeres. This analysis validates the previously Mann–Whitney test applied. The contingency table was able to show that healthy controls retained longer telomeres compared to the PBC patients. In particular, the TRF-disease stagedependence is indeed much more significant when comparing patients with each of the patients in the early stage and advanced stage (p-value ea-adv = 0.045), whereas the comparison between patients in the early stages and controls, and patients in the advanced stage and controls, does not seem to show a particular dependency (p-value ea-ctr = 0439; p-value adv-ctr = 0143) (Table 2 and Fig. 2B). It is to note that telomere shortening did not differ in subgroups of the PBC patients according to the presence of concomitant autoimmune disorders, such as autoimmune thyroid disease, rheumatoid arthritis (RA), and systemic lupus
Table 2 Association between telomerase length in primary biliary cirrhosis patients and different stage disease status and controls. Fig. 1. Representative example of telomere restriction fragments. Southern blot in peripheral-blood mononuclear cells of patients with primary biliary cirrhosis (Lanes 4–5) and control subjects (Lanes 6–9). Control-DNA with short telomeres (Lane 2) and long telomeres (Lane 3) were included in each gel. DNA molecular weight standards (Lane 1) served as a reference for samples. Telomere length, in relation to DNA molecular weight standards, was determined by calculating the mean TRF length using Southern blot method (Telo TAGGG telomere length assay, Roche) described in detail in the text.
Short telomere Long telomere Total
Early disease
Advanced disease
Controls
Total
6 (26%) 17 (74%) 23 (100%)
14 (56%) 11 (44%) 25 (100%)
20 (36%) 35 (64%) 55 (100%)
40 (39%) 63 (61%) 103 (100%)
Short and long telomeres are originated from the average telomere length of samples corresponding to a value of 5.8 kb which leads to split the set of these variables.
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Fig. 2. Telomere shortening in peripheral-blood mononuclear cells from patients with primary biliary cirrhosis. (A) The length of telomere restriction fragments in peripheralblood mononuclear cells was determined in 48 patients with primary biliary cirrhosis, 12 with hepatitis C virus and 55 age-matched controls. Telomere length was determined by calculating the mean TRF length in relation to DNA molecular weight standards using Southern blot method (Telo TAGGG telomere length assay, Roche). Telomere length did not differ between primary biliary cirrhosis, hepatitis C virus and control group. Statistical analysis was performed with Mann–Whitney test: p > 0.05. (B) Comparison of telomere length in clinical status of disease: early and late stage primary biliary cirrhosis patients. Results demonstrated a progressive shortening with advancing pathology (Mann–Whitney test: *p < 0.05, **p < 0.01). (PBMC, peripheral-blood mononuclear cells; PBC, primary biliary cirrhosis; HCV, hepatitis C virus; CTR, control).
erythematous (SLE) (data not shown). Moreover, no significant differences were revealed with and without UDCA treatment (data not shown). Furthermore, in both early and in the advanced PBC disease, non-significance was found between increased albumin and INR levels and telomere shortening in our study population (data not shown). 3.2. Enhanced telomerase activity in early-stage of primary biliary cirrhosis In order to deepen the role of telomere in PBC initiation and progression, we measured the PBMC telomerase activity using a highly sensitive and specific TRAP ELISA detection system in 30 PBC patients in either early stage (n = 15) or advanced stage (n = 15), and in 12 healthy controls. As expected and similarly to the telomere length, PBMC telomerase activity did not differ between PBC patients and healthy controls (1.0 ± 2.6 OD vs. 0.6 ± 0.8 OD, p = 0.79; Fig. 3A), although a trend to an increased enzymatic activity in the PBC group can be observed. Interestingly, PBC patients with advanced disease retained significantly less telomerase activity than those with early-stage disease (0.6 ± 0.9 OD vs. 1.5 ± 3.7 OD, p = 0.03; Fig. 3B), thus well-fitting with our finding of a reduced telomere length in advanced stage PBC. Although telomerase activity is increased in early-stage PBC patients, we speculated that late stage has progressively exhausted its activity accelerating the process of telomeric loss. 4. Discussion There has been mounting evidence of a causal role for telomere dysfunction in a number of degenerative disorders [12,14,25]. It has been increasingly suggested that telomere dysfunction is
implicated in immunosenescence phenomenon reflecting premature ageing of immune system [16,26–29]. Many studies have observed that accelerated telomere loss in different lymphocyte subpopulations is a common feature of autoimmune disease such as SLE, RA, scleroderma, atopic dermatitis and psoriasis [15,30–36]. For the first time, our study investigated the clinical significance of telomere erosion in peripheral immune system from PBC patients. Since female patients with PBC and other autoimmune diseases are characterized by an enhanced X monosomy rate in PBMC, particularly T and B lymphocytes, a marker of chromosomal instability [3,18], our study were specifically designed on an entire female population. Moreover, since lymphocytes telomere length is known to change with age, we carefully selected age-matched controls. Interestingly, we reported that telomere shortening affected PBMCs of advanced stage PBC patients. Based on these data, it seems that telomere shortening is an “acquired” feature and not a “constitutive” property of PBMC in PBC disease. We suggest that a progressively acquired chromosomal instability due to telomere dysfunction may be involved in the pathogenesis of PBC (mainly in advanced disease) by inducing senescence of immune-related cells. In order to clarify the role of telomere erosion in PBC pathogenesis, we next examined PBMC telomerase activity in PBC patients and controls. PCR-based TRAP assay could not detect clear difference between these two groups, but the study revealed that telomerase activity was higher in patients with early stage than in advanced phase. Similar findings were found in previous investigation on telomere dysfunction in biliary epithelial cells in PBC and other hepatobiliary diseases [19,37,38]. Indeed, Sasaki et al. showed that telomere shortening and accumulation of DNA damage characterized cellular senescence of bile ducts, the specific target-tissue cells [19]. Furthermore, it has been described that other types of autoimmune diseases, such as inclusion body myositis, telomere shorten
Fig. 3. Mean telomerase activity in peripheral-blood mononuclear cells. (A) Telomerase activity is reported as optical density (OD). Bar graph showed no difference in telomerase activity between primary biliary cirrhosis patients and controls (Mann–Whitney test: p > 0.05). (B) Telomerase activity in peripheral-blood mononuclear cells from early and late stage patients with primary biliary cirrhosis. Telomerase activity is reported as optical density (OD). Mean OD was higher in early disease than in advanced disease (Mann–Whitney test: p < 0.05). (PBC, primary biliary cirrhosis; CTR, control).
P. Invernizzi et al. / Digestive and Liver Disease 46 (2014) 363–368
in affected primary muscular cells, the target-tissue cells [39]. Moreover, beside autoimmune pathologies, telomere shortening has been reported in other organs subject to chronic inflammation, such as Barrett’s oesophagus and ulcerative colitis [40,41]. The causal relation between autoimmunity and telomere shortening is still enigma [16,26–29]. However, the immune system is in constant self-renewal and, consequently, is highly dependent on efficient telomere maintenance. Indeed, cellular component of the immune system is derived from hematopoietic stem cells which actively divide and differentiate throughout life [42]. The telomere shortening in PBC could be due to the chronic activation/proliferation occurring in autoimmune conditions and could lead to premature immune senescence, involving exit from the mitotic cycle and/or cell death. Based on results of analyses of telomerase activity and telomerase length, we speculate that in patients with early-stage PBC, many lymphocytes cells divide continuously and their telomerase activity is higher than that in healthy subjects, but not so high to prevent telomere shortening. Further, telomerase activity was elevated in early-stage disease but not in the advanced. Many possible explanations could be proposed for these telomere dysfunctions. At the core of this investigation was the concept that diminished TRF length in PBC could reflect a secondary phenomenon resulting from the telomerase that was not expressed in advanced disease. At this regard, it might be possible that in advanced-stage PBC patients, telomerase activity is progressively exhausted and not sufficient to prevent telomere erosion during liver disease progression. Indeed, advanced PBC is characterized by evident telomere loss. In line with this hypothesis some studies have reported that telomere shortening is present in cirrhosis induced by viral hepatitis, toxic liver damage, autoimmunity, and cholestasis [38]. Moreover, several reports have demonstrated that immunosenescence of biliary epithelial cells, the specific target-tissue cells, might be important in the development of PBC [8,19,43,44]. Furthermore, it has been shown that hepatocyte telomere shortening and senescence correlate with progression of cirrhosis. The telomere hypothesis of human cirrhosis indicates that hepatocellular telomere shortening and senescence represents a molecular mechanism in the evolution of human cirrhosis in early advanced PBC patients [38]. In relation to other studies carried out on liver tissue, the novelty of our investigation is based on the correlation between telomere loss and PBC disease progression, testing the peripheral immune system. We suggest that telomerase activity might be sufficient to compensate for the telomere loss that occurs during proliferation and thus prevent accelerated ageing of the immune system in those patients with early-stage disease. In advanced-stage disease the telomerase might not prevent telomere loss and this fact supports the hypothesis that cirrhosis in PBC might be the predominant factor promoting telomere shortening. We highlighted that other variables are also involved in PBC initiation and progression such as: age, duration of pathology, treatment with drugs, presence of associated disease, disease stage, in addition to TRF and telomerase activity. In conclusion, in contrast with previous studies on telomere dysfunction in other autoimmune and degenerative diseases, we could not demonstrate a significant reduction in telomere length in PBMCs from PBC patients compared to healthy controls. However, we were able to recapitulate the association with disease stages, showing that patients with advanced-stage PBC are characterized by extremely short telomeres and a significantly decreasing telomerase activity. In this respect, the data we obtained are of clinical interest also because of the current possibility to therapeutically target the telomere system [9]. In view of our data, we can speculate that telomere dysfunction in PBC may neither be related to
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Digestive and Liver Disease journal homepage: www.elsevier.com/locate/dld
Liver, Pancreas and Biliary Tract
Telomere dysfunction in peripheral blood mononuclear cells from patients with primary biliary cirrhosis Pietro Invernizzi a , Francesca Bernuzzi a , Ana Lleo a , Vanila Pozzoli b , Monica Bignotto b,c , Paola Zermiani b , Andrea Crosignani b , Pier Maria Battezzati b , Massimo Zuin b , Mauro Podda a , Chiara Raggi a,∗ a
Liver Unit and Center for Autoimmune Liver Diseases Humanitas Clinical and Research Center, Rozzano, Italy Gastroenterology and Liver Unit, San Paolo Hospital Medical School, University of Milan, Milan, Italy c Department of Human Morphology and Biomedical Sciences, University of Milan, Italy b
a r t i c l e
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Article history: Received 29 August 2013 Accepted 19 November 2013 Available online 27 December 2013 Keywords: Autoimmune disease Primary biliary cirrhosis Telomere dysfunction
a b s t r a c t Background: Chromosomal instability in peripheral blood mononuclear cells has a role in the onset of primary biliary cirrhosis. We hypothesized that patients with primary biliary cirrhosis may harbour telomere dysfunction, with consequent chromosomal instability and cellular senescence. Aim: To evaluate the clinical significance of telomerase activity and telomere length in peripheral blood mononuclear cells from patients with primary biliary cirrhosis. Study design: In this population-based case control study, 48 women with primary biliary cirrhosis (25 with cirrhosis), 12 with chronic hepatitis C matched by age and severity of disease, and 55 age-matched healthy women were identified. Mononuclear cells from the peripheral blood of patients and controls were isolated. Telomere length and telomerase activity were measured. Results: Telomere length and telomerase activity did not differ between cases (5.9 ± 1.5 kb) and controls (6.2 ± 1.4 kb, pc = 0.164). Telomere shortening and advanced-stage disease strongly correlated with telomerase activity. Patients with advanced disease retained significantly less telomerase activity than those with early-stage disease (0.6 ± 0.9 OD vs. 1.5 ± 3.7 OD, p = 0.03). Telomere loss correlated with age, suggesting premature cellular ageing in patients with primary biliary cirrhosis. Conclusion: Our data strongly support the telomere hypothesis of human cirrhosis, indicating that telomere shortening and telomerase activity represent a molecular mechanism in the evolution of human cirrhosis in a selected population of patients. © 2013 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.
1. Introduction Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by progressive destruction of intrahepatic bile ducts with cholestasis, portal inflammation, and fibrosis [1,2]. The origin of PBC may be associated with sex chromosome defects mainly based on the observation that X chromosome contains a considerable number of sex- and immune-related genes [3–6]. It has been suggested that environmental factors may trigger immune-mediated aggression against small and medium intrahepatic bile ducts in genetically predisposed individuals [7,8]. The chronic immune characteristic of PBC, similarly to the majority
∗ Corresponding author at: Liver Unit and Center for Autoimmune Liver Diseases, Humanitas Clinical and Research Center, via A. Manzoni 56, 20089 Rozzano, Milan, Italy. Tel.: +39 02 8224 5127; fax: +39 02 8224 5191. E-mail address: [email protected] (C. Raggi).
of autoimmune disorders, can affect the stability, replication and function of chromosomes [5,7,8]. Telomere dysfunction is a hallmark of chromosomal instability [9–11]. Telomeres are chromatin structures formed by kilobases of tandem repeats of the sequences TTAGGG and specific proteins, which cap and protect the end of chromosomes [12]. These telomeric repeats are elongated by telomerase, a ribonucleoprotein enzyme that adds TTAGGG repeats into pre-existent telomeres and extends the 3 end of telomeres. In humans the telomerase is expressed only in germ and stem cells, and prevents critical shortening of their telomeres. Studies in somatic cells have demonstrated that there is a telomere progressive shortening with every cell division and it is believed to be a biological clock, which leads to cellular senescence. Stably capped telomeres contribute greatly to genome stability and a healthy lifespan. Telomerase can counteract replicative senescence by maintaining telomeres and genomic stability. Telomerase also plays a role in ageing and longevity of organisms via its telomere stabilizing function as demonstrated in a cancer-protected mouse model [13].
1590-8658/$36.00 © 2013 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.dld.2013.11.008
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Telomere dysfunction has been described in many human pathologies [12,14] mainly in cancers but also in inflammatory disorders such as autoimmune [15,16] and liver diseases [17]. Indeed, telomerase activity is constantly present in malignant cells which are able to escape senescence and to have unlimited replication potential [10,14]. Accelerated telomere loss (with a reactive, but ineffective, enhanced telomerase activity) has also been observed in different lymphocyte subset from patients with autoimmune diseases, reflecting premature ageing of the immune system [15,16]. To this end, it is important to note that women with PBC and other autoimmune diseases are characterized by an enhanced X monosomy rate in peripheral blood mononuclear cells (PBMCs), particularly T and B lymphocytes [3,18], possibly due to chromosomal instability. Finally, a number of reports showed that telomere dysfunction is a disease-dependent sign of liver cirrhosis thus suggesting that hepatocellular telomere shortening and senescence represent a molecular mechanism in the evolution of human cirrhosis [17]. Similarly, a significant decrease in telomere length was recently found in the biliary epithelial cells of patients with PBC, the target organ of the disease [19]. Remarkably, there are several studies analysing telomeres in liver tissue of patients with liver disorders of different origin but hardly any studies analysing telomere function in PBMC. In this regard, Hoare and colleagues showed the relation between lymphocyte telomere length, age and clinical outcome in non-hereditary liver disorder such as chronic hepatitis C virus (HCV) infection [20]. Since telomere shortening may be a trigger of senescence of immune-related cells and autoimmunity, we here tested the hypothesis that patients with PBC bear telomere dysfunction in PBMCs, by estimating telomere length and telomerase activity.
2. Materials and methods 2.1. Study population Blood samples were obtained from 48 women with PBC. The diagnosis of PBC was based on the internationally accepted criteria, which include the presence of cholestatic liver disease for at least six months, a liver biopsy compatible with the diagnosis, a positive test for anti-mitochondrial antibodies (AMA), serum alkaline phosphatase levels of at least one and a half times more than the upper normal limit, and the absence of biliary obstruction as assessed by ultrasonography, computed tomography or endoscopic cholangiography [2,21]. The characteristics of PBC patients are shown in Table 1. Serum autoantibodies were determined using indirect immunofluorescence [22]. All PBC patients were AMA positive [23]. The following were considered disease-related symptoms: pruritus, jaundice, major complications of portal hypertension, i.e. hepatic encephalopathy, variceal bleeding or ascites requiring diuretic therapy. Disease duration was calculated as the time from the date of the earliest suspected evidence of liver disease to the date of blood collection. Twenty-three patients with no fibrosis on liver biopsy fibrosis (Ludwig’s stage I and II) [24] were considered as having early-stage disease, while the remaining 25 with fibrosis or cirrhosis (stage III or IV) as having advanced disease. At the time of enrolment, 38 (79%) of the PBC patients were receiving ursodeoxycholic acid (UDCA) treatment. The control populations consisted of 12 women with chronic hepatitis C (CHC) who attended our Liver Unit during the same enrolment period as the PBC patients, and 55 age-matched healthy women. The CHC patients were matched with the PBC patients in terms of age (10 five-year age classes ranging from 34 years or younger to 75 years or older) and the severity of liver disease (absence of cirrhosis, presence of compensated cirrhosis, development of major complications of portal hypertension). CHC patients
underwent blood sampling before receiving any anti-viral medication. All subjects provided written consent after being informed about the nature of the study. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki (6th revision, 2008) as reflected in a priori approval by the institution’s human research committee. 2.2. Cell separation PBMCs obtained from a total of 48 female PBC patients were used for this study. PBMCs were isolated from 40 ml of blood with ethylenediaminetetraacetic acid (EDTA) by density-gradient centrifugation using Histopaque-1077 (Sigma). After centrifugation, cells were washed with phosphate-buffered saline (PBS) and the viability of cells was determined using trypan blue. Part of these PBMCs was stored for the study on telomerase activity. 2.3. Telomere length measurement DNA was purified from PBMC using the QIAamp DNA blood Midi Kit (Qiagen, and stored in aliquots at −20 ◦ C. The mean terminal restriction fragment (TRF) length was determined by the use of the TeloTAGGG Telomere Length Assay (Roche). Briefly, 2 g of purified DNA was digested in a final volume of 20 l reaction mixture with HinfI/RsaI enzyme mixture (1 U/l for each enzyme) at 37 ◦ C for 2 h and electrophoresed on 0.8% agarose MP gel in 1× Tris–acetate–EDTA buffer (0.04 M Tris–acetate, 0.001 M EDTA, pH 8) for 2.30 h at 150 V cm. Gels were then denatured, neutralized, and Southern transferred to a positively charged nylon membrane, according to the protocol described by the supplier (Roche). The blotted DNA fragments were hybridized to a digoxigenin (DIG)labelled probe specific for telomeric repeats and incubated with a DIG-specific antibody covalently coupled to alkaline phosphate. To determine the TRF length, the hybridized probe was visualized by chemiluminescent detection which detected TRF DNA on Hyperfilm (Amersham Biosciences). The exposed X-ray film was scanned and analysed densitometrically by Gel Doc with QuantityOne software (Biorad). The amount of telomeric DNA was calculated by integrating the area highest signal intensity of each smear. The TRF length was estimated relative to molecular weight standards that was run in each gel. For quantitative measurement and valid comparison of telomere length in different samples, control-DNA with short telomeres (L) and long telomeres (H) were included in each gel. Fig. 1 shows a representative Southern blotting gel. 2.4. Telomerase activity assay Telomerase activity was measured in PBMCs from PBC patients and controls using the TeloTAGGG Telomerase PCR ELISA kit (Roche), which combines a telomere repeat amplification protocol (TRAP) assay with detection by ELISA. In brief, 2 × 105 PBMCs were added to an elongation/amplification mixture and distilled water in a total volume of 50 l. The resulting elongation products were amplified by polymerase chain reaction (PCR), and PCR products were hybridized to a DIG-labelled telomeric repeat-specific probe bound to a streptavidin-coated 96-well plate. The binding reaction was detected with an anti-DIG-peroxidase antibody, visualized by a colour reaction product, and quantified photometrically. The absorbance of each sample was measured at 450 nm reading against the blank (reference wavelength 690 nm) using an ELISA microplate reader (Labsystems iEMS) within 30 min after addition of the stop reagent. Each sample was measured in duplicates. Each negative sample was obtained by heat treatment (10 min at 80 ◦ C). The samples were regarded as telomerase-positive if the difference
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Table 1 Clinicopathologic characteristics of study population. PBC
No. females Age (years) UDCA treatment Associated diseasesb Disease duration (years)
All
Early diseasea
Advanced diseasea
48 64 ± 11 38 (79%) 15 (31%) 12.7
23 62 ± 11 17 (74%) 5 (22%) 12.3
25 66 ± 11 21 (84%) 10 (40%) 12.8
Controls
CHC
55 55 ± 11 – – –
12 68 ± 8 – – –
Notes: UDCA, ursodeoxycholic acid; CHC, chronic hepatitis C; PBC, primary biliary cirrhosis. a Disease status. b Autoimmune diseases most frequently associated with primary biliary cirrhosis (autoimmune thyroid disease, scleroderma, rheumatoid arthritis and systemic lupus erythematosus).
in absorbance is higher than the twofold background activity. All criteria were fulfilled according to the kit requirements (Roche).
2.5. Statistical analysis The chi-square or Fisher’s exact test were used to compare categorical variables. For the analysis of continuous variables, the Mann–Whitney test was used to compare two groups and the Kruskall–Wallis test, a non-parametric analysis of variance, was used to compare more than two groups. When the analysis of variance, or the overall chi-square test, gave a significant (p < 0.05) result, pairwise comparisons were carried out using Mann–Whitney or Fisher’s exact tests to assess which group differed from the others. In order to control for multiple testing, p-values from such pairwise comparisons were corrected (pc ) by the number of comparisons done, according to the Bonferroni inequality method. Linear regression analysis was used to study the relationship between age, duration of disease and the telomere length. The data in the text and figures are expressed as mean ± SD. The statistical comparisons were made using GraphPad PRISM version 5 (GraphPad). All of the analyses were two-tailed.
3. Results 3.1. Telomere length shortening in advanced stage of primary biliary cirrhosis To test whether premature telomeric loss occurred in PBC patients possibly due to telomere dysfunction, telomere shortening in the PBMCs was analysed on a large selection of PBC patients. The length of telomere restriction fragment (TRF) was analysed using DNA extracted from PBMC samples. The mean TRF length of PBMC did not differ between the PBC (5.9 ± 1.5 kb) and the control group (6.2 ± 1.4 kb, pc = 0.164) or the CHC group (5.8 ± 1.0 kb, pc = 0.095) (Fig. 2A). Interestingly, the mean TRF of PBMC in PBC was significantly shorter in the subgroup with advanced liver disease (n = 25) than that with early-stage disease (n = 23) (5.4 ± 0.8 kb vs. 6.4 ± 1.8 kb, p = 0.03) (Fig. 2B). Furthermore, there was a significant difference between PBC patients with advanced disease and the normal controls (n = 55) (pc = 0.008), while the TRF was similar in the PBC group with early-stage disease compared to the control group (pc = 0.46). To confirm our data we have compared two classes of variables, the first containing the values of the length of telomeres, (collected in two subsets created based on the average of the total), the second containing the stages of the disease (early stage, advanced) with controls. Applying the Fisher test on the two classes of values using a cut off 5.8 kb, we were able to show a dependent relationship between the stages of pathology and the lengths of the telomeres. This analysis validates the previously Mann–Whitney test applied. The contingency table was able to show that healthy controls retained longer telomeres compared to the PBC patients. In particular, the TRF-disease stagedependence is indeed much more significant when comparing patients with each of the patients in the early stage and advanced stage (p-value ea-adv = 0.045), whereas the comparison between patients in the early stages and controls, and patients in the advanced stage and controls, does not seem to show a particular dependency (p-value ea-ctr = 0439; p-value adv-ctr = 0143) (Table 2 and Fig. 2B). It is to note that telomere shortening did not differ in subgroups of the PBC patients according to the presence of concomitant autoimmune disorders, such as autoimmune thyroid disease, rheumatoid arthritis (RA), and systemic lupus
Table 2 Association between telomerase length in primary biliary cirrhosis patients and different stage disease status and controls. Fig. 1. Representative example of telomere restriction fragments. Southern blot in peripheral-blood mononuclear cells of patients with primary biliary cirrhosis (Lanes 4–5) and control subjects (Lanes 6–9). Control-DNA with short telomeres (Lane 2) and long telomeres (Lane 3) were included in each gel. DNA molecular weight standards (Lane 1) served as a reference for samples. Telomere length, in relation to DNA molecular weight standards, was determined by calculating the mean TRF length using Southern blot method (Telo TAGGG telomere length assay, Roche) described in detail in the text.
Short telomere Long telomere Total
Early disease
Advanced disease
Controls
Total
6 (26%) 17 (74%) 23 (100%)
14 (56%) 11 (44%) 25 (100%)
20 (36%) 35 (64%) 55 (100%)
40 (39%) 63 (61%) 103 (100%)
Short and long telomeres are originated from the average telomere length of samples corresponding to a value of 5.8 kb which leads to split the set of these variables.
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Fig. 2. Telomere shortening in peripheral-blood mononuclear cells from patients with primary biliary cirrhosis. (A) The length of telomere restriction fragments in peripheralblood mononuclear cells was determined in 48 patients with primary biliary cirrhosis, 12 with hepatitis C virus and 55 age-matched controls. Telomere length was determined by calculating the mean TRF length in relation to DNA molecular weight standards using Southern blot method (Telo TAGGG telomere length assay, Roche). Telomere length did not differ between primary biliary cirrhosis, hepatitis C virus and control group. Statistical analysis was performed with Mann–Whitney test: p > 0.05. (B) Comparison of telomere length in clinical status of disease: early and late stage primary biliary cirrhosis patients. Results demonstrated a progressive shortening with advancing pathology (Mann–Whitney test: *p < 0.05, **p < 0.01). (PBMC, peripheral-blood mononuclear cells; PBC, primary biliary cirrhosis; HCV, hepatitis C virus; CTR, control).
erythematous (SLE) (data not shown). Moreover, no significant differences were revealed with and without UDCA treatment (data not shown). Furthermore, in both early and in the advanced PBC disease, non-significance was found between increased albumin and INR levels and telomere shortening in our study population (data not shown). 3.2. Enhanced telomerase activity in early-stage of primary biliary cirrhosis In order to deepen the role of telomere in PBC initiation and progression, we measured the PBMC telomerase activity using a highly sensitive and specific TRAP ELISA detection system in 30 PBC patients in either early stage (n = 15) or advanced stage (n = 15), and in 12 healthy controls. As expected and similarly to the telomere length, PBMC telomerase activity did not differ between PBC patients and healthy controls (1.0 ± 2.6 OD vs. 0.6 ± 0.8 OD, p = 0.79; Fig. 3A), although a trend to an increased enzymatic activity in the PBC group can be observed. Interestingly, PBC patients with advanced disease retained significantly less telomerase activity than those with early-stage disease (0.6 ± 0.9 OD vs. 1.5 ± 3.7 OD, p = 0.03; Fig. 3B), thus well-fitting with our finding of a reduced telomere length in advanced stage PBC. Although telomerase activity is increased in early-stage PBC patients, we speculated that late stage has progressively exhausted its activity accelerating the process of telomeric loss. 4. Discussion There has been mounting evidence of a causal role for telomere dysfunction in a number of degenerative disorders [12,14,25]. It has been increasingly suggested that telomere dysfunction is
implicated in immunosenescence phenomenon reflecting premature ageing of immune system [16,26–29]. Many studies have observed that accelerated telomere loss in different lymphocyte subpopulations is a common feature of autoimmune disease such as SLE, RA, scleroderma, atopic dermatitis and psoriasis [15,30–36]. For the first time, our study investigated the clinical significance of telomere erosion in peripheral immune system from PBC patients. Since female patients with PBC and other autoimmune diseases are characterized by an enhanced X monosomy rate in PBMC, particularly T and B lymphocytes, a marker of chromosomal instability [3,18], our study were specifically designed on an entire female population. Moreover, since lymphocytes telomere length is known to change with age, we carefully selected age-matched controls. Interestingly, we reported that telomere shortening affected PBMCs of advanced stage PBC patients. Based on these data, it seems that telomere shortening is an “acquired” feature and not a “constitutive” property of PBMC in PBC disease. We suggest that a progressively acquired chromosomal instability due to telomere dysfunction may be involved in the pathogenesis of PBC (mainly in advanced disease) by inducing senescence of immune-related cells. In order to clarify the role of telomere erosion in PBC pathogenesis, we next examined PBMC telomerase activity in PBC patients and controls. PCR-based TRAP assay could not detect clear difference between these two groups, but the study revealed that telomerase activity was higher in patients with early stage than in advanced phase. Similar findings were found in previous investigation on telomere dysfunction in biliary epithelial cells in PBC and other hepatobiliary diseases [19,37,38]. Indeed, Sasaki et al. showed that telomere shortening and accumulation of DNA damage characterized cellular senescence of bile ducts, the specific target-tissue cells [19]. Furthermore, it has been described that other types of autoimmune diseases, such as inclusion body myositis, telomere shorten
Fig. 3. Mean telomerase activity in peripheral-blood mononuclear cells. (A) Telomerase activity is reported as optical density (OD). Bar graph showed no difference in telomerase activity between primary biliary cirrhosis patients and controls (Mann–Whitney test: p > 0.05). (B) Telomerase activity in peripheral-blood mononuclear cells from early and late stage patients with primary biliary cirrhosis. Telomerase activity is reported as optical density (OD). Mean OD was higher in early disease than in advanced disease (Mann–Whitney test: p < 0.05). (PBC, primary biliary cirrhosis; CTR, control).
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in affected primary muscular cells, the target-tissue cells [39]. Moreover, beside autoimmune pathologies, telomere shortening has been reported in other organs subject to chronic inflammation, such as Barrett’s oesophagus and ulcerative colitis [40,41]. The causal relation between autoimmunity and telomere shortening is still enigma [16,26–29]. However, the immune system is in constant self-renewal and, consequently, is highly dependent on efficient telomere maintenance. Indeed, cellular component of the immune system is derived from hematopoietic stem cells which actively divide and differentiate throughout life [42]. The telomere shortening in PBC could be due to the chronic activation/proliferation occurring in autoimmune conditions and could lead to premature immune senescence, involving exit from the mitotic cycle and/or cell death. Based on results of analyses of telomerase activity and telomerase length, we speculate that in patients with early-stage PBC, many lymphocytes cells divide continuously and their telomerase activity is higher than that in healthy subjects, but not so high to prevent telomere shortening. Further, telomerase activity was elevated in early-stage disease but not in the advanced. Many possible explanations could be proposed for these telomere dysfunctions. At the core of this investigation was the concept that diminished TRF length in PBC could reflect a secondary phenomenon resulting from the telomerase that was not expressed in advanced disease. At this regard, it might be possible that in advanced-stage PBC patients, telomerase activity is progressively exhausted and not sufficient to prevent telomere erosion during liver disease progression. Indeed, advanced PBC is characterized by evident telomere loss. In line with this hypothesis some studies have reported that telomere shortening is present in cirrhosis induced by viral hepatitis, toxic liver damage, autoimmunity, and cholestasis [38]. Moreover, several reports have demonstrated that immunosenescence of biliary epithelial cells, the specific target-tissue cells, might be important in the development of PBC [8,19,43,44]. Furthermore, it has been shown that hepatocyte telomere shortening and senescence correlate with progression of cirrhosis. The telomere hypothesis of human cirrhosis indicates that hepatocellular telomere shortening and senescence represents a molecular mechanism in the evolution of human cirrhosis in early advanced PBC patients [38]. In relation to other studies carried out on liver tissue, the novelty of our investigation is based on the correlation between telomere loss and PBC disease progression, testing the peripheral immune system. We suggest that telomerase activity might be sufficient to compensate for the telomere loss that occurs during proliferation and thus prevent accelerated ageing of the immune system in those patients with early-stage disease. In advanced-stage disease the telomerase might not prevent telomere loss and this fact supports the hypothesis that cirrhosis in PBC might be the predominant factor promoting telomere shortening. We highlighted that other variables are also involved in PBC initiation and progression such as: age, duration of pathology, treatment with drugs, presence of associated disease, disease stage, in addition to TRF and telomerase activity. In conclusion, in contrast with previous studies on telomere dysfunction in other autoimmune and degenerative diseases, we could not demonstrate a significant reduction in telomere length in PBMCs from PBC patients compared to healthy controls. However, we were able to recapitulate the association with disease stages, showing that patients with advanced-stage PBC are characterized by extremely short telomeres and a significantly decreasing telomerase activity. In this respect, the data we obtained are of clinical interest also because of the current possibility to therapeutically target the telomere system [9]. In view of our data, we can speculate that telomere dysfunction in PBC may neither be related to
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