Abstract
Accumulating studies suggest that senescent biliary epithelial cells (BECs) produce senescence-associated secretory phenotypes (SASPs) and play various roles in the pathogenesis of primary biliary cholangitis (PBC) and other cholangiopathies. We examined comprehensive profiles of senescent BECs and its contribution to the pathogenesis of PBC taking advantage of microarray analysis. cDNA microarray analysis revealed that 1841 genes including CCL2, IFIT3, CPQ were commonly up-regulated in senescent BECs cultured in serum depleted media or media with glycochenodeoxycholic acid. Knockdown of IFIT3 significantly suppressed cellular senescence (p < 0.01) and significantly increased apoptosis (p < 0.01) in BECs treated with serum depletion or glycochenodeoxycholic acid. Significantly increased expression of IFIT3 was seen in senescent BECs in small bile ducts showing cholangitis and in ductular reactions in PBC, compared to control livers (p < 0.01). An inadequate response to UDCA was inversely correlated to the increased expression of IFIT3 in small bile duct in PBC (p < 0.05). In conclusion, the expression of various genes related to immunity and inflammation including SASPs were increased in senescent BECs. Upregulated IFIT3 in senescent BECs may be associated with the pathogenesis of PBC and may be a possible therapeutic target in PBC.
Similar content being viewed by others
Introduction
Primary biliary cholangitis (PBC) is an autoimmune cholestatic liver disease characterized by a unique chronic non-suppurative destructive cholangitis (CNSDC) in small bile ducts and serum anti-mitochondrial antibodies (AMAs)1,2,3. PBC presents progressive chronic cholestasis and biliary fibrosis, which subsequently develop liver failure. There still remains issues to be clarified in the pathogenesis of PBC, so far1,2,3. The only accepted first-line agent is ursodeoxycholic acid (UDCA) for the treatment of PBC1,2,3,4. Approximately one-third of patients treated with UDCA are “nonresponders” to UDCA at risk of disease progression prompting the need for additional therapeutic strategies1,2,3,4.
PBC is characterized by biliary epithelial senescence in small bile duct and bile ductules5,6,7,8,9,10,11,12. Definition of cellular senescence is a condition in which a cell no longer has the ability to proliferate9,13,14. Irreversible G1/S arrest of cell cycle is a feature of senescent cells9,13,14. Senescent cells do not respond to various external stimuli, although they remain metabolically active9,13,14. Cellular senescence is observed in the damaged small bile duct involved in CNSDC and also bile ductular cells in ductular reactions in our previous studies5,6. Although exact mechanisms inducing cellular senescence in PBC remains unknown, so far, cellular senescence can be induced by treatments with oxidative stress, serum depletion or glycochenodeoxycholic acid (GCDC) in cultured BECs in our previous studies5,8,12,15. It is conceivable that senescent BECs may participate in the pathogenesis of PBC and other various cholangiopathies by secreting senescence-associated secretory phenotypes (SASPs), such as augmented inflammation, progression of fibrosis and bile duct loss8,9,13,14,15,16.
Although accumulating data suggest important roles of senescent BECs in hepatobiliary diseases5,6,7,8,9,10,11,12, features of senescent BECs have not been comprehensively studied, so far. Taking advantage of cDNA microarray analysis, we examined RNA expression profiles of senescent BECs and its contribution to cholangiopathies. Results of cDNA microarray analysis showed that some of the commonly up-regulated genes in senescent BECs following in vitro incubation in serum depleted media or treatment with GCDC included CCL2, IFIT3 CPQ. We chose to focus on the interferon (IFN)-induced protein with tetratricopeptide repeats 3 (IFIT3), since it showed the greatest increase in levels of gene expression. IFIT3 is an IFN-induced antiviral protein acting as an inhibitor of cellular and viral processes, cell migration, proliferation, signaling, and viral replication17,18,19. A participation of IFN pathways in the pathogenesis of PBC was previously reported20,21,22,23,24, so we selected IFIT3 for further examination.
In this study, we examined the effects of knockdown of IFIT3 on cellular senescence, proliferation and apoptosis in cultured BECs. We also examined the expression of IFIT3 and its association with senescent markers p16INK4a and p21WAF1/Cip1 in human PBC and control livers.
Results
Culture study
A number of genes was upregulated in senescent BECs induced by serum depletion and GCDC treatment
Upregulated genes in senescent BECs, which show more than twofold change compared to control, were 2870 and 2789 genes in serum depletion and GCDC treatment for 7 days, respectively. 1841 genes were commonly upregulated in both 2 conditions. Major genes commonly upregulated in senescent BECs induced by serum depletion and GCDC treatment included CCL2, IFIT3, CPQ, NUPR1 and CIB2 (Table 1, Supplementary table S1). Various inflammatory genes (chemokines and cytokines) including CCL2, CCL20, IL3, IL11, IL15 were upregulated in senescent BECs as SASPs (data not shown) in accordance with our previous study8,16. We put focus on IFIT3 and further examined its role in cell proliferation, apoptosis and cellular senescence. GSEA was also performed on top 500 up-regulated genes derived from senescent BECs induced by serum depletion and GCDC treatment. Various gene-sets were upregulated in each condition and a gene set: GO_RESPONSE_TO_BIOTIC_STIMULUS was commonly upregulated in both condition and IFIT3 was included in several gene sets including this gene set (Supplementary Table S2).
IFIT3 expression was increased in senescent cells in mRNA and protein level
We examined the mRNA expression of IFIT3 in cultured BECs treated with serum depletion and GCDC for 7 days. The expression of IFIT3 mRNA was significantly increased in senescent BECs treated with serum depletion and GCDC, compared with control (p < 0.01) (Fig. 1A). The expression of IFIT3 at protein level was also significantly increased in senescent BECs treated with serum depletion and GCDC, compared with control (p < 0.01) (Fig. 1B).
IFIT3 knockdown increased cell proliferation, apoptosis and decreased cellular senescence in BECs treated with serum depletion and GCDC
Effective knockdown of IFIT3
We examined the effect of IFIT3 knockdown on BECs. An effective knockdown of IFIT3 using siRNA was confirmed in mRNA and protein levels (Fig. 2A,B).
IFIT3 knockdown increased cell proliferation activity
Cell proliferation activity detected by BrdU assay after the induction of cellular senescence (serum depletion and GCDC) for 4 days with or without knockdown of IFIT3 using siRNA. BrdU-labelling index was significantly low in BECs treated with serum depletion and GCDC for 4 days (p < 0.05) (Fig. 2C). The knockdown of IFIT3 significantly increased cell proliferation activity in BECs treated with serum depletion and GCDC (p < 0.05) (Fig. 2C).
IFIT3 knockdown did not change the growth curve assessed by cell number
Cell growth was significantly arrested in BECs by the treatment with serum depletion or GCDC (p < 0.01). IFIT3 knockdown did not change the growth curve in each condition in BECs (Fig. 2D). The increased proliferation and apoptosis may result in no change in the growth curve by treatments with serum depletion and GCDC.
IFIT3 knockdown resolved G1/S arrest
Cell cycle was analyzed using Cell-Clock cell cycle assay on BECs by the treatment with serum depletion or GCDC for 4 days with or without knockdown of IFIT3 using siRNA. G1/S arrest was induced by the treatment with serum depletion or GCDC (p < 0.05) (Fig. 2E). G1/S arrest was significantly resolved by IFIT3 knockdown in BECs treated with serum depletion or GCDC (p < 0.01) (Fig. 2E).
IFIT3 knockdown increased apoptosis
Caspase-3/7 activity was detected for assessment of apoptosis at 4 days after the induction of cellular senescence with or without knockdown of IFIT3 using siRNA. Caspase-3/7 activity with green fluorescence was detected in apoptotic cells (Fig. 2F). Apoptotic cells were significantly increased in senescent BECs with knockdown of IFIT3 (p < 0.01) (Fig. 2F).
IFIT3 knockdown decreased cellular senescence
Cellular senescence was assessed by the activity of SA-β-gal after treatment with serum deprivation or GCDC (500 nM) for 4 days with or without knockdown of IFIT3 using siRNA (Fig. 2G). SA-β-gal labelling index, a marker of cellular senescence, was significantly increased by treatment with serum depletion or GCDC. Cellular senescence was significantly decreased by knockdown of IFIT3 using siRNA in BECs treated with serum depletion or GCDC for induction of senescence (p < 0.01) (Fig. 2G).
Human study
Increased expression of IFIT3 in senescent BECs in damaged small bile ducts in PBC
IFIT3 was expressed in the cytoplasm in BECs, when present (Fig. 3A–D). The expression of IFIT3 was significantly increased in BECs in small bile ducts involved in cholangitis in PBC (Fig. 3B,C). Table 2 is a summary of the extent of IFIT3 expression in small bile ducts in PBC and control livers. The expression of IFIT3 was significantly more extent in small bile ducts in PBC, compared with control livers (p < 0.01). The expression of IFIT3 was significantly correlated with cholangitis activity in PBC (p < 0.01). Similar tendency was confirmed in another cohort of 15 patients with PBC (data not shown). The increased expression of IFIT3 in small bile duct was inversely correlated to inadequate response to UDCA in PBC (p < 0.05).
Increased expression of IFIT3 in senescent bile ductular cells in ductular reaction in PBC
The expression of IFIT3 was significantly increased in bile ductular cells in ductular reaction in PBC (Fig. 3D). Table 3 is a summary of the extent of IFIT3 expression in bile ductular cells in PBC and control livers. The expression of IFIT3 was significantly more extent in bile ductular cells in PBC, compared with control livers (p < 0.01). Similar tendency was confirmed in another cohort of 15 patients with PBC (data not shown).
Increased expression of IFIT3 in senescent BECs in damaged small bile ducts and bile ductules in PBC
Double immunostaining revealed that the expression of IFIT3 was frequently increased in BECs in the senescent small bile ducts showing expression of p21WAF1/Cip1 or p16INK4a in PBC (Fig. 4A). The increased expression of IFIT3 was also seen in senescent BECs with expression of p16INK4a and p21WAF1/Cip1 in ductular reactions in PBC (Fig. 4B). Since intracytoplasmic localization of p21WAF1/Cip1 (nucleus) or p16INK4a (nucleus and cytoplasm) are different from IFIT3 (cytoplasm), the co-localization was not indicated by yellow color in the merged images (Fig. 4A,B). However, the expression was seen in same BECs. Figure 4C shows a view of double fluorescent stain in control normal liver.
Discussion
The findings are summarized as follows; (1) Senescent BECs induced by both serum depletion or a treatment with GCDC for 7 days showed common upregulation of 1841 genes (fold change > 2), which included CCL2, IFIT3, CPQ and a number of chemokines and cytokines; (2) Cell proliferation and apoptosis were significantly increased (p < 0.01) and cellular senescence was significantly decreased (p < 0.01) by knockdown of IFIT3 in BECs treated with serum depletion or GCDC; (3) Small bile ducts showing cholangitis and in ductular cells in ductular reactions expressed IFIT3 in significantly higher level in PBC, compared to control livers; (4) Cholangitis activity was significantly correlated with the expression of IFIT3 in small bile ducts in PBC (p < 0.01); (5) Senescent BECs showing the expression of p16INK4a and p21WAF1/Cip1 in bile duct lesions in PBC expressed IFIT3 in high level; (6) Inadequate response to UDCA was inversely correlated to the increased expression of IFIT3 in small bile duct in PBC (p < 0.05).
In this study, we examined comprehensive profiles of senescent BECs relating to cholangiopathies. The results showed that the expression of various chemokines and cytokines are increased as SASPs in agreement with our previous studies8,15,16. For example, CCL2, which we reported an increased expression and possible roles in the pathogenesis of PBC8,16, is included in the top 5 commonly-upregulated genes in senescent BECs in this study. It is now well known that senescent BECs express various chemokines and cytokines as SASPs in PBC and PSC, which may modulate inflammatory cell infiltration and fibrosis in cholangiopathies8,15,16,25. Altered expression of various inflammatory factors is also known in senescent cells9,13,14,26.
Among these inflammatory factors, we put focus on one of the top5 commonly-upregulated genes; IFIT317,18,19 in senescent BECs in this study. In vitro study confirmed the increased expression of IFIT3 in mRNA and protein levels in this study, confirming the data of mRNA array study. In the present study, we found for the first time the increased expression of IFIT3 relating to cellular senescence in BECs in PBC. Increased expression of IFIT3 and its association with pathophysiology were reported in several diseases such as SLE27, RA28 and degenerative annulus fibrosus29. IFIT3 is an IFN-induced protein and upregulates cGMP-AMP synthase (cGAS)-signal via stimulator of IFN genes protein (STING) pathway27,29. cGAS-STING pathway is known to play roles in the process of cellular senescence such as the expression of SASPs30. Taken together, IFIT3 may also contribute to the overactivation of cGAS/STING signaling pathway in BECs and may participate in the pathophysiology of PBC. Further studies are mandatory to confirm an involvement of IFIT3 on signal transduction pathways.
Accumulating evidences suggest that IFN pathways may participate in the pathogenesis of PBC20,21,22,23,24, however, there has been no report regarding IFIT3 in PBC to our knowledge. Recent integrated analysis using GWAS, and mRNA microarray data sets predicted that IFNG and CD40L are the central upstream regulators in both disease susceptibility and activity of PBC24. The interplay of types I and II IFN is also implicated as a cause of human PBC23 and a murine model of autoimmune cholangitis22. A previous paper implicated type I IFN signaling as a necessary component of the sex bias in the murine model of autoimmune cholangitis with chronic IFN–γ stimulating22. The previous paper also suggested that drugs that target the type I IFN signaling pathway would have potential benefit in the earlier stages of PBC22. IFN‐γ but not IFN‐β, TGF‐β or TNFα was found to up‐regulate STING expression in keratinocytes31,32. Taken together, the present study suggests that IFIT3 may be a key molecule, which participates in the crossroads of IFN signaling, cGAS-STING pathway and cellular senescence in PBC.
In the present study, cellular senescence was decreased, and cell proliferation was increased by a knockdown of IFIT3 in BECs. Furthermore, G1/S arrest was resolved by a knockdown of IFIT3 in BECs treated with serum depletion or GCDC. A previous study reported significant accumulation of cells at G1/S transition in human monocytic cells with ectopic expression of IFIT318. IFIT3 has been shown to have anti-proliferative activity by enhancing the expression of cell cycle negative regulators such as p27 and p21 by downregulating c-Myc18. The findings in the present study agree with previous study, suggesting that IFIT3 may contribute to induce cellular senescence.
A knockdown of IFIT3 in BECs treated with serum depletion or GCDC significantly increased apoptosis in the present study. Previous study suggested that upregulation of IFIT3 plays a protective role in lung epithelial cells in dengue virus infection by an inhibition of apoptosis33. IFIT3 knockdown induced apoptosis and suggested that the apoptotic effects of IFIT2 could be negatively regulated by IFIT334. The findings in the present study agree these previous studies and confirmed anti-apoptotic roles of IFIT3 overexpression. It is well known that senescent cells are resistant to apoptosis26,35. “Senescent cells and anti-apoptotic pathways” (SCAPs) including Bcl-xL are thought to have responsibility for this resistant mechanism26,35. Since IFIT3 is upregulated in senescent cells and inhibits apoptosis33,34, increased expression of IFIT3 in bile duct lesions in senescent BECs in PBC may contribute to the anti-apoptosis as one of SCAPs. Taken together, IFIT3 may serve as a novel therapeutic target for clearance of senescent cells and for blocking the production of type I IFN and other proinflammatory cytokines by the cGAS/STING signaling pathway in PBC.
In conclusion, senescent BECs showed increased expression of various genes related to immunity and inflammation including SASPs. The increased expression of IFIT3 in BECs may be involved in the pathogenesis of PBC and could be a therapeutic target in PBC.
Methods
Culture study
Cell culture and treatments
Mouse intrahepatic BECs were isolated from 8-week-old female BALB/c mice and were purified and cultured as described previously36,37. All methods were carried out in accordance with relevant guidelines and regulations. The cell density of the cells was less than 80% during experiments. Cellular senescence was induced in BECs cultured in vitro incubation in serum depleted media or treatment with media containing 200 μM GCDC for 4–7 days, as described previously37,38,39. A treatment with GCDC causes cellular senescence via the induction of endoplasmic reticulum stress and dysregulated autophagy, as demonstrated in previous studies15,37,38,39.
RNA extraction and cDNA microarray
Total RNA was extracted from the cells with a QIAGEN RNeasy Mini kit (QIAGEN, Hilden, Germany), as described previously37 according to the manufacturer’s protocol. Genome-wide expression profiling was performed using a 3D-Gene scanner with 3D-Gene Oligo chip 24 k (Toray Industries, Inc., 23,522 distinct genes) and the supplier's protocol. Hybridization signals were scanned 3D-Gene Scanner (Toray Industries) and processed by 3D-Gene Extraction software (Toray Industries). The raw data of each spot was normalized by subtraction with a mean intensity of the background signal determined by all blank spots' signal intensities of 95% confidence intervals. The raw data intensities greater than 2 standard deviations (SD) of the background signal intensity were considered to be valid. cDNA microarray was performed using pooled RNA samples from 2 independent experiments for each condition. Second cDNA microarray was performed using different sets of samples for each condition and major up-regulated genes including IFIT3 were confirmed. Data analysis and functional analysis was performed by Gene Ontologies and KEGG pathway analysis. GSEA was performed according to the instructions.
Data deposition
Microarray data are deposited in the National Center for Biotechnology Information Gene Expression Omnibus database (accession number GSE168052).
Knockdown of Ifit3 by small interfering RNA (siRNA)
Validated siRNA for Ifit3 and negative control siRNA were purchased from Santa-Cruz biotech (Santa-Cruz, CA, USA) and QIAGEN, respectively. One day before transfection, BECs were plated in 35 mm-dishes (5 × 105 cells), 96-well plate (1 × 104 cells/well) or 12-well plate (5 × 104 cells/well), and then the cells were transiently transfected with either Ifit3 or control siRNA (100 nM) using Lipofectamine 3000 (Invitrogen, Carlsbad, CA), as described previously37 according to the manufacturer’s protocol.
Real-time quantitative reverse transcriptase-polymerase chain reaction
After cDNA was synthesized, quantitative real-time PCR was performed to measure Ifit3 and β-actin mRNA, as described previously37 according to a standard protocol using the SYBR Green PCR Master Mix (Toyobo, Tokyo, Japan). Forward and reverse primers 5′-GAGTGCTGCTTATGGGGAGA and 5′-AGAGCAGTTTGTCAGCAATCC, respectively, were used for Ifit3 and 5′-CCACCGATCCACACAGAGTA and 5′-GGCTCCTAGCACCATGAAGA for β-actin as an internal control. Each experiment was performed twice in triplicate, and the mean was calculated for each of the experiments.
Immunoblotting
The cell lysate samples (10 μg) were resolved by SDS-PAGE and transferred to a nitrocellulose membrane as described previously37. After transfer, the membranes were processed for immunoblotting as described previously37. The primary antibodies used were shown in supplementary table S2. Densitometry of the resulting bands was performed using Image-J software and normalized to the loading control.
Assay for cell proliferation
Cell proliferation activity was assessed on day 4 after treatment by using a 5-bromo-2′-deoxy-uridine (BrdU) Labeling and Detection Kit (Roche, Nonenwald, Germany), according to manufactures’ protocol. The nuclei were simultaneously stained with DAPI. At least 1 × 103 total cells were checked and counted to assess the BrdU-labeling index with a conventional fluorescence microscope (Olympus).
Assay for cell number
BECs were seeded into 96-well microplates (1 × 104 cells/well) and incubated in a final volume of 100 μl medium. The cell number was assessed on days 1, 2, 4 and 7 after treatment using a Cell Proliferation Reagent WST-1 (Roche, Basel, Switzerland) according to manufacturer’s recommendation.
Assay for cell cycle
The cell cycle alteration was detected on day 4 after treatment by a Cell‐Clock Assay Kit (Biocolor, Northern Ireland, UK), according to manufacturer’s protocol. Images of the cells were acquired using a conventional microscope (Olympus) and analyzed using Image J software.
Assay for apoptosis
The apoptotic cells in each condition were assessed after the induction of cellular senescence and a treatment with senolytic reagents by using CellEvent Caspase-3/7 Green Detection Reagent (Life Technologies, Carlsbad, CA) as described previously39 according to manufacturer’s protocol. The nuclei were simultaneously stained with DAPI. At least 1 × 103 total cells were checked and counted to assess the percentage of apoptotic cells showing Caspase-3/7 activity with a conventional fluorescence microscope.
Assay for cellular senescence
The activity of senescence-associated β-galactosidase (SA-β-gal) was detected after the induction of cellular senescence and a treatment with senolytic reagents by using the senescence detection kit (Bio Vision, Mountain View, CA) according to manufacturer’s protocol40. The proportion of senescent cells was assessed by counting SA-β-gal-positive cells in at least 1 × 103 total cells.
Human study
Classification of intrahepatic biliary tree
The intrahepatic biliary tree is classified into intrahepatic large and small bile ducts (septal and interlobular bile ducts) by their size and distributions in the portal tracts41. Bile ductules, which are characterized by tubular or glandular structures with a poorly defined lumen and located at the periphery of the portal tracts41,42, are not included in the small bile ducts and evaluated separately.
Liver tissue preparation
A total of 171 liver tissue specimens (all were biopsied or surgically resected) were collected from the liver disease file of our laboratory and affiliated hospitals. All methods were carried out in accordance with the Declaration of Helsinki, relevant guidelines and regulations. The Ethics Committee of Kanazawa University approved this study. The liver specimens in this study were 70 PBC, 61 chronic viral hepatitis (CVH), 12 PSC, 10 extrahepatic biliary obstruction (EBO) and 18 “histologically normal” livers. All PBC specimens were from patients fulfilling the clinical, serological and histological characteristics consistent with the diagnosis of PBC2 and histologically classified according to Nakanuma classification43. Table 4 is a summary of the clinicopathological features of the PBC patients included in the present study.
Seventeen PBC livers were after UDCA therapy and 17 were UDCA non-responders. Thirty-eight and 23 CVH patients were regarded as F0-2 and as F3, 4, respectively44. Ten and 61 of CVH cases were serologically positive for hepatitis B surface antigen and anti-hepatitis C viral antibody, respectively. Causes of EBO were obstruction of the bile duct at the extrahepatic bile ducts or the hepatic hilum due to stone or carcinoma, and the duration of jaundice was less than 1 month. “Histologically normal” livers were obtained from surgically resected livers for metastatic liver tumor or traumatic hepatic rupture. Normal liver tissues were obtained from an area apart from the tumor and carcinoma tissues were not evaluated.
Liver tissue samples were fixed in 10% neutral-buffered formalin and embedded in paraffin. More than twenty serial sections, 4 μm-thick, were cut from each block. Several sections were processed routinely for histologic study, and the remainder was processed for the subsequent immunohistochemistry.
Immunohistochemistry
The expression of IFIT3 and senescence-related markers p16INK4a, p21WAF1/Cip1 were examined, as described previously12. The primary antibodies used are shown in supplementary table S3. A similar dilution of the control mouse or rabbit Immunoglobulin G (Dako) was applied instead of the primary antibody as a negative control. Positive and negative controls were routinely included. Histological analysis was performed in a blinded manner. BECs in small bile ducts and bile ductules were separately evaluated.
Extent of IFIT3 expression in small bile ducts and bile ductules
The extent of expression was evaluated as follows: 1+, focal, positive cells are detected in one third or fewer portal tracts; 2+, moderate, positive cells are detected in small bile ducts of more than one third of portal tracts; 3+, extensive, positive cells are detected in small bile ducts of more than two thirds of portal tracts.
Double immunofluorescence
Double immunofluorescence for IFIT3 with senescent markers (p16INK4a and p21WAF1/Cip1) was also performed. In brief, either of p16INK4a or p21WAF1/Cip1 was detected using Vector Red Alkaline Phosphatase Substrate Kit (Vector Lab, Burlingame, CA), followed by second staining for Ifit3 using Alexa-488-labeled anti-rabbit IgG. The sections were counterstained with DAPI and evaluated under a conventional fluorescence microscope.
Statistical analysis
Statistical analysis of differences was performed using the Kruskal–Wallis test with Dunn’s posttest. When the number of groups is 2, statistical analysis of difference was performed using the Mann–Whitney test. The Chi-square test or Fisher’s exact test was used to analyze categorical data. The correlation coefficient of 2 factors was evaluated using Spearman’s rank correlation test. When the P value was less than 0.05, the difference was regarded as significant. All analyses were performed using the GraphPad Prism software (GraphPad Software, San Diego, CA, USA).
References
Kaplan, M. & Gershwin, M. Primary biliary cirrhosis. N. Engl. J. Med. 353, 1261–1273 (2005).
Portmann, B. & Nakanuma, Y. In Pathology of the Liver (eds Burt, A. D. et al.) 491–562 (Churchill Livingstone, 2011).
Lindor, K. D., Bowlus, C. L., Boyer, J., Levy, C. & Mayo, M. Primary biliary cholangitis: 2018 practice guidance from the American association for the study of liver diseases. Hepatology 69, 394–419. https://doi.org/10.1002/hep.30145 (2019).
Tanaka, A. Emerging novel treatments for autoimmune liver diseases. Hepatol. Res. 49, 489–499. https://doi.org/10.1111/hepr.13347 (2019).
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. 205, 451–459 (2005).
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 48, 186–195 (2008).
Sasaki, M., Miyakoshi, M., Sato, Y. & Nakanuma, Y. Increased expression of mitochondrial proteins associated with autophagy in biliary epithelial lesions in primary biliary cirrhosis. Liver Int. 33, 312–320. https://doi.org/10.1111/liv.12049 (2013).
Sasaki, M., Miyakoshi, M., Sato, Y. & Nakanuma, Y. Modulation of the microenvironment by senescent biliary epithelial cells may be involved in the pathogenesis of primary biliary cirrhosis. J. Hepatol. 53, 318–325. https://doi.org/10.1016/j.jhep.2010.03.008 (2010).
Sasaki, M. & Nakanuma, Y. Cellular senescence in biliary pathology. Special emphasis on expression of a polycomb group protein EZH2 and a senescent marker p16INK4a in bile ductular tumors and lesions. Histol. Histopathol. 30, 267–275 (2015).
Sasaki, M. et al. Bile ductular cells undergoing cellular senescence increase in chronic liver diseases along with fibrous progression. Am. J. Clin. Pathol. 133, 212–223. https://doi.org/10.1309/AJCPWMX47TREYWZG (2010).
Nakanuma, Y., Sasaki, M. & Harada, K. Autophagy and senescence in fibrosing cholangiopathies. J. Hepatol. 62, 934–945. https://doi.org/10.1016/j.jhep.2014.11.027 (2015).
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. 169, 831–845 (2006).
Hoare, M., Das, T. & Alexander, G. Ageing, telomeres, senescence, and liver injury. J. Hepatol. 53, 950–961. https://doi.org/10.1016/j.jhep.2010.06.009 (2010).
Collado, M., Blasco, M. A. & Serrano, M. Cellular senescence in cancer and aging. Cell 130, 223–233 (2007).
Sasaki, M. & Nakanuma, Y. Bile acids and deregulated cholangiocyte autophagy in primary biliary cholangitis. Dig. Dis. 35, 210–216. https://doi.org/10.1159/000450913 (2017).
Sasaki, M., Miyakoshi, M., Sato, Y. & Nakanuma, Y. Chemokine-chemokine receptor CCL2-CCR2 and CX3CL1-CX3CR1 axis may play a role in the aggravated inflammation in primary biliary cirrhosis. Dig. Dis. Sci. 59, 358–364. https://doi.org/10.1007/s10620-013-2920-6 (2014).
Liu, X. Y., Chen, W., Wei, B., Shan, Y. F. & Wang, C. IFN-induced TPR protein IFIT3 potentiates antiviral signaling by bridging MAVS and TBK1. J. Immunol. 187, 2559–2568. https://doi.org/10.4049/jimmunol.1100963 (2011).
Xiao, S. et al. RIG-G as a key mediator of the antiproliferative activity of interferon-related pathways through enhancing p21 and p27 proteins. Proc. Natl. Acad. Sci. USA 103, 16448–16453. https://doi.org/10.1073/pnas.0607830103 (2006).
Pidugu, V. K., Pidugu, H. B., Wu, M. M., Liu, C. J. & Lee, T. C. Emerging functions of human IFIT proteins in cancer. Front. Mol. Biosci. 6, 148. https://doi.org/10.3389/fmolb.2019.00148 (2019).
Bae, H. R. et al. Chronic expression of interferon-gamma leads to murine autoimmune cholangitis with a female predominance. Hepatology 64, 1189–1201. https://doi.org/10.1002/hep.28641 (2016).
Shimoda, S. et al. Natural killer cells regulate T cell immune responses in primary biliary cirrhosis. Hepatology 62, 1817–1827. https://doi.org/10.1002/hep.28122 (2015).
Bae, H. R. et al. The interplay of type I and type II interferons in murine autoimmune cholangitis as a basis for sex-biased autoimmunity. Hepatology 67, 1408–1419. https://doi.org/10.1002/hep.29524 (2018).
Takii, Y. et al. Enhanced expression of type I interferon and toll-like receptor-3 in primary biliary cirrhosis. Lab Investig. 85, 908–920. https://doi.org/10.1038/labinvest.3700285 (2005).
Ueno, K. et al. Integrated GWAS and mRNA microarray analysis identified IFNG and CD40L as the central upstream regulators in primary biliary cholangitis. Hepatol. Commun. 4, 724–738. https://doi.org/10.1002/hep4.1497 (2020).
Tabibian, J. H., O’Hara, S. P., Splinter, P. L., Trussoni, C. E. & LaRusso, N. F. Cholangiocyte senescence by way of N-ras activation is a characteristic of primary sclerosing cholangitis. Hepatology 59, 2263–2275. https://doi.org/10.1002/hep.26993 (2014).
Kirkland, J. L. & Tchkonia, T. Cellular senescence: a translational perspective. EBioMedicine 21, 21–28. https://doi.org/10.1016/j.ebiom.2017.04.013 (2017).
Wang, J. et al. Association of abnormal elevations in IFIT3 with overactive cyclic GMP-AMP synthase/stimulator of interferon genes signaling in human systemic lupus erythematosus monocytes. Arthritis Rheumatol. 70, 2036–2045. https://doi.org/10.1002/art.40576 (2018).
Yokoyama-Kokuryo, W. et al. Identification of molecules associated with response to abatacept in patients with rheumatoid arthritis. Arthritis Res. Ther. 22, 46. https://doi.org/10.1186/s13075-020-2137-y (2020).
Kazezian, Z. et al. Gene expression profiling identifies interferon signalling molecules and IGFBP3 in human degenerative annulus fibrosus. Sci. Rep. 5, 15662. https://doi.org/10.1038/srep15662 (2015).
Dou, Z. et al. Cytoplasmic chromatin triggers inflammation in senescence and cancer. Nature 550, 402–406. https://doi.org/10.1038/nature24050 (2017).
Nishikawa, Y. et al. Modulation of stimulator of interferon genes (STING) expression by interferon-gamma in human keratinocytes. Biochem. Genet. 56, 93–102. https://doi.org/10.1007/s10528-017-9832-7 (2018).
Flood, B. A., Higgs, E. F., Li, S., Luke, J. J. & Gajewski, T. F. STING pathway agonism as a cancer therapeutic. Immunol. Rev. 290, 24–38. https://doi.org/10.1111/imr.12765 (2019).
Hsu, Y. L., Shi, S. F., Wu, W. L., Ho, L. J. & Lai, J. H. Protective roles of interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) in dengue virus infection of human lung epithelial cells. PLoS ONE 8, e79518. https://doi.org/10.1371/journal.pone.0079518 (2013).
Reich, N. C. A death-promoting role for ISG54/IFIT2. J. Interferon Cytokine Res. 33, 199–205. https://doi.org/10.1089/jir.2012.0159 (2013).
Zhu, Y. et al. The Achilles’ heel of senescent cells: from transcriptome to senolytic drugs. Aging Cell 14, 644–658. https://doi.org/10.1111/acel.12344 (2015).
Katayanagi, K., Kono, N. & Nakanuma, Y. Isolation, culture and characterization of biliary epithelial cells from different anatomical levels of the intrahepatic and extrahepatic biliary tree from a mouse. Liver 18, 90–98 (1998).
Sasaki, M., Sato, Y. & Nakanuma, Y. An impaired biliary bicarbonate umbrella may be involved in dysregulated autophagy in primary biliary cholangitis. Lab Investig. 98, 745–754. https://doi.org/10.1038/s41374-018-0045-4 (2018).
Sasaki, M., Yoshimura-Miyakoshi, M., Sato, Y. & Nakanuma, Y. A possible involvement of endoplasmic reticulum stress in biliary epithelial autophagy and senescence in primary biliary cirrhosis. J. Gastroenterol. 50, 984–995. https://doi.org/10.1007/s00535-014-1033-0 (2015).
Sasaki, M., Sato, Y. & Nakanuma, Y. Increased p16(INK4a)-expressing senescent bile ductular cells are associated with inadequate response to ursodeoxycholic acid in primary biliary cholangitis. J. Autoimmun. 107, 102377. https://doi.org/10.1016/j.jaut.2019.102377 (2020).
Dimri, G. P. et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc. Natl. Acad. Sci. USA 92, 9363–9367 (1995).
Nakanuma, Y. & Sasaki, M. Expression of blood-group-related antigens in the intrahepatic biliary tree and hepatocytes in normal livers and various hepatobiliary diseases. Hepatology 10, 174–178 (1989).
Roskams, T. A. et al. Nomenclature of the finer branches of the biliary tree: canals, ductules, and ductular reactions in human livers. Hepatology 39, 1739–1745 (2004).
Nakanuma, Y. et al. Application of a new histological staging and grading system for primary biliary cirrhosis to liver biopsy specimens: interobserver agreement. Pathol. Int. 60, 167–174. https://doi.org/10.1111/j.1440-1827.2009.02500.x (2010).
Desmet, V., Gerber, M., Hoofnagle, J., Manns, M. & Scheuer, P. Classification of chronic hepatitis: diagnosis, grading and staging. Hepatology 19, 1513–1520 (1994).
Acknowledgements
This study was supported in part by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports and Science and Technology of Japan (18K06985), the Haazami (Acanthus) Foundation and the Hokkoku Cancer Foundation.
Author information
Authors and Affiliations
Contributions
M.S. contributed to the study conception and design. Material preparation, data collection and analysis were performed by M.S., Y.S. and Y.N. The first draft of the manuscript was written by M.S. and Y.S. and Y.N. commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Sasaki, M., Sato, Y. & Nakanuma, Y. Interferon-induced protein with tetratricopeptide repeats 3 may be a key factor in primary biliary cholangitis. Sci Rep 11, 11413 (2021). https://doi.org/10.1038/s41598-021-91016-6
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41598-021-91016-6
This article is cited by
-
Cellular senescence in the cholangiopathies: a driver of immunopathology and a novel therapeutic target
Seminars in Immunopathology (2022)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.