Galectin-3 and prohibitin 1 are autoantigens in IgG4-related cholangitis without clear-cut protective effects against toxic bile acids

Background and aimsIgG4-related cholangitis (IRC) is the hepatobiliary manifestation of IgG4-related disease, a systemic B cell-driven fibro-inflammatory disorder. Four autoantigens have recently been described in IgG4-RD: annexin A11, galectin-3, laminin 511-E8, and prohibitin 1. We have previously reported a protective role of annexin A11 and laminin 511-E8 in human cholangiocytes against toxic bile acids. Here, we explored the potentially protective role of the carbohydrate-binding lectin galectin-3 and the scaffold proteins prohibitins 1 and 2.MethodsAnti-galectin-3, anti-prohibitin 1 and 2 autoantibody positivity in IRC and healthy and disease (primary sclerosing cholangitis (PSC)) control sera was assessed by ELISA/liquid chromatography–tandem mass spectrometry (LC-MS/MS). Human H69 cholangiocytes were subjected to short hairpin RNA (shRNA) knockdown targeting galectin-3 (LGALS3), prohibitin 1 (PHB1), and prohibitin 2 (PHB2). H69 cholangiocytes were also exposed to recombinant galectin-3, the inhibitor GB1107, recombinant prohibitin 1, and the pan-prohibitin inhibitor rocaglamide. Protection against bile acid toxicity was assessed by intracellular pH (pHi) measurements using BCECF-AM, 22,23-3H-glycochenodeoxycholic acid (3H-GCDC) influx, and GCDC-induced apoptosis using Caspase-3/7 assays.ResultsAnti-galectin-3 autoantibodies were detected in 13.5% of individuals with IRC but not in PSC. Knockdown of LGALS3 and galectin-3 inhibition with GB1107 did not affect pHi, whereas recombinant galectin-3 incubation lowered pHi. LGALS3 knockdown increased GCDC-influx but not GCDC-induced apoptosis. GB1107 reduced GCDC-influx and GCDC-induced apoptosis. Recombinant galectin-3 tended to decrease GCDC-influx and GCDC-induced apoptosis. Anti-prohibitin 1 autoantibodies were detected in 61.5% and 35.7% of individuals with IRC and PSC, respectively. Knockdown of PHB1, combined PHB1/2 KD, treatment with rocaglamide, and recombinant prohibitin 1 all lowered pHi. Knockdown of PHB1, PHB2, or combined PHB1/2 did not alter GCDC-influx, yet knockdown of PHB1 increased GCDC-induced apoptosis. Conversely, rocaglamide reduced GCDC-influx but did not attenuate GCDC-induced apoptosis. Recombinant prohibitin 1 did not affect GCDC-influx or GCDC-induced apoptosis. Finally, anti-galectin-3 and anti-prohibitin 1 autoantibody pretreatment did not lead to increased GCDC-influx.ConclusionsA subset of individuals with IRC have autoantibodies against galectin-3 and prohibitin 1. Gene-specific knockdown, pharmacological inhibition, and recombinant protein substitution did not clearly disclose a protective role of these autoantigens in human cholangiocytes against toxic bile acids. The involvement of these autoantibodies in processes surpassing epithelial secretion remains to be elucidated

Galectin-3 and prohibitin 1 are autoantigens in IgG4-related cholangitis without clear-cut protective effects against toxic bile acids

BACKGROUND AND AIMS: IgG4-related cholangitis (IRC) is the hepatobiliary manifestation of IgG4-related disease, a systemic B cell-driven fibro-inflammatory disorder. Four autoantigens have recently been described in IgG4-RD: annexin A11, galectin-3, laminin 511-E8, and prohibitin 1. We have previously reported a protective role of annexin A11 and laminin 511-E8 in human cholangiocytes against toxic bile acids. Here, we explored the potentially protective role of the carbohydrate-binding lectin galectin-3 and the scaffold proteins prohibitins 1 and 2. METHODS: Anti-galectin-3, anti-prohibitin 1 and 2 autoantibody positivity in IRC and healthy and disease (primary sclerosing cholangitis (PSC)) control sera was assessed by ELISA/liquid chromatography-tandem mass spectrometry (LC-MS/MS). Human H69 cholangiocytes were subjected to short hairpin RNA (shRNA) knockdown targeting galectin-3 (LGALS3), prohibitin 1 (PHB1), and prohibitin 2 (PHB2). H69 cholangiocytes were also exposed to recombinant galectin-3, the inhibitor GB1107, recombinant prohibitin 1, and the pan-prohibitin inhibitor rocaglamide. Protection against bile acid toxicity was assessed by intracellular pH (pHi) measurements using BCECF-AM, 22,23-3H-glycochenodeoxycholic acid (3H-GCDC) influx, and GCDC-induced apoptosis using Caspase-3/7 assays. RESULTS: Anti-galectin-3 autoantibodies were detected in 13.5% of individuals with IRC but not in PSC. Knockdown of LGALS3 and galectin-3 inhibition with GB1107 did not affect pHi, whereas recombinant galectin-3 incubation lowered pHi. LGALS3 knockdown increased GCDC-influx but not GCDC-induced apoptosis. GB1107 reduced GCDC-influx and GCDC-induced apoptosis. Recombinant galectin-3 tended to decrease GCDC-influx and GCDC-induced apoptosis. Anti-prohibitin 1 autoantibodies were detected in 61.5% and 35.7% of individuals with IRC and PSC, respectively. Knockdown of PHB1, combined PHB1/2 KD, treatment with rocaglamide, and recombinant prohibitin 1 all lowered pHi. Knockdown of PHB1, PHB2, or combined PHB1/2 did not alter GCDC-influx, yet knockdown of PHB1 increased GCDC-induced apoptosis. Conversely, rocaglamide reduced GCDC-influx but did not attenuate GCDC-induced apoptosis. Recombinant prohibitin 1 did not affect GCDC-influx or GCDC-induced apoptosis. Finally, anti-galectin-3 and anti-prohibitin 1 autoantibody pretreatment did not lead to increased GCDC-influx. CONCLUSIONS: A subset of individuals with IRC have autoantibodies against galectin-3 and prohibitin 1. Gene-specific knockdown, pharmacological inhibition, and recombinant protein substitution did not clearly disclose a protective role of these autoantigens in human cholangiocytes against toxic bile acids. The involvement of these autoantibodies in processes surpassing epithelial secretion remains to be elucidated

IgG4-related disease of the biliary tract and pancreas: clinical and experimental advances

Purpose of reviewIgG4-related disease (IgG4-RD) is an immune-mediated disease of unknown cause. It predominantly affects the biliary tract [IgG4-associated cholangitis (IAC)] and pancreas [autoimmune pancreatitis (AIP)] of mostly elderly men. Accurate diagnostic tests are lacking. Patients benefit from predniso(lo)ne treatment. However, disease relapse is often seen. This review will address pathophysiological aspects and advances in diagnostic and therapeutic strategies.Recent findingsThe role of IgG1 and IgG4 in the pathophysiology of IgG4-RD was studied in mice which showed more intense organ damage of pancreas and salivary glands when IgG1 rather than IgG4 of patients with IgG4-RD was injected. Coadministration of IgG1+IgG4 led to dampening of IgG1-mediated injury supporting the view that IgG4 exerts immune-dampening effects. IgG4+ B-cell receptor clones identified by next-generation sequencing and the IgG4/IgG RNA ratio in human blood assessed by quantitative PCR were able to accurately distinguish IAC/AIP from primary sclerosing cholangitis or pancreatobiliary malignancies. Long-term treatment with low-dose prednisolone was safe and reduced the number of flare-ups in patients with AIP.SummaryEarly diagnosis by a novel accurate and easy-to-use qPCR test may prevent life-threatening complications, unnecessary interventions and fatal course because of misdiagnosis. Prednisolone treatment remains the standard of care in patients with IgG4-R

On the role of IgG4 in inflammatory conditions: lessons for IgG4-related disease

The pathophysiology of immunoglobulin G4-related disease (IgG4-RD) and its most common manifestations, IgG4-associated (sclerosing) cholangitis and autoimmune pancreatitis, remains largely unknown, but IgG4 is presumably involved. IgG4 is a promiscuous antibody, which could be directly pathogenic, fulfill a protective role, or could just be a fortuitous marker of an aberrant inflammatory response. IgG4 antibodies possess exclusive structural and functional characteristics suggesting anti-inflammatory and tolerance-inducing effects. By studying the role of IgG4 in other inflammatory conditions, namely hypersensitivity and allergies, autoimmune and immune-mediated diseases, infections and malignancies, new insights can be obtained increasing our understanding of the role of IgG4 antibodies in IgG4-RD. Beekeepers, animal laboratory workers and individuals undergoing allergen immunotherapy possess high serum levels of allergen-specific IgG4, which exhibit immunosuppressive functions, protecting the individual from anaphylactic reactions. In autoimmune/immune-mediated diseases, such as pemphigus vulgaris, pemphigus foliaceus and MuSK-myasthenia gravis, IgG4 autoantibodies are pathogenic. Regarding malignancies such as melanoma and cholangiocarcinoma or helminthic infections, IgG4 antibodies inhibit clearance of tumor cells or the invader, respectively. Translating these findings to IgG4-RD, IgG4 alone can implement pathogenic effects and structural damage, but may also function as a protective antibody dampening the more harmful effects of IgG1 when directed against the same epitopes. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Janse

IgG4-Associated Cholangitis in Patients Resected for Presumed Perihilar Cholangiocarcinoma: A 30-Year Tertiary Care Experience

Background: Distinguishing perihilar cholangiocarcinoma (PHC) from benign forms of sclerosing cholangitis affecting the hilar bile ducts is challenging, since histological confirmation of PHC is difficult to obtain and accurate non-invasive diagnostic tests are not available. IgG4-associated cholangitis (IAC), an imitator of PHC, may present with clinical and radiographical signs of PHC. IAC can be accurately diagnosed with a novel qPCR test. The aim of this study was to investigate the incidence and long-term activity of IAC in patients resected for PHC in a single tertiary center over a period of 30 years. Methods: All patients with benign disease who underwent surgery for presumed PHC in our institute between 1984 and 2015 were identified. Benign liver and bile duct specimens were re-evaluated by a pathologist and scored according to international consensus pathology criteria for IgG4-related disease (IgG4-RD). Patients with benign disease still alive were followed-up and a clinical diagnosis of IAC was made using a combination of the HISORt group C (response to steroids) criteria and elevated serum IgG4 levels and/or the novel IgG4/IgG RNA ratio. Also, recurrent symptomatic disease at any time after surgery requiring immunosuppression was assessed. Results: Out of 323 patients who underwent surgery for presumed PHC, 50 patients (15%) had benign disease. In 42% (n = 21/50) of these patients a histological (n = 17) or clinical (n = 4) diagnosis of IAC was established. The remaining patients were diagnosed with unclassified sclerosing inflammation, cystadenoma, or sclerosing hemangioma. Nine out of 12 IAC patients who were followed-up showed episodes of recurrent disease requiring immunosuppressive treatment. Conclusions: Liver and bile duct resections for PHC during three decades disclosed in 15% benign biliary disorders mimicking PHC of which 42% were definitely diagnosed as IAC. IgG4-RD remains active in the majority of patients with IAC years after surgery. Novel diagnostic tests for IAC might reduce misdiagnosis, unnecessary surgery, and life-threatening complications

IgG4-Associated Cholangitis--A Mimic of PSC

IgG4-associated cholangitis (IAC) is an inflammatory disorder of the biliary tract representing a major manifestation of IgG4-related disease (IgG4-RD) often with elevation of serum IgG4 levels, infiltration of IgG4+ plasma cells in the affected tissue and good response to immunosuppressive treatment. Its first description may go back to 150 years ago. The clinical presentation of IAC is often misleading, mimicking other biliary diseases such as primary sclerosing cholangitis (PSC) or cholangiocarcinoma. The HISORt criteria--histopathological, imaging, and serological features (sIgG4), other organ manifestations of IgG4-RD and response to treatment--are the standard for the diagnosis of IAC. In this overview of a recent lecture, we summarize our original findings on IgG4-RD that (i) dominant IgG4+ B-cell clones identified by advanced next generation sequencing (NGS) are highly specific for IgG4-RD (meanwhile confirmed by others), are a highly accurate diagnostic marker to distinguish IgG4-RD from PSC and biliary/pancreatic malignancies and may be crucial in unravelling the pathophysiology of IgG4-RD; (ii) sIgG4/sIgG1 >0.24 have additional diagnostic value in comparison to sIgG4 in differentiating IAC from PSC; (iii) blood IgG4 mRNA is a highly accurate diagnostic marker comparable to NGS and may become an easily available and affordable diagnostic standard for distinguishing IgG4-RD from PSC and biliary/pancreatic malignancies; and (iv) 'blue collar work' with long-term exposure to solvents, paints, oil products or industrial gases may be a risk factor for development of IgG4-RD. These findings may contribute to the understanding of the pathophysiology and to the early diagnosis and adequate treatment of IgG4-R

Annexin A11 is targeted by IgG4 and IgG1 autoantibodies in IgG4-related disease

Immunoglobulin G4-related disease (IgG4-RD) is a multiorgan immune-mediated disease that predominantly affects the biliary tract (IgG4-associated cholangitis, IAC) and pancreas (autoimmune pancreatitis, AIP). We recently identified highly expanded IgG4+ B-cell receptor clones in blood and affected tissues of patients with IAC/AIP suggestive of specific (auto)antigenic stimuli involved in initiating and/or maintaining the inflammatory response. This study aimed to identify (auto)antigen(s) that are responsible for the clonal expansion of IgG4+ B cells in IgG4-RD. We screened sera of patients with IAC/AIP (n=50), in comparison to control sera of patients with primary sclerosing cholangitis (PSC) and pancreatobiliary malignancies (n=47), for reactivity against human H69 cholangiocyte lysates on immunoblot. Subsequently, target antigens were immunoprecipitated and analysed by mass spectrometry. Prominent reactivity against a 56 kDa protein was detected in human H69 cholangiocyte lysates exposed to sera of nine patients with IAC/AIP. Affinity purification and mass spectrometry analysis identified annexin A11, a calcium-dependent phospholipid-binding protein. Annexin A11-specific IgG4 and IgG1 antibodies were only detected in serum of patients with IgG4-RD of the biliary tract/pancreas/salivary glands and not in disease mimickers with PSC and pancreatobiliary malignancies. Epitope analysis showed that two annexin A11 epitopes targeted by IgG1 and IgG4 autoantibodies were shared between patients with IAC/AIP and IgG4 antibodies blocked binding of IgG1 antibodies to the shared annexin A11 epitopes. Our data suggest that IgG1-mediated pro-inflammatory autoreactivity against annexin A11 in patients with IgG4-RD may be attenuated by formation of annexin A11-specific IgG4 antibodies supporting an anti-inflammatory role of IgG4 in IgG4-R

Blue-collar work is a risk factor for developing IgG4-related disease of the biliary tract and pancreas

Background & Aims: Immunoglobulin G4-related disease (IgG4-RD) of the biliary tract and pancreas is a fibroinflammatory disease of unknown origin with striking male predominance. We aimed to investigate whether blue-collar work and occupational contaminant exposure are risk factors for IgG4-RD of the biliary tract and pancreas. Method: We performed an age-/sex-matched case-control study in the largest academic medical centers of the Netherlands. Occupational history was surveyed by questionnaires. The International Standard Classification of Occupations (ISCO88) was used to classify jobs. Job exposure matrices ALOHA and DOM were utilized to assess the years individuals were exposed to compounds. The disease control cohort consisted of patients from 6 equally sized groups. Conditional logistic regression was used to assess effects of blue-collar work and exposure to occupational contaminants on developing IgG4-RD of the biliary tract and pancreas. Results: Overall, 101 patients with IgG4-RD of the biliary tract and pancreas were matched 1:3 to 303 controls. Patients with IgG4-RD had a lower level of education (p = 0.001). Individuals who at least once performed blue-collar work (>1 year), had higher odds of developing IgG4-RD than individuals that only performed white-collar work (odds ratio [OR] 3.66; CI 2.18–6.13; p 1 year) to industrial ALOHA (e.g. mineral dust; vapors-dust-gases-fumes) and DOM compounds (e.g. asbestos) resulted in higher odds of IgG4-RD (OR 2.14; 95% CI 1.26–3.16; p <0.001 and OR 2.95; 95% CI 1.78-4.90; p <0.001, respectively). Conclusion: Blue-collar work is a risk factor for developing IgG4-RD of the biliary tract and pancreas putatively driven by exposure to selected industrial compounds; this may explain the striking male predominance among patients. Lay summary: Immunoglobulin G4-related disease (IgG4-RD) causes tumor-like lesions and typically affects middle-aged to elderly men. The background and cause of this disease remain relatively unknown. In this study, we identified blue-collar work as a risk factor for developing IgG4-RD of the biliary tract and pancreas, which may explain the striking male predominance among patients. Furthermore, these results suggest that toxic exposure to occupational contaminants may drive autoimmunity in IgG4-RD of the biliary tract and pancreas

Blue-collar work is a risk factor for developing IgG4-related disease of the biliary tract and pancreas

Background & Aims: Immunoglobulin G4-related disease (IgG4-RD) of the biliary tract and pancreas is a fibroinflammatory disease of unknown origin with striking male predominance. We aimed to investigate whether blue-collar work and occupational contaminant exposure are risk factors for IgG4-RD of the biliary tract and pancreas. Method: We performed an age-/sex-matched case-control study in the largest academic medical centers of the Netherlands. Occupational history was surveyed by questionnaires. The International Standard Classification of Occupations (ISCO88) was used to classify jobs. Job exposure matrices ALOHA and DOM were utilized to assess the years individuals were exposed to compounds. The disease control cohort consisted of patients from 6 equally sized groups. Conditional logistic regression was used to assess effects of blue-collar work and exposure to occupational contaminants on developing IgG4-RD of the biliary tract and pancreas. Results: Overall, 101 patients with IgG4-RD of the biliary tract and pancreas were matched 1:3 to 303 controls. Patients with IgG4-RD had a lower level of education (p = 0.001). Individuals who at least once performed blue-collar work (>1 year), had higher odds of developing IgG4-RD than individuals that only performed white-collar work (odds ratio [OR] 3.66; CI 2.18–6.13; p 1 year) to industrial ALOHA (e.g. mineral dust; vapors-dust-gases-fumes) and DOM compounds (e.g. asbestos) resulted in higher odds of IgG4-RD (OR 2.14; 95% CI 1.26–3.16; p <0.001 and OR 2.95; 95% CI 1.78-4.90; p <0.001, respectively). Conclusion: Blue-collar work is a risk factor for developing IgG4-RD of the biliary tract and pancreas putatively driven by exposure to selected industrial compounds; this may explain the striking male predominance among patients. Lay summary: Immunoglobulin G4-related disease (IgG4-RD) causes tumor-like lesions and typically affects middle-aged to elderly men. The background and cause of this disease remain relatively unknown. In this study, we identified blue-collar work as a risk factor for developing IgG4-RD of the biliary tract and pancreas, which may explain the striking male predominance among patients. Furthermore, these results suggest that toxic exposure to occupational contaminants may drive autoimmunity in IgG4-RD of the biliary tract and pancreas

DataSheet_1_Galectin-3 and prohibitin 1 are autoantigens in IgG4-related cholangitis without clear-cut protective effects against toxic bile acids.pdf

Background and aimsIgG4-related cholangitis (IRC) is the hepatobiliary manifestation of IgG4-related disease, a systemic B cell-driven fibro-inflammatory disorder. Four autoantigens have recently been described in IgG4-RD: annexin A11, galectin-3, laminin 511-E8, and prohibitin 1. We have previously reported a protective role of annexin A11 and laminin 511-E8 in human cholangiocytes against toxic bile acids. Here, we explored the potentially protective role of the carbohydrate-binding lectin galectin-3 and the scaffold proteins prohibitins 1 and 2.MethodsAnti-galectin-3, anti-prohibitin 1 and 2 autoantibody positivity in IRC and healthy and disease (primary sclerosing cholangitis (PSC)) control sera was assessed by ELISA/liquid chromatography–tandem mass spectrometry (LC-MS/MS). Human H69 cholangiocytes were subjected to short hairpin RNA (shRNA) knockdown targeting galectin-3 (LGALS3), prohibitin 1 (PHB1), and prohibitin 2 (PHB2). H69 cholangiocytes were also exposed to recombinant galectin-3, the inhibitor GB1107, recombinant prohibitin 1, and the pan-prohibitin inhibitor rocaglamide. Protection against bile acid toxicity was assessed by intracellular pH (pHi) measurements using BCECF-AM, 22,23-3H-glycochenodeoxycholic acid (3H-GCDC) influx, and GCDC-induced apoptosis using Caspase-3/7 assays.ResultsAnti-galectin-3 autoantibodies were detected in 13.5% of individuals with IRC but not in PSC. Knockdown of LGALS3 and galectin-3 inhibition with GB1107 did not affect pHi, whereas recombinant galectin-3 incubation lowered pHi. LGALS3 knockdown increased GCDC-influx but not GCDC-induced apoptosis. GB1107 reduced GCDC-influx and GCDC-induced apoptosis. Recombinant galectin-3 tended to decrease GCDC-influx and GCDC-induced apoptosis. Anti-prohibitin 1 autoantibodies were detected in 61.5% and 35.7% of individuals with IRC and PSC, respectively. Knockdown of PHB1, combined PHB1/2 KD, treatment with rocaglamide, and recombinant prohibitin 1 all lowered pHi. Knockdown of PHB1, PHB2, or combined PHB1/2 did not alter GCDC-influx, yet knockdown of PHB1 increased GCDC-induced apoptosis. Conversely, rocaglamide reduced GCDC-influx but did not attenuate GCDC-induced apoptosis. Recombinant prohibitin 1 did not affect GCDC-influx or GCDC-induced apoptosis. Finally, anti-galectin-3 and anti-prohibitin 1 autoantibody pretreatment did not lead to increased GCDC-influx.ConclusionsA subset of individuals with IRC have autoantibodies against galectin-3 and prohibitin 1. Gene-specific knockdown, pharmacological inhibition, and recombinant protein substitution did not clearly disclose a protective role of these autoantigens in human cholangiocytes against toxic bile acids. The involvement of these autoantibodies in processes surpassing epithelial secretion remains to be elucidated.</p