Abstract
Annexin A11 was identified as autoantigen in IgG4-related cholangitis (IRC), a B-cell driven disease. Annexin A11 modulates calcium-dependent exocytosis, a crucial mechanism for insertion of proteins into their target membranes. Human cholangiocytes form an apical 'biliary bicarbonate umbrella' regarded as defense against harmful hydrophobic bile acid influx. The bicarbonate secretory machinery comprises the chloride/bicarbonate exchanger AE2 and the chloride channel ANO1. We aimed to investigate the expression and function of annexin A11 in human cholangiocytes and a potential role of IgG1/IgG4-mediated autoreactivity against annexin A11 in the pathogenesis of IRC. Expression of annexin A11 in human liver was studied by immunohistochemistry and immunofluorescence. In human control and ANXA11 knockdown H69 cholangiocytes, intracellular pH, AE2 and ANO1 surface expression, and bile acid influx were examined using ratio microspectrofluorometry, cell surface biotinylation, and 22,23-3H-glycochenodeoxycholic acid permeation, respectively. The localization of annexin A11-mEmerald and ANO1-mCherry was investigated by live-cell microscopy in H69 cholangiocytes after incubation with IRC patient serum containing anti-annexin A11 IgG1/IgG4-autoantibodies or disease control serum. Annexin A11 was strongly expressed in human cholangiocytes, but not hepatocytes. Knockdown of ANXA11 led to reduced plasma membrane expression of ANO1, but not AE2, alkalization of intracellular pH and uncontrolled bile acid influx. High intracellular calcium conditions led to annexin A11 membrane shift and colocalization with ANO1. Incubation with IRC patient serum inhibited annexin A11 membrane shift and reduced ANO1 surface expression. Cholangiocellular annexin A11 mediates apical membrane abundance of the chloride channel ANO1, thereby supporting biliary bicarbonate secretion. Insertion is inhibited by IRC patient serum containing anti-annexin A11 IgG1/IgG4-autoantibodies. Anti-annexin A11 autoantibodies may contribute to the pathogenesis of IRC by weakening the 'biliary bicarbonate umbrella'. We previously identified annexin A11 as a specific autoantigen in immunoglobulin G4-related cholangitis (IRC), a B-cell driven disease affecting the bile ducts. Human cholangiocytes are protected against harmful hydrophobic bile acid influx by a defense mechanism referred to as the 'biliary bicarbonate umbrella'. We found that annexin A11 is required for the formation of a robust bicarbonate umbrella. Binding of patient-derived annexin A11 autoantibodies inhibits annexin A11 function, possibly contributing to bile duct damage by weakening the biliary bicarbonate umbrella in patients with IRC.
Highlights
Immunoglobulin G4 (IgG4)-related cholangitis (IRC) is the hepatobiliary manifestation of IgG4related disease, a systemic immune-mediated disorder characterized by typical histopathological findings in affected organs and often elevated IgG4 plasma levels [1, 2]
Annexin A11 is detected in the cytoplasm and on the apical and basolateral surface of human cholangiocytes To assess whether annexin A11 is expressed in cholangiocytes, the cell type targeted in IgG4-related cholangitis (IRC), paraffin-embedded human liver tissue was stained for annexin A11 using immunohistochemistry
To further investigate the localization of annexin A11 in cholangiocytes, human SV40transformed cholangiocytes (H69 cholangiocytes) were transduced with short hairpin ribonucleic acid (RNA) against ANXA11, the coding gene of annexin A11, or non-targeting shRNA, and surface biotinylation followed by immunoblotting was performed
Summary
Immunoglobulin G4 (IgG4)-related cholangitis (IRC) is the hepatobiliary manifestation of IgG4related disease, a systemic immune-mediated disorder characterized by typical histopathological findings in affected organs and often elevated IgG4 plasma levels [1, 2]. ANO1 mediates Cl- secretion, which allows the exchange of extracellular Cl- with HCO3- by the anion exchanger 2 (AE2) on the apical surface of human cholangiocytes [24]. This exchange is indispensable for the maintenance of a cholangiocellular defense mechanism against the toxic effects of glycine-conjugated bile acids in human bile described by us as ‘biliary HCO3umbrella’ [25,26,27]. A11 in human cholangiocytes and a potential role of IgG1/IgG4-mediated autoreactivity against annexin A11 in the pathogenesis of IRC
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