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Identifying a Novel Biomarker of Primary Sclerosing Cholangitis

Primary sclerosing cholangitis (PSC) is a chronic, progressive autoimmune disease, where ducts that transport bile within the liver and between the liver, gallbladder, and intestines become narrowed due to inflammation and fibrosis.1 The resulting accumulation of bile within the liver damages the tissue and ultimately leads to organ failure. Clinicians use non-specific testing including imaging, liver biopsy, and serum biochemical assays to diagnose patients with PSC.1 Moreover, patients with other liver diseases exhibit overlapping symptoms, which makes it difficult to differentiate between the diseases and correctly diagnose the patients. Clinicians require PSC-specific biomarkers to improve diagnosis, and researchers from Kyoto University have identified a potential diagnostic biomarker, anti-integrin αvβ6 autoantibodies.2

Elucidating the Target of PSC Autoantibodies

Although researchers know very little about its pathogenesis, previous studies have associated PSC with inflammatory bowel disease (IBD), with approximately 70% of patients with PSC also affected by IBD.3 Additionally, scientists have detected autoantibodies against biliary epithelial cells, which line the bile ducts, in patients with PSC and hypothesize that these antibodies may contribute to PSC pathogenesis.4 However, it is unknown which surface antigens these autoantibodies specifically target.

Earlier work by Hiroshi Seno’s laboratory team from Kyoto University revealed that patients with ulcerative colitis—the most common form of IBD—contained autoantibodies against a member of the integrin family, integrin αvβ6.5 Integrins are heterodimeric surface receptors, which are each composed of an α and β subunit. Researchers have already identified 18 α subunits and eight β subunits in mammals, which together assemble into 24 different integrins.2 Epithelial cells, such as biliary epithelial cells, express integrin αvβ6 receptors on their surface, where this heterodimer interacts with extracellular matrix proteins including fibronectin. This integrin is also necessary for biliary epithelial cell proliferation after bile duct injury.6 Moreover, people with mutations in a subunit of integrin αvβ6, β6, have both IBD- and PSC-like symptoms, which suggests that this integrin may play an important role in both diseases.2,7

Because of the known association between PSC and IBD, Seno, Hiroyuki Yoshida, and their colleagues hypothesized that patients with PSC may also have autoantibodies in their sera against integrin αvβ6 and potentially other integrin heterodimers.2 To test this hypothesis, the researchers extracted sera from patients with PSC, as well as patients with other liver-associated diseases and healthy controls, which they collectively considered control participants. Yoshida and his team then examined which integrin proteins the autoantibodies target by employing enzyme-linked immunosorbent assays (ELISAs). Using ELISA buffers and substrate from Bethyl Laboratories, a Fortis Life Sciences® company, 23 recombinant integrin proteins as the immobilized antigens, and a labeled secondary antibody to detect the autoantibodies, the authors found that 88.2% of the patients with PSC were positive for autoantibodies against integrin αvβ6, while 64.7% of the patients contained anti-integrin αvβ3 autoantibodies.

Additionally, they ascertained that very few of the patients with PSC had antibodies against the other 21 integrin heterodimers and none of the control participants possessed any integrin-binding autoantibodies.

Based on the results of their ulcerative colitis study and the fact that biliary epithelial cells express integrin αvβ6, Yoshida and his colleagues decided to examine only anti-integrin αvβ6 autoantibodies moving forward. To confirm their preliminary findings using a larger cohort, they used ELISA to assess additional control and PSC sera samples. In total, they determined that 89.1% of patients with PSC contained autoantibodies against integrin αvβ6, in contrast to 3.3% of the control participants.

Testing the Autoantibodies’ Blocking Activity

Seno and his team previously revealed that autoantibodies from patients with ulcerative colitis could block fibronectin from binding to integrin αvβ6.5 To assess if the PSC-associated autoantibodies were also capable of inhibiting this interaction, they employed solid-phase binding assays. After coating microplate wells with integrin αvβ6, the researchers added IgG that they purified from patient or control sera and confirmed the purity of the IgG fractions using Bethyl’s Human IgA ELISA kit, Human IgM ELISA kit, Human IgE ELISA kit, and SDS-PAGE with Coomassie Brilliant Blue staining to test for IgA, IgM, IgE, and protein contaminants respectively. The samples were then incubated in the microplate wells with fibronectin. Through antibody-based detection of fibronectin, they observed which wells the fibronectin was free to bind to integrin αvβ6. The authors determined that 40.5% of the purified IgG samples from patients with PSC inhibited fibronectin from binding to integrin αvβ6 and that the detected blocking activity directly correlated with the concentration of anti-integrin αvβ6 autoantibodies in the patient’ serum.

Advancing PSC Diagnosis

As researchers search for effective therapeutic options, there is an increasing need for established biomarkers to allow clinicians to diagnose and treat patients with PSC at an earlier stage.8 Through the analysis of patient sera, Yoshida and his colleagues established that anti-integrin αvβ6 autoantibodies could serve as a non-invasive diagnostic biomarker.2 Although researchers have yet to characterize how integrin αvβ6 and its targeting autoantibodies contribute to healthy bile duct repair and disease pathogenesis, the authors hypothesized that the autoantibodies could prevent integrin αvβ6-expressing biliary epithelial cells from functioning correctly, which may lead to PSC development.

References

  1. Dyson JK, et al. Primary sclerosing cholangitis. The Lancet. 2018;391(10139):2547-2559.
  2. Yoshida H, et al. Anti-integrin αvβ6 autoantibodies in patients with primary sclerosing cholangitis. J Gastroenterol. 2023;58(8):778-789.
  3. Palmela C, et al. Inflammatory bowel disease and primary sclerosing cholangitis: A review of the phenotype and associated specific features. Gut Liver. 2018;12(1):17-29.
  4. Xu B, et al. High frequency of autoantibodies in patients with primary sclerosing cholangitis that bind biliary epithelial cells and induce expression of CD44 and production of interleukin 6. Gut. 2002;51(1):120-127.
  5. Kuwada T, et al. Identification of an anti–integrin αvβ6 autoantibody in patients with ulcerative colitis. Gastroenterology. 2021;160(7):2383-2394.e21.
  6. Guillot A, et al. Bile acid-activated macrophages promote biliary epithelial cell proliferation through integrin αvβ6 upregulation following liver injury. J Clin Invest. 2021;131(9):e132305, 132305.
  7. Weil P, et al. β6 integrinosis: A new lethal autosomal recessive ITGB6 disorder leading to impaired conformational transitions of the αVβ6 integrin receptor. Gut. 2020;69(7):1359-1361.
  8. Vesterhus M, Karlsen TH. Emerging therapies in primary sclerosing cholangitis: Pathophysiological basis and clinical opportunities. J Gastroenterol. 2020;55(6):588-614.
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