The Role Of IL-35 And P4HA2 In Autoimmune Liver Diseases

Part Ⅰ IL-35 promotes Th9 cell-mediated Ig G4 immunoglobulin class switch in Ig G4-related diseaseBackgrounds & Aims: Ig G4-related disease(Ig G4-RD)is a multi-organ affected fibroinflammatory disease,which is characterized by elevated serum levels of Ig G4,intense infiltration of Ig G4-positive plasma cells in liver tissue and pancreas,obliterative phlebitis and storiform fibrosis.However,the mechanisms acting in the immune responses in Ig G4-RD are not fully understood.Patients with Ig G4-related sclerosing cholangitis with/without autoimmune pancreatitis in the hepatobiliary-pancreatic system,suffer from refractory jaundice and biliary infection,even progress to cirrhosis and liver failure.The aim of this study was to identify the proinflammatory signatures and activities associated with Ig G4-RD in the hepatobiliary-pancreatic system,and to dissect the mechanism underlying the immunoglobulin class switch in Ig G4-RD by addressing the crosstalk between IL-35-producing plasma cells and Th9 cells.Methods: Expression levels of 24 proinflammatory cytokines and 13 chemokines were examined in plasma samples from patients with hepatobiliary and/or pancreatic manifestations of Ig G4-RD.Our data demonstrate that Ig G4-RD patients exhibit significantly high-level productions of IL-35 as compared to disease and healthy controls.We detected the two subunits of IL-35,EBI3 and IL12p35 in the two major affected organs,liver and pancreatic tissue from patients with Ig G4-related hepatobiliary-pancreatic disease.The effect of IL-35 in Th9 differentiation was then assessed.The frequency of Th9 cells in peripheral blood and affected tissue was detected by flow cytometry,immunohistochemistry and confocal,the effect of IL-9 in production of immunoglobulin was explored in vitro.Results: The expression level of IL-35 was significantly elevated in Ig G4-RD.The EBI3 and IL-12p35 positive cells were significantly higher in affected organs in Ig G4-RD as compared to disease controls.The colocalization of EBI3 with CD19 and CD38,markers for B cells suggest the presence of IL-35-producing B cells in affected organs in Ig G4-RD.Surprisingly,IL-35 treatment promoted na?ve CD4 T cell differentiating towards Th9 cells through IRF4 signaling.Th9 cells accumulated in peripheral blood and affected tissue of patients with Ig G4-RD.As a consequence,IL-9 secreted by Th9 cells promoted the differentiation of plasma cells and production of Ig G1 and Ig G4,predominantly Ig G4.Conclusions: our data demonstrate that IL-35 actively participates in the process of inflammation and plays an important role in Th9 differentiation resulting in an immunoglobulin class switch towards Ig G4.Part Ⅱ P4HA2: A new regulator critical for ductular reaction and biliary fibrosis in autoimmune liver diseasesBackgrounds & Aims: Primary biliary cholangitis(PBC)and primary sclerosing cholangitis(PSC)are autoimmune-mediated chronic cholestatic liver diseases,which are characterized by non-suppurative bile duct injury,may progress to biliary fibrosis,cirrhosis and liver failure.During the process of chronic cholestatic liver injury,ductular reaction is closely associated with biliary fibrosis.Prolyl4-hydroxylase subunit alpha 2(P4HA2)is mainly expressed in ductular reactive cells.The purpose of this study was to explore the role and mechanism of P4HA2 promoting ductular reaction and biliary fibrosis.Methods: The expression of P4HA2 in liver tissue was detected by immunohistochemistry and immunofluorescence.P4HA2-/-mice were constructed and murine chronic cholestatic liver injury induced by DDC was established for our study.The level of ductular reaction and biliary fibrosis was detected using H&E staining,Masson staining,immunohistochemistry,q PCR and other methods between wild type mice and P4HA2-/-mice in DDC-induced chronic cholestatic liver injury mice models.The mechanism of P4HA2 regulating cholangiocyte proliferation was explored in vitro by using RNA-seq,q PCR,Western blot and co-immunoprecipitation.Results: Compared with healthy control and other disease control,the level of P4HA2 in liver tissue was significantly elevated in patients with PBC and PSC.Besides,patients with more serious liver injury,higher levels of inflammation,and fibrotic stages expressed higher levels of P4HA2 in liver tissue.Deficiency of P4HA2 in mice significantly alleviated ductular reaction and biliary fibrosis compared with wild type mice in DDC-induced chronic cholestatic liver injury mice models.In vitro studies showed that P4HA2 interacted with SAV1,a key element of the Hippo signaling pathway,which promoted hydroxylation of SAV1 and degradation through the ubiquitin-proteasome system,resulting in activation of YAP and cholangiocyte proliferation.Conclusions: P4HA2 promotes ductular reaction and biliary fibrosis through interaction with SAV1,indicating that P4HA2 may be a potential therapeutic target for autoimmune liver diseases including PBC and PSC.