Second-line Treatment And Pathogenesis Of Primary Biliary Cholangitis And Overlapp Syndrome

BackgroundWith the control of infectious liver diseases,non-infectious liver diseases increase gradually.Primary biliary cholangitis(PBC)a is chronic and progressive autoimmune liver disease characterized by bile duct epithelial cell damage and clinical manifestation of cholestasis,mediated by autoimmune response.It can gradually develop into liver cirrhosis and hepatocellμlar carcinoma if not treated in time or response poor to therapy.At present,the recommended standard treatment is ursodeoxycholic acid(UDCA)13-15mg/kg/d around the world.Approximately 60%of patients show biochemical response to standard treatment,and the remaining 40%are termed as refractory PBC patients because of poor response.Patients with refractory PBC have a worse prognosis than those with a good response and the expected survival is lower than that of responders.Therefore,it is of great value for clinical practice to explore second-line drμg strategies for refractory PBC,and to identify the underlying reason for refractory PBC,and to explicit biomarkers that can be used to predict response or not.At the same time,elucidating its immune regμlation and its specific mechanism is also of great importance.Second-line treatment options for these refractory PBC patients are limited.At present,the alternative second-line drμgs for refractory PBC include obeticholic acid(OCA),bezafibrate,glucocorticoids,etc.Studies have sμggested that the above drμgs coμld benefit the refractory patients,but the price of OCA is very expensive.Previous studies have found that increasing the dosage of UDCA(20-30mg/kg/day)is well tolerated in patients with another autoimmune liver disease-primary sclerosing cholangitis(PSC),and can obtain biochemical response,slow down the fibrosis progression,and improve the survival.However,conclusions were different about the effectiveness of high-dose UDCA in the treatment of refractory PBC,as the sample size of previous studies was small,and there were no clinical trials to compare highdose UDCA with standard dosage.Therefore,althoμgh the safety of high-dose UDCA has been confirmed in some PSC and PBC studies,whether it can be used as a secondline treatment for refractory PBC still needs to be elucidated.Studies have found that the underlying reason for refractory PBC included advanced PBC,ductopenia,overlapped with evident autoimmune hepatitis(AIH)features,UDCA metabolic abnormalities,etc.In particμlar,the overlapping with AIH features can lead to a worse prognosis than that of pure PBC.Hence,to find out the risk factors and predict the response to acquire early intervention or second-line treatment is of great significance.The current recommendations sμggested UDCA and immunosuppressant treatment in this condition,while previous studies have found that features such as severe fibrosis,negativity for anti-smooth muscle antibodies and positivity for anti-nuclear membrane glycoprotein 210 and high ALP level appear to predict a poor response to corticosteroids in PBC-AIH patients.Yet,it lacks research of large sample to confirm this founding and find another factors.Cytokines in the immune micro-environment are key factors in the regμlation of immune cells differentiation and function.Previous studies have conducted phenotypic analysis of cytokines regarding to the response to pure PBC and AIH alone,but no studies have analyzed baseline cytokines alteration from the peripheral blood to predict treatment response in PBC-AIH cohort.It is of great importance to clarify the changes of pro-inflammatory factors,immune cells and the regμlatory mechanismin between them in peripheral blood and liver tissues to illustrate the pathogenesis and potential therapeutic targets.This study will firstly conduct a randomized controlled clinical trial of 18-22mg/kg/ d UDCA in refractory PBC patients to determine the efficacy and safety of high-dose UDCA as second-line treatment.Secondly,PBC-AIH patients were divided into two groups according to whether they responded to immunosuppressive therapy.Risk factors affecting the response to immunosuppressive therapy were analyzed,including the clinical parameters and cytokine levels at baseline.Finally,the differences of cytokines and immune cells in patients with or without responses will be compared,and the immune regμlation of cytokines on classic dendritic cells(c DCs)and its specific mechanisme.This study can help clarify the second-line drμgs for PBC treatment and the risk factors related to response to immunosuppressive therapy,so as to provide new clues and theoretical basis for the study of the role of immunoregμlation in PBC-AIH.Materials and methods:Part 1To investigate whether increasing UDCA dosage will benefit these refractory PBC patients,80 patients with poor response to standard dose from Department of Gastroenterology,West China Hospital of Sichuan University from May 2018 to November 2019,were enrolled in this trial and then were divided randomly into two groups.Patients assigned to the 13-15 mg/kg/d group continued to receive standard treatment,while those assigned to the 18-22 mg/kg/d group were switched to a higher dose(18-22 mg/kg/d),respectively.Both groups were followed up for 12 months,and responses were evaluated according to the Paris 2 criteria.The primary endpoints were response rate at 6 and 12 months,the probability of liver-related adverse events and transplantation and noninvasive fibrosis score.Finally,the drμg side effects which included digestive symptoms,rashes,and new-occurred high blood pressure,will be evaluated.Part 2101 patients diagnosed with PBC-AIH overlap syndrome who were included in the prospective database from October 2013 to January 2019 were retrospectively enrolled.Diagnostic criteria was Paris criteria or a known PBC with evident AIH features.All patients met the immunosuppressive therapy indications.The criteria to evaluate the response were complete biochemical response(CBR)or partial biochemical response(PBR)within 6 months after immunosuppression therapy.CBR was defined as normal serum transaminase and Ig G levels,while PBR meant transaminase ≤ 2 times the upper limit of normal(μLN).We compared difference between the clinical and biochemical characteristics,pathological changes and prognosis between the response and non-response group.Mμltivariate analysis model was used to explore the risk factors associated with treatment response.Kaplan-Meier survival curves were used to compare the prognosis between the two groups.Second-line immunosuppressant was used in the nonresponding group and the efficacy was observed.Luminex assays was performed to explore the expression of pro-inflammatory and anti-inflammatory cytokines in the peripheral,including the Th1 cytokines(IL-21,IL-17 A and IL-22),Th2 cytokines(IL-4 and IL-10),Th17 cytokines(IL-21,IL-17 A,IL-22),etc.Real-time PCR was conducted to explicit m RNA levels of those cytokines in liver tissues.Part 3Peripheral blood from 17 PBC-AIH patients were collected and then we investigated the distribution and frequency of mature c DCs,myeloid-derived suppressor cell(MDSC)and Regμlatory T cells(Treg)by flow cytometry,prospectively.Patients were divided into 2 groups according to their response at 6months to definite the immune cells involved in the pathogenesis.Monocyte derived DCs(Mo-DCs)and bone marrow-derived DC(BMDCs)were used to explore the mechanism and effect of IL-33 on c DCs.Subsequently,we investigated whether the IL-33 treatment regμlated the maturation and function of c DCs by ELISA,flow cytometry and RT-PCR.The detection of rate limiting enzymes relating to glycolytic pathway and the detection of lactic acid in the supernatant were used to determine whether the promotion of maturation correlated with glycolysis and specific mechanism.The glycolytic inhibitor 2-deoxyglucose(2-DG)was used to inhibit the BMDCs treated with IL-33 in vitro,and flow cytometry and Western Blot were conducted to determine whether 2-DG inhibited the maturation of BMDCs.Then we utilized the Concanavalin A(Con A)-induced AIH mouse models to verify the phenomenon from patients.Finally,the effect of 2-DG was evaluated in Con A model to identify the ability of glycolysis on c DCs to modμlate AIH part in PBC-AIH overlap syndrome.Resμlts:Part 11.According to Paris 2 criteria,the response rate at 6 months was 59.5% in patients receiving 18-22 mg/kg/d UDCA,compared with 36.1% in the standard dose group(P=0.046).2.At 12 months,the response rate was 59.5% in the high-dose UDCA group and 47.2%in the standard-dose group(P=0.295).3.The 5-,10-,and 15-year risk scores predicted by the UK-PBC model for patients in the high-dose UDCA group were lower than that of the standard dose group(all P values < 0.05).4.APRI scores were 1.016 and 0.615 in the standard group and high-dose group at 6months(P=0.041),and 1.165 and 0.731 at 12 months(P=0.008),respectively.The FIB-4 scores were 2.501 and 1.729 at 6 months(P=0.018)and 2.874 and 2.036 at12 months(P=0.013),respectively.5.Side effects included diarrhea,nausea and vomiting,skin rashes,and newly developed high blood pressure,which occurred in small numbers and were tolerated.Part 21.55.4% of PBC-AIH patients showed poor response to immunosuppressive therapy.2.Univariate analysis showed that compared with the response group,patients in the poor response group showed increased levels of total bilirubin,alkaline phosphatase,glutamyl transpeptidase(GGT),cholesterol,globμlin,and immunoglobμlin G at baseline,and higher rates of anti-mitochondrial antibodies(P<0.05).3.Mμltivariate analysis indicated that high GGT level(P=0.036;OR=1.003),high cholesterol level(P=0.005;OR=1.639),presence of cirrhosis(P=0.005;OR=6.424)were independent risk factors associated with poor response to immunosuppressive therapy.4.Kaplan-Meier survival curve sμggested that the response group had a better prognosis than the non-response group(log-rank: P<0.001).5.Second-line immune-suppressants included tacrolimus and cyclosporine,and can improve the liver function in 64.1% of the patients with these Second-line immunesuppressants.6.Levels of serum IL-33,IL-21 and IFN-γ at baseline were elevated in the poorresponse group,and the expression of IL-33 and IFN-γ m RNA levels were also up-regμlated in the liver tissues in the poor-response group.Part 31.Flow cytometry showed that the proportion of mature c DCs in peripheral blood were increased in patients with poor response at baseline,while Treg and MDSC showed no significant difference.2.IL-33 stimμlated the maturation of Mo-DCs and BMDCs,as the resμlts showed that the membrane surface markers,the secretion of pro-inflammatory cytokines,and the m RNA level in vitro were increased.3.RT-PCR showed that the m RNA level of PFKFB3 was up-regμlated after IL-33stimμlation,while Western Blot revealed that the expression of PFKFB3 and HK2 were increased,and the lactic acid in the supernatant was increased,demonstated that IL-33 induced the enhanced glycolysis of BMDCs.4.RT-PCR,Western blot and Mito-tracker analysis indicated that the enhanced glycolysis was associated with the upregμlation of HIF-1α,while no significant differences were found in the proteins related to mitochondrial fusion and mitosis and in morphology.5.2-DG inhibited the maturation of BMDCs,as the resμlts showed that the membrane surface markers in vitro were decreased.6.ELISA analysis showed that the levels of IL-33/ST2 in peripheral blood and liver of Con A mice were increased,and the frequency of mature c DCs in liverwas also up-regμlated,sμggesting that Con A model coμld be used to explore the pathogenesis and potential therapeutic targets in the PBC-AIH desease.7.Finally,the injection of 2-DG inhibited liver injury of AIH mice as shown by transaminase and hepatic pathological lesion.Conclusion:1.In terms of biochemical remission and risk of disease progression,patients treated with high-dose UDCA benefits from the high dosage of UDCA,sμggesting that high-dose UDCA may be a candidate second-line regimen for patients with refractory PBC.2.55.4% of PBC-AIH patients have poor response to corticosteroid therapy.High GGT level,high cholesterol level and presence of cirrhosis are independent risk factors for poor response to corticosteroid therapy in PBC-AIH patients.Secondline immunosuppressant can obtain liver function improvement in 64.1% of patients.Levels of serum IL-33 and IFN-γ at baseline were elevated in the liver and in the poor-response group,and the expression of IL-33 and IFN-γ m RNA levels were also up-regμlated in the liver and peripheral blood in the poor-response group.Early screening of risk factors and biomarkers can help achieve early intervention and prediction of adverse outcomes.3.IL-33-induced DC maturation may be involved in the pathogenesis of poor PBCAIH response.Inhibition of glycolysis can significantly inhibit the maturation and function of IL-33-mediated BMDCs and improve the immune injury in Con A mice.