The Clinical Characteristics Of Patients With Primary Biliary Cirrhosis And Th17 Cells In The Study Of The Effect Of The Disease

Part1Clinical profiles and risk factors for hepatic decompensation in patients with primary biliary cirrhosis in ChinaAims:The clinical profiles and prognosis of primary biliary cirrhosis (PBC) have not been well-studied prospectively in China. In the current study, we will examine the clinical features and analyze prognostic factors in a prospective study of a large cohort of PBC patients.Methods:From database of our research group, PBC patients without hepatic decompensation were enrolled. The risk factors for hepatic decompensation and survival were assessed by Cox regression and Kaplan-Meier analysis, respectively.Results:Two hundred and sixty-two PBC patients were enrolled with a median follow-up of73.5months (range,21-201). Two hundred forty patients (91.6%) were female with an age of51.5±10.2yr at diagnosis. Two hundred and forty-five (93.5%) were seropositive for anti-mitochondrial antibodies (AMA). At presentation,170patients (64.9%) were symptomatic, while96patients had extra-hepatic autoimmune disease. During the follow-up period,62(23.7%) patients developed hepatic decompensation of whom4underwent liver transplantation and17(6.5%) died. One (0.4%) patient died of interstitial lung disease (ILD), while one (0.4%) died of multiple organ failure on account of lymphoma. The cumulative survival rates were96.4%and83.9%at5-year and10-year, respectively. Compared to patients with hepatic free-decompensation, the age, antinuclear antibodies (ANAs) positivity, prevalence of clinical manifestations, anticentromere antibody (ACA) positivity, the levels of aspartate aminotransferase (AST), alanine aminotransferase ratio (AAR), alkaline phosphatase (ALP), y-glutamyl transpeptidase (GGT), total bilirubin (TBil), direct bilirubin (DBil) and IgA were higher at the baseline, while the incidence of good response to ursodeoxycholic acid (UDCA) treatment and early patients, and the levels of albumin (ALB) were lower. Cox regression analysis revealed that incomplete UDCA response or inconsistent treatment (P<0.001; HR95%CI=2.423-7.541), ACA positivity (P<0.001; HR95%CI=2.516-7.137), AAR elevations (P<0.001; HR95%CI=1.357-2.678), level of TBil (P=0.004; HR95%CI=1.002-1.009), and level of ALP (P=0.008; HR95%CI=1.000-1.002) were predictors. Histological advanced liver disease was also a risk factor for hepatic decompensation(P=0.006, HR95%CI=1.481-10.847) as determined by Cox regression analysis.Conclusions:1. The clinical features and survival of PBC in China were consistent with those described in Western countries. However, the symptomatic patients were more.2. Compared to patients with hepatic free-decompensation, the age, ANA and ACA positivity, prevalence of clinical manifestations, the levels of AST, AAR, ALP, GGT, TBil, DBil and IgA were higher at the baseline, while the incidence of good response to UDCA treatment and early patients, and the levels of ALB were lower.3. Incomplete UDCA response or inconsistent treatment, ACA positivity, AAR elevations, ALP elevations, TBil elevations, and advanced histological stage were independent predictors of hepatic decompensation. Part2The role of T help17cells and interleukin-17in PBCAims:To investigate the levels, subsets, and distrubition of Th17cells in peripheral blood mononuclear cells (PBMCs), and interleukin-17(IL-17)-positive cells in liver in patients with PBC and healthy controls (HC). We will explore the reasons why Th17cells elevated, and the role of IL-17in PBC fibrosis.Methods:1. We used flow cytometry to compare the percentage of circulating CD4+IL-17+cells between patients and HC. IL-17-positive cells that infiltrated the liver were examined by immunohistochemistry.2. We used flow cytometry to compare the percentage of circulating CD4+IL-17+Foxp3+cells, CD4+IL-17+IFN-γ+cells, and Treg cells between patients and HC. Treg cells were cultured with IL-2, IL-6, and TGF-β for seven days, and the expressions of IL-17in these cells were measured by flow cytometry.3. We used flow cytometry to compare the percentage of circulating CD4+CD161+cells between PBC patients and HC. IL-17expressions from stimulated CD4+CD161+cells were determined by enzyme linked immunosorbent assay (ELISA). The capcitity of proliferation of CD4+CD161+cells was indenified by cell counting kit8(CCK8). And serum IL-23levels were measured by ELISA.4. We used flow cytometry to compare the percentage of circulating CD4+CCR6+cells between PBC patients and HC. Serum CCL20levels were measured by ELISA, and CCL20levels in liver were examined by immunohistochemistry.5. Hepatic stellate cells (HSCs) were cultured with0ng/mL,1ng/mL,5ng/mL and10ng/mL IL-17. The capcitity of proliferation of HSCs was indenified by CCK8at24,72h and144h, respectively. The expressions of a-smooth muscle actin (SMA) were determined by real-time polymerase chain reaction (PCR) at144h. The levels of IL-8in culture supernatant were measured by ELISA at24,72h and144h, respectively.1μg/mL anti-IL-17were added to culture medium to neutralize above reactions.Results:1. Circulating Th17cells were elevated in PBC patients compared to HCs (1.03± 0.22%and0.55±0.20%, P<0.0001), and higher than those in chronic hepatic B (1.03±0.22%and0.67±0.20%, P<0.0001). IL-17-positive cells that infiltrated the liver were higher in PBC patients compared to HCs.2. Circulating CD4+IL-17+IFN-y+cells (0.39±0.16%and0.14±0.07%, P<0.0001), and CD4+IL-17+Foxp3+cells (0.21±0.13%and0.06±0.04%, P<0.0001) were all increased in PBC patients. The percentage of CD4+IL-17+Foxp3+cells were positively related with Th17cells (r2=0.441, P<0.0001); however, the relationship between CD4+IL-17+Foxp3+cells and Treg cells was not statistically significant. Treg cells can produced more IL-17after stimulation with IL-2, IL-6and TGF-β in PBC patients compared to HCs (4.46±0.60%to6.6±0.5%, P=0.0003in PBC;3.32±0.85%to4.18±1.29%, P=0.2491in HCs). What’s more, we found the percentage of circulating Treg cells were decreased in patients (2.37±1.24%and3.57±1.57%, P=0.0068). The relationship between CD4+IL-17+IFN-γ+cells and Thl or Th17cells was not statistically significant.3. After stimulation with IL-23and IL-1β, the increased progenitor of Th17cells, CD4+CD161+cells from PBC patients (22.24±5.33%and13.89±3.92%, P<0.0001) expressed more IL-17(45.28±35.73pg/mL and17.03±16.78pg/mL, P=0.0556). The relationship between CD4+CD161+cells and Th17cells was not statistically significant (P=0.279). The capcitities of proliferation of CD4+CD161+cells were similar in PBC and HCs (1.079±0.093%and1.074±0.034%, P=0.9089) after stimulation with IL-23and IL-1β. Accordingly, the levels of serum IL-1β and IL-23(35.8±8.34pg/mL and23.5±5.35pg/mL, P=0.023) were increased in PBC patients.4. Early PBC presented with more Th17cells in periphery blood (1.09±0.23%and0.93±0.16%, P=0.0351) than advanced patients. In contrast, IL-17was higher in the liver in advanced PBC patients. Accordingly, the levels of serum CCL20were higher in PBC patients (53.23±38.58pg/mL and23.54±21.95pg/mL, P=0.001), especially in advanced disease (85.88±32.11pg/mL and28.98±13.57pg/mL, P=0.001). More importantly, the levels of CCL20were more obvious in liver in PBC, especially around portal area. Whereas, the percentages of CD4+CCR6+cells were not statistically significant different between PBC patients and HCs (17.69±7.11%and17.62±6.71%, P=0.975).5. IL-17can promote capcitity of proliferation of HSCs in a dose dependent way (P <0.001). And IL-17can also increase the IL-8expression of HSCs in both a dose and a time dependent way (P<0.001). However, the production of a-SMA may not be influenced by IL-17(P=0.0847). Anti-IL-17neutralizes above reactions.Conclusions:1. Compared to HCs and chronic hepatic B, the levels of circulating Th17cells and IL-17-positive cells infiltrated liver are higher in PBC patients.2. Proimflammatory cytokines-stimulated Treg cells can secrete more IL-17in PBC patients, resulting in decreased Treg cells and increased CD4+IL-17+Foxp3+cells.3. Increased CD4+CD161+cells produce more IL-17after stimulation with IL-1β and IL-23in PBC patients. Therefore, CD4+CD161+cells are a source of increased Th17cells in PBC.4. The increased Th17population is greater in circulation and less in liver in early PBC patients. With disease progression, enhanced serum and hepatic CCL20induce IL-17-expressing cells to migrate to liver. Therefore, Th17cells are lower in circulation and enhance in liver in advanced patients.5. IL-17can promote capcitity of proliferation of HSCs in a dose dependent way, and increase the IL-8expression of HSCs in both a dose and a time dependent way. Therefore, anti-IL-17is a promosing therapy for PBC fibrosis.