Junliang Fu

Circulating and Liver Resident CD4+CD25+ Regulatory T Cells Actively Influence the Antiviral Immune Response and Disease Progression in Patients with Hepatitis B

CD4+CD25+ regulatory T cells (Treg) have been shown to maintain immune tolerance against self and foreign Ags, but their role in persistent viral infection has not been well-defined. In this study, we investigated whether and where CD4+CD25+ Treg contribute to the development of chronic hepatitis B (CHB). One hundred twenty-one patients were enrolled, including 16 patients with acute hepatitis B, 76 with CHB, and 29 with chronic severe hepatitis B. We demonstrated that in chronic severe hepatitis B patients, the frequencies of CD4+CD25+ Treg in both PBMC and liver-infiltrating lymphocytes were significantly increased and there was a dramatic increase of FoxP3+-cell and inflammatory cell infiltration in the liver compared with healthy controls. In CHB patients, circulating CD4+CD25+ Treg frequency significantly correlates with serum viral load. In acute hepatitis B patients, circulating CD4+CD25+ Treg frequency was initially low and with time, the profile reversed to exhibit an increased number of circulating Treg in the convalescent phase and restored to normal levels upon resolution. In PBMC taken from infected patients, depletion of CD4+CD25+ Treg led to an increase of IFN-γ production by HBV-Ag-stimulated PBMC. In addition, CD4+CD25+ Treg were capable of suppressing proliferation of autologous PBMC mediated by HBV Ags, which probably reflects the generation of HBV-Ag-specific Treg in circulation and in the liver of HBV-infected patients. Together, our findings suggest that CD4+CD25+ Treg play an active role not only in modulating effectors of immune response to HBV infection, but also in influencing the disease prognosis in patients with hepatitis B.

Interleukin-17–producing CD4+ T cells increase with severity of liver damage in patients with chronic hepatitis B†

Interleukin‐17 (IL‐17)‐producing CD4+ T cells (Th17)‐mediated immune response has been demonstrated to play a critical role in inflammation‐associated disease; however, its role in chronic hepatitis B virus (HBV) infection remains unknown. Here we characterized peripheral and intrahepatic Th17 cells and analyzed their association with liver injury in a cohort of HBV‐infected patients including 66 with chronic hepatitis B (CHB), 23 with HBV‐associated acute‐on‐chronic liver failure (ACLF), and 30 healthy subjects as controls. The frequency of circulating Th17 cells increased with disease progression from CHB (mean, 4.34%) to ACLF (mean, 5.62%) patients versus healthy controls (mean, 2.42%). Th17 cells were also found to be largely accumulated in the livers of CHB patients. The increases in circulating and intrahepatic Th17 cells positively correlated with plasma viral load, serum alanine aminotransferase levels, and histological activity index. In vitro, IL‐17 can promote the activation of myeloid dendritic cells and monocytes and enhance the capacity to produce proinflammatory cytokines IL‐1β, IL‐6, tumor necrosis factor (TNF)‐α, and IL‐23 in both CHB patients and healthy subjects. In addition, the concentration of serum Th17‐associated cytokines was also increased in CHB and ACLF patients. Conclusion: Th17 cells are highly enriched in both peripheral blood and liver of CHB patients, and exhibit a potential to exacerbate liver damage during chronic HBV infection. (HEPATOLOGY 2009.)

Human umbilical cord mesenchymal stem cells improve liver function and ascites in decompensated liver cirrhosis patients.

Decompensated liver cirrhosis (LC), a life‐threatening complication of chronic liver disease, is one of the major indications for liver transplantation. Recently, mesenchymal stem cell (MSC) transfusion has been shown to lead to the regression of liver fibrosis in mice and humans. This study examined the safety and efficacy of umbilical cord‐derived MSC (UC‐MSC) in patients with decompensated LC. A total of 45 chronic hepatitis B patients with decompensated LC, including 30 patients receiving UC‐MSC transfusion, and 15 patients receiving saline as the control, were recruited; clinical parameters were detected during a 1‐year follow‐up period. No significant side‐effects and complications were observed in either group. There was a significant reduction in the volume of ascites in patients treated with UC‐MSC transfusion compared with controls (P < 0.05). UC‐MSC therapy also significantly improved liver function, as indicated by the increase of serum albumin levels, decrease in total serum bilirubin levels, and decrease in the sodium model for end‐stage liver disease scores. UC‐MSC transfusion is clinically safe and could improve liver function and reduce ascites in patients with decompensated LC. UC‐MSC transfusion, therefore, might present a novel therapeutic approach for patients with decompensated LC.

Human Mesenchymal Stem Cell Transfusion Is Safe and Improves Liver Function in Acute-on-Chronic Liver Failure Patients

Acute‐on‐chronic liver failure (ACLF) is a severe, life‐threatening complication, and new and efficient therapeutic strategies for liver failure are urgently needed. Mesenchymal stem cell (MSC) transfusions have been shown to reverse fulminant hepatic failure in mice and to improve liver function in patients with end‐stage liver diseases. We assessed the safety and initial efficacy of umbilical cord‐derived MSC (UC‐MSC) transfusions for ACLF patients associated with hepatitis B virus (HBV) infection. A total of 43 ACLF patients were enrolled for this open‐labeled and controlled study; 24 patients were treated with UC‐MSCs, and 19 patients were treated with saline as controls. UC‐MSC therapy was given three times at 4‐week intervals. The liver function, adverse events, and survival rates were evaluated during the 48‐week or 72‐week follow‐up period. No significant side effects were observed during the trial. The UC‐MSC transfusions significantly increased the survival rates in ACLF patients; reduced the model for end‐stage liver disease scores; increased serum albumin, cholinesterase, and prothrombin activity; and increased platelet counts. Serum total bilirubin and alanine aminotransferase levels were significantly decreased after the UC‐MSC transfusions. UC‐MSC transfusions are safe in the clinic and may serve as a novel therapeutic approach for HBV‐associated ACLF patients.

Functional impairment in circulating and intrahepatic NK cells and relative mechanism in hepatocellular carcinoma patients.

Functional defects in natural killer (NK) cells have been proposed to be responsible for the failure of anti-tumor immune responses. Whether and how NK cells are impaired in hepatocellular carcinoma (HCC) patients remain unknown. In this study, we found that HCC patients displayed a dramatic reduction in peripheral CD56(dim)CD16(pos) NK subsets compared with healthy subjects. A significant reduction of CD56(dim)CD16(pos) NK subsets was also found in tumor regions compared with non-tumor regions in the livers of these HCC patients. Both these peripheral and tumor-infiltrating NK cells exhibited poorer capacity to produce IFN-gamma and kill K562 targets, which was further found to be associated with increased CD4(+)CD25(+) T regulatory cells as we previously-described in HCC patients. Addition of Tregs from HCC patients efficiently inhibited the anti-tumor ability of autologous NK cells in vitro. These findings are helpful for understanding the mechanism of NK cell-mediated anti-tumor immune responses in HCC patients.

Hypercytolytic activity of hepatic natural killer cells correlates with liver injury in chronic hepatitis B patients.

Natural killer (NK) cells are abundant in the liver and serve as a major innate immune component against microbial infection. Although NK cells have been implicated in inducing hepatocellular damage in patients with chronic hepatitis virus infections, the roles that hepatic NK cells play in chronic hepatitis B virus (HBV) infections remain obscure. In this study, we comprehensively characterized intrahepatic and peripheral NK cells and investigated their impact on liver pathology in a cohort of HBV‐infected individuals; this cohort included 51 immune‐activated (IA) patients, 27 immune‐tolerant (IT) carriers, and 26 healthy subjects. We found that NK cells expressing NK receptors (activation receptors) preferentially accumulated in the livers of IA patients, in which they were activated and skewed toward cytolytic activity but without a concomitant increase in interferon‐γ production, in comparison with those of IT carriers and healthy subjects. Further analysis showed that the livers of IA patients, in comparison with those of IT and healthy subjects, expressed higher levels of interleukin‐12 (IL‐12), IL‐15, and IL‐18 in situ and lower levels of IL‐10, which in vitro can induce the activation and degranulation of NK cells from healthy individuals. Finally, hepatic NK cells displayed more cytolytic activity than peripheral NK cells, and this was found to be positively correlated with the liver histological activity index and serum alanine aminotransferase levels in these IA patients. Conclusion: In IA patients, hepatic NK cells are activated and preferentially skew toward cytolytic activity, which depends on an imbalanced cytokine milieu and correlates with liver injury during chronic HBV infection. (HEPATOLOGY 2011)

Severe dendritic cell perturbation is actively involved in the pathogenesis of acute-on-chronic hepatitis B liver failure

BACKGROUND/AIMS Functionally impaired dendritic cells (DCs) play important roles in suppressing host immune responses and facilitating viral persistence in chronic hepatitis B virus (HBV) infection. However, little is known regarding the status of intrahepatic DCs in HBV infection. METHODS Based on availability, 11 recipient liver samples were obtained from acute-on-chronic hepatitis B liver failure (ACHBLF) patients who had undergone liver transplantation. The frequencies, phenotypes, and functions of intrahepatic DC subsets were analyzed. RESULTS Both plasmacytoid dendritic cells (pDCs) and myeloid dendritic cells (mDCs) extensively infiltrated the liver of the ACHBLF patients and expressed mature phenotypes therein. In particular, activated hepatic pDCs produced interferon (IFN)-alpha, which subsequently induced interleukin (IL)-12 and IL-10 production via toll-like receptor-9 ligation in liver-infiltrating lymphocytes cultured in vitro. However, blockade of IFN-alpha production significantly reduced the cytokine production of the LILs. Further, a significantly low frequency of peripheral pDCs and highly reduced IFN-alpha production were observed in a large cohort of the ACHBLF patients, particularly in the non-survivors. Moreover, a persistently upregulated expression of hepatic IFN-alpha-associated genes was observed in the ACHBLF patients during disease progression. CONCLUSIONS Activated pDCs accumulated in large numbers in the liver of the ACHBLF patients and regulated local immune responses in chronic HBV infection.

Imbalanced intrahepatic cytokine expression of interferon-gamma, tumor necrosis factor-alpha, and interleukin-10 in patients with acute-on-chronic liver failure associated with hepatitis B virus infection.

Goals This study attempts to determine expressions of intrahepatic proinflammatory and anti-inflammatory cytokines and their secreting immunocytes to evaluate their roles in the pathogenesis of acute-on-chronic liver failure (ACLF) in chronically hepatitis B virus (HBV)-infected patients. Background ACLF generally affects patients with established, compensated chronic liver diseases who develop an acute deterioration in liver function. In China, HBV-associated ACLF patients account for more than 80% of ACLF patients owing to a high prevalence of chronic HBV infection. Clinical observation showed that the deterioration of this disease may correlate with host immune responses, but related underlying mechanism remains largely unknown. Study In situ expressions of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), and their secreting CD4, CD8 T cells, and Kupffer cells (KCs) were analyzed in the livers of patients with ACLF, chronic hepatitis B (CHB), and normal controls (NC) using immunohistochemistry. Results Intrahepatic proinflammatory IFN-γ and TNF-α expressions were markedly up-regulated in ACLF compared with CHB and NC. However, similar anti-inflammatory IL-10 expressions were observed in ACLF and CHB. IFN-γ overexpression correlated significantly with increased CD4 and CD8 T-cell accumulation. TNF-α up-regulation also correlated significantly with increased KCs. Conclusions The imbalanced expression of proinflammatory and anti-inflammatory cytokines and increased accumulation of CD4, CD8 T cells, and KCs may contribute to immunopathogenesis in HBV-infected ACLF.

The decrease of regulatory T cells correlates with excessive activation and apoptosis of CD8+ T cells in HIV-1-infected typical progressors, but not in long-term non-progressors

Persistent HIV infection results in a decrease in absolute counts of CD4+ CD25+ regulatory T cells (Treg). To investigate the role of decreased Treg counts in the regulation of excessive activation and apoptosis of CD8+ T cells in human immunodeficiency virus (HIV)‐1 infection, we characterized Treg in 83 HIV‐1‐infected individuals, including 19 long‐term non‐progressors (LTNPs) and 51 typical progressors (TPs) who were treatment‐naïve, and 13 AIDS patients on highly active antiretroviral therapy (HAART), of whom nine were complete responders (CRs) and the remaining four were non‐responders (NRs) to the treatment. TPs but not LTNPs had a significant decrease in absolute counts of circulating Treg, which was inversely correlated with the activation and apoptosis of CD8+ T cells. Efficient HAART was found to increase Treg counts in CR patients and temper the excessive activation and apoptosis of CD8+ T cells. Moreover, isolated Treg significantly inhibited the spontaneous and anti‐CD3‐induced apoptosis of CD8+ T cells in a dose‐dependent manner in vitro. Thus, our findings indicate that the decrease in Treg closely correlates with the increase in apoptotic CD8+ T cells and disease progression in chronic HIV‐1 infection, and that Treg may play a key role in maintaining the balance between the amount and quality of CD8+ T cells in HIV‐1 infection. Manipulation of Treg function may be a promising strategy for immune therapy of this disease.

A pilot study of umbilical cord‐derived mesenchymal stem cell transfusion in patients with primary biliary cirrhosis

BACKGROUND AND AIM Ursodeoxycholic acid (UDCA) treatment is an effective medical therapy for patients with primary biliary cirrhosis (PBC); however, 40% of PBC patients show an incomplete response to the UDCA therapy. This study aimed to investigate the safety and efficacy of umbilical cord-derived mesenchymal stem cell (UC-MSC) transfusion in PBC patients with an incomplete response to UDCA. METHODS We conducted a single-arm trial that included seven PBC patients with a suboptimal response to UDCA treatment. UC-MSCs were first cultured, and then 0.5 × 10(6) cells/kg body weights were infused through a peripheral vein. UC-MSCs were given three times at 4-week intervals, and patients were followed up for 48 weeks. Primary outcomes were to evaluate the safety and feasibility of UC-MSC treatment, and secondary outcomes were to evaluate liver functions and patients quality of life. RESULTS No obvious side-effects were found in the patients treated with UC-MSCs. Symptoms such as fatigue and pruritus were obviously alleviated in most patients after UC-MSC treatment. There was a significant decrease in serum alkaline phosphatase and γ-glutamyltransferase levels at the end of the follow-up period as compared with baseline. No significant changes were observed in serum alanine aminotransferase, aspartate aminotransferase, total bilirubin, albumin, prothrombin time activity, international normalized ratio, or immunoglobulin M levels. The Mayo risk score, a prognostic index, was also stable during the treatment and follow-up period. CONCLUSIONS UC-MSC transfusion is feasible and well tolerated in patients with PBC who respond only partially to UDCA treatment, thus representing a novel therapeutic approach for patients in this subgroup. A larger, randomized controlled cohort study is warranted to confirm the clinical efficacy of UC-MSC transfusion.