Elsevier

Journal of Hepatology

Volume 52, Issue 3, March 2010, Pages 315-321
Journal of Hepatology

Research Article
Abundant numbers of regulatory T cells localize to the liver of chronic hepatitis C infected patients and limit the extent of fibrosis

https://doi.org/10.1016/j.jhep.2009.12.013Get rights and content

Background & Aims

Weak hepatitis C virus (HCV) specific immunity in peripheral blood has been shown to be partially controlled by regulatory T cells (Treg). However, little is known about Treg present in livers of HCV-infected patients, their association with clinical parameters, and immunopathology resulting in disease progression.

Methods

The frequency and phenotype of CD4+FoxP3+ Treg, conventional CD4+ T cells, and the distribution of lymphocytes and leukocytes were studied by multi-color flowcytometry in liver and peripheral blood of 43 chronic HCV patients at different phases of liver disease. Comparisons between healthy blood and liver and correlations with disease parameters were made.

Results

An extensive lymphocyte infiltration containing abundant numbers of CD4+FoxP3+ Treg was present in HCV-infected livers, while absent from healthy liver. Moreover, in all patients, intrahepatic CD4+FoxP3+ Treg showed a fully differentiated and highly activated phenotype on the basis of the surface markers CD45RO, CCR7, CTLA-4 and HLA-DR. These Treg were more numerous in those HCV-infected livers showing only limited fibrosis. However, HCV RNA loads or alanine transaminase levels did not correlate with CD4+FoxP3+ Treg frequencies.

Conclusions

Our data demonstrate that large numbers of highly activated and differentiated CD4+FoxP3+ Treg localize to the infiltrated chronic HCV-infected liver and may result in limiting the extent of fibrosis. This suggests that CD4+FoxP3+ Treg play a pivotal role in limiting collateral damage by suppressing excessive HCV-induced immune activation.

Introduction

Following infection with the hepatitis C virus (HCV), immunity fails to successfully eradicate the virus in the majority of individuals [1], [2], [3]. As a consequence, an estimated 120–170 million individuals are currently chronically infected worldwide [4]. Due to ongoing immunopathology, these patients are at increased risk of developing cirrhosis, and subsequent liver decompensation and/or hepatocellular carcinoma. However, without accelerating factors, such as co-infections and co-morbidities, disease progression is slow and it typically takes over a decade before serious health problems occur.

Patients chronically infected with HCV generally show a weak peripheral blood T cell response against HCV, which is insufficient to eradicate the virus [5], [6], [7], [8]. It has been convincingly shown that peripheral blood regulatory T cells (Treg) from HCV-infected patients suppress both HCV-specific T cell proliferation and IFN-gamma production [9], [10], [11], [12], [13]. Thus, these cells may hamper the immune response against HCV during chronic infection, although other mechanisms have been proposed as well (reviewed in [1], [2], [3], [14], [15]). The role of intrahepatic Treg may be especially important to understand the chronic nature of the disease, since HCV predominantly infects hepatocytes. Although Treg have been detected before in livers of chronic HCV patients by performing immunohistochemical stainings [16], [17], [18], [19], data on the phenotype of Treg at the primary site of infection is still lacking and their role in immunopathology remains unclear.

Regulation of the magnitude of the effector response may result in failure to eliminate the pathogen, and thus in the case of HCV infection, may lead to the establishment of a persistent infection. It has been demonstrated in many experimental infections that Treg act by dampening excessive inflammatory responses, and consequently help to limit tissue damage associated with the inflammatory reaction (reviewed in [20]). In chronic HCV infections, a higher suppressive capacity of peripheral blood Treg was observed in patients showing a relatively low level of hepatocyte death, as reflected by alanine transaminase (ALT) levels [10]. However, Treg have not yet been implicated in controlling the outcome of immunopathology, i.e. liver fibrosis.

In this study, we characterized intrahepatic Treg in chronic HCV patients and their impact on disease progression. An extensive lymphocyte infiltration containing abundant numbers of CD4+FoxP3+ Treg was present in HCV-infected livers, while absent from the healthy liver. Moreover, these intrahepatic CD4+FoxP3+ Treg of patients at diverse stages of liver disease showed a fully differentiated and highly activated phenotype, and were more numerous in HCV-infected livers with mild fibrosis, suggesting an important role for intrahepatic Treg during chronic HCV infection.

Section snippets

Patients and healthy controls

Intrahepatic cells were obtained from 43 chronic HCV-infected patients (Table 1 for clinical characteristics) by fine needle aspiration biopsy (FNAB; n = 28) or percutaneous core needle biopsy (n = 15). Paired venous blood samples were collected from all patients. All patients had detectable HCV RNA levels in serum. Patients co-infected with human immunodeficiency virus or hepatitis B virus were excluded from the study. Diagnostic core biopsy specimens from all 43 patients, obtained within 3 months

Extensive inflammation of the liver is observed in patients with chronic HCV infections

To determine the degree and nature of inflammation in the liver of patients with chronic HCV infections, flow cytometric analyses were performed. As expected, vast numbers of CD45-expressing inflammatory cells were detected in the liver of patients with chronic HCV infections (Fig. 1). The degree of inflammation, defined as the fraction of CD45-positive leukocytes out of the total number of liver cells [22], was on average 15% for HCV-infected livers whereas in control livers, only an average

Discussion

In patients chronically infected with HCV, the virus replicates at high levels within the liver for decades. In order to maintain this viral persistence, regulatory mechanisms are in place that balance an ineffective protective HCV-specific immunity and mild immune-mediated liver damage. The present study shows that high numbers of CD4+FoxP3+ Treg accumulate in the HCV-infected liver. These CD4+FoxP3+ Treg display a highly activated and differentiated effector/memory phenotype and may be

Acknowledgements

The authors who have taken part in this study state that they do not have anything to declare regarding funding from industry or conflict of interest with respect to this manuscript. This work was supported by ZonMW (VIDI-Grant 917.56.329) for H.J. The authors thank Janneke Samsom and Jaap Kwekkeboom for helpful discussions and Duygu Turgut for excellent technical assistance.

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