Research paper
Induction of inducible CD4+CD25+Foxp3+ regulatory T lymphocytes by porcine reproductive and respiratory syndrome virus (PRRSV)

https://doi.org/10.1016/j.vetimm.2009.07.012Get rights and content

Abstract

Increases in numbers or activities of regulatory T lymphocytes (Tregs) have been linked to the establishments of several persistent infections. It has been previously shown that porcine reproductive and respiratory syndrome virus (PRRSV) can negatively modulate the host immune responses, resulting in persistent infection and secondary immunodeficiency. Recently, the existence of porcine CD4+CD25+ Tregs has been demonstrated. We investigated the effect of PRRSV on the CD4+CD25+ Tregs. The CD4+CD25+Foxp3+ T lymphocytes in the peripheral blood mononuclear cells (PBMCs) were identified, using the anti-human anti-Foxp3 monoclonal antibody. In vitro culture of porcine PBMC in the presence of PRRSV, but not classical swine fever virus, significantly increased the numbers of Foxp3+ lymphocytes, particularly in the CD4+CD25high subpopulation. The time-course study revealed that PRRSV significantly increased the numbers of viral-specific CD4+CD25highFoxp3+ subpopulation in the culture starting from 12 h through the end of the observation period. Consistent to the results obtained by flow cytometry, enhanced Foxp3 gene expression was observed in the PBMC cultured with PRRSV in a time-course manner. The presence of monocyte-derived DC in the co-culture significantly enhanced the induction of CD4+CD25+ Foxp3+ T lymphocytes. The PRRSV-induced CD4+CD25high T lymphocytes exhibited suppressive activity when co-cultured with PHA-activated, autologous peripheral blood leukocytes, indicating the suppressive activity of the PRRSV-specific Tregs. In addition, PRRSV exposure significantly increased the numbers of PRRSV-specific CD4+CD25+Foxp3+ subpopulation in the PBMC of infected pigs at 10 days post-infection. In summary, the results indicated that PRRSV could increase the numbers of viral-specific, inducible regulatory T lymphocytes in the porcine PBMC, both in vitro and in vivo. The findings suggested the novel immunomodulatory mechanism induced by PRRSV.

Introduction

Over the past few years, numerous lines of evidence from human and mouse models indicate that CD4+CD25+Foxp3+ regulatory T cells (Tregs) are crucial for establishing self-tolerance, controlling inflammatory activities and maintaining host homeostasis (Sakaguchi, 2004, Shevach et al., 2006). It is believed that Tregs play an important role in limiting immunopathology caused by the activation of the pathogen-specific effector T cells (Bacchetta et al., 2007, Baecher-Allan and Hafler, 2006, Belkaid, 2007, Belkaid et al., 2006). Interestingly, several types of pathogens preferentially induce Tregs to benefit their chance of survival within the infected hosts (Belkaid, 2007). The role of Tregs has been implicated in several persistent or chronic viral infections in humans, as increased Tregs numbers have been observed in several chronic infected subjects, including human immunodeficiency virus, hepatitis C virus and human cytomegalovirus infections (Humphreys et al., 2007, Rehermann, 2007, Rouse et al., 2006, Suvas and Rouse, 2006). In some cases, aberrant function of Tregs can lead to generalized immunosuppression, resulting in an inability of the host to clear infections, interference with vaccine efficacy, secondary complications and reduced resistance to tumors (Vahlenkamp et al., 2005).

Since its emergence in the late 1980s, porcine reproductive and respiratory syndrome virus (PRRSV) has been recognized worldwide as one of the most economically important pathogens of swine. PRRSV, an enveloped positive-stranded RNA virus, is a member of the family Arteriviridae in the order Nidovirales (Cavanagh, 1997). The major characteristics of porcine reproductive and respiratory syndrome (PRRS) include the reproductive failure of sows and respiratory disease in pigs of all ages. Several studies suggest that PRRSV negatively modulates the host immune responses. PRRSV-infected pigs usually demonstrate prolonged viraemia and persistent infection (Murtaugh et al., 2002). In addition, weak innate immunity followed by delayed and inefficient specific immunity is usually observed following PRRSV infection (Lopez and Osorio, 2004, Meier et al., 2003). PRRSV infection weakens respiratory defense mechanisms, leading to secondary complications, which are known as the porcine respiratory disease complex (Halbur, 1998). Moreover, several reports indicate that active infections with PRRSV confer a negative impact on the host immune system that leads to inadequate response to vaccination (De Bruin et al., 2000, Li and Yang, 2003, Suradhat et al., 2006, Thacker et al., 2000). We postulated that the observed immunomodulatory role of PRRSV might partly relate to the induction of Tregs activities within the infected pigs.

In humans and mice, the CD4+CD25+ Tregs represent between 5 and 10% of the CD4+ T cell subset in the peripheral blood (Baecher-Allan et al., 2004). It has been well accepted that the Forkhead family transcriptional factor; Foxp3 is the best marker for identifying the CD4+CD25+ Treg population (Wang and Wang, 2007, Yi et al., 2006). Foxp3 is constitutively expressed at high levels in both natural and adaptive CD4+CD25+ Tregs in both humans and mice (Ziegler, 2006). Furthermore, Foxp3 also serves as a master regulator for the development and functions of Tregs (Hori et al., 2003, Hori and Sakaguchi, 2004). Although, Foxp3 genes, with a high degree of sequence homology, have been identified in many placental vertebrates, including monkeys and several domestic species (Aluvihare and Betz, 2006), there has been limited information regarding the existence and functions of Tregs in other species apart from humans and mice. Nevertheless, the role of CD4+CD25+Foxp3+ Tregs has recently been reported in macaques (Hartigan-O’Connor et al., 2007) and chimpanzees (Manigold et al., 2006). In addition, there are reports describing the CD4+CD25+ T cells with Tregs-like activities in cats (Joshi et al., 2004, Joshi et al., 2005, Vahlenkamp et al., 2004), and dogs (Biller et al., 2007). In pigs, CD4+CD25high regulatory T cells with a strong Foxp3 gene expression and suppressive capacity have recently been reported, suggesting the existence of porcine Tregs (Käser et al., 2008b). Characterization of porcine CD4+CD25+Foxp3+ T lymphocytes, using rat anti-mouse/rat Foxp3 antibody, has been recently reported (Käser et al., 2008a). As previously reported information suggests the possibility of Treg induction by PRRSV, we investigated the in vitro and in vivo effects of PRRSV on the induction of CD4+CD25+Foxp3+ lymphocyte subpopulation.

Section snippets

Viruses and cells

The US genotype, Thai PRRSV strain 01NP1 (passage no. 15), isolated from PRRSV-infected pigs in Thailand (Thanawongnuwech et al., 2004), was provided by The Veterinary Diagnostic Laboratory, The Faculty of Veterinary Science, Chulalongkorn University. The virus was cultured and titrated in MARC-145 cells as previously described (Thanawongnuwech et al., 1998), and stored at −80 °C until needed.

The classical swine fever virus (CSFV) reference strain; ALD was a gift from the National Institute of

In vitro effect of PRRSV on porcine CD4+CD25+Foxp3+ lymphocytes

The effect of PRRSV on porcine Foxp3+ lymphocytes was investigated following the 48 h in vitro culture. The results revealed that PRRSV significantly increased the number of CD4+CD25+Foxp3+ lymphocytes in the cultured PBMC (Fig. 1d). The induction of the Foxp3+ lymphocyte subpopulation was unique to PRRSV, since the enhancement was not observed in the PBMC cultured with the other RNA virus, CSFV (Fig. 2). It should be noted that the increased Foxp3+ lymphocytes were observed in both the PBMC,

Discussion

In humans and mice, CD4, CD25 and the transcriptional factor; Foxp3 are the important markers for the identification of Tregs (Baecher-Allan et al., 2004, Belkaid et al., 2006, Jiang et al., 2006, Shevach et al., 2006). Several species, including pigs, have been shown to have Foxp3 gene in their genomes, with a high degree of sequence homology (Aluvihare and Betz, 2006). Recently, the existence of porcine Tregs, characterized by strong Foxp3 gene expression, moderate interleukin-10 (IL-10)

Acknowledgements

The authors are grateful to Drs. J.A. Roth and R. Platt (Iowa State University, Ames, IA, USA) for providing the custom-conjugated mAbs and their critical review of the manuscript, Dr. K. Ruxrungtham, and Dr. P. Hansasuta (The Faculty of Medicine, Chulalongkorn University) for providing the reagents and facilities for cellular sorting and flow cytometric analyses, the staffs of the Department of Veterinary Pathology and the Veterinary Diagnostic Laboratory, Faculty of Veterinary Science,

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