Intraperitoneal Echinococcus multilocularis infection in mice modulates peritoneal CD4+ and CD8+ regulatory T cell development

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Abstract

Intraperitoneal proliferation of the metacestode stage of Echinococcus multilocularis in experimentally infected mice is followed by an impaired host immune response favoring parasite survival. We here demonstrate that infection in chronically infected mice was associated with a 3-fold increase of the percentages of CD4+ and CD8+ peritoneal T (pT) cells compared to uninfected controls. pT cells of infected mice expressed high levels of IL-4 mRNA, while only low amounts of IFN-γ mRNA were detected, suggesting that a Th2-biased immune response predominated the late stage of disease. Peritoneal dendritic cells from infected mice (AE-pDCs) expressed high levels of TGF-β mRNA and very low levels of IL-10 and IL-12 (p40) mRNA, and the expression of surface markers for DC-maturation such as MHC class II (Ia) molecules, CD80, CD86 and CD40 was down-regulated. In contrast to pDCs from non-infected mice, AE-pDCs did not enhance Concanavalin A (ConA)-induced proliferation when added to CD4+ pT and CD8+ pT cells of infected and non-infected mice, respectively. In addition, in the presence of a constant number of pDCs from non-infected mice, the proliferation of CD4+ pT cells obtained from infected animals to stimulation with ConA was lower when compared to the responses of CD4+ pT cells obtained from non-infected mice. This indicated that regulatory T cells (Treg) may interfere in the complex immunological host response to infection. Indeed, a subpopulation of regulatory CD4+ CD25+ pT cells isolated from E. multilocularis-infected mice reduced ConA-driven proliferation of CD4+ pT cells. The high expression levels of Foxp3 mRNA by CD4+ and CD8+ pT cells suggested that subpopulations of regulatory CD4+ Foxp3+ and CD8+ Foxp3+ T cells were involved in modulating the immune responses within the peritoneal cavity of E. multilocularis-infected mice.

Introduction

Human alveolar echinococcosis (AE) is a hepatic parasitic disease that is largely characterized by uncontrolled proliferation of the larval stage (metacestode) of the fox tapeworm Echinococcus multilocularis. Besides small rodents, which act as natural intermediate hosts, humans may accidentally acquire infection by ingestion of parasite eggs that have been shed with the feces of definitive hosts such as foxes or dogs. The increasing rural and urban fox population intensities especially in Central Europe [1], [2] resulted not only in an increased environmental contamination with E. multilocularis eggs, but also led to an increase of annual incidence of AE such as recently reported in Switzerland [3], [4]. Mice, being the natural intermediate hosts, represent an excellent model to study the pathogenesis of AE and characteristics of the respective host–parasite interplay. Experimental infection, leading to secondary AE, is performed by inoculation of E. multilocularis metacestode vesicles into the peritoneal cavity. These metacestodes have already formed, and are protected by, the surface-associated and carbohydrate-rich laminated layer (LL). Immunocompetent mice subsequently react parasite-specifically i.e. by producing a largely T cell-independent humoral immune response to the major carbohydrate antigen Em2, which is associated with the LL [5], [6]. In addition, a relatively inefficient cell-mediated immune response is mounted, which appears to impair, but does not inhibit, the proliferation of the developing metacestode [7]. Thus, immunosuppression is the hallmark of intraperitoneal infection with E. multilocularis metacestodes, and this process needs to be addressed to further understand the biology of AE.

Surface molecules as well as excretory/secretory (E/S) metabolic products of metacestodes are considered to be important key players in the host–parasite interplay [8] of many helminthiasis, as they are implicated in immune evasion strategies through mechanisms including shedding surface-bound ligands of cells, modulation of host inflammatory responses and alteration of lymphocyte, macrophage and granulocyte functions [8]. In human AE, studies on peripheral blood mononuclear cells (PBMC) of AE patients showed that E. multilocularis antigens modulate both regulatory and inflammatory cytokine and chemokine expression [9]. In the mouse model for secondary AE, studies on spleen cells showed that the initial expression of Th1-oriented IFN-γ and IL-2 cytokines observed early in the infection were, at later stages of infection, gradually replaced by IL-4 and IL-5 cytokines of the Th2 lineage. This indicated a gradual Th2 shift during chronic E. multilocularis infection (reviewed in [6], [7], [10]). We therefore hypothesize that the proliferating metacestode itself specifically activates and concurrently modulates the murine immune response within the peritoneal cavity, the site of infection.

In the present work, we addressed the role of the first line of the immune response, by investigating antigen-presenting dendritic cells (DCs) and regulatory subsets of T cells that may develop in the peritoneal cavity of E. multilocularis-infected mice. DCs originate from hematopoietic precursors in the bone marrow, and migrate to the periphery where they reside as immature cells. They subsequently mature in the presence of exogenous stimuli and proinflammatory cytokines such as IL-12, IL-6 and TNF-α, or by interacting with CD40L-expressing T cells [11], [12]. During an infectious and/or inflammatory event, immature DCs capture and process foreign antigens and then differentiate into mature DCs. Mature DCs are characterized by expression of high levels of MHC II co-stimulatory molecules such as CD40, CD80, and CD86 as well as by the production of large amounts of the Th1 cytokine IL-12 [13].

The maturation of DCs following the cascade of events described above is somehow dogmatic because it involves mostly viral, bacterial and protozoan stimuli and concerns the essential role of DCs in Th1 and/or TH17 cell-mediated immunity. For the role of DCs in Th2 differentiation, such as in the case of helminthiases, there have been no mirror image signatures of cytokine or characteristic surface ligands described so far [14]. However, several helminthic infections have been shown to generate production of T reg cells, which synthesize down-regulatory cytokines (IL-10 and TGF-β) that switch off inflammatory and putatively protective immune responses, and interfere with effector T cell activation in a contact-dependent manner [16]. It is therefore conceivable, that in experimental murine AE the intraperitoneal immunological microenvironment is also characterized by the development of humoral and cellular components that mediate immunosuppression, such as T reg cells. In turn, these might be implicated in the reduction of inflammatory responses as earlier reported (reviewed in [7], [17], [18]). Jenne et al. [15] showed that DCs lack in vitro maturation upon exposure to unfractionated crude E. multilocularis antigen. In addition, while DCs were isolated from the peritoneum of mice experimentally infected with E. multilocularis metacestodes, their phenotype and their stimulatory role remained unclear.

The purpose of this study was to investigate the role of DCs in the complex events that orchestrate the immune response during secondary infection of mice with E. multilocularis metacestodes. First, this was addressed by assessing the level of peritoneal maturation and functional properties of DCs in E. multilocularis-infected mice (AE-pDCs) in comparison to non-infected control mice. This included determination of the relative cytokine expression levels of TGF-β, IL-10 and IL-12, and the expression levels of co-stimulatory molecules, and studies on the capacity of AE-pDCs to enhance Con A-driven proliferation of different populations of peritoneal T (pT) cells. Secondly, the expression profiles of selected Th2 cytokines such as TGF-β and IL-10, and Th1 cytokines including IL-2, IL-4 and IFN-γ in pT cells of mice infected with E. multilocularis (AE-pT cells) were analyzed. Third, we investigated whether T reg cells develop within the peritoneal cavity during secondary AE. In this context, expression levels of Foxp3, a transcription factor that drives T reg cell development [19], were determined in peritoneal CD4+ and CD8+ T cells of infected and non-infected mice.

Section snippets

Mice

Female 6–10 week-old C57BL/6 mice purchased from Charles River GmbH, Germany, were used for intraperitoneal infection with E. multilocularis as previously described [17], [18]; littermates were used as mock-infected control mice. All mice were raised, housed and handled according to the rules of the Swiss regulations for animal experimentation.

Parasite and experimental infection

The parasite used in this study was a cloned E. multilocularis (KF5) isolate maintained by serial passages (vegetative transfer) in C57BL/6 mice [20].

mRNA expression of TGF-β, IL-10 and IL-12 (p40) in (AE-pDCs)

pDCs obtained from non-infected mice and from mice at the early and late chronic stage of secondary AE were analyzed for respective gene expression levels of selected cytokines by quantitative RT-PCR. Fig. 1 shows that AE-pDCs expressed substantially increased levels of TGF-β mRNA, while IL-10 and IL-12 (p40) mRNA expression remained at the levels of non-infected mice.

Secondary AE impairs AE-pDC surface expression of MHC class II (Ia) molecules and accessory molecules (CD80), (CD86) and CD40

Flow cytometry analysis showed that the surface expression of MHC class II molecules (Ia) was down-regulated in AE-pDCs in

Discussion

Previous experiments had shown earlier that cellular immunity induced by a Th1 cytokine secretion profile provided some means of control of metacestode development and/or proliferation at the initial stage of infection in relatively resistant hosts [24]. On the other hand, a Th2-oriented immune response, which occurred at the later stage of infection, resulted in more rapid metacestode growth. The control of metacestode proliferation appeared to be predominantly T cell dependent, as revealed by

Acknowledgements

Markus Spiliotis (Institute of Parasitology, University of Bern) is greatly acknowledged for his helpful comments and discussion. This work was supported by the Swiss National Science Foundation (grant no. 3100A0-111780).

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