Effect of cysteine peptidase inhibitor of Eudiplozoon nipponicum (Monogenea) on cytokine expression of macrophages in vitro

Highlights

• Recombinant EnStef downregulates the expression of TNF-α and IL-10 by macrophages.

• rEnStef affects activity of cells derived from non-fish species.

• rEnStef is the first monogenean molecule tested on in vitro cell cultures.

Abstract

The gills of the common carp, whose mucosal surface belongs to the key defence mechanisms of piscine immunity, can be infested with both the larval and adult stage of Eudiplozoon nipponicum (Monogenea). Although on their own, monogeneans do not considerably compromise their hosts’ health status, fish with epithelial barriers damaged in parasite feeding and attachment sites are at an increased risk of bacterial challenge with possible harmful consequences. Several studies suggest that helminth parasites of teleost fish evade and manipulate host immune system via their excretory-secretory products, but our knowledge of these processes in the monogeneans is limited. Cysteine peptidase inhibitors (CPI), which are found in the secretions of numerous parasites, often induce immunosuppression by subverting Th1 mechanisms and drawing the immune system towards a Th2/Treg response. We employed the qPCR to test the effect of recently characterised CPI of E. nipponicum (rEnStef) on the mRNA expression of pro-inflammatory cytokine TNF-α and anti-inflammatory cytokine IL-10 produced by porcine macrophages in vitro. After an initial preincubation with rEnStef, we stimulated the macrophages using LPS. By inducing a Th1 pro-inflammatory response, we imitated the immune reaction during a bacterial challenge in tissue damaged by the feeding and attachment of E. nipponicum. We observed a significant dose-dependent downregulation of the expression of TNF-α and IL-10 cytokines. The observed suppression of TNF-alpha expression by rEnStef could result in decreased pathogen control, which might in turn lead to increased rates of secondary bacterial infections in fish infected by E. nipponicum.

Introduction

Monoxenous blood-feeding flatworms of the Diplozoidae family (Monogenea: Polyopisthocotylea) are obligatory ectoparasites of cyprinid fish. Diplozoids, such as Eudiplozoon nipponicum Goto, 1891, stand out by their peculiar mating strategy where two larval individuals (diporpae) during maturation fuse and form an X-like structure [1]. Unlike infectious bacteria and protists, which usually cause acute infections, helminths have a remarkable ability to circumvent host immune system and reduce pathology. By recruiting a vast range of immunomodulatory excretory-secretory products, such as inhibitors of cysteine proteases (CPIs), they induce hyporesponsiveness of the immune system to the parasite’s or bystander antigens. The hallmark of most helminth infections is an induction of a modified Th2/Treg immune response as a defence against inflammatory reaction of the host. On the cytokine level, it is characterised by a downregulation of pro-inflammatory cytokines (e.g. INFγ, IL-1, TNF-α) and upregulation of anti-inflammatory cytokines (mainly IL-10 and TGF-β) [2]. A downregulation of Th1 pro-inflammatory response impairs host’s defence against bacteria and viruses, which in case of monogenean-infested fish can possibly lead to an increased microbial burden.

The dynamics of immune reaction during fish infection by ectoparasitic Monogenea seems to deviate from this pattern. A generally strong pro-inflammatory defence, characterised by the induction of TLRs and downstream Th1 cytokines TNF-α, ß and IL-1ß, has been reported in both natural and experimental infection [[3], [4], [5], [6]]. Nevertheless, research focused on the role of particular molecules of parasite origin in the abovementioned process is missing.

CPIs belong to parasite-derived modulators which facilitate and maintain hosts’ tolerance of parasites and enable their long-term and often asymptomatic coexistence [2]. Recently, we characterised a type I cysteine peptidase inhibitor (stefin) secreted by E. nipponicum, which has the extraordinary ability to inhibit not only papain-like proteases (cathepsin B, L) but also asparaginyl endopeptidases (legumains) [7]. This ability was confirmed also in type II cysteine peptidases from several parasitic nematodes. These molecules have been intensively studied and numerous reports confirm their role in the suppression of host immune system, e.g. by interfering with the MHCII pathway of antigen processing, maintaining the dendritic cells in immature state, regulating the IL-10 production of PBMC, supressing T cell proliferation, or by recruiting IL-10 secreting macrophages (reviewed in Klotz et al. [8]). It has been demonstrated for cystatin produced by Acanthocheilonema vitae that after being taken up by host macrophages, it induces a phosphorylation of mitogen-activated protein kinases (MAPK) ERK1/2 and p38 and thus triggers the expression of IL-10 [9]. The IL-10 is then responsible for subsequent inhibition of antigen-specific proliferation of T cells and other downstream anti-inflammatory processes.

Our goal was to perform a preliminary study of immunomodulatory properties of recombinant stefin of E. nipponicum (rEnStef) in vitro, while focusing on the expression of cytokines TNF-α and IL-10. We have previously demonstrated that rEnStef can inhibit papain-like proteases and asparaginyl endoproteases of different origin (e.g. mouse, Ixodes ricinus) [7]. By using LPS-stimulated porcine alveolar macrophages (PAMs), we mimicked the conditions of bacterial infections which often accompany monogenean infestation. The present study is the first attempt to assess the effect of monogenean-secreted protein on cytokine gene expression of immune cells in vitro.