Research paper
The response of monocyte derived dendritic cells following exposure to a nematode larval carbohydrate antigen

https://doi.org/10.1016/j.vetimm.2012.06.024Get rights and content

Abstract

The glycolipid CarLA (carbohydrate larval antigen) is present on the epicuticle of the infective-stage larvae of gastrointestinal nematode parasites infecting livestock. The molecule is lost from the surface of the larvae in the few days post-ingestion by a host animal, and the resulting anti-CarLA antibody response has been demonstrated to be protective in vivo. Both the anti-CarLA response, and anti-parasite immunity in general, are slow to develop, and several months of natural exposure to ingested larvae is required. The current study was designed to provide information on how the anti-CarLA response develops, and focuses on the initial recognition of the molecule by human monocyte derived dendritic cells (mdDC) in vitro.

Immunofluorescence and flow cytometry demonstrated that mdDC recognise and internalise both the purified and the native form of CarLA, in the case of the latter once it is shed from the larval surface. However, the recognition of CarLA did not result in classical maturation of DC, while there was only transient or minor up-regulation of CD86, CD83, HLA-DR and CD40.

Exposure of mdDC to purified CarLA resulted in the increased production of the pro-inflammatory cytokines IL-6 and to a lesser extent of IL-8 and TNF-α, and a reduced production of the anti-inflammatory cytokine IL-1RA. CarLA therefore has little ability to mature and functionally alter monocyte derived dendritic cell function.

Introduction

Long-term chronic infection is characteristic of many parasitic helminth infections in mammals. This can be partly attributed to an ability of the worm to evade the host immune system, through such strategies as migration, surface shedding, antigenic variation, anti-oxidant enzyme production and/or immunomodulation (Behnke et al., 1992, Maizels et al., 2004). In the case of the latter, a range of helminth glycans has been identified as immunomodulators during infection (Harn et al., 2009, Kuijk and van Die, 2010, Van Die and Cummings, 2006, van Die and Cummings, 2010).

Previous studies in this laboratory have identified a glycolipid antigen (CarLA) on the surface of infectious-stage larvae (L3) of Trichostrongylid nematodes which infect grazing livestock (Harrison et al., 2003b). While the structure of the CarLA molecule remains unknown, preliminary biochemical analysis detected no amino acids but found evidence for the presence of sugars, amino sugars and fatty acids (Maass et al., 2009), constituents which it shares with many other parasite glycans (Harn et al., 2009).

CarLA appears to have diverse and, occasionally, opposing effects in the host/parasite relationship. For example, the presence of several distinct antigenic forms were proposed to contribute to immune evasion by worms (Maass et al., 2009). By contrast, antibodies expressed against CarLA have been shown to contribute to protective mucosal antibody responses in sheep (Harrison et al., 2003a, Harrison et al., 2008, Maass et al., 2007). This suggested that purified CarLA may be a suitable anti-nematode vaccine candidate, as epitopes were conserved between the native (as it occurs on larvae) and the purified form of the antigen. However, the use of purified CarLA in vaccination studies failed to induce either mucosal or serum antibody responses (Harrison, personal communication). The diverse role of CarLA in the host/parasite interaction, as well as the discrepancy between native and purified CarLA in the ability to induce protective antibody responses in vivo, highlight the need to investigate the fundamental mechanisms that underlie the recognition of CarLA by the immune system.

The present study was designed to study whether CarLA is recognised by DC, key sentinel cells whose roles include recognition and processing of self and non-self antigens, including those elicited during parasite infections (Everts et al., 2010, Mendlovic and Flisser, 2010). It is well established, however, that exposure of DC to parasite antigens, including those derived from helminths, can result in a limited maturation of these cells (Carvalho et al., 2009, Segura et al., 2007), and this effect can be mediated by carbohydrate recognition receptors (Kuijk and van Die, 2010).

Given the central role for DC in parasite glycan recognition, and the involvement of CarLA in anti-parasite immunity, the interaction between DC and CarLA was investigated using a combination of immunofluorescence microscopy, flow cytometry and cytokine-specific assays.

Section snippets

Experimental design

A set of experiments was designed to examine if mdDC recognise and internalise the purified or native nematode carbohydrate antigen CarLA, and whether or not the recognition of CarLA leads to maturation and changes in functional properties of DC. Immature human day 7 mdDC were either exposed to purified CarLA or incubated with live, exsheathed L3 of the nematode parasite Trichostrongylus colubriformis (TcL3). The response of mdDC to exposure to either purified CarLA or TcL3 was evaluated by

MdDC recognise CarLA

Flow cytometry revealed that the nematode glycan CarLA is recognised by immature mdDC. Surface binding of purified CarLA was evident as early as 1 h after exposure, with levels increasing after 4 h (in 3 out of 4 experiments; Fig. 1); internalisation was detectable as early as after 4 h of exposure (in 3 out of 4 experiments; Fig. 1) with detection in all experiments and fluorescence intensity increasing after 8 h (data not shown).

Recognition of native CarLA by mdDC was evident after incubation

Discussion

The current study describes the effect of the nematode-derived glycan antigen CarLA on immature human mdDC, and demonstrates that the antigen was recognised and internalised by a subset of cells. This was followed by limited maturation and minor changes in the expression of a number of co-stimulatory molecules. Broadly similar results were observed following exposure to either the native or purified molecule. We employed a well established human DC culture system that enabled the

Conflict of interest

The authors declare that there is no conflict of interest.

Acknowledgments

We are grateful to Dr. John Koolard for performing the statistical analysis, to Richard Shaw for providing the monoclonal antibody against CarLA, to Sheralee Cleland for providing the infective larvae, to Emma Ringqvist for performing the CD209 and CD206 binding ELISAs, to Natalie Parlane and Richard Shaw for reviewing the manuscript, and to the Ministry of Science and Innovation (MSI; contract C10X1004) for funding this programme.

References (39)

  • L. Carvalho et al.

    Review series on helminths, immune modulation and the hygiene hypothesis: mechanisms underlying helminth modulation of dendritic cell function

    Immunology

    (2009)
  • M. Eberl et al.

    Antibodies to glycans dominate the host response to schistosome larvae and eggs: is their role protective or subversive?

    J. Infect. Dis.

    (2001)
  • B. Everts et al.

    Helminths and dendritic cells: sensing and regulating via pattern recognition receptors, Th2 and Treg responses

    Eur. J. Immunol.

    (2010)
  • C.G. Figdor et al.

    C-type lectin receptors on dendritic cells and langerhans cells

    Nat. Rev. Immunol.

    (2002)
  • J.J. García-Vallejo et al.

    Endogenous ligands for C-type lectin receptors: the true regulators of immune homeostasis

    Immunol. Rev.

    (2009)
  • P. Geldhof et al.

    Presence of the LDNF glycan on the host-protective H-gal-GP fraction from Haemonchus contortus

    Parasite Immunol.

    (2005)
  • L. Gómez-García et al.

    Carbohydrate components of Taenia crassiceps metacestodes display Th2-adjuvant and anti-inflammatory properties when co-injected with bystander antigen

    Parasitol. Res.

    (2006)
  • D.A. Harn et al.

    Modulation of host immune responses by helminth glycans

    Immunol. Rev.

    (2009)
  • G.B. Harrison et al.

    Antibodies to surface epitopes of the carbohydrate larval antigen CarLA are associated with passive protection in strongylid nematode challenge infections

    Parasite Immunol.

    (2008)
  • Cited by (5)

    • Efferent intestinal lymph protein responses in nematode-resistant, -resilient and -susceptible lambs under challenge with Trichostrongylus colubriformis

      2014, Journal of Proteomics
      Citation Excerpt :

      The ability to limit nematode infection also involves the innate immune response, although the mode of action is not entirely understood. Several antigens displayed by T. colubriformis have been characterized but their direct involvement in acquired immunity or resistance to parasite infection has only been demonstrated for the carbohydrate larval allergen CarLA [14,15]. Nonetheless, ovine afferent lymph contains a diverse repertoire of antigen presenting cells (APCs) including dendritic cells (DC), macrophages and monocytes amongst other leukocytes [16,17].

    • The stimulatory effect of different CpG oligonucleotides on the maturation of chicken bone marrow-derived dendritic cells

      2014, Poultry Science
      Citation Excerpt :

      There are several different methods which are often used to generate DC in vitro. Each method uses a different cell of origin, including stem cells-derived DC (Gorczynski and Boudakov, 2007), monocyte-derived DC (Hartmann et al., 1999; Pernthaner et al., 2012), and isolation of DC from peripheral blood and lymphoid tissues (Del Cacho et al., 2009). Recently, studies about bone marrow-derived DC (BM-DC) have attracted major attention.

    1

    Current address: University of Calgary, Department of Comparative Biology and Experimental Medicine, Calgary, Alberta, Canada, T2N 4N1.

    View full text