Neospora caninum and bone marrow-derived dendritic cells: parasite survival, proliferation, and induction of cytokine expression M. STROHBUSCH, 1 N. MÜLLER, 1 A. HEMPHILL, 1 M. MARGOS, 1 D. GRANDGIRARD, 2 S. LEIB, 2 G. GREIF 3 & B. GOTTSTEIN 1 1 Institute of Parasitology and 2 Institute for Infectious Diseases, University of Berne, Bern, Switzerland, 3 Bayer HealthCare GmbH, Leverkusen, Germany SUMMARY Dendritic cells (DCs) represent the first line defence of the innate immune system following infection with pathogens. We exploratively addressed invasion and survival ability of Neospora caninum, a parasite causing abortion in cattle, in mouse bone marrow DCs (BMDCs), and respective cyto- kine expression patterns. Immature BMDCs were exposed to viable (untreated) and nonviable parasites that had been inactivated by different means. Invasion and ⁄ or internaliza- tion, as well as intracellular survival and proliferation of tachyzoites were determined by NcGRA2-RT-PCR and transmission electron microscopy (TEM). Cytokine expres- sion was evaluated by reverse transcription (RT)-PCR and cytokine ELISA. Transmission electron microscopy of DCs stimulated with untreated viable parasites revealed that N. caninum was able to invade and proliferate within BMDCs. This was confirmed by NcGRA2-RT-PCR. On the other hand, no viable parasite organisms were revealed by TEM when exposing BMDCs to inactivated parasites (non- viability demonstrated by NcGRA2-RT-PCR). Cytokine expression analysis (as assessed by both RT-PCR and ELISA) demonstrated that both viable and nonviable para- sites stimulated mBMDCs to express IL-12p40, IL-10 and TNF-a, whereas IL-4 RNA expression was not detected. Thus, exposure of mBMDCs to both viable and nonviable parasites results in the expression of cytokines that are rele- vant for a mixed Th1 ⁄ Th2 immune response. Keywords mBMDC, NcGRA2-RT-PCR, Neospora cani- num, Th1 ⁄ Th2 cytokines INTRODUCTION Current evidence strongly indicates that Neospora caninum is the protozoan pathogen most commonly associated with bovine abortion worldwide, causing serious veterinary health and economic problems in livestock production (1). In an early phase of infection, N. caninum invades almost all organs, whereas at later time points, it is found prefer- entially in the brain. So far, nothing is known about the migratory ability of the parasite within the host body or the interaction with immune-competent cells. Dendritic cells (DCs) are the most potent professional antigen-pre- senting cells (APC). Both mouse classical myeloid and plasmacytoid DCs generated from bone marrow as well as lymphoid tissue DCs can direct Th1 or Th2 responses depending on the dose of antigen and their state of maturation together with the stimulation of DCs by path- ogen-derived products (2). Dendritic cells directly sense pathogenic components via Toll-like receptors (TLRs) and respond to this recognition by upregulation of surface co-stimulatory molecules, secretion of cytokines and chemokines, which will enhance antigen presentation and migration to secondary lymphoid tissues (3). Extracts of the apicomplexan parasite Toxoplasma gondii were found to activate DCs and to stimulate IL- 12 production by DCs (4). By contrast, active T. gondii invasion of immature DCs suppressed the ability of these cells to participate in innate immunity and to induce adaptive immune responses (5). It was shown that T. gondii induced a state of hypermotility in infected DCs in vitro and that parasite-infected DCs pro- moted dissemination of Toxoplasma in vivo (6), thus allowing the parasites to be carried away from the site of infection and to undergo steady-state migration to draining lymphoid organs. This process could be impor- tant for T. gondii in terms of reaching the brain tissue and skeletal muscle, where tachyzoites undergo differenti- ation into the encysted bradyzoite forms. This establishes Correspondence: Bruno Gottstein, Institute of Parasitology, Vetsuisse Faculty, University of Berne, Laenggass-Strasse 122, CH-3001 Bern, Switzerland (e-mail: bruno.gottstein@ipa.unibe.ch). Disclosures: None Received: 9 December 2008 Accepted for publication: 2 March 2009 Parasite Immunology, 2009, 31, 366–372 DOI: 10.1111/j.1365-3024.2009.01112.x 366 Ó 2009 Blackwell Publishing Ltd