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Veterinary Parasitology 117 (2003) 73–83 Immunological role of the endosymbionts of Dirofilaria immitis: the Wolbachia su
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Veterinary Parasitology 117 (2003) 73–83
Immunological role of the endosymbionts of Dirofilaria immitis: the Wolbachia surface protein activates canine neutrophils with production of IL-8 C. Bazzocchi, C. Genchi, S. Paltrinieri, C. Lecchi, M. Mortarino, C. Bandi∗ Dipartimento di Patologia Animale Igiene e Sanità Pubblica Veterinaria, Sezione di Patologia Generale e Parassitologia, Università di Milano, Via Celoria 10, 20133 Milan, Italy Received 20 March 2003; received in revised form 11 July 2003; accepted 19 July 2003
Abstract Filarial nematodes, including Dirofilaria immitis and D. repens, harbour intracellular bacteria belonging to the genus Wolbachia. These bacteria have been implicated in the pathogenesis of filarial diseases, possibly through their endotoxins. Recent studies have shown that a major surface protein of Wolbachia (WSP) induces a specific IgG response in hosts infected by D. immitis. WSP from the Wolbachia of D. immitis was produced in recombinant form. The purified protein was used in stimulation assays on canine neutrophils. The assays performed using a modified Boyden chamber showed that WSP stimulates neutrophil chemokinesis. In addition, RT-PCR revealed increased production of chemokine IL-8 by cells incubated with this protein. Neutrophils have been shown to play a major role in the pathogenesis of river blindness, and to accumulate in the nodules of onchocerciasis patients. In dogs infected by D. immitis, neutrophils accumulate in kidneys and in the wall of pulmonary arteries. As shown by our studies, Wolbachia could contribute to these inflammatory phenomena through its surface protein WSP, independently from its endotoxin component. © 2003 Elsevier B.V. All rights reserved. Keywords: Dirofilaria immitis; Immune response; Wolbachia spp.; Neutrophils
1. Introduction Dirofilaria immitis is the main filariasis agent in dogs and cats, causing heartworm disease. D. immitis, like other filarial nematodes, harbours intracellular endosymbiotic bacte∗ Corresponding author. Tel.: +39-0250-318-093; fax: +39-0250-318-095. E-mail address: [email protected] (C. Bandi).
0304-4017/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2003.07.013
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ria belonging to the genus Wolbachia (Rickettsiales) (Bandi et al., 2001). The association between filarial nematodes and Wolbachia is considered an obligatory (perhaps mutualistic) symbiosis (e.g. see Casiraghi et al., 2002). Indeed, treatment with antibiotics which are effective against Rickettsiales (e.g. tetracycline) have detrimental effects on those filariae that harbour Wolbachia (Bandi et al., 2001). Wolbachia-associated molecules may interact with cells of the innate immune system such as macrophages and neutrophils (PMNs), thus contributing to the pathogenesis and immunology of filarial diseases (Bandi et al., 2001; Brattig et al., 2001; Taylor et al., 2001). Indeed, extracts of Brugia malayi and Onchocerca volvulus (filarial species which harbour Wolbachia) induce the production of cytokines by macrophages, while extracts of Acanthocheilonema viteae (a species of filaria without Wolbachia) do not (Brattig et al., 2000; Taylor et al., 2000). Furthermore, Wolbachia molecules are thought to be responsible for PMNs accumulation in the subcutaneous nodules induced by adult O. volvulus in human patients (Brattig et al., 2001). The Wolbachia surface protein (WSP) has recently been implicated in the immunology of heartworm infection: antibodies against this protein are present in sera of animals and humans infected by D. immitis (Bazzocchi et al., 2000; Simon et al., 2003). In addition, WSP seems to interact with the host immune system, possibly polarising the response towards a Th1-type (Marcos-Atxutegi et al., 2003). However, no data have yet been published on the effects of this protein following stimulation of immune cells. Bacteria typically attract PMNs. These cells thus appear as a suitable target for investigating the possible stimulatory effects of WSP. In canine dirofilariasis, PMNs infiltration has been observed in various organs and tissues, including kidney and arterial walls (Grauer et al., 1989; Forrester and Lees, 1995). The goal of this study is to verify the role of WSP from the Wolbachia of D. immitis in the activation of canine PMNs.
2. Materials and methods 2.1. Animals and study design Nine healthy dogs and six dogs naturally infected by D. immitis were included in this study. Uninfected dogs were owned animals sampled during routine clinical examinations. They were all clinically healthy, and routine haematology and clinical chemistry tests did not show any sub-clinical abnormality. D. immitis infection was excluded by negative serology using a commercial ELISA kit (SNAP, IDEXX, Westbrook, MA, USA). Dogs infected by D. immitis had positive ELISA tests and, in two cases, circulating microfilariae (100 and 2 microfilariae per 20 l of blood, respectively). Two out of six infected dogs had clinical signs consistent with class II heartworm disease such as poor athletic performance and occasional coughing after exercise (Di Sacco and Vezzoni, 1992). An amount of 6–10 ml of blood was taken from the jugular vein of each dog. Blood was collected in EDTA-coated tubes and processed for routine haematology and for PMNs purification within 2 h from sampling.
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2.2. Preparation of PMNs PMNs were purified from peripheral blood of each dog using a Percoll discontinuous gradient following a procedure described for dogs (Zwahlen et al., 1994). PMNs were suspended in 1 ml of Hanks’ balanced salt solution (HBSS) + HEPES (5 mg/ml) + bovine serum albumin (BSA, 10 mg/ml). The percentage of PMNs in the preparation was verified through May Grunwald–Giemsa staining of the cytocentrifuged cell suspension. Cell viability was evaluated using the Trypan blue exclusion dye test (Metcalf et al., 1986). Finally, purified PMNs were counted in an automated cell counter (Hemat 8, SEAC, Firenze, Italy) and diluted at the concentration of 2 × 106 cells/ml in HBSS + HEPES + BSA. All purification procedures were done on sterile plastic tubes under a vertical flow laminar hood to avoid PMNs activation due either to contact with glass tubes or to bacterial contamination. 2.3. Preparation of recombinant WSP WSP of the Wolbachia of D. immitis was produced in recombinant form according to Bazzocchi et al. (2000). Endotoxin contamination of WSP preparation and HBSS + HEPES + BSA was tested by Limulus Amebocyte Lysate (LAL) test (QCL 1000; BioWhittaker, Walkersville, MD, USA). WSP was than filtered over an anionic exchanger membrane (Sartorius, Goettingen, Germany) to remove endotoxins. The recombinant protein was filtered three times, leading to endotoxin levels