© 2006 DIPAV-Università degli Studi di Milano 73 Journal compilation © 2006 Blackwell Publishing Ltd Parasite Immunology , 2007, 29, 73–79 DOI: 10.1111/j.1365-3024.2006.00915.x Blackwell Publishing Ltd ORIGINAL ARTICLE WSP inhibits PMNs apoptosis Wolbachia surface protein (WSP) inhibits apoptosis in human neutrophils C. BAZZOCCHI, S. COMAZZI, R. SANTONI, C. BANDI, C. GENCHI & M. MORTARINO Dipartimento di Patologia Animale Igiene e Sanità Pubblica Veterinaria, Sezione di Patologia Generale e Parassitologia, Università di Milano, Milano, Italy SUMMARY Polymorphonuclear cells (PMNs) are essential for the innate immune response against invading bacteria. At the same time, modulation of PMNs’ apoptosis or cell death by bacteria has emerged as a mechanism of pathogenesis. Wolbachia bacteria are Gram-negative endosymbionts of filarial nematodes and arthropods, phylogenetically related to the genera Anaplasma, Ehrlichia and Neorickettsia ( family Anaplasmataceae). Although several pathogens are known to interfere with apoptosis, there is only limited information on specific proteins that modulate this phenomenon. This is the first evidence for the anti-apoptotic activity of a surface protein of Wolbachia from filarial nematode parasites (the Wolbachia surface protein, WSP). The inhibition of apoptosis was demonstrated on purified human PMNs in vitro by different methods. TUNEL assay showed that the percentage of dead cells was reduced after stimulation with WSP; Annexin V-FITC binding assay confirmed that cell death was due mainly to apoptosis and not to necrosis. Reduced caspase-3 activity in stimulated cells also confirmed an inhibition of the apoptotic process. Keywords apoptosis, filariasis, immunopathology, PMN, Wolbachia surface protein INTRODUCTION Polymorphonuclear cells (PMNs) are essential in initiation and execution of the acute inflammatory response and subsequent resolution of bacterial infection. PMNs are the first cells that migrate into tissues in response to invading organisms, and they phagocytose microbial pathogens, trigger- ing a cytokine network resulting in the development of inflammatory and specific immune responses. PMNs are known to have the shortest in vivo half-life among leucocytes and die rapidly in vitro, showing changes characteristic of cells undergoing programmed cell death or apoptosis (1). The exposure of PMNs to agents known to regulate inflammatory reactions (such as cytokines and bacterial products) can influence cell survival and the physiological process of apoptosis (2). Inflammatory mediators delay apoptosis of PMNs, which contributes to the persistence of inflammation (3). Furthermore, agonists of toll-like receptors (TLRs), such as lipopolysaccharide (LPS, TLR4 ligand), peptidoglycan (TLR2 ligand) and CpG motives (TLR9 ligand), are shown to delay the apoptosis of PMNs both in whole blood and in purified cells (4). Bacterial synthetic lipopeptides, derived from major constituents of the wall of diverse bacteria that are implicated in the pathogenesis of human sepsis, have also recently been shown to delay apoptosis in human neutrophils, through inhibition of caspase-3 activity (5). Wolbachia is a Gram-negative bacterial endosymbiont of filarial nematodes (6). These parasites infect more than 150 million people in tropical areas and cause diseases of eyes, skin and the lymphatic system, such as river blindness and elephantiasis. Wolbachia are abundant in all developmental stages of filarial nematodes, including the lateral cords of both sexes and the reproductive apparatus of adult females (7). Furthermore, the obligatory relationship between filariae and their abundant endobacteria has been exploited to develop a novel chemotherapeutic approach using tetra- cycline derivates that cause developmental block, long-term worm sterility and adult worm death (8–10). Correspondence: Dr Michele Mortarino, DIPAV, Sezione di Patologia Generale e Parassitologia, Università di Milano, Via Celoria 10, 20133 Milano, Italy (e-mail: michele.mortarino@unimi.it). Received: 15 February 2006 Accepted for publication: 3 August 2006