In Vitro effects of tamoxifen on equine neutrophils C. Borlone a , N. Morales a , C. Henriquez a , H. Folch b , C. Olave a , J. Sarmiento c , B. Uberti d , G. Moran a, ⁎ a Department of Pharmacology, Faculty of Veterinary Science, Universidad Austral de Chile, Valdivia, Chile b Department of Immunology, Faculty of Medicine, Universidad Austral de Chile, Valdivia, Chile c Department of Physiology, Faculty of Medicine, Universidad Austral de Chile, Valdivia, Chile d Department of Veterinary Clinical Sciences, Faculty of Veterinary Science, Universidad Austral de Chile, Valdivia, Chile abstract article info Article history: Received 28 June 2016 Received in revised form 26 October 2016 Accepted 3 November 2016 Available online xxxx Neutrophils participate in innate immunity as the first line of host defense against microorganisms. However, ex- acerbated neutrophil activity can be harmful to surrounding tissues; this is important in a range of diseases, in- cluding allergic asthma and chronic obstructive pulmonary disease in humans, and equine asthma (also known as recurrent airway obstruction (RAO). Tamoxifen (TX) is a non-steroidal estrogen receptor modulator with ef- fects on cell growth and survival. Previous preliminary studies showed that TX treatment in horses with induced acute pulmonary inflammation promoted early apoptosis of blood and BALF neutrophils, reduction of BALF neu- trophils, and improvement in animals' clinical status. The aim of this study was to evaluate the in vitro effect of TX on functional tests in equine peripheral blood neutrophils. Chemotaxis, respiratory burst production and phago- cytosis assays were performed on neutrophils isolated from peripheral blood samples from 10 healthy horses. Re- sults showed that IL-8 stimulated cells decrease their chemotactic index when treated with TX (1 and 10 μM). Respiratory burst production was also dampened after treatment with TX. In conclusion, these results confirm that tamoxifen has a direct action on equine peripheral blood neutrophils. However, more in vivo and in vitro studies are required to fully understand the mechanisms of action of TX on neutrophils, in order to elucidate if it can be used as treatment in disorders such as allergic asthma in humans and horses. © 2016 Elsevier Ltd. All rights reserved. Keywords: Asthma Recurrent airway obstruction Tamoxifen Horses Chemotaxis Respiratory burst 1. Introduction Neutrophils play a central role in innate immunity, acting as the first line of host defense against invading organisms. They are the predomi- nant cell type involved in the cellular phase of acute inflammation (Cassatella, 1999). They migrate to the site of infection or inflammation for containment and clearance of invading agents (Nathan, 2006). At this site, several locally-generated messenger molecules attract leukocytes from blood and direct their migration towards microbes, in a process termed chemotaxis (Baggiolini et al., 1993). This is regulated by chemokines, pro-inflammatory mediators that regulate leukocyte trafficking, among other functions (Sanz and Kubes, 2012). Once neutrophils encounter the inciting pathogen, they engulf them into a phagosome that fuses with intracellular granules to form a phagolysosome. Within the phagolysosome, pathogens are killed through exposure to enzymes, antimicrobial peptides and reactive oxy- gen species (ROS) (Mayer-Scholl et al., 2004). ROS are produced in what is called “respiratory burst”, during which the NADPH oxidase complex assembles at the phagosomal membrane and produces O 2 - , which is readily converted to hydrogen peroxide by the enzyme superoxide dis- mutase (Chapman et al., 2002). It is believed that neutrophils in inflamed tissues preferentially undergo apoptosis after completing their function. This process would prevent release of cytotoxic products –such as ROS or proteases– that would contribute to tissue damage (Nathan, 2006). Neutrophil apoptosis leads to the expression of cell surface “eat me” signals such as phosphatidylserine, enabling neutro- phils to be recognized and cleared by scavenger macrophages (Savill et al., 1989). When phagocytosis of apoptotic neutrophils is impaired, neutrophils undergo secondary necrosis and release their cytotoxic products, aggravating tissue injury and amplifying the inflammatory response (Filep and El Kebir, 2009). Therefore, in terms of resolution of inflammation, neutrophil apoptosis holds a central position as it brings sustained neutrophil recruitment to an end, while the phagocytic clearance of apoptotic neutrophils reprograms macrophages to an anti- inflammatory phenotype, which is characterized by the release of mediators that suppress the inflammatory response, such as transforming growth factor-β (TGF-β) and IL-10, thus contributing to the restora- tion of homeostasis after tissue injury or infection (Soehnlein and Lindbom, 2010). One of the diseases in which neutrophils play an important role in the equine airways is asthma, previously termed recurrent airway obstruction (RAO) (Bullone and Lavoie, 2015, Couetil et al., 2016; Pirie et al., 2016). Equine asthma develops in mature horses following sta- bling and exposure to dusty hay and straw (Robinson, 2001). The disease is characterized by pulmonary neutrophilia and excessive Research in Veterinary Science 110 (2017) 60–64 ⁎ Corresponding author. E-mail address: gmoran@uach.cl (G. Moran). http://dx.doi.org/10.1016/j.rvsc.2016.11.003 0034-5288/© 2016 Elsevier Ltd. All rights reserved. Contents lists available at ScienceDirect Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc