Intracellularly survived Staphylococcus aureus after phagocytosis are more virulent in inducing cytotoxicity in fresh murine peritoneal macrophages utilizing TLR-2 as a possible target Ajeya Nandi, Biswadev Bishayi * Department of Physiology, Immunology Laboratory, University of Calcutta, University Colleges of Science and Technology, 92 APC Road, Calcutta 700009, West Bengal, India article info Article history: Received 15 April 2016 Received in revised form 30 May 2016 Accepted 1 June 2016 Available online 4 June 2016 Keywords: Antioxidant inhibitors Murine peritoneal macrophage Reactive oxygen species Staphylococcus aureus infection Toll like receptor-2 abstract Staphylococcus aureus with high virulence potential is contributing to a current public health crisis in both hospital and community settings. TLR-2 and generation of reactive oxygen species (ROS) by phagocytic cells is thought to be an important component of the host’s immunity against S. aureus infection. However, response of S. aureus against modulation of host-derived ROS in absence of TLR-2 during acute staphylococcal infection is still remains unclear. Peritoneal macrophages were pretreated with either inhibitors of superoxide dismutase (SOD) or catalase in presence or absence of anti TLR-2 antibody and were infected with S. aureus strain AG-789. Bacteria were recovered after time depen- dent phagocytosis; intracellular killing, level and expression of SOD and catalase were measured. Phagocytosed bacteria from respective groups were further used for infection to fresh peritoneal mac- rophages as well as for in vivo infection. Levels of ROS, cytokine, lysozyme, antioxidant enzymes activity and TLR-2 expression were measured. Results revealed that more bacteria were escaped killing in SOD and catalase inhibitor pretreated TLR-2 neutralized macrophages, found to express more catalase and are antibiotic resistant. Infection of fresh macrophages with S. aureus, recovered from SOD and catalase inhibited TLR-2 neutralized macrophages induced lower ROS, lysozyme and cytokine production and caused increased bacterial count. Furthermore, bacterial antioxidants by modulating host-derived ROS could regulate the cell surface TLR-2 expression in murine peritoneal macrophages. So, in the early phase of infection, TLR-2 participates in the innate immune response and targeting bacterial antioxidants might be useful in the alleviation of Staphylococcus aureus infection. © 2016 Elsevier Ltd. All rights reserved. 1. Introduction Staphylococcus aureus is a dangerous opportunistic human pathogen, carried by about 30% of the population and a leading etiologic agent of nosocomial and community acquired infectious diseases. The pathogenesis of staphylococcal disease usually pro- ceeds from a local infection (e.g., wound infection, furuncle and cellulitis) to systemic dissemination (bacteremia) and then to metastatic infections (e.g., endocarditis, osteomyelitis and septic arthritis) [1]. A large number of virulence factors are known to contribute to staphylococcal pathogenesis and helps to accommo- date a diversity of niches in its host [2]. However, bacterial pathogenesis in the case of S. aureus is multifactorial since a single virulence factor is not sufficient to cause a staphylococcal infection. Therefore, it is important to understand the host response mech- anism after exposure to the whole S. aureus cells. S. aureus has been reported to persist inside phagocytes for prolonged periods. How- ever, a more thorough characterization of the different mechanisms for intracellular persistence is lacking. It has been reported that protection from primary staphylococcal infection is mainly dependent on innate rather than adaptive immune responses [3]. To persist in a hostile host environment, pathogen should be able to evade or subvert the host immune system aiming to control or eliminate them. Successful pathogenic organism that disables host defenses targets preferentially innate immunity. In the innate immune response, Toll like receptors (TLRs) which are predomi- nantly expressed in cells involved in immune-inflammatory re- sponses, play pivotal roles in the host defense against microbial * Corresponding author. E-mail addresses: biswa_dev2@yahoo.com, biswadevbishayi4@gmail.com (B. Bishayi). Contents lists available at ScienceDirect Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath http://dx.doi.org/10.1016/j.micpath.2016.06.007 0882-4010/© 2016 Elsevier Ltd. All rights reserved. Microbial Pathogenesis 97 (2016) 131e147