Interferon-γ Restricts Toxoplasma gondii Development in Murine Skeletal Muscle Cells via Nitric Oxide Production and Immunity-Related GTPases

Lu der CGK (2012) Interferon-c Restricts Toxoplasma gondii Development in Murine Skeletal Muscle Cells via Nitric Oxide Production and Immunity-Related GTPases. PLoS ONE 7(9): e45440. doi:10.1371/journal.pone.0045440 Interferon-c Restricts Toxoplasma gondii Development in Murine Skeletal Muscle Cells via Nitric Oxide Production and Immunity-Related GTPases Anna C. Taka cs 0 Izabela J. Swierzy 0 Carsten G. K. Lu der 0 Ira Blader, University of Oklahoma Health Sciences Center, United States of America 0 Institute for Medical Microbiology, University Medical Center, Georg-August-University , Go ttingen , Germany The apicomplexan parasite Toxoplasma gondii is regularly transmitted to humans via the ingestion of contaminated meat products from chronically infected livestock. This route of transmission requires intracellular development and long-term survival of the parasite within muscle tissue. In this study, we determined the cell-autonomous immunity of mature primary embryonic or C2C12 skeletal muscle cells (SkMCs) to infection with T. gondii. Non-activated SkMCs and control fibroblasts sustained parasite replication; however, interferon (IFN)-c significantly inhibited parasite growth in SkMCs but not in fibroblasts. Intracellular parasite replication was diminished by IFN-c whereas host cell invasion was not affected. Tumor necrosis factor (TNF) did not further increase the IFN-c-triggered host defense of SkMCs against Toxoplasma. Remarkably, IFN-c alone or in combination with TNF decreased the high level of T. gondii bradyzoite formation being observed in nonactivated SkMCs. Stimulation of SkMCs with IFN-c strongly triggered expression of inducible nitric oxide synthase (iNOS) transcripts, and induced significantly higher levels of nitric oxide (NO) in SkMCs than in fibroblasts. Consequently, pharmacological inhibition of iNOS partially abrogated the IFN-c-induced toxoplasmacidal activity of SkMCs. In addition, SkMCs strongly up-regulated immunity-regulated GTPases (IRGs) following stimulation with IFN-c. IRGs accumulated on Toxoplasma-containing vacuoles in SkMCs in a parasite strain-dependent manner. Subsequent vacuole disruption and signs of degenerating parasites were regularly recognized in IFN-c-treated SkMCs infected with type II parasites. Together, murine SkMCs exert potent toxoplasmacidal activity after stimulation with IFN-c and have to be considered active participants in the local immune response against Toxoplasma in skeletal muscle. - Funding: This study was supported by the Federal Ministry of Education and Research (BMBF; TOXONET consortium, grant numbers 01KI0766 and 01KI1002B to CGKL). Publication of the study was supported by the Open-Access-Publication Fund of the Georg-August-University and the German Research Council (DFG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Skeletal muscle plays a critical role in the transmission of the zoonotic parasite Toxoplasma gondii to humans. Between 30% and 63% of human infections have been related to the consumption of undercooked or cured meat products as revealed by a multi-centre study involving acutely infected pregnant women and non-infected controls [1]. Although infection is mostly asymptomatic or benign, the parasite is a significant threat for individuals with a premature or suppressed immune system and can lead to severe and lifethreatening toxoplasmosis. Transmission of T. gondii to humans via the ingestion of contaminated meat products may depend on the development and long-term survival of parasites in skeletal muscle cells (SkMCs) of chronically infected livestock and poultry. We have shown previously that these cells, after differentiation in vitro to mature myotubes, indeed provide a niche which sustains intracellular development and differentiation to the bradyzoite stage of the parasite [2]. During embryogenesis or following muscle injury, SkMCs transform from proliferating and fusogenic stem cells, i.e. myoblasts to multinucleated myotubes which further differentiate to large syncytial muscle fibers [3]. Mature SkMCs provide a unique immunological environment for the development of pathogens, with no detectable expression of major histocompatibility complex (MHC) class I and class II expression under physiological conditions [4]. Furthermore, expression of HLA-G or the B7 homologue B7-H1 (PD-L1) by human myoblasts fulfils tolerizing or even suppressive functions within muscle tissue [5,6]. Limited immune reactions in skeletal muscle may thus facilitate long-term survival of Toxoplasma and make this organ to one of the preferred body sites where tissue cysts persist until orally ingested by a new host [7]. Under certain conditions, i.e. after activation by proinflammatory cytokines in vitro or during inflammatory myopathies in vivo, however, muscle cells can express MHC class I and II antigens and present antigens to T cells [8,9]. In addition, they express a variety of cell surface receptors and soluble immune mediators and thus fulfil requirements for immunocompetence. During reactivation of chronic toxoplasmosis or during acute disease following recent infection, the occurrence of polymyositis has been well established in both humans and animals [10,11,12,13,14]. Although immune cells infiltrating the infected muscle tissue certainly mediate much of the local inflammatory response, this supports the view that SkMCs contribute to the immune response to T. gondii within muscle tissue and may be pivotal during toxoplasmic myopathies. However, the impact of SkMCs in the local host response to Toxoplasma and host factors or molecular mechanisms which might limit parasite development in SkMCs have not yet been determined. Resistance to infection with obligate intracellular Toxoplasma parasites largely depends on Th1-type cell-mediated immune responses. Interferon (IFN)-c released from CD4+ and CD8+ T lymphocytes is the most critical mediator of immunity against T. gondii [15,16,17,18]. Early during infection, natural killer (NK) cells are the main producers of IFN-c and are important mediators of innate immunity [19]. IFN-c activates effector cells of both hematopoietic and non-hematopoietic origin to exert anti-Toxoplasma activity [20]. Tumor necrosis factor (TNF), interleukin (IL)1 and IL-6 synergize with IFN-c to strengthen the anti-parasitic response [21,22]. They exert anti-parasitic activity by upregulating the expression of effector molecules in various cell types. Depending on the host species, control of intracellular T. gondii is mediated by production of nitric oxide (NO) by the inducible NO synthase (iNOS) [23,24], disruption of the parasitophorous vacuole by immunity-related GTPases (IRGs; formerly called p47 GTPases) and p65 guanylate-binding proteins (GBPs; also called p65 GTPases) [25,26,27], tryptophan st (...truncated)