Elsevier

Experimental Parasitology

Volume 169, October 2016, Pages 77-80
Experimental Parasitology

Research brief
Cytokine response of human THP-1 macrophages to Trichomonas tenax

https://doi.org/10.1016/j.exppara.2016.07.011Get rights and content

Highlights

  • Human THP-1 macrophages were incubated with live or sonicated Trichomonas tenax for 4–16 h.

  • Live T. tenax did not induce TNFα, IL-1β, IL-8, or IL-10 synthesis by THP-1 cells.

  • The highest concentration of T. tenax lysate stimulated IL-8 synthesis after 16 h.

Abstract

Trichomonas tenax is a protozoan that inhabits the oral cavity of humans, most often those with poor oral hygiene. Although T. tenax is widely considered a commensal, recent studies have suggested a pathogenic role for the protozoan in persons with periodontitis. Here we investigated the capacity of T. tenax to induce pro-inflammatory cytokine secretion in human macrophages, with the idea that elicitation of inflammation may be one mechanism by which T. tenax contributes to oral pathology. Human THP-1 cells differentiated to the macrophage phenotype (dTHP-1) were incubated with live or sonicated T. tenax at trophozoite:dTHP-1 ratios of 1:5, 1:10, and 1:20. Culture media removed from the wells after 4, 8, and 16 h of stimulation were assayed by ELISA for tumor necrosis factor alpha, interleukin-1 beta, interleukin-8, and the immunoregulatory cytokine interleukin-10. Live T. tenax trophozoites failed to induce production of any of the cytokines tested, regardless of trophozoite:dTHP-1 cell ratio or length of co-incubation. T. tenax lysates stimulated interleukin-8 synthesis, but only after 16 h of incubation at the 1:5 trophozoite:dTHP-1 cell ratio. These results suggest that pro-inflammatory cytokine synthesis by human macrophages in direct response to T. tenax contributes little to oral pathology.

Introduction

Trichomonas tenax (O.F. Müller) is a flagellated, aerotolerant, anaerobic protozoan that colonizes the human oral cavity, where it is found in and around diseased gums and teeth (Hersh, 1985). It has a world-wide distribution, with a prevalence that varies greatly among nations, e.g., 4–53% in Germany, 10% in England, and 16–30% in the United States (summarized by Honigberg and Burgess, 1994). Although often described as a commensal organism, T. tenax has been implicated as an etiological agent of periodontitis (Feki et al., 1981, Kurnatowska et al., 2004, Marty et al., 2015). Periodontitis is the most common cause of tooth loss in the world (Darveau, 2010). It is an inflammatory process directed by cytokines produced by host cells in response to oral microorganisms (Silva et al., 2015). Cytokines induce the loss of epithelial attachment, connective tissue, and alveolar bone (Sahingur and Yeudall, 2015). The current study examined the ability of T. tenax trophozoites to stimulate pro-inflammatory cytokine production by macrophages, information that may be relevant to treatment decisions for persons colonized by the protozoan.

Pro-inflammatory cytokines implicated in periodontitis include interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα), both of which promote bone and tooth resorption by stimulating osteoclast development and production of matrix metalloproteinases (Assuma et al., 1998). Equally important is interleukin-8 (IL-8), a potent chemoattractant for neutrophils that generate reactive oxygen species and tissue-damaging proteolytic enzymes (Jayaprakash et al., 2015). Human cells produce IL-1β, TNFα, and IL-8 in response to the pathogenic trichomonad Trichomonas vaginalis (Fichorova et al., 2006, Fiori et al., 2013, Ryu et al., 2004, Seo et al., 2014, Vilela and Benchimol, 2013). Thus, it is reasonable to speculate that T. tenax may likewise stimulate production of IL-1β, IL-8, and TNFα, and in this way contributes to the progression of periodontitis.

In this study, we incubated live or sonicated T. tenax trophozoites with human THP-1 cells differentiated to the macrophage phenotype (Schwende et al., 1996, Tsuchiya et al., 1982), and then measured IL-1β, IL-8, and TNFα released by the THP-1 cells into the culture medium. We also included interleukin-10 (IL-10) in our analysis, since both T. vaginalis and Tritrichomonas foetus can stimulate its production, and because the immunoregulatory activity of IL-10 may protect the protozoans from other elements of the immune system (Fichorova, 2009, Song et al., 2015, Vilela and Benchimol, 2013). Differentiated THP-1 (dTHP-1) cells were chosen as responder macrophages based on their ability to alter cytokine synthesis when challenged with such protozoans as Trichomonas vaginalis (Fiori et al., 2013), Toxoplasma gondii (Quan et al., 2013), Plasmodium falciparum (Kumaratilake et al., 1997), Entamoeba histolytica (Campbell et al., 2000), and Leishmania spp. (Cheekatla et al., 2012). The ability of dTHP-1 cells to synthesize TNFα, IL-1β, IL-8, and IL-10 after appropriate stimulation is well documented (Sun et al., 2014).

Section snippets

Cell lines and culture conditions

THP-1 cells and the Hs-4:NIH strain of T. tenax were purchased from the American Type Culture Collection (ATCC) (Manassas, Virginia) and maintained according to ATCC recommendations. THP-1 cells were cultured at 37 °C under 5% CO2 in complete RPMI-1640 medium supplemented with 10% fetal bovine serum and 0.05 mM 2-mercaptoethanol. Trophozoites were cultured at 35 °C in borosilicate glass screw-cap tubes in modified LYI Entamoeba medium supplemented with 15% adult bovine serum and 2% Diamond's

Results and discussion

This study yielded little evidence to support the hypothesis that T. tenax contributes to oral pathology by directly inducing a pro-inflammatory cytokine response in macrophages. The ELISA results pooled from three (TNFα, IL-8, IL-10) or four (IL-1β) independent experiments for each cytokine are presented in Fig. 1. Incubation of dTHP-1 cells in serum-free RPMI medium containing no added stimulus resulted in negligible production of the four cytokines over the 16 h assay period (Fig. 1A–D).

Acknowledgments

This research was funded by the Biomedical Sciences Graduate Program at the Kirksville College of Osteopathic Medicine, A.T. Still University of Health Sciences.

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