The Role of Reactive Oxygen Species and Nitric Oxide in the Inhibition of Trichophyton rubrum Growth by HaCaT Cells

Abstract

Trichophyton rubrum (T. rubrum) is one of the most important agents of dermatophyte infection in humans. The aim of this experiment was to evaluate the effect of HaCaT cells on T. rubrum, investigate the responsible mechanism of action, and explore the role of reactive oxygen species (ROS) and nitric oxide (NO) in the inhibition of T. rubrum growth by HaCaT cells. The viability of fungi treated with HaCaT cells alone and with HaCaT cells combined with pretreatment with the NADPH oxidase inhibitor (DPI) or the nitric oxide synthase (NOS) inhibitor L-NMMA was determined by enumerating the colony-forming units. NOS, ROS, and NO levels were quantified using fluorescent probes. The levels of the NOS inhibitor asymmetric dimethylarginine (ADMA) were determined by enzyme-linked immunosorbent assay (ELISA). Micromorphology was observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In addition, fungal keratinase activity was assessed by measuring dye release from keratin azure. In vitro fungal viability, keratinase activity, and ADMA content decreased after HaCaT cell intervention, whereas the levels of ROS, NO, and NOS increased. The micromorphology was abnormal. Fungi pretreated with DPI and L-NMMA exhibited opposite effects. HaCaT cells inhibited the growth and pathogenicity of T. rubrum in vitro. A suggested mechanism is that ROS and NO play an important role in the inhibition of T. rubrum growth by HaCaT cells.

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“…On the other hand, excess ROS reacts with NO to generate highly toxic RNS, further enhancing the therapeutic effects [ 30 , 38 ]. The intracellular NO and RNS levels were investigated after treatments with each agent.…”

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“…zeigten, dass die immunologische Funktion von Makrophagen nach einer T. rubrum ‐Infektion verloren ging oder durch Pilz‐Exoantigene und Mannan geschwächt wurde [52]. Vor kurzem wurde beschrieben, dass der Toll‐like ‐Rezeptor (TLR) 2 eine wichtige Rolle bei der Phagozytose von T. rubrum in Monozyten spielt, und dass reaktive Sauerstoffspezies und Stickstoffmonoxid das Wachstum von T. rubrum in Zelllinien in vitro hemmten [49, 53]. Lysin‐M‐Proteine von T. rubrum könnten den Pilz abzuschirmen und es ihm ermöglichen, sich der Immunüberwachung zu entziehen [54].…”

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“…showed that the immunological function of macrophages was lost following T. rubrum infection or weakened by fungal exoantigens and mannan [52]. Toll‐like receptor (TLR) 2 was recently shown to play an important role in the phagocytosis of T. rubrum in monocytes, and reactive oxygen species and nitric oxide inhibited the growth of T. rubrum in cell lines in vitro [49, 53]. T. rubrum lysin M proteins may help to shield the fungus and allow it to escape immune surveillance [54].…”