We have demonstrated
that IL-17A can enhance NO production in BCG-infected Silmitasertib price macrophages (Fig. 1). As a result, we are also interested in whether IL-17A can enhance the clearance of intracellular BCG by macrophages. We pre-treated the macrophages with IL-17A for 24 hr, followed by BCG infection. Intracellular BCG was recovered after 2 hr or 48 hr of infection and plated onto 7H10 agar for the determination of phagocytosis or intracellular survival of BCG, respectively. Our data revealed that IL-17A had no effects on phagocytosis of BCG by macrophage (Fig. 5a). On the other hand, the survival of intracellular BCG in macrophages with IL-17A pre-treatment was significantly reduced by 30% after 48 hr of infection (Fig. 5b). The results indicated that IL-17A has anti-mycobacterial effects towards
intracellular BCG. To investigate whether enhanced clearance of intracellular BCG in IL-17A-treated macrophages correlates with NO production, we used AG, a specific iNOS inhibitor, to suppress the enzymatic activity of iNOS. The macrophages were incubated with AG for 1 hr, followed by IL-17A pre-treatment for 24 hr and then BCG infection. The viabilities of macrophages in the presence of AG were analysed by LDH assay. We observed that there was no significant difference in LDH release among the groups, suggesting that the viabilities of macrophages among the groups were similar (Fig. 6a). With the addition of click here AG, we observed that the production of NO in infected macrophages was abolished, regardless of the presence of IL-17A (Fig. 6b). Incubation of macrophages with AG resulted in a 24% increase of intracellular BCG. The same inhibitor also abolished IL-17A-enhanced clearance of intracellular BCG, producing a c.49% increase in intracellular BCG (Fig. 6c). Our Calpain data confirmed that IL-17A-enhanced clearance of intracellular BCG is mediated through an NO-dependent mechanism. In response to microbial infection, macrophages eliminate the phagocytosed pathogens through innate defence mechanisms. The bactericidal effects
of NO on intracellular mycobacteria have long been appreciated in murine models.[12, 33, 34] Unlike murine macrophages, which readily produce NO in response to infections or stimulation, activated human macrophages fail to produce detectable levels of NO in vitro despite iNOS protein expression. Such observations were controversial when regarding the use of NO by human macrophages as an anti-mycobacterial effector. However, several lines of evidence have demonstrated that macrophages isolated from patients with tuberculosis, but not healthy donors, express iNOS and release substantial amounts of NO.[36-38] Further support is provided by studies that use iNOS inhibitors to abolish the killing effects of human macrophages isolated from patients towards intracellular pathogens including BCG and Klebsiella pneumoniae.