Decrease of cytokine production after IL-32 silencing in THP-1-derived macrophages infected with <i>Leishmania</i> species.

<p>PMA-differentiated THP-1 cells (2.5 x 10⁶ cells/800 μL) were electroporated by using Amaxa Nucleofector Technology with IL-32 siRNA (for IL-32 knockdown) and control siRNA according to the protocol described in [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005413#pntd.0005413.ref038" target="_blank">38</a>]. The final concentration per well was 3 x 10⁵ cells/100 μL. After 24 h of transfection, cells were infected with promastigote forms (1.5 x 10⁶ parasites) in the growth stationary phase of <i>L</i>. (<i>L</i>.) <i>amazonensis</i> (<i>L</i>. <i>amaz</i>) or metacyclic promastigote forms (1.5 x 10⁶ parasites) of <i>L</i>. (<i>V</i>.) <i>braziliensis</i> (<i>L</i>. <i>braz</i>). After 4 h, non-internalized parasites were washed out and cells were incubated for 24 h. (A) mRNA expression of IL-32γ isoform and all isoforms were determined by quantitative real-time PCR. mRNA expression and protein levels of (B) TNFα and IL-8, (C) IL-1β and IL-1Ra and (D) IL-10 were determined by quantitative real-time PCR and ELISA in supernatants, respectively. Values are expressed as means ± SEM of three independent experiments. *p < 0.05 (Control SiRNA vs IL-32 SiRNA); #p < 0.05 (<i>L</i>. <i>amaz</i>, <i>L</i>. <i>braz</i>).