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ATP6V0d2 upregulated by intracellular parasites does not participate in parasite resistance to IFN-γ/LPS-activated macrophages.

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posted on 2019-06-14, 17:42 authored by Carina Carraro Pessoa, Luiza Campos Reis, Eduardo Milton Ramos-Sanchez, Cristina Mary Orikaza, Cristian Cortez, Erica Valadares de Castro Levatti, Ana Carolina Benites Badaró, Joyce Umbelino da Silva Yamamoto, Vânia D’Almeida, Hiro Goto, Renato Arruda Mortara, Fernando Real

A. ATP6V0d2 mRNA expression relative to expression of isoform d1 (ATP6V0d1) in nonsilenced or ATP6V0d2-KD macrophages activated or not with IFN-γ/LPS and infected or not by L. amazonensis for 48 hours. The results are representative of 5 independent experiments. *p< 0.05; ns = nonsignificant. B. Expression of NOS2 (left panel) and arginase (right panel) mRNA after 6 and 72 hours of L. amazonensis infection, respectively, in nonsilenced and ATP6V0d2-KD macrophages activated or not with IFN-γ/LPS. NOS2 and arginase mRNA expression was calculated relative to β-actin mRNA expression. The asterisks indicate statistical significance (p<0.05) between nonsilenced and ATP6V0d2-KD measurements. ns = nonsignificant. The results are representative of 2 independent experiments. C-D. Dynamic quantification of parasite numbers in macrophage cultures recorded by live imaging using image segmentation and automatic counting algorithms. In C, upper panel, images of GFP-expressing macrophages (green) merged with images of DsRed2-expressing parasites (red) at the start (0d00:00) and after 20 hours and 30 minutes (0d:20:30) of image acquisition. In the lower panel, the results of image segmentation processing, which identifies parasites (red spots) and macrophages using a color scale ranging from cyan (noninfected macrophage) to magenta (macrophage sheltering >8 parasites). Bar = 10 μm. In D, the number of macrophages per recorded field (upper graph) and the number of parasites per macrophage (lower graph) were assessed throughout 36-hour live imaging recordings of infected nonsilenced or ATP6V0d2-KD macrophages activated or not with IFN-γ/LPS. The data are represented as the means and standard errors of 8 different microscopic fields per condition. The results are representative of 2 independent experiments. E. Infection indexes obtained from nonsilenced or ATP6V0d2-KD macrophages activated or not with IFN-γ/LPS and infected with L. amazonensis for 72 hours. The results are representative of 3 independent experiments.

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