Effect of <i>Mycobacterium avium</i> infection on intramacrophagic ferritin.

Abstract

<div><p>Bone marrow-derived macrophages were obtained from C57Bl/6 mice and infected with <i>M. avium</i>, as described in Material and Methods, or left uninfected. A - At different time points, macrophages were lysed and the amount of ferritin was quantified by ELISA. Data are presented as ng of ferritin per mg of total protein. The results are shown as average ± SD from one experiment performed in triplicate out of four independent experiments. Superscripts indicate statistical significance between M. avium-infected and uninfected, within the correspondent time-point, as follows: *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001. B – BMM uninfected or infected with <i>M. avium</i> for 24h were incubated for 6h with (⁵⁵Fe) ferric ammonium citrate. Total protein (18 µg) was loaded (in duplicates) in native PAGE and exposed to autoradiography to analyze protein-bound iron. A single band was detected corresponding to cytosolic H/L ferritin. The values indicate the average relative band intensity for each condition. C – BMM infected with <i>M. avium</i> for 4h, 1 and 3 days and respective uninfected controls were tested for IRP-IRE binding activity, by gel retardation assay. 2% of 2-mercaptoethanol (2-ME) fully activates IRP binding activity and shows equal loading. BMM treated with iron or deferoxamine (DFO) were tested in a separated gel to confirm the reliability of the assay. D – BMM were treated with the transcriptional inhibitor actinomycin D or with vehicle. After an 8h-infection with <i>M. avium</i>, the BMM were lysed and H- and L-ferritin were quantified by ELISA. Results show the average + SD from one experiment performed in triplicate out of three independent experiments. ***<i>p</i><0.001, NS not significant. </p> <p>E – At different time points, total RNA was collected from macrophages and the expression levels of ferritin genes was quantified by qRT-PCR, and normalized to <i>Hprt1</i>. Results are shown as fold increase in <i>M. avium</i>-infected macrophages in comparison with uninfected ones. Data are presented as average ± SE from one experiment performed in triplicate from a total of two independent experiments. </p></div