Mycolactone is present in the mononuclear cells of <i>M. ulcerans</i>-infected animals.

<p>C57BL/6 mice (n = 3) were infected by footpad injection of 10⁴ wtMu or mup045Mu bacilli. The distribution of mycolactone in PBMCs and in the mononuclear cell fraction of draining lymph nodes (DLNs), inguinal lymph nodes (ILNs) and spleens (Spleen) is shown 6 weeks post infection. Mononuclear cells were isolated from pooled samples of whole blood (1 ml), pooled DLNs (n = 3), or from the ILNs and spleens of 3 individual mice. Acetone-soluble lipid were then extracted from cell pellets and mycolactone concentrations determined by quantitative LC/MS-MS analysis. Means and SD are shown for ILNs and spleens. C) IL-2 production after whole blood stimulation with anti-CD3 and -CD28 antibodies for 24 h. Data are mean and SD of IL-2 concentrations, as measured in duplicate for pooled blood samples (n = 4) after 2, 4 and 6 weeks of infection, and are representative of three independent experiments. Differences in IL-2 concentration between groups were analyzed by one-way ANOVA (*: p<0,05; **: p<0,01).</p

Mycolactone targets mononuclear cells in blood and spleen cell suspensions.

<p>The distribution of mycolactone in the gradient density fractions of whole blood or spleen cell suspensions is shown. Mycolactone was added to whole blood (20 µg/ml) or spleen cell suspensions (20 µg/spleen) and incubated for 4 h or 1 h, respectively. The amount of mycolactone distributing in each gradient fractions was then determined by ESI-LC-MS quantitative analysis of their acetone-soluble lipid extracts. Data are mean percentages and SD of duplicate measurements, and are representative of two independent experiments.</p

Presence of mycolactone in serum during <i>M. ulcerans</i> infection.

<p><i>A)</i> The dose-dependent immunosuppressive activity of mycolactone on the IL-2 production of Jurkat T cells is shown. IL-2 concentration was measured in culture supernatants of Jurkat T cells incubated with mycolactone A/B for 6 h prior to stimulation with PMA/ionomycin for 16 h. <i>B)</i> The effect of control mouse sera on stimulation-induced production of IL-2 by Jurkat T cells is shown, by comparison to cells incubated in the absence of serum. The graph shows that this inhibitory effect can be neutralized by heating the control mouse sera at 90°C for 10 min (HT), prior to addition onto Jurkat T cells. Controls include cells in the absence of stimulation (NS), and cells activated in the presence of sera spiked with mycolactone (400 ng/ml), then heat-treated. <i>C)</i> Immunosuppressive activity of sera harvested from mice infected with wild-type <i>M. ulcerans</i> (wtMu) or the mycolactone deficient mutant mup045Mu, as compared to sera from uninfected animals. Data are mean and SD of duplicate measurements of IL-2 production by Jurkat T cells activated in the presence of mouse sera (n = 4), and are representative of two independent experiments.</p

<i>M. ulcerans</i>–produced mycolactone diffuses in internal organs of experimentally infected mice.

<p>The distribution of mycolactone is shown in internal organs of mice ten weeks post sc infection with 10⁴<i>M. ulcerans</i> bacilli. The amount of mycolactone was determined by ESI-LC-MS analysis of acetone-soluble lipid extracts prepared from pools of 5 homogenized organs. Data correspond to the calculated amount of total mycolactones (A/B and C forms) per organ, nd: not detected.</p

Pharmacokinetics of mycolactone<i> in vivo.</i>

<p>Panel A shows the kinetics of mycolactone concentration in the circulating blood of mice following an intravenous (IV), intraperitoneal (IP) or subcutaneous (SC) injection of 300 µg of ¹⁴C-labeled mycolactone. Panel B shows radioactivity levels in various internal organs 24 h post injection of mycolactone via these three administration routes. The left axis indicates cpm levels and the right one the corresponding mycolactone concentrations, for a 300 cpm/µg activity. Data were acquired on pools of blood samples or homogenized organs (n = 3). Panel C illustrates the preferential distribution of mycolactone in the spleen, versus kidney and liver, following injection via the IV (n = 5) or the SC (n = 5) route in three independent experiments. Radioactivity levels in each organ were compared with the Friedman Test (Nonparametric Repeated Measures ANOVA) and Dunn's multiple comparison post-test (*: p<0,05; **: p<0,01).</p