Zinc/copper imbalance reflects immune dysfunction in human leishmaniasis: an ex vivo and in vitro study-2

<p><b>Copyright information:</b></p><p>Taken from "Zinc/copper imbalance reflects immune dysfunction in human leishmaniasis: an ex vivo and in vitro study"</p><p>BMC Infectious Diseases 2004;4():50-50.</p><p>Published online 17 Nov 2004</p><p>PMCID:PMC534101.</p><p>Copyright © 2004 Van Weyenbergh et al; licensee BioMed Central Ltd.</p> ng/ml), Cu (10 μM CuCl) or both. Bars represent mean (+/-SEM) IFN-γ concentration in supernatants from six different donors. Unstimulated cells or cells stimulated with Cu alone did not produce IFN-γ. *p < 0.05 . anti-CD3-stimulated cells (Wilcoxon signed rank test)

Zinc/copper imbalance reflects immune dysfunction in human leishmaniasis: an ex vivo and in vitro study-1

<p><b>Copyright information:</b></p><p>Taken from "Zinc/copper imbalance reflects immune dysfunction in human leishmaniasis: an ex vivo and in vitro study"</p><p>BMC Infectious Diseases 2004;4():50-50.</p><p>Published online 17 Nov 2004</p><p>PMCID:PMC534101.</p><p>Copyright © 2004 Van Weyenbergh et al; licensee BioMed Central Ltd.</p>niasis before treatment (LCL), mucosal leishmaniasis (ML) and visceral leishmaniasis (VL) (Spearman r = 0.65, p = 0.0028)

Zinc/copper imbalance reflects immune dysfunction in human leishmaniasis: an ex vivo and in vitro study-0

<p><b>Copyright information:</b></p><p>Taken from "Zinc/copper imbalance reflects immune dysfunction in human leishmaniasis: an ex vivo and in vitro study"</p><p>BMC Infectious Diseases 2004;4():50-50.</p><p>Published online 17 Nov 2004</p><p>PMCID:PMC534101.</p><p>Copyright © 2004 Van Weyenbergh et al; licensee BioMed Central Ltd.</p>endemic (E) controls; and patients with mucosal leishmaniasis (ML), localized cutaneous leishmaniasis before (LCL0) and after 3 months of treatment (LCL3), and visceral leishmaniasis (VL). *p < 0.05, **p < 0.01, ***p < .001 for LCL0 and ML . E and for VL . U (Mann-Whitney test)

Dose-dependent leishmanicidal effect of DETC in <i>Leishmania</i>-infected human macrophages.

<p>Human monocyte-derived macrophages were infected with <i>Leishmania amazonensis</i> promastigotes (5∶1 ratio). (A) After infection, cells were treated with increasing concentrations of DETC and the number of intracellular amastigotes (symbols) was quantified as described in Material and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014394#s2" target="_blank">Methods</a>. Each bar represents the mean ± SEM of 100 cells counted in duplicate (One-way ANOVA, ***<i>p</i> = 0.0001; post test for linear trend, ***<i>p</i><0.001) representative of two different donors. (B) and (C) <i>Leishmania amazonensis</i>-infected macrophages were treated 48 h after infection with 2 mM of DETC. (B) The number of intracellular amastigotes was quantified as described in Material and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014394#s2" target="_blank">Methods</a>. Results are expressed as number of amastigotes/100 cells. Each bar represents the mean ± SEM of 4 donors (Paired <i>t</i> test, *<i>p</i> = 0.046). (C) Intracellular survival of <i>Leishmania amazonensis</i> amastigotes was quantified by transformation of proliferating extracellular motile promastigotes in Schneider's medium. Each bar represents the mean ± SEM of 4 donors (Paired <i>t</i> test, ***<i>p</i> = 0.0002).</p

Leishmanicidal effect of DETC in murine macrophages <i>in vitro</i>.

<p>(A) Murine (BALB/c) macrophages were infected with <i>Leishmania braziliensis</i> (5∶1 ratio) and treated with DETC (2 mM). Number of intracellular amastigotes was quantified as described in Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014394#s2" target="_blank">Methods</a>. Each bar represents the mean ± SEM of six (Mann Whitney test, **<i>p</i> = 0.0049). (B) Murine (BALB/c) macrophages were treated with DETC (2 mM) in the presence of hydroxylamine (0.5 mM) and supernatant was collected at 48 h. Superoxide production was quantified as described in Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014394#s2" target="_blank">Methods</a>. Each bar represents the mean ± SEM of six (Mann Whitney test, **<i>p</i> = 0.0043).</p

Oxidative damage of DETC is restricted to amastigotes in the phagosome.

<p>Transmission electron microscopy of human macrophages infected with <i>Leishmania amazonensis</i>. Human macrophages were infected with <i>Leishmania amazonensis</i> (5∶1 ratio) for 4 h and then treated with DETC (2 mM) for 10 h. Untreated macrophages displayed numerous well-preserved parasites (A and B). DETC-treated macrophages presented remarkably damaged parasites (C and D), whereas host cell and parasite mitochondria were not injured (white and black arrows, respectively). P indicates intracellular parasites and N indicates host cell nucleus, bars represent 2 µm (A), 1 µm (B and C) or 0.5 µm (D). Images are representative of 2 independent experiments.</p

Oxidative damage of DETC in axenic promastigotes is restricted to mitochondria.

<p>Transmission electron microscopy of promastigotes form of <i>Leishmania amazonensis</i> in axenic culture treated with DETC. Promastigotes were treated with DETC (0.2 mM) for 1 h. (A) Untreated promastigotes displayed a well-preserved cytoplasm and electron-dense mitochondria with normal cristae. (B) DETC-treated promastigotes presented a well-preserved cytoplasm, besides enlarged mitochondria with reduced electron density, often with parallel or circular cristae (thin arrow). Dense core-containing mitochondria reacted positively for calcium oxalate (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014394#s2" target="_blank">Methods</a>) by cytochemical detection (large arrow). (C) The use of NAC partially reverted the effects of DETC upon parasite mitochondria (large arrows with white margins). Images are representative of 2 independent experiments.</p

Gene Expression Profile of High IFN-γ Producers Stimulated with <i>Leishmania braziliensis</i> Identifies Genes Associated with Cutaneous Leishmaniasis

<div><p>Background</p><p>The initial response to <i>Leishmania</i> parasites is essential in determining disease development or resistance. <i>In vitro</i>, a divergent response to <i>Leishmania</i>, characterized by high or low IFN-γ production has been described as a potential tool to predict both vaccine response and disease susceptibility <i>in vivo</i>.</p><p>Methods and findings</p><p>We identified uninfected and healthy individuals that were shown to be either high- or low IFN-γ producers (HPs and LPs, respectively) following stimulation of peripheral blood cells with <i>Leishmania braziliensis</i>. Following stimulation, RNA was processed for gene expression analysis using immune gene arrays. Both HPs and LPs were shown to upregulate the expression of <i>CXCL10</i>, <i>IFI27</i>, <i>IL6</i> and <i>LTA</i>. Genes expressed in HPs only (<i>CCL7</i>, <i>IL8</i>, <i>IFI44L</i> and <i>IL1B)</i> were associated with pathways related to IL17 and TREM 1 signaling. In LPs, uniquely expressed genes (for example <i>IL9</i>, <i>IFI44</i>, <i>IFIT1</i> and <i>IL2RA</i>) were associated with pathways related to pattern recognition receptors and interferon signaling. We then investigated whether the unique gene expression profiles described here could be recapitulated in vivo, in individuals with active Cutaneous Leishmaniasis or with subclinical infection. Indeed, using a set of six genes (<i>TLR2</i>, <i>JAK2</i>, <i>IFI27</i>, <i>IFIT1</i>, <i>IRF1</i> and <i>IL6</i>) modulated in HPs and LPs, we could successfully discriminate these two clinical groups. Finally, we demonstrate that these six genes are significantly overexpressed in CL lesions.</p><p>Conclusion</p><p>Upon interrogation of the peripheral response of naive individuals with diverging IFN-γ production to <i>L</i>. <i>braziliensis</i>, we identified differences in the innate response to the parasite that are recapitulated <i>in vivo</i> and that discriminate CL patients from individuals presenting a subclinical infection.</p></div

Genes modulated in High- and Low- IFN-γ producers discriminate subclinical <i>L</i>. <i>braziliensis</i> infection from active CL.

<p>(A) PBMCs from CL patients (<i>n</i> = 5) and SC individuals (<i>n</i> = 8) were stimulated with <i>L</i>. <i>braziliensis</i> for 72h. Relative expression of <i>IFI27</i>, <i>IFIT1</i>, <i>TLR2</i>, <i>IRF1</i>, <i>JAK2</i> and <i>IL6</i> was evaluated by qRT-PCR. Gene expression is represented as fold change of stimulated over unstimulated cultures, normalized to a housekeeping gene and each symbol represents one individual. (B) A heat map was designed to depict the pattern of gene expression [shown in (A)] of SC individuals) vs. active CL and two-way hierarchical cluster analysis (Ward’s method) of differentially expressed genes was performed. Expression scale for each gene represents the log2-fold change from the mean. (C) Principal component analysis of the differentially expressed genes [depicted in (A)] showing PC1 (x axis), PC2 (y axis) and PC3 (z axis).</p

Expression profile of genes uniquely modulated in Low- IFN-γ producers.

<p>(A) PBMCs from Low IFN-γ Producers (LPs) (<i>n</i> = 3) were stimulated with <i>L</i>. <i>braziliensis</i> for 72h. Gene expression was determined using total RNA and immune gene arrays. Fold expression of the genes modulated in HPs or in LPs only, as evidenced by PCR array (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005116#sec005" target="_blank">materials and methods</a>). (B) Top canonical pathways identified in LPs by Ingenuity Pathway Analysis. Levels of significance are given by the right-tailed Fisher exact test. The negative log <i>P</i> value (blue bars), along the x-axis, increases as a pathway is more significantly associated with the set of genes expressed in LPs. The ratio (orange line) indicates the proportion of upregulated genes relative to all the genes present in a pathway.</p