Human Macrophage Response to <em>L. (Viannia) panamensis</em>: Microarray Evidence for an Early Inflammatory Response

<div><h3>Background</h3><p>Previous findings indicate that susceptibility to <em>Leishmania (Viannia) panamensis</em> infection of monocyte-derived macrophages from patients and asymptomatically infected individuals were associated with the adaptive immune response and clinical outcome.</p> <h3>Methodology/Principal Findings</h3><p>To understand the basis for this difference we examined differential gene expression of human monocyte-derived macrophages following exposure to <em>L. (V.) panamensis</em>. Gene activation profiles were determined using macrophages from healthy volunteers cultured with or without stationary phase promastigotes of <em>L. (V.) panamensis</em>. Significant changes in expression (>1.5-fold change; p<0.05; up- or down-regulated) were identified at 0.5, 4 and 24 hours. mRNA abundance profiles varied over time, with the highest level of activation occurring at earlier time points (0.5 and 4 hrs). In contrast to observations for other <em>Leishmania</em> species, most significantly changed mRNAs were up- rather than down-regulated, especially at early time points. Up-regulated transcripts over the first 24 hours belonged to pathways involving eicosanoid metabolism, oxidative stress, activation of PKC through G protein coupled receptors, or mechanism of gene regulation by peroxisome proliferators via PPARα. Additionally, a marked activation of Toll-receptor mediated pathways was observed. Comparison with published microarray data from macrophages infected with <em>L. (Leishmania) chagasi</em> indicate differences in the regulation of genes involved in signaling, motility and the immune response.</p> <h3>Conclusions</h3><p>Results show that the early (0.5 to 24 hours) human monocyte-derived macrophage response to <em>L. (Viannia) panamensis</em> is not quiescent, in contrast to published reports examining later response times (48–96 hours). Early macrophage responses are important for the developing cellular response at the site of infection. The kinetics and the mRNA abundance profiles induced by <em>L. (Viannia) panamensis</em> illustrate the dynamics of these interactions and the distinct biologic responses to different <em>Leishmania</em> species from the outset of infection within their primary host cell.</p> </div

Variation of up- and down-regulated transcripts with time post-infection.

<p>A) The number of gene transcripts up- or down-regulated at 0.5, 4 and 24 hours post-infection/interaction with <i>Leishmania (Viannia) panamensis</i> promastigotes are schematically summarized. The decreasing response of the up-regulated gene transcripts with time is evident; the number of down-regulated genes is relatively consistent across the time points. B) The numbers of gene transcripts in common or differentially expressed at each time point. Gene transcript expression comparisons were made between infected and uninfected macrophages for each individual at the different time points. Gene transcripts with average intensity values less than 2× background intensity for both comparison groups, or fold change less than 1.5 fold (up or down-regulated) were excluded in further statistical analysis. A paired <i>t</i> test using p-value cutoff 0.05 was applied to determine if a gene was statistically differentially expressed.</p

Validation of transcript expression of <i>L. (V.) panamensis</i>-infected human monocyte-derived macrophages.

<p>Comparison of transcript expression in human macrophages infected with <i>L. (Viannia) panamensis</i> by quantitative real time PCR versus microarray analyses. Relative expression is expressed as a ratio of transcripts in infected cells over uninfected cells at 30 minutes or 4 hours after infection/interaction was determined. The values presented for the quantitative reverse transcriptase real time PCR analyses are the geometric mean of the ratios of macrophage transcripts from 6 to 7 different donors. The gene expression at each time point was normalized by the geometric mean of GAPDH and 39 S ribosomal protein L18 <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001866#pntd.0001866-Vandesompele1" target="_blank">[36]</a>. The genes listed are: IL1-β – interleukin-1β, TNFα – tumor necrosis factor-α, GMCSF2 –colony-stimulating factor 2 (granulocyte-macrophage), PTGS2 – prostaglandin-endoperoxide synthase 2, and IL6 – interleukin 6. The 95%CI are indicated between brackets.</p