Chemokine receptor expression on cultured medullar macrophages from six single donors as analyzed by flow cytometry.

<p>The MFI values of CD14⁺⁺CD16⁺ monocytes before plating served as control ( = 100%).</p><p>Chemokine receptor expression on cultured medullar macrophages from six single donors as analyzed by flow cytometry.</p

Characterization of CD14⁺⁺CD16⁺ bone marrow monocytes by flow cytometry.

<p>(A) Representative contour plot of pooled BMC sample from three single donors showing a clear separation of two populations: CD14⁺⁺CD16⁺ (Q1) and CD14⁻CD16⁺⁺ (Q4). Gating was performed first in FSC/SSC dot plot following gating on CD45⁺ events in a SSC/CD45 dot plot. Finally, the expression of CD14 and CD16 was evaluated inside the CD45⁺ population. (B,C) Expression of monocyte-related antigens and representative histogram overlays with isotype control (gray filled) of crucial chemokine receptors on CD14⁺⁺CD16⁺ BMCs as analyzed by flow cytometry. Data are from 3 to 6 individual donors.</p

Functional analyses of CD14⁺⁺CD16⁺ BMC- and PBMC-derived macrophages.

<p>Representative flow cytometry histogram overlays for uptake of FITC-conjugated beads (A–C) and hydroethidine staining (D–F) in macrophages cultured from CD14⁺⁺CD16⁺ BMCs, fresh or cryopreserved PBMCs of three single donors. The gray filled histogram shows cells without beads or dye, respectively. (G,H) Phase contrast and immunofluorescent overlay of CD14⁺⁺CD16⁺ BMC-derived macrophages which were cultured in chamber slides and stained for α-tubulin-FITC/rhodamine-phalloidin after 7 days as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112140#s2" target="_blank">methods</a>. Cell nuclei were counterstained with DAPI. The scale bar indicates 40 µm. (I) Cluster formation and shape change of CD14⁺⁺CD16⁺ BM cells on Matrigel after 72 hours. Data in (G–I) are representative images of triplicates from pooled sample of three independent donors.</p

Side-by-side comparison of chemokine receptor expression on medullar with frozen or fresh blood monocytes.

<p>PBMCs from different donors were separated by density gradient centrifugation. Chemokine receptor expression was analyzed and compared on fresh monocyte subsets (gray bars) versus cryopreserved cells of the same donor (open bars) or unpaired medullar monocytes (black bars). *p<0.05, n = 6–10.</p

Percentages of blood monocyte subsets before and after freezing/thawing (n = 7–10).

<p>Percentages of blood monocyte subsets before and after freezing/thawing (n = 7–10).</p

Gene Expression Profiling in <i>Entamoeba histolytica</i> Identifies Key Components in Iron Uptake and Metabolism

<div><p><i>Entamoeba histolytica</i> is an ameboid parasite that causes colonic dysentery and liver abscesses in humans. The parasite encounters dramatic changes in iron concentration during its invasion of the host, with relatively low levels in the intestinal lumen and then relatively high levels in the blood and liver. The liver notably contains sources of iron; therefore, the parasite's ability to use these sources might be relevant to its survival in the liver and thus the pathogenesis of liver abscesses. The objective of the present study was to identify factors involved in iron uptake, use and storage in <i>E. histolytica.</i> We compared the respective transcriptomes of <i>E. histolytica</i> trophozoites grown in normal medium (containing around 169 µM iron), low-iron medium (around 123 µM iron), iron-deficient medium (around 91 µM iron), and iron-deficient medium replenished with hemoglobin. The differentially expressed genes included those coding for the ATP-binding cassette transporters and major facilitator transporters (which share homology with bacterial siderophores and heme transporters) and genes involved in heme biosynthesis and degradation. Iron deficiency was associated with increased transcription of genes encoding a subset of cell signaling molecules, some of which have previously been linked to adaptation to the intestinal environment and virulence. The present study is the first to have assessed the transcriptome of <i>E. histolytica</i> grown under various iron concentrations. Our results provide insights into the pathways involved in iron uptake and metabolism in this parasite.</p></div

A model summarizing our hypothesis for iron uptake and metabolism in <i>Entamoeba histolytica</i>.

<p>The pathways described in (I), (II), (III), and (IV) refer to potential routes for iron entry into cells. (I) Trophozoites are able to obtain iron from host proteins such as holotransferrin (H-Tf), hololactoferrin (H-Lf), ferritin (F), and Hb that bind to cell surface proteins. The complexes are internalized in clathrin-coated vesicles (for ferritin and holotransferrin) or caveolin-like coated vesicles (for hololactoferrin). The ligand-receptor complexes dissociate in the endosomes (early endosome; EE and late endosome; LE) and the iron bound to proteins is released in the lysosomes. It is not clear whether iron transporter proteins are degraded by cysteine proteinases (as has been suggested by López-Soto et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107102#pone.0107102-LpezSoto1" target="_blank">[13]</a>) or whether these transporters and cognate receptors are shuttled to the plasma membrane for reuse. (II) <i>E. histolytica</i> also acquires iron from Hb via heme internalization during the phagocytosis of human erythrocytes, which are degraded by hemolysin and phospholipases. It has been proposed that hemoglobinases and cysteine proteinases degrade Hb <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107102#pone.0107102-LpezSoto1" target="_blank">[13]</a>. The monooxygenase heme oxygenase degrades heme and releases iron; we suggest that a monooxygenase (EHI_009840) could be responsible for heme degradation in <i>E. histolytica.</i> (III) Heme can also be scavenged by secreted hemophores such as hemoglobin-binding proteins 45 and 26 (Ehhmbp 45 and Ehhmbp 26), which are able to bind Hb and heme <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107102#pone.0107102-CruzCastaeda1" target="_blank">[14]</a>. However, the mechanisms of hemophore export and heme-hemophore complex uptake are unknown (blue dashed arrows). An MFT (EHI_173950) and an ABC transporter (EHI_178050) may be involved in heme-hemophore uptake in <i>E. histolytica</i>. Once heme has entered the cell, it can be degraded by monooxygenase. (IV) Iron may be taken up by siderophores, since the ABC transporters, P-glycoprotein 5 transporters and the MFT family share homology with bacteria siderophore export systems. Lastly, a trans-plasma membrane electron transport (trans-PMET) system capable of transferring electrons from cytosolic electron donors to non-permeable electron acceptors may have an important role in iron reduction and acquisition (gray dashed arrow) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107102#pone.0107102-Bera1" target="_blank">[61]</a>. The above-listed mechanisms deliver iron to cells. The iron could then be used (for example) in iron-sulfur cluster (ISC) biosynthesis in the mitosomes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107102#pone.0107102-Maralikova1" target="_blank">[16]</a>. Heme synthesis has not been described in <i>E. histolytica</i>. Both GluRS and SAMS may be key sensors of iron status in this pathway. Ferroportin has not yet been identified in <i>E. histolytica</i>.</p

Differentially expressed genes relevant to oxidoreductase activity and sulfur-containing amino acid metabolism.

<p>For definition of columns please refer to footnote of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107102#pone-0107102-t002" target="_blank">Table 2</a>.</p><p>Differentially expressed genes relevant to oxidoreductase activity and sulfur-containing amino acid metabolism.</p

Differentially expressed genes relevant to other notable molecular functions.

<p>For definition of columns please refer to footnote of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107102#pone-0107102-t002" target="_blank">Table 2</a>.</p><p>Differentially expressed genes relevant to other notable molecular functions.</p

IRE-like sequences in differentially expressed genes.

<p>NM: non-modulated; CD: coding region; 5′UTR: 5′ untranslated region; H: high stringency; M: medium stringency, and L: low stringency.</p><p>IRE-like sequences in differentially expressed genes.</p