Phenotyping epithelial cells of normal rhesus macaques.

<p>(<b>A</b>) Representative expression of cytokeratin (CK; epithelial cell marker) and CD45 (leukocyte marker) in jejunum intestinal cells isolated from lower layer (“lymphocyte enriched”) of percoll gradient after treating with DTT and EDTA solution (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g001" target="_blank">Fig. 1</a>; Step IX). Note that 2.8% of cells were double positive for CK and CD45 receptors. Fractions of double negative CK−CD45− cells were also evident from isolated cells. Live cells were gated first from all acquired cells and plotted based on CK and CD45 markers. Visualization of epithelial cells both in (<b>B</b>) colon at 20× and (<b>C</b>) jejunum at 5× resolution were detected by immunohistochemistry staining using anti-CK monoclonal antibody with hematoxylin counterstain.</p

SIV infection induces apoptosis and upregulation of CD54 expression on intestinal epithelial cells (ECs).

<p>(<b>A</b>) Epithelial cells apoptosis was detected in jejunum by multi-labeled immunofluorescent confocal microscopy. Note that increased apoptosis of jejunum ECs was detected during acute (AV85; 21 days post infection) and chronic (HG58; 288 days post infection) SIV infection (as indicated by the white arrows). (<b>B</b>) Mean frequencies (± standard deviation) of surface CD54, CD80, CD86 and HLA-DR expression are shown for both jejunum ECs and CD45+ leukocytes from normal (n = 6) and chronically SIV-infected (n = 4) macaques. Note that a significant increase in CD54 expression on jejunum ECs was detected in SIV-infected macaques. However, increased expression of CD54, CD80 and HLA-DR on CD45+ leukocytes was observed in SIV-infected RMs. Statistically significant differences between each group of cells are shown.</p

Phenotyping colon and jejunum epithelial cells (ECs) by confocal microscopy.

<p>Essentially all colon (<b>A–E</b>) ECs (cytokeratin positive) were negative for (<b>A</b>) Ham56 (macrophage marker), and (<b>B</b>) CD11c (dendritic cell marker). Very few colon ECs were double positive for (<b>C</b>) both CD45 (leukocyte marker) and cytokeratin expression. A major population of colon ECs was positive for (<b>D</b>) HLA-DR expression. However, very few colon ECs were positive for (<b>E</b>) IL-10R expression which was evident in apical regions. Jejunum ECs were also negative for (<b>F</b>) CD54 (ICAM-1, cell adhesion marker). White arrow denotes the presence of double positive cells (cytokeratin and CD45/HLA-DR/IL-10R) for the specified sample and fluorochrome.</p

IL-10R expression in intestinal epithelial cells (ECs).

<p>(<b>A</b>) Left contour plot shows the IL-10R positive cells in the gated region from a normal healthy jejunum ECs. Jejunum ECs (open histogram) and isotype control (filled histogram) were also shown as histograms for expression of anti-IL-10R in the right panel. (<b>B</b>) Mean percentages (± standard deviation) of surface IL-10R expression are shown for both jejunum ECs and CD45+ leukocytes isolated from normal healthy rhesus macaques (n = 5). Statistical significant differences between each group of cells are shown.</p

Isolation of epithelial cells and leukocytes from intestinal tissues with EDTA and collagenase treatment.

<p>Representative dot plots of total cells from (<b>A</b>) upper layer of percoll density gradient isolated after EDTA treatment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g002" target="_blank">Fig. 2</a>, step VI); (<b>B</b>) lower layer of percoll density gradient isolated after EDTA treatment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g002" target="_blank">Fig. 2</a>, step VI); (<b>C</b>) upper layer of percoll density gradient isolated after collagenase treatment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g002" target="_blank">Fig. 2</a>, step X); and (<b>D</b>) lower layer of percoll density gradient isolated after collagenase treatment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g002" target="_blank">Fig. 2</a>, step X); in jejunum tissue showing distribution of epithelial cells (ECs; cytokeratin as an epithelial cell marker) and CD45 leukocytes in a normal uninfected healthy rhesus macaque. Each quadrant shows percentages of specified populations. Note increased percentage of ECs were isolated from both upper and lower layer cells of percoll density gradient isolated after EDTA treatment compared to other methods examined. Mean frequencies of cytokeratin (CK) positive, CD45 positive, double positive and double negative for CK and CD45 cell subsets are shown as box and whisker vertical bars for different isolation protocols as specified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g002" target="_blank">Fig. 2</a>. In summary all the specified bars represent (<b>E</b>) upper layer cells from percoll density gradient isolated after EDTA treatment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g002" target="_blank">Fig. 2</a>, step VI); (<b>F</b>) lower layer cells from percoll density gradient isolated after EDTA treatment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g002" target="_blank">Fig. 2</a>, step VI); (<b>G</b>) upper layer cells from percoll density gradient isolated after collagenase treatment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g002" target="_blank">Fig. 2</a>, step X); and (<b>H</b>) lower layer cells from percoll density gradient isolated after collagenase treatment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g002" target="_blank">Fig. 2</a>, step X); from jejunum in healthy, normal, uninfected rhesus macaques (n = 5). Note that in all isolation protocols, there was a variable amount of CD45+ and double negative CK−CD45− cells contamination observed. * Indicates significant differences between CK+CD45− and other different subsets of total cells within the specified isolation protocol.</p

Changes in chemokine levels in plasma in acute SHIVsf162p3 (filled circles) and SIVmac251-infected RMs (open squares).

<p>Means±SEM are shown. *Asterisks indicate significant differences between groups (P<0.05).</p

Schematic representation of epithelial cell and leukocyte isolation protocols from intestinal tissues after different enzymatic treatments.

<p>Note that this protocol explains cell isolation procedures with initial EDTA treatment. No prior DTT treatment has been used in this protocol.</p

Schematic representation of epithelial cell and leukocyte isolation protocols from intestinal tissues after different enzymatic treatments.

<p>Note that this protocol explains cell isolation procedures with initial DTT treatment.</p

Isolation of epithelial cells and leukocytes from intestinal tissues with DTT, EDTA and collagenase treatments.

<p>Representative dot plots of total cells from (<b>A</b>) DTT wash (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g001" target="_blank">Fig. 1</a>, step IV); (<b>B</b>) lower layer of percoll density gradient, isolated after EDTA treatment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g001" target="_blank">Fig. 1</a>, step IX); (<b>C</b>) lower layers of percoll density gradient, isolated after collagenase treatment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g001" target="_blank">Fig. 1</a>, step XIII) from jejunum tissue showing distribution of epithelial cells (ECs; cytokeratin as an epithelial cell marker) and CD45 leukocytes in a normal uninfected healthy rhesus macaque. Each quadrant shows percentages of specified cell populations. Note increased percentage of ECs were isolated from DTT wash compared to other methods examined. Mean frequencies of cytokeratin (CK) positive, CD45 positive, double positive and double negative for CK and CD45 cell subsets are shown as box and whisker vertical bars for different isolation protocols as specified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g001" target="_blank">Fig. 1</a>. In summary all the specified bars represent (<b>D</b>) cells isolated from DTT wash (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g001" target="_blank">Fig. 1</a>, step IV); (<b>E</b>) upper layer cells from percoll density gradient isolated after EDTA treatment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g001" target="_blank">Fig. 1</a>, step IX); (<b>F</b>) lower layer cells from percoll density gradient isolated after EDTA treatment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g001" target="_blank">Fig. 1</a>, step IX); (<b>G</b>) upper layer cells from percoll density gradient isolated after collagenase treatment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g001" target="_blank">Fig. 1</a>, step XIII); and (<b>H</b>) lower layer cells from percoll density gradient isolated after collagenase treatment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g001" target="_blank">Fig. 1</a>, step XIII) from jejunum in healthy, normal, uninfected rhesus macaques (n = 5). Note that in all isolation protocols, there was a variable amount of CD45+ and double negative CK−CD45− cells contamination observed. * Indicates significant differences between CK+CD45− and other different subsets of total cells within the specified isolation protocol.</p

Percentages of epithelial cells retrieved from different isolation protocols.

<p>Mean percentages (± standard deviation) of isolated cytokeratin (CK)+ CD45− cells from jejunum either after DTT, EDTA or collagenase treatment with or without prior DTT wash (as discussed in details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g001" target="_blank">Figs. 1</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g002" target="_blank">2</a>) are shown from uninfected normal healthy rhesus macaques (n = 5). Note that there was increased yield of ECs either by DTT or EDTA only treatment compared to other protocols followed in this experiment. Statistical significant differences between each group of cells are shown. * Indicates significant differences in ECs isolated from either DTT or EDTA only treatment compared to other group of cells.</p