Rhesus macaque-specific primers for real-time PCR

<p>Rhesus macaque-specific primers for real-time PCR</p

Relative CCL3 (A), CCL4 (B) and CCL5 (C) expression levels in GALT tissues at different stages of SHIV_SF162P4 infection (n = 5).

<p>PID 0 was used as the calibrator (value = 1).</p

Macrophages were also identified as CCL4+ cells.

<p>Macrophages (CD68+) are red, CCL4+ cells are green, overlap of red and green fluorescence is seen as yellow.</p

The proportions of peripheral blood CD3+CD4+CCL4+ and CD3+CD8+CCL4+ T lymphocytes were compared at PID 0 and PID 14 with SHIV_SF162P4 by flow cytometry (A).

<p>Gating was performed via CD3+ lymphocyte population. Statistically significant differences between CD3+CD4+CCL4+ and CD3+CD8+CCL4+ cells over CD3+ lymphocytes at PID 0 vs. PID 14 (n = 5) are shown (B).</p

SHIV_SF162P4 loads in plasma (A), CD4+ T cell counts in peripheral blood (B), relative CCL3 (C), CCL4 (D) and CCL5 (E) gene expression levels in PBMCs at PID 0-180 with SHIV_SF162P4.

<p>Each point on the graph represents the mean fold change in gene expression relative to pre-infection level±SE (n = 5).</p

Comparison of SHIV_SF162P4and SHIV_Ku1 primary infection in peripheral blood.

<p>Viral loads in plasma (A), CD4+ T cell counts (B), Fold-changes of CCL3, CCL4 and CCL5 gene expression levels in SHIV_SF162P4- and SHIV_Ku1–infected macaques at PID 0 vs. PID 14 (C) (n = 5 for each group). Negative sample cut-off for viral load measurements in plasma was <30 copies/ml e.g. PID 0 value.</p

Absolute (counts) and relative (%) levels of CCL4+ cells in MLN following SHIV_SF162P4 inoculation

a<p>Number of cells/0.5 mm² section.</p

Reduced Expression of CD27 by Collagenase Treatment: Implications for Interpreting B Cell Data in Tissues

<div><p>Surface markers have been used to identify distinct cell subpopulations and to delineate various stages of maturation or activation of lymphocytes. In particular CD27 is used for delineation of naïve and memory B cell populations, and is readily detected by flow cytometry. We here used flow cytometry to examine the expression of CD27 on lymphocytes isolated from various tissues of rhesus macaques, and found its expression was consistently low to absent on intestinal cell suspensions. However, immunohistochemistry revealed abundant CD27+ cells in intestinal tissue sections. Further investigation showed the marked loss of CD27 expression on processed intestinal cells was due to collagenase digestion of intestinal tissues, yet CD27 expression was recoverable within hours of cell isolation. By combining confocal microscopy, we confirmed that only a fraction of B cells express CD27, in contrast to expression on all T cells from tissues examined including the gut. Taken together, our results suggest that CD27 may be a memory marker for B cells, but not for T cells, since essentially all CD3 T cells expressed CD27. In summary, it is important to consider the influence of isolation procedures on cell surface expression of phenotypic markers, especially when examining tissue-resident lymphocytes by flow cytometry.</p></div

Immune response analysis using microarrays, RT-qPCR, cytokine assay and confocal microscopy.

<p>(<b>A</b>)<b>. Microarray</b>. Venn diagram shows the total number of genes perturbed in <i>Mtb</i>-H or <i>Mtb</i>-A infected- relative to uninfected-Rh-BMDMs. Total 226 genes (164 genes induced (UP), 62 genes repressed (DN) upon infection with <i>Mtb</i>-A; 2996 genes (1474 genes induced (UP), 1522 genes repressed (DN) upon infection with <i>Mtb</i>-H). Common genes (from up- or down-regulated gene dataset in both <i>Mtb</i>-H and -A group) are shown with overlap. For a description of the common genes e.g. RIPK4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095220#pone.0095220-Adams1" target="_blank">[42]</a>, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095220#pone.0095220.s002" target="_blank">Supplement S2</a>. Heat-map clusters; green, lower expression; red, higher expression. The data are shown from independent experiments with Rh-BMDMs isolated from two Rhesus macaques. (<b>B</b>) <b>RT-qPCR.</b> The relative fold change in transcripts <b>(</b><i>Mtb</i>-H infected Rh-BMDM to <i>Mtb</i>-A infected Rh-BMDM) in microarray (grey bars) and RT-qPCR (white bars) is shown. The relative fold change values (<i>Mtb</i>-H to <i>Mtb</i>-A) microarray and RT-qPCR (within bracket) are shown below; TNF, 5.3 (14.42); IL5, 3.49 (1.38); CASP8AP2, 2.93 (2.71); CXCL10, −5.7 (−8.7). (<b>C</b>) <b>Cytokine Assay and Multilabel confocal microscopy.</b> Measurements of TNF in supernatants, <i>Mtb</i>-H (red) or <i>Mtb</i>-A (green). Experiment was performed in triplicate and values were plotted using GraphPad Prism version 6.0b. The data is statistically significant; Student’s t-test, **P = 0.0027. Confocal microscopy shows secretion of TNF (green signal) detected only in the Rh-BMDMs (blue signal) infected with <i>Mtb</i>-H (red signal) (top panels) at 24 hr and 72 hr. The results are shown from Rh-BMDMs derived from two rhesus macaques.</p

Role of TNF in the Altered Interaction of Dormant <i>Mycobacterium tuberculosis</i> with Host Macrophages

<div><p><i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) persists within lung granulomas, despite being subjected to diverse stress conditions, including hypoxia. We hypothesized that the response of host phagocytes to <i>Mtb</i> experiencing hypoxia is radically altered and designed <i>in vitro</i> experiment to study this phenomenon. Hypoxia-stressed (<i>Mtb</i>-H) and aerobically grown <i>Mtb</i> (<i>Mtb</i>-A) were used to infect <u>Rh</u>esus Macaque <u>B</u>one <u>M</u>arrow <u>D</u>erived <u>M</u>acrophages (Rh-BMDMs) and the comparative host response to <i>Mtb</i> infection studied. Mechanistic insights were gained by employing RNAi. <i>Mtb</i>-H accumulated significantly lower bacterial burden during growth in Rh-BMDMs, concomitantly generating a drastically different host transcriptional profile (with only <2% of all genes perturbed by either infection being shared between the two groups). A key component of this signature was significantly higher TNF and apopotosis in <i>Mtb</i>-H- compared to <i>Mtb</i>-A-infected Rh-BMDMs. Silencing of TNF by RNAi reversed the significant control of <i>Mtb</i> replication. These results indicate a potential mechanism for the rapid clearance of hypoxia-conditioned bacilli by phagocytes. In conclusion, hypoxia-conditioned <i>Mtb</i> undergo significantly different interactions with host macrophages compared to <i>Mtb</i> grown in normoxia. These interactions result in the induction of the TNF signaling pathway, activation of apoptosis, and DNA-damage stress response. Our results show that <i>Mtb</i>-H bacilli are particularly susceptible to killing governed by TNF.</p></div