Plasma membrane protein signatures of myeloid cells identify unique cell functions.

<p>Gene ontology analysis of plasma membrane proteins enriched in M1 macrophages, M2 macrophages, all macrophage types (BmM, M1, and M2), and BmDCs identifies functional categories of proteins enriched in each cell type (<i>p</i><0.05 with Benjamini-Hochberg correction). The top three functional annotations are presented for each cell type along with three representative proteins.</p

The plasma membrane proteome of macrophages.

<p><b><i>Panel A:</i></b> Bone marrow-derived macrophages (BmM) were derived from bone marrow precursor cells of C57BL/6 mice cultured with M-CSF. Classically activated macrophages (M1) and alternatively activated macrophages (M2) were derived from BmMs by treatment with IFN-γ and LPS or with IL-4. <b><i>Panel B:</i></b> qRT-PCR of markers used to detect M1 and M2 macrophages. Results (means and SEMs, N = 6) were standardized to 18S, expressed relative to the cell type with the highest expression of each gene, and are representative of 3 independent analyses. <b><i>Panel C:</i></b> LC-ESI-MS/MS analysis of plasma membrane proteins isolated from differentially activated macrophages. Proteins were quantified by spectral counting (total number of peptides identified for a given protein) and subjected to sequential criteria to identify 192 plasma membrane proteins that were reproducibly detected with high confidence. <b><i>Panel D:</i></b> Quantification of the membrane proteomes of M1 and M2 macrophages. Differentially expressed proteins (red, upregulated; green, downregulated; gray, not significantly different) were identified based on <i>t-</i>test and <i>G-</i>test statistics. Significance cutoffs (dashed lines; <i>p</i><0.05 and <i>G-</i>statistic >1.5 or <−1.5) were determined based on permutation analysis (estimated FDR<5%). <b><i>Panel E:</i></b> Quantification of the membrane proteomes of M1 macrophages and BmMs. Proteins differentially expressed by M1 cells relative to both BmMs and M2 cells are indicated with colored dots (red, upregulated; green, downregulated). Proteins differentially expressed by M1 and M2 cells (<i>Panel D</i>) but not differentially expressed by M1 and BmMs are indicated by gray dots. <b><i>Panel F:</i></b> Examples of proteins that distinguish M1 cells from both BmM and M2 cells (CSF1R, ITGAL). Results (N = 6 per group) are means and SDs. <b><i>Panel G:</i></b> Examples of proteins that fail to distinguish M1 cells from both BmM and M2 cells (CD14, ITGAV). <b><i>Panel H:</i></b> Plasma membrane proteins differentially expressed by M1 cells (36 proteins), M2 cells (35 proteins), and BmMs (17 proteins).</p

Immunocytochemical detection of plasma membrane protein markers.

<p>Expression levels of widely used plasma membrane protein markers (<b><i>Panels A–B</i></b>) and newly identified markers (<b><i>Panels C–D</i></b>) of M1 cells, M2 cells, BmMs, and BmDCs were assessed by mass spectrometry (<b><i>Panels A,C</i></b>) and immunocytochemistry (<b><i>Panels B,D</i></b>). For MS/MS, proteins were quantified by spectral counting and expressed relative to the cell type with the highest expression level for each protein. Results are means and SDs. Cells were stained with antibodies specific to each protein (red channel), counterstained with DAPI to visualize nuclei (blue-channel), and examined by confocal microscopy. Immunostaining and microscopy were performed on the same day with identical microscope settings. Results are representative of 3 independent analyses.</p

Analysis of eMPCs harvested from wild-type and GM-CSF-deficient (<i>Csf2−/−</i>) mice.

<p>eMPCs isolated from wild-type (<i>wt</i>) and <i>Csf2</i>−/− mice were interrogated for cell number, function, and protein expression. <b><i>Panel A–B:</i></b> Accumulation of eMPCs 3 days (<i>Panel A</i>) and 5 days (<i>Panel B</i>) following intraperitoneal injection with thioglycolate. Results (N = 6) are means and SEMs. <b><i>Panel C:</i></b> Plasma membrane proteomic analysis of eMPCs isolated from <i>Csf2</i>−/− and wild-type mice. Differentially-expressed proteins were identified using the <i>t-</i>test and <i>G-</i>test (<i>p</i><0.05 and <i>G-</i>statistic >1.5) and quantified using the spectral index. <b><i>Panel D:</i></b> Proteins differentially expressed by eMPCs isolated from <i>Csf2</i>−/− mice (see <i>Panel C</i>) were measured in BmMs and BmDCs and quantified using the spectral index. <b><i>Panel E:</i></b> Cell surface CD11c and F4/80 expression on eMPCs was assessed by flow cytometry. Results are presented as contour plots with 10% probability increments. <b><i>Panel F:</i></b> Phagocytosis of fluorescein-labeled <i>E. coli</i> by eMPCs. Results (arbitrary units, AU; N = 4) are means and SEMs. <b><i>Panel G–H:</i></b> Antigen cross-presentation by eMPCs. Ovalbumin (0.2 mg/mL)-treated eMPCs were incubated with CFSE-labeled spleen cells isolated from OT-I transgenic mice. Levels of CFSE were assessed in OT-I T cells selected by flow cytometry and expression levels of CD8 and Vb5 (<i>Panel G</i>). The division index was calculated using FlowJo software. Results (N = 4) are means and SEMs (<i>Panel H</i>). Where applicable, <i>p</i>-values were derived using a two-tailed Student's <i>t-</i>test. Results obtained for eMPC quantification, flow cytometry, phagocytosis and antigen cross-presentation are representative of 3 independent analyses.</p

Mass spectrometric and immunohistochemical staining of thioglycolate-elicited peritoneal cells (eMPCs), polarized macrophages, and DCs.

<p><b><i>Panels A–B:</i></b> Hierarchical cluster analysis of eMPCs. Cells were harvested from the peritoneal cavity of C57BL/6J-<i>Ldlr^tm1Her</i> mice 5 days after intraperitoneal injection with thioglycolate. Isolated plasma membrane proteins detected by LC-MS/MS analysis of eMPCs were subjected to hierarchical cluster analysis, using the 107 proteins identified as differentially expressed by myeloid cells generated <i>in vitro</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033297#pone-0033297-g002" target="_blank"><b>Fig. 2</b></a>). <b><i>Panel B:</i></b> Relationships among eMPCs, M1 cells, M2 cells, BmMs, and DCs, as determined by cluster analysis. <b><i>Panel C:</i></b> Protein expression in eMPCs, M1 cells, M2 cells, BmMs, and BmDCs. Protein levels were quantified by MS/MS and spectral counting. Data are presented as means and SDs. <b><i>Panels D–E:</i></b> Flow cytometric analysis of CD11b, CD11c, and F4/80 in eMPCs. Results are presented as contour plots with 10% probability increments. <b><i>Panel F:</i></b> qRT-PCR analysis of M1 marker genes (<i>Nos2</i>, <i>Il12b</i>, <i>Tnfa</i>) in M1 macrophages, BmDCs, and eMPCs. Results (means and SEMs; N = 6) were standardized to 18S levels and expressed relative to M1 macrophages. <b><i>Panel G:</i></b> Immunostaining of eMPCs. Cells were stained with antibodies (red channel) to plasma membrane proteins differentially expressed by BmDCs (MBC2, FER1L), BmMs (ITGA6, STAB1), M2 cells (TFRC, ITGB5), and M1 cells (CD11a, CD40). Nuclei were visualized by DAPI staining (blue channel). Immunostaining and microscopy were performed on the same day and with identical microscope settings to experiments presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033297#pone-0033297-g003" target="_blank"><b>Fig. 3D</b></a> and <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033297#pone.0033297.s003" target="_blank">Fig. S3</a></b>. Results obtained for flow cytometry, qRT-PCR, and immunocotyochemistry are representative of 3 independent analyses.</p

The plasma membrane proteome of bone marrow-derived dendritic cells (DCs).

<p><b><i>Panel A:</i></b> Bone marrow-derived dendritic cells (BmDCs) were obtained by culturing bone marrow cells with GM-CSF. <b><i>Panel B:</i></b> Flow cytometric analysis of CD11c and F4/80 expression in BmDCs and BmMs. Results are presented as a contour plot with 10% probability increments. <b><i>Panel C:</i></b> Cell-surface exp ression of MHC-II by BmDCs and BmMs as assessed by flow cytometry. <b><i>Panel D:</i></b> The plasma membrane proteome of DCs. <i>Upper Panel:</i> Proteins expressed at similar levels by DCs and either M1 cells, M2 cells, or BmMs. <i>Lower Panel:</i> Proteins differentially expressed by DCs relative to M1 cells, M2 cells, and BmMs (<i>G-</i>test>1.5 or <−1.5 and <i>t-</i>test: <i>p</i><0.05). Red, upregulated; green, downregulated. <b><i>Panel E:</i></b> Examples of proteins expressed at similar levels by DCs and either M1 cells, or M2 cells. Results (N = 6 per group) are means and SDs. <b><i>Panel F:</i></b> Examples of plasma membrane proteins differentially expressed by DCs. Flow cytometry experiments are representative of 3 independent analyses.</p

100 µg/day/mouse L4F mimetic peptide and apoA-I overexpression do not affect plasma total cholesterol or triglycerides.

<p>Total plasma cholesterol and triglycerides are shown for the peptide (A,C; n = 5) and the apoA-I overexpression study (B,D, n = 9–13) after 4 weeks on the diet and at the end of the study protocol. FPLC cholesterol analysis on pooled plasma samples from the peptide study (E) and the apoA-I overexpression study (F). Differences in cholesterol eluting times, as noted by the different starting peaks for cholesterol, are due to a newer FPLC column used with the apoA-I overexpression study. No significant differences were identified. *P<0.05 4 week versus 12 weeks on diet peptide study, ***P<0.0001 4 week vs 16 weeks on diet apoA-I study by paired two-way ANOVA.</p

100 µg/day/mouse L4F did not attenuate inflammation, and neither L4F nor apoA-I overexpression improved glucose tolerance.

<p>SAA was measured in the plasma (A, n = 5) and inflammatory gene expression was analyzed in epididymal adipose tissue extracts (B, n = 4–5). GTTs are shown for the peptide study (C: n = 5) and the apoA-I overexpression study (D: n = 10–12). Plasma was collected at the 30 minute time point of the GTT and insulin was measured in both studies (E, F). No significant differences were noted.</p

100 µg/day/mouse L4F mimetic peptide and apoA-I overexpression do not affect weight gain.

<p>Body weights for the peptide study (A; n = 5) and the apoA-I overexpression study (B; n = 15–17). No significant differences were identified.</p

L4F mimetic peptide blocks palmitate-induced chemokine factor expression in 3T3-L1 adipocytes.

<p>MCP-1 (A) and SAA3 (B) gene expression in 3T3-L1 cells exposed to control (fatty acid free albumin) or palmitate (250 µmol/l). Prior to fatty acid exposure cells were pre-incubated with control, HDL (50 µg/ml), and L4F (25 µg/ml) containing media. Fold gene expression was determined using the ΔΔCT fold change method normalized to GAPDH. Each graph is representative of three independent experiments. ***P<0.001 vs control in palmitate treated cells using 2-way ANOVA and Bonferroni adjustment for multiple comparisons.</p