Name: integrate imaging flow cytometry and transcriptomic profiling to evaluate altered endocytic cd1d trafficking
Uploaded: 2018-10-29T13:00:00
Description: Watch this Scientific Journal Video about Integrate Imaging Flow Cytometry and Transcriptomic Profiling to Evaluate Altered Endocytic CD1d Trafficking at JoVE.com

The authors thank Robert Giulitto (Hoxworth blood center) for human blood samples and Dr. Liang Niu for the normalization of gene expression reads. We also thank grant support from the National Institute of Environmental Health Sciences (ES006096), Center for Environmental Genetics (CEG) pilot project (S.H.), National Institute of Allergy and Infectious Diseases (AI115358) (S.H.), University of Cincinnati University Research Council award (S.H.), and University of Cincinnati College of Medicine Core Enhancement Funding (X. Z.).

Human protocols in this study were approved by the Institutional Review Board of the University of Cincinnati and all methods were performed in accordance with the relevant guidelines and regulations. Blood samples from healthy donors were obtained from the Hoxworth Blood Center at the University of Cincinnati Medical Center.
1. Transcriptomic Profiling of Pollutant-exposed Human Monocyte-derived DCs
<ol>
	<li>Total RNA extraction of BaP-exposed DCs
	<ol>
		<li>Differentiate human DCs using ...

<ol>
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Functional confirmation of an altered gene pathway is challenging, because of widely impacted gene expression involving multiple pathways and the difficulty to&#160;integrate individual and populational cellular activities. We employed imaging flow cytometry to specifically test CD1d trafficking in endocytic compartments. Imaging flow cytometry integrates the populational measurement of cells and the individual demonstration of subcellular colocalization of multiple proteins. Confocal microscopy has previously provided h...

Antigen presentation typically involves intracellular protein trafficking, which has been often investigated using morphological characterization and phenotypic profiling of antigen presenting cells1,2,3. To integrate the advantages of imaging and phenotyping methods, we describe an imaging analysis platform at both single cell and population levels to demonstrate an altered protein colocalization in human dendritic cells (DCs). In peptide antigen presentation, major histocompatibility complex (MHC) class I molecules bind a short peptide (8-10 residues) in the endoplasmic reticulum to activate conventional CD8+ T cells, while MHC class II molecules bind a relatively longer peptide (~20 residues) in endocytic compartments to activate conventional CD4+ T cells1,4. In contrast, lipid-specific T cells are activated by CD1 proteins with lipid antigens loaded mainly in endocytic compartments5,6. Lipid antigen presentation requires the supply of lipid metabolites produced in lipid metabolism7,8,9,10 and the loading of functional lipid metabolites to CD1 proteins in endocytic compartments5,6. In this context, various cellular factors modulating lipid antigen presentation, especially in environmental exposure of lipophilic pollutants and immune disorders, are critical to be defined. In this study, we used transcriptomic analysis, image profiling, and cellular and populational imaging analysis of human monocyte-derived DCs to determine the endocytic protein trafficking contributing to lipid antigen presentation in pollutant exposure. Certainly, this combined platform can be applied to studying subcellular protein trafficking and colocalization in different biological processes.
Technically, subcellular protein localization was usually demonstrated using confocal microscopy and statistically analyzed in a limited number of detected cells1,2,3,11. Moreover, flow cytometry has been broadly applied to gate cell populations with co-stained signals of multiple proteins at a cellular level12; however, this lacks a detailed visualization of subcellular protein colocalization. To achieve comprehensive and statistical analyses of percentage protein colocalization at both cellular and population levels, we incorporate imaging profiling and analysis approaches to determine the features of protein colocalization with biological relevance. Specifically, we use imaging flow cytometry to detect the colocalization of CD1d and lysosomal-associated membrane protein 1 (Lamp1) proteins in this study. Quantitative analysis of colocalized molecules was previously difficult to be performed at a populational scale. In this study, we adapted the ImageJ-Fiji program to examine the percentage of protein colocalization with a large number of co-stained cells at both populational and individual cellular levels. Specifically, we measured co-localized areas, intensity, and populational size to support the conclusion that the CD1d protein was largely retained in late endocytic compartments of human DCs in exposure to a lipophilic pollutant benzo[a]pyrene (BaP)13. This combined cellular and population imaging analysis provided highly reproducible, comprehensive, and statistically significant results of CD1d-Lamp1 colocalization relevant to inhibited lipid antigen presentation.
The transcriptome of BaP-exposed human DCs strongly supported the hypothesis that endocytic lipid metabolism and CD1d endocytic trafficking were impaired in BaP exposure. To test this hypothesis, we applied imaging flow cytometry to profile the images of DC population that were co-stained with multiple proteins including CD1d, endocytic markers, and DC markers. Finally, co-stained cells were statistically analyzed to demonstrate the percentage of intensity and areas of CD1d and Lamp1 colocalization.

<table border="0" cellpadding="0" cellspacing="0" style="width:1340px;" width="1340">
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:284px;">Transcriptomics</td>
			<td style="width:448px;">Illumina</td>
			<td style="width:417px;">HiSeq 2500 v4</td>
			<td style="width:191px;">Illumina HiSeq system</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:284px;">ImagingStream X</td>
			<td style="width:448px;">Millipore</td>
			<td>100220</td>
			<td>Imaging flow cytometry</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">mirVana miRNA Isolation Kit</td>
			<td style="width:448px;">Thermo Fisher</td>
			<td></td>
			<td>Total RNA extraction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Agilent RNA 6000 Pico Kit</td>
			<td style="width:448px;">Agilent</td>
			<td></td>
			<td>Total RNA QC analysis</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Veriti 96-Well Fast Thermal Cycler</td>
			<td style="width:448px;">Thermo Fisher</td>
			<td></td>
			<td>PCR, enzyme reaction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">NEBNext Poly(A) mRNA Magnetic Isolation Module&#160;</td>
			<td style="width:448px;">New England Biolab</td>
			<td></td>
			<td>PolyA RNA purification</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Automated SMARTer Apollo system</td>
			<td style="width:448px;">Takara</td>
			<td></td>
			<td>PolyA RNA purification</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">NEBNext Ultra RNA Library Prep Kit for Illumina</td>
			<td style="width:448px;">New England Biolab</td>
			<td></td>
			<td>Library preparation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Agencourt AMPure XP magnetic beads</td>
			<td style="width:448px;">Beckman Coulter</td>
			<td></td>
			<td>DNA purification&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">2100 Bioanalyzer</td>
			<td style="width:448px;">Agilent</td>
			<td></td>
			<td>Library QC, size distribution analysis</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Agilent High Sensitivity DNA Kit</td>
			<td style="width:448px;">Agilent</td>
			<td></td>
			<td>Library QC, size distribution analysis</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">QuantStudio 5 Real-Time PCR System (Thermo Fisher)</td>
			<td style="width:448px;">Thermo Fisher</td>
			<td></td>
			<td>Library quantification</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">NEBNext Library Quant Kit</td>
			<td style="width:448px;">New England Biolab</td>
			<td></td>
			<td>Library quantification</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">cBot</td>
			<td style="width:448px;">Illumina</td>
			<td></td>
			<td>Library cluster generation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">TruSeq SR Cluster Kit v3 - cBot &#8211; HS</td>
			<td style="width:448px;">Illumina</td>
			<td></td>
			<td>Library cluster generation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">HiSeq 1000</td>
			<td style="width:448px;">Illumina</td>
			<td></td>
			<td>Sequencing</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">TruSeq SBS Kit v3 - HS (50-cycles)</td>
			<td style="width:448px;">Illumina</td>
			<td></td>
			<td>Sequencing</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:284px;">Phycoerythrin/Cyanine 7 (PE/Cy7)</td>
			<td style="width:448px;">Bio Legend</td>
			<td style="width:417px;">L243</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:284px;">Phycoerythrin/Cyanine 5 (PE/Cy5)</td>
			<td style="width:448px;">Bio Legend</td>
			<td align="right" style="width:417px;">3.9</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:284px;">Brilliant violet 421</td>
			<td style="width:448px;">Bio Legend</td>
			<td style="width:417px;">L161</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:284px;">PE-anti-mouse IgG2b</td>
			<td style="width:448px;">Bio Legend</td>
			<td align="right" style="width:417px;">51.1</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:284px;">Alexa Fluor 647 (AF647)</td>
			<td style="width:448px;">Bio Legend</td>
			<td style="width:417px;">CD107a(H4A3)</td>
			<td></td>
		</tr>
	</tbody>
</table>

integrate imaging flow cytometry and transcriptomic profiling to evaluate altered endocytic cd1d trafficking

Populational analyses of the morphological and functional alteration of endocytic proteins are challenging due to the demand of image capture at a single cell level and statistical image analysis at a populational level. To overcome this difficulty, we used imaging flow cytometry and transcriptomic profiling (RNA-seq) to determine altered subcellular localization of the cluster of differentiation 1d protein (CD1d) associated with impaired endocytic gene expression in human dendritic cells (DCs), which were exposed to the common lipophilic air pollutant benzo[a]pyrene. The colocalization of CD1d and endocytic marker Lamp1 proteins from thousands of cell images captured with imaging flow cytometry was analyzed using IDEAS and ImageJ-Fiji programs. Numerous cellular images with co-stained CD1d and Lamp1 proteins were visualized after gating on CD1d+Lamp1+ DCs using IDEAS. The enhanced CD1d and Lamp1 colocalization upon BaP exposure was further demonstrated using thresholded scatterplots, tested with Mander&#39;s coefficients for co-localized intensity, and plotted based on the percentage of co-localized areas using ImageJ-Fiji. Our data provide an advantageous instrumental and bioinformatic approach to measure protein colocalization at both single and populational cellular levels, supporting an impaired functional outcome of transcriptomic alteration in pollutant-exposed human DCs.

The lipophilic pollutant BaP alters endocytic gene clusters in human DCs. Human monocyte-derived DCs from each donor (n=3) were incubated with BaP and sorted for&#160;conventional DCs, which were further used for RNA extraction and transcriptomic analysis as described. Upon the normalization of gene expression, altered genes between BaP-exposed and non-exposed groups were clustered according to the functional correlation of differentially expressed genes. We input the altered gene list to...

Watch this Scientific Journal Video about Integrate Imaging Flow Cytometry and Transcriptomic Profiling to Evaluate Altered Endocytic CD1d Trafficking at JoVE.com

Integrate Imaging Flow Cytometry and Transcriptomic Profiling to Evaluate Altered Endocytic CD1d Trafficking

Imaging flow cytometry provides an ideal approach to detect the morphological and functional alteration of cells at individual and populational levels. Disrupted endocytic function for lipid antigen presentation in pollutant-exposed human dendritic cells was demonstrated with a combined transcriptomic profiling of gene expression and morphological demonstration of protein trafficking.

Populational analyses of the morphological and functional alteration of endocytic proteins are challenging due to the demand of image capture at a single ...

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