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

Summary

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.

Abstract

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'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.

Introduction

Antigen presentation typically involves intracellular protein trafficking, which has been often investigated using morphological characterization and phenotypic profiling of antigen presenting cells^1,2,3. 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 ret....

Protocol

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

1. Total RNA extraction of BaP-exposed DCs
   1. Differentiate human DCs using cytokines GM-CSF and IL-4, and expose DCs to BaP for 4 days¹³.
   2. Sort BaP-exposed DCs using flow cytometry based on t....

Results

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 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 the .......

Discussion

Functional confirmation of an altered gene pathway is challenging, because of widely impacted gene expression involving multiple pathways and the difficulty to 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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Transcriptomics	Illumina	HiSeq 2500 v4	Illumina HiSeq system
ImagingStream X	Millipore	100220	Imaging flow cytometry
mirVana miRNA Isolation Kit	Thermo Fisher		Total RNA extraction
Agilent RNA 6000 Pico Kit	Agilent		Total RNA QC analysis
Veriti 96-Well Fast Thermal Cycler	Thermo Fisher		PCR, enzyme reaction
NEBNext Poly(A) mRNA Magnetic Isolation Module	New England Biolab		PolyA RNA purification
Automated SMARTer Apollo system	Takara		PolyA RNA purification
NEBNext Ultra RNA Library Prep Kit for Illumina	New England Biolab		Library preparation
Agencourt AMPure XP magnetic beads	Beckman Coulter		DNA purification
2100 Bioanalyzer	Agilent		Library QC, size distribution analysis
Agilent High Sensitivity DNA Kit	Agilent		Library QC, size distribution analysis
QuantStudio 5 Real-Time PCR System (Thermo Fisher)	Thermo Fisher		Library quantification
NEBNext Library Quant Kit	New England Biolab		Library quantification
cBot	Illumina		Library cluster generation
TruSeq SR Cluster Kit v3 - cBot – HS	Illumina		Library cluster generation
HiSeq 1000	Illumina		Sequencing
TruSeq SBS Kit v3 - HS (50-cycles)	Illumina		Sequencing
Phycoerythrin/Cyanine 7 (PE/Cy7)	Bio Legend	L243
Phycoerythrin/Cyanine 5 (PE/Cy5)	Bio Legend	3.9
Brilliant violet 421	Bio Legend	L161
PE-anti-mouse IgG2b	Bio Legend	51.1
Alexa Fluor 647 (AF647)	Bio Legend	CD107a(H4A3)