tissue: Type II alveolar epithelial cells genotype: C57BL/6 background age: 8-12 weeks
Treatment protocol
Anesthetized mouse strains C57BL/6, Foxp3EGFP (C57BL/6 background), or Foxp3DTR (C57BL/6 background) received Escherichia coli LPS O55:B5 (3mg/kg) instilled intratracheally into the lungs. Type II alveolar epithelial cells from single cell suspension from the lungs were enriched by depleting CD31+ and CD45+ cells before sorted using a FACSAria (Becton Dickinson, San Jose, CA). Briefly, we utilized two transgenic strains, (Foxp3EGFP; Foxp3DTR Jackson Laboratory) to study differential transcript expression of type II alveolar epithelial cells in the presence (Foxp3EGFP) or absence (Foxp3DTR) of Foxp3+ Tregs. Foxp3EGFP mice express green fluorescent protein (GFP) downstream of the endogenous Foxp3 stop codon, resulting in fluorescence of all Foxp3+ Tregs. The Foxp3DTR mice express the human diphtheria toxin receptor (DTR) along with GFP, whose genes have been inserted into the 3’ untranslated region of the Foxp3 locus. These mice allow specific elimination of Foxp3+ Tregs in vivo through intraperitoneal (i.p.) administration of diphtheria toxin (DT). This results in apoptosis of Treg without significant systemic toxicity. By examining the effects of Treg depletion in the presence of other lymphocytes, we can better understand the cellular interactions that govern resolution of lung injury. At 7 days post LPS-induced injury, single cell suspensions were prepared from enzymatically digested and dissected mouse lungs from these three strains. The cells were washed with PBS containing 2 mM EDTA and 1.5% bovine serum albumin (FACS buffer). The cells were filtered through a 100 mM filter, and then the filter samples were subjected to negative selection by depleting CD31+ and CD45+ cells using MojoSort biotin nanobeads per manufacturer's protocols (Biolegend, San Diego, CA). The CD31-CD45- cell populations were resuspended in FACS buffer at a concentration of 2 x 107 cells/mL, and then subjected to cell sorting for CD326+ MHCII+CD104-CD24-T1a- cells, which are type II alveolar epithelial cells. Sorted cells were collected for mRNA analysis. Cells were kept at 4-8°C during staining and sorting.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using Zymo Direct-zol RNA MiniPrep with TRI Reagent miRNeasy kits from Zymo Research (Irvine, CA, USA) according to the manufacturer's instruction with the following modifications: 1) snap-frozen cells were lysed in at least 125 mL of TRI reagent without thawing cells; 2) cells were not centrifuged to collect debris or DNaseI-treated before applying to column. ALl samples were eluted in 25 mL of DNase/RNase-Free water.
Label
biotin
Label protocol
Total RNA (225 ng) was used to synthesize fragmented and labeled sense-strand cDNA and hybridize onto Affymetrx arrays. The Affymetrix HT WT User Manual was followed to prepare the samples. Briefly, the WT Expression HT Kit for Robotics (Ambion) was used to generate sense-strand cDNA from total RNA. Following synthesis of sense-strand cDNA, the cDNA was fragmented and labeled with the Affymetrix GeneChip HT Terminal Labeling Kit. The Beckman Coulter Biomek FXP Laboratory Automation Workstation with the Target Express set up was used to prepare the samples with these two kits.
Hybridization protocol
Fragmented and labeled cDNA was used to prepare a hybridization cocktail with the Affymetrix GeneTitan Hybridization Wash and Stain Kit for WT Arrays. Hybridization, washing, staining and scanning of the Affymtrix peg plate arrays was carried out using the Affymetrin GeneTitan MC Instrument. GeneChip Command Console Software (AGCC) was used for GeneTitan Instrument control.
Scan protocol
Scanning of the Affymetrix peg plate arrays was carried out using the Affymetrix GeneTitan MC Instrument. GeneChip Command Console Software (AGCC) was used for GeneTitan Instrument control.
Description
Control AT2 rep4 mRNA expression data from mouse lung regulatory T cells
Data processing
Affymetrix Expression Console Software was used for basic data analysis and quality control. Microarray data were preprocessed by RMA (Robust Multiarray Average) background correction, GC content and sequence correction, quantile normalization, and median polish summarization, and expression levels compared using ANOVA on log2 intensities to identify transcripts that were differentially expressed between genotype and treatment groups. Differentially expressed (DE) mRNAs were filtered at Benjamini-Hochberg FDR < 0.1, and fold change > 1.5, and expression patterns of DE transcripts were visualized using hierarchical clustering. Expression analyses were performed using Partek Genomics Suite (Partek Inc., St. Louis. MO, USA). Gene Set Enrichment Analysis (GSEA) was performed to identify significant Gene Ontology (GO) biological processes.
Transcriptional Analysis of Type II Alveolar Epithelial Cells in the Presence or Absence of Foxp3+ Regulatory T Cells during Experimental Acute Lung Injury Resolution
