DOI: 10.5061/dryad.8931zcrzx
title: Single cell transcriptomics unveiled that early life BDE-99 exposure reprogrammed the gut-liver axis to promote a pro-inflammatory metabolic signature in male mice at late adulthood (Part 2/2)

description: Polybrominated diphenyl ethers (PBDEs) are legacy flame retardants that
 bioaccumulate in the environment.  The gut microbiome is an
 important regulator of liver functions including xenobiotic
 biotransformation and immune regulation.  We recently showed that
 neonatal exposure to BDE-99, a human breast milk-enriched PBDE congener,
 up-regulated pro-inflammation- and down-regulated drug metabolism-related
 genes predominantly in males in young adulthood.  However, it
 remains unknown regarding the persistence of dysregulation into late
 adulthood, differential impact of hepatic cell types, and the involvement
 of the gut microbiome from neonatal BDE-99 exposure.  To address
 these knowledge gaps, male C57BL/6 mouse pups were orally exposed to corn
 oil (10 ml/kg) or BDE-99 (57 mg/kg) once daily from postnatal days
 2-4.  At 15 months of age, neonatal BDE-99 exposure
 down-regulated key xenobiotic- and lipid metabolizing enzymes and
 up-regulated genes involved in microbial influx in hepatocytes.
  Neonatal BDE-99 exposure also persistently increased the hepatic
 proportion of neutrophils and led to a predicted increase of macrophage
 migration inhibitory factor signaling. This was associated with decreased
 intestinal tight junction protein (Tjp) transcripts, persistent dysbiosis,
 and dysregulation of inflammation-related metabolites.  ScRNA-seq
 using germ-free (GF) mice demonstrated the necessity of a normal gut
 microbiome in maintaining hepatic immunotolerance. Microbiota transplant
 to GF mice using large intestinal microbiome from adults neonatally
 exposed to BDE-99 down-regulated Tjp transcripts and up-regulated several
 cytokines in the large intestine.  In conclusion, neonatal BDE-99
 exposure reprogrammed cell type-specific gene expression and communication
 in liver towards pro-inflammation, and BDE-99-mediated pro-inflammatory
 signatures may be partly due to the dysregulated gut environment.
description: <strong>Chemicals and dosing
 regimen</strong> 2,2’,4,4’,5-pentabromodiphenyl
 ether (BDE-99 [CAS No. 60348-60-9]) was purchased from AccuStandard, Inc.
 (New Haven, Connecticut).  Corn oil was purchased from Sigma-Aldrich (St.
 Louis, MO).  BDE-99 was dissolved in corn oil and filtered using a 0.22 μm
 Millipore Express Plus Membrane filter (EMD Millipore, Temecula,
 California).  All mice used in this study were housed according to the
 Association for Assessment and Accreditation of Laboratory Animal Care
 International guidelines.  The study was approved by the University of
 Washington Institutional Animal Care and Use Committee.  Eight-week-old
 specific pathogen-free C57BL/6J mice were purchased from the Jackson
 Laboratory (Bay Harbor, Maine), and were acclimated to the animal housing
 facility at the University of Washington for at least three breeding
 generations.  Mice were housed in standard air-filtered cages using
 autoclaved bedding (autoclaved Enrich-N’Pure, Andersons, Maumee, OH). 
 Mice had <em>ad libitum</em> access to non-acidified
 autoclaved water, as well as standard rodent chow (LabDiet No. 5021 for
 breeding pairs or to LabDiet No. 5010 for weaned pups) (LabDiet, St.
 Louis, MO).  From postnatal day (PND) 2 to PND 4, male pups were
 supralingually exposed to BDE-99 (57 mg/kg, n = 5) or corn oil (vehicle
 control, 10 ml/kg, n = 3) once daily for three consecutive days.  Litters
 and cages were randomly assigned to each exposure group.  Pups were weaned
 at PND 21.  At 15 months of age, serum, liver, small and large intestines,
 and fresh stool were collected. The remaining livers were subject to
 dissociation.  To determine the role of the microbiome in regulating
 hepatic cell types, livers from 7~8-month-old conventional (CV) or
 germ-free (GF) mice were collected. 
 <strong>Whole liver dissociation  </strong>
 Fresh whole livers rinsed in Dulbecco's phosphate-buffered
 saline (DPBS) (Cat# 14190136, Thermo Fisher Scientific, Waltham, MA) were
 minced to 1 – 3 mm pieces using surgical scissors.  The minced livers were
 then placed in 15 mL conical tubes containing 10 mL of DPBS.  Using
 serological pipettes, the DPBS was replaced by 10 mL of dissociation
 enzyme mixtures containing Liberase (Cat# 540115001, Sigma Aldrich, St.
 Louis, MO) and Dispase II (Cat# D4693-1G, Sigma Aldrich, St. Louis, MO)
 dissolved in Hanks' Balanced Salt Solution (Cat# 14025-092, Thermo
 Fisher Scientific, Waltham, MA).  The tubes with the livers and enzyme mix
 were incubated at 37 °C for 40 minutes using a Roto-Therm H2020 incubator
 (Benchmark Scientific Inc., Sayreville, NJ).  After the incubation, the
 cells and un-dissociated fragments were strained using a 40-micron cell
 strainer.  Strained cells were centrifuged at 300 g for 5 minutes at 4 °C
 and re-suspended in 5 mL red blood cell lysis buffer on ice for 8
 minutes.  Cells were centrifuged again at 300 g for 5 minutes at 4 °C. 
 Dead and dying cells were filtered using the Dead Cell Removal Kit
 (Miltenyi Biotec, Cologne, Germany) following the manufacturer’s
 instructions.  The viability was checked using a hemocytometer under a
 light microscope (Labophot-2, Nikon, Tokyo, Japan).  Samples with greater
 than 85% viability were then cryopreserved using 10% Dimethylsulfoxide
 (Cat# BP231-100, Thermo Fisher Scientific, Waltham, MA) and 90% fetal
 bovine serum (Cat# F2442-50ML, Sigma Aldrich, St. Louis, MO) until further
 analysis.  <strong>Single cell RNA
 sequencing</strong> Cryopreserved cells were
 thawed using a water bath at 37 °C for 2 minutes, followed by serial
 dilution in DPBS until 32 mL was reached.  Cells were centrifuged and
 re-suspended in DPBS until a concentration of approximately 100 cells/ μL
 was reached.  The re-suspended cells (n=3), targeting 10,000 cells per
 sample, were then subject to scRNA-seq using a Chromium Next GEM single
 cell 3’ v3.1 kit and a Chromium X controller (10X Genomics, Pleasanton,
 CA) following the manufacturer’s instructions.  The created libraries were
 then sequenced using the NovaSeq platform at paired-end 150 bp (~11 M
 reads per sample).  <strong>Data analysis of
 single cell RNA sequencing </strong> Raw data
 were processed using the Cell Ranger v7.0 (10X Genomics, Pleasanton, CA). 
 Processed data were read into R version 4.2.2 (R Core Team,
 2022) for further analyses.  Filtering and normalization were
 performed using the default parameters using Seurat v4 (Hao
 <em>et
 al.</em>,
 2021).  Clustering was performed using the first 35 principal
 components, and the standard deviation obtained through principal
 component analysis was 1.5.  Cell type labeling was performed using the
 differentially expressed genes using the function FindAllMarkers with
 default parameters in Seurat.  Cell-cell communication analysis was done
 using the CellChat (v. 1.5) package (Jin,
 2022).  Gene ontology enrichment was done using the TopGO
 (v.2.48.0) package (Alexa A,
 2022) using all detected genes as the background.  Heatmaps were
 made using the ComplexHeatmaps (v. 2.13.1) package.  All plots other than
 heatmaps were created using ggplot2 (v. 3.3.6).   
 <strong>Liver Hematoxylin and Eosin
 staining</strong> A fraction of the liver from
 each mouse was preserved in 4% formalin (Cat# SF100-4, Thermo Fisher
 Scientific, Waltham, MA) followed by 70% ethanol for histology. 
 Hematoxylin and Eosin (H&E) staining was performed by the
 University of Washington Histology Core in the Department of Comparative
 Medicine.  Briefly, all staining procedures were performed in a
 well-ventilated area with personal protective equipment.  Liver slides
 were prepared, dried overnight, and placed in the oven at 60˚C for
 approximately 30 minutes.  The slides were stained in the following
 protocol: xylene for 5 minutes, rinsed in 100% ethanol for 4 minutes, 95%
 ethanol for 2 minutes, de-ionized (DI) water for 1 minute, hematoxylin for
 3.5 minutes, DI water for 1 minute, clarifier for 30 seconds, DI water for
 1 minute, bluing for 1 minute, DI water for 1 minute, eosin for 15
 seconds, 95% ethanol for 1.5 minutes, 100% ethanol for 2 minutes, and
 xylene for 4 minutes.  Images were then taken (Nanozoomer HT-9600,
 Hamamatsu Photonics, Japan), and histological incidence and severity
 scoring was performed by a board-certified veterinary
 pathologist.  <strong>Metagenomic shotgun
 sequencing</strong> DNA from large intestinal
 content was extracted using the EZNA Stool DNA kit (Omega Bio-Tek Inc.,
 Norcross, GA).  Shallow shotgun metagenomic sequencing was performed at 2
 million reads (Diversigen, New Brighton, MN).  DNA sequences were aligned
 to a curated database containing all representative genomes in RefSeq for
 bacteria with additional manually curated mouse-specific Metagenomically
 Assembled Genomes (MAGs) and cell-cultured genomes. Only high-quality MAGs
 (Completeness &gt; 90% & Contamination &lt; 5% via checkm)
 were considered.  Alignments were made at 97% identity against all
 reference genomes.  Every input sequence was compared to every reference
 sequence in the Diversigen DivDB-Mouse database using fully gapped
 alignment with BURST.  Ties were broken by minimizing the overall number
 of unique Operational Taxonomic Units (OTUs).  For taxonomy assignment,
 each input sequence was assigned the lowest common ancestor that was
 consistent across at least 80% of all reference sequences tied for best
 hit.  Taxonomies are based on Genome Taxonomy Database (GTDB r95). 
 Samples with fewer than 10,000 sequences were discarded.  OTUs accounting
 for less than one-millionth of all strain-level markers and those with
 less than 0.01% of their unique genome regions covered (and &lt; 0.1%
 of the whole genome) at the species level were discarded. 
             For functional analysis, Kyoto Encyclopedia of Genes
 and Genomes Orthology groups (KEGG KOs) were used with alignment at 97%
 identity against a gene database derived from the strain database used
 above (DivDB-Mouse).  KOs were collapsed to level-2 (phylum) and -3
 (class) KEGG pathways and KEGG Modules. Count tables for taxonomic,
 enzyme, and pathway data were transformed using the centered-log ratio
 (CLR) method (Quinn
 <em>et
 al.</em>,
 2019).  Statistical testing for all count data was analyzed
 using ANCOM-BC2 (Lin and Peddada,
 2020) with BH-FDR &lt; 0.05.  Significant taxa were plotted
 as heatmaps using the R package ComplexHeatmap (v.2.13.1).  Bar plots were
 created using ggplot2 (v 3.3.6).
 <strong>Quantification of short-chain and medium-chain
 fatty acids</strong> Short-chain and medium-chain
 fatty acids and their intermediate precursors were quantified as
 previously described (Gu
 <em>et al.</em>, 2021; Dutta <em>et al.</em>, 2022; Gomez <em>et al.</em>, 2021).  Briefly, approximately 50 mg of each tissue sample were homogenized in a mixture of 20 μl hexanoic acid-6,6,6-d3 (IS; 200 µM in H2O), 20 μl sodium hydroxide solution (NaOH, 0.5 M in water), and 480 μl methanol (MeOH).  Then 400 μl of methanol was added.  The pH of the mixture was adjusted to approximately 10.  Samples were stored under −20°C for 20 min and then centrifuged at 21,694 × g for 10 minutes.  A final volume of 800 μl of supernatant was collected. Samples were then evaporated to dryness, reconstituted in 40 μl of methoxyamine hydrochloride in pyridine (20 mg/ml), and stored at 60°C for 90 minutes.  Subsequently, 60 μl of N-Methyl-N-tert-butyldimethylsilyltrifluoroacetamide was added and samples were heated to 60°C for 30 minutes.  Samples were then vortexed for 30 seconds and centrifuged at 21,694 × g for 10 min.  Finally, 70 μl of supernatant was collected from samples for gas chromatography-mass spectrometry (GC-MS) analysis.             GC-MS experiments were performed on an Agilent 88760 GC-5977B MSD system (Santa Clara, California) by injecting 1 µl of prepared samples.  Helium was used as the carrier gas with a constant flow rate of 1.2 ml/min.  The separation of metabolites was achieved using an Agilent HP-5ms capillary column (30 m × 250 × 0.25 µm).  The column temperature was maintained at 60°C for 1 minute, and then increased at a rate of 10°C/minute to 325°C and held at this temperature for 10 min.  The injector temperature was 250°C, and the operating temperatures for the transfer line, source, and quadruple were 290°C, 230°C, and 150°C, respectively.  Mass spectral signals were recorded after a 4.9 min solvent delay.  One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed for each metabolite in R for the analysis of liver metabolites (adjusted <em>p</em>-value &lt; 0.05). <strong>Fecal microbiota transplantation</strong> To investigate the functional role of changes in microbial composition in the large intestine, fecal microbiota transplant (FMT) was administered to 8-12 week-old male germ-free adult mice using the large intestinal content of adult conventional mice that were neonatally exposed to vehicle or BDE-99.  The FMT procedure was based on previous publications (Turnbaugh <em>et al.</em>, 2006; Ridaura <em>et al.</em>, 2013).  The intestinal content of donors was flushed out of the large intestine with sterile PBS.  The intestinal content was then diluted to approximately 50 mg/mL in sterile PBS.  Each sample was mixed with a 1 mL pipette thoroughly.  200 μl of the diluted homogenate was orally gavaged to the germ-free recipients (n = 3-5).  After one month of colonization, tissues were collected from the inoculated ex-germ-free mice and stored at -80 °C until further analysis.
description: # Single cell transcriptomics unveiled that early life BDE-99 exposure
 reprogrammed the gut-liver axis to promote a pro-inflammatory metabolic
 signature in male mice at late adulthood (Part 2/2) Description of the
 data and file structure The repository contains part 1 of 2 data of mouse
 single cell RNA sequencing fastq files which can be processed using the
 cell ranger pipeline (10X Genomics).  Part 1 contains 6 samples comparing
 the cell type-specific gene expression profiles using adult mouse livers
 neonatally exposed to BDE-99: DOI: 10.5061/dryad.4mw6m90gm  Part 2 (this
 dataset) contains 6 samples to investigate the necessity of the microbiome
 in regulating the hepatic transcriptome in mice: DOI:
 10.5061/dryad.8931zcrzx  The Dryad file upload contains the raw single
 cell RNA sequencing (scRNA-seq) data comparing conventional (i.e., with
 microbiome) and germ-free (i.e., without microbiome) wild type (C57BL/6)
 mice. Mice were 7-8 month old and scRNA-seq was performed using
 dissociated livers. Fastq files containing CV1, CV2, and CV3, and GF1,
 GF2, and GF3 represent conventional and germ-free mouse livers,
 respectively.