We visualized the expression levels of APC subset signature genes with
neurotransmitter receptor genes (expression >1 TPM in at least one
sample) by creating heat maps with hierarchically clustered rows and
columns (using Morpheus (https://software.broadinstitute.org/mor-
pheus/)) and the ternary plot (ggtern v3.1.0).
Collection of faecal samples and bacterial DNA isolation
Faecal samples were collected sequentially on days 0 (pre) and 2 (sham
or HVx) after surgery from each mouse. In this particular experiment,
each mouse was housed individually in a separate cage. Bacterial DNA
was prepared as described previously^63. In brief, bacterial DNA was
isolated by the enzymatic lysis method using lysozyme and achromo-
peptidase. DNA samples were then purified by treating with RNase
A, followed by precipitation with 20% polyethylene glycol solution
(PEG6000 in 2.5 M sodium chloride). DNA was then pelleted by cen-
trifugation, rinsed with 75% ethanol, and dissolved in tris-EDTA buffer.
Sequencing and processing of bacterial 16S rRNA gene in faecal
DNA
The hypervariable V3–V4 region of the 16S gene was amplified
using Ex Taq Hot Start (Takara Bio) and subsequently purified using
AMPure XP (Beckman Coulter). Mixed samples were prepared by
pooling approximately equal amounts of each amplified DNA and
sequenced using the Miseq Reagent Kit V3 (600 Cycle) and Miseq
sequencer (Illumina), according to the manufacturer’s instructions.
Sequences were analysed using the QIIME software package version
1.9.1^64 ,^65. Paired-end sequences were joined using a fastq-join tool in the
ea-utils software package (https://github.com/ExpressionAnalysis/
ea-utils). High-quality sequences per sample (15,000) were randomly
chosen from the quality filter-passed sequences. After trimming off
both primer sequences using cutadapt^66 followed by chimaeras detec-
tion by the USEARCH^67 de novo method, the sequences were assigned
to operational taxonomic units using the UCLUST algorithm^68 with a
sequence identity threshold of 96%. Taxonomic assignments of each
operational taxonomic unit were made by similarity searching against
the publicly available 16S (RDP version 10.27 and CORE update 2 Sep-
tember 2012) and the NCBI genome database using the GLSEARCH
program. The data were rarefied to 10,000 sequences per sample,
as determined by the rarefaction curves. Relative abundances of the
community members were determined using the rarefied data. UniFrac
analysis was performed as described previously^69.
Gnotobiotic mice
Faecal samples from sham-operated and HVx mice were collected and
suspended in equal volumes (w/v) of PBS containing 40% glycerol,
snap-frozen and stored at −80 °C until use. The frozen stocks were
thawed, suspended in fivefold volumes of PBS and passed through a
100-μm cell strainer. GF mice were orally inoculated with 200 μl of the
suspensions using a sterile stainless-steel feeding needle. Phenotypes
of colonic immune cells were analysed after a colonization period of
3 weeks.
Quantitative PCR with reverse transcription
We isolated and purified RNA from colon tissues and cells using a
RNeasy Mini Kit. Reverse transcription was carried out with an iScript
cDNA Synthesis Kit. Real-time PCR amplification was performed using
a Thermal Cycler Dice Real Time System (Takara Bio). Gene expression
levels were normalized to 18S ribosomal mRNA. See Supplementary
Table 2 for information about the primers used.
Histology and immunohistochemistry
Liver, colon, NG and DRG were fixed in 10% formalin and embedded in
paraffin. Spinal cords (Th4–7 and 13) and colon were cryoprotected
in a 30% sucrose solution for 24 h and preserved in OCT compound.
Paraffin-embedded colon sections were stained with haematoxylin
and eosin and then examined. For immunohistochemistry, antigens
were activated by autoclaving and blocked using Block Ace. Primary
antibody reactions were performed at room temperature for 4 h (dilu-
tion ratio; PGP9.5 (1/1,000), pERK1/2 (1/500), TUBB3 (1/200), TRPV1
(1/1,000), TH (1/1,000)) or overnight at 4 °C (I-A/I-E (1/200), TUBB3
(1/200)). After washing with PBS, the sections were incubated with Alexa
Fluor 488- or Alexa Fluor 647-labelled secondary antibodies (1/400)
at room temperature for 2 h. Tissue samples were observed under a
BX53 microscope (Olympus) and an LSM 710 confocal laser scanning
microscope (Carl Zeiss). The images were analysed using Imaris (Oxford
Instruments), ZEN (Carl Zeiss) and ImageJ (NIH). See Supplementary
Table 1 for information about the antibodies.Immunohistochemistry for pERK1/2 and c-Fos
pEKR1/2 and c-Fos expression were analysed immunohistochemically
as reported^1. DSS-treated mice were transcardially perfused with PBS
including 4% paraformaldehyde and 0.2% picric acid under anaesthesia.
The NGs and brains were collected, post-fixed in the same fixative for 2 h
to overnight at 4 °C and then incubated in phosphate buffer containing
30% sucrose for 48 h. Longitudinal sections (8 μm) of NGs were cut at
48-μm intervals using a precision cryostat (Leica Microsystems). Coro-
nal sections (40 μm) of hindbrain were cut at 120-μm intervals using
a freezing microtome. Rabbit polyclonal antibody against pERK1/2
(1/500) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (1/500)
were used. Fluorescence images were acquired with a BX50 micro-
scope and DP50 digital camera (Olympus). In c-Fos staining, anti-c-Fos
antisera (1/10,000) were used as the primary antibody. Colour was
developed with nickel-diaminobenzidine. Neurons immunoreactive
to pERK1/2 and c-Fos in the medial NTS were counted.c-Fos immunostaining in the myenteric nerve plexus
To prepare the myenteric nerve plexus, 3 cm of colon obtained from
sham-operated, VGx or HVx mice in a fed state were cut longitudinally
and soaked in a plastic plate containing ice-cold PBS. The mucosal
layer was removed and the myenteric nerve plexus was dropped into
4% PFA overnight, and washed at room temperature with cold PBS.
Samples were blocked for 1 h at room temperature with blocking solu-
tion. Then samples were incubated overnight at room temperature
with the primary antibodies (HuC/HuD, 1/500; c-Fos, 1/500) diluted
in the antibody diluent solution, washed 3 times with PBS, incubated
for 90 min at room temperature with the secondary antibodies (1/400)
and washed 3 times with PBS. Samples were mounted with fluorescent
mounting medium. The fluorescence of different tissues was measured
on confocal Zeiss Laser Scanning Microscope LSM-710.Measurement of calcitonin gene-related peptide, acetylecholine
and noradrenaline levels in colon
Neurotransmitter levels in colon were determined as previously
described^70 –^72. Colon tissues were washed with PBS and homogenized.
The homogenates were centrifuged at 15,000g for 10 min at 4 °C and
supernatants were collected. Samples were kept at −80 °C until use.
Protein concentrations were determined by BCA assay (Thermo Fisher
Scientific). Calcitonin gene-related peptide (CGRP) (Phoenix Pharma-
ceuticals), acetylcholine (Abcam) and noradrenaline (LsBio) levels in
homogenates were measured by ELISA.Western blot analysis
Proteins were extracted from liver tissue using T-PERincluding protease
inhibitor and PhosSTOP (Sigma). Western blotting was performed
as previously described using Clarity Western ECL Substrate and the
ChemiDoc Imaging System (Bio-Rad)^73.Knockdown of Raptor in the liver by RNA interference
Si-negative control (Si-Cont) and Si-Raptor (In-VivoReady grade) were
complexed with Invivofectamine 2.0 Reagent (Invitrogen) exactly