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Supplementary materials
Eosinophil-derived corticotrophin releasing hormone links impact of psychological stress
to gut epithelial barrier dysfunction
Peng-Yuan Zheng§, Bai-Sui Feng*§†, Christine Oluwole*†, Stevie Struiksma*†, Xiao
Chen*†, Ping Li*†, Shang-Guo Tang†, Johan D. Söderholm¶, Ping-Chang Yang*†
*McMaster Brain Body Institute, St. Joseph Health Care and †Department of Pathology
& Molecular Medicine, McMaster University, Hamilton, ON, Canada. §Department of
Gastroenterology, Zhengzhou University, Zhengzhou, China. ¶Divisions of Surgery,
Faculty of Health Sciences, Linköping University, Linköping, Sweden.
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Methods
Ussing chamber studies. Full thickness of small intestinal segments were taken from
mice and were mounted in Ussing chambers that was bathed with oxygenated Krebs
buffer containing (in mmol L−1): 115 NaCl, 1.25 CaCl2, 1.2 MgCl2, 2.0 KH2PO4 and 25
NaHCO3 (pH 7.35 ± 0.02, at 37 °C). In the Ussing chambers, an area of 0.6 cm2 of tissue
was exposed to 8 mL of buffer circulating on each side. In addition, the serosal buffer
contained 10 mmol L−1 glucose as an energy source, osmotically balanced by 10 mmol
L−1 mannitol in the mucosal buffer. Tissues were short-circuited at 0 V using a World
Precision Instruments automated voltage clamp (Narco Scientific, Mississauga, ON,
Canada). Conductance (G) was calculated according to Ohm's law, using potential
difference and short-circuit current (Isc) values. After a 15-min equilibration period,
reagents were added to the serosal buffer.
Assessment of permeability. The transepithelial passage of horseradish peroxidase
(HRP) (44 kDa) was determined by flux. HRP (type II) was added to the mucosal buffer
in the Ussing chamber at a concentration of 4.5 × 10−5 mol L−1, 500 μL samples were
obtained at 30-min after the addition of HRP from the serosal buffer and replaced with
Kreb's buffer to maintain a constant volume in the chambers. The amount of intact HRP
crossing the intestine was determined by measuring HRP activity using a modified
Worthington method 2 using a kinetic assay in a 96-well plate reader. Mucosal-to-serosal
fluxes of HRP were calculated according to standard formulae and expressed as
percentage of the original HRP added to the serosal side.
Real time quantitative reverse transcription polymerase chain reaction (qPCR).
Total RNA was extracted from Jejunal segments using an RNeasy Mini kit. cDNA was
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synthesized using iScriptTMcDNA Synthesis Kit. The resulting cDNA was subjected to
qPCR that was performed with a LightCycler using a SuperScript III Platinum SYBR
Green Two-Step qPCR Kit. The amplified product was detected by the presence of an
SYBR green fluorescent signal. The standard curve was designed with β-actin cDNA.
The resulted amplicon was quantified with the standard curve. The primers and qPCR
conditions included: CRH, forward: 5’-gagggagagagggagagagc-3’; reverse: 5’ctttggctcagaccacaaca-3’ (Annealing temperature: 60ºC, for 30 seconds, 39 cycles. 219 bp.
NCBI, NM_000756). β-actin: forward, 5’-ggacttcgagcaagagatgg-3’; reverse, 5’agcactgtgttggcgtacag-3’; 234 bp. NCBI, NM_001101).
Experimental design
Chronic stress increases the expression of CRH in eosinophils of the intestine.
Balb/c mice (6 mice/group) were treated with restraint stress 1 h daily for 10 days.
Jejunal cryosections were double stained for CRH and major basic protein, or mast cell
protease-1, or CD3, or CD11c by immunohistochemistry to identify the CRH-producing
cells. Mice were sacrificed at 0-30 days after stress treatment to determine the decline
tendency of CRH in the intestine.
Identification of stress-mediators that induce the expression of CRH in
eosinophils. Using eosinophil cell line, EoL-1 cells as a study platform, to identify which
stress mediator *[Cortisol (500 nMol), ACTH (10 nMol), methocholine (100 µMol) and
noradrenaline (100 µMol)] was involved in the expression of CRH in eosinophils.
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*Dosage of the mediators was determined in preliminary studies in a range of
doses that included: Cortisol (10~1000 nMol), ACTH (0.1~10 nMol), methocholine
(1~100 µMol) and noradrenaline (1~100 µMol).
To determine the subtype of SP receptor in eosinophils that is responsible for
CRH expression. Using SP as a stimulant, the roles of NK1 receptor antagonist CP96345 (2.5 μg/ml), or CP-99994 (5 μg/ml), or NK2 receptor antagonist GR-159897 (0.12
μg/ml), or NK3 receptor antagonist SB-222200 (2 μg/ml) on CRH expression/release in
EoL-1 cells were determined. The expression of CRH was assessed by qRT-PCR and
Western blotting. Level of CRH in culture media was determined by ELISA.
The dosage of SP receptor antagonists was determined in preliminary studies that
included: NK1: CP-96345 (1~2.5 μg/ml); CP-99994 (1~5 μg/ml); NK2: GR-159897
(0.01~0.2 μg/ml) and NK3: SB-222200 (0.1~2 μg/ml).
To determine whether eosinophil-derived CRH induces intestinal epithelial
barrier dysfunction via activation of mast cells. Transwell system was employed in
this section. T84 monolayer was prepared. TER and permeability to HRP were used as
markers of epithelial barrier function. Mast cell line HMC-1 cells were stimulated by
EoL-1 cells (activated by SP) in transwells. Then, TER and HRP flux were recorded.
In an alternative approach, Balb/c (or mast cell deficient) mice were treated with
restraint stress (1 h daily) for 10 days. Epithelial barrier function of the jejunum was
tested in Ussing chambers. SP was added to Ussing chambers directly. Isc and HRP flux
were recorded as markers of epithelial barrier function.
In another approach, Balb/c mice were stressed for 10 days. Based on our previous
studies (Teitelbaum et al. Am J Physiol Gastrointest Liver Physiol. 2008;295:G452-9),
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some mice were treated with CRH antagonist, α-helical CRH (20 μg/mouse), or CRH-R1
antagonist, antalarmin (30 μg/mouse), or CRH-R2 antagonist, antisauvagine-30 (50
μg/mouse) prior to each stress session to determine the subtypes of CRH receptors in
intestinal epithelial barrier dysfunction caused by eosinophil-derived CRH.
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Fig.S1. Substance P (SP) is involved in stress impairing intestinal epithelial barrier
function. Balb/c mice were treated with restraint stress 1 h daily for 10 days. Jejunal
segments were flattened and mounted on Ussing chambers. A, Western blots show CRH
protein in Ussing chambers. The total proteins in Ussing chambers were precipitated with
ammonium sulfate and then subjected to Western blotting. B, bars show the short circuit
current of the intestinal epithelial preparations that was determined in Ussing chambers.
C, bars indicate the horseradish peroxidase flux of the intestinal epithelial barrier that was
determined in Ussing chambers. Naïve: naïve mice. Stressed: stressed mice. SP:
stimulated in Ussing chambers with SP (10-6 Mol). TTX: epithelial tissue was treated
with tertrodotoxin in Ussing chambers prior to the addition of SP. Data were expressed as
mean ± SD. *, p<0.05, compared with naïve mice.
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Fig.S2. Stress induces increase in SP in the intestine. Balb/c mice were treated with
restraint stress 1 h daily for 10 days. Jejunal tissue extracts were analyzed by ELISA to
evaluate the content of SP. Bars indicate the content of SP in the jejunal. Naïve: naïve
mice. Stressed: mice were treated with restraint stress. Day 0 (Day 1), samples were
taken immediately (or one day) after last stress session. Data were expressed as mean ±
SD. *, p<0.05, compared with naïve mice. Each group consisted of 6 mice.
Fig.3. Colonic immune cells express CRH. Mononuclear cells were isolated from
colonic segments of stressed mice and stained with antibodies against macrophage (Mφ),
5-hydroxytryptamine, CD3 and major basic protein (MBP). The stained cells were
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analyzed by flow cytometry. A, dot plots show CRH+ cells (gated cells). B~E, flow
cytometry histograms show the percentage of macrophage (B), enterochromaffin cells
(C), T cells (D) and eosinophils within gated CRH+ cells.
Fig.S4. Both CRH-R1 and CRH-R2 are involved in SP-mediated intestinal epithelial
barrier dysfunction. Balb/c mice were treated with restraint stress for 10 days. Jejunal
epithelial segments were stimulated by SP in Ussing chambers. A, bars indicate Isc of
jejunal epithelium. B, bars indicate the epithelial permeability to HRP. *, p<0.05,
compared with naïve group. #, p<0.05, compared with those stressed, but not treated with
any CRH receptor antagonists. aCRH-R1 or aCRH-R2: antagonist of CRH-R1
(antalarmin, 30 μg/mouse) or antagonist of CRH-R2 (antisauvagine-30, 50 μg/mouse).
Each group consisted of 6 mice.
Table S1. Immunogen information of the antibodies using in the present study
Antibodies against
Immunogen Information
CRH
Immunogen sequence:
SEEPPISLDLTFHLLREVLEMARAEQLAQQAHSNRKLMEIIGK
CD11c
complement component 3 receptor 4 subunit
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MBP
Human eosinophil major basic protein
MMCP-1
Mouse mast cell protease-1
CD3
T-cell surface glycoprotein CD3 epsilon chain