Accepted Manuscript

Psychological stress promotes neutrophil infiltration in colon tissue through

adrenergic signaling in DSS-induced colitis model

Que Deng, Hongyu Chen, Yanjun Liu, Fengjun Xiao, Liang Guo, Dan Liu,
Xiang Cheng, Min Zhao, Xiaomeng Wang, Shuai Xie, Siyong Qi, Zhaoyang
Yin, Jiangping Gao, Xintian Chen, Jiangong Wang, Ning Guo, Yuanfang Ma,
Ming Shi

PII: S0889-1591(16)30107-6
DOI: http://dx.doi.org/10.1016/j.bbi.2016.04.016
Reference: YBRBI 2864

To appear in: Brain, Behavior, and Immunity

Received Date: 20 January 2016

Revised Date: 13 April 2016
Accepted Date: 26 April 2016

Please cite this article as: Deng, Q., Chen, H., Liu, Y., Xiao, F., Guo, L., Liu, D., Cheng, X., Zhao, M., Wang, X.,
Xie, S., Qi, S., Yin, Z., Gao, J., Chen, X., Wang, J., Guo, N., Ma, Y., Shi, M., Psychological stress promotes
neutrophil infiltration in colon tissue through adrenergic signaling in DSS-induced colitis model, Brain, Behavior,
and Immunity (2016), doi: http://dx.doi.org/10.1016/j.bbi.2016.04.016

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Psychological stress promotes neutrophil infiltration in colon tissue

through adrenergic signaling in DSS-induced colitis model

Que Deng1†, Hongyu Chen1†, Yanjun Liu 2†, Fengjun Xiao3, Liang Guo1, Dan Liu1,

Xiang Cheng1, Min Zhao1, Xiaomeng Wang1, Shuai Xie2, Siyong Qi4, Zhaoyang Yin4,

Jiangping Gao 4, Xintian Chen5, Jiangong Wang5, Ning Guo1*, Yuanfang Ma2*, Ming

Shi1*

1
Institute of Basic Medical Sciences, Beijing 100850, P.R. China
2
Laboratory of Cellular and Molecular Immunology, Medical School of Henan

University, Kaifeng 475004, P.R. China

3
Department of Experimental Hematology, Beijing Institute of Radiation Medicine,

Beijing 100850, P.R. China

4
Department of Urology, the First Affiliated Hospital, General Hospital of PLA,

Beijing, 100048, P.R. China

5
Department of Cancer Biotherapy, Cancer Institute, Tangshan People’s Hospital,

Tangshan 063001, P.R. China

Running title: Psychological stress promotes neutrophil infiltration

†
Contributed equally

* Correspondence: [email protected]; [email protected]; [email protected]

1
Abstract
Inflammatory bowel disease (IBD) is a chronic intestinal inflammatory condition.
Psychological stress has been postulated to affect the clinical symptoms and
recurrence of IBD. The exact molecular mechanisms are not fully understood. In the
present study, we demonstrate that psychological stress promotes neutrophil
infiltration into colon tissues in dextran sulfate sodium (DSS)-induced colitis model.
The psychological stress resulted in abnormal expression of the proinflammatory
cytokines (IL-1β, IL-6, IL-17A, and IL-22) and neutrophil chemokines (CXCL1 and
CXCL2) and overactivation of the STAT3 inflammatory signaling pathway. Under
chronic unpredictable stress, the adrenergic nervous system was markedly activated,
as the expression of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine
biosynthesis, in bone marrow and colonic epithelium was enhanced, especially in the
myenteric ganglia. The β-AR agonist isoproterenol mimicked the effects of
psychological stress on neutrophilia, neutrophil infiltration, and colonic damage in
DSS-induced colitis. The β1-AR/β2-AR inhibitor propranolol reduced the numbers of
the neutrophils in the circulation, suppressed neutrophil infiltration into colonic
tissues, and attenuated the colonic tissue damage promoted by chronic stress.
Propranolol also abolished stress-induced upregulation of proinflammatory cytokines
and neutrophil chemokines. Our data reveal a close linkage between the
β1-AR/β2-AR activation and neutrophil trafficking and also suggest the critical roles
of adrenergic nervous system in exacerbation of inflammation and damage of colonic
tissues in experimental colitis. The current study provides a new insight into the
mechanisms underlying the association of psychological stress with excessive
inflammatory response and pathophysiological consequences in IBD. The findings
also suggest a potential application of neuroprotective agents to prevent relapsing
immune activation in the treatment of IBD.

Keywords: Inflammatory bowel disease; neutrophil; β-AR; catecholamine

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1. Introduction
Inflammatory bowel disease (IBD) is a chronic, relapsing, and remitting disease
characterized by chronic irritation and inflammation in the gastrointestinal tract. The
causes leading to chronic intestinal inflammation in IBD remain elusive. A defective
mucosal immune mechanism has been indicated in the pathogenesis of chronic
intestinal inflammation (Goldberg et al., 2015; Maloy and Powrie, 2011). Emerging
evidence suggests that stress may affect clinical symptoms of idiopathic IBD (such as
ulcerative colitis and Crohn’s disease) (Goodhand and Rampton, 2008; Mawdsley and
Rampton, 2005).
The previous studies showed that experimental psychological stress contributed to
both the initiation and reactivation of gastrointestinal inflammation in animal models
of colitis. For example, chronic psychosocial stress increases the severity of 2, 4,
6-trinitrobenzenesulfonic acid (TNBS)-induced colitis in rat. Reactivation of mucosal
inflammation occurred in rats, which had recovered from TNBS-induced colitis, in
response to restraint stress (Mawdsley and Rampton, 2005). Clinical studies revealed
that psychological stress exacerbated the disease course and caused relapses of IBD
patients. On the other hand, IBD was also associated with an increased risk of primary
psychiatric diseases such as depression and anxiety disorders (Gerbarg et al., 2015;
Reichmann et al., 2015). However, the underlying mechanisms have not been fully
understood.
Stress response is natural response or reaction to environmental demands or
pressures. In response to chronic stressors, autonomic nervous system and
hypothalamic-pituitary-adrenal (HPA) axis, which is the central stress response
system, are activated, resulting in sustained release of catecholamine [such as
epinephrine and norepinephrine (NE)] from sympathetic neurons and adrenal medulla
and cortisol from adrenal cortex (Chrousos, 2009). Over-release of a variety of
neurotransmitters (such as catecholamines and vasoactive intestinal peptide)
influences the activities of lymphocytes, macrophages, and neutrophils. Immune cells
express adrenergic receptors (ARs), through which locally released NE or circulating
catecholamines regulate the production of cytokines and antibodies and the functional
activities of immune cells (Irwin and Cole, 2011).
Multiple lines of evidence indicate that NE and epinephrine inhibit the production
of pro-inflammatory cytokines but stimulate the production of anti-inflammatory

3
cytokines, resulting in a selective suppression of Th1 response and promotion of Th2
polarization (Evans et al., 2015). However, catecholamines may also boost regional
immune response by inducing the production of pro-inflammatory cytokines under
certain conditions. In the patients with rheumatoid arthritis, the inhibitory effects of
catecholamines on IFN-γ production or Th2 shift were impaired or lost (de Brouwer
et al., 2014; Straub, 2014). In IBD, abnormal amplification and persistence of
inflammation by chronic stress can cause severe tissue injuries (Mawdsley and
Rampton, 2005). The triggering factors initiating the inflammatory response are
mostly unknown.
The present study set out to explore the mechanistic basis of psychological
stress-induced colonic tissue injuries in DSS-induced colitis model. We hypothesized
that the activation of the stress-associated signaling lowers the threshold for triggering
an inflammatory response in acute colitis, resulting in exacerbated colon mucosal
damage. Therefore, we investigated the effects of chronic stress on the expression of
proinflammatory cytokines and on inflammatory cell recruitment/infiltration in
colonic tissues. We further explored the stress-triggered signaling pathways that are
involved in the pathogenesis of IBD.

2. Materials and methods

2.1. Mice
Seven to eight-week-old male C57BL/6 mice were housed in a pathogen-free
facility and the animal studies were approved by the Animal Care and Use Committee
of Institute of Basic Medical Sciences.

2.2. Chronic restraint stress (CRS) and chronic unpredictable stress (CUS)
After acclimation for one week, the mice were individually placed in a
well-ventilated and transparent plastic mouse restraint system (Thaker et al., 2006) for
four hours per day for consecutive seven days. The mouse restraint system was
cleaned and sterilized between each restraint cycles. Food- and water-deprived but not
restrained mice were used as control animals, since stressed mice in the restraint
system did not have access to food and water during this time period.
For CUS, the mice were exposed to chronic variable stressors, including cage tilt,
isolation, crowding, rapid light-dark changes, damp bedding, and overnight

4
illumination (Supplementary Table 1). All stressors were randomly shuffled in
consecutive five days. The detailed procedure was described previously (Heidt et al.,
2014).

2.3. Induction of colitis

Experimental acute colitis was induced by giving mice ad libitum access to
drinking water containing 2.5% dextran sulfate sodium (DSS, MP Biomedicals, US)
for seven days. To investigate the effects of stress on the severity of experimental
colitis, DSS was added to the drinking water after exposure to CRS or CUS for five
days. To determine the effects of catecholamines and activation of stress-related
signaling pathways, mice were injected intraperitoneally with 10 mg/kg/day
isoproterenol (ISO), 10 mg/kg/day propranolol (Sigma-Aldrich, UK), 5 mg/kg/day the
beta 3-selective adrenoceptor antagonist (SR 59230A, Sigma-Aldrich, UK), or the
same volume of solvent (Azhdarinia et al., 2013; Heidt et al., 2014; Lin et al., 2015;
Thaker et al., 2006).

2.4. Flow cytometry

Peripheral blood samples were collected from the tail vein of the mice into tubes
containing heparin. The blood cells were resuspended in PBS containing 1% bovine
serum albumin (BSA) and then stained with fluorescein isothiocyanate-conjugated rat
anti-mouse CD11b, APC-conjugated rat anti-mouse Ly6G, and PE-conjugated rat
anti-mouse Ly6C (Biolegend, US) for 30 minutes in dark at room temperature. Then
the samples were treated with erythrocyte lysis buffer (BD, Bioscience, UK)
following the manufacturer’s instructions. After washing with PBS twice, the Ly6C+
cells were gated and analyzed for the expression of CD11b and Ly6G on a FACScan
flow cytometer using CellQuest software (BD, Bioscience, UK). The experiments
were repeated at least twice.
For evaluating the infiltration of neutrophils in colon tissues, the animals were
perfused with saline to wash out circulating blood cells under anesthesia. After
perfusion, the colon tissues were collected, carefully cleaned in ice-cold PBS, and
then mechanically chopped. The colon tissues were digested for 30 min at 37°C in 1
mg/ml collagenase type IV (Gibco, US) and 1 mg/ml dispase II (Sigma-Aldrich, UK)
in serum-free RPMI 1640 medium. The enzyme activities were neutralized by
addition of cold RPMI 1640 medium containing 8% fetal bovine serum (FBS). The
5
suspension was dispersed through a 150 µm cell strainer. The sing-cell suspensions
were stained with PerCP-conjugated rat anti-mouse CD45, fluorescein
isothiocyanate-conjugated rat anti-mouse CD11b, and APC-conjugated rat anti-mouse
Ly6G (Biolegend, US). The CD45+ cells were gated and analyzed for the expression
of CD11b and Ly6G. The experiments were repeated at least twice.

2.5. Bacterial translocation

Bacterial translocation assay was performed as previously described (Yang et al.,
2002). Briefly, the skin was cleaned, the abdominal cavity opened, and the viscera
exposed using sterile techniques. The mesenteric lymph nodes were removed,
weighed, and homogenized. The homogenates (100 µl) were plated onto
Luria-Bertani agar (OXOID) and incubated at 37°C under aerobic conditions for 24
hours. The colonies were counted and results expressed as colony-forming units (CFU)
per gram of tissue.

2.6. Immunohistochemistry
The mice were perfused with saline to wash out circulating blood cells under
anesthesia. Subsequently, the colon tissues were collected from mice and the length
was measured in a relaxed position without stretching. Then the colon tissues were
cleaned with ice-cold PBS and divided longitudinally into two parts. One part was
used for subsequent Western blot or real-time RT-PCR analysis and another part was
fixed in 10% buffered formaldehyde and embedded in paraffin. Immunohistochemical
staining was performed as previously described (Liu et al., 2016). The sections were
dewaxed in xylene and gradually hydrated in a decreasing ethanol series ending in
distilled water. Endogenous peroxidase activity was quenched using 3% hydrogen
peroxide in distilled water and then washed in PBS. After antigen retrieval by boiling
the slides in 1 mM EDTA buffer (pH 8.0) for 10 minutes, the sections were blocked
with 5% goat serum in PBS for 30 minutes at 37°C. Then, the sections were incubated
with the anti-p-STAT3 (Cell signaling) or anti-Ly6B.2 (AbD Serotec, UK) antibodies
in PBS containing 1% BSA overnight at 4°C. Following washing with PBS, the
sections were incubated with horseradish peroxidase-conjugated secondary antibodies
(ZSGB-BIO, CN). The color was developed by incubation with 3,
3’-diaminobenzidine solution. The sections were then counterstained with
hematoxylin, dehydrated, and mounted. Omission of the primary antibodies and
6
substitution by non-specific rabbit or rat IgG at the same concentration was used as
negative controls. Images were taken on an Olympus BX51 microscope (Olympus,
Tokyo, JP) using the Spot insight image capture system CCD camera. Staining was
assessed microscopically by two independent pathologists in a blinded manner.

2.7. Histological scoring

For histological scoring, over 100 fields were photographed for each group. Each
field was evaluated for tissue damage and inflammatory cell infiltration by two
independent pathologists in a blinded manner. Tissue damage was assessed as follows:
1 (normal), no mucosal damage; 2 (mild), punctuate mucosal erosions; 3 (moderate),
focal ulceration or surface mucosal erosion; 4 (severe), extensive mucosal damage
and extension into deeper structures of the bowel wall. Inflammatory cell infiltration
was assessed as follows: 1 (normal), occasional presence of inflammatory cells in the
lamina propria; 2 (mild), increased presence of inflammatory cells in the lamina
propria; 3 (moderate), confluence of inflammatory cells infiltrating into the
submucosa; 4 (severe), transmural extension of the inflammatory cells. The data
represent the percentage of fields for Normal, Mild, Moderate, and Severe in all fields
for each group.

2.8. Western blot

The lysates were prepared from HT29 cells or mouse colon tissues and total protein
content was quantified. 50 µg protein from each sample was loaded onto a
SDS-PAGE gel and separated by electrophoresis. Then the proteins were transferred
to nitrocellulose membranes. After blocking, blots were probed with the appropriate
primary antibodies overnight at 4°C. The antibodies used include anti-p-STAT3
(1:2000 dilution; Cell Signaling Technology Inc., US), anti-E-cadherin (1:1000
dilution; Cell Signaling Technology Inc., US), and anti-glyceraldehyde-3-phosphate
dehydrogenase monoclonal antibodies (1:1000 dilution; Cell Signaling Technology
Inc., US). The blots were then washed and incubated with horseradish
peroxidase-conjugated secondary antibodies (1:2000 dilution; Cell Signaling
Technology Inc., US). The bands were detected by enhanced chemiluminesence
(Thermo Scientific, US).

2.9. Real-time RT-PCR

7
 Total RNA was isolated from mouse intestinal tissue homogenates, HT29 cells, or
CT26 cells using TRIzol reagent (Invitrogen, US) following the manufacturer’s
instructions and quantified by spectrophotometry. cDNA was synthesized by reverse
transcription kit (Toyobo Inc. JP) in accordance with the manufacturer’s instructions.
Real-time PCR was performed using SYBR Green Supermix (TransGen Biotech, CN)
on Real-Time PCR Detection System (Agilent Technologies, US) as recommended by
the manufacturer. The results were analyzed using the comparative threshold cycle
method with β-actin as an internal control. Specific primers (Supplementary Table 2)
were used to screen the mRNA expression of IL-1β, IL-6, IL-17A, IL-22, CXCL1,
CXCL2, CXCL5, and CXCL15. The experiments were performed three times
independently.

2.10. Cell culture and treatment

CT26 mouse colon carcinoma cells and HT29 human colon carcinoma cells are
obtained from the American Type Culture Collection. The cells were cultured in
Dulbecco's Modified Eagle’s medium (DMEM) supplemented with 10% FBS. For the
treatment with β-AR agonist and IL-17A, CT26 cells were incubated overnight in a
serum-free DMEM medium and then treated with 10 µM ISO (Sigma–Aldrich, UK),
10 µM norepinephrine (NE) (Sigma–Aldrich, UK) or 200 ng/ml mouse IL-17A (Sino
Biological Inc. CN). HT29 cells were starved and then treated with 5 or 10 µM NE or
100 ng/ml human IL-17A (Sino Biological Inc. CN).

2.11. ELISA
HT29 cells were planted in the 10 cm-diameter plates. When the culture reaches
80-90% confluence, the cells were incubated overnight in a serum-free DMEM
medium and then treated with 10 µM NE (Sigma–Aldrich, UK), 100 ng/ml human
IL-17A (Sino Biological Inc. CN) or both for 1 hour. Phosphorylated STAT3 was
detected by using Phospho-STAT3 (Tyr705) sandwich ELISA kit (Cell Signaling
Technology Inc., US) following the manufacturer’s instructions.

2.12. Neutrophil migration assay

HT29 cells were placed in the lower compartment of the Transwell chambers
(Croning, Inc. US) and incubated overnight in a serum free medium. Subsequently,
8
HT29 cells were pre-treated with 50 µM Janus kinase 2 (JAK2) inhibitor AG490
(Sigma-Aldrich, UK) for 1 hour and then treated with 10 µM NE, 100 ng/ml human
IL-17A or both for 2 hours. Neutrophils were isolated from human peripheral blood
using neutrophil isolation kit (TBD Science, Inc. CN) according to the manufacturer’s
protocol. 1 × 106 isolated neutrophils were placed in the upper compartment of the
Transwell chambers (Croning, Inc. US). After incubation for 1 hour, neutrophils that
migrated to the bottom wells were collected and counted.

2.13. Statistical analysis

The data were presented as the means ± SEM (n ≥ 5) or the means ± SD (n ≤ 4) and
analyzed by comparing the means using one-way ANOVA followed by Dunnett’s test,
two-way ANOVA followed by a Bonferroni’s post-hoc test for multiple comparisons
or t test. The distribution of different severities of tissue damage or inflammatory cell
infiltration between two and four groups was analyzed by Wilcoxon two-sample test
and CMHχ2 test, respectively. P < 0.05 was considered statistically significant.

3. Results
3.1. Psychological stress aggravates colonic inflammation induced by DSS
We employed a well characterized chemical model to initiate experimental IBD in
mice by giving mice access to 2.5% DSS-containing drinking water ad libitum for
seven days. In order to investigate the roles of psychological stress in
colonic inflammation, the mice were subjected to CUS (Supplementary Table 1). The
CUS mouse model has been used to study the role of psychological stress in the
inflammation-related diseases (Heidt et al., 2014). Our data show that both
non-stressed (DSS administration only) and stressed animals developed severe
diarrhea and colitis characterized by loss of weight, extensive ulceration of the
epithelial layer, edema, crypt damage of bowel wall, and leukocyte infiltration into
the mucosa. However, the stressed mice displayed more severe weight loss, colon
shortening, and deterioration of the mucosal architecture with an almost complete loss
of crypts and dense infiltrates of neutrophils (Figure 1A to 1D). The histology scores
were significantly higher in the stressed group than in the non-stressed group (P<0.01;
Figure 1E). In addition, an increased bacterial translocation in the mesenteric lymph
nodes was observed in the DSS+CUS group (Figures 1F and 1G), suggesting that the

9
colonic mucosal barrier function was damaged and the colonic mucosal permeability
increased.
To further verify the relationship between psychological stress and colonic
inflammation, the mice were exposed to CRS (Wu et al., 2014). The
DSS+CRS-treated mice suffered from more severe colonic inflammation, compared
with the non-stressed mice (Supplementary Figures 1A to 1C). There was more severe
body weight loss and shrinking of the inflamed colon in the DSS+CRS-treated mice
than in the DSS-treated mice. Accordingly, histological scores were significantly
higher in the DSS+CRS group then in the DSS group (P<0.01; Supplementary Figure
1D and 1E). These data demonstrate that chronic psychological stress aggravates
DSS-induced colonic inflammation and tissue damage. We noticed that repeated
restraint stress caused a rapid weight loss of the mice within three days
(Supplementary Figure 1A), while it did not occur in CUS model. Therefore, the CUS
model was applied to the following experiments.

10
Figure 1 Psychological stress aggravates colonic inflammation induced by DSS. A to C, The
mice were given access to 2.5% dextran sulfate sodium (DSS)-containing drinking water ad
libitum and exposed to chronic variable stressors, including cage tilt, isolation, crowding, rapid
light-dark changes, damp bedding, and overnight illumination. All stressors were randomly
shuffled. Each animal was weighed daily (A, Data are presented as means ± SEM. One-way
ANOVA, n=5, F(15,64)=32.04, P<0.0001; Dunett’s t test, * P<0.05, ** P<0.01). At end of the
experiment, colon tissues were removed and the colon length was measured (B and C, Data are

11
expressed as means ± SEM. T-test, n=5, t=3.522, P=0.0065). D and E, the colon tissues were
fixed in formalin and embedded in paraffin. Colonic tissue sections were stained with
hematoxylin/eosin and observed under a light microscope (D). Bar = 400 µm. 179 and 145 fields
were photographed for the DSS and DSS+CUS groups, respectively (5 mice per group). The
histopathological score for tissue damage and inflammatory cell infiltration of colon was
determined by two pathologists in a blinded manner (E, Wilcoxon two-sample test, for
inflammatory cell infiltration, Z=4.9318, P<0.0001; for tissue damage, Z=5.5391, P<0.0001). F
and G, The mesenteric lymph nodes of the mice were removed, weighed, and homogenized. 100 µl
homogenates were plated onto Luria-Bertani agar and incubated at 37°C under aerobic conditions
for 24 hours. The colonies were counted and results expressed as colony-forming units per gram
of tissue (Data are presented as means ± SEM. T-test, n=5, t=2.453, P=0.0397). * P<0.05, **
P<0.01

3.2. Psychological stress induces neutrophila and amplifies the inflammatory

response in DSS-induced colitis
Neutrophils are key inflammatory cells in the innate defense against invading
pathogens. The egress and recruitment of neutrophils to the site of inflammation are
tightly controlled in physiological and pathological conditions. Excessive neutrophil
infiltration contributes to tissue damage in inflammatory disorders (Kolaczkowska
and Kubes, 2013). It has been proposed that neutrophil recruitment/infiltration is
directly related to increased pathological damage in the experimental colitis, although
their relative contributions to the pathogenesis of IBD are still controversial (Davies
and Abreu, 2015; Koelink et al., 2014).
To test the effects of psychological stress on neutrophils, the mice were divided into
four groups, including control, CUS, DSS and DSS+CUS groups (Figure 2A). Each
group contains five mice. The mice (CUS and DSS+CUS groups) were exposed to
CUS for 12 days. On day 5, the peripheral blood samples were taken from the tail
vein of the mice in the control and CUS groups. The effects of CUS on neutrophils
were evaluated by flow cytometry. The results show that exposure of the mice to CUS
resulted in an increase in the proportion of CD11b +Ly6C+Ly6G+ neutrophils in
peripheral blood in the absence of DSS (Supplementary Figure 2A). On day 6, the
mice (DSS and DSS+CUS groups) were given with the access to 2.5%
DSS-containing drinking water ad libitum for seven days. After DSS treatment for 2
days, the peripheral blood samples of the DSS and DSS+CUS groups were taken and
analyzed. Compared with the DSS group, the increase of neutrophils in the DSS+CUS
12
group was more pronounced (Supplementary Figure 2B), suggesting that CUS was
also associated with neutroplilia in the presence of DSS. On day 12, the peripheral
blood and colonic tissues from all mice were collected. The CD11b +Ly6C+Ly6G+
neutrophils were measured by flow cytometry. Figure 2B shows that a substantially
higher abundance of the CD11b +Ly6C+Ly6G+ cells in the circulation was observed in
the DSS+CUS group than in the DSS group, implicating that CUS may augment the
effects of DSS on myeloid-derived cell mobilization. Noticeably, in the absence of
DSS, the percentage of CD11b+Ly6C+Ly6G+ cells in the CUS group was not
increased further (compared with the data of day 5), but returned to a normal level,
which may be explained by the adaption to chronic stress.

13
14
Figure 2 Psychological stress induces neutrophila and amplifies the inflammatory response
in DSS-induced colitis. A, A Schematic representation of CUS and DSS administration to induce
colitis in mice. The mice were given ad libitum access to drinking water containing 2.5% DSS
after exposure to CUS for 5 days. The peripheral blood of the mice were collected on day 5, 8 and
12 (black arrows). The colonic tissues were collected on day 12. B, The proportion of
CD11b+Ly6C+Ly6G+ neutrophils was analyzed by flow cytometry (Data are presented as means ±
SD. Two-way ANOVA, n=4, Fmodel(3,12)=29.32, P<0.0001; FDSS(1,12)=64.35, P<0.0001;
FCUS(1,12)=6.06, P=0.03; FDSS × CUS(1,12)=17.57, P=0.0013. Bonferroni post-hoc test, *P<0.05,
**P<0.01). C, The cells were isolated from the colonic tissues of the mice.
+
CD45 marrow-derived cells were gated and analyzed for the expression of CD11b and Ly6G by
flow cytometry (Data are presented as means ± SD. Two-way ANOVA, n=4, Fmodel(3,12)=32.2,
P<0.0001; FDSS(1,12)=59.78, P<0.0001; FCUS(1,12)=18.18, P=0.0011; FDSS × CUS(1,12)=18.65,

P=0.0010. Bonferroni post-hoc test, *P<0.05, **P<0.01). D and E, Immunohistochemical

staining was performed on the paraffin-embedded colonic tissues using an antibody against
Ly6B.2. The positive cells were counted in the proximal (Data are presented as means ± SEM.
T-test, n=15 fields, 3 fields/mouse colon tissue, 5 mice/group, t=6.103, P<0.0001), middle
(t=7.406, P<0.0001), and distal (t=2.504, P=0.0184) regions of the mouse colon (*P<0.05,
**P<0.01).

Then the cells isolated from the colonic tissues were gated on the population of
CD45+ marrow-derived cells for further analysis of the CD11b and Ly6G expression
by flow cytometry. Figure 2C demonstrates that DSS administration caused the
neutrophil infiltration, but the numbers of infiltrated neutrophils in the colon tissues
were significantly higher in the DSS+CUS group, implicating that chronic stress
promoted the migration of neutrophils from peripheral blood to colon tissue. The data
were further confirmed by immunohistochemical staining on the paraffin-embedded
sections of the colonic tissues using the antibody against Ly6B.2, a neutrophil marker
(Figures 2D and 2E and Supplementary Figures 2C and 2D). There was no obvious
neutrophil infiltration in the colon tissues in the CUS group. The data suggest that
psychological stress induces peripheral neutrophilia and amplifies the inflammatory
response in IBD mouse model.

3.3. Psychological stress enhances DSS-induced inflammatory signaling and

neutrophil recruitment
Numerous proinflammatory cytokines have been suggested to be important
mediators involved in the initiation and perpetuation of intestinal inflammation in IBD.
15
Increased secretion of the pro-inflammatory cytokines by infiltrating inflammatory
cells during intestinal inflammation has been reported (Neurath, 2014). To
characterize the effect of psychological stress on the inflammatory signaling in
DSS-induced colitis, we analyzed the expression of interleukin-6 (IL-6), IL-1β,
IL-17A, and IL-22 in the colonic tissues at the mRNA levels by real-time RT-PCR.
Figure 3A shows that the levels of IL-6, IL-1β, and IL-17A were significantly
elevated in the DSS group, whereas CUS further promoted the expression of all the
cytokines tested. However, in the absence of DSS, CUS had no effect on the
expression of the pro-inflammatory cytokines.
STAT3 is a key signaling molecule for many inflammatory cytokines and
hyperactivation of the STAT3 pathway is critically involved in the pathogenesis of
IBD (Nguyen et al., 2015). IL-6 activates STAT3 directly. Although STAT3 is not a
direct target of IL-1β, IL-1β stimulates the expression of IL-6 and initiates the
activation of the IL-6/STAT3 pathway. Figure 3B shows that the phosphorylation
level of STAT3 was increased in colonic tissues of the DSS-treated mice, whereas the
STAT3 phosphorylation was much more intensive in the DSS+CUS group.
Immunohistochemical staining also demonstrated a marked increase of the
phosphorylated STAT3 positive cells in the proximal and middel colonic tissues in the
DSS+CUS group, compared with the DSS group (Figure 3C and Supplementary
Figure 3A). Treatment by CUS alone did not result in the activation of STAT3 in
colonic tissues (Supplementary Figure 3B). The data implicate that CUS aggravates
DSS-induced colonic injuries by promoting proinflammatory cytokine expression and
activating the STAT3 pathway. It has been shown that proinflammatory cytokines
may alter barrier function, which is regulated in large part by the tight junction, during
intestinal inflammation. Neutrophil transmigration in IBD has been associated with
decreased expression of E-cadherin (a tight junction protein) in the lateral membranes
of epithelial cells (Sumagin and Parkos, 2015). We detected the expression of
E-cadherin in mouse colonic tissues. Western blot analysis revealed that DSS
administration appeared to downregulate E-cadherin only slightly. A clear reduction
in the E-cadherin expression was observed in the DSS+CUS group (Figure 3B), but
the E-cadherin expression was not affected by CUS treatment alone.

16
17
Figure 3 Psychological stress enhances DSS-induced inflammatory signaling and neutrophil
recruitment. The mice were treated with DSS, CUS, or both. A, The expression of IL-6, IL-1β,
IL-17A, and IL-22 at the mRNA levels in the colonic tissues was analyzed by real-time RT-PCR
(Data are presented as means ± SEM. Two-way ANOVA, n=5 ~ 6; IL-6, Fmodel(3,20)=18.99,
P<0.0001; FDSS(1,20)=39.73, P<0.0001; FCUS(1,20)=14.26, P=0.0012; FDSS × CUS(1,20)=2.98,

P=0.0997. IL-1β, Fmodel(3,19)=14.04, P<0.0001; FDSS(1,19)=23.86, P<0.0001; FCUS(1,19)=9.01,

P=0.0073; FDSS × CUS(1,19)=8.34, P=0.0094. IL-17A, Fmodel(3,20)=18.2, P<0.0001; FDSS(1,20)=41.78,
P<0.0001; FCUS(1,20)=7.76, P=0.0114; FDSS × CUS(1,20)=5.06, P=0.0359. IL-22, Fmodel(3,18)=7.83,
P=0.0015; FDSS(1,18)=6.09, P=0.0239; FCUS(1,18)=8.27, P=0.0101; FDSS × CUS(1,18)=10.47, P=0.0046.
Bonferroni post-hoc test, NS (no significance), *P<0.05, **P<0.01). B, The phosphorylated
STAT3 and E-cadherin in colon tissue lysates were analyzed by Western blot. C, The
phosphorylated STAT3 was detected in the paraffin-embedded sections of colon tissues by
immunohistochemical staining. Percentage of p-STAT3+ cells in total cells were analyzed by using
Image analysis software Image Pro Plus Version 6.0 (Data are presented as means ± SEM. T-test,
n=14 ~ 15 (fields), 5 mice/group, t=3.245, P=0.0031). Bar = 200 µm D, The expression of
CXCL1, CXCL2, CXCL5, and CXCL15 in colonic tissues was analyzed by real-time RT-PCR
(Data are presented as means ± SEM. Two-way ANOVA, n=5 ~ 6; CXCL1, Fmodel(3,20)=29.03,
P<0.0001; FDSS(1,20)=70.33, P<0.0001; FCUS(1,20)=7.52, P=0.0126; FDSS × CUS(1,20)=9.26, P=0.0065.
CXCL-2, Fmodel(3,20)=13.37, P<0.0001; FDSS(1,20)=25.94, P<0.0001; FCUS(1,20)=8.68, P=0.008; FDSS
× CUS(1,20)=5.49, P=0.0296. CXCL-5, Fmodel(3,20)=1, P=0.4125. CXCL15, Fmodel(3,20)=2.05,
P=0.1391. Bonferroni post-hoc test, NS (no significance), *P<0.05, **P<0.01).

Chemokines CXCL1, CXCL2, CXCL5, and CXCL15 play important roles in

homing of neutrophils at early stage of tissue inflammation (Kolaczkowska and
Kubes, 2013). It has been reported that STAT3 regulates the expression of some
chemokines, including CXCL1, CXCL2, and CXCL5, and that STAT3 controls
neutrophil migratory response to CXCL2 (Fielding et al., 2008; Nguyen-Jackson et al.,
2012; Traber et al., 2015). It was also demonstrated that phosphorylated STAT3
interacted with the human il-8 (mouse CXCL15 homolog) gene promoter, regulating
the expression of IL-8 (Gharavi et al., 2007). To determine the effects of chronic
stress on the expression of chemokines, we detected the chemokines responsible for
neutrophil recruitment (cxcl1, cxcl2, cxcl5, and cxcl5 transcripts) in colonic tissues by
real-time RT-PCR. Figure 3D shows that DSS administration upregulated the
expression of CXCL1 and CXCL2, whereas combined treatment with DSS and CUS
produced stronger effect. Upregulation of CXCL5 and CXCL15 was not observed in
all groups. CUS alone did not cause the changes in the chemokine expression. The

18
data indicate that CUS enhances inflammatory signaling-triggered activation of the
STAT3 pathway and promotes neutrophil recruitment and transmigration by inducing
the expression of CXCL1/CXCL2 in DSS-induced IBD model.

3.4. Catecholamines play a key role in chronic stress-induced neutrophil infiltration

It has been suggested that the activation of stress-related signaling pathway plays a
critical role in the mobilization and altered distribution of leukocytes. Several recent
studies implicated that the stress-induced neutrophilia and neutrophil egress from
bone marrow were associated with elevated NE level (Hanoun et al., 2014; Katayama
et al., 2006; Mendez-Ferrer et al., 2008). We assumed that neutrophilia may be related
to the increased catecholamine synthesis in the bone marrow of the stressed mice. To
explore the possible mechanisms, we first analyzed the expression of tyrosine
hydroxylase (TH), the rate-limiting enzyme in the synthesis process of NE, using
immunohistochemistry. Figures 4A to 4C show that TH was highly expressed in the
bone marrow of the DSS+CUS-treated mice, as determined by measuring the
TH-positive areas and cell numbers. However, the expression level of TH was much
lower in the mice treated with DSS alone. We also examined the expression of TH in
the colonic tissues. Figures 4D and 4E clearly show that strong immunostaining of TH
was predominantly found in the myenteric ganglia. TH positive nerve fiber-like
structures were also observed in the muscular wall of the colon. The
immunoreactivity intensity in the DSS+CUS group was much stronger than that in the
DSS group. In addition, TH positive colonic epithelial cells were also detectable and,
similarly, stronger staining was observed in the DSS+CUS group (Figures 4D and 4E).
The data suggest that CUS treatment led to enhanced synthesis of catecholamines in
colonic tissues.

19
20
Figure 4 Catecholamine plays a key role in chronic stress-induced neutrophil infiltration.
The mice (6 mice/group) were treated with DSS, CUS, or both. A to C, The expression of tyrosine
hydroxylase (TH) in bone marrow of mice was assessed by immunohistochemistry using the
antibody against TH and observed under a light microscope (A). The positive area densities (B,
Data are presented as means ± SEM. T-test, n = 12 (fields), t=11.76, P<0.0001) and cell numbers
(C, t=7.867, P<0.0001) were analyzed by using Image analysis software Image Pro Plus Version
6.0. D and E, The expression of TH in colonic tissues was analyzed by immunohistochemistry (D).
The optical density of the positive staining was analyzed by Image Pro Plus Version 6.0 (E, Data
are presented as means ± SEM. T-test, n=36 (fields), t=3.928, P=0.0002). F to K, The mice (6
mice/group) were given ad libitum access to drinking water containing 2.5% DSS after exposure
to ISO (10 mg/kg) for 5 days and the peripheral blood and colonic tissues of the mice collected on
day 12. The colon length was measured (F and G, Data are presented as means ± SEM. T-test, n=5,
t=7.253, P<0.0001). The proportion of CD11b+Ly6C+Ly6G+ neutrophils was analyzed by flow
cytometry (H and I, Data are presented as means ± SEM. T-test, n=5, t=2.587, P=0.0323). The
paraffin-embedded sections of the proximal (Data are presented as means ± SEM. T-test, n=12,
t=4.715, P<0.0001), middle (t=4.849, P<0.0001), and distal (t=2.825, P=0.0099) regions of
colon tissues were stained using the anti-Ly6B.2 antibody (J and K).

To further determine the roles of catecholamines in DSS-induced colitis, we treated

the mice with DSS and ISO. ISO treatment aggravated DSS-induced colitis, as
demonstrated that the inflammatory colonic damage, colon shortening, and
inflammatory cell infiltration were more severe in the DSS+ISO group than in the
DSS group (P<0.01; Figures 4F and 4G, Supplementary Figures 4A and 4B).
Moreover, the percentage of peripheral blood neutrophils was significantly higher
(Figures 4H and 4I) and the neutrophil infiltration in colonic tissues was also more
pronounced in the DSS+ISO group (Figures 4J and 4K). Additionally, ISO-induced
upregulation of the IL-6 and CXCL1 mRNAs in colonic tissues was more remarkable
in the DSS+ISO group (Supplementary Figures 4C and 4D). Interestingly, the
expression of IL-17A, which was significantly upregulated in the DSS+CUS-treated
mice, appeared to be unaltered in the DSS+ISO-treated mice (Supplementary Figure
4E).

3.5 Catecholamines enhanced IL-17A-induced neutrophil infiltration through

activating the STAT3 signaling pathway
Emerging data suggest that IL-17 is crucial for the induction of neutrophilic
chemokines (CXCL1 and CXCL2) and therefore influences neutrophil migration and
21
function in sterile inflammation (Herjan et al., 2013; Sun et al., 2011). To assess if
catecholamines are responsible for the increased abundance of neutrophilic
chemokines, we treated CT26 murine colon carcinoma cells and HT29 human colon
cancer cells with ISO or NE in the presence and absence of IL-17A and analyzed the
expression of CXCL1 and CXCL2. When given independently, IL-17A induced the
expression of CXCL1 and CXCL2, whereas ISO alone had no effect. NE seemed to
be a weak stimulus for the expression of the chemokines (Supplementary Figure 5A).
Noticeably, a profound synergistic effect on the expression of CXCL1 and CXCL2
was observed when NE or ISO and IL-17A were added simultaneously to the cells.
Previous studies including ours demonstrated the association of catecholamines with
the STAT3 activation in some tumor cells. It has been reported that STAT3 directly
regulates the expression of the il17 gene. On the other hand, IL-17 induces the
production of IL-6, which in turn activates STAT3 (Meng et al., 2012; Pan et al.,
2015). Our data show that both NE and IL-17A could induce the phosphorylation of
STAT3 in HT29 cells, but combination of NE and IL-17A produced stronger effect
(Supplementary Figures 5B and 5C). The expression of CXCL1 and CXCL2 induced
by NE/IL-17A in HT29 cells could be completely abrogated by the JAK2 inhibitor
AG490 (Supplementary Figure 5D). To further confirm that NE/IL-17A-mediated
STAT3 signaling pathway participates in chemoattractant-directed migration of
neutrophils, we preformed neutrophil migration assays by placing 1 × 106 neutrophils
in the upper chamber of the Transwell and 2.5 × 10 5 HT29 cells treated with NE or
IL-17A or both in the chamber below. Either NE or IL-17A exerted a chemotactic
effect on neutrophil migration. Maximal effect occurred when NE and IL-17A were
added simultaneously (Supplementary Figure 5E). Blockade of the STAT3 pathway
by AG490 not only significantly inhibited the migration of neutrophils in the absence
of NE or IL-17A, but also abolished NE/IL-17A-induced chemotactic effect. These
results support that catecholamines enhanced IL-17A-induced neutrophil infiltration
through activating the STAT3 signaling pathway.

3.6. Psychological stress enhances DSS-induced inflammatory response through

activating β1-AR/β2-AR
A previous study showed that chronic stress stimulated hematopoietic stem cell
proliferation through activating β3-AR, leading to an increased output of neutrophils
(Heidt et al., 2014). On the other hand, several recent studies suggested that
22
stress-induced mobilization of lymphocytes from stores (such as marginal pools or the
spleen) into peripheral circulation was dependent on β2-AR and that signals from the
sympathetic nervous system regulated hematopoietic progenitor cell egress from bone
marrow through activation of β-ARs, including β1-, β2-, and β3-AR (Hanoun et al.,
2014).
To determine whether β-ARs regulate the CD11b +Ly6C+Ly6G+ neutrophil
mobilization in DSS-induced mice, we first treated the mice with the β3-AR inhibitor
SR59230A. Inhibition of the β3-AR activation did not impair either DSS- or
DSS+CUS-induced colonic damage and inflammatory cell infiltration in colonic
tissues (Supplementary Figures 6A to 6C). Elevation of the CD11b +Ly6C+Ly6G+
neutrophils in peripheral blood of the DSS+CUS-induced mice was not inhibited by
SR59230A (Supplementary Figure 6D).
Then, we utilized propranolol, a β1-AR/β2-AR blocker to test whether the
β1-AR/β2-AR activation is involved in exacerbated colonic inflammatory response by
CUS. Although propranolol did not attenuate DSS-induced inflammatory cell
infiltration and colonic tissue damage, the severity of DSS-induced colitis enhanced
by CUS was ameliorated (P<0.01; Figures 5A to 5C and Supplementary Figure 7A).
Additionally, stress-induced CD11b+Ly6C+Ly6G+ cells (37.2% vs. 19.4%) in
peripheral blood was reversed by propranolol (Figure 5D and Supplementary Figure
7B). Correspondingly, the numbers of infiltrated neutrophils in colon tissues were
also significantly reduced (Figure 5E and Supplementary Figures 7C and 7D). The
upregulation of the IL-6 and IL-1β expression in colonic tissues of the
DSS+CUS-treated mice was reversed by propranolol (Figure 5F). The effect of CUS
on the STAT3 phosphorylation was completely abrogated by propranolol (Figure 5G).
Additionally, the expression of the CXCL1 and CXCL2 mRNAs in the colonic tissues
was also repressed (Figure 5H). The data support that psychological stress enhances
DSS-induced inflammatory response through activating β1-AR/β2-AR. However,
propranolol did not repress the upregulation of the IL-17A and IL-22 expression
induced by CUS (Supplementary Figure 7E), suggesting that other stress-related
signaling pathways may also be involved.

23
24
Figure 5 Psychological stress enhances DSS-induced inflammatory response through
activating β1-AR/β2-AR. The mice were given propranolol (10 mg/kg) 1 hour prior to exposure
to CUS. After CUS exposure for 5 days, the mice were given ad libitum access to drinking water
containing 2.5% DSS in consecutive 7 days. The peripheral blood and colonic tissues of the mice
were collected on day 12. A and B, The colon length was measured (Data are presented as means
± SEM. Two-way ANOVA, n=4 ~ 5, Fmodel(3,15)=14.04, P<0.0001; FCUS(1,15)=2.26, P=0.1536;
FPro(1,15)=12.27, P=0.0032; FCUS × Pro(1,15)=22.5, P=0.0003. Bonferroni post-hoc test, NS (no
significance), *P<0.05, **P<0.01). C, The histopathological score was determined (CMHχ2 test,
DSS group, 4 mice, n=104 (fields); DSS+Pro group, 4 mice, n=105 (fields); DSS+CUS group, 5
mice, n=109; DSS+CUS+Pro group, 5 mice, n=147. For inflammatory cell infiltration, χ2
Pro(1)=4.7057, P=0.0301; For tissue damage, χ2Pro(1)=6.8219, P=0.009). D, The proportion of
CD11b+Ly6C+Ly6G+ peripheral blood neutrophils was analyzed by flow cytometry (Data are
presented as means ± SEM. T-test, n=7 ~ 8, t=3.347, P=0.0052). E, The CD45+CD11b+Ly6G+
cells in the colon tissues were analyzed by flow cytometry (Data are presented as means ± SD.
T-test, n=4, t=4.578, P=0.006). F, The expression of IL-6 and IL-1β mRNAs in colon tissues was
evaluated by real-time RT-PCR (Data are presented as means ± SEM. T-test, n=4 ~ 6. For IL-6
assay, t=3.297, P=0.0109; For IL-1β assay, t=2.464, P=0.0359). G, The phosphorylation of
STAT3 was analyzed by Western blot. H, The expression of CXCL1 and CXCL2 mRNAs was
analyzed by real-time RT-PCR (Data are presented as means ± SEM. T-test, n=6; For CXCL1
assay, t=3.299, P=0.008; For CXCL-2 assay, t=3.397, P=0.0068).

4. Discussion
Psychological stress has long been associated with gastrointestinal dysfunction.
Chronic psychosocial stress has also been proposed to be a risk factor for the
development and subsequent relapse of IBD (Mawdsley and Rampton, 2005;
Vanuytsel et al., 2014). In the course of chronic visceral inflammation, stress is
suggested as a crucial factor that initiates and reactivates local inflammation.
Stress-sensitive ascending and descending neural pathways trigger the activation of
the HPA axis and the sympathetic nervous system, which communicates with the
enteric nervous system, in response to stressors, thus linking emotional control center
of brain with peripheral intestinal functions. The complicate network has been termed
the brain-gut-axis. The precise mechanisms underlying brain-gut-axis
communications are unclear (Bonaz and Bernstein, 2013).
In the present study, we show that psychological stress aggravated DSS-induced
colitis with exaggerated mucosa erosions and ulcerations, massive neutrophil
infiltration, and increased epithelial permeability. The psychological stress resulted in
25
abnormal expression of the proinflammatory cytokines (IL-1β, IL-6, IL-17A, and
IL-22) and chemokines (CXCL1, CXCL2, CXCL5, and CXCL15) and overactivation
of the STAT3 signaling pathway. The robust inflammatory response has been
associated with the epithelial barrier disruption, mucosal injury, and neutrophil
infiltration in IBD. The dysregulation of immune response was worsened by the
activation of catecholamines-mediated signaling pathways, as β-AR agonist ISO
exerted similar effects on colonic damage and neutrophil infiltration in DSS-induced
colitis and the β1-AR/β2-AR inhibitor propranolol effectively suppressed the
expression of the proinflammatory cytokines and chemokines and phosphorylation of
STAT3 induced by CUS. We noticed that the expression of TH, the rate-limiting
enzyme in catecholamine biosynthesis was significantly increased in the myenteric
ganglia, reflecting elevated activities of adrenergic nervous system. Although the
chronic stress evoked a short-lasting increase of neutrophil level in circulation in the
absence of DSS, neutrophil infiltration in the colonic tissues did not occur. The
expression of the proinflammatory cytokines and phosphorylation of STAT3 in colon
tissues were not affected by CUS alone. With prolonged exposure to stress, the
neutrophil level returned to normal, implicating the development of adaptation to
chronic stress.
Several recent publications have documented that catecholamines regulated
hematopoietic progenitor cell proliferation, mobilization, and trafficking through
β-ARs (Hanoun et al., 2014; Katayama et al., 2006; Mendez-Ferrer et al., 2008). In
atherosclerosis-prone ApoE−/− mice, increased output of neutrophils was associated
with elevated hematopoietic stem cell proliferation through the activation of the
β3-AR signaling during chronic stress (Heidt et al., 2014). In our study, treatment
with β3-AR selective blocker SR59230A failed to alleviate stress-induced colonic
damage and neutrophil response in DSS-induced mice, but the β1-AR/β2-AR
inhibitor propranolol effectively reduced the numbers of the CD11b +Ly6C+Ly6G+
cells in the circulation, suppressed neutrophil infiltration into colonic tissues, and
attenuated the colonic tissue damage caused by chronic stress, revealing a close
linkage between the β1-AR/β2-AR activation and neutrophil trafficking in
experimental colitis. In addition, propranolol also abolished CUS-induced
upregulation of proinflammatory cytokines and neutrophil chemokines. The data
indicate that chronic stress triggers enhanced inflammatory response through

26
catecholamines-mediated activation of the β1-AR/β2-AR signaling pathways in
DSS-induced colitis.
IL-17 and IL-22 have been considered as major effector cytokines that trigger
inflammatory responses and thereby contribute to chronic inflammation in the
presence of pathogens. Synergism between IL-17 and IL-22 has been demonstrated in
the enhancement of the certain inflammatory cytokine expression in mucosal
responses (Goldberg et al., 2015). Recruitment of neutrophils to the inflammatory site
depends on the specific chemokines (such as CXCL1). It has been proposed that
IL-17A has a stabilizing effect on the CXCL1 mRNA and promotes the expression of
CXCL2 and CXCL5, which were also related to neutrophil recruitment and
trafficking (Herjan et al., 2013; Mizutani et al., 2014; Sun et al., 2011). Our data
demonstrate that IL-17A and IL-22 were upregulated under chronic stress,
concomitant with the upregulation of the neutrophil chemokines. Co-treatment of
CT26 and HT29 cells with IL-17 and ISO produced an enhanced effect on induction
of CXCL1 and CXCL2. NE also amplified the effects of IL-17A on the expression of
chemokines and neutrophil migration, indicating the cooperativity of the stress-related
hormone and IL-17 in driving neutrophil recruitment and trafficking in IBD.
Surprisingly, propranolol did not antagonize the effect of CUS on the expression of
IL-17A and IL-22. A recent study showed that psychological stress-derived prolactin
increased the production of IL-17 from regulatory T cells (Wu et al., 2014). It is
possible that the changes of the IL-17 and IL-22 expression observed in the present
study may be attributed to the activation of other stress-associated pathways. However,
this hypothesis is not tested in our current study, because chronic stress may cause
dysfunction of central nervous system, sympathetic nervous system, and
neuroendocrine system. Multiple stress hormones may be involved. Nevertheless, the
adrenergic nervous system plays an important role in promoting neutrophil response
in DSS-induced colitis under chronic stress.
In conclusion, the current study reveals that chronic psychological stress
upregulates the levels of proinflammatory cytokines and neutrophil chemokines,
stimulates neutrophil mobilization, and promotes colonic neutrophil infiltration
through catecholamines-mediated β-adrenergic signaling in DSS-induced model of
IBD. The sustained presence of the driving stimulus may enhance the vulnerability to
experimental colitis, resulting in colonic hypersensitivity, dysfunction, and
leukocyte-mediated tissue injury. Our data provide a new insight into the mechanisms
27
underlying the association of psychological stress with excessive inflammatory
response and pathophysiological consequences in IBD. The findings also suggest a
potential application of neuroprotective agents to prevent relapsing immune activation
in the treatment of IBD.

Conflict of interest
The authors have no conflict of interest to declare.

Acknowledgments
This work is supported by National Key Technologies R&D Program for New
Drugs (2013ZX09102056), the National High-Tech Research and Development Plan
(863 Program, No. 2014AA020604), National Natural Science Foundation of China
(No. 31370825, 81272232, 81402562, 81572845, and 31500702), Beijing Natural
Science Foundation (No. 7162144, 7132163 and 7122124), and China Postdoctoral
Science Foundation (No. 2015T81095).

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Highlights
• Chronic psychological stress promotes neutrophil infiltration through β-adrenergic
signaling.
• Sustained stressors may enhance the vulnerability to colitis and amplify
inflammatory response.
• Neuroprotective agents may be applied to prevent relapsing immune activation in
IBD.

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