Psychological Stress Promotes Neutrophil Infiltration in Colon Tissue Through DSS-induced Colitis
Psychological Stress Promotes Neutrophil Infiltration in Colon Tissue Through DSS-induced Colitis
Psychological Stress Promotes Neutrophil Infiltration in Colon Tissue Through DSS-induced Colitis
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
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
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Psychological stress promotes neutrophil infiltration in colon tissue
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
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.
2
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.
2.2. 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).
2.6. 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.
2.11. 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.
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
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,
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.
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,
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.
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).
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).
References
Azhdarinia, A., Daquinag, A.C., Tseng, C., Ghosh, S.C., Ghosh, P., Amaya-Manzanares, F.,
Sevick-Muraca, E., Kolonin, M.G., 2013. A peptide probe for targeted brown adipose tissue imaging.
Nature communications 4, 2472.
Bonaz, B.L., Bernstein, C.N., 2013. Brain-gut interactions in inflammatory bowel disease.
Gastroenterology 144, 36-49.
Chrousos, G.P., 2009. Stress and disorders of the stress system. Nature reviews. Endocrinology 5,
374-381.
Davies, J.M., Abreu, M.T., 2015. The innate immune system and inflammatory bowel disease.
Scandinavian journal of gastroenterology 50, 24-33.
de Brouwer, S.J., van Middendorp, H., Stormink, C., Kraaimaat, F.W., Joosten, I., Radstake, T.R., de
Jong, E.M., Schalkwijk, J., Donders, A.R., Eijsbouts, A., van de Kerkhof, P.C., van Riel, P.L., Evers, A.W.,
2014. Immune responses to stress in rheumatoid arthritis and psoriasis. Rheumatology (Oxford,
England) 53, 1844-1848.
Evans, S.S., Repasky, E.A., Fisher, D.T., 2015. Fever and the thermal regulation of immunity: the
immune system feels the heat. Nature reviews. Immunology 15, 335-349.
Fielding, C.A., McLoughlin, R.M., McLeod, L., Colmont, C.S., Najdovska, M., Grail, D., Ernst, M., Jones,
S.A., Topley, N., Jenkins, B.J., 2008. IL-6 regulates neutrophil trafficking during acute inflammation via
STAT3. Journal of immunology (Baltimore, Md. : 1950) 181, 2189-2195.
Gerbarg, P.L., Jacob, V.E., Stevens, L., Bosworth, B.P., Chabouni, F., DeFilippis, E.M., Warren, R.,
Trivellas, M., Patel, P.V., Webb, C.D., Harbus, M.D., Christos, P.J., Brown, R.P., Scherl, E.J., 2015. The
Effect of Breathing, Movement, and Meditation on Psychological and Physical Symptoms and
28
Inflammatory Biomarkers in Inflammatory Bowel Disease: A Randomized Controlled Trial.
Inflammatory bowel diseases 21, 2886-2896.
Gharavi, N.M., Alva, J.A., Mouillesseaux, K.P., Lai, C., Yeh, M., Yeung, W., Johnson, J., Szeto, W.L., Hong,
L., Fishbein, M., Wei, L., Pfeffer, L.M., Berliner, J.A., 2007. Role of the Jak/STAT pathway in the
regulation of interleukin-8 transcription by oxidized phospholipids in vitro and in atherosclerosis in
vivo. The Journal of biological chemistry 282, 31460-31468.
Goldberg, R., Prescott, N., Lord, G.M., MacDonald, T.T., Powell, N., 2015. The unusual suspects--innate
lymphoid cells as novel therapeutic targets in IBD. Nature reviews. Gastroenterology & hepatology 12,
271-283.
Goodhand, J., Rampton, D., 2008. Psychological stress and coping in IBD. Gut 57, 1345-1347.
Hanoun, M., Zhang, D., Mizoguchi, T., Pinho, S., Pierce, H., Kunisaki, Y., Lacombe, J., Armstrong, S.A.,
Duhrsen, U., Frenette, P.S., 2014. Acute myelogenous leukemia-induced sympathetic neuropathy
promotes malignancy in an altered hematopoietic stem cell niche. Cell stem cell 15, 365-375.
Heidt, T., Sager, H.B., Courties, G., Dutta, P., Iwamoto, Y., Zaltsman, A., von Zur Muhlen, C., Bode, C.,
Fricchione, G.L., Denninger, J., Lin, C.P., Vinegoni, C., Libby, P., Swirski, F.K., Weissleder, R.,
Nahrendorf, M., 2014. Chronic variable stress activates hematopoietic stem cells. Nature medicine 20,
754-758.
Herjan, T., Yao, P., Qian, W., Li, X., Liu, C., Bulek, K., Sun, D., Yang, W.P., Zhu, J., He, A., Carman, J.A.,
Erzurum, S.C., Lipshitz, H.D., Fox, P.L., Hamilton, T.A., 2013. HuR is required for IL-17-induced
Act1-mediated CXCL1 and CXCL5 mRNA stabilization. Journal of immunology (Baltimore, Md. : 1950)
191, 640-649.
Irwin, M.R., Cole, S.W., 2011. Reciprocal regulation of the neural and innate immune systems. Nature
reviews. Immunology 11, 625-632.
Katayama, Y., Battista, M., Kao, W.M., Hidalgo, A., Peired, A.J., Thomas, S.A., Frenette, P.S., 2006.
Signals from the sympathetic nervous system regulate hematopoietic stem cell egress from bone
marrow. Cell 124, 407-421.
Koelink, P.J., Overbeek, S.A., Braber, S., Morgan, M.E., Henricks, P.A., Abdul Roda, M., Verspaget, H.W.,
Wolfkamp, S.C., te Velde, A.A., Jones, C.W., Jackson, P.L., Blalock, J.E., Sparidans, R.W., Kruijtzer, J.A.,
Garssen, J., Folkerts, G., Kraneveld, A.D., 2014. Collagen degradation and neutrophilic infiltration: a
vicious circle in inflammatory bowel disease. Gut 63, 578-587.
Kolaczkowska, E., Kubes, P., 2013. Neutrophil recruitment and function in health and inflammation.
Nature reviews. Immunology 13, 159-175.
Lin, J.Z., Martagon, A.J., Cimini, S.L., Gonzalez, D.D., Tinkey, D.W., Biter, A., Baxter, J.D., Webb, P.,
Gustafsson, J.A., Hartig, S.M., Phillips, K.J., 2015. Pharmacological Activation of Thyroid Hormone
Receptors Elicits a Functional Conversion of White to Brown Fat. Cell reports 13, 1528-1537.
Liu, D., Yang, Z., Wang, T., Chen, H., Hu, Y., Hu, C., Guo, L., Deng, Q., Liu, Y., Yu, M., Shi, M., Du, N., Guo,
N., 2016. beta2-AR signaling controls trastuzumab resistance-dependent pathway. Oncogene 35,
47-58.
Maloy, K.J., Powrie, F., 2011. Intestinal homeostasis and its breakdown in inflammatory bowel disease.
Nature 474, 298-306.
Mawdsley, J.E., Rampton, D.S., 2005. Psychological stress in IBD: new insights into pathogenic and
therapeutic implications. Gut 54, 1481-1491.
Mendez-Ferrer, S., Lucas, D., Battista, M., Frenette, P.S., 2008. Haematopoietic stem cell release is
regulated by circadian oscillations. Nature 452, 442-447.
29
Meng, F., Wang, K., Aoyama, T., Grivennikov, S.I., Paik, Y., Scholten, D., Cong, M., Iwaisako, K., Liu, X.,
Zhang, M., Osterreicher, C.H., Stickel, F., Ley, K., Brenner, D.A., Kisseleva, T., 2012. Interleukin-17
signaling in inflammatory, Kupffer cells, and hepatic stellate cells exacerbates liver fibrosis in mice.
Gastroenterology 143, 765-776 e761-763.
Mizutani, N., Nabe, T., Yoshino, S., 2014. IL-17A promotes the exacerbation of IL-33-induced airway
hyperresponsiveness by enhancing neutrophilic inflammation via CXCR2 signaling in mice. Journal of
immunology (Baltimore, Md. : 1950) 192, 1372-1384.
Neurath, M.F., 2014. Cytokines in inflammatory bowel disease. Nature reviews. Immunology 14,
329-342.
Nguyen-Jackson, H.T., Li, H.S., Zhang, H., Ohashi, E., Watowich, S.S., 2012. G-CSF-activated STAT3
enhances production of the chemokine MIP-2 in bone marrow neutrophils. Journal of leukocyte
biology 92, 1215-1225.
Nguyen, P.M., Putoczki, T.L., Ernst, M., 2015. STAT3-Activating Cytokines: A Therapeutic Opportunity
for Inflammatory Bowel Disease? Journal of interferon & cytokine research : the official journal of the
International Society for Interferon and Cytokine Research 35, 340-350.
Pan, B., Shen, J., Cao, J., Zhou, Y., Shang, L., Jin, S., Cao, S., Che, D., Liu, F., Yu, Y., 2015. Interleukin-17
promotes angiogenesis by stimulating VEGF production of cancer cells via the STAT3/GIV signaling
pathway in non-small-cell lung cancer. Scientific reports 5, 16053.
Reichmann, F., Hassan, A.M., Farzi, A., Jain, P., Schuligoi, R., Holzer, P., 2015. Dextran sulfate
sodium-induced colitis alters stress-associated behaviour and neuropeptide gene expression in the
amygdala-hippocampus network of mice. Scientific reports 5, 9970.
Straub, R.H., 2014. Rheumatoid arthritis: Stress in RA: a trigger of proinflammatory pathways? Nature
reviews. Rheumatology 10, 516-518.
Sumagin, R., Parkos, C.A., 2015. Epithelial adhesion molecules and the regulation of intestinal
homeostasis during neutrophil transepithelial migration. Tissue barriers 3, e969100.
Sun, D., Novotny, M., Bulek, K., Liu, C., Li, X., Hamilton, T., 2011. Treatment with IL-17 prolongs the
half-life of chemokine CXCL1 mRNA via the adaptor TRAF5 and the splicing-regulatory factor SF2 (ASF).
Nature immunology 12, 853-860.
Thaker, P.H., Han, L.Y., Kamat, A.A., Arevalo, J.M., Takahashi, R., Lu, C., Jennings, N.B., Armaiz-Pena, G.,
Bankson, J.A., Ravoori, M., Merritt, W.M., Lin, Y.G., Mangala, L.S., Kim, T.J., Coleman, R.L., Landen,
C.N., Li, Y., Felix, E., Sanguino, A.M., Newman, R.A., Lloyd, M., Gershenson, D.M., Kundra, V.,
Lopez-Berestein, G., Lutgendorf, S.K., Cole, S.W., Sood, A.K., 2006. Chronic stress promotes tumor
growth and angiogenesis in a mouse model of ovarian carcinoma. Nature medicine 12, 939-944.
Traber, K.E., Hilliard, K.L., Allen, E., Wasserman, G.A., Yamamoto, K., Jones, M.R., Mizgerd, J.P.,
Quinton, L.J., 2015. Induction of STAT3-Dependent CXCL5 Expression and Neutrophil Recruitment by
Oncostatin-M during Pneumonia. American journal of respiratory cell and molecular biology 53,
479-488.
Vanuytsel, T., van Wanrooy, S., Vanheel, H., Vanormelingen, C., Verschueren, S., Houben, E., Salim
Rasoel, S., Tomicronth, J., Holvoet, L., Farre, R., Van Oudenhove, L., Boeckxstaens, G., Verbeke, K.,
Tack, J., 2014. Psychological stress and corticotropin-releasing hormone increase intestinal
permeability in humans by a mast cell-dependent mechanism. Gut 63, 1293-1299.
Wu, W., Sun, M., Zhang, H.P., Chen, T., Wu, R., Liu, C., Yang, G., Geng, X.R., Feng, B.S., Liu, Z., Yang,
P.C., 2014. Prolactin mediates psychological stress-induced dysfunction of regulatory T cells to
facilitate intestinal inflammation. Gut 63, 1883-1892.
30
Yang, R., Gallo, D.J., Baust, J.J., Uchiyama, T., Watkins, S.K., Delude, R.L., Fink, M.P., 2002. Ethyl
pyruvate modulates inflammatory gene expression in mice subjected to hemorrhagic shock. American
journal of physiology. Gastrointestinal and liver physiology 283, G212-221.
31
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.
32