Discussion
Psychological disorders have long been related to gastrointestinal
dysfunction. For example, patients with IBD have more opportunity to
suffer from anxiety and depression, in contrast, children with autism
spectrum disorders have a higher prevalence of CD and
UC7,34,35.
Evidence have now been published to support the hypothesis that
intestinal disorders can lead to psychological disorders, including
depression and anxiety, and vice versa . This mutual effect is
mediated by bidirectional communications involving reciprocal signaling
between the brain and gut. In our experiments, DSS-induced colitis
invoked an abnormal stress response in mice. Compared with mice in the
control group, the IBD mice were immobile for long periods and were less
motivated to perform self-care activities. While immobilization reflects
a reduced willingness to explore, mobilization and grooming reflect
motivation and beneficial self-care. The cause of stress-related
behavior in mice with colitis appears to be the activation of nNOS/ NO
pathway. In a previous report, an elevation in the nNOS/NO activity in
the hippocampus was shown to account for stress-induced anxiety behavior28. As a consequence of
the increased nNOS/ NO activity, increase in OTR, ERK and BDNF
expression may be expected in order to alleviate the stress response. In
addition, previous research has demonstrated that BDNF mRNA and protein
expression in the hippocampus increased after chronic restraint stress29. The increased NO
level leads to neurodegeneration, the induction of acidosis, and
inflammation. Morris et al . reported that the increased NO
impaired mitochondrial function, contributing to depression36. In the central
nervous system, nNOS neurons co-exist with OT in the hypothalamus37. Therefore, a
decrease in the NO level after intranasal OT administration may
facilitate recovery from intestinal inflammation. In addition to the
above mechanisms, other brain regions, and other genes, may be involved
in the behavioral changes observed after intranasal OT administration.
However, the importance of additional mechanisms has not been explored.
In the present study, we confirmed that DSS-induced colitis could
activate the HPA axis. Moreover, we show that subsequent intranasal OT
administration can inhibit this HPA axis activation. Considering the
anti-inflammatory properties of glucocorticoids, the observed elevation
in plasma cortisol levels is likely a manifestation of the restoration
of homeostasis. However, superabundant cortisol levels have been shown
to increase vulnerability to psychological
disorders38. Indeed,
hypercortisolism is a common finding in patients with depression and
anxiety 3940.
Thus, the inflammatory response
caused an increase of cortisol. Elevated cortisol crossed the
blood-brain barrier to enter the brain, which leads to increase of
negative factor (nNOS/NO) and compensatory protective factors (BDNF).
Intranasal OT administration also increased the concentration of OT
level in the hypothalamus. This externally administered OT may activate
the hypothalamus neurons to release more OT through an autofeedback
mechanism41. OT can
also prevent transcription of CRH in the hypothalamus by interfering
with promotor
activity42. Thus,
changes in the HPA axis and alleviation of intestinal inflammation may
be attributed to intranasal OT.
Increased serum OT caused by DSS-induced colitis may be a compensatory
response of the body considering the protective role of OT in intestinal
inflammation. Importantly, our results showed that increased OT
expression in the hypothalamus did not result in increased OT levels in
the periphery. Since we can also exclude the possibility that
intranasally administered OT was released into the blood, OT must exert
its influences through another pathway.
The induction and development of IBD disturbs the balance between the
sympathetic and vagal nervous systems which are responsible for the
maintenance of homeostasis43,44.
In IBD patients, this autonomic dysfunction manifests itself in higher
concentrations of serum catecholamine and low heart rate variability4,45,46. As
a component of the sympathetic nervous system, catecholamine plays a
crucial role in maintaining homeostasis in the
gut47. Our results show
a fluctuation in catecholamine levels consistent with the changes in HPA
activity. We hypothesize that that intranasal OT activates neurons in
the dorsal vagal complex (DVC), the primary center of the CAP that
regulates gastrointestinal immunity. The DVC is known to express OTR and
microinjection of OT into the DVC can enhance gut motility48-50. In future
studies, we aim to investigate whether intranasal OT can enhance the
activity of DVC and if so, to determine the underlying mechanism.
An elevation in the M1 and M2 macrophages in the spleen of the IBD mice
and a decrease in the M1/M2 ratio have both been reported in earlier
research51. The M2
macrophages, which possess anti-inflammatory activity, are known to
reside in the gut and to counteract intestinal inflammation52. However, not much
is known about the macrophage activity in lymphoid organs outside the
gut (e.g. spleen). For the first time, we reported here that intranasal
OT administration could shift the M1/M2 equilibrium in the spleen of
DSS-treated mice toward the M2 type. The classic mechanism in the
vago-splenic pathway involved the release of ACh from T lymphocytes
residing in the spleen, Ach activation α7nAChR channels on macrophages,
and inhibition of release of
TNF-α53. In the gut,
ACh promotes macrophage polarization to the M2 phenotype in the
gut54,55.
The observed decrease in TNF-α level and increase in Ach level in the
spleen after intranasal OT administration are consistent with the
intranasal OT activation of the vago-splenic pathway (part of the CAP).
NKp46(+) NK cells have been reported to be increased in the intestinal
mucosa of patients with CD compared with controls56. However, the role
of splenic NKp46+NK cells in the regulation of gut inflammation is
unknown. For the first time, our results indicate that DSS-induced
colitis did not change the expression of peripheral NKp46+NK cells.
Moreover, we specifically demonstrated that intranasal OT administration
down-regulated NKP46+NK cells in the spleen. We speculate that the
decrease in NKp46+NK cells in the spleen was related to activation of
the vago-splenic pathway. DSS-induced colitis did reduce the percentage
of Treg cells in the spleen. Similarly, patients with IBD exhibited
reduced numbers of peripheral Treg
cells57. Peripheral
Treg cells have been reported to serve as a protective mechanism in IBD58. In a model of viral
myocarditis, activation of the cholinergic anti-inflammatory pathway
increased the portion of Treg cell in the spleen59. The observed
increase in Treg cells in DSS-treated mice after intranasal OT
administration may be related to the decrease of TNF-α and increase of
ACh2760.
Splenectomy did not affect DSS-induced colitis, which means colitis is
independent from spleen involvement. This observation was in accord with
the hypothesis that intestinal immune tissue, but not the spleen,
contributes to the development of colitis61. In the DSS-treated
mice, who had undergone a splenectomy, attenuation of colitis severity
or of abnormal behaviors was not observed after intranasal OT
administration. Furthermore, while inflammation is initiated from the
gut, regulation of circulating immune cells and the release of cytokine
are mediated by the spleen. The beneficial effects of intranasal
OT administration on DSS-induced colitis mice were lost following
splenectomy in our mice model. Thus, intranasal OT required spleen
mediation. Elimination of cholinergic anti-inflammatory efficacy in mice
following splenectomy has previously been demonstrated in both DSS- and
DNBS- induced intestinal inflammation, lethal endotoxemia, polymicrobial
sepsis and kidney ischemia-reperfusion
injury62636465. It should also be
acknowledged that the vagus nerve innervates the proximal colon in
addition to the spleen. Therefore, it is theoretically possible that
intranasal OT administration altered enteric nervous system activity in
the remainder of the colon66, and that this, in
turn, may result in alterations in immune cell activities, cytokines
release, and suppression of inflammation. This could also account for
the effects of vagus nerve signaling on colitis that were observed in
this and other studies6768.
However, the speculation about colon participation should be proven. In
future studies, we aim to investigate the effects of intranasal OT
administration on the enteric nervous system.
Further, our study has several limitations. Firstly, a direct
correlation between changes in OT and neuron activity in the DVC has not
been shown. Moreover, we did not investigate the relationship between
intranasal OT administration and the enteric nervous system. Secondly,
the route, timing, dosage and side effects of intranasal OT treatment
should be elucidated. Finally, our study only reports data from an IBD
mouse model. Few studies have investigated intranasal OT administration
in IBD patients. Thus, additional research in IBD patients is required.
In conclusion, our study demonstrates that intranasal OT administration
significantly attenuates DSS-induced abnormal stress-related behavior
and intestinal inflammation. We conclude that intranasal OT
administration may provide a basis for the treatment of both exaggerated
stress responses and intestinal diseases in the future.
Abbreviations
IBD: inflammatory bowel disease
CD: Crohn’s disease
UC: ulcerative colitis
HPA axis: hypothalamic-pituitary-adrenal axis
OT: oxytocin
OTR: oxytocin receptor
Treg: regulatory T
DSS: dextran sodium sulfate
WAST: water avoidance stress test
SAM axis: sympathetic-adrenal medulla axis
CRH: corticotropin-releasing hormone
ACTH: adreno-cortico-tropic-hormone
ELISA: enzyme-linked immunosorbent assay
Ach: acetylcholine
CAP: cholinergic anti-inflammation pathway
DVC: dorsal vagal complex