| Abstract |
The term inflammatory bowel disease refers to chronic inflammatory
conditions, characterized by chronic relapsing immune activation and inflammation
of the gastrointestinal tract. The precise etiology of IBD is unclear, although many
contributing factors have been identified. These factors are mostly linked to the
maintenance of homeostasis. In the current study, we aim to evaluate the role of
CRH in the process of tissue regeneration under pathological conditions and to
clarify putative interactions with other homeostatic mechanisms, such as the
immune response, epithelial regeneration and autophagy. For this purpose, we
used the model of DSS induced colitis. In this model, Crh -/- mice exhibit decreased
survival rates, characterized by increased protein levels of proinflammatory
cytokines TNFalpha, IL1beta and IL12p70 and particularly IL6, which was elevated
even before the initiation of DSS administration (baseline).
In terms of tissue regeneration, histological and gene expression analysis
imply that CRH deficiency leads to downregulation of Wnt / Notch pathways.
Disruption of the number, the morphology of epithelial types and the downstream
targets of Wnt / Notch pathways, is also observed. Wnt / beta catenin pathway,
which acts upstream of Wnt / Notch pathways, is also downregulated due to CRH
deficiency. The direct effect of CRH was further confirmed with the utilization of
model organisms, such as zebrafish (Danio rerio), revealing a conserved function of
the stress hormone, CRH.
It has been established that during the stress response, beta catenin
interacts with the autophagic machinery due to lack of nutrients, while Wnt / beta
catenin pathway is a crucial negative regulator of autophagy. We hypothesized that
excessive beta catenin in Crh -/- mice along with the subsequent Wnt / beta
catenin inhibition could act as an input signal in the autophagic regulation. Indeed,
beta catenin immunoreactivity is colocalized with the autophagic marker, LC3, in
the intestine of Crh -/- mice. Further analysis of the regulation of macroautophagy
in Crh -/- colon, suggested that CRH deficiency results in upregulated autophagic
activation under baseline conditions compared to WT mice, which is observed
through the course of experimental colitis. Administration of the autophagic
xviii
inhibitor, 3-Methyladenine, increased life expectancy of Crh -/- mice following DSS
administration and limited the excessive cytokines secretion. In vitro, aminoacid
deprivation in Mouse Embryonic Fibroblasts (MEFs) and LPS-induced autophagy in
murine macrophages, Raw264.7, confirmed the hypothesis that there is a negative
correlation between autophagy and CRH.
Considering the known interaction between autophagy and inflammasomes,
we searched for a putative interaction between CRH deficiency and inflammasome
activation. NLRPs gene expression is upregulated due to CRH deficiency throughout
the inflammatory response and especially during the repair phase. Broad spectrum
antibiotics administration, and the subsequent microbiome elimination, for 10 days
prior to and during DSS administration, resulted in the amelioration of the
inflammatory response in Crh -/- mice, along with high survival rates characterized
by low body weight loss and complete tissue regeneration.
In order to test whether the upregulated protein levels of IL6 in Crh -/- mice under
baseline conditions, are responsible for the subsequent dysregulation of the
inflammatory response and the individual homeostatic mechanisms, we used the
pharmacological inhibitor of IL6 receptor (IL6R), tocilizumab. The inhibition of the
biological effect of IL6, resulted in the amelioration of the histopathological features
and increased the survival rate of Crh -/- mice. Our experimental data imply the
synergistic effect of CRH and IL6, which is revealed when one of these two factors
is absent or dysfunctional, leading to development of colitis.
For the first time, we provide a generalized approach for the understanding
of CRH participation in IBD pathogenesis and the orchestration of the individual
homeostatic mechanisms through tissue response to inflammatory stress. Based on
our findings, CRH contributes in the host immune response both under baseline
conditions and following an inflammatory stimulus, by controlling, among others,
the abundance and the composition of the microbiome. The clarification of the exact
order of events that follow CRH deficiency may provide therapeutic targets, based
on the stage of the disease and the inflammatory response. Our findings provide
further evidence to study inflammatory pathological conditions, through the
understanding of the effect of environmental stressors on the progress of the
disease.
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