4 Discussion
Previous studies have shown that hypoxia leads to abnormal systemic
immune function, which may be closely related to the development of
various chronic diseases [10,11]. TLRs [12], NOD-like receptors
[13] and HIN200 [14,15] family members expressed by intestinal
epithelial cells are widely involved in limiting the invasion of
intestinal pathogenic microorganisms and preventing the deterioration of
infectious diseases.The oxygen-sensing mechanism in the body can
regulate the expression of genes under exposure to different oxygen
concentrations [16]. An important cellular metabolic feature of
hypoxia is the elevated expression of hypoxia-inducible factor (HIF-1),
which participates in human physiological and pathological processes by
regulating a variety of related genes [17].Inflammatory bowel
disease (IBD) caused by C. rodentium can produce a large amount
of proinflammatory cytokines and severely damage the layer of epithelial
cells [18,19]. The levels of the proinflammatory cytokines TNF-α,
IL-6 and IL-1β in colon tissues are significantly increased with the
occurrence of IBD [20,21]. MPO plays an important role in bacterial
infections [22].
Based on the experimental results, we found that in each group, the
colon length of mice infected with C. rodentium in a normoxic
environment was shortened, which was more pronounced under hypoxic
exposure. The stained colon tissues of mice exposed to hypoxia showed
obvious damage to or loss of colonic epithelial mucosa and erosion. The
arrangement of intrinsic glands on the intestine was more disordered. A
large amount of inflammatory cell infiltration could be seen in the
mucosa and submucosa. According to pathological observation and scoring,
it was clear that hypoxia exposure could aggravate the occurrence and
development of the colitis inflammatory response.This study proved for
the first time that a hypoxic environment can reduce the innate immune
function of mice by affecting the chemotaxis level of phagocytes,
resulting in an increase in bacterial excretion in C.
rodentium -infected mice, weight loss, diarrhoea, blood in the stool,
and shortened colon length, which confirmed that hypoxic exposure can
exacerbate the inflammatory response in colitis. Neutrophils mainly rely
on respiratory oxidative bursts [23] and the production of reactive
oxygen species [24] to destroy pathogens. However, hypoxia causes
insufficient oxygen pressure in the environment, resulting in
insufficient recruitment of NOX2 in neutrophils and insufficient
production of reactive oxygen species, which can lead to a decrease in
the ability to clear pathogens.
Macrophages are the main phagocytic cells in the intestine and are
closely related to the pathogenesis of IBD [25].In the process of
maturation, phagosomes need to further fuse with lysosomes to generate
phagolysosomes [26]. In the acute phase, the number of macrophages
significantly increased in the intestinal mucosa in IBD. The expression
of lymphocyte costimulatory molecules and TLR was upregulated, and the
receptor for myeloid cell triggering-1 (TREM-1) was coexpressed
[27,28]. TREM-1 can activate the synthesis and secretion of TNF,
IL-1β, IL-6 and other inflammatory factors, which can cause damage to
local tissues.
In our study, it was found that although a hypoxic environment can
increase the chemotaxis of macrophages, macrophage activation is reduced
due to the lack of oxygen. It has been reported in the literature that
glycolytic capacity is significantly increased after the induction of
macrophages by LPS and IFN-γ [29]. HIF-1, a heterodimeric
transcription factor, played a key role in the adaptation of macrophages
to changes in oxygen tension, and HIF-1α, as the active subunit of
HIF-1, was the main functional factor [30]. HIF-1α has a very wide
range of target genes, including GLUT1, GAPDH, LDHA, PKM and a variety
of glycolytic enzymes [31]. After macrophages were activated, it
adapted to the stimulation of inflammatory factors through the
transformation of metabolic pathways. The hypoxic environment in our
experiment significantly upregulated HIF-1α expression, which resulted
in an increase in the glycolysis pathway [32], but the massive
production of inflammatory factors can cause a more severe inflammatory
response at the same time.
Previous studies have found that MPO can participate in the regulation
of various inflammatory responses and pathological effects in the body
[22], so we focused on the changes in MPO levels on the pathological
response of C. rodentium -induced colitis in mice in a hypoxic
environment.
MPO activity is an indicator commonly used to monitor IBD activity
[33]. MPO in epithelial cells can release a variety of
proinflammatory substances, such as HOCl and
H2O2 [34]. After infecting WT mice
and MPO-/- mice with C. rodentium , we found
that MPO-/- mice excreted more bacteria and lost more
weight. This is mainly because neutrophils in MPO-/-mice cannot function to clear bacteria normally in the absence of MPO.
Subsequently, we further analysed the results and found that the
pathological changes of C. rodentium -induced enteritis in
MPO-/- mice in a normoxic environment were more
severe, mainly manifested as obvious submucosal oedema and a large
amount of neutrophil and macrophage infiltration. Goblet cell numbers
decreased significantly, and intestinal epithelial integrity was
significantly damaged. In addition, we found that a large number of
inflammatory factors and chemokines were produced in large quantities
with the occurrence and development of infection, but although the
secretion of inflammatory factors and chemokines in
MPO-/- mice increased, the intestinal inflammatory
pathological damage was relatively mild in hypoxic mice compared with
that in WT mice.In addition, changes in cytokine levels in IBD patients
deserve attention. IL-1β, TNF-α and IFN-γ participate in and aggravate
the progression of IBD. In the NF-κB pathway, the lack of MPO inhibits
the activation of NF-κB after inhibiting ROS production, resulting in a
decrease in the mRNA transcription of IL-1β, TNF-α and IFN-γ [35].
In conclusion, in the C. rodentium -induced colitis mouse model,
MPO can not only affect bacterial clearance but also participate in
pathological damage to tissues. On the one hand, MPO may have a direct
effect itself, and on the other hand, MPO may modulate histopathological
damage through the ROS pathway.