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.