3.4 Expression of inflammatory chemokines
Comparing the expression of inflammatory chemokines in the mucosa of the CRS group and the ABRS group, the neutrophil related chemokines IL-6 and IL-8 were significantly increased in the ABRS group (P<0.05), but there was no statistical difference in eosinophil related chemokines between the two groups (Table 3, Fig. 2). Comparison of chemokine expression in mucosa and polyps on the affected side of CRS showed that the neutrophil-associated chemokines IL-6, IL-8 and MCP-1 were significantly increased in polyps (Table 3, Fig. 3).
In all the samples, the contents of IL-5 and GM-CSF were lower than the detection limit (10pg/ml). Considering the low content of them in tissues, no valid data could be displayed.
Discussion
ABRS is a common condition and it is difficult to estimate its exact incidence. About 0.5-2% of children with ARS develop secondary bacterial infections that lead to ABRS, but most of them can be cured by anti-infective treatment 1, 10. Studies have found that when people are stimulated by rhinovirus, local inflammatory reaction will be caused, IL-6 and IL-8 in nasal lavage fluid will increase11. RV-1B infection can promote the endocytosis of Staphylococcus aureus by lung cells cultured with semi-permeable membranes, and induce the release of IL-6 and IL-8 and the overexpression of ICAM-112. In our study, the results of bacterial culture of nasal sinus secretion showed that the most common bacteria were Staphylococcus (12 cases), followed by Streptococcus (5 cases), and the rest were multiple infections.
In order to rule out the presence of primary CFTR dysfunction caused by potential CF patients in the CRS group, SCLs and WES were performed, and parental generation verification was performed in patients with non-SNPs. CF diagnosis was ruled out after analyzing the results. In other words, children with these mutations do not normally affect the amount or function of CFTR protein in the body. However, in the case of exposure to allergens or invasion of microorganisms such as viruses, the anti-strike ability and recovery ability of CFTR may be weakened13.
Acquired CFTR dysfunction is mostly associated with epithelial mucosal injury caused by acute and chronic infection, inflammation, hypoxia and environmental pollution8. Virus invasion causes nasal mucosal epithelial injury, and stimulates the immune response and repair mechanism to prevent the virus from causing persistent airway injury14. However, when the immunity of the body is not enough to clear the virus or the viral load is too high and the virulence is too strong, the CFTR function of mucosal epithelium cannot be restored in time, and the mucus fluidity under the airway mucosa is reduced, which leads to acute and chronic infection.
The immune response of the nasal mucosal epithelium is not limited to release of antimicrobial surfactants and mucus to delay the spread of pathogens in the airway, but it also expresses and secretes various cytokines and chemokines to drive the immune response against invading pathogens in the airway14. In an in vitro study, infection of human primary nasal epithelial cells with H3N7 resulted in an exponential increase in IFN α, IL 28A, and IL 29. The chemokines and the inflammatory markers such as CXCL11, RANTES, IL-1, IL-6, IL-8, are rapidly produced and released 14. In this study, the expression of CFTR mRNA in the ABRS group was lower than that in the CRS group, and the IL-6 and IL-8 associated with neutrophils were significantly increased in the ABRS group. When activated in an inflammatory response, neutrophils can kill pathogens directly by phagocytosis or degranulation and indirectly by releasing TNF-α, IL-1, IL-8, IL-6, IFN-γ 11, 15. These factors stimulate the production of downstream factors and recruit more neutrophils or other leukocytes to the site of infection or inflammation, exerting anti-inflammatory effects 16, 17. In combination with the results of this study, we speculate that the extremely low CFTR mRNA caused by acute inflammation of nasal mucosa makes CFTR function seriously impaired, which not only easily aggravates the proliferation of pathogens such as bacteria, but also plays a certain role in the chemotaxis of neutrophil.
The etiology of nasal polyps is complex, which is a chronic inflammatory lesion of nasal mucosa. Eosinophils, macrophages and dendritic cells can participate in the activation, release, induction and regulation of a variety of cytokines to mediate inflammatory processes. Tissue remodeling caused by chronic inflammation and immune response eventually leads to nasal polyps 18. In the CRS group, we compared the expression of CFTR mRNA and inflammatory chemokines in the ipsilateral mucosa and polyps of the same patient. It was found that the expression of CFTR mRNA in polyps was significantly lower than in mucosa. Thi et al. showed the expression of CFTR in nasal polyps was significantly lower than that in nasal mucosa, which was also supported by our results19. The formation of nasal polyps, epithelial barrier damage and rupture is an important feature, and colonized bacteria and other pathogens, such as Staphylococcus aureus, etc., aggravate the epithelial barrier damage20. Moreover, the hypoxic environment of the sinuses in CRS patients may also cause locally acquired CFTR dysfunction when polyps block the nasal or sinus drainage pathways, and may initiate a cascade of persistent fluid and electrolyte abnormalities in the sinuses8. Some findings suggest that local persistent hypoxia may lead to acquired CFTR dysfunction in nasal mucosa, resulting in mucociliary dysfunction in CRS21, 22.
In recent years, more and more data show that the endotype of CRS in different races is not the same. CRSwNP is mainly eosinophilic inflammation in western countries. However, CRS is characterized by non-eosinophilic inflammation in Asia23, 24. In this study, we also observed that the neutrophil-mediated chemokines IL-6, IL-8 and MCP-1 were significantly increased in polyps, while the expression of eosinophil-associated chemokines was not significantly different between mucosa and polyps. Some studies found that IL-6 was elevated in CRS polyps and GRO, IL-8 were positively correlated with IL-6, suggesting that IL-6 may enhance neutrophil recruitment to the site of infection25. Therefore, we hypothesized that chronic inflammation of the nasal mucosa and pathogen infection not only cause CFTR dysfunction, but also cause neutrophil chemotaxis. And because of local mucosal hyperplasia or swelling, resulting in local hypoxia microenvironment, which further lead to CFTR dysfunction: The intracellular H2O could not be discharged out of the cell along with Cl-, which participated in the formation of nasal polyps.
This study is a single-center study, so the sample size is limited. Due to the limitation of sample collection, local mucosa and polyps may not represent the inflammation status of all mucosa. Besides, baseline data on CFTR expression in the nasal mucosa of children are lacking due to the unavailability of nasal mucosa tissue from healthy children. However, we still observed differences in CFTR expression in different inflammatory states of nasal mucosa. In this study, the detection results of eosinophil chemokines IL-5 and CSF were below the lower limit of detection. Considering their low amount of content in the sample, the data were unreliable, so they could not be included in the analysis and discussion. Future studies with larger sample sizes and more sensitive reagents and assays will be considered to explore the role of neutrophil chemotactic factor and eosinophil chemotactic factor in rhinosinusitis.
Conclusion
Acquired CFTR dysfunction and neutrophil chemokine play a role in the development of acute and chronic rhinosinusitis in children. CFTR dysfunction and neutrophil chemokines IL-6 and IL-8 play more important roles in ABRS than in chronic inflammation of nasal mucosa and may play a role in the occurrence and development of nasal polyps.