Increased Expressions And Roles Of CCL21 And CCL25 In Chronic Rhinosinusitis With Nasal Polyps

BACKGROUND: Chronic rhinosinusitis(CRS)is a local inflammation of the nasal mucosa and sinus that persists for more than 12 weeks.CC chemokine ligand(CCL)19,CCL21 and CCL25 share the same receptor called atypical chemokine receptor(ACKR)4,and CC chemokine ligand(CCL)19 expression is known to be elevated in CRS.In this study we focused on CCL21 and CCL25,and explored their expressions in different types of CRS and their significance in CRS development.Methods: 116 patients were randomly enrolled for this study: there were 23 cases in the control group,32 cases in the chronic rhinosinusitis without nasal polyps(CRSs NP)group,and 62 cases in the chronic rhinosinusitis with nasal polyps(CRSw NP)group.The samples used in the study were the inferior turbinate(IT)mucosa,uncoated(UT)mucosa or nasal polyp(NP)taken from patients with CRS or other diseases during surgery.We used immunohistochemistry(IMC)and western blotting(WB)to detect the expressions of CCL21 and CCL25 protein in each nasal tissue sample.Real-time PCR was used to detect the m RNA levels of CCL21 and CCL25.We further used THP-1,a human peripheral blood mononuclear cell line,to induce macrophages,and further explored the correlation between macrophages and two chemokines.Enzyme linked immunosorbent assay(ELISA)was used to check the expression of CCL25 protein in the serum of each group.Results: IMC results showed that the expressions of CCL21 and CCL25 were significantly increased only in CRSw NP group(all p < 0.05),but there was no significant difference between the control group and CRSs NP group(CCL21: p = 0.23;CCL25: p = 0.15).WB results showed that expressions of the two chemokines were gradually increased in patients in control group,CRSs NP group and CRSw NP group(all p<0.05).Real-time PCR results showed that the m RNA levels of CCL21 and CCL25 were also gradually increased in the control group,CRSs NP group and CRSw NP group(all p<0.05).Further analysis found that the protein levels of the two chemokines were positively correlated with the severity of CRS.In addition,their upregulations were not significantly related to the degree of eosinophil infiltration in nasal polyps.IF results showed that CCL25 + cells and CD68 + macrophages co-localized in nasal polyp tissue,while CCL21+ cells and CD68 + macrophages had no obvious colocalization.THP-1 cells culture showed that when they were stimulated by phorbol12-myristate 13-acetate(PMA)and differentiated into macrophages,the expression of CCL25 m RNA was up-regulated(p < 0.05),while the expression of CCL21 was slightly down-regulated(p<0.05),all of them were statistically significant.And when macrophages were co-cultured with IFN-? and differentiated into M1 macrophages,the expression of CCL25 m RNA was obviously up-regulated(p < 0.05),and the upregulation reached a peak at 48 hours.When macrophages were co-cultured with IL-4and IL-13 transformed into M2 macrophages or co-cultured with TNF-?,the expressions of CCL25 m RNA were lower than that co-cultured with IFN-? and transformed into M1 macrophages(p < 0.05).The ELISA results showed that the protein levels of CCL25 in the serum of patients in the CRSw NP group were significantly higher than those in control group and CRSs NP group(p<0.05),and there were no significantly difference between the atopic subgroup and non-atopic subgroup(p=0.87).WB result showed there were no significantly difference between atopic subgroup and non-atopic subgroup in protein levels of CCL25 in the nasal polyps(p=0.82).It was suggested that the serum CCL25 level has certain significance for further classification of patients with CRS.Conclusion: CCL21 and CCL25 are significantly up-regulated in CRSw NP patients,and their up-regulations are positively correlated with the severity of the disease,suggesting that it may be involved in the development of the disease.CCL21 is not related to macrophages obviously.The up-regulation of CCL25 is closely related to macrophages in CRSw NP patients,and M1 macrophages can significantly secrete CCL25.CCL25 was significantly up-regulated in peripheral serum of CRSw NP patients,which can be used as a serological marker for further classify CRS patients.