Expression And Significance Of Tfh Cells And Its Rela Ted Factors Bcl-6,IL-21 In Pa Tients With CRSwNP

Part1Expression and significance of Tfh cells related factors Bcl-6,IL-21 in patients with CRSwNPObjectives:Chronic rhinosinusitis with nasal polyps (CRSwNP) is a common clinical syndrome characterized by Th2-skewed mucosal inflammation with marked tissue eosinophilia. Aside from mucosal eosinophilia, increased accumulation of B cells and excessive immunoglobulin (Ig) production (IgA and IgE) are also observed in polyp tissues of CRSwNP patients when compared with CRSsNP patients or healthy controls, suggesting that local B cell accumulation, maturation and IgA and IgE production may be of great importance to promote mucosal inflammation of CRSwNP patients. However, few data was available to understand the local B cell immunity in polyp tissues. T follicular helper (Tfh) cells represent the subpopulation of CD4+T cells that provides help for antigen-specific B cells in the germinal center (GC) response. However, whether T follicular cells (Tfh cells) were involved in this process has not been documented. To address this issue, we examined the expression of Bcl-6 and IL-21 in polyp tissues of CRSwNP patients in this study.Methods:Adult CRSwNP patients (n=22) were recruited from the Department of Otorhinolaryngology, Shandong Provincial Hospital Affiliated to Shandong University Twelve non-atopic patients undergoing transnasal surgery because of cerebrospinal fluid leak,orbital wall fracture were considered as controls. Polyp size was scored on a scale of 1-3 as follows:1, polyps in the middle meatus but not reaching below the inferior border of the middle turbinate; 2, polyps reaching below the inferior border of the middle turbinate but not to the inferior border of the inferior turbinate; and 3, extensive large polyps congesting the inferior meatus. None of the subjects used oral or nasal corticosteroids 4 weeks before sampling. The polyp tissues in CRSwNP patients and sinus tissues in normal controls were sampled during endoscopic inspection. Bcl-6 (the key transcription factor for Tfh cell differentiation) immunoreactivity was examined by immunohistochemical staining, the mRNA and protein expression of Bcl-6 and IL-21 was examined using qPCR, ELISA and western blot, respectively.Results:The existence of Bcl-6+cells in polyp tissues was significantly higher than those in the normal controls (P＜0.01). The number of Bcl-6+cells in polyp tissues of score 3 was significantly greater than those in polyp tissues of score 2 (P<0.01,).Both the Bcl-6 and IL-21 mRNA levels were significant upregulated in polyp tissues compared with normal controls (P＜0.01). The level of Bcl-6 mRNA correlated significant with the level of IL-21mRNA in polyp tissues (P＜0.01). Accordingly, The protein levels of Bcl-6 and IL-21 in polyp tissues were significant higher than those in normal controls (P＜0.01, Fig 2d-e).CD20+cells were extensively distributed in the polyp lamina propria of CRSwNP patients. The number of CD20+cells in polyp tissues was significantly increased compared to the normal controls (P<0.01, Fig 4b). The protein levels of IgA and IgE in polyp tissues, we found both IgA and IgE levels in polyp tissues were significant higher than those in normal controls (P＜0.01). In 27.2%(6 of 22) of CRSwNP patients, we observed prominent GC-like structure.Conclutions:Our results showed that Bcl-6, IL-21 expression was significantly upregulated in CRSwNP patients in polyp tissues were associated with enhanced B cell accumulation, IgA and IgE production. These findings thus added a new insight into the molecular mechanisms underlying CRSwNP and raise the possibility that Tfh cells could be a novel therapeutic target for difficult-to-treat CRSwNP.Part 2Flow cytometric analysis of Tfh cells in patients with CRSwNPObjectives:Based on our and other findings, the severity of CRSwNP has been suggested to be associated with tissue eosinophilia, asthma co-morbidity and recurrence after surgery in CRSwNP patients, which implied a difficult-to-treat. But we don’t know if the expression and significance of Tfh cells are associated with tissue eosinophilia, asthma co-morbidity and recurrence after surgery. Therefore we investigated the expression and significance of Tfh cells in different subgroup patients with CRSwNP.Methods:Nasal tissue samples were cut into pieces of approximately 1 to 2 mm and digested. Nasal cells were incubated with anti-CD4 at 4℃ in the dark for 30 min. Following fixation and permeabilization with Permeabilization/Fixation buffer, these cells were stained with conjugated mAbs for Bcl-6. Then the samples were analyzed by flow cytometry to get the proportion of Bcl6+CD4+cells in nasal polyp tissues.Results:The frequencies of Bcl-6+CD4+cells were significantly higher in CRSwNP patients with GC-like structure (GC+NP), tissue eosinophilia, asthma co-morbidity and recurrence after surgery compared with those without GC-like structure (GC-NP), tissue eosinophilia, asthma co-morbidity and initial cases.Conclutions:These finding collectively suggested Tfh cells may present a useful surrogate biomarker of disease severity by promoting local B cell activation, Ig production, eosinophil accumulation and concomitant asthma in CRSwNP patients.