| [BACKGROUND]Chronic rhinosinusitis (CRS) showed persistent inflammation of nasal and sinus mucosa, and its incidence in the United States was about14%, and this data showed an increased trend in China. CRS without nasal polyps (NP) was defined as CRSsNP, and CRS with NP was named as CRSwNP. Eosinophilic CRSwNP (Eos CRSwNP) and non-eosinophilic CRSwNP (Non-Eos CRSwNP) was distinguished by infiltration of eosinophils. Increasing data indicates that eosinophilic and non-eosinophilic CRSwNP are heterogenous in many aspects, but studies on what resulting in difference are rare.[OBJECTIVE]To research causative factors leading to increased IgE in Eos CRSwNP and its associated molecular events, to add new content for elucidating the pathogenesis of CRS and to provide a new approach for the treatment of CRS.Therefore the purposes of this study are as the follows:1. To detect existence of the ectopic lymphoid follicles like structure in nasal mucosa and its potential function; 2. To study level of immunoglobulins and its subtypes;3. To analyze the expression of key genes associated TFH cells and B cells;4. To evaluate the expression of TFH cells and B cells and their subtypes;5. To analyze the location of TFH cells in nasal mucosa;6. To investigate the correlation between TFH cells and B cells and relevance between TFH cells and immunoglobulins.[METHODS]1. Imunohistochemistry and fluorescence immunoassay were used to detect the presence of ectopic lymphoid follicle-like structures;2. Multiples was employed to analyze the level of immunoglobulins and its subtypes;3. Real-time RT-PCR was used to evaluate key genes associated with TFH cells and B cells;4. The proportion of TFH cells and B cells and their subtypes was analyzed by flow cytometry. The level of IL-21, IL-4, IL-17A, IFN γ produced by TFH cells or PBMC or NMC was detected by intracellular cytokine staining;5. Immunofluorescence was employed to evaluate the location of TFH cells in nasal mucosa.[RESULTS]1. There were ectopic lymphoid follicle-like structures in nasal mucosa detected by imunohistochemistry and fluorescence immunoassay, which character as CD20+B cells and CD138+plasma cells infiltration. However, this stuctures did not show FDC, but AID posive cells were detected in the ectopic follicle-like structures. The proportion of these structures in NP was higher compared with normal control;2. Multiplex analysis showed that IgA was significantly increased in Eos CRSwNP and Non-Eos CRSwNP, and IgE was markly elevated in Eos CRSwNP than that in Non-Eos CRSwNP and normal control, and IgG4was dramtically increased in Eos CRSwNP than that in normal control. There were no significance of IgG1, IgG2, IgG3, total IgG, IgM among normal control, Eos CRSwNP and Non-Eos CRSwNP; 3. Real-time RT-PCR results show that BCL-6was increased in CRSsNP, Eos CRSwNP and Non-Eos CRSwNP than that in normal control, and BCL-6was increased in Non-Eos CRSwNP compared with CRSsNP; C-maf was increased in CRSsNP, Eos CRSwNP and Non-Eos CRSwNP than that in normal control; There was no significance of BATF and ICOS among the three groups; Blimp-1, which was antagonist of BCL-6was also increased in Eos CRSwNP and Non-Eos CRSwNP when compared with normal control; NRP1, CD40L, IL21R and CXCR5was dramatically decreased in normal control than that in CRSsNP, Eos CRSwNP and Non-Eos CRSwNP; BTLA was markly increased in Eos CRSwNP when compared with normal control; IL-21was increased in Eos CRSwNP than that in normal control, CRSsNP, Non-Eos CRSwNP;4. Flow cytometry detection show that proportion of CD4+CXCR5+TFH/NMC, CD4+CXCR5+ICOS+TFH/NMC, CD4+CXCR5+ICOS+TFH/CD4+CXCR5+TFH, CD4+CXCR5+ICOS+PD1+TFH/NMC and CD4+CXCR5+PD1+TFH/NMC in nasal mucosa was respectively increased in Eos CRSwNP and Non-Eos CRSwNP than that in normal control; there was no significance of CD4+CXCR5+PD1+TFH/CD4+CXCR5+TFH among three group; the frequece of CD4+CXCR5+ICOS+PD1+TFH/CD4+CXCR5+TFH was increased in Eos CRSwNP than that in normal control and Non-Eos CRSwNP; the ratio of CD4+CXCR5+ICOS+TFH/CD4+CXCR5+PD1+TFH was dramatically increased in Eos CRSwNP and Non-Eos CRSwNP than that in normal control;5. Flow cytometry detection show that proportion of CD4+CXCR5+ICOS+TFH/PBMC, CD4+CXCR5+ICOS+TFH/CD4+CXCR5+TFH, in blood was respectively increased in Eos CRSwNP and Non-Eos CRSwNP than that in normal control; there was no significance of CD4+CXCR5+TFH/PBMC, CD4+CXCR5+PD1+TFH/CD4+CXCR5+TFH. CD4+CXCR5+ICOS+PD1+TFH/PBMC and CD4+CXCR5+PD1+TFH/PBMC. and CD4+CXCR5+ICOS+PD1+TFH/CD4+CXCR5+TFH among three group; the ratio of CD4+CXCR5+ICOS+TFH/CD4+CXCR5+PD1+TFH was dramatically increased in Eos CRSwNP and Non-Eos CRSwNP than that in normal control;6. Flow cytometry analysis show that proportion IL-21+cells/NMC was no dramatic change in three groups;the frequece of CD4+CXCR5+IL-21+TFH/CD4+CXCR5+TFH was increased in Eos CRSwNP than that in normal control; the proportion of CD4+CXCR5+IL-21+TFH/NMC was markly increased in EosCRSwNP and Non-Eos CRSwNP compared with normal control; the proportion of IL-4+cells/NMC, CD4+CXCR5+IL-4+TFH/CD4+CXCR5+TFH, and CD4+CXCR5+IL-4+TFH/NMC was significantly increased in Eos CRSwNP than that in normal control and Non-Eos CRSwNP, respectively. The proportion of CD4+CXCR5+IL-4+TFH/CD4+CXCR5+TFH, and CD4+CXCR5+IL-4+TFH/NMC was increased in Non-Eos CRSwNP when compared with control. The frequence of IFNy+cells/NMC, CD4+CXCR5+IFNy+FH/CD4+CXCR5+TFH, and CD4+CXCR5+IFNy+TFH/NMC was increased in Non-Eos CRSwNP and Eos CRSwNP compared with normal control. The proportion of IL-17A+cells/NMC, CD4+CXCR5+IL-17A+TFH/CD4+CXCR5+TFH, and CD4+CXCR5+IL-17A+TFH/NMC was increased in Eos CRSwNP and Non-Eos CRSwNP than that in normal control.7. Flow cytometry detection showed that there was no significance in proportion of IL-21+cells/PBMC, IL-4+cells/PBMC, IFNy+cells/PBMC, IL-17A+cells/PBMC, CD4+CXCR5+IL-21+TFH/CD4+CXCR5+TFH, CD4+CXCR5+IL-4+TFH/CD4+CXCR5+TFH, CD4+CXCR5+IFNy+TFH/CD4+CXCR5+TFH-CD4+CXCR5+IL-17A+TFH/CD4+CXCR5+TFH, CD4+CXCR5+IL-21+TFH/PBMC, CD4+CXCR5+IL-4+TFH/PBMC, CD4+CXCR5+IFNy+TFH/PBMC, and CD4+CXCR5+IL-17A+TFH/PBMC among three groups, respectively.8. Flow cytometry detection showed that there was CD3-CD19+B cell and its subtypes in nasal mucosa. The proportion of CD3-CD19+B cells/NMC CD3-CD19+IgD-CD38-B cells/NMC and CD3-CD19+IgD-CD38+B cells/NMC was increased in Eos CRSwNP and Non-Eos CRSwNP; the frequence of CD3-CD19+IgD+CD38-B cells/NMC and CD3-CD19+IgD-CD38-B cells/CD3-CD19+Bcells was increased in Eos CRSwNP than that in normal control; the proportion of CD3-CD19+IgD+CD38-B cells/CD3-CD19+B cells, CD3-CD19+IgD+CD38+B cells/CD3-CD19+cells, and CD3-CD19+IgD+B cells/CD3-CD19+B cells was decreased in Eos CRSwNP and Non-Eos CRSwNP, respectively.9. Flow cytometry detection showed that there was no significance in proportion of CD3-CD19+B cells/PBMC, CD3-CD19+IgD-CD38-B cells/PBMC, CD3-CD19+ IgD+CD38+B cells/PBMC, CD3-CD19+IgD+B cells/PBMC, CD3-CD19+IgD-CD38+B cells/PBMC among groups; The proportion of CD3-CD19+IgD+CD38-B cells/PBMC was increased in Eos CRSwNP than that in normal control; the proportion of CD3-CD19+IgD+CD38-B cells/CD3-CD19+B cells was decreased in normal control than that in Eos CRSwNP and Non-Eos CRSwNP; The proportion of CD3-CD19+IgD+CD38+B cells/CD3-CD19+B cells was decreased in Eos CRSwNP and Non-Eos CRSwNP compared with normal control; The proportion of CD3-CD19+IgD+Bcells/CD3-CD19+B cells was markly increased in normal control compared with Eos CRSwNP; the proportion of CD3-CD19+IgD-CD38-Bcells/CD3-CD19+B cells was increased in Eos CRSwNP than that in normal control;10. Flow cytometry analysis showed that the proportion of CD3-CD138+plasma cells/NMC was significantly increased in Eos CRSwNP and Non-Eos CRSwNP than that in normal control;11. Immunofluorescence detection showed that CD4+BCL-6+TFH cells were expressed in nasal mucosa, especially in lymphoid follicles like structures, while nasal interstitial also express few TFH cells;12. There was a significant positive correlation between CD4+CXCR5+TFH/NMC (%) and CD3-CD19+BC/NMC (%), CD3-CD19+IgD-CD38-BC/BC (%), CD3-CD19+IgD-CD38+BC/BC (%), CD3-CD138+IgD+CD38+PC/NMC (%), respectively. We found a dramatically negative correlation between CD4+CXCR5+TFH/NMC (%) and CD3-CD19+IgD+CD38+BC/BC (%) and CD3-CD19+IgD+BC/BC (%), respectively. There was no significant correlation between CD4+CXCR5+TFH/NMC (%) and CD3-CD19+IgD+CD38-BC/BC (%); IgE in nasal mucosa detected by multiplex and CD4+CXCR5+ICOS+TFH/TFH (%), or CD4+CXCR5+ICOS+PD1+TFH/TFH (%), or CD4+CXCR5+ICOS+PD1+TFH/NMC (%) was positively correlated. We did not found a dramatic correlation between IgE and CD4+CXCR5+PD1+TFH/TFH (%), or CD4+CXCR5+TFH/NMC (%), or CD4+CXCR5+ICOS+TFH/NMC (%), or CD4+CXCR5+PD1+TFH/NMC (%); We found a positive correlation between IgA in nasal mucosa and CD4+CXCR5+ICOS+TFH/TFH (%), or CD4+CXCR5+ICOS+PD1+TFH/TFH (%), or CD4+CXCR5+TFH/NMC (%), or CD4+CXCR5+ICOS+PD1+TFH/NMC (%), or CD4+CXCR5+ICOS+TFH/NMC (%). We did not found a dramatic correlation between IgA and CD4+CXCR5+PD1+TFH/TFH (%), or CD4+CXCR5+PD1+TFH/NMC (%).[CONCLUSION]1. This research found that there were ectopic lymphoid follicle-like structures in nasal mucosa, which character as CD20+B cells and CD138+plasma cells infiltration. However, this stuctures did not show FDC, but AID posive cells were detected in the ectopic follicle-like structures. These structures may have specific function, but may different from typically lymphoid follicles;2. This study demonstrated the TFH cells associated genes and its subtypes were increased in Eos CRSwNP and Non-Eos CRSwNP, which may lead to different subtypes of immunoglobulins generation, and thus the final performance of nasal mucosa;3. This research was the first prove different B cells subtypes in nasal mucosa, and TFH cells may assist apecific subtype B cells to produce antibody, and contributing to onset of CRS;4. Our topic was the first time demonstrated the localization and expression of TFH cells in nasal mucosa. TFH cells may be involved in the pathogenesis of CRS, which provides bright for treatment and research of CRS. |
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