Effect Of RA/RARasignaling Pathway And BATF On Class Switch Recombination To IgA In IgA Nephropathy

IgA nephropathy (immunoglobulin A nephrology, IgAN) is a group of chronic glomerular diseases with multiple causes but the same patholodical features,the predominance of IgA deposits in the glomerular mesangium. The major clinical manifestations are hematuria, proteinuria, hypertension and renal damage. IgAN is the most prevalent primary glomerular disease worldwide. Approximately20%of the patients progress to the end-stage renal disease. The pathogenesis of IgA nephropathy remain unclear. Current data indicate that the mesangial IgA is exclusively of the IgAl subclass. As important sites of IgA synthesis and secretion,mucosal associated lymphoid tissue immunity is closely related to IgAN. Tonsil is a major component of mucosal immunity system. Studies have demonstrated that the IgA antibody eluted form the kidney of IgA nephropathy patients can bind to the tonsil cells,indicating that at least a part of deposited IgA may originate form tonsil.Under the exposure to activating stimuli such as bacterial or viral antigen, the B cells increase the expression of activation-induced cytidine deaminase (AID) and undergo class-switch recombination (CSR). This molecular event generates secondary IgG, IgA and IgE isotypes that have the same antigen specificity as IgM and IgD, but different effector functions.IgA class-switching in tonsil is a significant sourece of IgAl production in the circulation and focal site,which may promote the possibility of mesangial IgAl deposition.A variety of cytokines such as TGF-β, IL-5and IL-6are found to be involved in IgA switching.Retinoic acid,a vitamin A-derived, small lipophilic molecule that acts as ligand for nuclear RA receptors (RARs,RXR), has gained attention due to its multiple effects on innate and adaptive immunity.RA can be secreted by dendritic cells and gut epithelia cells. GALT-DC-derived RA plays an important role in the B-cell proliferation,the gut-homing receptors expression, and the transformation of IgA secreting plasma cells (PC). It remains unclear the expression of RA/RAR in tonsil and how this signaling pathway may affect the IgA nephropathy.BATF (Basic leucine zipper transcription factor, ATF-like) is a basic leucine zipper (b-Zip) transcription factor of the AP-1protein family. BATF is widely expressed and is required for the generation of Thl7cells Th2cells and Tfh cells and class switch recombination in B cells. It has also been reported BATF is required for optimal expression of CCR9and a4β7by gut-homing CD4+T cells in response to the RA signal. Yet, no study has been carried out to clarify the BATF expression in tonsil and the underlying interaction between BATF and RA in the CSR in tonsil mononuclear cells in IgA nephropathy.Clinically,IgAN patients show episodic macrohematuria, which coincides with mucosal infection especially in the upper respiratory tract or gastrointestinal tract. Some results provided a link between abnormal immune reaction to infection in MALT and IgAN. In our previous study, we identified a-hemolytic streptococcus (HS) as the most common Gram-positive bacteria isolated from tonsils of IgAN patients and chronic tonsillitis ones. Lipopolysaccharide (LPS) is the major component of the outer membrane of Gram-negative bacteria, so we used HS and LPS respectively to stimulate the tonsil mononuclear cells (TMCs) to imitate mucosal infection in IgAN. To detect the expression of BATF and RAR,analyse the role of BATF and RAR in CSR in tonsil mononuclear cells Part I The expression of RA/RARα,and the relationship with IgAl secretion in tonsil and clinical indices in IgA nephropathy patientsObejective:To detect the expression levels of RA/RARain tonsil components and mononuclear cells, analyze the correlation between RA/RARa pathway, and IgAl、proteinuria、hematuria and eGFR.in IgA nephropathy.Methods:Tonsillar tissue specimens were obtained from20patients with IgA nephropathy (IgAN) who had been diagnosed based on immunofluorescent and light microscopic findings from percutaneous renal biopsy. The tonsils from22patients with chronic tonsillitis (CT) lacking renal diseases, were used as controls. Immunohistochemical stains were performed to detect the expression of RARαin tonsil components; Mononuclear cells were separated by density-gradient centrifugation using Lymphocyte Separation Medium, and the expression of RARain tonsil mononuclear cells (TMCs) were tested by RT-PCR and Western-blot. IgAl content in the supernatant was measured in duplicate using ELISA. Collect the data of patients’ proteinuria,hematuria and eGFR.Results:1. The expression of RARawere significantly higher compared to CT group. They localized in all tonsil components (including germinal center and tonsillar crypt epithelium) of IgAN patients.2. The mRNA and protein expression of RARαin the tonsil derived from IgAN patients was significantly higher than that of CT patients.3. The concentration of RA and IgAl in supernatants of cultured cells from IgAN patients was higher than control ones.4. The level of Ra and RARaexpression were negtively correlated with renal function as expressed by eGFR, and positively correlated with IgAl and daily proteinuria and hematuria.Conclusion:RA/RARasignaling pathway was highly activated in IgAN group; RA/RARawas related to IgAN patients’clinical indices.. It seems that RA/RARamay play important role in IgAl secretion in IgAN. Part II. The relationship between BATF、RARaand AID、Ia-Ca in tonsil of patients with IgA nephropathyObejective:To detect the expression levels of BATF in tonsil components and mononuclear cells, analyze the correlation between RA/RARa pathway, BATF and IgAl、AID、Ia-Ca in IgA nephropathy.Methods:Tonsillar tissue specimens were obtained from patients with IgA nephropathy and patients with chronic tonsillitis (the same as chapte Ⅰ). Immunohistochemical stains were performed to detect the expression of BATF in tonsil components; Mononuclear cells were separated by density-gradient centrifugation using Lymphocyte Separation Medium, and the expression of BATF、AID and Ia-Ca in tonsil mononuclear cells (TMCs) were tested by RT-PCR and protein lever of BATF、AID were tested by Western-blot. IgAl content in the supernatant was measured in duplicate using ELISA.Results:1. The expression of BATF were significantly higher compared to CT group. They localized in all tonsil components (including germinal center and tonsillar crypt epithelium) of IgAN patients.2. The mRNA amount of BATF、AID and Ia-Ca in the tonsil derived from IgAN patients was significantly higher than that of CT patients. The protein expression of BATF and AID in the IgAN patients was significantly higher than that of CT patients3. The level of RARa(data in chapter I) was significantly positively correlated with AID、Ia-Ca.The BATF mRNA expression was significantly positively correlated with AID、Ia-Ca and IgAl.Conclusion:RARawas highly expressed in IgAN group and was related to some key factors in CSR,indicating RA/RARasignaling may promote the IgAl secretion through IgAl CSR. And BATF was highly activated in IgAN group and related to some CSR key factors. It seems that BATF may interact with RA/RARa in the IgAl CSR. Part III. Effects of LPS and HS on RA/RARα、BATF、 AID、Ia-Ca expression and IgAl secretionObjective:To detect the effects of LPS and HS on RA/RARα、BATF、 AID、Ia-Ca expression and IgAl secretion.Methods:Tonsillar lymphocyte were cultured with HS (1×108cfu/ml) or LPS (10ug/ml) for72hours. The mRNA lever of RARα、BATF. AID and Ia-Ca in tonsil mononuclear cells (TMCs) were tested by RT-PCR and protein lever of RARα BATF、AID were tested by Western-blot. IgAl and RAcontent in the supernatant was measured in duplicate using ELISA.Results:1. The levels of mRNA encoding RARα、BATF、AID and Ia-Ca in cells coincubated with LPS, HS were significantly higher than that without stimulation after72h in IgAN group. The protein expression of RARα、BATF and AID show the same trend as the mRNA. The IgAl and RA production of TMCs stimulated with LPS or HS was significantly higher compared to the groups without stimulation. And thte change were more remarkable in cells stimulated with HS than the ones cultured with LPS.2. The levels of mRNA encoding RARα、BATF、AID and Ia-Ca in cells coincubated with LPS, HS were significantly higher than that without stimulation after72h in CT group. The protein expression of RARα、BATF and AID show the same trend as the mRNA. The IgA1and RA production of TMCs stimulated with LPS or HS was higher compared to the groups without stimulation but lower than those in the IgAN group.Conclusion:RA/RARαpathway in tonsil of IgA nephropathy can be activated by LPS and HS, which can together promoting the production of BATF.All the above factors promote CSR and lead to the production of IgA1. Part IV. The effect of silencing RARaor BATF gene expression in IgAl CSR and secretionObjective:RARaor BATF were highly activated in tonsil of IgAN group.And they have a close relationship with IgAl CSR and secretion. In this chapter, we silenced the gene expression of RARaor BATF to observe the influence on the IgAl CSR and secretion.Methods:To assess the percentage of apoptotic and viable cell fractions after transfection of RARa siRNA or BATF siRNA.A human Annexin Ⅴ-FITC/PI staining kit was used. The mRNA of RARα、BATF、AID and Ia-Ca in tonsil mononuclear cells (TMCs) were tested by RT-PCR and the protein lever of AID were tested by Western-blot. IgAl content in the supernatant were measured in duplicate using ELISA.Results:1. Our results showed that the expression of AID protein and mRNA and Ia-Ca mRNA were decreased significantly in IgAN-RARa siRNA-treated group with or without LPS stimulation or HS stimulation.2. The expression of AID protein and mRNA and Ia-Ca mRNA were decreased significantly in IgAN-BATFsiRNA-treated group with or without LPS stimulation or HS stimulation.The level of IgAl is hardly detectable after BATFsiRNA transfection. And the inhibition effect of BATFsiRNA is more obvious than RARasiRNA. 3. The expression of AID、Ia-Ca mRNA and IgAl lever were decreased significantly in IgAN-BATFsiRNA-treated group with RA stimulation。Conclusion:RARa-siRNA and BATF-siRNA negatively regulate LPS and HS induced IgA CSR and IgAl secretion. The IgAl suppressing effect of BATF-siRNA is more potent than RARa-siRNA. BATF-siRNA can suppress the IgAl secretion induced by RA/RARα.Thus, these results suggest RARaand BATF may play key roles in IgA CSR in IgAN.And BATF may be indispensable in IgA CSR downstream of RA/RARasignaling in IgAN.