Effect Of IL-4/STAT6Signaling Pathway And HIPK2in Under-O-glycosylation IgA1by Tonsil From Patients With IgA Nephropathy And Its Possible Mechanism

Background:IgA nephropathy (IgAN) is recognized as the most common immune complex related to the cause of glomerulonephritis worldwide. Approximately20%of patients progress to end-stage renal disease within20years. However, the pathogenesis and mechanisms of IgAN remain to be elucidated. The disease is characterized by the predominant deposition of IgA1in the mesangial area of glomeruli. IgAl differs from IgA2, particularly at the hinge region; IgA1contains an extended polyproline peptide bearing multiple serine and threonine residues and distinctive O-glycans. Considerable evidence has revealed that abnormalities of IgA1O-glycosylation may be one of the key pathogeneses of IgAN over the past10years. The IgAl O-glycans are based on a core N-acetyl galactosamine (GalNAc) usually extended with galactose (Gal) under the effect of a j31,3Gal transferase (C1GALT1) working with its chaperone Cosmc (core I β3-Gal-T-specific molecular chaperone). β1,3-galacto-syltransferase (β1,3GT) synthesis activity was remarkably lower in peripheral B lymphocyte of IgAN patients when compared with that of controls, and the Cosmc mRNA expression level in IgAN patients was significantly decreased and correlated with IgA glycosylation abnormality degree as well as clinical manifestations. Yet, no study has been carried out to clarify the underlying basis of the expression suppression.Although the pathogenesis of this disease is unclear, some researchers suggested that it might not be genetic disorders but external suppression that causes the low COSMC and C1GALT1C1mRNA expression in IgAN. LPS could significantly inhibit COSMC and C1GALT1C1expression. Recent work using a surface IgA1-positive human B lymphoma cell line has shown that the T-helper2cytokine interleukin-4may play a key role in controlling O-glycosylation of the IgAl hinge region. Interleukin-4stimulation significantly decreased the mRNA levels of both and Cosmc. In parallel, C1GALT1C1activity was reduced and the synthesized IgAl O-glycoforms were poorly galactosylated. Moreover, IgAN patients with severe renal dysfunction are more likely to hyperproduce Th2cytokine and synthesize more IL-4compared to patients with mild disease. These findings suggest that Th2polarity in systemic immune response may induce dysregulation of systemic tolerance, followed by glomerular IgA deposition and injury. The T cell-derived cytokines IL-4specifically activate STAT6, which plays important roles in Th2differentiation and inflammation. Yet, no study has been carried out to clarify the underlying relationship between the expression suppression of C1GALT1and COSMC with IL-4/STAT6signaling in IgA nephropathy.More recently, high-throughput sequencing of chromatin immunoprecipitated DNA has identified genes bound by STAT6. One study compared genes bound by STAT6in wild-type and Stat6-/-Th2cells and found that some of the STAT6-bound regions coincided with various permissive epigenetic marks, and the corresponding genes include IL-4and home-odomain-interacting protein kinase2(HIPK2). IL-4induced STAT6-dependent expression of a selection of genes identified by GeneChip analysis, including HIPK2was confirmed by Northern blot. STAT6deficiency clearly inhibited the IL-4-induced up-regulation of HIPK2which is most likely a primary target gene of STAT6. HIPK2is a nuclear serine-threonine kinase participating in transcriptional regulation and growth control.While O-glycans of IgAl are synthesized in a step-wise manner, beginning with attachment of GalNAc to Ser or Thr catalyzed by a member of the UDP-GalNAc-transferase enzyme family. We hypothesized and experimentally validated that HIPK2, a previously unrecognized kinase in the context of IgA nephropathy, contributes to increased synthesis of aberrantly galactosylated IgAl.We know that many IgAN patients show episodic macrohematuria, which coincides with mucosal infection especially in the upper respiratory tract or gastrointestinal tract. Two groups have reported tonsillectomy to be an effective treatment in retrospective and prospective studies. The results of immunization studies in IgAN patients support the notion that mucosal immunization with neoantigen results in impaired mucosal and systemic IgA responses but normal IgG and IgM responses. There is a growing body of evidence to suggest that impaired mucosal IgA response may result in impaired elimination of mucosal antigens. In our previous study, we identified that a-hemolytic streptococcus (HS) was the most common Gram-positive bacteria isolated from tonsils of patients with IgAN or chronic tonsillitis. Lipopolysaccharide (LPS) is the major component of the outer membrane of Gram-negative bacteria, so we used both HS and LPS to stimulate the tonsil mononuclear cells (TMCs) to imitate mucosal infection in IgAN. Chapter I. The expression of IL-4/STAT6, HIPK2, C1GALT1, COSMC and its relationship with IgAl secretion and O-Glycosylation in tonsil from patients with IgA nephropathyObejective:To detect the expression levels of IL-4, STAT6, C1GALT1C1, COSMC, HIPK2in tonsil components and mononuclear cells, and analyze the correlation between IL-4/STAT6pathway and HIPK2with aberrantly glycosylated IgA1in IgA nephropathy.Methods:Tonsillar tissue specimens were obtained from22patients with IgA nephropathy (IgAN) who had been diagnosed based on immunofluorescent and light microscopic findings from percutaneous renal biopsy. The tonsils from24patients with chronic tonsillitis (CT) lacking renal diseases, were used as controls. Immunofluorescence and immunohistochemical stains were performed to detect the expression of IL-4, STAT6, HIPK2, C1GALT1C1, COSMC in tonsil components; Mononuclear cells were separated by density-gradient centrifugation using Lymphocyte Separation Medium, and the expression of IL-4, STAT6, C1GALT1C1, Cosmc, HIPK2in tonsil mononuclear cells (TMCs) were tested by RT-PCR and Western-blot. IgAl content in the supernatant was measured in duplicate using ELISA. The levels of IgAl O-glycosylation were determined by Vicia villosa (VV) lectin-binding assay.Results:1. The expression of IL-4, STAT6and HIPK2were significantly higher compared to CT group. They localized in all tonsil components (including germinal center and tonsillar crypt epithelium) of IgAN patients, but the expression of C1GALT1and COSMC decreased significantly in patients with IgAN in comparison with those of CT.2. The mRNA and protein amount of IL-4, STAT6and HIPK2in the tonsil derived from IgAN patients was significantly higher than that of CT patients. While the expression of C1GALT1and COSMC decreased significantly in patients with IgAN3. The concentration of IgA1in supernatants from cells cultured was higher than control cells. The OD value of VV lectin binding ELISA was significantly higher than that of CT patients.4. The level of HIPK2mRNA expression significantly negatively correlated with renal function as expressed by eGFR, and also significantly positively correlated with daily proteinuria, IgAl concentration and its VVL-binding ELISA OD value.Conclusion:IL-4/STAT6signaling pathway and HIPK2was highly activated in IgAN group; the baseline levels of O-glycosylation of patients with IgAN were significantly lower than CT group. It seems that HIPK2plays a key role in in IgAl secretion and O-Glycosylation. Chapter Ⅱ. Effects of LPS and HS stimulation on TH1, TH2and TH1/TH2ratio in tonsillar lymphocyteObjective:To detect Th1/Th2polarization in palatine tonsil of IgA nephropathy; observe the effects of LPS and HS stimulation on TH1, TH2and TH1/TH2ratio in tonsillar lymphocyteMethods:Tonsillar lymphocyte were cultured with or without LPS (lOug/ml) or HS (1*108/ml) for72hours. PMA/Ionomycin mixture and BFA/Monensin mixture were added as lymphocyte stimulating agent before Flow cytometry test.The determination of CD4+cells was inferred by differential gatering in a flow cytometer, considering CD3+CD8-T cells. Cell populations were defined as follows, TH1: CD3-positive, CD8-negative, IFN-y-positive and IL-4-negative; TH2: CD3-positive, CD8-negative, IFN-y-negative and IL-4-positive.Results:1. Flow cytometry analyses showed percentage of TH1population in IgAN group vs CT group were5.73±2.14%vs4.71±1.98%without stimulation,1.24±0.55%vs0.37±0.22%, in the TH2population. The mean±SD Th1:Th2ratio for the IgAN group without situmlation was4.62±2.12, and in CT group was12.73±5.78.2. Percentage of TH1population in IgAN group vs. CT group were13.30±4.15%vs7.39±3.78%after LPS stimulation,1.20±0.78%vs0.58±0.33%, in the TH2population. The mean±SD values for LPS stimulation group were11.08±4.33vs12.74±5.01. The value in CT group was significantly higher than that in IgAN group without or with stimulation.3. Percentage of TH1population in IgAN group vs CT group were7.03±3.14%vs3.52±2.55%after HS stimulation,8.11±3.98%vs0.77±0.41%, in the TH2population. The mean±SD values for HS stimulation group in IgAN group vs CT group were0.87±0.25vs4.57±1.92.4. The values of the Th1:Th2ratio varied widely among the patients in IgAN group, and we selected those patients who had undergone proteinuria. The Th1:Th2ratio significantly negatively correlated with proteinuria.Conclusion:The value of Thl/Th2in CT group was significantly higher than that in IgAN group without or with stimulation. We hypothesized that a polarization toward TH2response at the stimulated lymphocyte level may lead to immune abnormalities in IgAN. THO cells are differentially mobilized during contact sensitization and by adjuvants such as LPS that induce T helper typel (Th1) responses, or HS that induces T helper type2(Th2) responses. Chapter Ⅲ. Effects of IL-4, LPS and HS on STAT6, HIPK2, C1GALT1and COSMC expression, IgAl secretion and O-GlycosylationObjective:To detect the Effects of IL-4, LPS and HS on IL-4/STAT6, HIPK2, C1GALT1and COSMC expression, IgAl secretion and O-Glycosylation.Methods:Tonsillar lymphocyte were cultured with or without10ng/mL of recombinant human IL-4or HS (1*108/ml) or LPS (10ug/ml) for72hours. The expression of STAT6, HIPK2, C1GALT1and COSMC were examined by RT-PCR and Western blot. IgAl content in the supernatant was measured in duplicate using ELISA. The levels of IgA1O-glycosylation were determined by Vicia villosa (VV) lectin-binding assayResults:1. The levels of mRNA and protein encoding IL-4, STAT6and HIPK2in cells coincubated with IL-4, LPS, HS were significantly higher than that in control without stimulation after72h in IgAN group. Whereas the levels of mRNA and protein encoding C1GALT1and COSMC was significantly lower compared to that without stimulation in IgAN group. The C1GALT1and COSMC mRNA and protein expression from cells cultured with HS was significantly lower than cells cultured with IL-4or LPS in IgAN group (P<0.05). Moreover, the C1GALT1and COSMC expression of TMCs in IgAN patients after stimulation was significantly lower than that in CT patients. 2. The levels of mRNA and protein encoding IL-4, STAT6and HIPK2in cells coincubated with IL-4, LPS, HS were significantly higher than that in control in CT group. While no significantly difference could be seen in C1GALT1and COSMC expression of CT group among different stimulations compared to control group.3. The IgAl production of TMCs stimulated with IL-4, LPS or HS was significantly higher compared to that without stimulation in both groups. Mean IgAl levels of supernatants were remarkable higher after IL-4or HS stimulation compared with cells cultured with LPS. As expected, VV lectin binding to IgAl derived from cells stimulated by HS, IL-4and LPS was significantly higher than that from unstimulated cells in IgAN group. No significant increase in lectin binding was observed for IgA stimulated with IL-4, LPS or HS relative to the unstimulated cultures in CT group.Conclusion:IL-4/STAT6signaling pathway in tonsil of IgA nephropathy can be activated by IL-4, LPS and HS, which can together promoting the production of HIPK2, and downregulation the expression of C1GALT1and COSMC. All the above lead to the production of Under-O-glycosylation IgAl Chapter IV. The effect of silencing HIPK2gene expression in IgAl secretion and O-GlycosylationObjective:HIPK2was highly activated in tonsil of IgAN group, and it has a close relationship with IgAl secretion and O-Glycosylation. In this chapter, we silenced the gene expression of HIPK2to observe the influence in IgAl secretion and O-Glycosylation by HIPK2-siRNA.Methods:To assess the percentage of apoptotic and viable cell fractions after transfection of HIPK2siRNA, a human Annexin V-FITC/PI staining kit was used. The expression of C1GALT1C1and COSMC in tonsil mononuclear cells (TMCs) were tested by RT-PCR and Western-blot. IgAl content and the levels of O-glycosylation in the supernatant were measured in duplicate using ELISA.Results:1. We detected the apoptosis of mononuclear cells after24hours since transfection of HIPK2siRNA. The early stage of apoptosis is (13.801±4.371)%, while the late stage of apoptosis is (10.204±3.441)%. The apoptosis of mononuclear cells increased after HIPK2-siRNA transfection.2. Our results showed that the expression of C1GALT1and COSMC protein and mRNA was increased significantly in IgAN-HIPK2siRNA-treated group.While in CT group the expression of C1GALT1and COSMC protein and mRNA remains stable after transfection of HIPK2siRNA. 3. IgAl protein detected by ELISA decreased after HIPK2-siRNA transfection, while in CT group, IgAl level maintain stable. In IgAN-HIPK2siRNA-treated group, VV lectin binding to IgAl was significantly lower than that from normal controls. While in CT group the OD level of VV lectin binding ELISA remains stable.Conclusion:HIPK2-siRNA negatively regulates IL-4/STAT6pathway-induced IgA secretion. Importantly, HIPK2-siRNA attenuates the aberrant glycosylation of IgAl secretion. Thus, these results suggest that HIPK2is indispensable for IgA production and may play a key role in IgA glycosylation.