Loss of the Golgi Matrix Protein 130 Cause Aberrant IgA1 Glycosylation in IgA Nephropathy

2019 ◽  
Vol 49 (4) ◽  
pp. 307-316 ◽  
Author(s):  
Chang Wang ◽  
Muyao Ye ◽  
Qiulan Zhao ◽  
Ming Xia ◽  
Di Liu ◽  
...  
Keyword(s):  
Iga Nephropathy ◽  
Matrix Protein ◽  
Mononuclear Cells ◽  
Lectin Binding ◽  
Underlying Mechanism ◽  
Sirna Transfection ◽  
Peripheral Blood Mononuclear ◽  
Vicia Villosa Lectin ◽  
Iga1 Glycosylation ◽  
Golgi Matrix

Background: Aberrant O-glycosylation IgA1 production is a major factor in the pathogenesis of IgA nephropathy, but the underlying mechanism is still unclear. IgA1 glycosylation modification is in Golgi, and downregulation of the Golgi peripheral membrane protein Golgi matrix protein 130 (GM130) could lead to glycosylation deficiency. In this study, we aimed to explore the role of GM130 in glycosylate deficiency IgA1 (Gd-IgA1) production. Methods: We enrolled 27 IgA nephropathy patients, 12 patients with chronic tonsillitis, 15 non-IgAN chronic kidney disease patients, and 15 healthy volunteers as healthy control. We explored GM130 expression in Tonsillar tissue by immunofluorescence staining and Western blotting and expression in peripheral blood mononuclear cells (PBMCs) by flow cytometry. The concentration of IgA1 and level of O-glycosylation were determined by ELISA and Vicia Villosa lectin-binding assay. Real-time PCR and Western blot were used to analyze the levels of β1,3-Gal transferase (C1GALT1) and ST6GalNAC2, respectively. To explore the contribution of GM130 in IgA1 O-glycosylation modification, cells were subjected to experiments for evaluation of GM130 silencing by GM130-siRNA transfection. Results: GM130 expression was significantly decreased in tonsil tissues and PBMC of IgAN patients; the expression of C1GALT1 decreased and Gd-IgA1 level increased significantly in patients with IgAN patients. The expression of GM130 was negatively related to Gd-IgA1 production. By siRNA transfection, our results clearly indicated that the downregulation of GM130 can increase IgA1 O-glycosylation deficiency, which is thought to reduce C1GALT1 expression but not affect the expression of ST6GalNAC2. Conclusion: We identified and demonstrated that GM130 plays an important role in IgA1 O-glycans deficiency in IgAN patients, by negatively regulating C1GALT1 expression. We believe that this finding will provide theoretical foundations for a new mechanism of Gd-IgA1 production in IgAN patients.

scholarly journals Astragaloside IV Inhibits Galactose-Deficient IgA1 Secretion via miR-98-5p in Pediatric IgA Nephropathy

2021 ◽  
Vol 12 ◽  
Author(s):  
Caiqiong Liu ◽  
Xiaoyan Li ◽  
Lanjun Shuai ◽  
Xiqiang Dang ◽  
Fangrong Peng ◽  
...  
Keyword(s):  
Iga Nephropathy ◽  
Mononuclear Cells ◽  
Lectin Binding ◽  
Immunoglobulin A ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Enzyme Linked Immunosorbent Assay ◽  
Astragaloside Iv ◽  
Peripheral Blood Mononuclear ◽  
Vicia Villosa Lectin

Purpose: The factor associated with IgA nephropathy (IgAN) is an abnormality of IgA known as galactose-deficient IgA1 (Gd-IgA1). The purpose of this study was to determine the molecular role played by miRNAs in the formation of Gd-IgA1 in IgAN and investigate the regulatory role of Astragaloside IV (AS-IV) in miRNAs.Patients and methods: Bioinformatics analysis, along with functional and mechanistic experiments, were used to investigate the relationship and function of miRNA, β-1, 3-galactosyltransferase (C1GALT1), Gd-IgA1, and AS-IV. Analyses involved a series of tools, including quantitative real-time polymerase chain reaction (qRT-qPCR), Western blot, enzyme-linked immunosorbent assay (ELISA), Vicia Villosa lectin-binding assay (VVA), Cell counting kit-8 assay (CCK-8), and the dual-luciferase reporter assay.Results: miRNA screening and validation showed that miR-98-5p was significantly upregulated in the peripheral blood mononuclear cells (PBMCs) of pediatric patients with IgAN compared with patients diagnosed with mesangial proliferative glomerulonephritis (MsPGN) and immunoglobulin A vasculitis nephritis (IgAV-N), and healthy controls (p < 0.05). Experiments with the dual-luciferase reporter confirmed that miR-98-5p might target C1GALT1. The overexpression of miR-98-5p in DAKIKI cells decreased both the mRNA and protein levels of C1GALT1 and increased the levels of Gd-IgA1 levels; these effects were reversed by co-transfection with the C1GALT1 plasmid, and vice versa. In addition, AS-IV downregulated the levels of Gd-IgA1 level in DAKIKI cells by inhibiting miR-98-5p.Conclusions: Our results revealed that AS-IV could inhibit Gd-IgA1 secretion via miR-98-5p. Increased levels of miR-98-5p in pediatric IgAN patients might affect the glycosylation of IgA1 by targeting C1GALT1. In addition, our analyses suggest that the pathogenesis of IgAN may differ from that of IgAV-N. Collectively, these results provide significant insight into the pathogenesis of IgAN and identify a potential therapeutic target.

scholarly journals A New Vision of IgA Nephropathy: The Missing Link

2019 ◽  
Vol 21 (1) ◽  
pp. 189 ◽  
Author(s):  
Fabio Sallustio ◽  
Claudia Curci ◽  
Vincenzo Di Leo ◽  
Anna Gallone ◽  
Francesco Pesce ◽  
...  
Keyword(s):  
Iga Nephropathy ◽  
Mononuclear Cells ◽  
Serum Levels ◽  
High Serum ◽  
In Vitro Production ◽  
Peripheral Blood Mononuclear ◽  
Familial Form ◽  
New Vision ◽  
New Perspective

IgA Nephropathy (IgAN) is a primary glomerulonephritis problem worldwide that develops mainly in the 2nd and 3rd decade of life and reaches end-stage kidney disease after 20 years from the biopsy-proven diagnosis, implying a great socio-economic burden. IgAN may occur in a sporadic or familial form. Studies on familial IgAN have shown that 66% of asymptomatic relatives carry immunological defects such as high IgA serum levels, abnormal spontaneous in vitro production of IgA from peripheral blood mononuclear cells (PBMCs), high serum levels of aberrantly glycosylated IgA1, and an altered PBMC cytokine production profile. Recent findings led us to focus our attention on a new perspective to study the pathogenesis of this disease, and new studies showed the involvement of factors driven by environment, lifestyle or diet that could affect the disease. In this review, we describe the results of studies carried out in IgAN patients derived from genomic and epigenomic studies. Moreover, we discuss the role of the microbiome in the disease. Finally, we suggest a new vision to consider IgA Nephropathy as a disease that is not disconnected from the environment in which we live but influenced, in addition to the genetic background, also by other environmental and behavioral factors that could be useful for developing precision nephrology and personalized therapy.

Downregulating the Fibromodulin Gene by Short Interfering RNA Causes B-CLL Cell Death.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 176-176 ◽  
Author(s):  
Aniruddha Choudhury ◽  
Katja Derkow ◽  
Eva Mikaelsson ◽  
Parviz Kokhaei ◽  
Anders Österborg ◽  
...  
Keyword(s):  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Rt Pcr ◽  
Sirna Transfection ◽  
Peripheral Blood Mononuclear ◽  
Normal Peripheral Blood ◽  
Extracellular Matrix Molecule ◽  
Rnai Technology ◽  
Blood Mononuclear Cells

Abstract Development of targeted therapies against B-CLL is dependent on the identification of molecules that are essential for the proliferation and survival of the leukemic cells. One such molecule investigated by us as a putative leukemia-associated target is Fibromodulin (FIM). FIM is an extracellular matrix molecule belonging to the leucin-rich proteoglycan family. Our laboratory studies have verified that expression of FIM is upregulated in B-CLL cells. Moreover, this molecule is specifically overexpressed in B-CLL cells and not on normal peripheral blood mononuclear cells. Analysis of FIM expression on various other hematological tumor cells have revealed that with the exception of mantle cell lymphoma, FIM is not expressed in other hematological malignancies. RNAi technology has recently emerged as a powerful method to specifically silence the expression of a gene. We have generated three siRNA against various segments of FIM and tested the effects of these siRNA on B-CLL cells. Transfection of B-CLL cells with these siRNA significantly diminished, or completely abrogated the expression of FIM mRNA as detected by RT-PCR. The figure below shows silencing of the FIM gene following siRNA transfection, as assayed by RT-PCR. “Untrans” and “ctrl” represents untransfected and control siRNA-transfected B-CLL cells respectively. As noted, transfection with each of the three siRNA against FIM completely abrogated the expression of FIM siRNA. 24–48 hours after siRNA transfection, a substantial fraction (30–70%) of the B-CLL cells, compared to cells transfected with control non-silencing siRNA, went into apoptosis as assayed by Annexin-V-propidium iodide staining. Time kinetic studies revealed that siRNA mediated silencing and apoptosis occurred between 18 and 48 hours. No such effect was noted when peripheral blood mononuclear cells of healthy donors or FIM-positive fibroblast cell lines were transfected with siRNA. In reconstitution experiments, siRNA treated B-CLL cells were co-cultured with various numbers of FIM-positive fibroblasts. The presence of these fibroblasts greatly diminished the apoptosis of B-CLL cells induced after siRNA treatment. Our results indicate that overexpression of FIM in B-CLL cells are critical for their survival and FIM may serve as a leukemia-specific target for B-CLL therapy. Figure Figure

scholarly journals Sialylation of IgG inhibits the formation of galactose-deficient IgA1-containing immune complexes and protects mesangial cells from injury in IgA nephropathy

2022 ◽  
Vol 23 (1) ◽  
Author(s):  
Youxia Liu ◽  
Hongfen Li ◽  
Huyan Yu ◽  
Fanghao Wang ◽  
Junya Jia ◽  
...  
Keyword(s):  
Iga Nephropathy ◽  
B Lymphocytes ◽  
Immune Complexes ◽  
Mononuclear Cells ◽  
Mesangial Cells ◽  
Healthy Controls ◽  
Peripheral Blood Mononuclear ◽  
Tnf Α ◽  
Inflammatory State

Abstract Background The addition of sialic acid alters IgG from a pro-inflammatory state to an anti-inflammatory state. However, there is a lack of research on the changes of IgG sialylation in IgA nephropathy (IgAN). Methods This study included a total of 184 IgAN patients. The sialylated IgG (SA-IgG), IgG-galactose-deficient IgA1 complex (IgG-Gd-IgA1-IC), IL-6, TNF-α, and TGF-β were detected using commercial ELISA kits. SA-IgG, non-sialylated IgG (NSA-IgG), sialylated IgG-IgA1 complex (SA-IgG-IgA1), and non-sialylated IgG-IgA1 complex (NSA-IgG-IgA1) were purified from IgAN patients and healthy controls (HCs). Results The mean SA-IgG levels in plasma and B lymphocytes in IgAN patients were significantly higher than those of healthy controls. A positive correlation was found between SA-IgG levels in plasma and B lymphocytes. In vitro, the results showed that the release of IgG-Gd-IgA1-IC was significantly decreased in peripheral blood mononuclear cells (PBMCs) cultured with SA-IgG from both IgAN patients and healthy controls. The proliferation ability and the release of IL-6, TNF-α, and TGF-β in human mesangial cells (HMCs) were measured after stimulating with SA-IgG-IgA1-IC and NSA-IgG-IgA1-IC. The mesangial cell proliferation levels induced by NSA-IgG-IgA1-IC derived from IgAN patients were significantly higher than those caused by SA-IgG-IgA1-IC derived from IgAN patients and healthy controls. Compared with NSA-IgG-IgA1 from healthy controls, IgAN-NSA-IgG-IgA1 could significantly upregulate the expression of IL-6 and TNF-α in mesangial cells. The data showed that there weren’t any significant differences in the levels of IL-6, TNF-α, and TGF-β when treated with IgAN-SA-IgG-IgA1 and HC-NSA-IgG-IgA1. Conclusions The present study demonstrated that the sialylation of IgG increased in patients with IgA nephropathy. It exerted an inhibitory effect on the formation of Gd-IgA1-containing immune complexes in PBMCs and the proliferation and inflammation activation in mesangial cells.

Crystals of monosodium urate monohydrate enhance lipopolysaccharide-induced release of interleukin 1β by mononuclear cells through a caspase 1-mediated process

2008 ◽  
Vol 68 (2) ◽  
pp. 273-278 ◽  
Author(s):  
E J Giamarellos-Bourboulis ◽  
M Mouktaroudi ◽  
E Bodar ◽  
J van der Ven ◽  
B-J Kullberg ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Gouty Arthritis ◽  
Underlying Mechanism ◽  
Monosodium Urate ◽  
Peripheral Blood Mononuclear ◽  
Caspase 1 ◽  
Pyrin Domain ◽  
Factor Α ◽  
Msu Crystals ◽  
Urate Crystals

Objective:Recent studies suggest that crystals of monosodium urate (MSU), deposited in joints of patients with acute gouty arthritis, activate the NACHT domain, leucine-rich repeat and pyrin domain-containing protein (NALP)3 inflammasome. In the present study we have investigated whether production of proinflammatory cytokines by crystals was exacerbated during costimulation with Toll-like receptor (TLR) ligands.Methods:Mononuclear cells of 22 healthy donors were stimulated by various concentrations of MSU crystals in the absence or presence of lipopolysaccharide (LPS), Pam3Cys and flagellin. Production of tumour necrosis factor α (TNFα), interleukin (IL)1β and IL6, as well as the intracellular concentrations of proIL1β were measured by ELISA. mRNA transcripts of TNFα and IL1β were assessed by real-time PCR. Stimulation experiments were also performed with peripheral blood mononuclear cells (PBMCs) of one patient carrying a NALP3 mutation.Results:MSU induced a moderate release of IL1β and IL6, but not of TNFα. Urate crystals amplified IL1β production stimulated by the TLR4 ligand LPS, while no synergy was apparent for IL6 production. In addition, no synergy between urate crystals and Pam3Cys (TLR2 ligand) or flagellin (TLR5 ligand) was apparent. The synergy between urate crystals and LPS was directed at the level of the NALP3 inflammasome, as it was present only when active IL1β was measured, but not at the level of IL1 mRNA or proIL1β. The synergy between LPS and MSU crystals ceased to exist in the presence of a caspase 1 inhibitor.Conclusions:MSU crystals act in synergy with LPS for the induction of enhanced release of IL1β. Increased cleavage of proIL1β by urate-activated caspase 1 is proposed as the underlying mechanism.

scholarly journals Plasma ST6GAL1 regulates IgG sialylation to control IgA nephropathy progression

2021 ◽  
Vol 12 ◽  
pp. 204062232110486
Author(s):  
Youxia Liu ◽  
Huyan Yu ◽  
Sijing Wu ◽  
Xia Yang ◽  
Congcong Cao ◽  
...  
Keyword(s):  
Iga Nephropathy ◽  
Protective Factor ◽  
Mononuclear Cells ◽  
Continuous Variable ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Peripheral Blood Mononuclear ◽  
Tnf Α ◽  
Egfr Decline

Background: Our previous study revealed that plasma levels of a-2,6-sialyltransferase 1 (ST6GAL1) were increased in patients with IgA nephropathy (IgAN). ST6GAL1 catalyzes terminal sialylation of IgG to shift the antibody effector function to the anti-inflammatory pattern. However, the role of plasma ST6GAL1 in the progression of IgAN and underlying mechanisms are still unknown. Methods: A total of 180 IgAN patients were included. The kidney outcomes were defined as the eGFR decline or proteinuria remission. Peripheral blood mononuclear cells (PBMCs) were either stimulated with purified sialylated IgG (SA-IgG) or with non-sialylated IgG (NSA-IgG) from IgAN patients to detect the levels of interleukin (IL)-6 and tumor necrosis factor-α (TNF-α) in supernatant. Results: Compared with the lower ST6GAL1 (reference), the risk of eGFR decline decreased for the higher ST6GAL1 group after adjustment for baseline eGFR, systolic blood pressure (SBP), and proteinuria. The results showed that patients with higher ST6GAL1 levels had a higher rate of proteinuria remission. ST6GAL1, expressed as a continuous variable, was a protective factor for eGFR decline and proteinuria remission. An in vitro study showed that the administration of recombinant ST6GAL1 (rST6GAL1) decreased the levels of IL-6 and TNF-α in PBMCs. Furthermore, the administration of rST6GAL1 resulted in the enrichment of SA-IgG in a concentration-dependent manner. In addition, as compared to control, purified SA-IgG-treated PBMCs showed a significant decrease in the expression of IL-6 and TNF-α. Conclusion: Our study indicated that elevated ST6GAL1 was associated with a slower progression of IgAN, which may play a protective effect by increasing IgG sialylation to inhibit the production of proinflammatory cytokines in PBMCs.

scholarly journals IL-21 Rescues The Defect of IL-10-Producing Regulatory B Cells and Improves Allergic Asthma in DOCK8 Deficient Mice

2021 ◽  
Author(s):  
Jinqiu Jiang ◽  
Tao Qin ◽  
Liang Zhang ◽  
Qiao Liu ◽  
Jiabin Wu ◽  
...  
Keyword(s):  
B Cells ◽  
Mononuclear Cells ◽  
Allergic Diseases ◽  
Underlying Mechanism ◽  
Regulatory B Cells ◽  
Peripheral Blood Mononuclear ◽  
Asthma Model ◽  
Dock8 Deficiency ◽  
Immunodeficiency Syndrome

Abstract Purpose Mutations in human DOCK8 cause a combined immunodeficiency syndrome characterized by allergic diseases such as asthma and food allergy. However, the underlying mechanism is unclear. Regulatory B (Breg) cells that produce IL-10 exert potent immunosuppressive functions in patients with allergic and autoimmune disorders. DOCK8-deficient B cells have been revealed diminished responses to LPS stimulation, suggesting a possible defect in IL-10-producing Breg cells in those with DOCK8 deficiency, which may contribute to allergies.Methods we collected peripheral blood mononuclear cells from DOCK8-deficient patients and generated a DOCK8KO mouse model to study the effect of DOCK8 deficiency on the Regulatory B cells.Results We show that DOCK8-deficient patients and DOCK8KO mice harbor quantitative and qualitative defects in IL-10-producing Breg cells; these defects are caused by abnormal DOCK8-/- IL-21-producing CD4+ T cells. We found that recombinant murine (rm)IL-21 restores the function of Bregs both in vitro and in DOCK8KO mice, leading to reduced inflammatory cell infiltration of the lungs in a murine asthma model. Conclusion Overall, the results provide new insight into the potential design of Breg-based or IL-21-based therapeutic strategies for allergic diseases, including asthma with DOCK8 deficiency.

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