The TLR4-MyD88-NF-κB Pathway Is Involved in sIgA-Mediated IgA Nephropathy

Background: IgA nephropathy (IgAN) is the most common primary glomerulonephritis worldwide. Previous studies have shown that secretory IgA (sIgA; an important antibody in mucosal immunity) was critically involved in IgAN immune responses. Toll-like receptors (TLRs), especially TLR4 which participates in mucosal immunity, may be involved in the pathogenesis of IgAN. However, it remains unclear whether sIgA and TLRs interact to mediate the pathogenesis of IgAN. The purpose of this study was to investigate whether sIgA and TLR4 interact to mediate kidney damage in IgAN patients. Methods: IgAN patients with positive sIgA deposition in renal tissues were screened by immunofluorescence assay. Patient salivary sIgA (P-sIgA) was collected and purified by jacalin affinity chromatography. Salivary sIgA from healthy volunteers was used as a control (NsIgA). Immunohistochemistry was used to assess expression of TLR4, MyD88, NF-κB, tumour necrosis factor (TNF)-α, interleukin (IL-6), and monocyte chemoattractant protein (MCP)-1 in renal tissues of IgAN patients. Human renal mesangial cells (HRMCs) were cultured in vitro and binding of sIgA to HRMCs was assessed by flow cytometry. HRMCs were cultured in the presence of sIgA (400 μg/mL) for 24 h. Transcript and protein levels of TLR4, MyD88, NF-κB and cytokines in HRMCs were determined by qPCR, western blotting and ELISA, respectively. Finally, shRNA lentiviral particles were used to silence TLR4 expression and a small-molecule inhibitor (BAY 11-7082) was used to block NF-κB signalling in HRMCs and changes in cytokine production were observed. Results: Flow cytometry showed that P-sIgA bound HRMCs (85% positive) significantly better than N-sIgA (P< 0.05). Expression of TLR4, MyD88, NF-κB, TNF-α, IL-6, and MCP-1 were detected in the mesangial area of IgAN patients. Expression levels in renal tissue in patients with positive sIgA deposition were higher than that in patients with negative sIgA deposition. Compared with cells cultured with N-sIgA, HRMCs cultured in vitro with P-sIgA showed enhanced expression of TLR4, increased secretion of TNF-α, IL-6, and MCP-1, and increased expression of MyD88/NF-κB (P<0.05). TLR4 shRNA silencing and NF-κB inhibition both reduced the ability of HRMCs to synthesize TNF-α, IL-6, and MCP-1. Conclusions: Our results indicate that sIgA may induce high expression of TLR4 in HRMCs and further activate downstream signalling pathways, prompting HRMCs to secrete multiple cytokines and thereby mediating kidney damage in IgAN patients. Funding Statement: This work was supported by grants from the National Natural Science Foundation of China (No. 81570645/H0509), the Innovation Scientists and Technicians Troop Construction Projects of Henan Province (2018JR0014), the Sponsored by Program for Science & Technology Innovation Talents in Universities of Henan Province (18HASTIT043), and the Major Project of Henan Medical Science and Technology Research Program (201501018). Declaration of Interests: The authors have no conflicts or financial interests to declare. Ethics Approval Statement: The study was reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University, and was carried out according to the principles laid out in the World Medical Association’s Declaration of Helsinki.