Galactose-deficient IgA1 has been proposed as an important effector molecule in IgA nephropathy (IgAN). We previously showed that the galactose-deficient IgA1-specific monoclonal antibody KM55 can detect circulating galactose-deficient IgA1 in patients with IgAN, enabling us to study the molecular roles of galactose-deficient IgA1. Herein, we further examined the pathophysiological significance of galactose-deficient IgA1 in glomerular deposits of patients with IgAN by immunohistochemistry using KM55. Immunostaining of galactose-deficient IgA1 with KM55 was performed in paraffin-embedded sections of renal biopsy specimens from 48 patients with IgAN and 49 patients with other renal diseases such as lupus nephritis, HCV-related nephropathy, IgA vasculitis with nephritis (IgA-VN), and membranous nephropathy. Glomerular galactose-deficient IgA1 was specifically detected in IgAN and IgA-VN but not in the other renal diseases. Galactose-deficient IgA1 was localized predominantly in the mesangial region as IgA deposition. However, galactose-deficient IgA1 was not detected in patients with lupus nephritis accompanied by glomerular IgA deposition. Thus, our study strongly suggests that IgAN and IgA-VN have a shared feature regarding galactose-deficient IgA1-oriented pathogenesis.

Galactose-deficient IgA1 (Gd-IgA1) plays a crucial role in the development of IgA nephropathy (IgAN). However, the pathogenic mechanisms driving Gd-IgA1 production have not been fully elucidated. Innate-immune activation via Toll-like receptor 9 (TLR9) is known to be involved in Gd-IgA1 production. A proliferation inducing ligand (APRIL) and IL-6 are also known to enhance Gd-IgA1 synthesis in IgAN. With this as background, we investigated how TLR9 activation in IgA secreting cells results in overproduction of nephritogenic IgA in the IgAN-prone ddY mouse and in human IgA1-secreting cells. Injection of the TLR9 ligand CpG-oligonucleotides increased production of aberrantly glycosylated IgA and IgG-IgA immune complexes in ddY mice that, in turn, exacerbated kidney injury. CpGoligonucleotide-stimulated mice had elevated serum levels of APRIL that correlated with those of aberrantly glycosylated IgA and IgG-IgA immune complexes. In vitro, TLR9 activation enhanced production of the nephritogenic IgA as well as APRIL and IL-6 in splenocytes of ddY mice and in human IgA1-secreting cells. However, siRNA knockdown of APRIL completely suppressed overproduction of Gd-IgA1 induced by IL-6. Neutralization of IL-6 decreased CpG-oligonucleotide-induced overproduction of Gd-IgA1. Furthermore, APRIL and IL-6 pathways each independently mediated TLR9-induced overproduction of Gd-IgA1. Thus, TLR9 activation enhanced synthesis of aberrantly glycosylated IgA that, in a mouse model of IgAN, further enhanced kidney injury. Hence, APRIL and IL-6 synergistically, as well as independently, enhance synthesis of Gd-IgA1.

BackgroundRecent studies have shown that galactose-deficient IgA1 (GdIgA1) has an important role in the pathogenesis of IgA nephropathy (IgAN). Although emerging data suggest that serum GdIgA1 can be a useful non-invasive IgAN biomarker, the localization of nephritogenic GdIgA1-producing B cells remains unclear. Recent clinical and experimental studies indicate that immune activation tonsillar toll-like receptor (TLR) 9 may be involved in the pathogenesis of IgAN. Here we assessed the possibility of GdIgA1 production in the palatine tonsils in IgAN patients.MethodsWe assessed changes in serum GdIgA1 levels in IgAN patients with clinical remission of hematuria and proteinuria following combined tonsillectomy and steroid pulse therapy. Further, the association between clinical outcome and tonsillar TLR9 expression was evaluated.ResultsPatients (n = 37) were divided into two groups according to therapy response. In one group, serum GdIgA1 levels decreased after tonsillectomy (59%) alone, whereas in the other group most levels only decreased after the addition of steroid pulse therapy to tonsillectomy (41%). The former group showed significantly higher tonsillar TLR9 expression and better improvement in hematuria immediately after tonsillectomy than the latter group.ConclusionsThe present study indicates that the palatine tonsils are probably a major sites of GdIgA1-producing cells. However, in some patients these cells may propagate to other lymphoid organs, which may partially explain the different responses observed to tonsillectomy alone. These findings help to clarify some of the clinical observations in the management of IgAN, and may highlight future directions for research.

In IgAN patients, expression levels of tonsillar TLR9 and TLR9 SNP were well correlated with the efficacy of tonsillectomy with SPT.

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Dysregulation of mucosal immunity may play a role in the pathogenesis of IgA nephropathy (IgAN). However, it is unclear whether the nasal-associated lymphoid tissue (NALT) or gut-associated lymphatic tissue is the major induction site of nephritogenic IgA synthesis. To examine whether exogenous mucosal antigens exacerbate the pathogenesis of IgAN, we assessed the disease phenotypes of IgAN-onset ddY mice housed germ-free. These mice were transferred to a specific pathogen-free environment and divided into three groups: challenged with the Toll-like receptor 9 (TLR9) ligand CpG-oligodeoxynucleotide, fecal transplantation, and the untreated control group. The levels of aberrantly glycosylated IgA and IgG-IgA immune complexes were measured in the serum and supernatant of cultured cells purified from the NALT, mesenteric lymph nodes, and Peyer's patch. Although the germ-free IgANonset ddY mice did not develop IgAN, they showed aggravation of kidney injury with mesangial IgA deposition after transfer to the specific pathogen-free state. The NALT cells produced more aberrantly glycosylated IgA than those from the mesenteric lymph node and Peyer's patch, resulting in induction of IgG-IgA immune complexes formation. Additionally, TLR9 enhanced the production of nephritogenic IgA and IgG-IgA immune complexes by nasal-associated lymphoid but not gut-associated lymphatic cells. Furthermore, the germ-free IgAN-onset ddY mice nasally immunized with CpG-oligonucleotide showed aggravation of kidney injury with mesangial IgA deposition, whereas those that received fecal transplants did not develop IgAN. Thus, NALT is the major induction site of the production of aberrantly glycosylated IgA in murine IgAN.

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These results indicate that CD19(+) B cells, which can regulate nephritogenic IgA production in a T-cell-independent manner, are responsible for the disease and are disseminated in peripheral lymphoid organs.