IKK-β inhibitor TPCA-1 increases Gd-IgA1 production and decreases C1GALT1 expression in EBV-immortalized B cells from human donors

IgA nephropathy (IgAN) is a common primary glomerulonephritis characterized by glomerular immune-complex deposits with (co)dominant IgA. These deposits are enriched for IgA1 glycoforms with some O- glycans deficient in galactose (Gd-IgA1). Circulating Gd-IgA1 is bound by IgG autoantibodies forming immune complexes, some of which deposit in glomeruli and induce kidney injury. Genomic and immunologic studies indicate involvement of pro-inflammatory signaling pathways in the production of Gd-IgA1 in IgAN. Genomic studies identified multiple genetic loci associated with IgAN and suggested a convergence on the NF-κB pathway, including RELA , the gene encoding the NF-κB subunit p65. However, the mechanisms by which NF-κB pathways may affect O -glycosylation in IgA1-producing cells are unknown. Using EBV-immortalized B cells derived from peripheral-blood mononuclear cells of IgAN patients and healthy controls, we report that inhibition of p65 NF-κB by an IKK-β inhibitor, TPCA-1, reduced phosphorylation of p65 NF-κB and decreased production of IgA1 and, conversely, increased Gd-IgA1 production. This effect on Gd-IgA1 was likely related to reduced expression of C1GALT1 gene that encodes the enzyme responsible for galactosylation of IgA1 O -glycans. Flow-cytometry imaging revealed changes in nuclear translocation and co-localization of p65 NF-κB with co-transcriptional factor SP1, a transcriptional activator of C1GALT1 , suggesting that NF-κB pathway and SP1 association affect IgA1 O -glycosylation through transcriptional control of C1GALT1 expression. Furthermore, prolonged IKK-β inhibition altered B-cell subpopulations, enhancing generation of cells with a plasmablast-like phenotype, characterized by high SSC MFI and CD138 expression. Together, these findings implicate p65 NF-κB and its transcriptional partners in IgA1 O -glycosylation.