Inhibition of p65 NF-κB enhances production of galactose-deficient IgA1 through suppression of C1GALT1 and SP1 in plasmablast-like cell subpopulations

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 to form immune complexes, some of which deposit in glomeruli. 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 that have constitutively activated NF-κB, we report that inhibition of NF-κB/p65 by a selective IKKβ inhibitor TPCA-1 reduced phosphorylation of NF-κB/p65 at S536 and decreased production of IgA1 and, conversely, increased Gd-IgA1 production. This 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 the NF-κB/p65 with co-transcriptional factor SP1, a transcriptional activator of C1GALT1, suggesting that NF-κB pathway affects IgA1 O-glycosylation via SP1 transcriptional control of C1GALT1 expression. Furthermore, prolonged IΚΚβ 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 provide functional evidence for involvement of NF-κB/p65 and its transcriptional partners in IgA1 O-glycosylation.