Brigham and Women's Hopsital Boston, MA, United States
Bo Sun1, Benjamin Geary2, Santanu Mondal3, Ronak Tilvawala4, Konstantin Tsoyi5, Ivan O. Rosas5, Paul Thompson3 and I-Cheng Ho2, 1Brigham and Women's Hospital, Lexington, MA, 2Brigham and Women's Hospital, Boston, MA, 3UMass Medical School, Worcester, MA, 4University of Kansas, Lawrence, KS, 5Baylor College of Medicine, Houston, TX
Background/Purpose: The secretion of antibody requires concerted action of several endoplasmic reticulum proteins (ERPs) in B cells. For example, marginal zone B and B1 cell-specific protein (MZB1), an ERP that is highly expressed in lymph nodes from patients with lupus or rheumatoid arthritis (RA), is required for the secretion of IgA. How the function of ERPs is regulated in B cells is still not fully understood. Approximately 60-70% of patients with RA have antibody against auto-antigens that have undergone citrullination, which is a unique form of post-translational modification converting peptidylarginine to peptidylcitrulline and is catalyzed by peptidylarginine deiminases (PADs), including PAD1-4 and PAD6 in mammals. Several major risk factors of RA are associated with hypercitrullination, which not only expands the pool of citrullinated auto-antigens but also alters the function of proteins and immune cells, including T cells, neutrophils, and macrophages. Surprisingly, very little is known regarding the regulatory role of PADs or citrullination in B cells, which produce anti-citrullinated protein antibodies and are very effective therapeutic targets.
Methods: An unbiased citrullinomic analysis was conducted to identify lung proteins that are preferentially citrullinated in RA-associated interstitial lung disease (RA-ID) compared to other chronic lung diseases. The status of citrullination of the proteins thus identified was examined with in vitro citrullination, mass spectrometry, PAD inhibitors, and in PAD2-deficient cells. Primary human and mouse peripheral B cells were differentiated into plasmablasts in vitro and their production of immunoglobulin was examined with FACS and ELISA. CRISPR was used to ablate genes of interest. Site-specific mutagenesis was used to convert arginine to lysine.
Results: MZB1 was preferentially enriched in the pool of citrullinated lung proteins obtained from patients with RA-ILD compared to that with idiopathic pulmonary fibrosis or chronic obstructive pulmonary diseases. MZB1 was a substrate of PADs and was citrullinated in both mouse and human primary B cells. Citrullination of MZB1 enhanced its interaction with IgA and this enhanced interaction was mediated mainly by one arginine residue. Pharmacological inhibition or genetic ablation of PAD2 in human and mouse primary plasmablasts inhibited the secretion of IgA but not the differentiation of plasmablasts, class switch of immunoglobulin, or expression of IgA, thereby phenocopying MZB1 deficiency.
Conclusion: PAD2 promotes IgA secretion through citrullinating MZB1. This finding expands the regulatory role of PAD-mediated citrullination to B cells and suggests a potential mechanism by which hypercitrullination contributes to the pathogenesis of RA and RA-ILD.
Disclosures: B. Sun, None; B. Geary, None; S. Mondal, None; R. Tilvawala, None; K. Tsoyi, None; I. Rosas, None; P. Thompson, None; I. Ho, None.
