Increase of MZB1 in B cells in systemic lupus erythematosus: proteomic analysis of biopsied lymph nodes

Systemic lupus erythematosus (SLE) is a systemic inflammatory autoimmune disease characterized by production of autoantibodies directed against nucleic acid-associated autoantigens that cause multiple organ damage, including skin, joints, kidney, and the central nervous system [1]. Aberrant innate immune responses play an important role in SLE, contributing tissue injury via release of proinflammatory cytokines and aberrant activation of T and B cells, with the latter leading to pathogenic autoantibody production [2]. Autoreactive B cells differentiate into pathogenic memory and plasma cells via germinal center responses [3]. B-cell depletion is an attractive therapeutic option in SLE. However, targeting human CD20 (rituximab), which is expressed on almost all B-cell lineages except early pro-B cells, plasmablasts, and plasma cells, has shown disappointing results in two randomized clinical trials of lupus [4, 5], although there has been a question about both trial design and clinical outcome measures [2]. Recent clinical trials using belimumab, a fully humanized monoclonal antibody directed against B-lymphocyte stimulator (BLyS; also known as BAFF), proved beneficial [6‐9]. Dual B-cell immunotherapy was confirmed to be superior to individual anti-CD20 depletion or BAFF blockade in a murine lupus model [10]. Upon binding to its receptors, TACI, BAFF receptor, and B-cell maturation antigen, BLyS activates signals for B-cell survival and maturation with belimumab effect via depletion of recently formed, rather than memory, B cells or long-lived plasma cells in SLE [6‐8]. Recently, proteasome inhibitors bortezomib, delanzomib, and carfilzomib have shown to be effective in refractory SLE patients [11] or lupus-prone mice by depleting plasma cells [12‐14]. Plasma cells are sensitive to proteasome inhibitors because of their high rate of antibody synthesis [11‐14].

In the study of murine lupus-prone models, the spleen is examined to understand the underlying mechanisms of lupus [15‐21]. Although localized or generalized lymphadenopathy is a common symptom, seen at some stage in the evolution of the disease in about 60% of SLE patients [22], for SLE patients lymphoid organs (e.g., spleen and lymph node) are rarely biopsied, only if malignancy should be ruled out. The pathology database in our institution contained five archives of biopsied specimens from patients with lymphadenopathy associated with SLE, which were collected to rule out malignancy. Formalin-fixed paraffin-embedded (FFPE) tissues archived in pathology department laboratories worldwide may provide an extremely valuable resource for biomarker discovery for rare diseases [23]. Recent developments in methodologies using FFPE tissue in mass spectrometry-based proteomics led us to examine potential biomarkers and new therapeutic targets of SLE [24].

Here, we applied liquid chromatography-tandem mass spectrometry (LC-MS) to analyze lymph node tissue from SLE patients and identified MZB1 as a potential biomarker of SLE.

MZB1, also known as pERp1, is an ER-associated protein that regulates B-cell receptor-driven calcium responses and is greatly induced during plasma cell differentiation. MZB1 regulates proper assembly and secretion of mature IgM. It is highly expressed in MZ B cells and B1 cells, with much lower expression in FoB cells in accordance with their role as prolific IgM secretors [28, 29, 31]. We found increased expression of MZB1 protein in SLE patient specimens as well as aged BWF1 mice specimens compared with respective controls, reflecting excessive autoantibody secretion in lupus. Our study confirmed MZB1 colocalized with CD138⁺ plasma cells and IRTA1⁺ MZ B cells in human tissues. In aged BWF1 mice, isolated splenic MZ B cells and plasma cells showed elevated levels of MZB1 compared with control mice. MZ B cells play important roles in the early phases of humoral immune responses. MZ B cells reside in follicles in lymphoid organs and efficiently transport blood-borne antigens to follicles, where they activate CD4⁺ T cells and rapidly differentiate into plasma cells [33‐35]. In contrast, the majority of conventional B cells, termed FoB cells, produce specific antibodies with much slower kinetics. MZ B cells also contain a large number of autoreactive clones and the expansion of this compartment has been associated with autoimmunity [19]. The MZ B-cell compartment is expanded in BWF1 mice [16] and other murine models of lupus [15, 17, 18, 21]. Autoreactive MZ B cells enter follicles and interact with CD4⁺ T cells in lupus-prone mice [19, 20]. Increased intrafollicular localization of MZB1⁺ cells was observed in specimens from SLE patients as well as BWF1 mice compared with respective controls, suggesting the role of MZ B cells within follicles in lupus pathogenesis.

Prominent ectopic lymphoid tissue in target organs was observed in aged BWF1 mice, which was not seen in young BWF1 mice. Long-lived autoantibody-secreting cells are reportedly found within inflamed kidneys of BWF1 mice as well as SLE patients [36]. Excessive MZB1 expression in specimens from SLE patients and BWF1 mice may indicate MZB1 is a potential marker of prolific autoantibody-secreting B cells in autoimmune disease. Approximately 31% of renal IgG-producing cells react with dsDNA, which is higher than cells from the spleen (15%) and bone marrow (21%) in aged BWF1 mice [36]. Autoantibodies secreted by long-lived plasma cells in target organs were shown to be pathogenic and major drivers of inflammation in a study using adoptive transfer of antibody-secreting cells from BWF1 mice to Rag^–/– mice [37]. In our study, the frequency of MZB1⁺ cells in an isolated splenic B-cell subset was relatively lower than expected (at maximum, 34% of plasma cells) in aged BWF1 mice, while MZB1⁺ cells were predominantly observed in target organs, suggesting that survival niches for plasma cells exist in target organs [36].

The ER functions to properly fold and process secreted and transmembrane proteins. Physiological and pathological stimuli can disrupt ER homeostasis, resulting in an accumulation of misfolded and unfolded proteins, known as ER stress [26]. ER stress activates a complex signaling network referred as the UPR pathway to reduce ER stress, resulting in increased transcription of ER resident chaperones, folding enzymes, and components of the protein degradation machinery [26]. If misfolded proteins cannot be properly refolded, then they are sent outside the ER and subsequently degraded by cytosolic 26S proteasomes [32, 38]. BiP is a central regulator of UPR stress sensors as well as an ER chaperone that assists in protein folding. MZB1 is found to associate with ER resident chaperones and oxidoreductases, including BiP, GRP94, ERp57, calnexin/calreticulin, and PDIA6, and assists in folding of Igμ heavy chain [28, 29, 31]. Moreover, MZB1 appears to act as a cochaperone of GRP94 [39, 40]. MZB1 also promotes cell surface expression of integrins and Toll-like receptors, which are GRP94 clients [31, 39].

It is known that the UPR signaling pathway is turned on when B cells differentiate into plasma cells to facilitate proper protein folding [32]; however, under unresolvable ER stress conditions, the UPR pathway promotes apoptosis [26]. Our study showed BiP expression peaked within 24 hours, followed by recovery on day 10, in BWF1 mice after TM treatment. MZB1 expression peaked at 48 hours. BiP is ubiquitously expressed in cells and plays a role as a UPR stress sensor as well as an ER chaperone to assist protein folding, in contrast to MZB1 which is a B-cell-specific protein [26, 39, 40]. This may explain the earlier upregulation of BiP than MZB1. In TUNEL staining of tissue sections from TM-treated BWF1 mice, the number of TUNEL-positive cells peaked on day 3 and recovered by day 7. Serum anti-dsDNA antibody titers decreased with TM treatment, which may be due to apoptosis of B cells caused by excessive ER stress. One BWF1 mouse showed an increased anti-dsDNA antibody titer on day 4 compared with that before treatment; however, the titer decreased by day 7. ER stress may stimulate antibody-secreting cells to release antibodies soon after TM treatment; however, cell death may be induced under excessive ER stress, resulting in the decreased anti-dsDNA antibody titers observed on day 7. Apoptosis was predominantly observed in MZB1⁺ cells, suggesting that TM treatment mainly affected antibody-secreting cells.

Because chaperones can associate with a number of proteins, including autoantigens, they can act as endogenous adjuvants in immune responses [41]. ER resident chaperones BiP, GRP94, calnexin, and HSP90 take part in MHC peptide loading in addition to their roles in folding and assembly of early intermediates of MHC class I and II molecules, and thus they may be involved in antigen recognition and presentation [42]. Some ER resident chaperones, such as GRP94, are released into extracellular fluid, likely by destruction of the plasma membrane that accompanies stress-induced apoptosis or necrosis [41]. Autoantibodies against BiP, GRP94, calnexin, and HSP90 are detected in SLE and RA patients [43‐45], although their pathophysiological role remains unknown. In this study, we determined serum anti-MZB1 antibody concentrations by ELISA and found no significant difference between SLE patients and healthy donors (data not shown). This may be because MZB1 is expressed at a lower concentration than ubiquitously expressed BiP, GRP94, and HSP90 [26].

The observations described in this study implicate an endogenous chaperone in the pathogenesis of SLE, and the selective nature and excessive expression of MZB1 in B-cell subsets make it an attractive therapeutic target for SLE treatment [46]. Knockdown of MZB1 slows down IgM polymerization and subsequently leads to reduced IgM secretion [29, 31]. Recently, HSP90 inhibitors were suggested to be effective against a variety of oncogene-addicted cancers [47]. Bortezomib treatment has been demonstrated to be effective in refractory SLE patients [11]. Bortezomib is a selective inhibitor of the 26S proteasome, which is present in all eukaryotic cells, and thereby it exhibits systemic toxicity [11, 48]. In contrast, MZB1 is limitedly expressed in MZ B cells, B1 cells, and plasma cells. MZB1 inhibitors may cause degradation of immunoglobulin, causing apoptosis of autoantibody-secreting cells in SLE and other autoimmune diseases.

MZB1, an ER resident molecular chaperone, was overexpressed in B-cell subsets in lymph nodes from SLE patients and lupus-prone mice. MZB1 may be a potential therapeutic target in excessive antibody-secreting cells in SLE.