Overexpression of Transmembrane TNF Drives Development of Ectopic Lymphoid Structures in the Bone Marrow and B Cell Lineage Alterations in Experimental Spondyloarthritis

Abstract

TNF is important in immune-mediated inflammatory diseases, including spondyloarthritis (SpA). Transgenic (tg) mice overexpressing transmembrane TNF (tmTNF) develop features resembling human SpA. Furthermore, both tmTNF tg mice and SpA patients develop ectopic lymphoid aggregates, but it is unclear whether these contribute to pathology. Therefore, we characterized the lymphoid aggregates in detail and studied potential alterations in the B and T cell lineage in tmTNF tg mice. Lymphoid aggregates developed in bone marrow (BM) of vertebrae and near the ankle joints prior to the first SpA features and displayed characteristics of ectopic lymphoid structures (ELS) including presence of B cells, T cells, germinal centers, and high endothelial venules. Detailed flow cytometric analyses demonstrated more germinal center B cells with increased CD80 and CD86 expression, along with significantly more T follicular helper, T follicular regulatory, and T regulatory cells in tmTNF tg BM compared with non-tg controls. Furthermore, tmTNF tg mice exhibited increased IgA serum levels and significantly more IgA+ plasma cells in the BM, whereas IgA+ plasma cells in the gut were not significantly increased. In tmTNF tg × TNF-RI−/− mice, ELS were absent, consistent with reduced disease symptoms, whereas in tmTNF tg × TNF-RII−/− mice, ELS and clinical symptoms were still present. Collectively, these data show that tmTNF overexpression in mice results in osteitis and ELS formation in BM, which may account for the increased serum IgA levels that are also observed in human SpA. These effects are mainly dependent on TNF-RI signaling and may underlie important aspects of SpA pathology.

Splenic Architecture and Function Requires Tight Control of Transmembrane TNF Expression

“…Overexpression of tmTNF resulted in a skewed class, switching in favor of IgA and IgM at the expense of the percentage of IgG + PCs, which decreased. This is in line with the increase in systemic IgA levels that we found earlier in tmTNF-tg mice [15]. It may be possible that due to the reduced proportion of GC B cells, there is a reduction of the IgG + PC output in the spleen.…”

“…This observation was in line with the observed decrease in Ki-67 expression, as the DZ is the site of GC proliferation. These effects of tmTNF overexpression on LZ and DZ B cell characteristics were similar to our previous findings in the ectopic lymphoid structures (ELS) present in the vertebrae of tmTNF-tg mice [15]. In contrast to the spleen, PLN LZ B cells decreased while DZ B cells increased in tmTNF-tg mice (Figure S2B), which is consistent with the systemic inflammation observed in these mice.…”

“…tmTNF-tg mice display clear signs of (systemic) inflammation and clinical features of SpA. In addition, they exhibit vertebral ELS formation [11,15]. Therefore, it is surprising that the spleen of these mice lacks mature splenic follicles and that even reduced proportions of GC B cells and follicular T cells are present.…”

“…For example, tmTNF expression in the gut is predictive for anti-TNF treatment response in Crohn's disease [51]. Moreover, we recently found that the overexpression of aberrant tmTNF expression in mice results in SpA features, including pathological bone formation, arthritis, ELS formation in the bone marrow of vertebrae, and increased serum IgA levels [11,15]. Further dissection of the role of tmTNF in these SpA features, together with the observed effects on SLOs and lymphoid cells, would be instrumental in advancing our understanding of the role of tmTNF in SpA and potentially other IMIDs.…”

“…Single-cell suspensions of spleen and PLNs were made by passing the tissue through a 100 µm cell mesh filter in PBS supplemented with 0.5% BSA and 2 mM EDTA). Next, 2 × 10 6 cells were incubated with antibody cocktails for intra-and extracellular markers and various controls [15]. Intracellular immunoglobulin staining was done by fixation and permeabilization of cells with BD Cytofix/Perm Buffer and BD Perm/Wash buffer (BD Biosciences, Franklin Lakes, NJ, USA), respectively.…”