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

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.

Soluble tumor necrosis factor (sTNF) is an important inflammatory mediator and essential for secondary lymphoid organ (SLO) development and function. However, the role of its transmembrane counterpart (tmTNF) in these processes is less well established. Here, the effects of tmTNF overxpression on SLO architecture and function were investigated using tmTNF-transgenic (tmTNF-tg) mice. tmTNF overexpression resulted in enlarged peripheral lymph nodes (PLNs) and spleen, accompanied by an increase in small splenic lymphoid follicles, with less well-defined primary B cell follicles and T cell zones. In tmTNF-tg mice, the spleen, but not PLNs, contained reduced germinal center (GC) B cell fractions, with low Ki67 expression and reduced dark zone characteristics. In line with this, smaller fractions of T follicular helper (Tfh) and T follicular regulatory (Tfr) cells were observed with a decreased Tfh:Tfr ratio. Moreover, plasma cell (PC) formation in the spleen of tmTNF-tg mice decreased and skewed towards IgA and IgM expression. Genetic deletion of TNFRI or -II resulted in a normalization of follicle morphology in the spleen of tmTNF-tg mice, but GC B cell and PC fractions remained abnormal. These findings demonstrate that tightly regulated tmTNF is important for proper SLO development and function, and that aberrations induced by tmTNF overexpression are site-specific and mediated via TNFRI and/or TNFRII signaling.

NIK-IKK complex interaction controls NF-κB-dependent inflammatory activation of endothelium in response to LTßR ligation.

NF-κB-inducing kinase (NIK; also known as MAP3K14) is a central regulator of non-canonical NF-κB signaling in response to stimulation of TNF receptor superfamily members, such as the lymphotoxin-ß receptor (LTßR), and is implicated in pathological angiogenesis associated with chronic inflammation and cancer. Here, we identify a previously unrecognized role of the LTßR-NIK axis during inflammatory activation of human endothelial cells (ECs). Engagement of LTßR-triggered canonical and non-canonical NF-κB signaling promoted expression of inflammatory mediators and adhesion molecules, and increased immune cell adhesion to ECs. Sustained LTßR-induced inflammatory activation of ECs was NIK dependent, but independent of p100, indicating that the non-canonical arm of NF-κB is not involved. Instead, prolonged activation of canonical NF-κB signaling, through the interaction of NIK with IκB kinase α and ß (also known as CHUK and IKBKB, respectively), was required for the inflammatory response. Endothelial inflammatory activation induced by synovial fluid from rheumatoid arthritis patients was significantly reduced by NIK knockdown, suggesting that NIK-mediated alternative activation of canonical NF-κB signaling is a key driver of pathological inflammatory activation of ECs. Targeting NIK could thus provide a novel approach for treating chronic inflammatory diseases.

Differential Contribution of NF-κB Signaling Pathways to CD4+ Memory T Cell Induced Activation of Endothelial Cells.

Endothelial cells (ECs) are important contributors to inflammation in immune-mediated inflammatory diseases (IMIDs). In this study, we examined whether CD4+ memory T (Tm) cells can drive EC inflammatory responses. Human Tm cells produced ligands that induced inflammatory responses in human umbilical vein EC as exemplified by increased expression of inflammatory mediators including chemokines and adhesion molecules. NF-κB, a key regulator of EC activation, was induced by Tm cell ligands. We dissected the relative contribution of canonical and non-canonical NF-κB signaling to Tm induced EC responses using pharmacological small molecule inhibitors of IKKß (iIKKß) or NF-κB inducing kinase (iNIK). RNA sequencing revealed substantial overlap in IKKß and NIK regulated genes (n=549) that were involved in inflammatory and immune responses, including cytokines (IL-1ß, IL-6, GM-CSF) and chemokines (CXCL5, CXCL1). NIK regulated genes were more restricted, as 332 genes were uniquely affected by iNIK versus 749 genes by iIKKß, the latter including genes involved in metabolism, proliferation and leukocyte adhesion (VCAM-1, ICAM-1). The functional importance of NIK and IKKß in EC activation was confirmed by transendothelial migration assays with neutrophils, demonstrating stronger inhibitory effects of iIKKß compared to iNIK. Importantly, iIKKß - and to some extent iNIK - potentiated the effects of currently employed therapies for IMIDs, like JAK inhibitors and anti-IL-17 antibodies, on EC inflammatory responses. These data demonstrate that inhibition of NF-κB signaling results in modulation of Tm cell-induced EC responses and highlight the potential of small molecule NF-κB inhibitors as a novel treatment strategy to target EC inflammatory responses in IMIDs.

A Straightforward Method for 3D Visualization of B Cell Clusters and High Endothelial Venules in Lymph Nodes Highlights Differential Roles of TNFRI and -II.

Whole mount tissue immunolabeling and imaging of complete organs has tremendous benefits in characterizing organ morphology. Here, we present a straightforward method for immunostaining, clearing and imaging of whole murine peripheral lymph nodes (PLNs) for detailed analysis of their architecture and discuss all procedures in detail in a step-by-step approach. Given the importance of tumor necrosis factor receptor (TNFR) signaling in development of PLNs we used TNFRI-/- and TNFRII-/- mice models as proof-of-concept for this technique by visualizing and analyzing structural changes in PLN B cell clusters and high endothelial venules (HEVs). Samples were subjected to de- and rehydration with methanol, labeled with antibodies for B cells, T cells and high endothelial venules (HEVs) and optically cleared using benzyl alcohol-benzyl benzoate. Imaging was done using LaVision light sheet microscope and analysis with Imaris software. Using these techniques, we confirmed previous findings that TNFRI signaling is essential for formation of individual B cell clusters. In addition, Our data suggest that TNFRII signaling is also to some extent involved in this process as TNFRII-/- PLNs had a B cell cluster morphology reminiscent of TNFRI-/- PLNs. Moreover, visualization and objective quantification of the complete PLN high endothelial vasculature unveiled reduced volume, length and branching points of HEVs in TNFRI-/- PLNs, revealing an earlier unrecognized contribution of TNFRI signaling in HEV morphology. Together, these results underline the potential of whole mount tissue staining and advanced imaging techniques to unravel even subtle changes in lymphoid tissue architecture.

The Role of Endothelial Cells and TNF-Receptor Superfamily Members in Lymphoid Organogenesis and Function During Health and Inflammation.

Lymph nodes (LNs) are crucial for the orchestration of immune responses. LN reactions depend on interactions between incoming and local immune cells, and stromal cells. To mediate these cellular interactions an organized vascular network within the LN exists. In general, the LN vasculature can be divided into two components: blood vessels, which include the specialized high endothelial venules that recruit lymphocytes from the bloodstream, and lymphatic vessels. Signaling via TNF receptor (R) superfamily (SF) members has been implicated as crucial for the development and function of LNs and the LN vasculature. In recent years the role of cell-specific signaling of TNFRSF members in different endothelial cell (EC) subsets and their roles in development and maintenance of lymphoid organs has been elucidated. Here, we discuss recent insights into EC-specific TNFRSF member signaling and highlight its importance in different EC subsets in LN organogenesis and function during health, and in lymphocyte activation and tertiary lymphoid structure formation during inflammation.

Silencing NIK potentiates anti-VEGF therapy in a novel 3D model of colorectal cancer angiogenesis.

Angiogenesis is essential for colorectal cancer (CRC) progression, as demonstrated by the beneficial clinical effects of therapeutics inhibiting VEGF signaling. However, alternative mechanisms of neovascularization can develop, resulting in treatment failure. Previously we demonstrated NF-κB-inducing kinase (NIK) contributes to pathological angiogenesis. Here, we investigate NIK as a therapeutic target in endothelial cells (EC) in CRC. To determine NIK expression levels in CRC tissues, we immunostained both primary colorectal tumors and tumors metastasized to the liver. Additionally, a 3D tumor-stromal cell interaction model was developed including EC, fibroblasts and CRC cells to study tumor angiogenesis. This model tested efficacy of NIK-targeting siRNA (siNIK) in EC alone or in combination with the anti-VEGF antibody, bevacizumab. Both primary CRC and liver metastases contained blood vessels expressing NIK. In patients receiving chemotherapy plus bevacizumab, immature NIK+ vessels (p < 0.05) were increased as compared to chemotherapy alone. Activation of NIK by lymphotoxin-beta receptor (LTßR) induced increases in pro-angiogenic mediators, including interleukin (IL)-6, IL-8, chemokine (C-X-C motif) ligand (CXCL)1 and CXCL5 in EC and fibroblasts, accompanied by sprouting in the 3D model, which was blocked by siNIK in EC. Treatment with bevacizumab plus siNIK in EC resulted in a synergistic effect and reduced VEGF and bFGF-induced sprouting (p < 0.05). Here, we demonstrate a role for NIK in CRC-associated angiogenesis. Targeting NIK in EC in combination with anti-VEGF antibody bevacizumab may hold therapeutic potential to increase efficiency in blocking tumor neovascularization, either to prevent treatment failure due to activation of accessory pathways such as NF-κB signaling or as a rescue treatment.