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Targeting non-canonical nuclear factor-κB signalling attenuates neovascularization in a novel 3D model of rheumatoid arthritis synovial angiogenesis

Objective: Angiogenesis is crucial in RA disease progression. Lymphotoxin β receptor (LTβR)-induced activation of the non-canonical nuclear factor-κB (NF-κB) pathway via NF-κB-inducing kinase (NIK) has been implicated in this process. Consequently, inhibition of this pathway may hold therapeutic potential in RA. We describe a novel three-dimensional (3D) model of synovial angiogenesis incorporating endothelial cells (ECs), RA fibroblast-like synoviocytes (RAFLSs) and RA synovial fluid (RASF) to further investigate the contributions of NF-κB in this process.

Methods: Spheroids consisting of RAFLSs and ECs were stimulated with RASF, the LTβR ligands LTβ and LIGHT, or growth factor bFGF and VEGF, followed by quantification of EC sprouting using confocal microscopy and digital image analysis. Next, the effects of anginex, NIK-targeting siRNA (siNIK), LTβR-Ig fusion protein (baminercept) and a novel pharmacological NIK inhibitor were investigated.

Results: RASF significantly promoted sprout formation, which was blocked by the established angiogenesis inhibitor anginex (P < 0.05). LTβ and LIGHT induced significant sprouting (P < 0.05), as did bFGF/VEGF (P < 0.01). siNIK pre-treatment of ECs led to reductions in LTβR-induced vessel formation (P < 0.05). LTβR-Ig not only blocked LTβ- or LIGHT-induced sprouting, but also RASF-induced sprouting (P < 0.05). The NIK inhibitor blocked angiogenesis induced by LTβ, LIGHT, growth factors (P < 0.05) and RASF (P < 0.01).

Conclusion: We present a novel 3D model of synovial angiogenesis incorporating RAFLSs, ECs and RASF that mimics the in vivo situation. Using this system, we demonstrate that non-canonical NF-κB signalling promotes neovascularization and show that this model is useful for dissecting relative contributions of signalling pathways in specific cell types to angiogenic responses and for testing pharmacological inhibitors of angiogenesis.

Authors

Chrissta X Maracle, Paulina Kucharzewska, Boy Helder, Corine van der Horst, Pedro Correa de Sampaio, Ae-Ri Noort, Katinka van Zoest, Arjan W Griffioen, Henric Olsson, Sander W Tas

Link

https://doi.org/10.1093/rheumatology/kew393

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Development of follicular dendritic cells in lymph nodes depends on retinoic acid-mediated signaling

Specialized stromal cells occupy and help define B- and T-cell domains, which are crucial for proper functioning of our immune system. Signaling through lymphotoxin and TNF receptors is crucial for the development of different stromal subsets, which are thought to arise from a common precursor. However, mechanisms that control the selective generation of the different stromal phenotypes are not known. Using in vitro cultures of embryonic mouse stromal cells, we show that retinoic acid-mediated signaling is important for the differentiation of precursors towards the Cxcl13pos follicular dendritic cell (FDC) lineage, and also blocks lymphotoxin-mediated Ccl19pos fibroblastic reticular cell lineage differentiation. Accordingly, at the day of birth we observe the presence of Cxcl13posCcl19neg/low and Cxcl13neg/lowCcl19pos cells within neonatal lymph nodes. Furthermore, ablation of retinoic acid receptor signaling in stromal precursors early after birth reduces Cxcl13 expression, and complete blockade of retinoic acid signaling prevents the formation of FDC networks in lymph nodes.

Authors

Jasper J Koning, Anusha Rajaraman, Rogier M Reijmers, Tanja Konijn, Junliang Pan, Carl F Ware, Eugene C Butcher, Reina E Mebius

Link

https://doi.org/10.1242/dev.199713

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Lymph node stromal cells: subsets and functions in health and disease

Lymph nodes (LNs) aid the interaction between lymphocytes and antigen-presenting cells, resulting in adequate and prolonged adaptive immune responses. LN stromal cells (LNSCs) are crucially involved in steering adaptive immune responses at different levels. Most knowledge on LNSCs has been obtained from mouse studies, and few studies indicate similarities with their human counterparts. Recent advances in single-cell technologies have revealed significant LNSC heterogeneity among different subsets with potential selective functions in immunity. This review provides an overview of current knowledge of LNSCs based on human and murine studies describing the role of these cells in health and disease.

Authors

C Grasso, C Pierie, R E Mebius, L G M van Baarsen

Link

https://doi.org/10.1016/j.it.2021.08.009

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Stromal cells and immune cells involved in formation of lymph nodes and their niches

Secondary lymphoid organs are critical for efficient interaction between innate antigen presenting cells and adaptive lymphocytes in order to start adaptive immune responses. The efficiency by which these cellular subsets meet is highly increased by the orchestrating role of stromal cells within the secondary lymphoid organs. These cells provide cytokines, chemokines and cell surface receptors necessary for survival and guided migration. This increases the likelihood that antigen specific adaptive immune responses occur. Already from initial formation of secondary lymphoid organs, the interaction of immune cells with stromal cells is crucial and this interaction continues during immune activation. With the recent discovery of many stromal cell subsets new immune micro-niches with specific functions that are orchestrated by stromal cells will be discovered. Here, we will discuss how the development of lymph nodes as well as their specific niches is guided by the interaction of immune cells and stromal cells.

Authors

Jasper J Koning, Reina E Mebius.

Link

https://doi.org/10.1016/j.coi.2020.03.003

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Lymph Node Stromal Cells Generate Antigen-Specific Regulatory T Cells and Control Autoreactive T and B Cell Responses

Within lymph nodes (LNs), T follicular helper (TFH) cells help B cells to produce antibodies, which can either be protective or autoreactive. Here, we demonstrate that murine LN stromal cells (LNSCs) suppress the formation of autoreactive TFH cells in an antigen-specific manner, thereby significantly reducing germinal center B cell responses directed against the same self-antigen. Mechanistically, LNSCs express and present self-antigens in major histocompatibility complex (MHC) class II, leading to the conversion of naive CD4+ T cells into T regulatory (TREG) cells in an interleukin-2 (IL-2)-dependent manner. Upon blockade of TREG cells, using neutralizing IL-2 antibodies, autoreactive TFH cells are allowed to develop. We conclude that the continuous presentation of self-antigens by LNSCs is critical to generate antigen-specific TREG cells, thereby repressing the formation of TFH cells and germinal center B cell responses. Our findings uncover the ability of LNSCs to suppress the early activation of autoreactive immune cells and maintain peripheral tolerance.

Authors

Reza Nadafi, Catarina Gago de Graça, Eelco D Keuning, Jasper J Koning, Sander de Kivit, Tanja Konijn, Sandrine Henri, Jannie Borst, Rogier M Reijmers, Lisa G M van Baarsen, Reina E Mebius.

Link

https://doi.org/10.1016/j.celrep.2020.03.007

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Nestin-Expressing Precursors Give Rise to Both Endothelial as well as Nonendothelial Lymph Node Stromal Cells

During embryogenesis, lymph nodes form through intimate interaction between lymphoid tissue inducer and lymphoid tissue organizer (LTo) cells. Shortly after birth in mice, specialized stromal cell subsets arise that organize microenvironments within the lymph nodes; however, their direct precursors have not yet been identified. In the bone marrow, mesenchymal stem cells are labeled with GFP in nestin-GFP mice, and we show that during all stages of development, nestin(+) cells are present within lymph nodes of these mice. At day of birth, both mesenchymal CD31(-) and endothelial CD31(+) LTo cells were GFP(+), and only the population of CD31(-) LTo cells contained mesenchymal precursors. These CD31(-)nestin(+) cells are found in the T and B cell zones or in close association with high endothelial venules in adult lymph nodes. Fate mapping of nestin(+) cells unambiguously revealed the contribution of nestin(+) precursor cells to the mesenchymal as well as the endothelial stromal populations within lymph nodes. However, postnatal tamoxifen induced targeting of nestin(+) cells in nes-creER mice showed that most endothelial cells and only a minority of the nonendothelial cells were labeled. Overall our data show that nestin(+) cells contribute to all subsets of the complex stromal populations that can be found in lymph nodes.

Authors

Jasper J Koning, Tanja Konijn, Kim A Lakeman, Tom O’Toole, Keane J G Kenswil, Marc H G P Raaijmakers, Tatyana V Michurina, Grigori Enikolopov, Reina E Mebius.

Link

https://doi.org/10.4049/jimmunol.1501162

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Stromal-derived interleukin 6 drives epithelial-to-mesenchymal transition and therapy resistance in esophageal adenocarcinoma

The tissue dynamics that govern maintenance and regeneration of the pancreas remain largely unknown. In particular, the presence and nature of a cellular hierarchy remains a topic of debate. Previous lineage tracing strategies in the pancreas relied on specific marker genes for clonal labeling, which left other populations untested and failed to account for potential widespread phenotypical plasticity. Here we employed a tracing system that depends on replication-induced clonal marks. We found that, in homeostasis, steady acinar replacement events characterize tissue dynamics, to which all acinar cells have an equal ability to contribute. Similarly, regeneration following pancreatitis was best characterized by an acinar self-replication model because no evidence of a cellular hierarchy was detected. In particular, rapid regeneration in the pancreas was found to be driven by an accelerated rate of acinar fission-like events. These results provide a comprehensive and quantitative model of cell dynamics in the exocrine pancreas.

Authors

Eva A. Ebbing, Amber P. van der Zalm, Anne Steins, Aafke Creemers, Simone Hermsen, Rosa Rentenaar, Michelle Klein, Cynthia Waasdorp, Gerrit K. J. Hooijer, Sybren L. Meijer, Kausilia K. Krishnadath, Cornelis J.A. Punt, Mark I. van Berge Henegouwen, Suzanne S. Gisbertz, Otto M. van Delden, Maarten C. C. M. Hulshof, Jan Paul Medema, Hanneke W. M. van Laarhoven and Maarten F. Bijlsma.

Link

https://doi.org/10.1073/pnas.1820459116

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Microdissected pancreatic cancer proteomes reveal tumor heterogeneity and therapeutic targets

The tissue dynamics that govern maintenance and regeneration of the pancreas remain largely unknown. In particular, the presence and nature of a cellular hierarchy remains a topic of debate. Previous lineage tracing strategies in the pancreas relied on specific marker genes for clonal labeling, which left other populations untested and failed to account for potential widespread phenotypical plasticity. Here we employed a tracing system that depends on replication-induced clonal marks. We found that, in homeostasis, steady acinar replacement events characterize tissue dynamics, to which all acinar cells have an equal ability to contribute. Similarly, regeneration following pancreatitis was best characterized by an acinar self-replication model because no evidence of a cellular hierarchy was detected. In particular, rapid regeneration in the pancreas was found to be driven by an accelerated rate of acinar fission-like events. These results provide a comprehensive and quantitative model of cell dynamics in the exocrine pancreas.

Authors

Tessa Y.S. Le Large, Giulia Mantini, Laura L. Meijer, Thang V. Pham, Niccola Funel, Nicole C.T. van Grieken, Bart Kok, Jaco Knol, Hanneke W.M. van Laarhoven, Sander R. Piersma, Connie R. Jimenez, G. Kazemier, Elisa Giovannetti, and Maarten F. Bijlsma

Link

https://doi.org/10.1172/jci.insight.138290

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Continuous clonal labeling reveals uniform progenitor potential in the adult exocrine pancreas

The tissue dynamics that govern maintenance and regeneration of the pancreas remain largely unknown. In particular, the presence and nature of a cellular hierarchy remains a topic of debate. Previous lineage tracing strategies in the pancreas relied on specific marker genes for clonal labeling, which left other populations untested and failed to account for potential widespread phenotypical plasticity. Here we employed a tracing system that depends on replication-induced clonal marks. We found that, in homeostasis, steady acinar replacement events characterize tissue dynamics, to which all acinar cells have an equal ability to contribute. Similarly, regeneration following pancreatitis was best characterized by an acinar self-replication model because no evidence of a cellular hierarchy was detected. In particular, rapid regeneration in the pancreas was found to be driven by an accelerated rate of acinar fission-like events. These results provide a comprehensive and quantitative model of cell dynamics in the exocrine pancreas.

Authors

Sophie C Lodestijn, Tom van den Bosch, Lisanne E Nijman, Leandro F Moreno, Sophie Schlingemann, Vivek M Sheraton, Sanne M van Neerven, Jasper J Koning, Felipe A Vieira Braga, Nanne J Paauw, Maria C Lecca, Kristiaan J Lenos, Edward Morrissey, Daniël M Miedema, Douglas J Winton, Maarten F Bijlsma, Louis Vermeulen

Link

https://doi.org/10.1016/j.stem.2021.07.004

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Marker-free lineage tracing reveals an environment-instructed clonogenic hierarchy in pancreatic cancer

Effective treatments for pancreatic ductal adenocarcinoma (PDAC) are lacking, and targeted agents have demonstrated limited efficacy. It has been speculated that a rare population of cancer stem cells (CSCs) drives growth, therapy resistance, and rapid metastatic progression in PDAC. These CSCs demonstrate high clonogenicity in vitro and tumorigenic potential in vivo. However, their relevance in established PDAC tissue has not been determined. Here, we use marker-independent stochastic clonal labeling, combined with quantitative modeling of tumor expansion, to uncover PDAC tissue growth dynamics. We find that in contrast to the CSC model, all PDAC cells display clonogenic potential in situ. Furthermore, the proximity to activated cancer-associated fibroblasts determines tumor cell clonogenicity. This means that the microenvironment is dominant in defining the clonogenic activity of PDAC cells. Indeed, manipulating the stroma by Hedgehog pathway inhibition alters the tumor growth mode, revealing that tumor-stroma crosstalk shapes tumor growth dynamics and clonal architecture.

Authors

Sophie C Lodestijn , Daniël M Miedema, Kristiaan J Lenos, Lisanne E Nijman, Saskia C Belt, Khalid El Makrini, Maria C Lecca, Cynthia Waasdorp, Tom van den Bosch, Maarten F Bijlsma, Louis Vermeulen.

Link

https://doi.org/10.1016/j.celrep.2021.109852