The contribution of NF-κB signalling to immune regulation and tolerance

Immune regulation is necessary to control inflammatory responses and to prevent autoimmune diseases. Therefore, mechanisms of central and peripheral tolerance have evolved to ensure that T cells recognize antigens as self- or non-self-antigens. The thymus is crucially important for central tolerance induction to self-antigens via negative selection of T cells. However, if T cells escape negative selection in the thymus and enter the periphery, peripheral mechanisms are active to warrant immune tolerance. Secondary lymphoid organs, as well as tolerogenic dendritic cells and regulatory T cells, play an important role in peripheral tolerance. In chronic inflammatory diseases, tertiary lymphoid organs are sometimes formed that may also be involved in the induction of peripheral tolerance. This review discusses the main processes that are involved in immune regulation and tolerance, and focuses on the contribution of NF-κB signalling to these processes. This narrative review is based on peer-reviewed publications listed on PubMed up to December 2014. The focus of our literature search was on studies investigating the role of (non)canonical NF-κB signalling in central and peripheral mechanisms of tolerance. Only studies published in English language were considered. This review discusses the immune phenotype of mutant mice with defective (non)canonical NF-κB signalling, corroborated with human data, and emphasizes the contribution of the noncanonical NF-κB pathway to immune regulation and tolerance induction. Noncanonical NF-κB signalling has an important immunoregulatory role in the immune system and contributes to both central and peripheral mechanisms of toleranc

Zebrafish enpp1 mutants exhibit pathological mineralization, mimicking features of generalized arterial calcification of infancy (GACI) and pseudoxanthoma elasticum (PXE)

In recent years it has become clear that, mechanistically, biomineralization is a process that has to be actively inhibited as a default state. This inhibition must be released in a rigidly controlled manner in order for mineralization to occur in skeletal elements and teeth. A central aspect of this concept is the tightly controlled balance between phosphate, a constituent of the biomineral hydroxyapatite, and pyrophosphate, a physiochemical inhibitor of mineralization. Here, we provide a detailed analysis of a zebrafish mutant, dragonfish (dgf), which is mutant for ectonucleoside pyrophosphatase/phosphodiesterase 1 (Enpp1), a protein that is crucial for supplying extracellular pyrophosphate. Generalized arterial calcification of infancy (GACI) is a fatal human disease, and the majority of cases are thought to be caused by mutations in ENPP1. Furthermore, some cases of pseudoxanthoma elasticum (PXE) have recently been linked to ENPP1. Similar to humans, we show here that zebrafish enpp1 mutants can develop ectopic calcifications in a variety of soft tissues - most notably in the skin, cartilage elements, the heart, intracranial space and the notochord sheet. Using transgenic reporter lines, we demonstrate that ectopic mineralizations in these tissues occur independently of the expression of typical osteoblast or cartilage markers. Intriguingly, we detect cells expressing the osteoclast markers Trap and CathepsinK at sites of ectopic calcification at time points when osteoclasts are not yet present in wild-type siblings. Treatment with the bisphosphonate etidronate rescues aspects of the dgf phenotype, and we detected deregulated expression of genes that are involved in phosphate homeostasis and mineralization, such as fgf23, npt2a, entpd5 and spp1 (also known as osteopontin). Employing a UAS-GalFF approach, we show that forced expression of enpp1 in blood vessels or the floorplate of mutant embryos is sufficient to rescue the notochord mineralization phenotype. This indicates that enpp1 can exert its function in tissues that are remote from its site of expression

Mature osteoblasts dedifferentiate in response to traumatic bone injury in the zebrafish fin and skull

Zebrafish have an unlimited capacity to regenerate bone after fin amputation. In this process, mature osteoblasts dedifferentiate to osteogenic precursor cells and thus represent an important source of newly forming bone. By contrast, differentiated osteoblasts do not appear to contribute to repair of bone injuries in mammals; rather, osteoblasts form anew from mesenchymal stem cells. This raises the question whether osteoblast dedifferentiation is specific to appendage regeneration, a special feature of the lepidotrichia bone of the fish fin, or a process found more generally in fish bone. Here, we show that dedifferentiation of mature osteoblasts is not restricted to fin regeneration after amputation, but also occurs during repair of zebrafish fin fractures and skull injuries. In both models, mature osteoblasts surrounding the injury downregulate the expression of differentiation markers, upregulate markers of the pre-osteoblast state and become proliferative. Making use of photoconvertible Kaede protein as well as Cre-driven genetic fate mapping, we show that osteoblasts migrate to the site of injury to replace damaged tissue. Our findings suggest a fundamental role for osteoblast dedifferentiation in reparative bone formation in fish and indicate that adult fish osteoblasts display elevated cellular plasticity compared with mammalian bone-forming cells

Proteomics analysis of the zebrafish skeletal extracellular matrix

The extracellular matrix of the immature and mature skeleton is key to the development and function of the skeletal system. Notwithstanding its importance, it has been technically challenging to obtain a comprehensive picture of the changes in skeletal composition throughout the development of bone and cartilage. In this study, we analyzed the extracellular protein composition of the zebrafish skeleton using a mass spectrometry-based approach, resulting in the identification of 262 extracellular proteins, including most of the bone and cartilage specific proteins previously reported in mammalian species. By comparing these extracellular proteins at larval, juvenile, and adult developmental stages, 123 proteins were found that differed significantly in abundance during development. Proteins with a reported function in bone formation increased in abundance during zebrafish development, while analysis of the cartilage matrix revealed major compositional changes during development. The protein list includes ligands and inhibitors of various signaling pathways implicated in skeletogenesis such as the Int/Wingless as well as the insulin-like growth factor signaling pathways. This first proteomic analysis of zebrafish skeletal development reveals that the zebrafish skeleton is comparable with the skeleton of other vertebrate species including mammals. In addition, our study reveals 6 novel proteins that have never been related to vertebrate skeletogenesis and shows a surprisingly large number of differences in the cartilage and bone proteome between the head, axis and caudal fin regions. Our study provides the first systematic assessment of bone and cartilage protein composition in an entire vertebrate at different stages of development

Proteomics Analysis of the Zebrafish Skeletal Extracellular Matrix

<div><p>The extracellular matrix of the immature and mature skeleton is key to the development and function of the skeletal system. Notwithstanding its importance, it has been technically challenging to obtain a comprehensive picture of the changes in skeletal composition throughout the development of bone and cartilage. In this study, we analyzed the extracellular protein composition of the zebrafish skeleton using a mass spectrometry-based approach, resulting in the identification of 262 extracellular proteins, including most of the bone and cartilage specific proteins previously reported in mammalian species. By comparing these extracellular proteins at larval, juvenile, and adult developmental stages, 123 proteins were found that differed significantly in abundance during development. Proteins with a reported function in bone formation increased in abundance during zebrafish development, while analysis of the cartilage matrix revealed major compositional changes during development. The protein list includes ligands and inhibitors of various signaling pathways implicated in skeletogenesis such as the Int/Wingless as well as the insulin-like growth factor signaling pathways. This first proteomic analysis of zebrafish skeletal development reveals that the zebrafish skeleton is comparable with the skeleton of other vertebrate species including mammals. In addition, our study reveals 6 novel proteins that have never been related to vertebrate skeletogenesis and shows a surprisingly large number of differences in the cartilage and bone proteome between the head, axis and caudal fin regions. Our study provides the first systematic assessment of bone and cartilage protein composition in an entire vertebrate at different stages of development.</p></div

Contribution of Type H Blood Vessels to Pathologic Osteogenesis and Inflammation in an Experimental Spondyloarthritis Model

Objective: Spondyloarthritis (SpA) is characterized by pathologic osteogenesis, inflammation, and extensive angiogenesis in axial and peripheral tissues. Current therapies effectively target inflammation, but these therapies lack efficacy in preventing pathologic osteogenesis. Transgenic mice overexpressing transmembrane tumor necrosis factor (tmTNF-Tg mice) exhibit SpA-like features. We hypothesized that type H blood vessels, which are implicated in osteogenesis, are increased and contribute to pathology in this experimental SpA model. Methods: We analyzed ankles, femora, and vertebrae of tmTNF-Tg mice and nontransgenic littermates and tmTNF-Tg mice on either a TNF receptor type I (TNFRI)–deficient or TNF receptor type II (TNFRII)–deficient background for osteogenesis, angiogenesis, and inflammation using advanced imaging technologies at various stages of disease. Results: Compared to nontransgenic littermates, tmTNF-Tg mice exhibited an increase in vertebral type H vessels and osteoprogenitor cells in subchondral bone. These features of increased angiogenesis and osteogenesis were already present before onset of clinical disease symptoms. Type H vessels and osteoprogenitor cells were in close proximity to inflammatory lesions and ectopic lymphoid structures. The tmTNF-Tg mice also showed perivertebral ectopic type H vessels and osteogenesis, an increased number of vertebral transcortical vessels, and enhanced entheseal angiogenesis. In tmTNF-Tg mice crossed on a TNFRI- or TNFRII-deficient background, no clear reduction in type H vessels was shown, suggesting that type H vessel formation is not exclusively mediated via TNFRI or TNFRII. Conclusion: The contribution of type H vessels to pathologic osteogenesis in experimental SpA advances our knowledge of the pathophysiology of this disease and may also provide a novel opportunity for targeted intervention

Composition of the zebrafish extracellular protein profile.

<p>(A) Distribution of molecular functions for the proteins identified as extracellular proteins. <i>^a</i> Gene Ontology information based on the human ortholog of a protein in case this information was absent in the zebrafish database. The diagram contains 351 entries corresponding to 262 different proteins of which some fall into multiple categories (B) Ten most abundant extracellular proteins within the obtained protein profile based on their intensity.</p

Quantitative analysis of the zebrafish craniofacial skeleton by MS-based proteomics.

<p>(A) Alcian blue/alizarin red stain of cartilage/bone structures in the craniofacial skeleton. Lateral (top) and ventral (bottom) images of all three time point used for protein extraction (left to right: 14 dpf, 28 dpf, 358 dpf). (B) VENN diagram of proteins selected for label-free quantification, with areas drawn to represent the number of proteins. Total number of proteins as well as distinct and common proteins are indicated for each time point. Proteins that did not qualify for label-free quantification are depicted in grey. (C-E) Ratio abundance plots showing log total iBAQ intensities versus log protein abundance ratio of the 28/14 dpf (C), the 358/28 dpf (D) and the 358/14 dpf (E) craniofacial skeleton ratios of all the proteins that met the strict criteria for label-free quantification (black circles, significantly differential abundant proteins at FDR =  0.01; dark grey circles, significantly differential abundant proteins at FDR =  0.05; light grey circles, no significant change in abundance). The numbers correspond to the proteins listed in 2F. (F) Table containing several of the significant differentially abundant proteins within the cranial skeleton. Specific proteins are discussed in the text.</p

Ingenuity Pathway Analysis (IPA).

<p>Proteins that differ in abundance are involved in connective tissue, skeletal and muscular development. Green shading indicates an increase in abundance during development whereas red shading indicates a decrease. Increased intensity in colors indicates a higher differential abundance.</p

Quantitative analysis of the zebrafish axial skeleton by MS-based proteomics.

<p>(A) Alcian blue/alizarin red stain of cartilage/bone structures in the axial skeleton. Lateral images of all three time point used for protein extraction (left to right: 14 dpf, 28 dpf, 358 dpf). (B) VENN diagram of proteins selected for label-free quantification, with areas drawn to represent the number of proteins. Total number of proteins as well as distinct and common proteins are indicated for each time point. Proteins that did not qualify for label-free quantification are depicted in grey. (C-E) Ratio abundance plots showing log total iBAQ intensities versus log protein abundance ratio of the 28/14 dpf (C), the 358/28 dpf (D) and the 358/14 dpf (E) axial skeleton ratios of all the proteins that met the strict criteria for label-free quantification (black circles, significantly differential abundant proteins at FDR =  0.01; dark grey circles, significantly differential abundant proteins at FDR =  0.05; light grey circles, no significant change in abundance). The numbers correspond to the proteins listed in 3F. (F) Table containing several of the significant differentially abundant proteins within the axial skeleton. Specific proteins are discussed in the text.</p