Sensing of endogenous nucleic acids by ZBP1 induces keratinocyte necroptosis and skin inflammation.

Aberrant detection of endogenous nucleic acids by the immune system can cause inflammatory disease. The scaffold function of the signaling kinase RIPK1 limits spontaneous activation of the nucleic acid sensor ZBP1. Consequently, loss of RIPK1 in keratinocytes induces ZBP1-dependent necroptosis and skin inflammation. Whether nucleic acid sensing is required to activate ZBP1 in RIPK1-deficient conditions and which immune pathways are associated with skin disease remained open questions. Using knock-in mice with disrupted ZBP1 nucleic acid-binding activity, we report that sensing of endogenous nucleic acids by ZBP1 is critical in driving skin pathology characterized by antiviral and IL-17 immune responses. Inducing ZBP1 expression by interferons triggers necroptosis in RIPK1-deficient keratinocytes, and epidermis-specific deletion of MLKL prevents disease, demonstrating that cell-intrinsic events cause inflammation. These findings indicate that dysregulated sensing of endogenous nucleic acid by ZBP1 can drive inflammation and may contribute to the pathogenesis of IL-17-driven inflammatory skin conditions such as psoriasis.

A standardized methodical approach to characterize the influence of key parameters on the in vitro efficacy of extracorporeal photopheresis.

Extracorporeal photopheresis (ECP) is an autologous immunomodulatory cell therapy that consists of the ex vivo collection of mononuclear cells (MNCs), which are irradiated with UVA in the presence of the photosensitizing agent 8-methoxypsoralen (8-MOP) to induce cell apoptosis. This photoactivated cell preparation is then reinfused into the patient. While the clinical benefits of ECP are well-demonstrated, no study has yet characterized the influence of variations in the composition of the cell preparation on the efficacy of ECP in vitro. Here, we describe a standardized methodology for the in vitro assessment of ECP that uses the human lymphoma T-cell line and mimics the clinical procedure. By quantifying cell apoptosis, inhibition of cell proliferation, and 8-MOP consumption, we used this approach to characterize the specific influence of key variables on the cellular response to ECP. We found that (i) increases in hematocrit and plasma concentrations attenuated the cellular response to ECP; (ii) plasma concentration was the only variable tested that influenced 8-MOP consumption; and (iii) the loss of efficacy due to variations in the concentration of certain blood components could be counteracted by modulating the UVA dose. This methodology may enable evaluation of other leukapheresis preparation protocols and better determination of the optimal working parameters for ECP.

Public media communications about H1N1, risk perceptions and immunization behaviours: A Quebec-France comparison.

During the H1N1 pandemic, governments tailored their communications plans in order to influence risk perception and promote public compliance with the public health plan measures. Considering the volume and the content of calls to flu information centres as indicators of the public risk perception, this mixed method study compares the relation between public communications, risk perception and immunization behaviour in Quebec and France. Results suggest that advocating for clear information and coordination between health authorities and the media promotes adherence to preventive behaviour. However, over-exaggerating the risks and minimizing the population's agency may undermine health authority credibility.

RIPK1 ensures intestinal homeostasis by protecting the epithelium against apoptosis.

Receptor interacting protein kinase 1 (RIPK1) has an essential role in the signalling triggered by death receptors and pattern recognition receptors. RIPK1 is believed to function as a node driving NF-κB-mediated cell survival and inflammation as well as caspase-8 (CASP8)-dependent apoptotic or RIPK3/MLKL-dependent necroptotic cell death. The physiological relevance of this dual function has remained elusive because of the perinatal death of RIPK1 full knockout mice. To circumvent this problem, we generated RIPK1 conditional knockout mice, and show that mice lacking RIPK1 in intestinal epithelial cells (IECs) spontaneously develop severe intestinal inflammation associated with IEC apoptosis leading to early death. This early lethality was rescued by antibiotic treatment, MYD88 deficiency or tumour-necrosis factor (TNF) receptor 1 deficiency, demonstrating the importance of commensal bacteria and TNF in the IEC Ripk1 knockout phenotype. CASP8 deficiency, but not RIPK3 deficiency, rescued the inflammatory phenotype completely, indicating the indispensable role of RIPK1 in suppressing CASP8-dependent apoptosis but not RIPK3-dependent necroptosis in the intestine. RIPK1 kinase-dead knock-in mice did not exhibit any sign of inflammation, suggesting that RIPK1-mediated protection resides in its kinase-independent platform function. Depletion of RIPK1 in intestinal organoid cultures sensitized them to TNF-induced apoptosis, confirming the in vivo observations. Unexpectedly, TNF-mediated NF-κB activation remained intact in these organoids. Our results demonstrate that RIPK1 is essential for survival of IECs, ensuring epithelial homeostasis by protecting the epithelium from CASP8-mediated IEC apoptosis independently of its kinase activity and NF-κB activation.

Ectodysplasin research--where to next?

Ectodysplasin (Eda) is the most studied tumor necrosis ligand in the field of developmental biology. In all vertebrates studied so far, inactivating germline mutations in Eda lead to the genetic disease called hypohidrotic ectodermal dysplasia (HED). In humans, HED is a life-threatening condition in particular in infants due to absent or severely reduced sweating leading to hyperthermia. HED is also characterized by sparse hair, and oligo- or anodontia. Research of the Eda pathway has not only increased our knowledge on ectodermal appendage development and etiology of developmental disorders, but also on evolution of several vertebrate species including humankind. Studies on mouse and dog models of HED has led to one of the most stunning breakthroughs in applied developmental biology research by showing that a short-term treatment of neonates with a synthetic ligand corrects many of the HED-associated traits. Eighteen years after the identification of EDA as the causative gene in HED, a phase II trial aiming at permanent correction of the disease is now ongoing. This review summarizes the latest discoveries in the Eda field and points to areas that need further investigation such as the possible involvement of Eda in cell migration, stem cell maintenance, or cancer.

Directional cell migration, but not proliferation, drives hair placode morphogenesis.

Epithelial reorganization involves coordinated changes in cell shapes and movements. This restructuring occurs during formation of placodes, ectodermal thickenings that initiate the morphogenesis of epithelial organs including hair, mammary gland, and tooth. Signaling pathways in ectodermal placode formation are well known, but the cellular mechanisms have remained ill defined. We established imaging methodology for live visualization of embryonic skin explants during the first wave of hair placode formation. We found that the vast majority of placodal cells were nonproliferative throughout morphogenesis. We show that cell compaction and centripetal migration are the main cellular mechanisms associated with hair placode morphogenesis and that inhibition of actin remodeling suppresses placode formation. Stimulation of both ectodysplasin/NF-κB and Wnt/ß-catenin signaling increased cell motility and the number of cells committed to placodal fate. Thus, cell fate choices and morphogenetic events are controlled by the same molecular pathways, providing the framework for coordination of these two processes.

Establishment of crown-root domain borders in mouse incisor.

Teeth are composed of two domains, the enamel-covered crown and the enamel-free root. The understanding of the initiation and regulation of crown and root domain formation is important for the development of bioengineered teeth. In most teeth the crown develops before the root, and erupts to the oral cavity whereas the root anchors the tooth to the jawbone. However, in the continuously growing mouse incisor the crown and root domains form simultaneously, the crown domain forming the labial and the root domain the lingual part of the tooth. While the crown-root border on the incisor distal side supports the distal enamel extent, reflecting an evolutionary diet adaptation, on the incisor mesial side the root-like surface is necessary for the attachment of the interdental ligament between the two incisors. Therefore, the mouse incisor exhibits a functional distal-mesial asymmetry. Here, we used the mouse incisor as a model to understand the mechanisms involved in the crown-root border formation. We analyzed the cellular origins and gene expression patterns leading to the development of the mesial and distal crown-root borders. We discovered that Barx2, En1, Wnt11, and Runx3 were exclusively expressed on the mesial crown-root border. In addition, the distal border of the crown-root domain might be established by cells from a different origin and by an early Follistatin expression, factor known to be involved in the root domain formation. The use of different mechanisms to establish domain borders gives indications of the incisor functional asymmetry.

Ectodysplasin regulates hormone-independent mammary ductal morphogenesis via NF-κB.

Ductal growth of the mammary gland occurs in two distinct stages. The first round of branching morphogenesis occurs during embryogenesis, and the second round commences at the onset of puberty. Currently, relatively little is known about the genetic networks that control the initial phases of ductal expansion, which, unlike pubertal development, proceeds independent of hormonal input in female mice. Here we identify NF-κB downstream of the TNF-like ligand ectodysplasin (Eda) as a unique regulator of embryonic and prepubertal ductal morphogenesis. Loss of Eda, or inhibition of NF-κB, led to smaller ductal trees with fewer branches. On the other hand, overexpression of Eda caused a dramatic NF-κB-dependent phenotype in both female and male mice characterized by precocious and highly increased ductal growth and branching that correlated with enhanced cell proliferation. We have identified several putative transcriptional target genes of Eda/NF-κB, including PTHrP, Wnt10a, and Wnt10b, as well as Egf family ligands amphiregulin and epigen. We developed a mammary bud culture system that allowed us to manipulate mammary development ex vivo and found that recombinant PTHrP, Wnt3A, and Egf family ligands stimulate embryonic branching morphogenesis, suggesting that these pathways may cooperatively mediate the effects of Eda.

Identification of ectodysplasin target genes reveals the involvement of chemokines in hair development.

Ectodysplasin (Eda), a member of the tumor necrosis factor (Tnf) family, regulates skin appendage morphogenesis via its receptor Edar and transcription factor NF-κB. In humans, inactivating mutations in the Eda pathway components lead to hypohidrotic ectodermal dysplasia (HED), a syndrome characterized by sparse hair, tooth abnormalities, and defects in several cutaneous glands. A corresponding phenotype is observed in Eda-null mice, where failure in the initiation of the first wave of hair follicle development is a hallmark of HED pathogenesis. In an attempt to discover immediate target genes of the Eda/NF-κB pathway, we performed microarray profiling of genes differentially expressed in embryonic skin explants after a short exposure to recombinant Fc-Eda protein. Upregulated genes included components of the Wnt, fibroblast growth factor, transforming growth factor-ß, Tnf, and epidermal growth factor families, indicating that Eda modulates multiple signaling pathways implicated in skin appendage development. Surprisingly, we identified two ligands of the chemokine receptor cxcR3, cxcl10 and cxcl11, as new hair-specific transcriptional targets of Eda. Deficiency in cxcR3 resulted in decreased primary hair follicle density but otherwise normal hair development, indicating that chemokine signaling influences the patterning of primary hair placodes only.

The expression of Visinin-like 1 during mouse embryonic development.

Visinin like 1 (Vsnl1) encodes a calcium binding protein which is well conserved between species. It was originally found in the brain and its biological functions in central nervous system have been addressed in several studies. Low expression levels have also been found in some peripheral organs, but very little information is available regarding its physiological roles in non-neuronal tissues. Except for the kidney, the expression pattern of Vsnl1 mRNA and protein has not yet been addressed during embryogenesis. By in situ hybridization and immunolabeling we have extensively analyzed the expression pattern of Vsnl1 during murine development. Vsnl1 specifies the cardiac primordia and its expression becomes restricted to the atrial myocardium after heart looping. However, in the adult heart, Vsnl1 is expressed by all four cardiac chambers. It also serves as a specific marker for the cardiomyocyte-derived structures in the systemic and pulmonary circulation. Vsnl1 is dynamically expressed also by many other organs during development e.g. taste buds, cochlea, thyroid, tooth, salivary and adrenal gland. The stage specific expression pattern of Vsnl1 makes it a potentially useful marker particularly in studies of cardiac and vascular morphogenesis.

Identification of dkk4 as a target of Eda-A1/Edar pathway reveals an unexpected role of ectodysplasin as inhibitor of Wnt signalling in ectodermal placodes.

The development of epithelial appendages, including hairs, glands and teeth starts from ectodermal placodes, and is regulated by interplay of stimulatory and inhibitory signals. Ectodysplasin-A1 (Eda-A1) and Wnts are high in hierarchy of placode activators. To identify direct targets of ectodysplasin pathway, we performed microarray profiling of genes differentially regulated by short exposure to recombinant Eda-A1 in embryonic eda(-/-) skin explants. Surprisingly, there were only two genes with obvious involvement in Wnt pathway: dkk4 (most highly induced gene in the screen), and lrp4. Both genes colocalized with Eda-A1 receptor Edar in placodes of ectodermal organs. They were upregulated upon Edar activation while several other Wnt associated genes previously suggested as Edar targets were unaffected. However, low dkk4 and lrp4 expression was retained in the absence of NF-kappaB signalling in eda(-/-) hair placodes. We provide evidence that this expression was dependent on Wnt activity present prior to Eda-A1/Edar signalling. Dkk4 was recently suggested as a key Wnt antagonist regulating lateral inhibition essential for correct patterning of hair follicles. Several pieces of evidence suggest Lrp4 as a Wnt inhibitor, as well. The finding that Eda-A1 induces placode inhibitors was unexpected, and underlines the importance of delicate fine-tuning of signalling during placode formation.

NMR investigation of the interaction between the neuronal protein tau and the microtubules.

Whereas the interaction between Tau and the microtubules has been studied in great detail both by macroscopic techniques (cosedimentation, cryo-electron microscopy, and fluorescence spectroscopy) using the full-length protein or by peptide mapping assays, no detailed view at the level of individual amino acids has been presented when using the full-length protein. Here, we present a nuclear magnetic resonance (NMR) study of the interaction between the full-length neuronal protein Tau and paclitaxel-stabilized microtubules (MTs). As signal disappearance in the heteronuclear 1H-15N correlation spectra of isotope-labeled Tau in complex with MTs is due to direct association of the corresponding residue with the solid-like MT wall, we can map directly the fragment in interaction with the MT surface, and obtain a molecular picture of the precise interaction zones. The N-terminal region projects from the microtubule surface, and the lack of chemical shift variations when compared with free Tau proves that this region can regulate microtubular separation without adopting a stable conformation. Amino acids in the four microtubule binding repeats (MTBRs) lose all of their intensity, underscoring their immobilization upon binding to the MTs. The same loss of NMR intensity was observed for the proline-rich region starting at Ser214, underscoring its importance in the Tau:MT interaction. Fluorescence resonance energy transfer (FRET) experiments were used to obtain thermodynamic binding parameters, and led to the conclusion that the NMR defined fragment indeed is the major player in the interaction. When the same Ser214 is phosphorylated by the PKA kinase, the Tau:MT interaction strength decreases by 2 orders of magnitude, but the proline-rich region including the phospho-Ser214 does not gain sufficient mobility in the complex to make it observable by NMR spectroscopy. The presence of an intramolecular disulfide bridge, on the contrary, does lead to a partial detachment of the C-terminus of Tau, and decreases significantly the overloading of Tau on the MT surface.