a b s t r a c tCutaneous innervation is increasingly recognized as a major element of skin physiopathology through the neurogenic inflammation driven by neuropeptides that are sensed by endothelial cells and the immune system. To investigate this process in vitro, models of innervated tissue-engineered skin (TES) were developed, yet exclusively with murine sensory neurons extracted from dorsal root ganglions. In order to build a fully human model of innervated TES, we used induced pluripotent stem cells (iPSC) generated from human skin fibroblasts. Nearly 100% of the iPSC differentiated into sensory neurons were shown to express the neuronal markers BRN3A and b3-tubulin after 19 days of maturation. In addition, these cells were also positive to TRPV1 and neurofilament M, and some of them expressed Substance P, TrkA and TRPA1. When stimulated with molecules inducing neuropeptide release, iPSC-derived neurons released Substance P and CGRP, both in conventional monolayer culture and after seeding in a 3D fibroblastpopulated collagen sponge model. Schwann cells, the essential partners of neurons for function and axonal migration, were also successfully differentiated from human iPSC as shown by their expression of the markers S100, GFAP, p75 and SOX10. When cultured for one additional month in the TES model, iPSCderived neurons seeded at the bottom of the sponge formed a network of neurites spanning the whole TES up to the epidermis, but only when combined with mouse or iPSC-derived Schwann cells. This unique model of human innervated TES should be highly useful for the study of cutaneous neuroinflammation. Statement of SignificanceThe purpose of this work was to develop in vitro an innovative fully human tissue-engineered skin enabling the investigation of the influence of cutaneous innervation on skin pathophysiology. To reach that aim, neurons were differentiated from human induced pluripotent stem cells (iPSCs) generated from normal human skin fibroblasts. This innervated tissue-engineered skin model will be the first one to show iPSC-derived neurons can be successfully used to build a 3D nerve network in vitro. Since innervation has been recently recognized to play a central role in many human skin diseases, such as psoriasis and atopic dermatitis, this construct promises to be at the forefront to model these diseases while using patient-derived cells.

Dynamic polarisation of tumour cells is essential for metastasis. While the role of polarisation during dedifferentiation and migration is well established, polarisation of metastasising tumour cells during phases of detachment has not been investigated. Here we identify and characterise a type of polarisation maintained by single cells in liquid phase termed single-cell (sc) polarity and investigate its role during metastasis. We demonstrate that sc polarity is an inherent feature of cells from different tumour entities that is observed in circulating tumour cells in patients. Functionally, we propose that the sc pole is directly involved in early attachment, thereby affecting adhesion, transmigration and metastasis. In vivo, the metastatic capacity of cell lines correlates with the extent of sc polarisation. By manipulating sc polarity regulators and by generic depolarisation, we show that sc polarity prior to migration affects transmigration and metastasis in vitro and in vivo.

Inhibition of excessive Toll-like receptor 4 (TLR4) signaling is a therapeutic approach pursued for many inflammatory diseases. We report that Mannoside Glycolipid Conjugates (MGCs) selectively blocked TLR4-mediated activation of human monocytes and monocyte-derived dendritic cells (DCs) by lipopolysaccharide (LPS). They potently suppressed pro-inflammatory cytokine secretion and maturation of DCs exposed to LPS, leading to impaired T cell stimulation. MGCs did not interfere with LPS and could act in a delayed manner, hours after LPS stimulation. Their inhibitory action required both the sugar heads and the lipid chain, although the nature of the sugar and the structure of the lipid tail could be modified. They blocked early signaling events at the cell membrane, enhanced internalization of CD14 receptors, and prevented colocalization of CD14 and TLR4, thereby abolishing NF-κB nuclear translocation. When the best lead conjugate was tested in a mouse model of LPS-induced acute lung inflammation, it displayed an anti-inflammatory action by suppressing the recruitment of neutrophils. Thus, MGCs could serve as promising leads for the development of selective TLR4 antagonistic agents for inflammatory diseases.

Osteosarcoma (OS) is the most common primary bone cancer, where the overall 5-year surviving rate is below 20% in resistant forms. Accelerating cures for those poor outcome patients remains a challenge. Nevertheless, several studies of agents targeting abnormal cancerous pathways have yielded disappointing results when translated into clinic because of the lack of accurate OS preclinical modeling. So, any effort to design preclinical drug testing may consider all inter-, intra-, and extra-tumoral heterogeneities throughout models mimicking extracellular and immune microenvironment. Therefore, the bioengineering of patient-derived models reproducing the OS heterogeneity, the interaction with tumor-associated macrophages (TAMs), and the modulation of oxygen concentrations additionally to recreation of bone scaffold is proposed here. Eight 2D preclinical models mimicking several OS clinical situations and their TAMs in hypoxic conditions are developed first and, subsequently, the paired 3D models faithfully preserving histological and biological characteristics are generated. It is possible to shape reproducibly M2-like macrophages cultured with all OS patient-derived cell lines in both dimensions. The final 3D models pooling all heterogeneity features are providing accurate proliferation and migration data to understand the mechanisms involved in OS and immune cells/biomatrix interactions and sustained such that engineered 3D preclinical systems will improve personalized medicine.

Background Immune modulation by vitamin D3 through dendritic cells (DCs) remains controversial. Human DCs exposed in vitro counteract type‐1 T‐helper (Th1) differentiation and induce regulatory T cells. However, cutaneous application on mice promotes Th2‐driven inflammation resembling atopic dermatitis and relying on thymic stromal lymphopoietin (TSLP) from keratinocytes and T‐cell orientation by TSLP‐stimulated skin DCs. We studied the effects of vitamin D3 in human skin, focusing on TSLP production and the role of skin DCs in T‐cell differentiation. Methods Human healthy skin explants were exposed in vitro to vitamin D3 analogs. Migrating DCs were analyzed and TSLP quantified in the supernatant. Allogeneic naïve CD4+ T cells were cocultured with DCs to assess their proliferation and cytokine production. Results Vitamin D3 induced skin DCs to differentiate Th2 cells producing IL‐4 and IL‐13. Vitamin D3 triggered TSLP release in ~30% of skin explants, correlating with IL‐13 detection in Th2 cells. In these donors, blocking TSLP receptor during skin explant cultures abrogated IL‐13 production, yet IL‐4+ Th2 cells were unaffected. Among skin DCs emerged CD14+ cells that had responded directly to vitamin D3 and differed from classical CD14+ dermal emigrants. Vitamin D3‐elicited CD14+ DCs sufficed to promote IL‐4+ Th2 cells in a TSLP‐independent manner. Conclusion Vitamin D3, despite inducing TSLP in some donors, had a direct influence on skin DCs, affecting their phenotype and ability to drive Th2 responses independently of TSLP. Our findings pave the way toward in vitro systems that accurately model human cutaneous Th2 responses, notably involved in atopic dermatitis.

Pseudomonas aeruginosa is a Gram‐negative bacterium causing morbidity and mortality in immuno‐compromised humans. It produces a lectin, LecB, that is considered a major virulence factor, however, its impact on the immune system remains incompletely understood. Here we show that LecB binds to endothelial cells in human skin and mice and disrupts the transendothelial passage of leukocytes in vitro. It impairs the migration of dendritic cells into the paracortex of lymph nodes leading to a reduced antigen‐specific T cell response. Under the effect of the lectin, endothelial cells undergo profound cellular changes resulting in endocytosis and degradation of the junctional protein VE‐cadherin, formation of an actin rim, and arrested cell motility. This likely negatively impacts the capacity of endothelial cells to respond to extracellular stimuli and to generate the intercellular gaps for allowing leukocyte diapedesis. A LecB inhibitor can restore dendritic cell migration and T cell activation, underlining the importance of LecB antagonism to reactivate the immune response against P. aeruginosa infection.

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Le système immunitaire et le système nerveux sensoriel sont responsables de la perception du danger, sous des formes distinctes mais complémentaires. Ces dernières années, les interactions neuro-immunes se sont imposées comme un axe de recherche important en dermatologie pour comprendre la cicatrisation, la dermatite atopique ou le psoriasis. Nous présentons ici une sélection de modèles tridimensionnels in vitro reproduisant la structure de la peau et intégrant une fonction immunitaire ou sensorielle. Les évolutions futures de ces modèles permettront d’obtenir une vision aussi complète que possible des influences réciproques entre système immunitaire et système nerveux sensoriel.