Human mesenchymal stromal cell-secreted lactate induces M2-macrophage differentiation by metabolic reprogramming

Human mesenchymal stromal cells (MSC) have been shown to dampen immune response and promote tissue repair, but the underlying mechanisms are still under investigation. Herein, we demonstrate that umbilical cord-derived MSC (UC-MSC) alter the phenotype and function of monocyte-derived dendritic cells (DC) through lactate-mediated metabolic reprogramming. UC-MSC can secrete large quantities of lactate and, when present during monocyte-to-DC differentiation, induce instead the acquisition of M2-macrophage features in terms of morphology, surface markers, migratory properties and antigen presentation capacity. Microarray expression profiling indicates that UC-MSC modify the expression of metabolic-related genes and induce a M2-macrophage expression signature. Importantly, monocyte-derived DC obtained in presence of UC-MSC, polarize naïve allogeneic CD4+ T-cells into Th2 cells. Treatment of UC-MSC with an inhibitor of lactate dehydrogenase strongly decreases lactate concentration in culture supernatant and abrogates the effect on monocyte-to- DC differentiation. Metabolic analysis further revealed that UC-MSC decrease oxidative phosphorylation in differentiating monocytes while strongly increasing the spare respiratory capacity proportional to the amount of secreted lactate. Because both MSC and monocytes are recruited in vivo at the site of tissue damage and inflammation, we propose the local increase of lactate concentration induced by UC-MSC and the consequent enrichment in M2-macrophage generation as a mechanism to achieve immunomodulation

Deep tissue penetration of bottle-brush polymers via cell capture evasion and fast diffusion

Drug nanocarriers (NCs) capable of crossing the vascular endothelium and deeply penetrating into dense tissues of the CNS could potentially transform the management of neurological diseases. In the present study, we investigated the interaction of bottle-brush (BB) polymers with different biological barriers in vitro and in vivo and compared it to nanospheres of similar composition. In vitro internalization and permeability assays revealed that BB polymers are not internalized by brain-associated cell lines and translocate much faster across a blood–brain barrier model compared to nanospheres of similar hydrodynamic diameter. These observations performed under static, no-flow conditions were complemented by dynamic assays performed in microvessel arrays on chip and confirmed that BB polymers can escape the vasculature compartment via a paracellular route. BB polymers injected in mice and zebrafish larvae exhibit higher penetration in brain tissues and faster extravasation of microvessels located in the brain compared to nanospheres of similar sizes. The superior diffusivity of BBs in extracellular matrix-like gels combined with their ability to efficiently cross endothelial barriers via a paracellular route position them as promising drug carriers to translocate across the blood–brain barrier and penetrate dense tissue such as the brain, two unmet challenges and ultimate frontiers in nanomedicine

Nanoparticle shell structural cues drive in vitro transport properties, tissue distribution and brain accessibility in Zebrafish

Zwitterion polymers with strong antifouling properties have been suggested as the prime alternative to polyethylene glycol (PEG) for drug nanocarriers surface coating. It is believed that PEG coating shortcomings, such as immune responses and incomplete protein repellency, could be overcome by zwitterionic polymers. However, no systematic study has been conducted so far to complete a comparative appraisal of PEG and zwitterionic-coating effects on nanoparticles (NPs) stealthness, cell uptake, cell barrier translocation and biodistribution in the context of nanocarriers brain targeting. Core-shell polymeric particles with identical cores and a shell of either PEG or poly(2-methacryloyloxyethyl phosphorylcholine (PMPC) were prepared by impinging jet mixer nanoprecipitation. NPs with similar size and surface potential were systematically compared using in vitro and in vivo assays. NPs behavior differences were rationalized based on their protein-particles interactions. PMPC-coated NPs were significantly more endocytosed by mouse macrophages or brain resident macrophages compared to PEGylated NPs but exhibited the remarkable ability to cross the blood-brain barrier in in vitro models. Nanoscale flow cytometry assays showed significantly more adsorbed proteins on PMPC-coated NPs than PEG-coated NPs. In vivo, distribution in zebrafish larvae, showed a strong propensity for PMPC-coated NPs to adhere to the vascular endothelium, while PEG-coated NPs were able to circulate for a longer time and escape the bloodstream to penetrate deep into the cerebral tissue. The stark differences between these two types of particles, besides their similarities in size and surface potential, points towards the paramount role of surface chemistry in controlling NPs fate likely via the formation of distinct protein corona for each coating

Adolescent idiopathic scoliosis associated POC5 mutation impairs cell cycle, cilia length and centrosome protein interactions

International audienceAdolescent Idiopathic Scoliosis (AIS) is a spinal deformity that affects approximately 3 percent of human adolescents. Although the etiology and molecular basis of AIS is unclear, several genes such as POC5 have been identified as possible causes of the condition. In order to understand the role of POC5 in the pathogenesis of AIS, we investigated the subcellular localization of POC5 in cilia of cells over-expressing either the wild type (wt) or an AIS-related POC5 variant POC5A429V. Mutation of POC5 was found to alter its subcellular localization and to induce ciliary retraction. Furthermore, we observed an impaired cell-cycle progression with the accumulation of cells in the S-phase in cells expressing POC5A429V. Using immunoprecipitation coupled to mass spectrometry, we identified specific protein interaction partners of POC5, most of which were components of cilia and cytoskeleton. Several of these interactions were altered upon mutation of POC5. Altogether, our results demonstrate major cellular alterations, disturbances in centrosome protein interactions and cilia retraction in cells expressing an AIS-related POC5 mutation. Our study suggests that defects in centrosomes and cilia may underlie AIS pathogenesis

Role of Chd7 in Zebrafish: A Model for CHARGE Syndrome

<div><p>CHARGE syndrome is caused by mutations in the <em>CHD7</em> gene. Several organ systems including the retina, cranial nerves, inner ear and heart are affected in CHARGE syndrome. However, the mechanistic link between mutations in <em>CHD7</em> and many of the organ systems dysfunction remains elusive. Here, we show that Chd7 is required for the organization of the neural retina in zebrafish. We observe an abnormal expression or a complete absence of molecular markers for the retinal ganglion cells and photoreceptors, indicating that Chd7 regulates the differentiation of retinal cells and plays an essential role in retinal cell development. In addition, zebrafish with reduced Chd7 display an abnormal organization and clustering of cranial motor neurons. We also note a pronounced reduction in the facial branchiomotor neurons and the vagal motor neurons display aberrant positioning. Further, these fish exhibit a severe loss of the facial nerves. Knock-down of <em>Chd7</em> results in a curvature of the long body axis and these fish develop irregular shaped vertebrae and have a reduction in bone mineralization. <em>Chd7</em> knockdown also results in a loss of proper segment polarity illustrated by flawed <em>efnb2a</em> and <em>ttna</em> expression, which is associated with later vascular segmentation defects. These critical roles for Chd7 in retinal and vertebral development were previously unrecognized and our results provide new insights into the role of Chd7 during development and in CHARGE syndrome pathogenesis.</p> </div

Knockdown of <i>chd7</i> affects eye and heart development.

<p>(<b>A–B</b>) Representative images of eye phenotypes induced by injection of <i>chd7</i>-MO in zebrafish embryos (<b>B</b>) compared with control embryos (<b>A</b>) at 72 hpf. Lateral view with anterior to right. <i>Chd7</i> morphants had a reduced ocular size. <i>Chd7</i>-MO-injected zebrafish exhibited cardiac anomalies such as dysmorphic heart and tube-like heart shape (<b>D</b>) compared with control embryos (<b>C</b>). Insets in (<b>C</b>) and (<b>D</b>) represent an illustration of the heart phenotype in control or <i>chd7</i> morphant embryos, respectively. (<b>E–F</b>) Bar graphs illustrating the prevalence of eye (<b>E</b>) and heart (<b>F</b>) phenotypes in wild-type (white bar), <i>chd7</i>-MO (black bar), control-MO-injected embryos (grey bar) and mRNA rescue (dark grey bar). <b>***</b><0.001, <b>*</b><0.01. <b>***</b><0.001, <b>*</b><0.01.</p

Chd7 is important for cranial ganglia development and proper projection of facial nerves.

<p>Dorsal view (rostal towards the top) of confocal fluorescent composite images of hindbrain branchiomotor neurons in control-MO injected (<b>A</b>) and <i>chd7</i>-MO-injected (<b>B</b>) 48 hpf Isl1-GFP transgenic embryos. (<b>C, D</b>) Confocal fluorescent composite images showing anti-3A10 antibody-stained axons of 48 hpf embryos. The broken lines indicate the area of significant loss of VII nerves in <i>chd7</i> morphants. nV, trigeminal motoneurons; Va, anterior trigeminal motoneurons; Vp, posterior trigeminal motoneurons; nVII, facial motoneurons; nX, Vagal motoneurons; V, trigeminal nerve; VII, facial nerve. Scale bar: 50 µm.</p