Turbinal bones are still one of the last frontiers of the tetrapod skull: hypotheses, challenges and perspectives.

Turbinals are bony or cartilaginous structures that are present in the nasal cavity of most tetrapods. They are involved in key functions such as olfaction, heat, and moisture conservation, as well as protection of the respiratory tract. Despite recent studies that challenged long-standing hypotheses about their physiological and genomic correlation, turbinals remain largely unexplored, particularly for non-mammalian species. Herein, we review and synthesise the current knowledge of turbinals using an integrative approach that includes comparative anatomy, physiology, histology and genomics. In addition, we provide synonyms and correspondences of tetrapod turbinals from about 80 publications. This work represents a first step towards drawing hypotheses of homology for the whole clade, and provides a strong basis to develop new research avenues.

Identification of monoclonal antibodies from naive antibody phage-display libraries for protein detection in formalin-fixed paraffin-embedded tissues.

Most antibodies used in immunohistochemistry (IHC) have been developed by animal immunization. We wanted to explore naive antibody repertoires displayed on filamentous phages as a source of full-length antibodies for IHC on Formalin-Fixed and Paraffin-Embedded (FFPE) tissues. We used two isogenic mouse fibroblast cell lines that express or not human HER2 to generate positive and negative FFPE pseudo-tissue respectively. Using these pseudo-tissues and previously described approaches based on differential panning, we isolated very efficient antibody clones, but not against HER2. To optimize HER2 targeting and tissue specificity, we first performed 3-4 rounds of in vitro panning using recombinant HER2 extracellular domain (ECD) to enrich the phage library in HER2 binders, followed by one panning round using the two FFPE pseudo-tissues to retain binders for IHC conditions. We then analyzed the bound phages using next-generation sequencing to identify antibody sequences specifically associated with the HER2-positive pseudo-tissue. Using this approach, the top-ranked clone identified by sequencing was specific to the HER2-positive pseudo-tissue and showed a staining pattern similar to that of the antibody used for the clinical diagnosis of HER2-positive breast cancer. However, we could not optimize staining on other tissues, showing that specificity was restricted to the tissue used for selection and screening. Therefore, future optimized protocols must consider tissue diversity early during the selection by panning using a wide collection of tissue types.

RIP140 regulates transcription factor HES1 oscillatory expression and mitogenic activity in colon cancer cells.

The transcription factor receptor-interacting protein 140 (RIP140) regulates intestinal homeostasis and tumorigenesis through Wnt signaling. In this study, we investigated its effect on the Notch/HES1 signaling pathway. In colorectal cancer (CRC) cell lines, RIP140 positively regulated HES1 gene expression at the transcriptional level via a recombining binding protein suppressor of hairless (RBPJ)/neurogenic locus notch homolog protein 1 (NICD)-mediated mechanism. In support of these in vitro data, RIP140 and HES1 expression significantly correlated in mouse intestine and in a cohort of CRC samples, thus supporting the positive regulation of HES1 gene expression by RIP140. Interestingly, when the Notch pathway is fully activated, RIP140 exerted a strong inhibition of HES1 gene transcription controlled by the level of HES1 itself. Moreover, RIP140 directly interacts with HES1 and reversed its mitogenic activity in human CRC cells. In line with this observation, HES1 levels were associated with a better patient survival only when tumors expressed high levels of RIP140. Our data identify RIP140 as a key regulator of the Notch/HES1 signaling pathway, with a dual effect on HES1 gene expression at the transcriptional level and a strong impact on colon cancer cell proliferation.

Three-dimensional imaging of vascular development in the mouse epididymis.

Long considered an accessory tubule of the male reproductive system, the epididymis is proving to be a key determinant of male fertility. In addition to its secretory role in ensuring functional maturation and survival of spermatozoa, the epididymis has a complex immune function. Indeed, it must manage both peripheral tolerance to sperm antigens foreign to the immune system and the protection of spermatozoa as well as the organ itself against pathogens ascending the epididymal tubule. Although our knowledge of the immunobiology of this organ is beginning to accumulate at the molecular and cellular levels, the organization of blood and lymphatic networks of this tissue, important players in the immune response, remains largely unknown. In the present report, we have taken advantage of a VEGFR3:YFP transgenic mouse model. Using high-resolution three-dimensional (3D) imaging and organ clearing coupled with multiplex immunodetections of lymphatic (LYVE1, PDPN, PROX1) and/or blood (PLVAP/Meca32) markers, we provide a simultaneous deep 3D view of the lymphatic and blood epididymal vasculature in the mature adult mouse as well as during postnatal development.

Design and selection of optimal ErbB-targeting bispecific antibodies in pancreatic cancer.

The ErbB family of receptor tyrosine kinases is a primary target for small molecules and antibodies for pancreatic cancer treatment. Nonetheless, the current treatments for this tumor are not optimal due to lack of efficacy, resistance, or toxicity. Here, using the novel BiXAb™ tetravalent format platform, we generated bispecific antibodies against EGFR, HER2, or HER3 by considering rational epitope combinations. We then screened these bispecific antibodies and compared them with the parental single antibodies and antibody pair combinations. The screen readouts included measuring binding to the cognate receptors (mono and bispecificity), intracellular phosphorylation signaling, cell proliferation, apoptosis and receptor expression, and also immune system engagement assays (antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity). Among the 30 BiXAbs™ tested, we selected 3Patri-1Cetu-Fc, 3Patri-1Matu-Fc and 3Patri-2Trastu-Fc as lead candidates. The in vivo testing of these three highly efficient bispecific antibodies against EGFR and HER2 or HER3 in pre-clinical mouse models of pancreatic cancer showed deep antibody penetration in these dense tumors and robust tumor growth reduction. Application of such semi-rational/semi-empirical approach, which includes various immunological assays to compare pre-selected antibodies and their combinations with bispecific antibodies, represents the first attempt to identify potent bispecific antibodies against ErbB family members in pancreatic cancer.

CDK8 and CDK19 act redundantly to control the CFTR pathway in the intestinal epithelium.

CDK8 and CDK19 form a conserved cyclin-dependent kinase subfamily that interacts with the essential transcription complex, Mediator, and also phosphorylates the C-terminal domain of RNA polymerase II. Cells lacking either CDK8 or CDK19 are viable and have limited transcriptional alterations, but whether the two kinases redundantly control cell proliferation and differentiation is unknown. Here, we find in mice that CDK8 is dispensable for regulation of gene expression, normal intestinal homeostasis, and efficient tumourigenesis, and is largely redundant with CDK19 in the control of gene expression. Their combined deletion in intestinal organoids reduces long-term proliferative capacity but is not lethal and allows differentiation. However, double-mutant organoids show mucus accumulation and increased secretion by goblet cells, as well as downregulation of expression of the cystic fibrosis transmembrane conductance regulator (CFTR) and functionality of the CFTR pathway. Pharmacological inhibition of CDK8/19 kinase activity in organoids and in mice recapitulates several of these phenotypes. Thus, the Mediator kinases are not essential for cell proliferation and differentiation in an adult tissue, but they cooperate to regulate specific transcriptional programmes.

SPACR Encoded by IMPG1 Is Essential for Photoreceptor Survival by Interplaying between the Interphotoreceptor Matrix and the Retinal Pigment Epithelium.

Several pathogenic variants have been reported in the IMPG1 gene associated with the inherited retinal disorders vitelliform macular dystrophy (VMD) and retinitis pigmentosa (RP). IMPG1 and its paralog IMPG2 encode for two proteoglycans, SPACR and SPACRCAN, respectively, which are the main components of the interphotoreceptor matrix (IPM), the extracellular matrix surrounding the photoreceptor cells. To determine the role of SPACR in the pathological mechanisms leading to RP and VMD, we generated a knockout mouse model lacking Impg1, the mouse ortholog. Impg1-deficient mice show abnormal accumulation of autofluorescent deposits visible by fundus imaging and spectral-domain optical coherence tomography (SD-OCT) and attenuated electroretinogram responses from 9 months of age. Furthermore, SD-OCT of Impg1-/- mice shows a degeneration of the photoreceptor layer, and transmission electron microscopy shows a disruption of the IPM and the retinal pigment epithelial cells. The decrease in the concentration of the chromophore 11-cis-retinal supports this loss of photoreceptors. In conclusion, our results demonstrate the essential role of SPACR in maintaining photoreceptors. Impg1-/- mice provide a novel model for mechanistic investigations and the development of therapies for VMD and RP caused by IMPG1 pathogenic variants.

In high-grade ovarian carcinoma, platinum-sensitive tumor recurrence and acquired-resistance derive from quiescent residual cancer cells that overexpress CRYAB, CEACAM6, and SOX2.

Most high-grade ovarian carcinomas (HGOCs) are sensitive to carboplatin (CBP)-based chemotherapy but frequently recur within 24 months. Recurrent tumors remain CBP-sensitive and acquire resistance only after several treatment rounds. Recurrences arise from a small number of residual tumor cells not amenable to investigation in patients. We developed patient-derived xenografts (PDXs) that allow the study of these different stages of CBP-sensitive recurrence and acquisition of resistance. We generated PDX models from CBP-sensitive and intrinsically resistant HGOC. PDXs were CBP- or mock-treated and tumors were sampled, after treatment and at recurrence. We also isolated models with acquired-resistance from CBP-sensitive PDXs. Tumors were characterized at the histological and transcriptome levels. PDX models reproduced treatment response seen in the patients. CBP-sensitive residual tumors contained nonproliferating tumor cell clusters embedded in a fibrotic mesh. In nontreated PDX tumors and treated CBP-resistant tumors, fibrotic tissue was not prevalent. Residual tumors had marked differences in gene expression when compared to naïve and recurrent tumors, indicating downregulation of the cell cycle and proliferation and upregulation of interferon response and the epithelial-mesenchymal transition. This gene expression pattern resembled that described in embryonal diapause and 'drug-tolerant persister' states. Residual and acquired-resistance tumors share the overexpression of three genes: CEACAM6, CRYAB, and SOX2. Immunostaining analysis showed strong CEACAM6, CRYAB, and SOX2 protein expression in CBP-sensitive residual and acquired-resistance PDX, thus confirming the RNA profiling results. In HGOC PDX, CBP-sensitive recurrences arise from a small population of quiescent, drug-tolerant, residual cells embedded in a fibrotic mesh. These cells overexpress CEACAM6, CRYAB, and SOX2, whose overexpression is also associated with acquired resistance and poor patient prognosis. CEACAM6, CRYAB, and SOX2 may thus serve as a biomarker to predict recurrence and emergence of resistant disease in CBP-treated HGOC patients. © 2022 The Pathological Society of Great Britain and Ireland.

Preliminary in vivo study of biodegradable PLA-PEU-PLA anti-adhesion membranes in a rat Achilles tendon model of peritendinous adhesions.

Peritendinous adhesions are complications known to occur up to 6 weeks after surgery and cause chronic pain and disability. Anti-adhesion barriers are currently the best option for prevention. In a previous study, we designed two biodegradable membranes, D-PACO1 and D-PACO2, based on new triblock copolymers and conducted in vitro evaluations. The membranes maintained filmogenic integrity, had degradation rates that promoted anti-adhesion and were biocompatible, suggesting their safe and effective use as anti-adhesion devices. To test this hypothesis, we conducted a preliminary in vivo study in a rat model of peritendinous adhesions and evaluated the membranes' degradation rates, tendon healing and anti-adhesion effect compared to non-surgical and surgical control groups 2 and 10 weeks after surgery. Macroscopic evaluation showed membranes were effective in reducing the extent and severity of adhesions. Membranes acted as physical barriers at 2 weeks and underwent a complete or significant biodegradation at 10 weeks. D-PACO2 had a longer degradation rate compared to D-PACO1, was more effective in reducing adhesions and is expected to be more effective in promoting tendon healing. The tendency of D-PACO1 to promote tendon healing while D-PACO2 did not interfere with healing highlights the need to redesign the porosity of the D-PACO membranes for optimal nutrient diffusion, while maintaining their anti-adhesion effect and clinical usability. Preliminary findings revealed that adhesions form beyond the 6 weeks cited in the literature. In this study, adhesion formation continued for up to 10 weeks, underlining the need to increase the experimental period and sample size of future experiments evaluating anti-adhesion membranes.

A 31-plex panel for high-dimensional single-cell analysis of murine preclinical models of solid tumors by imaging mass cytometry.

Currently, the study of resistance mechanisms and disease progression in cancer relies on the capacity to analyze tumors as a complex ecosystem of healthy and malignant cells. Therefore, one of the current challenges is to decipher the intra-tumor heterogeneity and especially the spatial distribution and interactions of the different cellular actors within the tumor. Preclinical mouse models are widely used to extend our understanding of the tumor microenvironment (TME). Such models are becoming more sophisticated and allow investigating questions that cannot be addressed in clinical studies. Indeed, besides studying the tumor cell interactions within their environment, mouse models allow evaluating the efficacy of new drugs and delivery approaches, treatment posology, and toxicity. Spatially resolved analyses of the intra-tumor heterogeneity require global approaches to identify and localize a large number of different cell types. For this purpose, imaging mass cytometry (IMC) is a major asset in the field of human immuno-oncology. However, the paucity of validated IMC panels to study TME in pre-clinical mouse models remains a critical obstacle to translational or basic research in oncology. Here, we validated a panel of 31 markers for studying at the single-cell level the TME and the immune landscape for discovering/characterizing cells with complex phenotypes and the interactions shaping the tumor ecosystem in mouse models.

The multifunctional protein E4F1 links P53 to lipid metabolism in adipocytes.

Growing evidence supports the importance of the p53 tumor suppressor in metabolism but the mechanisms underlying p53-mediated control of metabolism remain poorly understood. Here, we identify the multifunctional E4F1 protein as a key regulator of p53 metabolic functions in adipocytes. While E4F1 expression is upregulated during obesity, E4f1 inactivation in mouse adipose tissue results in a lean phenotype associated with insulin resistance and protection against induced obesity. Adipocytes lacking E4F1 activate a p53-dependent transcriptional program involved in lipid metabolism. The direct interaction between E4F1 and p53 and their co-recruitment to the Steaoryl-CoA Desaturase-1 locus play an important role to regulate monounsaturated fatty acids synthesis in adipocytes. Consistent with the role of this E4F1-p53-Steaoryl-CoA Desaturase-1 axis in adipocytes, p53 inactivation or diet complementation with oleate partly restore adiposity and improve insulin sensitivity in E4F1-deficient mice. Altogether, our findings identify a crosstalk between E4F1 and p53 in the control of lipid metabolism in adipocytes that is relevant to obesity and insulin resistance.

RIP140 Represses Intestinal Paneth Cell Differentiation and Interplays with SOX9 Signaling in Colorectal Cancer.

RIP140 is a major transcriptional coregulator of gut homeostasis and tumorigenesis through the regulation of Wnt/APC signaling. Here, we investigated the effect of RIP140 on Paneth cell differentiation and its interplay with the transcription factor SOX9. Using loss of function mouse models, human colon cancer cells, and tumor microarray data sets we evaluated the role of RIP140 in SOX9 expression and activity using RT-qPCR, immunohistochemistry, luciferase reporter assays, and GST-pull down. We first evidence that RIP140 strongly represses the Paneth cell lineage in the intestinal epithelium cells by inhibiting Sox9 expression. We then demonstrate that RIP140 interacts with SOX9 and inhibits its transcriptional activity. Our results reveal that the Wnt signaling pathway exerts an opposite regulation on SOX9 and RIP140. Finally, the levels of expression of RIP140 and SOX9 exhibit a reverse response and prognosis value in human colorectal cancer biopsies. This work highlights an intimate transcriptional cross-talk between RIP140 and SOX9 in intestinal physiopathology.

In the mouse, prostaglandin D2 signalling protects the endometrium against adenomyosis.

Adenomyosis is characterised by epithelial gland and mesenchymal stroma invasion of the uterine myometrium. Adenomyosis is an oestrogen-dependent gynaecological disease in which a number of factors, such as inflammatory molecules, prostaglandins (PGs), angiogenic factors, cell proliferation and extracellular matrix remodelling proteins, also play a role as key disease mediators. In this study, we used mice lacking both lipocalin and hematopoietic-PG D synthase (L- and H-Pgds) genes in which PGD2 is not produced to elucidate PGD2 roles in the uterus. Gene expression studied by real-time PCR and hormone dosages performed by ELISA or liquid chromatography tandem mass spectroscopy in mouse uterus samples showed that components of the PGD2 signalling pathway, both PGDS and PGD2-receptors, are expressed in the mouse endometrium throughout the oestrus cycle with some differences among uterine compartments. We showed that PGE2 production and the steroidogenic pathway are dysregulated in the absence of PGD2. Histological analysis of L/H-Pgds-/- uteri, and immunohistochemistry and immunofluorescence analyses of proliferation (Ki67), endothelial cell (CD31), epithelial cell (pan-cytokeratin), myofibroblast (α-SMA) and mesenchymal cell (vimentin) markers, identify that 6-month-old L/H-Pgds-/- animals developed adenomyotic lesions, and that disease severity increased with age. In conclusion, this study suggests that the PGD2 pathway has major roles in the uterus by protecting the endometrium against adenomyosis development. Additional experiments, using for instance transcriptomic approaches, are necessary to fully determine the molecular mechanisms that lead to adenomyosis in L/H-Pgds-/- mice and to confirm whether this strain is an appropriate model for studying the human disease.

Anti-tumoral activity of the Pan-HER (Sym013) antibody mixture in gemcitabine-resistant pancreatic cancer models.

Chemoresistance, particularly to gemcitabine, is a major challenge in pancreatic cancer. The epidermal growth factor receptor (EGFR) and human epidermal growth factor receptors 2 and 3 (HER2, HER3) are expressed in many tumors, and they are relevant therapeutic targets due to their synergistic interaction to promote tumor aggressiveness and therapeutic resistance. Cocktails of antibodies directed against different targets are a promising strategy to overcome these processes. Here, we found by immunohistochemistry that these three receptors were co-expressed in 11% of patients with pancreatic adenocarcinoma. We then developed gemcitabine-resistant pancreatic cancer cell models (SW-1990-GR and BxPC3-GR) and one patient-derived xenograft (PDX2846-GR) by successive exposure to increasing doses of gemcitabine. We showed that expression of EGFR, HER2 and HER3 was increased in these gemcitabine-resistant pancreatic cancer models, and that an antibody mixture against all three receptors inhibited tumor growth in mice and downregulated HER receptors. Finally, we demonstrated that the Pan-HER and gemcitabine combination has an additive effect in vitro and in mice xenografted with the gemcitabine-sensitive or resistant pancreatic models. The mixture of anti-EGFR, HER2 and HER3 antibodies is a good candidate therapeutic approach for gemcitabine-sensitive and -resistant pancreatic cancer.

Ki-67 regulates global gene expression and promotes sequential stages of carcinogenesis.

Ki-67 is a nuclear protein that is expressed in all proliferating vertebrate cells. Here, we demonstrate that, although Ki-67 is not required for cell proliferation, its genetic ablation inhibits each step of tumor initiation, growth, and metastasis. Mice lacking Ki-67 are resistant to chemical or genetic induction of intestinal tumorigenesis. In established cancer cells, Ki-67 knockout causes global transcriptome remodeling that alters the epithelial-mesenchymal balance and suppresses stem cell characteristics. When grafted into mice, tumor growth is slowed, and metastasis is abrogated, despite normal cell proliferation rates. Yet, Ki-67 loss also down-regulates major histocompatibility complex class I antigen presentation and, in the 4T1 syngeneic model of mammary carcinoma, leads to an immune-suppressive environment that prevents the early phase of tumor regression. Finally, genes involved in xenobiotic metabolism are down-regulated, and cells are sensitized to various drug classes. Our results suggest that Ki-67 enables transcriptional programs required for cellular adaptation to the environment. This facilitates multiple steps of carcinogenesis and drug resistance, yet may render cancer cells more susceptible to antitumor immune responses.

In Lyl1-/- mice, adipose stem cell vascular niche impairment leads to premature development of fat tissues.

Lyl1 encodes a hematopoietic- and endothelial-specific bHLH transcription factor. Lyl1-deficient mice are viable, but they display mild hematopoietic and vascular defects. Specifically, LYL1 is required for the maturation and stabilization of blood vessel endothelial adherens junctions. Here, we report that young adult Lyl1-/- mice exhibit transient overweight associated with general expansion of adipose tissue, without signs of metabolic disorder and unrelated to food intake. The increased fat tissue development in Lyl1-/- mice resulted from earlier differentiation of adipose stem cells (ASCs) into adipocytes through noncell autonomous mechanisms. Specifically, we found that in Lyl1-/- mice, the adipose tissue vascular structures are immature, as indicated by their high permeability, reduced coverage by pericytes, lower recruitment of VE-cadherin and ZO1 at cell junctions, and more prone to angiogenesis. Together, our data show that in Lyl1-/- mice, the impaired vascular compartment of the adipose niche promotes ASC differentiation, leading to early adipocyte expansion and premature ASC depletion. Our study highlights the major structural role of the adipose tissue vascular niche in coordinating stem cell self-renewal and differentiation into adipocytes.

Vegetable oil-based hybrid microparticles as a green and biocompatible system for subcutaneous drug delivery.

The aim of this study was to evidence the ability of vegetable oil-based hybrid microparticles (HMP) to be an efficient and safe drug delivery system after subcutaneous administration. The HMP resulted from combination of a thermostabilized emulsification process and a sol-gel chemistry. First of all, castor oil was successfully silylated by means of (3-Isocyanatopropyl)trimethoxysilane in solvent-free and catalyst-free conditions. Estradiol, as a model drug, was dissolved in silylated castor oil (ICOm) prior to emulsification, and then an optimal sol-gel crosslinking was achieved inside the ICOm microdroplets. The resulting estradiol-loaded microparticles were around 80 µm in size and allowed to entrap 4 wt% estradiol. Their release kinetics in a PBS/octanol biphasic system exhibited a one-week release profile, and the released estradiol was fully active on HeLa ERE-luciferase ERα cells. The hybrid microparticles were cytocompatible during preliminary tests on NIH 3T3 fibroblasts (ISO 10993-5 standard) and they were fully biocompatible after subcutaneous injection on mice (ISO 10993-6 standard) underlining their high potential as a safe and long-acting subcutaneous drug delivery system.

Description of the unusual digestive tract of Platax orbicularis and the potential impact of Tenacibaculum maritimum infection.

BACKGROUND: Ephippidae fish are characterized by a discoid shape with a very small visceral cavity. Among them Platax orbicularis has a high economic potential due to its flesh quality and flesh to carcass ratio. Nonetheless, the development of its aquaculture is limited by high mortality rates, especially due to Tenacibaculum maritimum infection, occurring one to three weeks after the transfer of fishes from bio-secure land-based aquaculture system to the lagoon cages for growth. Among the lines of defense against this microbial infection, the gastrointestinal tract (GIT) is less studied. The knowledge about the morphofunctional anatomy of this organ in P. orbicularis is still scarce. Therefore, the aims of this study are to characterize the GIT in non-infected P. orbicularis juveniles to then investigate the impact of T. maritimum on this multifunctional organ. METHODS: In the first place, the morpho-anatomy of the GIT in non-infected individuals was characterized using various histological techniques. Then, infected individuals, experimentally challenged by T. maritimum were analysed and compared to the previously established GIT reference. RESULTS: The overlapped shape of the GIT of P. orbicularis is probably due to its constrained compaction in a narrow visceral cavity. Firstly, the GIT was divided into 10 sections, from the esophagus to the rectum. For each section, the structure of the walls was characterized, with a focus on mucus secretions and the presence of the Na+/K+ ATPase pump. An identification key allowing the characterization of the GIT sections using in toto histology is given. Secondly, individuals challenged with T. maritimum exhibited differences in mucus type and proportion and, modifications in the mucosal and muscle layers. These changes could induce an imbalance in the trade-off between the GIT functions which may be in favour of protection and immunity to the disadvantage of nutrition capacities.

Zika Virus Infection Promotes Local Inflammation, Cell Adhesion Molecule Upregulation, and Leukocyte Recruitment at the Blood-Brain Barrier.

The blood-brain barrier (BBB) largely prevents toxins and pathogens from accessing the brain. Some viruses have the ability to cross this barrier and replicate in the central nervous system (CNS). Zika virus (ZIKV) was responsible in 2015 to 2016 for a major epidemic in South America and was associated in some cases with neurological impairments. Here, we characterized some of the mechanisms behind its neuroinvasion using an innovative in vitro human BBB model. ZIKV efficiently replicated, was released on the BBB parenchyma side, and triggered subtle modulation of BBB integrity as well as an upregulation of inflammatory and cell adhesion molecules (CAMs), which in turn favored leukocyte recruitment. Finally, we showed that ZIKV-infected mouse models displayed similar CAM upregulation and that soluble CAMs were increased in plasma samples from ZIKV-infected patients. Our observations suggest a complex interplay between ZIKV and the BBB, which may trigger local inflammation, leukocyte recruitment, and possible cerebral vasculature impairment.IMPORTANCE Zika virus (ZIKV) can be associated with neurological impairment in children and adults. To reach the central nervous system, viruses have to cross the blood-brain barrier (BBB), a multicellular system allowing a tight separation between the bloodstream and the brain. Here, we show that ZIKV infects cells of the BBB and triggers a subtle change in its permeability. Moreover, ZIKV infection leads to the production of inflammatory molecules known to modulate BBB integrity and participate in immune cell attraction. The virus also led to the upregulation of cellular adhesion molecules (CAMs), which in turn favored immune cell binding to the BBB and potentially increased infiltration into the brain. These results were also observed in a mouse model of ZIKV infection. Furthermore, plasma samples from ZIKV-infected patients displayed an increase in CAMs, suggesting that this mechanism could be involved in neuroinflammation triggered by ZIKV.

Study of Usutu virus neuropathogenicity in mice and human cellular models.

Usutu virus (USUV), an African mosquito-borne flavivirus closely related to West Nile virus, was first isolated in South Africa in 1959. USUV emerged in Europe two decades ago, causing notably massive mortality in Eurasian blackbirds. USUV is attracting increasing attention due to its potential for emergence and its rapid spread in Europe in recent years. Although mainly asymptomatic or responsible for mild clinical signs, USUV was recently described as being associated with neurological disorders in humans such as encephalitis and meningoencephalitis, highlighting the potential health threat posed by the virus. Despite this, USUV pathogenesis remains largely unexplored. The aim of this study was to evaluate USUV neuropathogenicity using in vivo and in vitro approaches. Our results indicate that USUV efficiently replicates in the murine central nervous system. Replication in the spinal cord and brain is associated with recruitment of inflammatory cells and the release of inflammatory molecules as well as induction of antiviral-responses without major modulation of blood-brain barrier integrity. Endothelial cells integrity is also maintained in a human model of the blood-brain barrier despite USUV replication and release of pro-inflammatory cytokines. Furthermore, USUV-inoculated mice developed major ocular defects associated with inflammation. Moreover, USUV efficiently replicates in human retinal pigment epithelium. Our results will help to better characterize the physiopathology related to USUV infection in order to anticipate the potential threat of USUV emergence.