[The viral protein NS1: a major player in the pathogenesis of orthoflaviviruses]. / La protéine virale NS1 : un acteur majeur de la pathogenèse des orthoflavivirus.

Orthoflaviviruses are enveloped positive-sense RNA viruses comprising numerous human pathogens transmitted by hematophagous arthropods. This includes viruses such as dengue virus, Zika virus, and yellow fever virus. The viral nonstructural protein NS1 plays a central role in the pathogenesis and cycle of these viruses by acting in two different forms: associated with the plasma membrane (NS1m) or secreted outside the cell (NS1s). The versatility of NS1 is evident in its ability to modulate various aspects of the infectious process, from immune evasion to pathogenesis. As an intracellular protein, it disrupts many processes, interfering with signaling pathways and facilitating viral replication in concert with other viral proteins. As a secreted protein, NS1 actively participates in immune evasion, interfering with the host immune system, inhibiting the complement system, facilitating viral dissemination, and disrupting the integrity of endothelial barriers. This review primarily aims to address the role of NS1 in viral pathogenesis associated with orthoflaviviruses.

Experimental Models to Study Endothelial to Mesenchymal Transition in Myocardial Fibrosis and Cardiovascular Diseases.

Fibrosis is a common feature of cardiovascular diseases and targets multiple organs, such as the heart and vessels. Endothelial to mesenchymal transition is a complex, vital process that occurs during embryonic formation and plays a crucial role in cardiac development. It is also a fundamental process implicated in cardiac fibrosis and repair, but also in other organs. Indeed, in numerous cardiovascular diseases, the endothelial-to-mesenchymal transition has been shown to be involved in the generation of fibroblasts that are able to produce extracellular matrix proteins such as type I collagen. This massive deposition results in tissue stiffening and organ dysfunction. To advance our understanding of this process for the development of new specific diagnostic and therapeutic strategies, it is essential to develop relevant cellular and animal models of this process. In this review, our aim was to gain an in-depth insight into existing in vitro and in vivo models of endothelial to mesenchymal transition in cardiovascular diseases with a focus on cardiac fibrosis. We discuss important parameters impacting endothelial to mesenchymal transition, and we give perspectives for the development of relevant models to decipher the underlying mechanisms and ultimately find new treatments specific to fibrosis happening in cardiovascular diseases.

One Health surveillance of West Nile and Usutu viruses: a repeated cross-sectional study exploring seroprevalence and endemicity in Southern France, 2016 to 2020.

BackgroundWest Nile virus (WNV) and Usutu virus (USUV), two closely related flaviviruses, mainly follow an enzootic cycle involving mosquitoes and birds, but also infect humans and other mammals. Since 2010, their epidemiological situation may have shifted from irregular epidemics to endemicity in several European regions; this requires confirmation, as it could have implications for risk assessment and surveillance strategies.AimTo explore the seroprevalence in animals and humans and potential endemicity of WNV and USUV in Southern France, given a long history of WNV outbreaks and the only severe human USUV case in France in this region.MethodsWe evaluated the prevalence of WNV and USUV in a repeated cross-sectional study by serological and molecular analyses of human, dog, horse, bird and mosquito samples in the Camargue area, including the city of Montpellier, between 2016 and 2020.ResultsWe observed the active transmission of both viruses and higher USUV prevalence in humans, dogs, birds and mosquitoes, while WNV prevalence was higher in horses. In 500 human samples, 15 were positive for USUV and 6 for WNV. Genetic data showed that the same lineages, WNV lineage 1a and USUV lineage Africa 3, were found in mosquitoes in 2015, 2018 and 2020.ConclusionThese findings support existing literature suggesting endemisation in the study region and contribute to a better understanding of USUV and WNV circulation in Southern France. Our study underlines the importance of a One Health approach for the surveillance of these viruses.

Differential neurovirulence of Usutu virus lineages in mice and neuronal cells.

BACKGROUND: Usutu virus (USUV) is an emerging neurotropic arthropod-borne virus recently involved in massive die offs of wild birds predominantly reported in Europe. Although primarily asymptomatic or presenting mild clinical signs, humans infected by USUV can develop neuroinvasive pathologies (including encephalitis and meningoencephalitis). Similar to other flaviviruses, such as West Nile virus, USUV is capable of reaching the central nervous system. However, the neuropathogenesis of USUV is still poorly understood, and the virulence of the specific USUV lineages is currently unknown. One of the major complexities of the study of USUV pathogenesis is the presence of a great diversity of lineages circulating at the same time and in the same location. METHODS: The aim of this work was to determine the neurovirulence of isolates from the six main lineages circulating in Europe using mouse model and several neuronal cell lines (neurons, microglia, pericytes, brain endothelial cells, astrocytes, and in vitro Blood-Brain Barrier model). RESULTS: Our results indicate that all strains are neurotropic but have different virulence profiles. The Europe 2 strain, previously described as being involved in several clinical cases, induced the shortest survival time and highest mortality in vivo and appeared to be more virulent and persistent in microglial, astrocytes, and brain endothelial cells, while also inducing an atypical cytopathic effect. Moreover, an amino acid substitution (D3425E) was specifically identified in the RNA-dependent RNA polymerase domain of the NS5 protein of this lineage. CONCLUSIONS: Altogether, these data show a broad neurotropism for USUV in the central nervous system with lineage-dependent virulence. Our results will help to better understand the biological and epidemiological diversity of USUV infection.

Sodium restriction modulates innate immunity and prevents cardiac remodeling in a rat model of metabolic syndrome.

In the view of the relationships between excessive sodium intake, immunity and target organ damage, we hypothesized that reduction in dietary sodium would be beneficial in the prevention of cardiac alterations through a restrained local immunity response in a rat model of metabolic syndrome. Sprague-Dawley rats were fed a 60% fructose diet with either a normal sodium (0.64% NaCl) or a low sodium content (<0.01% NaCl) for 8weeks. After 4weeks, rats were infused or not with angiotensin II (200ng·kg-1·min-1, sc) for 4weeks. Tail-cuff blood pressure was determined in conscious rats. Heart and left ventricle weight, cardiomyocyte size, and cardiac fibrosis were evaluated. We performed a transcriptomic analysis in order to identify differentially regulated cardiac mRNAs between normal and low sodium diets. We validated those results using qPCR and immunohistochemistry. Angiotensin II-induced blood pressure rise was blunted (~50%) in the low-sodium fed rats while cardiac hypertrophy and fibrosis were prevented. Transcriptomic analysis revealed 66 differentially regulated genes including 13 downregulated genes under the low sodium diet and implicated in the innate immune response. This was confirmed by reduced cardiac macrophages infiltration under the low sodium diet. Dietary sodium restriction prevents structural alterations of the heart of rats with fructose-induced insulin resistance and angiotensin II-hypertension. The reduction of cardiac inflammation and macrophage infiltration suggests that innate immunity has an important role in the beneficial effect of sodium restriction on cardiac remodeling.

Characterization of resident B cells of vascular walls in human atherosclerotic patients.

Animal models of atherosclerosis suggest that B cells have contradictory protective or proatherogenic effects that are also subset and context dependent. To further understand the pathophysiology of human atheroma, we characterized local Ig production and functional properties of resident B cells in human arterial lesions. Ig repertoires were analyzed by RT-PCR in carotid endarterectomy samples. Cytokine, differentiation marker and transcription factor mRNA expression was studied on arterial wall lymphocytes isolated by laser capture microdissection. Ig sequence analysis revealed that individual samples each contained a limited number of B cell clones. Functional α and γ mRNAs made up the majority of H chain mRNAs in the adventitia. Clonal evolution of Ig V regions, expression of activation-induced cytidine deaminase, clonal H chain switch, and an inverted λ/κ ratio of Ig L chain usage indicated that a local differentiation process was taking place in arterial walls. Clonotypic markers revealed different plaque and adventitia Ig repertoires and a B cell recirculation between adventitia and draining lymph nodes. Microdissected mononuclear cells had an activated phenotype expressing IL-6, GM-CSF, and TNF-α, whereas IL-2, IL-4, IL-10, M-CSF, and IFN-γ were not detected. Adventitial oligoclonal resident B cells of atherosclerotic patients are mainly mature B2 (conventional) CD20⁻ plasmablasts lacking markers of terminal differentiation to plasma cell (CD138 and Blimp-1). They present hallmarks of Ag-driven maturation and could act on inflammation and disease progression directly or by promoting polarization of other immune cells.

[B lymphocytes: a promising target to treat atherosclerosis?]. / Les lymphocytes B - Une cible prometteuse pour traiter l'athérosclérose ?

Atherosclerosis is a chronic inflammatory disease of the arterial wall. It is already well established that several immune cells (macrophages, T lymphocytes, etc.) modulate atherosclerosis progression whereas the role of the different subpopulations of B lymphocytes emerged only recently. B1 lymphocytes secrete protective IgM antibodies that act as scavenger of deleterious molecules whereas B2 lymphocytes probably worsen the disease by activating pro-inflammatory T lymphocytes. The outcome of these opposite functional properties of B lymphocytes on the evolution of arterial lesions may vary depending on their local environment during the different stages of the disease. In this review, we emphasize recent progresses in understanding the specific contribution of B lymphocytes to atherosclerosis and discuss the interest of targeting them to improve therapy.

[Serum autoantibodies profiling and early-stage cancer detection]. / Autoanticorps et diagnostic précoce des cancers.

It is now well established that an immune response to cancer is elicited in humans, as demonstrated in part by the identification of autoantibodies against a number of tumor-associated antigens in sera from patients with different types of cancer. During these past few years, proteomic approaches have been developed to identify tumor-associated antigens and their cognate autoantibodies. Detection of a panel of serum autoantibodies has thus been proposed as a new method for early cancer diagnosis. Early detection seems to be particularly adequate in high-risk populations, such as heavy smokers for lung cancer or in women with high mammographic density for breast cancer. In this review, we highlight the features of serum autoantibody biomarkers and outline the proteomic strategies employed to identify and validate their use in clinical practice for cancer screening and diagnosis. We particularly emphasize the clinical utility of autoantibody signatures, using the examples of lung and breast cancer. Finally, we discuss the challenges remaining for clinical validation.

Optimization of RNA extraction methods from human metabolic tissue samples of the COMET biobank.

Constitution of biobank of human tissues requires careful handling and storage of biological material, to guarantee the quality of samples. Tissue preparation is also critical for further applications such as transcriptomic profiling. In this study, our aim was to evaluate the impact of different disruption techniques (FastPrep-24 instrument, GentleMACS dissociator, and syringe/needle) and homogenizing buffers (RLT versus QIAzol) on RNA purity and quality of metabolic tissues (adipose tissues, liver and skeletal muscle) present in the COMET Biobank. For all homogenization methods used and tissue types, the A260/280 ratios reached values ≥ 1.8, which are in the range of what is found in human tissues and cell lines, while the A260/230 ratios were however ≤ 1.8, with the lowest value obtained with GentleMACS Dissociator. In addition, GentleMACS Dissociator combined with QIAzol reagent gave the highest RIN value and 28S/18S ratio for all tissues tested, except for muscle. Performing RT-qPCR, Ct values for different housekeeping genes can be influenced by extraction methods and RNA quality of samples. In conclusion, we have demonstrated that different disruption techniques and homogenizing buffers impact the purity and some quality markers of RNA, and can also impact quantification of mRNAs by RT-qPCR in human metabolic tissues.

Evidence of Exposure to USUV and WNV in Zoo Animals in France.

West Nile virus (WNV) and Usutu virus (USUV) are zoonotic arboviruses. These flaviviruses are mainly maintained in the environment through an enzootic cycle involving mosquitoes and birds. Horses and humans are incidental, dead-end hosts, but can develop severe neurological disorders. Nevertheless, there is little data regarding the involvement of other mammals in the epidemiology of these arboviruses. In this study, we performed a serosurvey to assess exposure to these viruses in captive birds and mammals in a zoo situated in the south of France, an area described for the circulation of these two viruses. A total of 411 samples comprising of 70 species were collected over 16 years from 2003 to 2019. The samples were first tested by a competitive enzyme-linked immunosorbent assay. The positive sera were then tested using virus-specific microneutralization tests against USUV and WNV. USUV seroprevalence in birds was 10 times higher than that of WNV (14.59% versus 1.46%, respectively). Among birds, greater rhea (Rhea Americana) and common peafowl (Pavo cristatus) exhibited the highest USUV seroprevalence. Infections occurred mainly between 2016-2018 corresponding to a period of high circulation of these viruses in Europe. In mammalian species, antibodies against WNV were detected in one dama gazelle (Nanger dama) whereas serological evidence of USUV infection was observed in several Canidae, especially in African wild dogs (Lycaon pictus). Our study helps to better understand the exposure of captive species to WNV and USUV and to identify potential host species to include in surveillance programs in zoos.

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.

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.

Peripheral blood T4 cell surface CCR5 density as a marker of activity in rheumatoid arthritis treated with anti-CD20 monoclonal antibody.

The chemokine (C-C motif) receptor CCR5 and its ligand CCL5 play key roles in the intra-articular recruitment of peripheral blood mononuclear cells (PBMC) in rheumatoid arthritis (RA). Therefore, using quantitative cytofluorometry, we followed T4 cell surface CCR5 density in 27 subjects with RA before and after treatment with the anti-CD20 monoclonal antibody rituximab. We observed low T4 cell surface CCR5 densities before treatment, which correlated positively with disease activity, as determined using a disease activity score evaluated on 28 joints (DAS 28), and negatively with CCL5 mRNA concentrations in PBMC, contrasting with a high proportion of intracellular CCR5 molecules, a pattern compatible with ligand-induced CCR5 internalization. At 3 months post-treatment, CCL5 mRNA expression in PBMC declined, whereas T4 cell surface CCR5 densities increased proportionally to the decrease in DAS 28. Thus, peripheral blood T4 cell surface CCR5 density is a good surrogate marker of RA activity and of the efficiency of anti-CD20 therapy.

Identifying autoantibody signatures in cancer: a promising challenge.

Biomarkers that show high sensitivity and specificity are needed for the early diagnosis and prognosis of cancer. An immune response to cancer is elicited in humans, as demonstrated, in part, by the identification of autoantibodies against a number of tumor-associated antigen (TAAs) in sera from patients with different types of cancer. Identification of TAAs and their cognate autoantibodies is a promising strategy for the discovery of relevant biomarkers. During the past few years, three proteomic approaches, including serological identification of antigens by recombinant expression cloning (SEREX), serological proteome analysis (SERPA) and, more recently, protein microarrays, have been the dominant strategies used to identify TAAs and their cognate autoantibodies. In this review, we aim to describe the advantages, drawbacks and recent improvements of these approaches for the study of humoral responses. Finally, we discuss the definition of autoantibody signatures to improve sensitivity for the development of clinically relevant tests.

FKBP4 connects mTORC2 and PI3K to activate the PDK1/Akt-dependent cell proliferation signaling in breast cancer.

Purpose: Among the FKBP family members, FKBP4 has been described to have a potential role in tumorigenesis, and as a putative tissue marker. We previously showed that FKBP4, an HSP90-associated co-chaperone, can elicit immune response as a tumor-specific antigen, and are overexpressed in breast cancer. Experimental design: In this study, we examined how loss of FKBP4 affect breast cancer progression and exploited protein interactomics to gain mechanistic insight into this process. Results: We found that FKBP4 expression is associated with breast cancer progression and prognosis, especially of ER-negative breast cancer. Furthermore, FKBP4 depletion specifically reduces cell growth and proliferation of triple negative breast cancer cell model and xenograft tumor model. Using specific protein interactome strategy by BirA proximity-dependent biotin identification, we demonstrated that FKBP4 is a novel PI3K-Akt-mTOR proximal interacting protein. Conclusion: Our results suggest that FKBP4 interacts with PI3K and can enhance Akt activation through PDK1 and mTORC2.

Zika virus induces strong inflammatory responses and impairs homeostasis and function of the human retinal pigment epithelium.

BACKGROUND: Zika virus (ZIKV) has recently re-emerged as a pathogenic agent with epidemic capacities as was well illustrated in South America. Because of the extent of this health crisis, a number of more serious symptoms have become associated with ZIKV infection than what was initially described. In particular, neuronal and ocular disorders have been characterized, both in infants and in adults. Notably, the macula and the retina can be strongly affected by ZIKV, possibly by a direct effect of the virus. This is supported by the detection of replicative and infectious virus in lachrimal fluid in human patients and mouse models. METHODS: Here, we used an innovative, state-of-the-art iPSC-derived human retinal pigment epithelium (RPE) model to study ZIKV retinal impairment. FINDINGS: We showed that the human RPE is highly susceptible to ZIKV infection and that a ZIKV African strain was more virulent and led to a more potent epithelium disruption and stronger anti-viral response than an Asian strain, suggesting lineage differences. Moreover, ZIKV infection led to impaired membrane dynamics involved in endocytosis, organelle biogenesis and potentially secretion, key mechanisms of RPE homeostasis and function. INTERPRETATION: Taken together, our results suggest that ZIKV has a highly efficient ocular tropism, which creates a strong inflammatory environment that could have acute or chronic adverse effects. FUND: This work was funded by Retina France, REACTing and La Région Languedoc-Roussillon.

Specific increase of human kallikrein 4 mRNA and protein levels in breast cancer stromal cells.

The kallikrein family (KLK) has been implicated in cancer and may be useful as tumor markers. Here, we compared the 15 KLK genes' expression in malignant and normal breast tissues using real-time quantitative PCR. Most KLKs were expressed at lower levels in breast cancer compared to normal breast tissue. The only exception was the eightfold increase level of KLK4 in breast cancer tissues (P=0.008). KLK4 level was strongly associated with tumor grade (P=0.0015). Interestingly, based on laser cell microdissection analysis and immunochemistry, the up-regulation of kallikrein 4 occurred in the surrounding stromal cells. Our findings suggest that KLK4 may be associated with the development and progression of breast cancer and suggest its potential use in breast cancer monitoring.

Proteomic profile determination of autosomal aneuploidies by mass spectrometry on amniotic fluids.

BACKGROUND: Prenatal diagnosis of chromosomal abnormalities by cytogenetic analysis is time-consuming, expensive, and requires highly qualified technicians. Rapid diagnosis of aneuploidies followed by reassurance of women with normal results can be performed by molecular analysis of uncultured foetal cells. In the present study, we developed a proteomic fingerprinting approach coupled with a statistical classification method to improve diagnosis of aneuploidies, including trisomies 13, 18, and 21, in amniotic fluid samples. RESULTS: The proteomic spectra obtained from 52 pregnant women were compiled, normalized, and mass peaks with mass-to-charge ratios between 2.5 and 50 kDa identified. Peak information was combined together and analysed using univariate statistics. Among the 208 expressed protein peaks, 40 differed significantly between aneuploid and non aneuploid samples, with AUC diagnostic values ranging from 0.71 to 0.91. Hierarchical clustering, principal component analysis and support vector machine (SVM) analysis were performed. Two class predictor models were defined from the training set, which resulted in a prediction accuracy of 92.3% and 96.43%, respectively. Using an external and independent validation set, diagnostic accuracies were maintained at 87.5% and 91.67%, respectively. CONCLUSION: This pilot study demonstrates the potential interest of protein expression signature in the identification of new potential biological markers that might be helpful for the rapid clinical management of high-risk pregnancies.

[Comparison of proteomic strategies to identify antibodies resulting from the humoral immune response to cancer]. / Etude de la réaction immunitaire humorale aux cancers par des approches protéomiques.

Discovery of new serum biomarkers exhibiting an increased sensitivity and specificity for cancer are of major importance for diagnosis improvement. There is considerable evidence for an immune response to cancer in humans, as demonstrated in part by the identification of autoantibodies against a number of tumour-associated antigens in sera from patients with different cancer types. Thus, identification of tumour-associated antigens and their associated antibodies is a promising strategy to find relevant biomarkers. Proteomic approaches such as SEREX and SERPA have allowed identification of great numbers of antigens and their cognate autoantibodies during these past few years. They show many advantages, and allowed identification of relevant autoantigens in different types of cancer. However, they are also time consuming, and lack sensibility and specificity. To circumvent these drawbacks, new proteomic techniques, based on protein or antibody arrays, allow high throughput analysis of multiple targets in a single experiment. Specific combinations of markers should thus be identified, theoretically being more efficient to detect a tumor compared to a single marker. In conclusion, these approaches promise great advances in the field of biomarkers for cancer. They also further need to be validated for clinical application on large populations.