Human red blood cells release microvesicles with distinct sizes and protein composition that alter neutrophil phagocytosis.

Extracellular vesicles (EVs) are membrane-bound structures released by cells and tissues into biofluids, involved in cell-cell communication. In humans, circulating red blood cells (RBCs), represent the most common cell-type in the body, generating daily large numbers of microvesicles. In vitro, RBC vesiculation can be mimicked by stimulating RBCs with calcium ionophores, such as ionomycin and A23187. The fate of microvesicles released during in vivo aging of RBCs and their interactions with circulating cells is hitherto unknown. Using SEC plus DEG isolation methods, we have found that human RBCs generate microvesicles with two distinct sizes, densities, and protein composition, identified by flow cytometry, and MRPS, and further validated by immune TEM. Furthermore, proteomic analysis revealed that RBC-derived microvesicles (RBC-MVs) are enriched in proteins with important functions in ion channel regulation, calcium homeostasis, and vesicular transport, such as of sorcin, stomatin, annexin A7, and RAB proteins. Cryo-electron microscopy identified two separate pathways of RBC-MV-neutrophil interaction, direct fusion with the plasma membrane and internalization, respectively. Functionally, RBC-MVs decrease neutrophil ability to phagocytose E. coli but do not affect their survival at 24 hrs. This work brings new insights regarding the complexity of the RBC-MVs biogenesis, as well as their possible role in circulation.

Global RNAseq of ocular cells reveals gene dysregulation in both asymptomatic and with Congenital Zika Syndrome infants exposed prenatally to Zika virus.

In 2015, Brazil reported an outbreak identified as Zika virus (ZIKV) infection associated with congenital abnormalities. To date, a total of 86 countries and territories have described evidence of Zika infection and recently the appearance of the African ZIKV lineage in Brazil highlights the risk of a new epidemic. The spectrum of ZIKV infection-induced alterations at both cellular and molecular levels is not completely elucidated. Here, we present for the first time the gene expression responses associated with prenatal ZIKV infection from ocular cells. We applied a recently developed non-invasive method (impression cytology) which use eye cells as a model for ZIKV studies. The ocular profiling revealed significant differences between exposed and control groups, as well as a different pattern in ocular transcripts from Congenital Zika Syndrome (CZS) compared to ZIKV-exposed but asymptomatic infants. Our data showed pathways related to mismatch repair, cancer, and PI3K/AKT/mTOR signaling and genes probably causative or protective in the modulation of ZIKV infection. Ocular cells revealed the effects of ZIKV infection on primordial neuronal cell genes, evidenced by changes in genes associated with embryonic cells. The changes in gene expression support an association with the gestational period of the infection and provide evidence for the resulting clinical and ophthalmological pathologies. Additionally, the findings of cell death- and cancer-associated deregulated genes raise concerns about the early onset of other potential pathologies including the need for tumor surveillance. Our results thus provide direct evidence that infants exposed prenatally to the Zika virus, not only with CZS but also without clinical signs (asymptomatic) express cellular and molecular changes with potential clinical implications.

Serum Proteomics Reveals Alterations in Protease Activity, Axon Guidance, and Visual Phototransduction Pathways in Infants With In Utero Exposure to Zika Virus Without Congenital Zika Syndrome.

In 2015, ZIKV infection attracted international attention during an epidemic in the Americas, when neurological disorders were reported in infants who had their mothers exposed to ZIKV during pregnancy. World Health Organization (WHO) epidemiological data show that 5 to 15% of neonates exposed to ZIKV in the uterus have complications included in abnormalities related to Congenital Zika Syndrome (CZS). The risk of complications after birth is not well documented, however, clinical evidence shows that 6% of infants exposed to ZIKV during pregnancy have complications present at birth, and this rate rises to 14% when medical monitoring is performed in all exposed infants, regardless of birth condition. Thus, the evaluation and monitoring of all exposed infants are of foremost importance as the development of late complications has been increasingly supported by clinical evidence. The identification of changes in protein profile of infants exposed to ZIKV without CZS could provide valuable findings to better understand molecular changes in this cohort. Here, we use a shotgun-proteomics approach to investigate alterations in the serum of infants without CZS symptoms but exposed to intrauterine ZIKV (ZIKV) compared to unexposed controls (CTRL). A complex pattern of differentially expressed proteins was identified, highlighting the dysregulation of proteins involved in axon orientation, visual phototransduction, and global protease activity in children exposed to ZIKV without CZS. These data support the importance of monitoring children exposed to ZIKV during gestation and without early CZS symptoms. Our study is the first to assess molecular evidence of possible late disorders in children victims of the ZIKV outbreak in the Americas. We emphasize the importance of medical monitoring of symptomatic and asymptomatic children, as apparently unexplained late neurological and eye disorders may be due to intrauterine ZIKV exposure.

Cellular Imprinting Proteomics Assay: A Novel Method for Detection of Neural and Ocular Disorders Applied to Congenital Zika Virus Syndrome.

Congenital Zika syndrome was first described due to increased incidence of congenital abnormalities associated with Zika virus (ZIKV) infection. Since the eye develops as part of the embryo central nervous system (CNS) structure, it becomes a specialized compartment able to display symptoms of neurodegenerative diseases and has been proposed as a noninvasive approach to the early diagnosis of neurological diseases. Ocular lesions result from defects that occurred during embryogenesis and can become apparent in newborns exposed to ZIKV. Furthermore, the absence of microcephaly cannot exclude the occurrence of ocular lesions and other CNS manifestations. Considering the need for surveillance of newborns and infants with possible congenital exposure, we developed a method termed cellular imprinting proteomic assay (CImPA) to evaluate the ocular surface proteome specific to infants exposed to ZIKV during gestation compared to nonexposure. CImPA combines surface cells and fluid capture using membrane disks and a large-scale quantitative proteomics approach, which allowed the first-time report of molecular alterations such as neutrophil degranulation, cell death signaling, ocular and neurological pathways, which are associated with ZIKV infection with and without the development of congenital Zika syndrome, CZS. Particularly, infants exposed to ZIKV during gestation and without early clinical symptoms could be detected using the CImPA method. Lastly, this methodology has broad applicability as it could be translated in the study of several neurological diseases to identify novel diagnostic biomarkers. Data are available via ProteomeXchange with identifier PXD014038.

Impression Cytology Is a Non-invasive and Effective Method for Ocular Cell Retrieval of Zika Infected Babies: Perspectives in OMIC Studies.

IMPORTANCE: Non-invasive techniques for retrieving ocular surface cells from babies infected by zika virus (ZIKV) during the gestational period remain to be determined. OBJECTIVES: The aim of this study was to describe an optimized impression cytology method for the isolation of viable cells from Zika infected babies with and without Congenital Zika Syndrome (CZS) in satisfactory amount and quality to enable easy adoption in the field and application in the context of genomic and molecular approaches. DESIGN SETTINGS AND PARTICIPANTS: Ocular surface samples were obtained with a hydrophilic nitrocellulose membrane (through optimized impression cytology method) from twelve babies referred to the Pediatric Service of the Antonio Pedro Hospital, Universidade Federal Fluminense (UFF), Niteroi, Rio de Janeiro, Brazil. After an authorized written informed consent from the parents, samples were collected from both eyes of 12 babies (4 babies with maternal ZIKV exposure during gestation and presence of clinical signs which included ocular abnormalities and microcephaly; 4 babies with maternal ZIKV exposure during gestation but no clinical signs; and 4 unaffected control babies with negative PCR for Zika virus and without clinical signs). Cells were used for microscopy analyses and evaluated for their suitability for downstream molecular applications in transcriptomic and proteomic experiments. RESULTS: Our optimized impression cytology protocol enabled the capture of a considerable number of viable cells. The microscopic features of the conjunctival epithelial cells were described by both direct analysis of the membrane-attached cells and analysis of cytospinned captured cells using several staining procedures. Epithelial basal, polyhedral and goblet cells were clearly identified in all groups. All cases of ZIKV infected babies showed potential morphological alterations (cell keratinization, pyknosis, karyolysis, anucleation, and vacuolization). Molecular approaches were also performed in parallel. Genomic DNA and RNA were successfully isolated from all samples to enable the establishment of transcriptomic and proteomic studies. CONCLUSIONS AND RELEVANCE: Our method proved to be a suitable, fast, and non-invasive tool to obtain ocular cell preparations from babies with and without Zika infection. The method yielded sufficient cells for detailed morphological and molecular analyses of samples. We discuss perspectives for the application of impression cytology in the context of ZIKV studies in basic and clinical research.