Evaluation of microRNAs in the immunopathogenesis of microcephaly caused by Zika virus in experimental models

O Zika Virus (ZIKV) e um flavivirus que causa comprometimento neurologico caracterizando a Sindrome Congenita do ZIKV (SCZ). Embora as celulas precursoras neuronais e os neurônios sejam as celulas mais afetadas, os astrocitos sao mais suscetiveis a infeccao pelo ZIKV, uma vez que toleram maiores cargas virais, sofrem menos apoptose e sao utilizados para replicação viral. Varios reguladores de processos biologicos estao envolvidos na suscetibilidade a SCZ, incluindo os microRNAs (miRNAs). Os miRNAs sao pequenos RNAs nao codificantes que interagem com sequencias alvo de mRNA complementares para repressao pos-transcricional. Nesse contexto, o objetivo principal e investigar as interacoes miRNAs-mRNA durante a transmissao vertical do ZIKV, com foco em astrocitos. Primeiramente, demonstramos que o ZIKV aumenta a expressao miRNAs no cerebro total de camundongos neonatos nascidos de maes infectadas, estimulando a autofagia. A regulacao positiva do miR-295 limita a expressão do gene regulador da autofagia Bcl2l11 in vivo e em astrocitos in vitro. Alem disso, comparando o perfil de miRNAs em astrocitos primarios de camundongos SJL, observamos o aumento da expressao dos miR-295 e miR-302d. Utilizando plataformas bioinformaticas, mimetizadores e inibidores dos miRNAs, e ensaio de luciferase, demonstramos que os genes Ahrr, Neurod4 e Neurod6 sao regulados negativamente pelos miR-295 e miR-302d. Curiosamente, os miR-295 e miR-302d sao transcritos juntamente com os genes Nlrp12 e Larp7, respectivamente. E para entender melhor o mecanismo usado pelo ZIKV para induzir estes miRNAs, avaliamos fatores que levam a transcricao dos genes Nlrp12 e Larp7. De modo interessante, o ZIKV aumentou a expressao dos fatores de transcricao CEBP/b e da subunidade p65 de NF-kB. Corroborando, o ZIKV induziu o recrutamento de CEBP/b para o promotor de Nlrp12 e Larp7 e p65 para o promotor de Larp7. Ainda, a inibicao de Rela e Cebpb por siRNAs, diminuiu Nlrp12, Larp7, premiR-295, premiR-302d, miR-295 e miR-302d, enquanto aumentou a expressao dos genes Ahrr, Neurod4 e Neurod6. Em seguida, demonstramos que a ativação dessa via descrita e um mecanismo de suscetibilidade a SCZ, uma vez que nao observamos aumento da expressao de Cebpb, Rela, Nlrp12, premiR-295, Larp7, premiR-302d, miR-295 e miR-302d, e dos seus genes alvos, Ahrr, Neurod4, Neurod6 em astrocitos de camundongos C57BL/6 WT resistentes a microcefalia causada pelo ZIKV. Dessa forma, demonstramos que o ZIKV ativa os fatores de transcricao C/EBPb e NF-kB para aumentar a transcricao de Nlrp12 e Larp7 e, consequentemente, dos miR-295 e miR-302d em astrocitos SJL. Estes miRNAs levam a diminuicao da expressao dos genes Ahrr, Neurod4 e Neurod6, contribuindo para o desenvolvimento de SCZ em camundongos SJL suscetiveis, mas nao em camundongos C57BL/6 WT resistentes. Em conjunto, sugerimos um mecanismo de suscetibilidade ao desenvolvimento da SCZ.The Zika Virus (ZIKV) is a flavivirus that leads to neurological impairment characterizing the Congenital ZIKV Syndrome (CZS). Although neuronal precursor cells and neurons are the most affected cells, astrocytes are more susceptible to ZIKV infection, once tolerate higher viral loads, suffer less apoptosis and are used for viral replication. Curiously, it is known that several regulators of biological processes are involved in the susceptibility to CZS, including microRNAs (miRNAs). MiRNAs are small non-coding RNAs, which work downstream of transcription factors by interacting with complementary mRNA target sequences for further post-transcriptional repression. In this context, the principal aim is investigated miRNAsmRNA interactions during vertical transmission of ZIKV, focusing on astrocytes. First, we demonstrated that ZIKV increases miRNAs expression in the brain of mice neonates from infected mothers, stimulating autophagy. Upregulation of miR-295 limits the expression of Bcl2l11, an autophagy regulator gene, in vivo and in astrocytes in vitro. Furthermore, comparing miRNAs profile in SJL astrocytes, we observed an increased expression of miR-295 and miR-302d. Using bioinformatics platforms, miRNAs mimics and inhibitors, and luciferase assay, we demonstrated that Ahrr, Neurod4 and Neurod6 genes are downregulated by miR- 295 and miR-302d. Interestingly, miR-295 and miR-302d are transcribed along with Nlrp12 and Larp7 genes, respectively. To better understand the mechanism used by ZIKV to induce miRNAs, we evaluated transcription factors for Nlrp12 and Larp7 transcription. Interestingly, ZIKV increased the expression of CEBP/b and NF-kB p65 subunit. Corroborating, ZIKV induced the recruitment of CEBP/b to Nlrp12 and Larp7 promote, p65 to Larp7 promoter. Moreover, Rela and Cebpb inhibition by siRNAs decreased Nlrp12, Larp7, premiR-295, premiR-302d, miR- 295 and miR-302d, while increased Ahrr, Neurod4 and Neurod6 gene expression. Following, we demonstrate that the activation of this described pathway is a susceptibility mechanism to CZS, since we did not observe increased expression of Cebpb, Rela, Nlrp12, premiR-295, Larp7, premiR-302d, miR-295 and miR-302d, and their target genes, Ahrr, Neurod4, Neurod6 in astrocytes from C57BL/6 WT resistant mice to microcephaly caused by ZIKV. Thus, we demonstrate that ZIKV activates transcription factors C/EBPb and NF-kB to increase transcription of Nlrp12 and Larp7, and consequently, miR-295 and miR-302d in SJL astrocytes. These miRNAs lead to decreased expression of Ahrr, Neurod4 and Neurod6 genes, contributing to CZS development in susceptible SJL mice, but not in resistant C57BL/6 WT mice. Together, we suggest a mechanism of susceptibility to CZS development

Evaluation of the regulatory capacity of endometrial mesenchymal stem cells in the experimental autoimmune encephalomyelitis model.

A esclerose múltipla é uma doença inflamatória crônica desencadeada por células T autorreativas contra antígenos proteicos da mielina. A encefalomielite experimental autoimune é o modelo murino mais utilizado para o estudo da EM. As tubas uterinas e o útero de camundongos fêmeas são órgãos ricos em células mesenquimais que são pouco utilizadas em estudos. Dessa forma, no presente projeto, caracterizamos a obtenção dessa população e avaliamos sua capacidade imunossupressora utilizando o modelo de EAE. Observamos que o tratamento é capaz de modular o perfil de linfócitos T CD4+ durante sua ativação nos linfonodos, induzindo o direcionamento para a subpopulação Tr1 e atenuando as Th1 e Th17. Assim, houve uma diminuição do número de células infiltrantes no SNC associado a uma menor ativação de células da microglia. Em conjunto, demostraramos que as meMSC utilizadas como tratamento são capazes de atrasar o desenvolvimento da EAE e, portanto, evidenciando o caráter imunomodulador das MSCs derivadas do endométrio, chamando a atenção para seu potencial terapêutico.Multiple sclerosis is a chronic inflammatory disease triggered by autoreactive T cells against myelin protein. Experimental Autoimmune Encephalomyelitis is the most commonly used murine model for the study of MS. The uterine tubes and uterus of female mice are organs rich in mesenchymal cells which are rarely used. Thus, in the present work, we characterized the extraction of this population and evaluated its immunosuppressive capacity using the EAE model. We observed that the meMSC treatment is capable of modulating the CD4 T lymphocyte profile during its activation in the lymph nodes, inducing the expansion of the Tr1 subpopulation and attenuating Th1 and Th17. Consequently, there was a decrease in the number of infiltrating cells in the CNS associated with a reduction of microglial activation. Taken together, our results demonstrated that the meMSCs used as treatment are capable of delaying the development of EAE, therefore, evidencing its immunomodulatory features drawing attention to its therapeutic potential

ZIKV Infection and miRNA Network in Pathogenesis and Immune Response

Over the years, viral infections have caused severe illness in humans. Zika Virus (ZIKV) is a flavivirus transmitted by mosquito vectors that leads to notable neurological impairment, whose most dramatic impact is the Congenital ZIKV Syndrome (CZS). ZIKV targets neuronal precursor cells leading to apoptosis and further impairment of neuronal development, causing microcephaly, lissencephaly, ventriculomegaly, and calcifications. Several regulators of biological processes are involved in CZS development, and in this context, microRNAs (miRNAs) seem to have a fundamental role. miRNAs are important regulators of protein translation, as they form the RISC silencing complex and interact with complementary mRNA target sequences to further post-transcriptional repression. In this context, little is known about their participation in the pathogenesis of viral infections. In this review, we discuss how miRNAs could relate to ZIKV and other flavivirus infections

Gut-licensed IFNγ + NK cells drive LAMP1 + TRAIL + anti-inflammatory astrocytes

Astrocytes are glial cells that are abundant in the central nervous system (CNS) and that have important homeostatic and disease-promoting functions1. However, little is known about the homeostatic anti-inflammatory activities of astrocytes and their regulation. Here, using high-throughput flow cytometry screening, single-cell RNA sequencing and CRISPR-Cas9-based cell-specific in vivo genetic perturbations in mice, we identify a subset of astrocytes that expresses the lysosomal protein LAMP12 and the death receptor ligand TRAIL3. LAMP1+TRAIL+ astrocytes limit inflammation in the CNS by inducing T cell apoptosis through TRAIL-DR5 signalling. In homeostatic conditions, the expression of TRAIL in astrocytes is driven by interferon-γ (IFNγ) produced by meningeal natural killer (NK) cells, in which IFNγ expression is modulated by the gut microbiome. TRAIL expression in astrocytes is repressed by molecules produced by T cells and microglia in the context of inflammation. Altogether, we show that LAMP1+TRAIL+ astrocytes limit CNS inflammation by inducing T cell apoptosis, and that this astrocyte subset is maintained by meningeal IFNγ+ NK cells that are licensed by the microbiome

Gut-licensed IFNγ+ NK cells drive LAMP1+TRAIL+ anti-inflammatory astrocytes

Astrocytes are glial cells that are abundant in the central nervous system (CNS) and that have important homeostatic and disease-promoting functions1. However, little is known about the homeostatic anti-inflammatory activities of astrocytes and their regulation. Here, using high-throughput flow cytometry screening, single-cell RNA sequencing and CRISPR–Cas9-based cell-specific in vivo genetic perturbations in mice, we identify a subset of astrocytes that expresses the lysosomal protein LAMP12 and the death receptor ligand TRAIL3. LAMP1+TRAIL+ astrocytes limit inflammation in the CNS by inducing T cell apoptosis through TRAIL–DR5 signalling. In homeostatic conditions, the expression of TRAIL in astrocytes is driven by interferon-γ (IFNγ) produced by meningeal natural killer (NK) cells, in which IFNγ expression is modulated by the gut microbiome. TRAIL expression in astrocytes is repressed by molecules produced by T cells and microglia in the context of inflammation. Altogether, we show that LAMP1+TRAIL+ astrocytes limit CNS inflammation by inducing T cell apoptosis, and that this astrocyte subset is maintained by meningeal IFNγ+ NK cells that are licensed by the microbiome.Fil: Sanmarco, Liliana Maria. Harvard Medical School; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Wheeler, Michael A.. Harvard Medical School; Estados UnidosFil: Gutiérrez Vázquez, Cristina. Harvard Medical School; Estados UnidosFil: Polonio Manganeli, Carolina. Harvard Medical School; Estados UnidosFil: Linnerbauer, Mathias. Harvard Medical School; Estados UnidosFil: Pinho Ribeiro, Felipe A.. Harvard Medical School; Estados UnidosFil: Li, Zhaorong. Harvard Medical School; Estados UnidosFil: Giovannoni, Federico. Harvard Medical School; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Batterman, Katelyn V.. University Of Boston. School Of Medicine.; Estados UnidosFil: Scalisi, Giulia. Harvard Medical School; Estados UnidosFil: Zandee, Stephanie E. J.. University of Montreal; CanadáFil: Heck, Evelyn Sabrina. Harvard Medical School; Estados Unidos. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Alsuwailm, Moneera. Harvard Medical School; Estados UnidosFil: Rosene, Douglas L.. University Of Boston. School Of Medicine.; Estados UnidosFil: Becher, Burkhard. Universitat Zurich; SuizaFil: Chiu, Isaac M.. Harvard Medical School; Estados UnidosFil: Prat, Alexandre. University of Montreal; CanadáFil: Quintana, Francisco Javier. Harvard Medical School; Estados Unido

AHR is a Zika virus host factor and a candidate target for antiviral therapy

Zika virus (ZIKV) is a flavivirus linked to multiple birth defects including microcephaly, known as congenital ZIKV syndrome. The identification of host factors involved in ZIKV replication may guide efficacious therapeutic interventions. In genome-wide transcriptional studies, we found that ZIKV infection triggers aryl hydrocarbon receptor (AHR) activation. Specifically, ZIKV infection induces kynurenine (Kyn) production, which activates AHR, limiting the production of type I interferons (IFN-I) involved in antiviral immunity. Moreover, ZIKV-triggered AHR activation suppresses intrinsic immunity driven by the promyelocytic leukemia (PML) protein, which limits ZIKV replication. AHR inhibition suppressed the replication of multiple ZIKV strains in vitro and also suppressed replication of the related flavivirus dengue. Finally, AHR inhibition with a nanoparticle-delivered AHR antagonist or an inhibitor developed for human use limited ZIKV replication and ameliorated newborn microcephaly in a murine model. In summary, we identified AHR as a host factor for ZIKV replication and PML protein as a driver of anti-ZIKV intrinsic immunity

Barcoded viral tracing of single-cell interactions in central nervous system inflammation.

Cell-cell interactions control the physiology and pathology of the central nervous system (CNS). To study astrocyte cell interactions in vivo, we developed rabies barcode interaction detection followed by sequencing (RABID-seq), which combines barcoded viral tracing and single-cell RNA sequencing (scRNA-seq). Using RABID-seq, we identified axon guidance molecules as candidate mediators of microglia-astrocyte interactions that promote CNS pathology in experimental autoimmune encephalomyelitis (EAE) and, potentially, multiple sclerosis (MS). In vivo cell-specific genetic perturbation EAE studies, in vitro systems, and the analysis of MS scRNA-seq datasets and CNS tissue established that Sema4D and Ephrin-B3 expressed in microglia control astrocyte responses via PlexinB2 and EphB3, respectively. Furthermore, a CNS-penetrant EphB3 inhibitor suppressed astrocyte and microglia proinflammatory responses and ameliorated EAE. In summary, RABID-seq identified microglia-astrocyte interactions and candidate therapeutic targets

Barcoded viral tracing of single-cell interactions in central nervous system inflammation.

Cell-cell interactions control the physiology and pathology of the central nervous system (CNS). To study astrocyte cell interactions in vivo, we developed rabies barcode interaction detection followed by sequencing (RABID-seq), which combines barcoded viral tracing and single-cell RNA sequencing (scRNA-seq). Using RABID-seq, we identified axon guidance molecules as candidate mediators of microglia-astrocyte interactions that promote CNS pathology in experimental autoimmune encephalomyelitis (EAE) and, potentially, multiple sclerosis (MS). In vivo cell-specific genetic perturbation EAE studies, in vitro systems, and the analysis of MS scRNA-seq datasets and CNS tissue established that Sema4D and Ephrin-B3 expressed in microglia control astrocyte responses via PlexinB2 and EphB3, respectively. Furthermore, a CNS-penetrant EphB3 inhibitor suppressed astrocyte and microglia proinflammatory responses and ameliorated EAE. In summary, RABID-seq identified microglia-astrocyte interactions and candidate therapeutic targets