RESUMO
BACKGROUND: Previous work showed that the microRNA (miRNA) miR-671-5p was upregulated in monocyte-derived dendritic cells (moDCs) stimulated with Bifidobacterium animalis subsp. lactis BB12 (BB12) with no increase in IL-10 after six hours of stimulation. In this work, we performed an in silico prediction of genes targeted by miR-671-5p and which are the terms and pathways involved with it. Also, miR-671-5p was transiently downregulated to assess its effect on IL-10 regulation. METHODS AND RESULTS: First, we performed a Gene Ontology enrichment analysis to predict immune response terms and pathways involved with miR-671-5p. Some of the terms and pathways found were related to the immune response promoted by the probiotic, as the terms "negative regulation of the inflammatory response to an antigenic stimulus" and "cancer" were highlighted. Then, to assess the role of miR-671-5p in IL-10 regulation, moDCs were derived from porcine peripheral blood and later transfected with miR-671-5p antisense oligonucleotide (ASO). Flow cytometry was employed to evaluate the transfection efficiency. Then, the moDCs were stimulated with BB12, and the expression of IL-10 was assessed by RT-qPCR and ELISA. An increase in IL-10 transcript in miR-671-5p-ASO-transfected moDCs stimulated with BB12 was observed compared with moDCs stimulated with BB12 but not transfected. These results suggest the participation of miR-671-5p as a negative regulator of IL-10. CONCLUSION: These findings suggest that miR-671-5p participates in the downregulation of IL-10, as previously predicted in silico by our work group. miR-671-5p could play an essential role in the immunomodulation promoted by the probiotic BB12.
Assuntos
MicroRNAs , Probióticos , Suínos , Animais , Interleucina-10/genética , Interleucina-10/metabolismo , Regulação para Baixo/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Monócitos/metabolismo , RNA Mensageiro/metabolismo , Probióticos/farmacologiaRESUMO
MicroRNAs (miRNAs) mediate the regulation of gene expression. Several reports indicate that probiotics induce miRNA-mediated immunomodulation at different levels, such as cytokine production and the up-regulation of several markers related to antigen presentation in antigen-presenting cells. The objective of this work was to identify target genes of miRNAs that are involved in the processing and presentation of antigens in monocyte-derived dendritic cells (moDCs) stimulated with the probiotic Bifidobacterium animalis ssp. lactis BB12 (BB12). First, an in silico prediction analysis for a putative miRNA binding site within a given mRNA target was performed using RNAHybrid software with mature sequences of differentially expressed miRNAs retrieved from a Genbank data set that included BB12-stimulated and unstimulated porcine monocytes. From them, 23 genes resulted in targets of 19 miRNAs, highlighting miR-30b-3p, miR-671-5p, and miR-9858-5p, whose targets were costimulatory molecules, and were overexpressed (p < 0.05) in BB12-stimulated moDCs. The analysis of moDCs showed that the percentage of cells expressing SLA-DR+CD80+ decreased significantly (p = 0.0081) in BB12-stimulated moDCs; interleukin (IL)-10 production was unchanged at 6 h but increased after 24 h of culture in the presence of BB12 (p < 0.001). In summary, our results suggest that SLA-DR and CD80 can be down-regulated by miRNAs miR-30b-3p, miR-671-5p, and miR-9858-5p, while miR-671-5p targets IL-10.
Assuntos
Apresentação de Antígeno/imunologia , Células Dendríticas/imunologia , MicroRNAs/efeitos dos fármacos , MicroRNAs/metabolismo , Monócitos/metabolismo , Probióticos/farmacologia , Animais , Antígeno B7-1 , Bifidobacterium animalis/fisiologia , Citocinas/metabolismo , Regulação para Baixo , Perfilação da Expressão Gênica , Imunomodulação , Interleucina-10/metabolismo , MicroRNAs/genética , RNA Mensageiro/metabolismo , Suínos , Regulação para CimaRESUMO
Human coronavirus (HCoV)-NL63 is an important contributor to upper and lower respiratory tract infections, mainly in children, while severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, can cause lower respiratory tract infections, and more severe, respiratory and systemic disease, which leads to fatal consequences in many cases. Using microscopy, immunohistochemistry (IHC), virus-binding assay, reverse transcriptase qPCR (RT-qPCR) assay, and flow cytometry, we compared the characteristics of the susceptibility, replication dynamics, and morphogenesis of HCoV-NL63 and SARS-CoV-2 in monolayer cultures of primary human respiratory epithelial cells (HRECs). Less than 10% HRECs expressed ACE2, and SARS-CoV-2 seemed much more efficient than HCoV-NL63 at infecting the very small proportion of HRECs expressing the ACE2 receptors. Furthermore, SARS-CoV-2 replicated more efficiently than HCoV-NL63 in HREC, which correlates with the cumulative evidence of the differences in their transmissibility.
Assuntos
Coronavirus Humano NL63 , Células Epiteliais , SARS-CoV-2 , Humanos , Enzima de Conversão de Angiotensina 2 , Linhagem Celular , Coronavirus Humano NL63/patogenicidade , COVID-19 , Células Epiteliais/virologia , Infecções Respiratórias , SARS-CoV-2/patogenicidadeRESUMO
The SARS-CoV-2 virus was detected for the first time in December 2019 in Wuhan, China. Currently, this virus has spread around the world, and new variants have emerged. This new pandemic virus provoked the rapid development of diagnostic tools, therapies and vaccines to control this new disease called COVID-19. Antibody detection by ELISA has been broadly used to recognize the number of persons infected with this virus or to evaluate the response of vaccinated individuals. As the pandemic spread, new questions arose, such as the prevalence of antibodies after natural infection and the response induced by the different vaccines. In Mexico, as in other countries, mRNA and viral-vectored vaccines have been widely used among the population. In this work, we developed an indirect ELISA test to evaluate S1 antibodies in convalescent and vaccinated individuals. By using this test, we showed that IgG antibodies against the S1 protein of SARS-CoV-2 were detected up to 42 weeks after the onset of the symptoms, in contrast to IgA and IgM, which decreased 14 weeks after the onset of symptoms. The evaluation of the antibody response in individuals vaccinated with Pfizer-BioNTech and CanSinoBio vaccines showed no differences 2 weeks after vaccination. However, after completing the two doses of Pfizer-BioNTech and the one dose of CanSinoBio, a significantly higher response of IgG antibodies was observed in persons vaccinated with Pfizer-BioNTech than in those vaccinated with CanSinoBio. In conclusion, these results confirm that after natural infection with SARS-CoV-2, it is possible to detect antibodies for up to 10 months. Additionally, our results showed that one dose of the CanSinoBio vaccine induces a lower response of IgG antibodies than that induced by the complete scheme of the Pfizer-BioNTech vaccine.