Analysis of single-cell gene expression of human lymphoid tissues infected by pandemic respiratory viruses: SARS-CoV-2 and Influenza

• Grant number: 22/08111-5
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: September 01, 2022
• End date: August 31, 2024
• Field of knowledge: Health Sciences - Medicine
• Principal Investigator: Eurico de Arruda Neto
• Grantee: Nilton Nascimento dos Santos Júnior
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated research grant: 19/26119-0 - Emerging and re-emerging viruses: biology, pathogenesis and prospection, AP.TEM

Abstract

Hypertrophy of adenoids and palatine tonsils is a highly prevalent chronic inflammatory condition of unknown etiology, with a strong impact on children's health, usually leading to surgery. As sentinels of the upper airways, the tonsils play important roles in the immune response to Respiratory Viruses (RVs), which are the most frequent causes of Acute Respiratory Infections (ARI). In previous studies, we reported a very high frequency (97%) of RVs in hypertrophic tonsils with chronic inflammation and respiratory secretions of children without AKI symptoms, with no apparent seasonality. Interestingly, flow cytometry and immunohistochemistry revealed that lymphomononuclear cells (LMNCs) can harbor various species and types of RVs. Together, these data indicate that there is long-term asymptomatic infection by respiratory viruses in LMNCs. To infect LMNCs in chronically inflamed tonsils, viruses must establish complex pathogen-host interactions to bypass immune and antiviral responses. Given the multiplicity of factors that can affect the establishment and maintenance of these infections, the application of single-cell analysis methods will be necessary to fully characterize this complex interaction between pathogen and host. This systematic approach can reveal how the viral community may be cooperating or interfering with host cell gene expression. Our objective is to carry out sequencing of LMNCs based on single-cell RNA and DNA of the main immunotypes (CD4+ and CD8+ T lymphocytes, B lymphocytes, macrophages, and dendritic cells), obtained from hypertrophic palatine tonsils and adenoids of children with tonsillar hypertrophy and of post-mortem tissues from adults. Sequence analysis will allow the segregation of SARS-CoV-2 and influenza infected cells from uninfected cells of the same immunophenotype and, therefore, will allow comparisons between them about various aspects, such as expression of antiviral and inflammatory genes, non-coders of different classes, and their epigenetic profiles. In addition, infected LMNCs will be cultured and evaluated for viability, and ability to divide in response to mitogens. Naturally uninfected MLNCs will be infected ex vivo with virus stocks to verify their permissiveness to infection. Human primary LMNCs and continuous cell lines will also be used for in vitro infection to validate candidate genes and selected pathways in single-cell analyses. For the present study, tonsils from children undergoing tonsillectomy will be used to treat obstructive complications related to tonsillar hypertrophy, and post-mortem tissues from adults will be used. LMNCs will be purified from newly dissociated lymphoid tissues by negative selection based on magnetic beads to minimize cellular activation during processing. In addition to tissue dissociation for single-cell analysis, tissue fragments will be preserved in RNAlater® for nucleic acid extraction and real-time PCR virus detection testing. Backup aliquots of dissociated tissues will also be kept for further testing or validation assays. (AU)