Immunological response of epididymis of K18-hACE2 transgenic mice infected with SARS-CoV-2, with emphasis on the clear cell

Abstract

The k18-hACE2 transgenic mice that express human ACE2, an essential protein for the infection of host cell by SARS-CoV-2, has been used to investigate the impact of the viral infection. The epididymis, an essential organ for male fertility, is responsible for spermatozoa maturation and protection of these cells against pathogens. The epithelial and stromal cells exert immunological responses, producing cytokines such as TNF-±, IL-1 e IL-6. Covid-19 induces the release of several cytokines, and male patients with Covid-19 have demonstrated hormonal and testicular disorders. Thus, we intend to localize the virus in the epididymal tissues of SARS-CoV-2-infected K18-hACE2 mice and to evaluate the morphofunctional integrity of this organ, focusing on the clear cells in an attempt to verify a possible immunological response of this cell to the viral infection. Twenty k18-hACE2 mice will be divided into two groups (n=10 each): control (CG) and 5-days post infection (5DG) mice. The G5D animals will be inoculated with the virus intranasally. The CG animals will be inoculated with DMEM culture medium. The epididymis will be processed and embedded in paraffin or Araldite (for transmission electron microscopy - TEM). The paraffin sections will be submitted to the immunohistochemistry or immunofluorescence reactions for detection of CD11c, F4/80 and CD8+ in an attempt to identify dendritic cells, macrophages and lymphocytes, respectively. The expression of estrogen receptors and the epithelial immunoexpression of testosterone and apoptotic proteins (Bax e Bcl2) will also be evaluate. Double immunoreactions for detection of NFk-², TLR-3 and TNF-± in the clear cells will also be performed using VATPase as clear cell marker. With the aim to verify possible inflammatory pathways in the epididymis to the viral infection, the expression of Cxcl 10, Cx3cl 1, INF-³, RIG-1,2, TLR-5, STAT 1 and 3, JAK 1 and 2, and the viral load will be evaluated by qPCR. The results will be statistically analyzed by Student`s-t test (pd0.05). With possible BEPE fellowship, we also intend to perform an mRNA screening of clear cells under viral infection in the laboratory of Dr. M.A. Battistone at Harvard Medical School (Boston, EUA).