Estabelecimento de metodologia de isolamento e cultivo primário de hepatócitos bovinos para estudos do efeito modulador da saliva do carrapato Rhipicephalus microplus sobre expressão de genes relacionados à imunidade inata

Abstract

Innate immunity is the body's first line of defense against injury and invasion of microorganisms. The complement system plays a key role in mediating acquired and innate responses in defense against microbial and parasitic infections. In cattle, the response against tick infestations leads the innate immunity cells in the injured tissue to produce pro-inflammatory cytokines, signaling the onset of a more specific response, such as the production of acute-phase proteins (APPs). Approximately 80 to 90% of the plasma components of complement and APPs are produced in the liver and released systemically for the control of inflammatory reactions. There are well-established methods of isolation and culture of hepatocytes for in vitro studies, as well as commercial lines for human and murine, however for bovines there are no well-established methodologies for isolation of hepatocytes or commercial cell lines available for cultivation of this subtype. The bovine tick, Rhipicephalus microplus, is a parasite that causes great economic losses for the world livestock. It is known that bovine breeds differ in tick resistance, with the Bos indicus breeds being more resistant compared to the Bos taurus breeds. Since there are differences in the production of complement and APP molecules among resistant and susceptible ticks, this work aimed to establish a methodology for the primary isolation and in vitro culture of bovine hepatocytes to verify the modulatory effect induced by the R. microplus tick saliva on the expression of genes related to innate immunity, such as complement factors (C1, C3, C4, C5, mannose binding lectin) and APPs (Alpha 1 acid glicoprotein, haptoglobin e serum amyloid A). For this purpose, several protocols of isolation and primary culture of hepatocytes were evaluated. We were able to establish the methodology with high cellular viability, morphological features and quality of the genetic material. Gene expression analysis suggested that hepatocytes cultured in vitro might be losing the physiological features, since the expression of some genes was not detected in cultured cells. Although the stimuli used as a positive control have upregulated the expression of the SAA encoding gene, the same behavior has not been observed in complement genes regulated by the same signaling pathway in murine model. In addition, no significant difference was observed between breeds in the expression of the evaluated genes against the stimuli of tick saliva and nuclear receptor modulating agent related to the hepatic control of murine inflammation. Our results suggest that the primary isolation and in vitro culture of bovine hepatocytes might be causing loss of function of these cells, and further investigation is needed to better understand the physiological processes performed by hepatocytes.