Avaliação in vitro da resposta inflamatória e metabolismo lipídico de macrófagos em animais taurinos e zebuínos estimulados com saliva do carrapto Rhipicephalus microplus

Abstract

The parasitism caused by Rhipicephalus microplus tick generates large losses in meat and milk production by cattle, as well as representing a unique health hazard due to the transmission of infectious diseases and the contamination of food and environment caused by acaricides, the main methodology adopted by producers for this hematophagous ectoparasite control. The phenotypic features of resistance and susceptibility to R. microplus are well defined between the zebu (Bos primigenius indicus) and taurine (Bos primigenius taurus) cattle breeds respectively, constituting a multifactorial genetic inheritance of variable heritability. Several papers have already identified the role of innate immunity and macrophage response against ticks. Despite this, little is known about the different phenotypes of macrophages M1 (proinflammatory activity, Th1 inducer) and M2 (regulatory activity, Th2 inducer), and its influences on tick resistance presented by taurine and zebu cattle. In this context, the present work aims to characterize the metabolic pathways involved in the activation of taurine and zebu macrophages to identify potential targets for metabolic reprogramming by immunomodulation and candidate genes for bovine genetic improvement programs. For this purpose, a macrophage differentiation protocol was established from in vitro isolation of peripheral blood monocytes of taurine (Holstein) and zebu (Gir) animals. The cells were treated with lipopolysaccharide (LPS), a potent inducer of M1 macrophages, and R. microplus tick saliva, which under parasitism conditions, is inoculated into the host to modulate the immune response and facilitate hematophagy. The RNA of differentiated macrophages were extracted and submitted to high throughput sequencing (RNA-Seq) for the identification of differentially expressed genes (DEGs). Gene ontology and metabolic pathways analysis showed that zebu non-stimulating macrophages have a greater activation of genes linked to the antigen processing and presentation, the activation of the NFκB transcription factor and the indirect control of the cell cycle through lipid metabolism. This macrophages implication, with more proinflammatory responses for zebu animals, seems to directly influence the M1 phenotype activation via LPS with a greater expression of genes related with nitric oxide synthesis, cytokines, such as IL-1 and IL-36, besides the NFκB2 factor, responsible in controlling the pro-inflammatory molecules transcription. The saliva of R. microplus tick induced significant differences in mitotic activation pathways and membrane potential regulated by oxidative stress. In addition, saliva demonstrated the ability in modulating the expression of genes encoding the nitric oxide synthase 2 enzyme (NOS2), the negative regulator of reactive oxygen species (NRROS), and the IL-10 cytokine in LPS-stimulated macrophages, evaluated by real time PCR. Taken together, these results, allowed us to identify candidate genes for tick resistance in breed genetic programs, as well as key biological process, which can be modulated for development of more effective strategies and less polluting tick control.