Análise em alta dimensão do desenvolvimento imune esplênico pós-natal utilizando uma nova técnica de microscopia confocal 7 canais

Abstract

The spleen is a unique secondary lymphoid organ that presents many important functions in immunity and blood homeostasis. Splenic architecture and its multicellular composition allow for blood surveillance and facilitate interactions between antigen-presenting cells (APCs) and lymphocytes, integrating innate and adaptive immune responses in an organized way. The spleen hosts all subtypes of leukocytes, including myeloid and lymphoid cells. These cells are key protectors of the organism because they identify blood borne pathogens and cellular stress, remove dying cells and foreign material, regulate tissue homeostasis and inflammatory responses, and shape adaptive immunity during postnatal development. Additionally, they can be key players in different parasitic diseases, such as bacterial and protozoan infections. However, how spleen leukocytes evolve across the developmental phase, and how they spatially organize and interact in vivo is still poorly understood. Using a novel and unique combination of high dimensional intravital microscopy, here we revealed how the splenic immune system evolves during life, describing the main immunologic changes during postnatal development. We observed a significant increase in the number of total leukocytes in the spleen between the 1st and 8th weeks of life; more specifically a 6-fold magnification. We also found that the B cell population is characterized as the largest immune cell population in the splenic environment at all ages studied, remaining proportionally stable throughout life. The T cell population, in turn, shows a significant increase in its frequency. Furthermore, we detected that the cellular composition and distribution of splenic leukocytes is quite different in neonates, and quite similar between infants and adults. Also, we exhibited how infections – using a model of malaria - might change the spleen immune profile in adults and infants, which could become the key to understanding different severity grades of infection. Therefore, we laid the foundations for future studies aiming to image cells under their native environment, developing not only a novel panel of seven different fluorophores that works in vivo, but also all the paths to use conventional confocal microscopes to work as multi- channel imaging platforms. Our new imaging solutions can be extremely useful for different groups in all areas of biological investigation, paving the way for new intravital approaches and advances.

Keywords: spleen, postnatal splenic development, immune cells, malaria, confocal microscopy.