Nuclear architecture of resting and LPS-stimulated porcine neutrophils by 3D FISH

Neutrophils are essential components of the innate immune system due to their ability to kill and phagocytose invading microbes. They possess a lobulated nucleus and are capable of extensive and complex changes in response to bacterial stimulation. The aim of our study was to investigate whether the 3D nuclear organization of porcine neutrophils was modified upon stimulation. The organization of centromeres, telomeres, and chromosome territories (chromosomes 2, 3, 7, 8, 12, 13, and 17) was studied on structurally preserved nuclei using 3D fluorescence in situ hybridization, confocal microscopy, and image analysis. By differential labeling of centromeres of acrocentric and metacentric/submetacentric chromosomes, we showed that centromeres associated to form chromocenters but did so preferentially between chromosomes with the same morphology. Upon activation, some of these chromocenters dispersed. Telomeres were also found to form clusters, but their number remained unchanged in lipopolysaccharide-stimulated neutrophils. The analysis of the position of chromocenters and telomere clusters showed a more internal location of the latter compared to the former. The analysis of chromosome territories revealed that homologs were distributed randomly among lobes whatever the cell's status. The volume of these territories was not proportional to chromosome length, and some chromosomes (chr 3, 12, 13, and 17) were more prone to decondensation when neutrophils were stimulated. Thus, our study demonstrated that activation of neutrophils resulted in several modifications of their nuclear architecture: a decrease in the number of non-acrocentric chromocenters and the decondensation of several chromosomes.