@phdthesis{Borkute2023,
  author    = {Rachana Borkute},
  title     = {Responses of bovine and human neutrophils to members of the Mycobacterium tuberculosis complex},
  journal    = {Reaktionen von Rinder- und menschlichen Neutrophilen auf Mitglieder des Mycobacterium tuberculosis-Komplexes},
  url       = {https://nbn-resolving.org/urn:nbn:de:gbv:9-opus-78766},
  pages     = {160},
  year      = {2023},
  abstract  = {PMN are one of the most important cells of the innate immune system and are responsible for fast clearance of invading pathogens in most circumstances. The role of human PMN during mycobacterial infection have been widely studied. Nevertheless, there are contradicting results regarding their role in protection or pathology during TB. Similar studies focusing on bovine PMN and their role in M. bovis infection remain understudied. Also, not much is known about attenuation of M. tb in cattle and responses of PMN to this MTBC member. The major aims of this study were to i) gain insights into bovine PMN biology and the cellular processes triggered by challenge with virulent mycobacteria and to ii) find out whether interspecies differences result in different outcomes upon in vitro challenge. In the first part of the work, a new isolation method for bovine PMN from whole blood was developed. Human and bovine PMN have different buoyant properties and hence need to be isolated using different procedures. The magnetic isolation method developed within this thesis is robust and results in very good yields of highly pure, viable bovine PMN populations. This is extremely advantageous and indispensable for downstream functional assays that are required to be performed on a single day. The second goal of this study was to compare and contrast the functional differences between bovine and human PMN upon BCG infection. The findings reveal for the first time that human PMN phagocytose more BCG in comparison to bovine counterparts. Non-opsonized bacteria were internalized via the lectin-like C-domain, require cholesterol and an active cytoskeleton in human PMN, whereas opsonized bacteria entered cells via the CR3 and, in particular, CD11b. It remains unresolved why bovine PMN reacted differently, notably phagocytosis remained unaltered, to various treatments, including blocking monoclonal antibodies to CD11b and chemical inhibitors altering the cell membrane. Nonetheless, the increased uptake of BCG by human PMN correlates to more potent response of these cells in functional assays in comparison to bovine PMN. No PMN intrinsic differences were found in the basal cholesterol content. Comparative assays with the virulent strains would be essential in order to generalize these observations. The third aim was to investigate the responses of bovine PMN to BCG, M. tb and M. bovis. While there was no difference in uptake between BCG and M. tb, serum opsonized BCG was taken up at a higher amount. This finding suggests differential binding of bacterial epitopes to host cell receptors which modulates mycobacteria uptake. However, between the virulent strains M. tb and M. bovis, the human-adapted bacillus was phagocytosed at a higher rate which hints towards the possibility of rapid recognition and clearance of M. tb in bovine host thereby possibly preventing pathology. The release of selective cytokines by PMN post infection with the virulent strains offers baseline information relevant for processes that probably occur in vivo. This work for the first time provides insights into responses of bovine PMN to mycobacteria in a two-tier approach: by cross-species analysis of PMN responses to selected mycobacterium and by head-to-head analysis of bovine PMN to animal-adapted and human-adapted mycobacteria. As a prospect for future research in bovine PMN biology in the context of mycobacterial infection, it would be highly advantageous to compare the subcellular localization of M. tb and M. bovis in bovine PMN using confocal and/or electron microscopy. This analysis would confer proof on attachment or internalization of mycobacteria by PMN and identify the features of the mycobacteria-containing compartments. Also, in-depth investigations of additional entry pathways for the pathogen in bovine cells would be informative for unlocking downstream cell signaling events. In addition, PMN viability studies will be meaningful particularly in bovine PMN challenged with M. bovis and M. tb, given the impact of death patterns on tissue pathology. Current results and follow up studies will contribute to the understanding of the roles of PMN in controlling elimination or growth of M. bovis and M. tb in cattle.},
  language  = {en}
}