Identifying Perivascular Macrophages as Crucial Mediators of Leukocyte Homing in Sterile Inflammation

Background: Numerous studies have elucidated key molecular mechanisms underlying leukocyte recruitment; however, little is known about the coordination between infiltrating leukocytes and perivascular immune cells during sterile inflammation. As perivascular macrophages (PVM) have been implicated in the pathogenesis of various sterile inflammatory diseases, it is noteworthy to explore their function in this setting. The recruitment of cytotoxic T lymphocytes (CTLs) has been implicated in shaping the immune milieu within various tissues. Although the Arp2/3 complex has been shown to be important for remodelling of the actin cytoskeleton during T cell migration, the role of Arp3, a subunit of Arp2/3, has not been extensively explored. Aim: The aim of this project is to evaluate the transmigration of leukocytes in context to PVM post-sterile inflammation. Moreover, we aim to investigate the effect of Arp3 deficiency on T cell migration and effector function. Methods: Intravital multiphoton microscopy was employed to visualise the homing behaviour of neutrophils and monocytes in post-capillary venules within the cremaster tissue post-inflammation. To assess the impact of Arp3 reduction on the morphology and migration mode of CTLs, Arp3-knockdown (Arp3-KD) T cells were imaged in vitro via confocal microscopy. Results: Flow cytometric analysis of the mice cremaster tissue revealed that TNF- induced inflammation resulted in a large influx of neutrophils at 4 hr and monocytes at 7 hr post- intrascrotal administration of the cytokine. Intravital imaging of the homing of these cells highlighted that neutrophils and monocytes predominantly extravasate adjacent to PVM. The majority of PVM in the cremaster tissue coincide with low expression regions (LERs), which are postulated to act as repositories for neutrophil- and monocyte-attracting chemokines. Reduction in Arp3 levels induced blebbing motility in CTLs and altered their homing pattern in peripheral tissues. Arp3-KD T cells also exhibited poor survival and impaired cytotoxicity. Conclusion: Our findings highlight PVM as mediators of leukocyte homing in sterile inflammation. Future studies to delineate potential mechanisms driving PVM-mediated recruitment could unveil novel therapeutic strategies for sterile inflammatory disorders