The mammalian sterile 20-like kinase 1 (MST1) is a critical regulator of neutrophil transmigration during inflammation
Anatomy and Physiology
Final Report Abstract
The results of the presented work expand our understanding of the underlying mechanisms by which endothelial cell interactions ‘educate’ neutrophils to penetrate the vascular basement membrane. So far, no signaling molecules had been associated with this step. In this study, the serine/threonine kinase MST1 (also known as STK4) was identified as the first signaling molecule indispensable for neutrophil BM penetration into inflamed tissue. However, the exact role of the laminin-binding integrins α3β1 integrin (VLA3), α6β1 integrin (VLA6) and the protease neutrophil elastase (NE) on neutrophils involved in penetration of the BM is still unclear. Since the vascular basement membrane is essential for maintaining vascular integrity, significant degradation of the basement membrane is unlikely. Enormous numbers of neutrophils penetrate the BM, but leave the BM morphologically intact. One of the hypothesis is that during transmigration, endothelial cell traction forces and/or surfaceassociated enzymatic activity might promote local reversible disassembly of the BM network and consequently increase local pore size to permit neutrophil transmigration. Using an NE-fluorescent activatable substrate (NE680FAST) we could reveal mild activity of NE on the surface of neutrophils interacting with the luminal site of venular endothelial cells in TNF-α stimulated cremaster muscles. This activity of NE was decreased in Mst1-/- mice, although the number of rolling and adherent neutrophils within postcapillary venules was similar compared to controls. Interestingly, we also found that the distribution and translocation of Rab27a was disrupted in Mst1-/- neutrophils. Rab27a is responsible for the mobilization of neutrophil vesicles, containing VLA-3, VLA-6 and NE. Rab GTPases are controlled by Rab effector proteins. So far, two Rab27a effector proteins, JFC1 and Munc13-4, could be associated with impaired release of granules in neutrophils. We found that MST1 mostly acts through modulating the function of JFC1, rather than Munc13-4, to regulate translocation of VLA-3, VLA-6 and NE. Taken together, we demonstrate that the serine/threonine-kinase MST1 is indispensable for neutrophil penetration of the basement membrane by regulating the mobilization of VLA-3, VLA-6 and NE from intracellular vesicles to the plasma membrane. These findings emphasize the importance of MST1 for innate immune responses and help to understand the clinical presentation of patients suffering from MST1 deficiency. As we had finished the core project earlier than anticipated, we expanded our work on MST1 in regulating neutrophil homeostasis. Key factor in regulating neutrophil homeostasis is the granulocyte colony-stimulating factor (G-CSF), which interacts with the G-CSF receptor leading to activation of the JAK-Stat signaling pathway. This includes the regulation of chemokine receptors CXCR2 and CXCR4 expression, which is critical for neutrophil trafficking between bone marrow and the vasculature. We found that i.v. injection of G-CSF failed to induce an increase of circulating neutrophils in the absence of MST1. Furthermore, we demonstrate by in vivo imaging defective mobility and intravasation of bone marrow resident neutrophils in Mst1-/- mice. We further provide evidence that this is potentially caused by impaired G-CSF signaling with diminished phosphorylation of Stat3 leading to impaired upregulation of CXCR2 expression on neutrophils, a necessary step for neutrophil mobilization into the blood circulation. Taken together, our recent findings show a critical role of MST1 in neutrophil homeostasis by regulating G-CSF receptor signaling facilitating mobilization and release of neutrophils into the circulation, which fulfils an important task in neutrophil homeostasis and innate immune defence.
Publications
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(2016). MST1-dependent vesicle trafficking regulates neutrophil transmigration through the vascular basement membrane. J Clin Invest 126, 4125-4139
Kurz, A.R., Pruenster, M., Rohwedder, I., Ramadass, M., Schafer, K., Harrison, U., Gouveia, G., Nussbaum, C., Immler, R., Wiessner, J.R., Margraf, A., Lim, D.S., Walzog, B., Dietzel, S., Moser, M., Klein, C., Vestweber, D., Haas, R., Catz, S.D., and Sperandio, M.
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(2018). Noncanonical Hippo Signalling in the Regulation of Leukocyte Function. Trends Immunol 39, 656-669
Kurz, A.R.M., Catz, S.D., and Sperandio, M.
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(2020). Neutrophil recruitment and intracellular vesicle transport: a short overview. Eur J Clin Invest 50, e13237
Masgrau-Alsina, S., Sperandio, M., and Rohwedder, I.
