Funktion der Protein Kinase D1 bei der Regulation der endothelialen Permeabilität nach stumpfem Throraxtrauma.
Zusammenfassung der Projektergebnisse
Major physical trauma triggers the release of damage- and pathogen-associated molecular patterns, cytokine release and an innate immune response by neutrophils that infiltrate the injury site. On one hand, neutrophil triggered immunity is important to protect the host from infection and foster healing, but if this trauma response turns unbalanced, systemic inflammation and endothelial barrier disruption can cause severe complications, such as multiple organ dysfunction syndrome (MODS). In our previous work, we have shown that Protein Kinase D (PKD) regulates neutrophil directional cell motility as well as cellular deformability to impact on extravasation and trans-endothelial passage of neutrophils from blood vessels towards an injury. Further investigation suggested that PKD can even affect post-traumatic inflammation at multiple levels and may be a good starting point to pharmacologically target an exaggerated immune response. In this grant proposal, we have therefore explored the role of PKD in the regulation of neutrophilendothelial interactions, endothelial barrier stability as well as in the generation of molecular gradients that facilitate neutrophil swarming behavior towards injuries. We have identified underlying molecular mechanisms and were able to show that PKD kinase activity does not only regulate neutrophil cell motility via actin cytoskeleton associated mechanisms, but also endothelial barrier stability in a complementary manner. Moreover, we have elucidated that PKD activity impacts on neutrophil adhesion at the endothelium and generation of molecular gradients that facilitate neutrophil swarming as well as activation. These effects are mediated by a complex crosstalk with the endothelium involving cytokines and the release of small extracellular vesicles (sEVs, exosomes) as instruments of intercellular communication. As part of our research in this topic, we have demonstrated that endothelial-derived post-traumatic sEVs are part of a hitherto unrecognized pathophysiological mechanisms that propagates inflammation after severe trauma in a neutrophil-dependent manner. We demonstrate that this effect is caused by different types of severe trauma, such as blunt-chest trauma (TxT) in mice, hemorrhagic shock in a swine, but also sEVs isolated after polytrauma from patients. We have demonstrated that a traumatic insult promotes a substantial increase in sEVs release with altered pro-inflammatory cargo. The pharmacological inhibition of sEV-secretion after TxT in mice significantly decreased overall plasma-sEV concentrations, reversed trauma-associated molecular signatures in lungs at the transcriptome and proteome level, significantly impaired neutrophil infiltration, systemic inflammation as well as endothelial barrier destabilization. Most importantly, blocking sEV secretion also prevented secondary organ damage in response to TxT. Mechanistically, we show in vitro that trauma-induced endothelial-derived sEVs propagate inflammation by systemically transferring mRNAs coding for endothelial adhesion molecules as well as pro-inflammatory cytokines (e.g., IL6, IL8) to other endothelial cells. In turn, this amplifies Interactions between neutrophils and the endothelium, driving both local, but also systemic inflammation. Moreover, disruption of the endothelial barrier by sEVs in vitro and in vivo was accomplished by destabilizing VE-cadherin-mediated cell-cell contacts via downregulation of CTNND1 (p120-catenin) through miRNA-298. We also demonstrate the clinical relevance of our findings for patients, as we were able to detect increased sEVs concentrations in the blood plasma of polytrauma patients and endothelial reprogramming by patient-derived sEVs, which induce expression of the same molecular targets identified in vitro and in TxT mice. Thus, research conducted as part of this grant proposal has characterized novel starting points that may be exploited in future translational efforts to ameliorate unbalanced post-traumatic inflammation.
Projektbezogene Publikationen (Auswahl)
- (2018) Thirty-eightnegative kinase 1 mediates trauma-induced intestinal injury and multi-organ failure. J Clin Invest 128, 5056-5072
Armacki, M., Trugenberger, A. K., Ellwanger, A. K., Eiseler, T., Schwerdt, C., Bettac, L., Langgartner, D., Azoltei, N., Halbgebauer, R., Groß, R.. Barth. T., Lechel. A., Walter, B. M., Kraus, J. M., Wiegreffe, C., Grimm, J., Scheffold, A., Schneider, M. R., Peuker, K., Zeißig, S., Britsch, S., Rose-John, S., VettorazzI, S.. Wolf. E.. Tannapfel, A., Steinestel, K., Reber, S. O., Walther, P., Kestler, H. A., Radermacher, P., Barth, T. F., Huber-Lang, M., Kleger, A., and Seufferlein, T.
(Siehe online unter https://doi.org/10.1172/jci97912) - (2021) SARS-CoV-2 infects and replicates in cells of the human endocrine and exocrine pancreas. Nat Metab 3,149-165
Müller, J. A., Groß, R., Conzelmann, C., Kruger, J., Merle, U., Steinhart, J., VVeil, T., Koepke, L, Bozzo, C. P., Read, C., Fois, G., Eiseler, T., Gehrmann, J., van Vuuren, J., Wessbecher, I. M., Frick, M., Costa, I. G., Breunig, M., Grüner, B., Peters, L., Schuster, M., Uebau, S., Seufferlein, T., Stenger, S., Stenzinger, A., MacDonald, P. E., KIrchhoff, F., Sparrer, K. M. J.. Walther, P., Uckert, H., Barth, T. F. E.. Wagner, M., Münch, J., Heller, S., and Kläger, A.
(Siehe online unter https://doi.org/10.1038/s42255-021-00347-1) - (2021) Small Extracellular Vesicles Propagate the Inflammatory Response After Trauma. Adv Sci (Weinh) 8, 62102381
Selbold, T., Schönfelder, J., Weeber, F., Lechel, A.. Armacki, M., Waldenmaier, M., Wille, C., Palmer, A., Halbgebauer, R., Karasu, E., Huber-Lang, M., Kalbitz, M., Radermacher, P., Paschke, S., Seufferlein, T. and Eiseler, T.
(Siehe online unter https://doi.org/10.1002/advs.202102381)
