Targeting metalloproteinases ADAM10 and ADAM17 : Effects on ADAM protein expression and antibacterial defense

• Targeting der Metalloproteinasen ADAM10 und ADAM17: Effekte auf die ADAM Proteinexpression und die antibakterielle Abwehr

Seifert, Anke Carmen; Ludwig, Andreas (Thesis advisor); Elling, Lothar (Thesis advisor)

Aachen : RWTH Aachen University (2021)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2021

Abstract

By shedding a large number of proteins including growth factors, receptors and mediators of inflammation such as TNFα, the metalloproteinases ADAM10 and ADAM17 critically contribute to development, cancer and inflammation. Several in vitro and in vivo studies showed that ADAM10 and ADAM17 play cell-specific roles in acute inflammatory processes. Therefore, targeting of these proteases is considered as a therapeutic approach. However, by the shedding of Toll-like receptors and scavenger receptors, ADAM10 and ADAM17 can potentially influence the innate immune defense against microorganisms. This necessitates to fully understand the regulation of ADAM10 and ADAM17 and to be aware of all possible consequences of the specific inhibition approaches. It is known, that the small molecule inhibitors GI254023X and TAPI-1 rapidly and efficiently inhibit the activity of ADAM10 or ADAM17/ADAM10, respectively. However, the long-term treatment effects on the proteases and their effect on bacterial phagocytosis representing a crucial step in the innate immune defense remain unknown. Hence, the first aim of the present study was to investigate the outcome of long-term treatment with inhibitors on the expression of ADAM10 and ADAM17 in vitro and in vivo. The second aim was to evaluate the effects of the proteases on bacterial phagocytosis by cultured murine and human phagocytes. In the first part of this thesis, it could be demonstrated that loss of proteolytic activity of ADAM10 by either inhibition or loss of function mutations induces removal of the protease from the cell surface and also from intracellular pools. This process was temperature-dependent, restricted to mature ADAM10 and associated with an increased internalization, lysosomal degradation and release of mature ADAM10 in extracellular vesicles. Recovery from this depletion required de novo synthesis. Functionally, this was reflected by loss and recovery of ADAM10 substrate shedding. Moreover, ADAM10 inhibition in mice reduced systemic ADAM10 levels in different tissues. Thus, ADAM10 activity is critically required for its surface expression in vitro and in vivo. In the second part of this study, multiple lines of evidence were provided that physiologic expression of ADAM17 in phagocytes attenuates bacterial uptake. Inhibition of metalloproteinase activity of ADAM17 led to increased phagocytosis of pHrodo-labelled Gram-negative and -positive bacteria (E. coli and S. aureus, respectively) by human and murine monocytic cell lines and primary phagocytes. Bone marrow-derived macrophages showed enhanced uptake of heat-inactivated and living E. coli when they lacked either ADAM17 or its adapter protein iRhom2 that is required for ADAM17 maturation, but not when they were ADAM10-deficient. In monocytic THP-1 cells, corresponding shRNA-mediated knockdown confirmed that reduction of ADAM17 but not of ADAM10 expression promoted phagocytosis of E. coli. The augmented bacterial uptake occurred in a cell autonomous manner and was accompanied by an increased release of the chemokine CXCL8. Inhibition experiments indicated that the enhanced bacterial phagocytosis after ADAM17 knockdown was partially dependent on TNFα activity. Thus, for further development of inhibition strategies for ADAM proteases it needs to be considered that ADAM10 inhibition will lead to long-term suppression of ADAM10 surface expression and that ADAM17 inhibition will promote bacterial phagocytosis. This work may stimulate further studies investigating whether the observed surface regulation also applies to other proteases than ADAM10 and whether ADAM17 inhibition would improve bacterial phagocytosis in vivo.

Institutions

• Department of Biology [160000]
• Chair of Biotechnology [162610]
• [528500-2]