A Disintegrin and Metalloproteinase : Control Elements in Infectious Diseases

Despite recent advances in treatment strategies, infectious diseases are still under the leading causes of death worldwide. Although the activation of the inflammatory cascade is one prerequisite of defense, persistent and exuberant immune response, however, may lead to chronicity of inflammation predisposing to a temporal or permanent tissue damage not only of the site of infection but also among different body organs. The initial response to invading pathogens is mediated by the recognition through various pattern-recognition receptors along with cellular engulfment resulting in a coordinated release of soluble effector molecules and cytokines aiming to terminate the external stimuli. Members of the ‘a disintegrin and metalloproteinase’ (ADAM) family have the capability to proteolytically cleave transmembrane molecules close to the plasma membrane, a process called ectodomain shedding. In fact, in infectious diseases dysregulation of numerous ADAM substrates such as junction molecules (e.g., E-cadherin, VE-cadherin, JAM-A), adhesion molecules (e.g., ICAM-1, VCAM-1, L-selectin), and chemokines and cytokines (e.g., CXCL16, TNF-α) has been observed. The alpha-cleavage by ADAM proteases represents a rate limiting step for downstream regulated intramembrane proteolysis (RIPing) of several substrates, which influence cellular differentiation, cell signaling pathways and immune modulation. Both the substrates mentioned above and RIPing crucially contribute to a systematic damage in cardiovascular, endocrine, and/or gastrointestinal systems. This review will summarize the current knowledge of ADAM function and the subsequent RIPing in infectious diseases (e.g., pathogen recognition and clearance) and discuss the potential long-term effect on pathophysiological changes such as cardiovascular diseases

Pseudomonas aeruginosa Alters Critical Lung Epithelial Cell Functions through Activation of ADAM17

Severe epithelial dysfunction is one major hallmark throughout the pathophysiological progress of bacterial pneumonia. Junctional and cellular adhesion molecules (e.g., JAMA-A, ICAM-1), cytokines (e.g., TNFα), and growth factors (e.g., TGFα), controlling proper lung barrier function and leukocyte recruitment, are proteolytically cleaved and released into the extracellular space through a disintegrin and metalloproteinase (ADAM) 17. In cell-based assays, we could show that the protein expression, maturation, and activation of ADAM17 is upregulated upon infection of lung epithelial cells with Pseudomonas aeruginosa and Exotoxin A (ExoA), without any impact of infection by Streptococcus pneumoniae. The characterization of released extracellular vesicles/exosomes and the comparison to heat-inactivated bacteria revealed that this increase occurred in a cell-associated and toxin-dependent manner. Pharmacological targeting and gene silencing of ADAM17 showed that its activation during infection with Pseudomonas aeruginosa was critical for the cleavage of junctional adhesion molecule A (JAM-A) and epithelial cell survival, both modulating barrier integrity, epithelial regeneration, leukocyte adhesion and transepithelial migration. Thus, site-specific targeting of ADAM17 or blockage of the activating toxins may constitute a novel anti-infective therapeutic option in Pseudomonas aeruginosa lung infection preventing severe epithelial and organ dysfunctions and stimulating future translational studies

Pseudomonas aeruginosa Triggered Exosomal Release of ADAM10 Mediates Proteolytic Cleavage in Trans

Pneumonia is a life-threatening disease often caused by infection with Streptococcus pneumo niae and Pseudomonas aeruginosa. Many of the mediators (e.g., TNF, IL-6R) and junction molecules (e.g., E-cadherin) orchestrating inflammatory cell recruitment and loss of barrier integrity are prote olytically cleaved through a disintegrin and metalloproteinases (ADAMs). We could show by Western blot, surface expression analysis and measurement of proteolytic activity in cell-based assays, that ADAM10 in epithelial cells is upregulated and activated upon infection with Pseudomonas aeruginosa and Exotoxin A (ExoA), but not upon infection with Streptococcus pneumoniae. Targeting ADAM10 by pharmacological inhibition or gene silencing, we demonstrated that this activation was critical for cleavage of E-cadherin and modulated permeability and epithelial integrity. Stimulation with heat-inactivated bacteria revealed that the activation was based on the toxin repertoire rather than the interaction with the bacterial particle itself. Furthermore, calcium imaging experiments showed that the ExoA action was based on the induction of calcium influx. Investigating the extracellular vesicles and their proteolytic activity, we could show that Pseudomonas aeruginosa triggered exosomal release of ADAM10 and proteolytic cleavage in trans. This newly described mechanism could consti tute an essential mechanism causing systemic inflammation in patients suffering from Pseudomonas aeruginosa-induced pneumonia stimulating future translational studies

In Silico and Experimental ADAM17 Kinetic Modeling as Basis for Future Screening System for Modulators

Understanding the mechanisms of modulators’ action on enzymes is crucial for optimizing and designing pharmaceutical substances. The acute inflammatory response, in particular, is regu lated mainly by a disintegrin and metalloproteinase (ADAM) 17. ADAM17 processes several disease mediators such as TNFα and APP, releasing their soluble ectodomains (shedding). A malfunction of this process leads to a disturbed inflammatory response. Chemical protease inhibitors such as TAPI-1 were used in the past to inhibit ADAM17 proteolytic activity. However, due to ADAM170 s broad expression and activity profile, the development of active-site-directed ADAM17 inhibitor was discontinued. New ‘exosite’ (secondary substrate binding site) inhibitors with substrate selectivity raised the hope of a substrate-selective modulation as a promising approach for inflammatory disease therapy. This work aimed to develop a high-throughput screen for potential ADAM17 modula tors as therapeutic drugs. By combining experimental and in silico methods (structural modeling and docking), we modeled the kinetics of ADAM17 inhibitor. The results explain ADAM17 inhibi tion mechanisms and give a methodology for studying selective inhibition towards the design of pharmaceutical substances with higher selectivity

The iRhom2/ADAM17 Axis Attenuates Bacterial Uptake by Phagocytes in a Cell Autonomous Manner

Uptake of bacteria by phagocytes is a crucial step in innate immune defence. Members of the disintegrin and metalloproteinase (ADAM) family critically control the immune response by limited proteolysis of surface expressed mediator molecules. Here, we investigated the significance of ADAM17 and its regulatory adapter molecule iRhom2 for bacterial uptake by phagocytes. Inhibition of metalloproteinase activity 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 or primary phagocytes. Bone marrow-derived macrophages showed enhanced uptake of heat-inactivated and living E. coli when they lacked either ADAM17 or iRhom2 but not upon ADAM10-deficiency. In monocytic THP-1 cells, corresponding short hairpin RNA (shRNA)-mediated knockdown confirmed that ADAM17, but not ADAM10, promoted phagocytosis of E. coli. The augmented bacterial uptake occurred in a cell autonomous manner and was accompanied by increased release of the chemokine CXCL8, less TNFα release and only minimal changes in the surface expression of the receptors TNFR1, TLR6 and CD36. Inhibition experiments indicated that the enhanced bacterial phagocytosis after ADAM17 knockdown was partially dependent on TNFα-activity but not on CXCL8. This novel role of ADAM17 in bacterial uptake needs to be considered in the development of ADAM17 inhibitors as therapeutics

Acute Downregulation but Not Genetic Ablation of Murine MCU Impairs Suppressive Capacity of Regulatory CD4 T Cells

By virtue of mitochondrial control of energy production, reactive oxygen species (ROS) generation, and maintenance of Ca2+ homeostasis, mitochondria play an essential role in modulating T cell function. The mitochondrial Ca2+ uniporter (MCU) is the pore-forming unit in the main protein complex mediating mitochondrial Ca2+ uptake. Recently, MCU has been shown to modulate Ca2+ signals at subcellular organellar interfaces, thus fine-tuning NFAT translocation and T cell activation. The mechanisms underlying this modulation and whether MCU has additional T cell subpopulationspecific effects remain elusive. However, mice with germline or tissue-specific ablation of Mcu did not show impaired T cell responses in vitro or in vivo, indicating that ‘chronic’ loss of MCU can be functionally compensated in lymphocytes. The current work aimed to specifically investigate whether and how MCU influences the suppressive potential of regulatory CD4 T cells (Treg). We show that, in contrast to genetic ablation, acute siRNA-mediated downregulation of Mcu in murine Tregs results in a significant reduction both in mitochondrial Ca2+ uptake and in the suppressive capacity of Tregs, while the ratios of Treg subpopulations and the expression of hallmark transcription factors were not affected. These findings suggest that permanent genetic inactivation of MCU may result in compensatory adaptive mechanisms, masking the effects on the suppressive capacity of Tregs

The metalloproteinase ADAM10 requires its activity to sustain surface expression

The metalloproteinase ADAM10 critically contributes to development, inflammation, and cancer and can be controlled by endogenous or synthetic inhibitors. Here, we demonstrate for the first time 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 the whole cell. This process is 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 requires de novo synthesis. Functionally, this is reflected by loss and recovery of ADAM10 substrate shedding. Finally, ADAM10 inhibition in mice reduces systemic ADAM10 levels in different tissues. Thus, ADAM10 activity is critically required for its surface expression in vitro and in vivo. These findings are crucial for development of therapeutic ADAM10 inhibition strategies and may showcase a novel, physiologically relevant mechanism of protease removal due to activity loss

Expression of the Metalloproteinase ADAM8 Is Upregulated in Liver Inflammation Models and Enhances Cytokine Release In Vitro

Acute and chronic liver inflammation is driven by cytokine and chemokine release from various cell types in the liver. Here, we report that the induction of inflammatory mediators is associated with a yet undescribed upregulation of the metalloproteinase ADAM8 in different murine hepatitis models. We further show the importance of ADAM8 expression for the production of inflammatory mediators in cultured liver cells. As a model of acute inflammation, we investigated liver tissue from lipopolysaccharide- (LPS-) treated mice in which ADAM8 expression was markedly upregulated compared to control mice. In vitro, stimulation with LPS enhanced ADAM8 expression in murine and human endothelial and hepatoma cell lines as well as in primary murine hepatocytes. The enhanced ADAM8 expression was associated with an upregulation of TNF-α and IL-6 expression and release. Inhibition studies indicate that the cytokine response of hepatoma cells to LPS depends on the activity of ADAM8 and that signalling by TNF-α can contribute to these ADAM8-dependent effects. The role of ADAM8 was further confirmed with primary hepatocytes from ADAM8 knockout mice in which TNF-α and IL-6 induction and release were considerably attenuated. As a model of chronic liver injury, we studied liver tissue from mice undergoing high-fat diet-induced steatohepatitis and again observed upregulation of ADAM8 mRNA expression compared to healthy controls. In vitro, ADAM8 expression was upregulated in hepatoma, endothelial, and stellate cell lines by various mediators of steatohepatitis including fatty acid (linoleic-oleic acid), IL-1β, TNF-α, IFN-γ, and TGF-β. Upregulation of ADAM8 was associated with the induction and release of proinflammatory cytokines (TNF-α and IL-6) and chemokines (CX3CL1). Finally, knockdown of ADAM8 expression in all tested cell types attenuated the release of these mediators. Thus, ADAM8 is upregulated in acute and chronic liver inflammation and is able to promote inflammation by enhancing expression and release of inflammatory mediators

Biochemical and transcriptomic evaluation of a 3D lung organoid platform for pre-clinical testing of active substances targeting senescence

Chronic lung diseases such as chronic obstructive pulmonary disease and cystic fibrosis are incurable. Epithelial senescence, a state of dysfunctional cell cycle arrest, contributes to the progression of such diseases. Therefore, lung epithelial cells are a valuable target for therapeutic intervention. Here, we present a 3D airway lung organoid platform for the preclinical testing of active substances with regard to senescence, toxicity, and inflammation under standardized conditions in a 96 well format. Senescence was induced with doxorubicin and measured by activity of senescence associated galactosidase. Pharmaceutical compounds such as quercetin antagonized doxorubicininduced senescence without compromising organoid integrity. Using single cell sequencing, we identified a subset of cells expressing senescence markers which was decreased by quercetin. Doxorubicin induced the expression of detoxification factors specifically in goblet cells independent of quercetin. In conclusion, our platform enables for the analysis of senescence-related processes and will allow the pre-selection of a wide range of compounds (e.g. natural products) in preclinical studies, thus reducing the need for animal testing

Transient Receptor Potential Vanilloid 6 (TRPV6) Proteins Control the Extracellular Matrix Structure of the Placental Labyrinth

Calcium-selective transient receptor potential Vanilloid 6 (TRPV6) channels are expressed in fetal labyrinth trophoblasts as part of the feto–maternal barrier, necessary for sufficient calcium supply, embryo growth, and bone development during pregnancy. Recently, we have shown a less- compact labyrinth morphology of Trpv6-deficient placentae, and reduced Ca2+ uptake of primary trophoblasts upon functional deletion of TRPV6. Trpv6-/- trophoblasts show a distinct calcium-dependent phenotype. Deep proteomic profiling of wt and Trpv6-/- primary trophoblasts using label-free quantitative mass spectrometry leads to the identification of 2778 proteins. Among those, a group of proteases, including high-temperature requirement A serine peptidase 1 (HTRA1) and different granzymes are more abundantly expressed in Trpv6-/- trophoblast lysates, whereas the extracellular matrix protein fibronectin and the fibronectin-domain-containing protein 3A (FND3A) were markedly reduced. Trpv6-/- placenta lysates contain a higher intrinsic proteolytic activity increasing fibronectin degradation. Our results show that the extracellular matrix formation of the placental labyrinth depends on TRPV6; its deletion in trophoblasts correlates with the increased expression of proteases controlling the extracellular matrix in the labyrinth during pregnancy