A cell-free, biomimetic hydrogel based on probiotic membrane vesicles ameliorates wound healing.

Probiotic bacteria, such as Lactobacilli, have been shown to elicit beneficial effects in various tissue regeneration applications. However, their formulation as living bacteria is challenging, and their therapeutic use as proliferating microorganisms is especially limited in immunocompromised patients. Here, we propose a new therapeutic avenue to circumvent these shortcomings by developing a bacteriomimetic hydrogel based on membrane vesicles (MVs) produced by Lactobacilli. We coupled MVs from Lactobacillus plantarum and Lactobacillus casei, respectively, to the surface of synthetic microparticles, and embedded those bacteriomimetics into a pharmaceutically applicable hydrogel matrix. The wound microenvironment changes during the wound healing process, including adaptions of the pH and changes of the oxygen supply. We thus performed proteomic characterization of the MVs harvested under different culture conditions and identified characteristic proteins related to the biological effect of the probiotics in every culture state. In addition, we highlight a number of unique proteins expressed and sorted into the MVs for every culture condition. Using different in vitro models, we demonstrated that increased cell migration and anti-inflammatory effects of the bacteriomimetic microparticles were dependent on the culture condition of the secreting bacteria. Finally, we demonstrated the bacteriomimetic hydrogel's ability to improve healing in an in vivo mouse full-thickness wound model. Our results create a solid basis for the future application of probiotic-derived vesicles in the treatment of inflammatory dispositions and stimulates the initiation of further preclinical trials.

Characterization of the Secreted Acid Phosphatase SapS Reveals a Novel Virulence Factor of Staphylococcus aureus That Contributes to Survival and Virulence in Mice.

Staphylococcus aureus possesses a large arsenal of immune-modulating factors, enabling it to bypass the immune system's response. Here, we demonstrate that the acid phosphatase SapS is secreted during macrophage infection and promotes its intracellular survival in this type of immune cell. In animal models, the SA564 sapS mutant demonstrated a significantly lower bacterial burden in liver and renal tissues of mice at four days post infection in comparison to the wild type, along with lower pathogenicity in a zebrafish infection model. The SA564 sapS mutant elicits a lower inflammatory response in mice than the wild-type strain, while S. aureus cells harbouring a functional sapS induce a chemokine response that favours the recruitment of neutrophils to the infection site. Our in vitro and quantitative transcript analysis show that SapS has an effect on S. aureus capacity to adapt to oxidative stress during growth. SapS is also involved in S. aureus biofilm formation. Thus, this study shows for the first time that SapS plays a significant role during infection, most likely through inhibiting a variety of the host's defence mechanisms.

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 pneumoniae 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 proteolytically 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 constitute an essential mechanism causing systemic inflammation in patients suffering from Pseudomonas aeruginosa-induced pneumonia stimulating future translational studies.

ADAM8 signaling drives neutrophil migration and ARDS severity.

Acute respiratory distress syndrome (ARDS) results in catastrophic lung failure and has an urgent, unmet need for improved early recognition and therapeutic development. Neutrophil influx is a hallmark of ARDS and is associated with the release of tissue-destructive immune effectors, such as matrix metalloproteinases (MMPs) and membrane-anchored metalloproteinase disintegrins (ADAMs). Here, we observed using intravital microscopy that Adam8-/- mice had impaired neutrophil transmigration. In mouse pneumonia models, both genetic deletion and pharmacologic inhibition of ADAM8 attenuated neutrophil infiltration and lung injury while improving bacterial containment. Unexpectedly, the alterations of neutrophil function were not attributable to impaired proteolysis but resulted from reduced intracellular interactions of ADAM8 with the actin-based motor molecule Myosin1f that suppressed neutrophil motility. In 2 ARDS cohorts, we analyzed lung fluid proteolytic signatures and identified that ADAM8 activity was positively correlated with disease severity. We propose that in acute inflammatory lung diseases such as pneumonia and ARDS, ADAM8 inhibition might allow fine-tuning of neutrophil responses for therapeutic gain.

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.

The Impact of Spironolactone on Markers of Myocardial Oxidative Status, Inflammation and Remodeling in Hyperthyroid Rats.

BACKGROUND: Hyperthyroidism promotes the development and progression of cardiovascular diseases (CVD). Aldosterone, a key mediator of myocardial inflammation, oxidative stress and fibrosis, may be activated in hyperthyroidism. OBJECTIVE: To assess the impact of hyperthyroidism on aldosterone levels and myocardial oxidative status, inflammatory and fibrotic markers in hyperthyroid rats, and to test if the use of spironolactone (an aldosterone antagonist) attenuates these changes. METHODS: Adult Wistar rats were randomly distributed into 4 groups; controls, spironolactone treated rats (Spir, 50mg/kg/day), hyperthyroid rats (Hyper, daily intraperitoneal levothyroxine 0.3mg/kg/day), and spironolactone treated hyperthyroid rats (Hyper+Spir) for 4 weeks. Blood pressure (Bp), and levels of serum and myocardial aldosterone, oxidants/antioxidants, inflammatory and fibrotic markers were measured. RESULTS: Levothyroxine increased serum thyroid hormones and increased Bp, heart rate and heart to bodyweight ratio. Relative to control, serum aldosterone levels were increased in Hyper and Hyper+ Spir groups. In parallel, cardiac lipid peroxides and serum endothelin-1 were increased whereas cardiac superoxide dismutase, catalase, glutathione, and matrix metalloproteinase -2 were reduced in the Hyper group. Spironolactone decreased serum thyroid hormones and improved cardiac lipid peroxides and metalloproteinase -2 levels. The use of spironolactone decreased serum nitrite levels and increased cardiac SOD and glutathione. Cardiac levels of aldosterone, endothelin-1, transforming growth factor-beta and nitrite were similar among all groups. CONCLUSION: Hyperthyroid status was associated with an increase in aldosterone and oxidant/ inflammatory biomarkers. The use of spironolactone enhanced antioxidant defenses. Aldosterone antagonists may serve as potential drugs to attenuate the development of cardiac disease in hyperthyroidism.