Biology subjects

Schuepbach, R. A.

Publications and source records attributed to Schuepbach, R. A..

5 recordsLinked to original sources

bioRxiv2023

Alpha-toxin elicited CX3CL1-release via ADAM10 in Staphylococcus aureus pneumoniaimpairs bactericidal function of human monocytes

Staphylococcus aureus is an important human pathogen causing severe invasive infections. Pathogenesis is attributed to a wide array of virulence factors, including several potent exotoxins such as the pore-forming alpha-toxin. In this study, we found that patients with S. aureus respiratory tract infections had elevated CX3CL1 levels in airway fluid and plasma. Using humanized organotypic lung models, we observed that stimulation of lung epithelium with alpha-toxin induce an intensified CX3CL1 expression apically in the epithelium as well as the release of CX3CL1. Blocking alpha-toxin or ADAM10 activity in organotypic lung using an alpha-toxin-blocking antibody or a specific ADAM-10 inhibitor confirmed their role in modulating CX3CL1 cleavage and release. Analyses of CD14+ human monocytes in combination with a CX3CR1 inhibitor revealed that alpha-toxin-mediated CX3CL1 release induce CX3CL1-dependent chemotaxis. In line with these data, lung tissue from patients with S. aureus respiratory tract infection showed elevated CX3CL1 and CD14 staining as compared to tissue from patients with non-infectious lung diseases. Functional studies of monocytes showed that CX3CL1 released by lung models resulted in upregulated CD83 and downregulated CD86, as well as impaired killing of phagocytosed S. aureus. Furthermore, stimulation of monocytes with soluble CX3CL1 hampered their reactive-oxygen and nitric-oxide production. Taken together, our data show that S. aureus triggers the release of lung epithelial CX3CL1; a process found to be dependent on the alpha-toxins effect on ADAM10 mediating cytotoxicity and resulting in impaired monocyte phagocytic killing. Hence, we identify an immunomodulatory effect of alpha-toxin involving the CX3CL1-ADAM10 axis extending beyond the cytolysis function.

microbiology↗

bioRxiv2022

Group A Streptococcus antibiotic tolerance in necrotizing fasciitis

ObjectivesGroup A Streptococcus (GAS) necrotizing fasciitis (NF) is a difficult-to-treat bacterial infection associated with high morbidity and mortality despite extensive surgery and targeted antibiotic treatment. Bacteria surviving prolonged antibiotic exposure without displaying genetic resistance, so-called persisters, are associated with difficult-to-treat infections, such as GAS-NF. In the present study, we investigated the presence of persistent GAS in tissue freshly debrided from three NF patients and examined more in depth the persisters phenomenon in GAS-NF clinical isolates. MethodsTime-lapse imaging of freshly isolated GAS-NF clinical isolates, the image analysis software ColTapp and antibiotic challenge-based persisters assays were used to assess the presence of persisters. ResultsWe show for the first time that GAS recovered directly from freshly debrided NF patients tissue are characterized by increased colony appearance time heterogeneity, indicating the presence of persisters. Acidic pH or nutrient stress exposure, mimicking the NF-like environment in vitro, similarly leads to phenotypic heterogeneity resulting in enhanced antibiotic survival and confirming the presence of GAS persisters. ConclusionsGAS persisters are present in the tissue freshly debrided from GAS-NF patients and might be one explanation for antibiotic treatment failure and surgery requirement in GAS-NF. Tailored treatment options, including the use of persisters-targeting drugs, need to be developed to increase GAS-NF therapy success.

microbiology↗

bioRxiv2021

Blunted Fas signaling favors RIPK1-driven neutrophil necroptosis in critically ill COVID-19 patients

Critically ill COVID-19 patients are characterized by a severely dysregulated cytokine profile and elevated neutrophil counts, which are thought to contribute to disease severity. However, to date it remains unclear how neutrophils contribute to pathophysiology during COVID-19. Here, we assessed the impact of the dysregulated cytokine profile on the tightly regulated cell death program of neutrophils. We show that in a subpopulation of neutrophils, canonical apoptosis was skewed towards rapidly occurring necroptosis. This phenotype was characterized by abrogated caspase-8 activity and increased RIPK1 levels, favoring execution of necroptosis via the RIPK1-RIPK3-MLKL axis, as further confirmed in COVID-19 biopsies. Moreover, reduction of sFas-L levels in COVID-19 patients and hence decreased signaling to Fas directly increased RIPK1 levels and correlated with disease severity. Our results suggest an important role for Fas signaling in the regulation of cell death program ambiguity via the ripoptosome in neutrophils during COVID-19 and a potential therapeutic target to curb inflammation and thus influence disease severity and outcome.

immunology↗

bioRxiv2020

Neutrophil and monocyte dysfunctional effector response towards bacterial challenge in critically-ill COVID-19 patients

COVID-19 displays diverse disease severities and symptoms. Elevated inflammation mediated by hypercytokinemia induces a detrimental dysregulation of immune cells. However, there is limited understanding of how SARS-CoV-2 pathogenesis impedes innate immune signaling and function against secondary bacterial infections. We assessed the influence of COVID-19 hypercytokinemia on the functional responses of neutrophils and monocytes upon bacterial challenges from acute and corresponding recovery COVID-19 ICU patients. We show that severe hypercytokinemia in COVID-19 patients correlated with bacterial superinfections. Neutrophils and monocytes from acute COVID-19 patients showed severely impaired microbicidal capacity, reflected by abrogated ROS and MPO production as well as reduced NETs upon bacterial challenges. We observed a distinct pattern of cell surface receptor expression on both neutrophils and monocytes leading to a suppressive autocrine and paracrine signaling during bacterial challenges. Our data provide insights into the innate immune status of COVID-19 patients mediated by their hypercytokinemia and its transient effect on immune dysregulation upon subsequent bacterial infections

immunology↗

bioRxiv2019

Thrombin cleaves and activates the protease-activated receptor 2 dependent on thrombomodulin co-receptor availability

IntroductionProtease-activated receptors (PARs) evolved to react to extracellular proteolytic activity. In mammals, three of the four PARs (PAR1, PAR3, and PAR4) that are expressed respond to the prototypical procoagulant enzyme thrombin, whereas PAR2 was assumed to resist activation by thrombin. To date, involvement of cell surface thrombin-recruiting co-receptors such as thrombomodulin (TM), which potentially facilitates PAR2 cleavage, has not been addressed. Thus, we examined whether TM-bound thrombin cleaved PAR2 and tested biological responses such as nuclear factor kappa B (NF-{kappa}B) DNA binding activity and cytokine release. Materials and MethodsWe examined 293T cells overexpressing PAR2 and TM for thrombin recruitment by TM promoting PAR2 cleavage. To test for the TM-thrombin interactions required for PAR2 cleavage and to map cleavage sites on PAR2, mutant constructs of TM or PAR2 were engineered. Biological effects because of PAR2 activation were investigated using an NF-{kappa}B reporter system and cytokine release. Results and ConclusionsWe identified that, at low to moderate concentrations, thrombin cleaved PAR2 in a TM co-receptor-dependent manner with cleavage efficiency comparable to that of trypsin. In TMs presence, thrombin efficiently cleaved both, PAR1 and PAR2, albeit kinetics differed. Whereas the majority of surface expressed PAR1 was immediately cleaved off, prolonged exposure to thrombin resulted in few additional cleavage. In contrast, PAR2 cleavage was sustained upon prolonged exposure to thrombin. However, TM EGF-like domain 5 was required and TM chondroitin sulfate (CS) proteoglycan sites serine 490 and serine 492 assisted in PAR2 cleavage, while thrombin preferentially cleaved at arginine 36 on PAR2s N-terminus. Note that thrombin-induced activation of NF-{kappa}B via PAR2 resulted in release of interleukin-8. Thus, we provide a novel concept of how thrombin efficiently cleaves PAR2 in a TM-dependent manner, resulting in pro-inflammatory interleukin-8 release. This unexpected pro-inflammatory role of TM, promoting cleavage and activation of PAR2 by thrombin, may lead to novel therapeutic options for treating inflammatory and malignant diseases.

molecular biology↗