cigarette smoke inhibits the nlrp3 inflammasome and leads to caspase 1 activation via the tlr4 trif caspase 8 axis in human macrophages

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In silico and in vivo combinatorial design of Octarellin VI, an artificial protein modeled on the (B/A)8 fold

One way to gain insight into the sequence-structure-function relationship in proteins is to perform de novo design of artificial proteins. The applications of such a study are varied. For example, in medicine and industry, it would give us the ability to precisely engineer proteins to perform a specific function under a wider range of conditions. Despite impressive successes in the de novo protein design, designing a folded protein of more than 100 amino acids remains a challenge. In our lab, four generations of Octarellins, de novo polypeptides of more than two hundred amino acids modelled on the (beta/alpha)8 barrel fold, have been built and structurally characterized using biophysical and spectroscopic methods. The last generation of Octarellins was designed following a hierarchical method combining the specificity of rational design and the power of computational design. The resulting artificial protein, named Octarellin VI, was expressed in E. coli and purified from inclusion bodies. The biophysical characterization showed a monomeric protein, with a secondary structure level similar to the computationally designed model and thermostability. However, the poor solubility in bacteria and low stability of the protein at long term make impossible determine its structure to criticize the model. To improve these negative features, we performed a directed evolution process over the Octarellin, following the improvement at solubility level in the bacteria, thanks to the fusion of Octarellin to the fluorescent folding reporter GFP. After 8 cycles of directed evolution by Error Prone PCR technique, we obtained a most soluble protein, with a 92% of sequence identity with the original protein. This soluble variant is under study to characterize its structural features. The combination between in silico design and directed evolution process emerges as a powerful tool for protein engineering, showing be complementaries techniques and the information obtained by the whole process of design and posterior comparison between 3D structure of Octarellin with the computational model will allow to improve the algorithms for protein design

Electrochemical sensor for evaluating oxidative stress in airway epithelial cells

Cigarette smoke exposure induces oxidative stress within the airways. Increased oxidative burden contributes to the pathogenesis of chronic lung disorders and is associated with aging and chronic inflammation. Airway epithelial cells highly contribute to Reactive Oxygen Species (ROS) generation within injured and inflamed lung tissues. Among ROS, hydrogen peroxide (H2O2) can be monitored in the extracellular space. Herein, we present an amperometric/voltammetric sensor based on gold nanoparticles and graphene oxide able to detect H2O2 with good sensitivity and selectivity. Using this sensor, H2O2 release was measured in conditioned medium from primary bronchial epithelial cells (PBEC), bronchial epithelial cell line, 16HBE, and adenocarcinoma alveolar basal epithelial cell line, A549, exposed to cigarette smoke extracts (CSE). 16HBE were also treated with resveratrol, an anti-oxidant compound. The results were compared with those obtained by flow cytometry using the same cells stained with Carboxy-H2DCFDA and MitoSOX Red, which detect intracellular ROS and mitochondrial superoxide, respectively. The exposure to CSE resulted in a significant increase of the cathodic current due to the reduction of H2O2 indicating an increased release. Addition of resveratrol decreased CSE-induced release of H2O2 in 16HBE. All the results paralleled those obtained by flow cytometry. The proposed sensor is highly sensitive and selective, fast and cost effective and can potentially be applied for real time and easy monitoring of oxidative stress

Development of a nanostructured sensor for monitoring oxidative stress in living cells

Oxidative burden is elevated in the lung of COPD patients and is associated with aging and chronic inflammation. When overcoming physiological levels, reactive oxygen species (ROS) cause cell damage and sustain inflammation. Both lung epithelium and alveolar macrophages contribute to ROS generation. Currently, ROS generation is measured using fluorescent probes and colorimetric/fluorimetric assays. We present an amperometric nanostructured sensor for real-time detection of hydrogen peroxide (H2O2) released by living cells. The H2O2 sensing performance was evaluated through the current vs time response of platinum rod at a working potential of −0.45 V vs saturated calomel electrode acting as a reference. The detection current was related to the bulk concentration of H2O2 in solution. Using this sensor, H2O2 release was measured in conditioned medium from THP-1 macrophages and 16-HBE bronchial epithelial cells exposed to different stimuli (lipopolysaccharide (LPS), cigarette smoke extract (CSE), nigericin). Results were compared with those obtained by flow cytometry using the same cells stained with Carboxy-H2DCFDA and MitoSOXTM Red. The addition of LPS, CSE and nigericin resulted in a significant increase of the cathodic current due to the reduction of H2O2 indicating an increase in H2O2 release. The results paralleled those obtained by flow cytometry. The proposed nanostructured sensor offers several advantages over current methods: (i) real time release of H2O2 is monitored without disturbing cell growth; (ii) quantitative data can be generated using a calibration curve; (iii) the method is highly sensitive, fast and cost effective; (iv) potential use for in vivo monitoring of oxidative stress

FbsC, a Novel Fibrinogen-binding Protein, Promotes Streptococcus agalactiae-Host Cell Interactions

International audienceStreptococcus agalactiae (group B Streptococcus or GBS) is a common cause of invasive infections in newborn infants and adults. The ability of GBS to bind human fibrinogen is of crucial importance in promoting colonization and invasion of host barriers. We characterized here a novel fibrinogen-binding protein of GBS, designated FbsC (Gbs0791), which is encoded by the prototype GBS strain NEM316. FbsC, which bears two bacterial immunoglobulin-like tandem repeat domains and a C-terminal cell wall-anchoring motif (LPXTG), was found to be covalently linked to the cell wall by the housekeeping sortase A. Studies using recombinant FbsC indicated that it binds fibrinogen in a dose-dependent and saturable manner, and with moderate affinity. Expression of FbsC was detected in all clinical GBS isolates, except those belonging to the hypervirulent lineage ST17. Deletion of fbsC decreases NEM316 abilities to adhere to and invade human epithelial and endothelial cells, and to form biofilm in vitro. Notably, bacterial adhesion to fibrinogen and fibrinogen binding to bacterial cells were abolished following fbsC deletion in NEM316. Moreover, the virulence of the fbsC deletion mutant and its ability to colonize the brain were impaired in murine models of infection. Finally, immunization with recombinant FbsC significantly protected mice from lethal GBS challenge. In conclusion, FbsC is a novel fibrinogen-binding protein expressed by most GBS isolates that functions as a virulence factor by promoting invasion of epithelial and endothelial barriers. In addition, the protein has significant immunoprotective activity and may be a useful component of an anti-GBS vaccine

PbsP, a cell wall-anchored protein that binds plasminogen to promote hematogenous dissemination of Group B Streptococcus.

International audienceStreptococcus agalactiae (Group B Streptococcus or GBS) is a leading cause of invasive infections in neonates whose virulence is dependent on its ability to interact with cells and host components. We here characterized a surface protein with a critical function in GBS pathophysiology. This adhesin, designated PbsP, possesses two Streptococcal Surface Repeat domains, a methionine and lysine-rich region, and a LPXTG cell wall-anchoring motif. PbsP mediates plasminogen (Plg) binding both in vitro and in vivo and we showed that cell surface-bound Plg can be activated into plasmin by tissue plasminogen activator to increase the bacterial extracellular proteolytic activity. Absence of PbsP results in a decreased bacterial transmigration across brain endothelial cells and impaired virulence in a murine model of infection. PbsP is conserved among the main GBS lineages and is a major plasminogen adhesin in non-CC17 GBS strains. Importantly, immunization of mice with recombinant PbsP confers protective immunity. Our results indicate that GBS have evolved different strategies to recruit Plg which indicates that the ability to acquire cell surface proteolytic activity is essential for the invasiveness of this bacterium

Caspase-8 activation by cigarette smoke induces pro-inflammatory cell death of human macrophages exposed to lipopolysaccharide

Abstract Cigarette smoking impairs the lung innate immune response making smokers more susceptible to infections and severe symptoms. Dysregulation of cell death is emerging as a key player in chronic inflammatory conditions. We have recently reported that short exposure of human monocyte-derived macrophages (hMDMs) to cigarette smoke extract (CSE) altered the TLR4-dependent response to lipopolysaccharide (LPS). CSE caused inhibition of the MyD88-dependent inflammatory response and activation of TRIF/caspase-8/caspase-1 pathway leading to Gasdermin D (GSDMD) cleavage and increased cell permeability. Herein, we tested the hypothesis that activation of caspase-8 by CSE increased pro-inflammatory cell death of LPS-stimulated macrophages. To this purpose, we measured apoptotic and pyroptotic markers as well as the expression/release of pro-inflammatory mediators in hMDMs exposed to LPS and CSE, alone or in combination, for 6 and 24 h. We show that LPS/CSE-treated hMDMs, but not cells treated with CSE or LPS alone, underwent lytic cell death (LDH release) and displayed apoptotic features (activation of caspase-8 and -3/7, nuclear condensation, and mitochondrial membrane depolarization). Moreover, the negative regulator of caspase-8, coded by CFLAR gene, was downregulated by CSE. Activation of caspase-3 led to Gasdermin E (GSDME) cleavage. Notably, lytic cell death caused the release of the damage-associated molecular patterns (DAMPs) heat shock protein-60 (HSP60) and S100A8/A9. This was accompanied by an impaired inflammatory response resulting in inhibited and delayed release of IL6 and TNF. Of note, increased cleaved caspase-3, higher levels of GSDME and altered expression of cell death-associated genes were found in alveolar macrophages of smoker subjects compared to non-smoking controls. Overall, our findings show that CSE sensitizes human macrophages to cell death by promoting pyroptotic and apoptotic pathways upon encountering LPS. We propose that while the delayed inflammatory response may result in ineffective defenses against infections, the observed cell death associated with DAMP release may contribute to establish chronic inflammation. CS exposure sensitizes human macrophages to pro-inflammatory cell death. Upon exposure to LPS, CS inhibits the TLR4/MyD88 inflammatory response, downregulating the pro-inflammatory genes TNF and IL6 and the anti-apoptotic gene CFLAR, known to counteract caspase-8 activity. CS enhances caspase-8 activation through TLR4/TRIF, with a partial involvement of RIPK1, resulting on the activation of caspase-1/GSDMD axis leading to increased cell permeability and DAMP release through gasdermin pores [19]. At later timepoints caspase-3 becomes strongly activated by caspase-8 triggering apoptotic events which are associated with mitochondrial membrane depolarization, gasdermin E cleavage and secondary necrosis with consequent massive DAMP release