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

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.

A multivariate statistical test for differential expression analysis.

Statistical tests of differential expression usually suffer from two problems. Firstly, their statistical power is often limited when applied to small and skewed data sets. Secondly, gene expression data are usually discretized by applying arbitrary criteria to limit the number of false positives. In this work, a new statistical test obtained from a convolution of multivariate hypergeometric distributions, the Hy-test, is proposed to address these issues. Hy-test has been carried out on transcriptomic data from breast and kidney cancer tissues, and it has been compared with other differential expression analysis methods. Hy-test allows implicit discretization of the expression profiles and is more selective in retrieving both differential expressed genes and terms of Gene Ontology. Hy-test can be adopted together with other tests to retrieve information that would remain hidden otherwise, e.g., terms of (1) cell cycle deregulation for breast cancer and (2) "programmed cell death" for kidney cancer.

The Sample Size Matters: To What Extent the Participant Reduction Affects the Outcomes of a Neuroscientific Research. A Case-Study in Neuromarketing Field.

The sample size is a crucial concern in scientific research and even more in behavioural neurosciences, where besides the best practice it is not always possible to reach large experimental samples. In this study we investigated how the outcomes of research change in response to sample size reduction. Three indices computed during a task involving the observations of four videos were considered in the analysis, two related to the brain electroencephalographic (EEG) activity and one to autonomic physiological measures, i.e., heart rate and skin conductance. The modifications of these indices were investigated considering five subgroups of sample size (32, 28, 24, 20, 16), each subgroup consisting of 630 different combinations made by bootstrapping n (n = sample size) out of 36 subjects, with respect to the total population (i.e., 36 subjects). The correlation analysis, the mean squared error (MSE), and the standard deviation (STD) of the indexes were studied at the participant reduction and three factors of influence were considered in the analysis: the type of index, the task, and its duration (time length). The findings showed a significant decrease of the correlation associated to the participant reduction as well as a significant increase of MSE and STD (p < 0.05). A threshold of subjects for which the outcomes remained significant and comparable was pointed out. The effects were to some extents sensitive to all the investigated variables, but the main effect was due to the task length. Therefore, the minimum threshold of subjects for which the outcomes were comparable increased at the reduction of the spot duration.

Stress Assessment by Combining Neurophysiological Signals and Radio Communications of Air Traffic Controllers.

Air Traffic Control (ATC) has been classified as the fourth most stressful job. In this regard, sixteen controllers were asked to perform ecological ATC simulation during which behavioral (Radio Communications with pilots - RCs), subjective (stress perception) and neurophysiological signals (brain activity and skin conductance - SC) were collected. All the considered parameters reported significant changes under high stress conditions. In particular, the theta, alpha, and beta brain rhythms increased significantly (all p<0.05) all over the brain areas, and both the SC components exhibited higher values (p<0.01). Additionally, the number of speech under high stress decreased significantly (p<10-4) while both the mean and median value of the F0 component of the RC increased (p<0.01). The results can be employed to objectively measure and track the controller's stress level while dealing with ATC activities to better tailoring the workshift and maintaining high safety levels.