Release of IL-1ß and IL-18 in human primary bronchial epithelial cells exposed to cigarette smoke is independent of NLRP3.

Cigarette smoke (CS) is a major risk factor for chronic lung diseases and promotes activation of pattern recognition receptors in the bronchial epithelium. NOD-like receptor family, pyrin domain-containing 3 (NLRP3) is a pattern recognition receptor whose activation leads to caspase-1 cleavage, maturation/release of IL-1ß and IL-18, and eventually pyroptosis. Whether the NLRP3 inflammasome participates in CS-induced inflammation in bronchial epithelial cells is still unclear. Herein, we evaluated the involvement of NLRP3 in CS-induced inflammatory responses in human primary bronchial epithelial cells. To this purpose, human primary bronchial epithelial cells were stimulated with CS extracts (CSE) and lytic cell death, caspase activation (-1, -8, -3/7), cytokine release (IL-1ß, IL-18, and IL-8), NLRP3, pro-IL-1ß/pro-IL-18 mRNA, and protein expression were measured. The impact of inhibitors of NLRP3 (MCC950), caspases, and the effect of the antioxidant N-acetyl cysteine were evaluated. We found that CSE increased pro-IL-1ß expression and induced activation of caspase-1 and release of IL-1ß and IL-18. These events were independent of NLRP3 and we found that NLRP3 was not expressed. N-acetyl cysteine reverted CSE-induced caspase-1 activation. Overall, our findings support that the bronchial epithelium may play a central role in the release of IL-1 family cytokines independently of NLRP3 in the lungs of smokers.

Cigarette smoke promotes inflammasome-independent activation of caspase-1 and -4 leading to gasdermin D cleavage in human macrophages.

Mechanisms and consequences of gasdermin D (GSDMD) activation in cigarette smoke (CS)-associated inflammation and lung disease are unknown. GSDMD is a downstream effector of caspase-1, -8, and -4. Upon cleavage, GSDMD generates pores into cell membranes. Different degrees of GSDMD activation are associated with a range of physiological outputs ranging from cell hyperactivation to pyroptosis. We have previously reported that in human monocyte-derived macrophages CS extract (CSE) inhibits the NLRP3 inflammasome and shifts the response to lipopolysaccharide (LPS) towards the TLR4-TRIF axis leading to activation of caspase-8, which, in turn, activates caspase-1. In the present work, we investigated whether other ASC-dependent inflammasomes could be involved in caspase activation by CSE and whether caspase activation led to GSDMD cleavage and other downstream effects. Presented results demonstrate that CSE promoted ASC-independent activation of caspase-1 leading to GSDMD cleavage and increased cell permeability, in the absence of cell death. GSDMD cleavage was strongly enhanced upon stimulation with LPS+CSE, suggesting a synergistic effect between the two stimuli. Noteworthy, CSE promoted LPS internalization leading to caspase-4 activation, thus contributing to increased GSDMD cleavage. Caspase-dependent GSDMD cleavage was associated with mitochondrial superoxide generation. Increased cleaved GSDMD was found in lung macrophages of smokers compared to ex-smokers and non-smoking controls. Our findings revealed that ASC-independent activation of caspase-1, -4, and -8 and GSDMD cleavage upon exposure to CS may contribute to macrophage dysfunction and feed the chronic inflammation observed in the smokers' lung.

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.

In Vitro Studies of Persister Cells.

Many bacterial pathogens can permanently colonize their host and establish either chronic or recurrent infections that the immune system and antimicrobial therapies fail to eradicate. Antibiotic persisters (persister cells) are believed to be among the factors that make these infections challenging. Persisters are subpopulations of bacteria which survive treatment with bactericidal antibiotics in otherwise antibiotic-sensitive cultures and were extensively studied in a hope to discover the mechanisms that cause treatment failures in chronically infected patients; however, most of these studies were conducted in the test tube. Research into antibiotic persistence has uncovered large intrapopulation heterogeneity of bacterial growth and regrowth but has not identified essential, dedicated molecular mechanisms of antibiotic persistence. Diverse factors and stresses that inhibit bacterial growth reduce killing of the bulk population and may also increase the persister subpopulation, implying that an array of mechanisms are present. Hopefully, further studies under conditions that simulate the key aspects of persistent infections will lead to identifying target mechanisms for effective therapeutic solutions.

Hfq-Assisted RsmA Regulation Is Central to Pseudomonas aeruginosa Biofilm Polysaccharide PEL Expression.

To appropriately switch between sessile and motile lifestyles, bacteria control expression of biofilm-associated genes through multiple regulatory elements. In Pseudomonas aeruginosa, the post-transcriptional regulator RsmA has been implicated in the control of various genes including those related to biofilms, but much of the evidence for these links is limited to transcriptomic and phenotypic studies. RsmA binds to target mRNAs to modulate translation by affecting ribosomal access and/or mRNA stability. Here, we trace a global regulatory role of RsmA to inhibition of the expression of Vfr-a transcription factor that inhibits transcriptional regulator FleQ. FleQ directly controls biofilm-associated genes that encode the PEL polysaccharide biosynthesis machinery. Furthermore, we show that RsmA alone cannot bind vfr mRNA but requires the assistance of RNA chaperone protein Hfq. This is the first example where a RsmA protein family member requires another protein for binding to its target RNA.