Amyloid Beta (Aβ) are a group of peptides present in several oligomeric states that upon misfolding cause prionopathies. Emerging evidence indicates that Pseudomonas aeruginosa infection and associated host immune responses in pulmonary microvascular endothelial cells (PMVECs) elicit extracellular release of Aβ. These Aβ transmit cytotoxicity independent of the primary infection propagating PMVEC barrier disruption and exacerbating lung injury. Inflammasomes are protein recognition receptors that assemble upon sensing danger molecules including Aβ, resulting in activation and extracellular secretion of caspase‐1. Caspase‐1 propagates innate immune responses by activating interleukins and causing pyroptosis, a programmed cell death. Higher levels of circulating caspase‐1 have been positively correlated with disease severity in critically ill septic patients. Paradoxically, evidence in experimental models indicates that caspase‐1 activation is protective for barrier and multiorgan function, however, the underlying mechanisms are yet to be determined. We therefore hypothesized that secreted caspase‐1 degrades Aβ maintaining PMVEC monolayer integrity during Aβ‐induced injury. Infection of PMVECs with P. aeruginosa strain PA103 stimulated release of Aβ to the culture medium as assessed by Thioflavin T (ThT), a benzothiazole dye that increases in fluorescence upon binding to Aβ fibrils/aggregates. Strikingly, Aβ accumulated in bacteria‐free culture supernatant is cytotoxic when added to naïve PMVECs. Pretreatment of Aβ‐containing bacteria‐free culture supernatant with recombinant caspase‐1 reduced Aβ levels as measured by western blotting and ablated cytotoxicity. In addition, pretreatment of recombinant Aβ with recombinant caspase‐1 prevented Aβ fibril/aggregate formation in vitro. Moreover, CRISPR/Cas9 caspase‐1 mutant PMVECs were enriched with cytotoxic Aβ following P. aeruginosa infection and caspase‐1 mutant PMVECs were more susceptible to damage when treated with Aβ‐containing bacteria‐free culture supernatant. Together with bioinformatics evidence of a caspase‐1 recognition motif (FRHD7), our data strongly suggest caspase‐1 cleaves Aβ as a protective response. To support our hypothesis, we collected plasma from 45 critically ill patients recruited from the University of South Alabama ICU and 7 control subjects under an IRB‐approved study for analysis of circulating caspase‐1 and Aβ. All critically ill patients demonstrated higher levels of Aβ compared to healthy controls. Pulmonary septic patients displayed higher levels of Aβ. Caspase‐1 measured by ELISA was found to be highest in extra‐pulmonary septic patients. Importantly, there was an inverse correlation between caspase‐1 levels and ThT across all critically ill patients. In conclusion, our data indicate that P. aeruginosa infection‐induced release of Aβ from PMVECs elicits a feedforward cytotoxic effect that is inhibited by caspase‐1. Thus, caspase‐1 constitutes a novel protective stress response that contracts Aβ danger molecules generated during infection.Support or Funding InformationNIH R01 HL118334 to D.F.A. and J.P.A.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Introduction: Loss of pulmonary microvascular endothelial cell barrier is a common finding in Pseudomonas aeruginosa -induced acute lung injury (ALI). Such a loss in barrier function occurs as a result of cell death and dysregulated inflammatory signaling. It is well known that inflammasome-mediated activation of caspase-1 results in a specific form of cell death (pyroptosis) and release of pro-inflammatory cytokines in immune cells. These cytokines have been implicated in causing epithelial barrier disruption in ALI. However, the role and the effect of caspase-1 in pulmonary microvascular endothelial cells (PMVECs) have not been studied. Considering that activation of caspase-1 has been shown to improve outcome measures in trauma and ischemia/reperfusion experimental models, we sought to determine if activation of caspase-1 is necessary to preserve PMVEC barrier integrity after infection with Pseudomonas aeruginosa strain PA103. Methods: To test this hypothesis, confluent monolayers of wild type PMVECs and caspase-1 inhibited PMVECs were inoculated with PA103 at multiplicities of infection of 10:1, 40:1, and 100:1. Barrier integrity was assessed by using a trans endothelial electrical resistance (TER) assay measured at the onset of the experiment and at 2 hour intervals for 12 hours. Cell death was assessed by TUNEL staining. Inhibition of caspase-1 was accomplished using 1) a molecular approach via an stable shRNA-mediated knock-down transfection and 2) a pharmacologic approach via the selective caspase-1 inhibitor, YVAD (10, 50, 100, 200 M). Activation of caspase-1 was determined by examining the cleaved/activated protein via western blot. Results: PMVECs treated with PA103 exhibited a progressive and significant loss in barrier integrity at 12 hours. Intriguingly, caspase-1 inhibited PMVECs displayed a statistically significant increase in the rate of barrier integrity loss compared to control over the 12-hour time course. Furthermore, we measured a significant increase in PA103-induced cell death in caspase-1 inhibited PMVECs (22% greater than control for YVAD-treated cells and 9% greater than control for shRNA-down-regulated cells). Conclusion: These data strongly suggest that caspase-1 activation deters PMVEC death and functions to preserve endothelial barrier function during infection with . Our results provide evidence for a role of caspase-1 in PMVECs that
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