Secreted Caspase‐1 Contracts Amyloid‐related Danger Molecules Released from Pulmonary Microvascular Endothelial Cell as a Novel Stress Response during Infection

Abstract

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.