Short Exposure to Ethanol Diminishes Caspase-1 and ASC Activation in Human HepG2 Cells In Vitro

Abstract

This paper discusses how the assembly of pro-caspase-1 and apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) in macromolecular protein complexes, inflammasomes, activates caspase-1. The present study investigates the molecular mechanisms of inflammasome activation in HepG2 cells and examines how short exposures to ethanol (EtOH) affect inflammasome activation. HepG2 cells were treated with lipopolysaccharide (LPS), ATP or nigericin (NIG) in a two-step model. After LPS priming, ATP or NIG were added. As inhibitors, sodium orthovanadate (general inhibitor of tyrosine phosphatases), AC-YVAD-CMK (caspase-1 inhibitor) or AZ10606120 (purinergic receptor P2X7R inhibitor) were applied after LPS priming. To monitor the inflammasome activation, the caspase-1 activity, ASC speck formation, reactive oxygen species (ROS) production and cell death were analyzed. To elucidate the mechanistical approach of EtOH to the inflammasome assembly, the cells were treated with EtOH either under simultaneous LPS administration or concurrently with ATP or NIG application. The co-stimulation with LPS and ATP induced a significant ASC speck formation, caspase-1 activation, cell death and ROS generation. The inhibition of the ATP-dependent purinoreceptor P2X7 decreased the caspase-1 activation, whereas sodium orthovanadate significantly induced caspase-1. Additional treatment with EtOH reversed the LPS and ATP-induced caspase-1 activation, ASC speck formation and ROS production. The ASC speck formation and caspase-1 induction require a two-step signaling with LPS and ATP in HepG2 cells. Inflammasome activation may depend on P2X7. The molecular pathway of an acute effect of EtOH on inflammasomes may involve a reduction in ROS generation, which in turn may increase the activity of tyrosine phosphatases.

Acute Alcohol Intoxication Modulates Monocyte Subsets and Their Functions in a Time-Dependent Manner in Healthy Volunteers

“…During and following the excessive alcohol intake, caspase-1 expression is upregulated but significantly less induced compared to non-intoxicated HV. This is in line with an in vitro study, that we have published recently showing that ethanol pre-treatment (170 mM) of human HepG2 liver cells downregulated caspase-1 expression following LPS and ATP administration ( 22 ). This may be caused by innate immune memory ( 40 ), whereby the first hit (here alcohol) may reprogram the innate immune cells resulting in an adaptation of the response to the subsequent second hit (here LPS and ATP).…”

“…50 µl blood and 240 µl RPMI 1640 media with supplements was transferred into polystyrene FACS tubes. Samples were incubated with 1 µg/ml LPS ( E. coli O127:B8 strain; Sigma Aldrich, Germany), 100 µM BzATP (ATP; Sigma-Aldrich, Germany) ( 22 ) and 10 µl 30X FAM-FLICA caspase-1 inhibitor) at 37°C and 5% CO 2 for 90 minutes. Subsequently, cells were washed with RPMI 1640 media supplemented with 10% FBS 2 times.…”

Pyroptosis-Mediated Periodontal Disease

“…Thus, inflammasomes are of central importance to inflammatory processes, as they promote the cleavage of pro-inflammatory cytokines, notably IL-1β and IL-18, through the maturation of caspase-1 [ 70 , 72 ]. Dysregulations or gene mutations of inflammasomes are associated with several auto-inflammatory diseases and cancer [ 33 , 73 ].…”

“…Caspase is an abbreviation for Cysteine-dependent ASPartate-specific proteASE or cysteinyl aspartate specific proteinase [ 9 ]. Caspase-1 is the leading enzyme to mediate the highly inflammatory process known as pyroptosis, which is characterised by rapid cell lysis and the release of pro-inflammatory cytokines [ 73 ]. The downstream processes, resulting from caspase-1 activation, are dictated by the cell type and the nature and magnitude of the stimulus received [ 34 , 42 ].…”

Moderate Beer Intake Downregulates Inflammasome Pathway Gene Expression in Human Macrophages

“…Moreover, polyphenols exhibited the capacity to suppress tumor growth by reducing NF-kβ activity and IL-1β secretion [36]. Other studies provide evidence that the alcoholic fraction (ethanol) inhibits the activation of the NLRP3 and AIM2 inflammasomes [19,37,38]. The different metabolites derived from the hydrolysis of alcohol seem to specifically inhibit the NLRP3 inflammasome by preventing potassium efflux and ASC oligomerization.…”