The central inflammasome adaptor protein ASC activates the inflammasome after transition from a soluble to an insoluble state.

Apoptosis-associated speck-like protein containing a caspase recruitment domain (CARD) (ASC) is a 22 kDa protein that functions as the central adaptor for inflammasome assembly. ASC forms insoluble specks in monocytes undergoing pyroptosis, and the polymerization of ASC provides a template of CARDs that leads to proximity-mediated autoactivation of caspase-1 in canonical inflammasomes. However, specks are insoluble protein complexes, and solubility is typically important for protein function. Therefore, we sought to define whether ASC specks comprise active inflammasome complexes or are simply the end stage of exhausted ASC polymers. Using a THP-1 cell-lysing model of caspase-1 activation that is ASC dependent, we compared caspase-1 activation induced by preassembled insoluble ASC specks and soluble monomeric forms of ASC. Unexpectedly, after controlling for the concentration dependence of ASC oligomerization, we found that only insoluble forms of ASC promoted caspase-1 autocatalysis. This link to insolubility was recapitulated with recombinant ASC. We show that purified recombinant ASC spontaneously precipitated and was functional, whereas the maltose-binding protein-ASC fusion to ASC (promoting enhanced solubility) was inactive until induced to insolubility by binding to amylose beads. This functional link to insolubility also held true for the Y146A mutation of the CARD of ASC, which avoids insolubility and caspase-1 activation. Thus, we conclude that the role of ASC insolubility in inflammasome function is inextricably linked to its pyrin domain-mediated and CARD-mediated polymerizations. These findings will support future studies into the molecular mechanisms controlling ASC solubility.

Inflammasome Activation in an In Vitro Sepsis Model Recapitulates Increased Monocyte Distribution Width Seen in Patients With Sepsis.

OBJECTIVES: Increased monocyte distribution width (MDW) has recently been shown to be a reliable indicator of early sepsis detection. This study therefore sought to determine if inflammasome activation can be linked to monocyte size changes in sepsis. DESIGN: An in vitro sepsis model using bacterial endotoxin (lipopolysaccharide [LPS]) to study the effect of inflammasome activation on monocyte cell size distribution by microscopy and MDW measurements using a standard clinical hematology analyzer. SETTING: University research laboratory. SUBJECTS: Healthy adult volunteers and cultured human monocyte cells in wild-type state and after clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 knockout of key inflammasome components (apoptosis-associated speck-like protein containing a caspase recruitment domain, caspase-1, gasdermin-D). INTERVENTIONS: In vitro treatment of specimens with bacterial LPS. MEASUREMENTS AND MAIN RESULTS: Wild-type THP1 cells demonstrated a significant increase in cell area (207 µm2 [159-400 µm2] vs 160 µm2 [134-198 µm2]; p < 0.001) and distribution width (198 vs 55 µm2; p < 0.0001) by microscopy following treatment with LPS. Increased MDW correlated with inflammasome activation as demonstrated by release of interleukin (IL)-1ß and with the presence of large distended pyroptotic cells by microscopy. All of these effects were blocked in the inflammasome knockout cells. Whole blood samples treated similarly also demonstrated IL-1ß release and increased MDW (median 24.7 U [22.2-27.2 U] vs 16.3 U [15.1-17.6 U]; p = 0.008) as measured using the Beckman-Coulter Unicel DxH900 analyzer. When peripheral blood mononuclear cells were isolated prior to treatment with LPS, microscopy confirmed the presence of large pyroptotic cells correlating to IL-1ß release in the human subject samples as well. CONCLUSIONS: The increased MDW seen in patients with sepsis can be reproduced in an in vitro sepsis model and blocked using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 technology to inactivate the inflammasome. These findings suggest that pyroptotic cellular swelling underlies changes in MDW in septic patients and connect MDW to early events in the inflammatory cascade of sepsis.

cAbl Kinase Regulates Inflammasome Activation and Pyroptosis via ASC Phosphorylation.

Inflammasome activation is regulated in part by the posttranslational modification of inflammasome proteins. Tyrosine phosphorylation is one possible modification. Having previously shown that the protein tyrosine kinase (PTK) inhibitor AG126 greatly inhibits inflammasome activation, we sought to uncover the target kinase. To do this, we screened a commercial tyrosine kinase library for inhibition of inflammasome-dependent IL-18/IL-1ß release and pyroptosis. THP-1 cells (human monocyte cell line) were incubated with PTK inhibitors (0.1, 1, and 10 µM) before stimulation with LPS followed by ATP. The PTK inhibitors DCC-2036 (Rebastinib) and GZD824, specific for Bcr-Abl kinase, showed the most severe reduction of IL-18 and lactate dehydrogenase release at all concentrations used. The suggested kinase target, cAbl kinase, was then deleted in THP-1 cells by CRISPR/Cas9 editing and then tested for its role in inflammasome function and potential to phosphorylate the inflammasome adaptor ASC. The cABL knockout not only significantly inhibited inflammasome function but also decreased release of phosphorylated ASC after LPS/ATP stimulation. One predicted target of cAbl kinase is tyrosine 146 in ASC. Complementation of ASC knockout THP-1 cells with mutated Y146A ASC significantly abrogated inflammasome activation and ASC oligomerization as compared with wild-type ASC complementation. Thus, these findings support cAbl kinase as a positive regulator of inflammasome activity and pyroptosis, likely via phosphorylation of ASC.

Inflammasome Adaptor ASC Is Highly Elevated in Lung Over Plasma and Relates to Inflammation and Lung Diffusion in the Absence of Speck Formation.

Rationale: Caspase-1 is a zymogen whose activation predominantly depends upon the assembly of ASC monomers into insoluble prion-like polymers (specks). ASC polymers support caspase-1 dimer formation inducing a proximity mediated auto-activation of caspase-1. Therefore, the amount and nature of ASC monomers and polymers in lung bronchoalveolar lavage fluid (BALF) might serve as a marker of lung inflammasome activity. Objectives: To determine whether lung ASC concentrations or oligomerization status predicts lung function or activity of lung inflammation. Methods: BALF ASC amount and oligomerization status was studied in three distinct cohorts: (1) young healthy non-smokers, vapers and smokers; (2) healthy HIV+ smokers who underwent detailed lung function studies; and (3) hospitalized patients with suspected pneumonia. We quantified cell free BALF ASC levels by ELISA and immunoblot. Oligomers (i.e., ASC specks) were identified by chemical crosslinking and ability to sediment with centrifugation. Measurement and Main Results: ASC levels are significantly higher in lung lining fluid than in plasma as well as higher in smoker lungs compared to non-smoker lungs. In this context, ASC levels correlate with macrophage numbers, smoking intensity and loss of lung diffusion capacity in a well-characterized cohort of healthy HIV+ smokers. However, only monomeric ASC was found in our BALF samples from all subjects, including patients with lung infections. Conclusions: Even though, most, if not all, extracellular ASC in BALF exists in the soluble, monomeric form, monomeric ASC concentrations still reflect the inflammatory status of the lung microenvironment and correlate with loss of lung function.

Inflammaging and Oxidative Stress in Human Diseases: From Molecular Mechanisms to Novel Treatments.

It has been proposed that a chronic state of inflammation correlated with aging known as inflammaging, is implicated in multiple disease states commonly observed in the elderly population. Inflammaging is associated with over-abundance of reactive oxygen species in the cell, which can lead to oxidation and damage of cellular components, increased inflammation, and activation of cell death pathways. This review focuses on inflammaging and its contribution to various age-related diseases such as cardiovascular disease, cancer, neurodegenerative diseases, chronic obstructive pulmonary disease, diabetes, and rheumatoid arthritis. Recently published mechanistic details of the roles of reactive oxygen species in inflammaging and various diseases will also be discussed. Advancements in potential treatments to ameliorate inflammaging, oxidative stress, and consequently, reduce the morbidity of multiple disease states will be explored.

Interplay between ROS and Antioxidants during Ischemia-Reperfusion Injuries in Cardiac and Skeletal Muscle.

Ischemia reperfusion (IR), present in myocardial infarction or extremity injuries, is a major clinical issue and leads to substantial tissue damage. Molecular mechanisms underlying IR injury in striated muscles involve the production of reactive oxygen species (ROS). Excessive ROS accumulation results in cellular oxidative stress, mitochondrial dysfunction, and initiation of cell death by activation of the mitochondrial permeability transition pore. Elevated ROS levels can also decrease myofibrillar Ca2+ sensitivity, thereby compromising muscle contractile function. Low levels of ROS can act as signaling molecules involved in the protective pathways of ischemic preconditioning (IPC). By scavenging ROS, antioxidant therapies aim to prevent IR injuries with positive treatment outcomes. Novel therapies such as postconditioning and pharmacological interventions that target IPC pathways hold great potential in attenuating IR injuries. Factors such as aging and diabetes could have a significant impact on the severity of IR injuries. The current paper aims to provide a comprehensive review on the multifaceted roles of ROS in IR injuries, with a focus on cardiac and skeletal muscle, as well as recent advancement in ROS-related therapies.