A Fluorescent Reporter Mouse for Inflammasome Assembly Demonstrates an Important Role for Cell-Bound and Free ASC Specks during In Vivo Infection

Abstract

Summary Inflammasome activation is associated with numerous diseases. However, in vivo detection of the activated inflammasome complex has been limited by a dearth of tools. We developed transgenic mice that ectopically express the fluorescent adaptor protein, ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain), and characterized the formation of assembled inflammasome complexes (“specks”) in primary cells and tissues. In addition to hematopoietic cells, we found that a stromal population in the lung tissues forms specks during the early phase of influenza infection whereas myeloid cells showed speck formation after two days. In a peritonitis and Group B streptococcus infection models, a higher percentage of neutrophils formed specks at early phases of infection, while dendritic cells formed specks at later time points. Furthermore, speck-forming cells underwent pyroptosis, and extensive release of specks to the extracellular milieu in vivo. These data underscore the importance of free specks during inflammatory processes in vivo.

The intra‐ and extracellular functions of ASC specks

“…Extracellular ASC specks were detected in tissues from patients and mice with chronic respiratory diseases, or systemically in the sera of patients with autoinflammatory diseases . These findings consolidate several earlier speculations, and extracellular ASC specks have now been observed in different studies, in vitro and in vivo . ASC specks were shown to be released by inflammasome‐activated macrophages and associated with ectopically delivered POP1, which prevented their inflammatory properties in recipient cells, or after their injection into mice .…”

“…ASC specks were shown to be released by inflammasome‐activated macrophages and associated with ectopically delivered POP1, which prevented their inflammatory properties in recipient cells, or after their injection into mice . In two independent studies, mice expressing a fluorescent ASC‐reporter construct revealed the release of ASC specks into the extracellular space and their accumulation in tissues upon challenge with inflammasome activators, bacterial, or viral infection . More recently, a study describing the generation of a Zebrafish ASC‐reporter model confirmed the release of ASC specks by keratinocytes, and showed their uptake by surrounding immune cells .…”

“…Of note, subcellular fractionation and microscopic analyses showed that neutrophils can carry ASC and caspase‐1 in secretory vesicles and tertiary granule compartments, which are released upon activation . Additionally, an ASC‐transgenic reporter mouse revealed that neutrophils are a relevant source of extracellular ASC specks during inflammasome activation in vivo . Finally, pyroptosis results in the release of large amounts of immature pro‐caspase‐1 and pro‐IL‐1β, which constitute the dominant bands in immunoblots of cell‐free supernatants from inflammasome‐activated cells .…”

Regulation and functions of NLRP3 inflammasome during influenza virus infection

“…In lung fibroblasts, caspase-1-dependent IL-1β secretion occurs independently of NLRP3 suggesting cell type-specific functions of NLRP3 in inflammasome activation (Ichinohe et al, 2009). Formation of ASC specks was also shown in tissue sections and lung fibroblasts from mice expressing fluorescent ASC protein further demonstrating inflammasome activity in non-hematopoietic cells (Tzeng et al, 2016). ASC speck formation was observed mainly in cells that stained positive for influenza virus nucleoprotein suggesting that inflammasome assembly occurs primarily in cells infected with influenza virus (Tzeng et al, 2016).…”

From atoms to physiology: what it takes to really understand inflammasomes

“…Detection of ASC specks in mouse primary cells will be simplified by reporter mice that express fluorescent fusions of ASC (Tzeng et al . ). Once inflammasome assembly has been observed, the involved inflammasome sensor could be detected by PLAs using antibodies or nanobodies against ASC as well as individual candidate sensors.…”