Calpain drives pyroptotic vimentin cleavage, intermediate filament loss, and cell rupture that mediates immunostimulation

Abstract

Pyroptosis is an inflammatory form of programmed cell death following cellular damage or infection. It is a lytic process driven by gasdermin D-mediated cellular permeabilization and presumed osmotic forces thought to induce swelling and rupture. We found that pyroptotic cells do not spontaneously rupture in culture but lose mechanical resilience. As a result, cells were susceptible to rupture by extrinsic forces, such as shear stress or compression. Cell analyses revealed that all major cytoskeleton components were disrupted during pyroptosis and that sensitivity to rupture was calpaindependent and linked with cleavage of vimentin and loss of intermediate filaments. Moreover, while release of lactate dehydrogenase (LDH), HMGB1, and IL-1β occurred without rupture, rupture was required for release of large inflammatory stimuli-ASC specks, mitochondria, nuclei, and bacteria. Importantly, supernatants from ruptured cells were more immunostimulatory than those from nonruptured cells. These observations reveal undiscovered cellular events occurring during pyroptosis, define the mechanisms driving pyroptotic rupture, and highlight the immunologic importance of this event.

Inflammasome‐associated cell death: Pyroptosis, apoptosis, and physiological implications

“…43,44 Calpains have been shown to promote the rupture of pyroptotic cell membranes by degrading vimentin intermediate filaments. 43 This is required for the release of proinflammatory/immunogenic macromolecules and organelles, such as ASC specks, mitochondrial DNA, mitochondria, and nuclei. Extracellular ASC specks may be taken up by phagocytes, in which the aggregates may activate caspase-1 to sustain and spread inflammation, indicating a role for calpains in the propagation of inflammasome responses.…”

Gasdermins and their role in immunity and inflammation

“…It is believed that pore formation results in loss of osmotic homeostasis, swelling of the cell, and death (Kayagaki et al, 2015; Shi et al, 2015a; Ding et al, 2016; Liu et al, 2016). However, newer studies raise some questions about the validity of this model (Chen et al, 2016; Davis et al, 2019; de Vasconcelos et al, 2019). Nevertheless, expression of the N-terminal domain by itself is sufficient to trigger pyroptosis.…”

“…GSDMD pores are often described in the literature as inducing an osmotic imbalance resulting in influx of water, cell swelling, and rupture. However, this view is not uniformly accepted (Chen et al, 2016; Davis et al, 2019; de Vasconcelos et al, 2019). If the pores were large enough to allow the free flow of ions and proteins across the membrane, this would prevent the formation of an osmotic gradient, limit the flux of water, and prevent swelling and rupture.…”

Type-3 Secretion System–induced pyroptosis protects Pseudomonas against cell-autonomous immunity

“…“Pore-induced Intracellular Traps” (PITs) entrap intracellular S . Typhimurium (11–13). As P.aeruginosa induced phagocytosis-independent activation of the NLRC4 inflammasome, we reasoned that such process might be a virulence strategy to escape bacterial caging into PITs.…”

“…Importantly, resulting IL-1β and IL-18 cytokine secretion mediates intracellular bacterial clearance by inducing, respectively, phagocyte recruitment and the production of the microbicidal cytokine interferon (IFN)-γ (1, 6). A recently discovered and understudied cell-autonomous immune process known as pyroptosis promotes intracellular bacteria entrapment and weakening in pore-induced intracellular trap structures (PITs) (11–13), which facilitates subsequent bacterial elimination through efferocytosis (13–16). However, host-adapted bacteria can inhibit PITs formation, which enables bacterial proliferation (14).…”