Abstract
Interleukin‐1 alpha (IL‐1α) is a powerful cytokine that modulates immunity, and requires canonical cleavage by calpain for full activity. Mature IL‐1α is produced after inflammasome activation and during cell senescence, but the protease cleaving IL‐1α in these contexts is unknown. We show IL‐1α is activated by caspase‐5 or caspase‐11 cleavage at a conserved site. Caspase‐5 drives cleaved IL‐1α release after human macrophage inflammasome activation, while IL‐1α secretion from murine macrophages only requires caspase‐11, with IL‐1β release needing caspase‐11 and caspase‐1. Importantly, senescent human cells require caspase‐5 for the IL‐1α‐dependent senescence‐associated secretory phenotype (SASP) in vitro, while senescent mouse hepatocytes need caspase‐11 for the SASP‐driven immune surveillance of senescent cells in vivo. Together, we identify IL‐1α as a novel substrate of noncanonical inflammatory caspases and finally provide a mechanism for how IL‐1α is activated during senescence. Thus, targeting caspase‐5 may reduce inflammation and limit the deleterious effects of accumulated senescent cells during disease and Aging.
INTRODUCTION
Inflammation has evolved to protect the host from acute insults such as infection or physical injury. However, chronic inflammation is associated with many age‐related diseases such as atherosclerosis, osteoarthritis and cancer. Senescent cells that drive inflammation also play pivotal roles in these diseases (Childs et al., 2016; Jeon et al., 2017; Kang et al., 2011), and naturally accumulate in tissues during ageing (Baker et al., 2016). Strikingly, removal of senescent cells can prevent the development of disease and also reverse natural features of ageing (Bakeretal., 2016, 2011; Childs et al., 2016; Jeon et al., 2017; Kang et al., 2011). Thus, understanding how senescence drives inflammation is of critical importance in health, disease and ageing.
Senescence is a protective mechanism that induces permanent cell cycle arrest to prevent transmission of defects to the next generation, particularly to stop malignant transformation. Replicative senescence occurs after repeated cell division critically shortens telomeres, while induced senescence occurs after oncogene activation, mitochondrial deterioration, oxidative stress or DNA damage (Munoz‐Espin & Serrano, 2014). As ageing drives tumorigenesis and telomere shortening, it induces senescence via both pathways. Most senescent cells develop altered secretory activities known as a senescence‐associated secretory phenotype (SASP). The SASP releases proinflammatory cytokines (e.g., IL‐1, IL‐6) and chemokines (e.g., IL‐8, GROα), growth factors (e.g., G‐CSF, bFGF), and proteases (e.g., MMPs, PAI‐1) (Coppe et al., 2008), conferring diverse activities. Thus, although cell cycle arrest during senescence limits cancer and the SASP instructs clearance of preneoplastic cells (Kang et al., 2011), this is balanced against establishment of a chronic inflammatory microenvironment that can damage tissue, drive disease and promote tumorigenesis if senescent cells persist (Grivennikov, Greten, & Karin, 2010). IL‐1α acts in an autocrine/paracrine fashion to drive the SASP (Gardner, Humphry, Bennett, & Clarke, 2015; Orjalo, Bhaumik, Gengler, Scott, & Campisi, 2009), with upstream expression controlled in part by ATM/ATR liberation of GATA4 from p62‐directed autophagy (Kang et al., 2015) and/or an mTORC1‐dependent pathway (Laberge et al., 2015). However, how IL‐1α is cleaved, activated or released during senescence to enable it to drive the SASP is unknown.
Interleukin‐1 (IL‐1) is an ancient cytokine that exerts effect on both innate and adaptive immunities. IL‐1α and IL‐1β are the principal ligands that bind to the type 1 IL‐1 receptor (IL‐1R1), causing recruitment of the IL‐1 receptor accessory protein (IL‐1RAP) and subsequent interaction with the signalling adapter MyD88 (Dinarello, 2009). A consequent phospho‐signalling cascade activates NF‐κB leading to multiple effects on immunity including cytokine secretion, upregulation of adhesion and/or MHC/costimulatory molecules, increased vascular permeability, TH17 cell differentiation, and effector T‐cell proliferation in the presence of regulatory T cells (Sims & Smith, 2010). These powerful actions of IL‐1 are countered by a receptor antagonist (IL‐1RA), a decoy receptor (IL‐1R2), and production of IL‐1α and IL‐1β as pro‐proteins that require cleavage for full biological activity. IL‐1α is canonically cleaved by calpain, which occurs upon necrosis in some cell types and significantly increases activity (Burzynski, Humphry, Bennett, & Clarke, 2015; Zheng, Humphry, Maguire, Bennett, & Clarke, 2013), while IL‐1β is activated by caspase‐1 (Black et al., 1988) after inflammasome engagement (Martinon, Burns, & Tschopp, 2002). How IL‐1α is released without necrosis is unknown and puzzling since IL‐1α release is inhibited in Casp1−/−mice (Kuida et al., 1995; Li et al., 1995), even though IL‐1α is not a caspase‐1 substrate. However, the original Casp1−/− mice have an inactivating passenger mutation in Casp11 (Kayagaki et al., 2011), suggesting caspase‐11 might activate IL‐1α. Murine caspase‐11 and the human orthologues caspase‐4 and caspase‐5 are required for noncanonical inflammasome activation in response to intracellular LPS (icLPS) from bacterial infection (Kayagaki et al., 2011; Shi et al., 2014), and control monocyte IL‐1 release (Vigano et al., 2015). Direct binding of LPS to caspase‐11, caspase‐4 or caspase‐5 leads to pyroptosis and/or NLRP3 inflammasome activation, with IL‐1β and/or IL‐1α release (Kayagaki et al., 2011; Shi et al., 2014). However, whether caspase‐4/5 or caspase‐11 requires caspase‐1 to mediate IL‐1α activation, if IL‐1α is only passively released, or if caspase‐4/5 or caspase‐11 can directly cleave and activate IL‐1α in any of these systems is unknown.
We show that IL‐1α is specifically cleaved and activated at a conserved site by caspase‐5 and caspase‐11, but not caspase‐4. Knockdown of caspase‐5 or expression of a caspase site mutant reduces release of IL‐1α after icLPS stimulation. IL‐1α cleavage and release from murine macrophages after icLPS require only caspase‐11, while IL‐1β needs both caspase‐11 and caspase‐1. Importantly, we show caspase‐5 and caspase‐11 are required for senescent cells to establish the IL‐1α‐dependent SASP. Thus, IL‐1α is a direct substrate for inflammatory caspases during noncanonical inflammasome activation and senescence.
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