Caspase 8 sounds the alarm for allergic inflammation

Alarmins are innate cytokines released by damaged or dying cells that act to initiate inflammatory immune responses. One prototypical alarmin is IL-33, a member of the IL-1 family of cytokines that is enriched in epithelial cells and plays a key role in the initiation of allergic immune responses.1Hammad H. Lambrecht B.N. Barrier epithelial cells and the control of type 2 immunity.Immunity. 2015; 43: 29-40Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar, 2Roan F. Obata-Ninomiya K. Ziegler S.F. Epithelial cell-derived cytokines: more than just signaling the alarm.J Clin Invest. 2019; 129: 1441-1451Crossref PubMed Scopus (242) Google Scholar, 3Cayrol C. Girard J.P. Interleukin-33 (IL-33): A nuclear cytokine from the IL-1 family.Immunol Rev. 2018; 281: 154-168Crossref PubMed Scopus (516) Google Scholar The 270–amino acid (AA) IL-33 protein has 3 main regions: an N-terminal nuclear domain (AAs 1-65), a protease-sensitive central domain (AAs 65-112), and a C-terminal IL-1–like domain (AAs 112-270) that binds to the IL-33 receptor ST2 and contains caspase cleavage sites that are thought to permit its degradation. The N-terminal nuclear domain localizes the full-length 270-AA precursor form (IL-331-270) in the nucleus, where it is normally sequestered. After cellular damage or necrosis, IL-331-270 is released into the extracellular milieu, where it can either directly bind ST2 or be cleaved into its more active mature form.3Cayrol C. Girard J.P. Interleukin-33 (IL-33): A nuclear cytokine from the IL-1 family.Immunol Rev. 2018; 281: 154-168Crossref PubMed Scopus (516) Google Scholar This extracellular cleavage step is mediated by endogenous proteases released from activated neutrophils and mast cells, which cleave at several sites in the central domain to yield C-terminal mature IL-33 (mIL-33) fragments (IL-3395-270, IL-33107-270, and IL-33109-270) that exhibit increased activity compared with IL-331-270. This cleavage step can also be performed by exogenous cysteine and serine proteases found in allergens such as house dust mite and Alternaria alternata.4Cayrol C. Duval A. Schmitt P. Roga S. Camus M. Stella A. et al.Environmental allergens induce allergic inflammation through proteolytic maturation of IL-33.Nat Immunol. 2018; 19: 375-385Crossref PubMed Scopus (215) Google Scholar These observations have led to our current understanding of IL-33 function, in which the full-length protein is released from the nucleus of necrotic cells into the extracellular milieu, where it has some minimal activity on its receptor ST2. After cleavage of its central domain by endogenous and exogenous proteases, free and highly active mIL-33 containing the intact C-terminal IL-1–like domain is released. This pathway is central to the initiation of allergic immune responses; however, fundamental questions about the cleavage and release of IL-33 remain. In response to allergen exposure, mIL-33 can be detected in the absence of necrosis and yet it drives the initiation of allergic inflammation.3Cayrol C. Girard J.P. Interleukin-33 (IL-33): A nuclear cytokine from the IL-1 family.Immunol Rev. 2018; 281: 154-168Crossref PubMed Scopus (516) Google Scholar If mIL-33 is detected before cell death and the influx of inflammatory cells, how is mIL-33 formed and released? Alternatively, is there another form of IL-33 that can initiate the inflammatory response? In a recent issue of Nature Immunology, Brusilovsky et al5Brusilovsky M. Rochman M. Rochman Y. Caldwell J.M. Mack L.E. Felton J.M. et al.Environmental allergens trigger type 2 inflammation through ripoptosome activation.Nat Immunol. 2021; 22: 1316-1326Crossref PubMed Scopus (34) Google Scholar illustrate a novel pathway for the allergen induced activation and release of mIL-33 (Fig 1). They found that incubation of the EPC2 epithelial cell line with a diverse group of animal and fungal, but not plant or food, allergens induced the rapid intracellular production of mIL-33 (∼21 kDa). This occurred concurrently with RIPK1 phosphorylation and caspase 8 cleavage, suggesting that allergen stimulation of epithelial cells activates the ripoptosome.5Brusilovsky M. Rochman M. Rochman Y. Caldwell J.M. Mack L.E. Felton J.M. et al.Environmental allergens trigger type 2 inflammation through ripoptosome activation.Nat Immunol. 2021; 22: 1316-1326Crossref PubMed Scopus (34) Google Scholar The ripoptosome is a spontaneously assembled macromolecular protein complex of RIPK1 (RIP1), procaspase 8, TRADD, FADD, and cFLIP. Activation of this complex by diverse stimuli, including genotoxic stress, loss of cellular inhibitor of apoptosis proteins (cIAPs), and Toll-like receptor 3 (TLR3) stimulation, leads to cleavage of procaspase 8 into its active form, ultimately culminating in caspase 8-mediated apoptosis and/or necrosis.6Feoktistova M. Geserick P. Kellert B. Dimitrova D.P. Langlais C. Hupe M. et al.cIAPs block ripoptosome formation, a RIP1/capase-8 containing intracellular cell death complex differentially regulated by cFLIP isoforms.Mol Cell. 2011; 43: 449-463Abstract Full Text Full Text PDF PubMed Scopus (678) Google Scholar,7Tenev T. Bianchi K. Darding M. Broemer M. Langlais C. Wallberg F. et al.The ripoptosome, a signaling platform that assembles in response to genotoxic stress and loss of IAPs.Mol Cell. 2011; 43: 432-448Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar Despite the role of caspase 8 in cell death, mIL-33 production and release appeared to precede or occur independently of cell death, indicating an alternative mechanism for IL-33 release and cleavage. Brusilovsky et al5Brusilovsky M. Rochman M. Rochman Y. Caldwell J.M. Mack L.E. Felton J.M. et al.Environmental allergens trigger type 2 inflammation through ripoptosome activation.Nat Immunol. 2021; 22: 1316-1326Crossref PubMed Scopus (34) Google Scholar found that caspase 8 activation led to downstream activation of caspases 3 and 7, which were capable of cleaving IL-331-270. Caspases 3 and 7 have previously been described as inactivating IL-33 by cleaving it in its IL-1–like cytokine domain after residue D175 or D178. Disruption of the C-terminal IL-1–like domain would be expected to inactivate IL-33, but Brusilovsky et al5Brusilovsky M. Rochman M. Rochman Y. Caldwell J.M. Mack L.E. Felton J.M. et al.Environmental allergens trigger type 2 inflammation through ripoptosome activation.Nat Immunol. 2021; 22: 1316-1326Crossref PubMed Scopus (34) Google Scholar showed instead that the IL-331-175 and IL-331-178 contained a minimal ST2-binding domain. As such, they were not only capable of binding to the ST2 receptor but were as potent as the unprocessed IL-331-270 at promoting inflammatory cytokine and chemokine release from the HMC-1 human mast cell line and primary murine eosinophils. Intriguingly, the ST2-mediated activity of the IL-331-175 and IL-331-178 fragments was augmented in the presence of histones that remained bound to the N-terminal chromatin-binding domain.5Brusilovsky M. Rochman M. Rochman Y. Caldwell J.M. Mack L.E. Felton J.M. et al.Environmental allergens trigger type 2 inflammation through ripoptosome activation.Nat Immunol. 2021; 22: 1316-1326Crossref PubMed Scopus (34) Google Scholar To establish a role for the caspase 8–dependent N-terminal fragment of IL-33 in vivo, Brusilovsky et al5Brusilovsky M. Rochman M. Rochman Y. Caldwell J.M. Mack L.E. Felton J.M. et al.Environmental allergens trigger type 2 inflammation through ripoptosome activation.Nat Immunol. 2021; 22: 1316-1326Crossref PubMed Scopus (34) Google Scholar used an A alternata–induced murine model of allergic airway inflammation. Intratracheal exposure to A alternata led to release of caspase 8–dependent IL-33 in the bronchial alveolar lavage fluid, with concomitant neutrophil and eosinophil influx that was blocked by a caspase 8–specific inhibitor or in mice lacking airway caspase 8. On the basis of the molecular weight of IL-33 and its detection before inflammatory cell infiltration, it is likely that this represented the IL-331-175/178 fragment, although ruling out the presence of the IL-33107-270 C-terminal fragment by using these assays is difficult. Likewise, whether caspase 8 was required for IL-331-270 cleavage or cell death is unclear; however, these data provide supporting in vivo evidence for the caspase 8–mediated pathway. To link these murine observations with human disease, the investigators examined esophageal biopsy samples from patients in remission or with active eosinophilic esophagitis. Active eosinophilic esophagitis was associated with increased staining for caspase 8 and caspase 3, IL-33, and eosinophils, thereby providing correlative evidence for their in vitro pathway in human samples.5Brusilovsky M. Rochman M. Rochman Y. Caldwell J.M. Mack L.E. Felton J.M. et al.Environmental allergens trigger type 2 inflammation through ripoptosome activation.Nat Immunol. 2021; 22: 1316-1326Crossref PubMed Scopus (34) Google Scholar These data reveal an unexpected new ripoptosome-mediated pathway for IL-33 maturation and release. However, many questions remain. The mechanism by which allergens activate this process remains unknown. Brusilovsky et al5Brusilovsky M. Rochman M. Rochman Y. Caldwell J.M. Mack L.E. Felton J.M. et al.Environmental allergens trigger type 2 inflammation through ripoptosome activation.Nat Immunol. 2021; 22: 1316-1326Crossref PubMed Scopus (34) Google Scholar show that fungal and animal allergens induced this pathway, whereas plant and food allergens did not. The inducing allergens include both protease-rich (eg, fungal and cockroach) and protease-poor (eg, cat dander) allergens, making protease activity an unlikely trigger. House dust mite extracts have been shown to contain double-stranded RNA with activity on TLR3, but TLR3 deficiency had no effect on downstream caspase activation or IL-331-175/178 production.8She L. Alanazi H.H. Yan L. Zou Y. Sun Y. Dube P.H. et al.Immune sensing of aeroallergen-associated double-stranded RNA triggers an IFN response and modulates type 2 lung inflammation.J Immunol. 2019; 203: 2520-2531Crossref PubMed Scopus (7) Google Scholar Thus, the mechanism of direct allergen sensing and the activation of ripoptosome triggered by allergens in the epithelium remains unclear. Beyond allergen sensing, the article by Brusilovsky et al5Brusilovsky M. Rochman M. Rochman Y. Caldwell J.M. Mack L.E. Felton J.M. et al.Environmental allergens trigger type 2 inflammation through ripoptosome activation.Nat Immunol. 2021; 22: 1316-1326Crossref PubMed Scopus (34) Google Scholar raises many questions about the roles of different IL-33 cleavage products. Cleavage within the protease-sensitive central region of IL-331-270 generates and frees the highly active and mature form IL-33112-270 containing the intact C-terminal IL-1–like domain. If IL-331-175/178 is also released after allergen exposure, what is the relative role or importance of each of these IL-33 cleavage products? The IL-331-175/178 fragment appeared to have activity similar to that of the full-length pIL-33, suggesting that IL-331-175/178 is less active than the IL-33112-270 fragment. Could these different IL-33 fragments play sequential roles in allergen-induced inflammation, with IL-331-175/178 initiating and IL-33112-270 amplifying local allergic inflammation? Alternatively, the IL-33 cleavage products may have different functions. IL-331-175/178 was released in complex with histones, which have innate antimicrobial activities and activate alternative inflammatory pathways.9Doolin T. Amir H.M. Duong L. Rosenzweig R. Urban L.A. Bosch M. et al.Mammalian histones facilitate antimicrobial synergy by disrupting the bacterial proton gradient and chromosome organization.Nat Commun. 2020; 11: 3888Crossref PubMed Scopus (39) Google Scholar It is tempting to speculate that allergen-induced release of IL-331-175/178 may have dual actions by activating innate inflammatory responses as well as by directly killing allergen-accompanying microbes. Given the similar size of both cleavage products (which differ in size by 2-3 kDa) and their dual detection by commercial antibodies, determining the relative contributions of IL-331-175/178 and IL-33112-270 may require the development of specific antibodies for detection. But as the article by Brusilovsky et al5Brusilovsky M. Rochman M. Rochman Y. Caldwell J.M. Mack L.E. Felton J.M. et al.Environmental allergens trigger type 2 inflammation through ripoptosome activation.Nat Immunol. 2021; 22: 1316-1326Crossref PubMed Scopus (34) Google Scholar indicates, understanding the complexity of IL-33 regulation and release may be central to understanding allergic immunity.