A549 cells for COVID-19 studies

A549 cells are commonly used for the study of respiratory infections. SARS-CoV-2, the causative agent of coronavirus disease-19 (COVID-19), gains entry into the human lung epithelium through the interaction of the virus Spike protein with the host ACE2 and TMPRSS2 receptors [1-3]. A549 cells express negligible levels of ACE2 and no TMPRSS2 and thus are poorly permissive to infection by SARS-CoV-2 [2-4, in-house data]. Stable transfection of A549 cells with the human ACE2 and TMPRSS2 genes increases their permissivity SARS-CoV-2 (in-house data).

A549 cells for inflammasome studies

A549 cells endogenously express proteins involved in the inflammasome signaling including ASC, caspase-1, and Gasdermin D/E. However, they are unable to mount inflammasome responses because of the lack of expression of some sensors/co-sensors. Notably, they lack expression of NLRP1 and NLRP3, two sensors that are strictly dependent on the ASC adaptor to bridge their interaction with pro-caspase-1 (in-house data) [5-7].

The ASC adaptor

ASC (apoptosis-associated speck-like protein containing a CARD domain, aka PYCARD is a protein adaptor important in canonical inflammasome responses [8,9]. ASC's bipartite composition, consisting of one PYD (pyrin domain) and one CARD (caspase recruitment domain) domain, allows the recruitment of the CARD-containing pro-caspase-1 to canonical inflammasome sensors [8]. In resting cells, ASC is present in a soluble and diffuse form, both in the cytoplasm and nucleus. Upon inflammasome activation, ASC molecules form a single large, micrometer-sized, ‘speck’ per cell, thus concentrating caspase-1 activation sites [9].

The NLRP1 sensor

NLRP1 (aka NALP1) was the first described inflammasome sensor of the Nod-Like Receptors (NLR) family [6-9]. Among other NLR, NLRP1 exhibit a unique structure with a PYRIN, NACHT and LRR in its N-terminal domain followed by a FIIND (function-to-find domain) and a CARD domain in its C-terminal region. The FIIND domain autocleaves NLRP1 into two proteins that persist connected in a non-covalent manner and in close association with the dipeptidyl peptidases DPP-8/-9, thereby maintaining NLRP1 in an auto-inhibitory state [6,10]. It took two decades to uncover the activation mechanisms of human NLRP1. The actual model relies on a “functional degradation” pathway [10], in which danger signals trigger proteasomal degradation of the NLRP1 N-terminal domain leading to the release of the C-terminal CARD containing domain, the active form of NLRP1. Subsequently, the ASC adaptor is recruited inducing the assembly of the NLRP1 inflammasome through oligomerization with pro-capase-1 [6-9].
So far, four activators of the NLRP1 pathway have been reported [5, 11-19]:
The firstly identified pathway relies on pharmacological inhibition of the cellular proteases DPP-8/-9 by Val‐boroPro. While the small molecule Val‐boroPro (Talabostat) was known since 2003 to stimulate anti-cancer immune responses [19], only recent work has identified its mechanism of action through activation of the NLRP1 inflammasome [11-13]. However, the physiological settings that trigger NLRP1 activation through DPP-8/DPP-9 inhibition are currently unknown.
The second pathway suggests that NLRP1 is acting as a decoy receptor aiming to detect viral proteases activities. Indeed, enteroviral 3C proteases [14-15] and coronaviral 3CL proteases [5], which are essential for maturation of viral proteins, have been shown to cleave NLRP1 within its N-terminal domain. Cleavage creates an unstable neo-N terminus that is rapidly degraded by the proteasome pathway, leading to NLRP1 inflammasome activation. In the context of SARS-CoV-2 infection, NLRP1 activation by the viral 3CL protease (NSP5) has been linked to lung epithelial cell death and inflammatory cytokine release, suggesting an important role of NLRP1 in COVID-19 pathophysiology [5].

Recent work has identified a third pathway by demonstrating that NLRP1 senses long dsRNA generated in the context of viral infection by Semliki Forrest Virus (SFV) [16]. Interestingly, this pathway can be mimicked by transfection of high molecular weight poly (I:C). It was found that long dsRNAs (>500 bp) bind directly to NLRP1 (NACHT-LRR domain). This interaction activates the ATPase, NACHT domain of NLRP1 leading to its activation following proteasome-dependent degradation of the N-terminal part of NLRP1 [16].

Lastly, Zhong’s team have discovered that human NLRP1 also senses the ultraviolet B (UVB)- and toxin-induced ribotoxic stress response (RSR). Mechanistically, RSR leads to the direct phosphorylation of NLRP1 N-terminal region by the MAP3K ZAK-α and the MAPK p38 hence driving NLRP1 destabilisation and inflammasome activation [17].
Additionally, NLRP1 polymorphism within human population is linked to several rare genetic diseases and autoimmune diseases such as psoriasis, vitiligo or type-1 diabetes [5-6, 20] and thus is considered an important therapeutic target.

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8. Mathur A. et al., 2017. Molecular mechanisms of inflammasome signaling. J. Leuk. Biol. 103:233.
9. Hoss F. et al., 2017. Assembly and regulation of ASC specks. Cell. Mol. Life Sci. 74:1211.
10. Sandstrom et al., 2019. Functional degradation: A mechanism of NLRP1 inflammasome activation by diverse pathogen enzymes. Science. 2019 Apr 5;364(6435):eaau1330. doi: 10.1126/science.aau1330. Epub 2019 Mar 14. PMID: 30872533; PMCID: PMC6532986.
11. Okondo et al., 2017. DPP8 and DPP9 inhibition induces pro-caspase-1-dependent monocyte and macrophage pyroptosis. Nat Chem Biol. 2017 Jan;13(1):46-53. doi: 10.1038/nchembio.2229. Epub 2016 Nov 7. PMID: 27820798; PMCID: PMC5477230.
12. Taabazuing et al., 2017. Pyroptosis and Apoptosis Pathways Engage in Bidirectional Crosstalk in Monocytes and Macrophages. Cell Chem Biol. 2017 Apr 20;24(4):507-514.e4. doi: 10.1016/j.chembiol.2017.03.009. Epub 2017 Apr 6. PMID: 28392147; PMCID: PMC5467448.
13. Zhong et al., 2018. Human DPP9 represses NLRP1 inflammasome and protects against autoinflammatory diseases via both peptidase activity and FIIND domain binding. J Biol Chem. 2018 Dec 7;293(49):18864-18878. doi: 10.1074/jbc.RA118.004350. Epub 2018 Oct 5. PMID: 30291141; PMCID: PMC6295727.Robinson et al., 2022.
14. Tsu et al., 2021. Diverse viral proteases activate the NLRP1 inflammasome. Elife. 2021 Jan 7;10:e60609. doi: 10.7554/eLife.60609. PMID: 33410748; PMCID: PMC7857732.
15. Robinson et al., 2020. Enteroviral 3C protease activates the human NLRP1 inflammasome in airway epithelia. Science. 2020 Dec 4;370(6521):eaay2002. doi: 10.1126/science.aay2002. Epub 2020 Oct 22. PMID: 33093214.
16. Bauernfried et al., 2022. Human NLRP1 is a sensor for double-stranded RNA. Science. 2021 Jan 29;371(6528):eabd0811. doi: 10.1126/science.abd0811. Epub 2020 Nov 26. PMID: 33243852.
17. Robinson et al., 2022. ZAKα-driven ribotoxic stress response activates the human NLRP1 inflammasome. Science. 2022 Jul 15;377(6603):328-335. doi: 10.1126/science.abl6324. Epub 2022 Jul 14. PMID: 35857590.
18. Walsh et al., 2003. Val-boroPro accelerates T cell priming via modulation of dendritic cell trafficking resulting in complete regression of established murine tumors. PLoS One. 2013;8(3):e58860. doi: 10.1371/journal.pone.0058860. Epub 2013 Mar 12. PMID: 23554941; PMCID: PMC3595211.
19. Adams et al., 2004. PT-100, a small molecule dipeptidyl peptidase inhibitor, has potent antitumor effects and augments antibody-mediated cytotoxicity via a novel immune mechanism. Cancer Res. 2004 Aug 1;64(15):5471-80. doi: 10.1158/0008-5472.CAN-04-0447. PMID: 15289357.
20. Levandowski et al., 2013. NLRP1 haplotypes associated with vitiligo and autoimmunity increase interleukin-1β processing via the NLRP1 inflammasome. Proc Natl Acad Sci U S A. 2013 Feb 19;110(8):2952-6. doi: 10.1073/pnas.1222808110. Epub 2013 Feb 4. PMID: 23382179; PMCID: PMC3581876.

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