SARS-CoV-2 targets the lysosome to mediate airway inflammatory cell death

ABSTRACT

As the coronavirus disease 2019 (COVID-19) pandemic continues to wreak havoc, researchers around the globe are working together to understand how the responsible agent - severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) damages the respiratory system and other organs. Macroautophagy/autophagy is an innate immune response against viral infection and is known to be manipulated by positive-strand RNA viruses, including SARS-CoV-2. Nevertheless, the link between autophagic subversion and cell death or inflammation in COVID-19 remains unclear. Emerging evidence suggests that SARS-CoV-2 could trigger pyroptosis, a form of inflammatory programmed cell death characterized by the activation of inflammasomes and CASP1 (caspase 1) and the formation of transmembrane pores by GSDMD (gasdermin D). In this connection, autophagic flux impairment is a known activator of inflammasomes. This prompted us to investigate if SARS-CoV-2 could target autophagy to induce inflammasome-dependent pyroptosis in lung epithelial cells.

Abbreviations: ATP6AP1: ATPase H+ transporting accessory protein 1; CASP1: caspase 1; COVID-19: coronavirus disease 2019; GSDMD: gasdermin D; IL1B: interleukin 1 beta; IL18: interleukin 18; KRT 18: keratin 18; NLRP3: NLR family pyrin domain containing 3; NOD: nucleotide oligomerization domain; NSP6: non-structural protein 6; TFEB: transcription factor EB; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2

KEYWORDS:

• Cell death
• Covid
• lung
• NSP
• pyroptosis

As the coronavirus disease 2019 (COVID-19) pandemic continues to wreak havoc, researchers around the globe are working together to understand how the responsible agent – severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) damages the respiratory system and other organs. Macroautophagy/autophagy is an innate immune response against viral infection and is known to be manipulated by positive-strand RNA viruses, including SARS-CoV-2. Nevertheless, the link between autophagic subversion and cell death or inflammation in COVID-19 remains unclear. Emerging evidence suggests that SARS-CoV-2 could trigger pyroptosis, a form of inflammatory programmed cell death characterized by the activation of inflammasomes and CASP1 (caspase 1) and the formation of transmembrane pores by GSDMD (gasdermin D). In this connection, autophagic flux impairment is a known activator of inflammasomes. This prompted us to investigate if SARS-CoV-2 could target autophagy to induce inflammasome-dependent pyroptosis in lung epithelial cells.

In our recent article [1], we report that NSP6 (non-structural protein 6) of SARS-CoV-2 binds to the inactive form of ATP6AP1 (an accessory subunit of the lysosomal proton pump) to inhibit its cleavage-dependent activation, leading to lysosome deacidification. The resulting autophagic flux impairment then instigates the NLRP3 (NLR family pyrin domain containing 3) inflammasomes to mediate CASP1 activation, IL1B (interleukin 1 beta) and IL18 (interleukin 18) maturation, and pyroptosis (Figure 1). Interestingly, NSP6 L37F, a variant associated with asymptomatic COVID-19, exhibits a weakened ability to interact with ATP6AP1 to deacidify the lysosome. In this respect, infection of cultured lung epithelial cells with live SARS-CoV-2 results in impairment of autophagic flux, activation of NLRP3 inflammasomes, and pyroptosis. With relevance to therapy, our study shows that pharmacological restoration of autophagic flux by 1α,25-dihyroxyvitamin D₃ (the active form of vitamin D₃), metformin (an AMP-activated protein kinase/AMPK-activating antidiabetic agent) and polydatin (a TFEB [transcription factor EB]-activating phytochemical) or inhibition/silencing of NLRP3 and CASP1 can mitigate NSP6-induced pyroptosis. Collectively, our data supports the hypothesis that NSP6 is a virulence factor of SARS-CoV-2 that can induce airway epithelial cell pyroptosis. Importantly, the pro-pyroptotic action of NSP6 is amenable to pharmacological exploitation.

Figure 1. Schematic diagram depicting the pro-pyroptotic mechanism of SARS-CoV-2 NSP6 in lung epithelial cells. NSP6 binds to the full-length ATP6AP1 to inhibit its cleavage-dependent activation, resulting in lysosome deacidification, autophagic flux impairment, NLRP3 inflammasome activation, and eventually pyroptotic cell death. 1α,25-dihyroxyvitamin D₃ (1α,25[OH]₂VD₃), metformin, and polydatin block the pro-pyroptotic action of NSP6 by restoring the autophagic flux. CT, C-terminal; NT, N-terminal; V-ATPase, vacuolar-type ATPase.

Given that the lysosome is a target of NSP6, it is pathologically plausible that any preexisting condition that diminishes lysosomal activity could exacerbate SARS-CoV-2-induced cell death. Concordantly, we found that disrupting the lysosomal acidic environment by chloroquine intensifies inflammasome activation and pyroptosis induced by NSP6. This may explain why COVID-19 patients treated with hydroxychloroquine, a chloroquine derivative, during the earlier waves of the pandemic exhibited a higher mortality. Besides the use of hydroxychloroquine, advanced age, obesity, and diabetes mellitus are known risk factors for severe disease and death in COVID-19. These three conditions are also associated with declined lysosomal activity. Further functional experiments are therefore warranted to examine the role of lysosomal dysfunction in the pathogenic interactions between SARS-CoV-2 and these risk factors.

From a mechanistic perspective, we think that autophagic flux impairment alone cannot fully explain the pro-pyroptotic action of NSP6. In this respect, NSP6 overexpression cannot further impair autophagic flux in the presence of bafilomycin A₁. However, it can result in additional CASP1 activation and IL1B and IL18 maturation. There is also a coordinated transcriptomic response characterized by generalized overexpression of different components in the inflammasome-related NOD-like receptor signaling pathway (e.g., NLRP3, CASP1, IL1B) upon SARS-CoV-2 infection. It is therefore plausible that NSP6 or other viral components could engage additional signaling mediators (e.g., transcription factors, histone modifiers, DNA methyltransferases/demethylases) to reprogram the host transcriptome to prepare the cells for entering a pyroptosis-prone state.

Pyroptosis is a type of programmed cell death used by the host to control pathogens. Intriguingly, bats, which are widely considered as the natural host of SARS-CoV-2, have evolved an overall diminished activation of the NLRP3 inflammasomes and CASP1 in response to viral infection. Such dampening of inflammatory signals might allow bats to serve as a reservoir for SARS-CoV-2 without developing any organ damage. In humans, the regulation of NLRP3 inflammasome activity is complex and involves both genetic and dietary factors. In our cohort of COVID-19 patients, severe cases distinctively show elevated serum levels of IL1B, IL18 and intact KRT18/CK18 (keratin 18; a marker of epithelial cell death). We therefore propose that excessive pyroptosis that occurs in severe COVID-19 is an overreaction to the hijacking of the host lysosome-autophagy system by SARS-CoV-2. Dampening of this inflammatory cell death cascade is thus expected to produce therapeutic effects in COVID-19 patients.

The Omicron variant of SARS-CoV-2 is an emerging variant of concern that is reportedly linked to higher transmissibility. Aside from more than 30 changes to the spike protein, two amino-acid changing mutations, namely Δ105-107 and I189V on NSP6, were identified in the Omicron variant. Given that SARS-CoV-2 can exploit the deacidified lysosome for egress, it is plausible that the NSP6 mutations found in the Omicron variant could alter NSP6’s ability to bind to ATP6AP1 for inhibiting the lysosome and therefore influence the rate of viral shedding and thus the transmissibility. Nevertheless, how the changes in NSP6 could affect the downstream pyroptotic pathway and their relationship to the possible changes in the virulence and clinical manifestation of this new variant remain to be fully assessed.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by The TUYF Charitable Trust and Shenzhen Science and Technology Programme [JCYJ20180508161604382].