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ACE2-containing defensosomes serve as decoys to inhibit SARS-CoV-2 infection [1]

['Krystal L. Ching', 'Kimmel Center For Biology', 'Medicine At The Skirball Institute', 'New York University Grossman School Of Medicine', 'New York', 'United States Of America', 'Maren De Vries', 'Department Of Microbiology', 'Juan Gago', 'Division Of Epidemiology']

Date: 2022-09 

Extracellular vesicles of endosomal origin, exosomes, mediate intercellular communication by transporting substrates with a variety of functions related to tissue homeostasis and disease. Their diagnostic and therapeutic potential has been recognized for diseases such as cancer in which signaling defects are prominent. However, it is unclear to what extent exosomes and their cargo inform the progression of infectious diseases. We recently defined a subset of exosomes termed defensosomes that are mobilized during bacterial infection in a manner dependent on autophagy proteins. Through incorporating protein receptors on their surface, defensosomes mediated host defense by binding and inhibiting pore-forming toxins secreted by bacterial pathogens. Given this capacity to serve as decoys that interfere with surface protein interactions, we investigated the role of defensosomes during infection by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiological agent of Coronavirus Disease 2019 (COVID-19). Consistent with a protective function, exosomes containing high levels of the viral receptor ACE2 in bronchoalveolar lavage fluid (BALF) from critically ill COVID-19 patients was associated with reduced intensive care unit (ICU) and hospitalization times. We found ACE2+ exosomes were induced by SARS-CoV-2 infection and activation of viral sensors in cell culture, which required the autophagy protein ATG16L1, defining these as defensosomes. We further demonstrate that ACE2+ defensosomes directly bind and block viral entry. These findings suggest that defensosomes may contribute to the antiviral response against SARS-CoV-2 and expand our knowledge on the regulation and effects of extracellular vesicles during infection.

Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: K.C. has received research support from Pfizer, Takeda, Pacific Biosciences, Genentech, and Abbvie. K.C. has consulted for or received honoraria from Vedanta, Genentech, and Abbvie. K.C. is an inventor on U.S. patent 10,722,600 and provisional patent 62/935,035 and 63/157,225. V.J.T. receives research support from Janssen Biotech Inc. V.J.T has consulted for Janssen Research & Development, LLC and have or received honoraria from Genentech and Medimmune. V.J.T. is an inventor on patents and patent applications filed by New York University, which are currently under commercial license to Janssen Biotech Inc. provides research funding and other payments associated with a licensing agreement.

Funding: This work was in part funded by National Institutes of Health (NIH) grants DK093668 (K.C.), HL123340 (K.C.), AI130945 (K.C.), AI140754 (K.C.), DK124336 (K.C.), AI121244 (K.C. and V.J.T.), AI099394 (V.J.T.), AI105129 (V.J.T.), AI137336 (V.J.T.), AI133977 (V.J.T.), AI140754 (V.J.T.), AI149350 (V.J.T.), AI143639 (M.D.), AI139374 (M.D.), CA244775 (L.N.S., NCI/NIH). Further funding was provided by Faculty Scholar grant from the Howard Hughes Medical Institute (K.C.), Crohn’s & Colitis Foundation (K.C.), Kenneth Rainin Foundation (K.C.), Judith & Stewart Colton Center of Autoimmunity (K.C.), NSF Graduate Research Fellowship (K.L.C.), NYU Grossman School of Medicine COVID-19 seed research funds to V.J.T, and a pilot funding from the NYU Langone Health Antimicrobial-resistant Pathogen Program (B.S. and V.J.T.). NYU Langone’s Cytometry and Sorting Laboratory, Microscopy Laboratory, Genome Technology Center, and Applied Bioinformatics Laboratory are shared resources partially supported by the Cancer Center Support Grant P30CA016087 at the Laura and Isaac Perlmutter Cancer Center. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the respiratory virus responsible for the ongoing global Coronavirus Disease 2019 (COVID-19) pandemic, can cause life-threatening tissue injury to the lung as well as extrapulmonary symptoms that are less understood [ 10 ]. Entry into host cells mainly depends on the binding of receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein to host receptor ACE2. The affinity of SARS-CoV-2 spike for ACE2 is 5 times higher than that of SARS-CoV spike [ 11 ] and 1,000 times higher than hemagglutinin of Influenza A for sialic acid [ 12 ]. Given this dependence on a high affinity cell surface interaction for viral entry, we hypothesized ACE2-containing defensosomes would inhibit SARS-CoV-2 infection through the binding of the spike protein.

In our previous work, we identified exosomes involved in host defense termed defensosomes that mediated protection against bacterial pore-forming toxins that kill target cells, such as α-toxin produced by Staphylococcus aureus [ 9 ]. Recognition of bacterial DNA by toll-like receptor 9 (TLR9) led to increased production of defensosomes decorated by ADAM10, the host cell surface target of α-toxin, in a manner dependent on ATG16L1 and other components of the membrane trafficking pathway of autophagy. We further demonstrated that ADAM10 + defensosomes serve as decoys that bind α-toxin and prevent cytotoxicity in cell culture and animal models [ 9 ]. Therefore, in addition to mediating intercellular communication by transferring signaling molecules between cells, exosomes can also promote host defense through a surface interaction with bacterial proteins. Whether defensosomes are deployed during a viral infection remains unclear.

Exosomes are a subgroup of single membraned vesicles 40 to 120 nm in diameter secreted by virtually every cell type. Cargo molecules ranging from noncoding RNAs to proteins involved in signal transduction are incorporated during exosome biogenesis, which involves exocytosis of endosomal structures [ 1 ]. As such, exosomes mediate intercellular communication events involved in cell proliferation, migration, and cancer [ 2 ]. A role in host defense is supported by the finding that exosomes can deliver nucleic acids and other immunogenic moieties from infected cells that elicit interferon (IFN) responses or promote antigen presentation by target cells [ 3 – 8 ]. However, how exosomes are regulated in response to infections remains unclear. A better understanding may inform strategies that seek to use extracellular vesicles as biomarkers or therapeutic agents for infectious disease.

Results

ACE2+ defensosomes neutralize SARS-CoV-2 virions Our finding that ACE2+ exosomes in patient BALF display an inverse correlation with length of hospital stay raised the possibility that they play a role during viral infection akin to our previous description of defensosomes during bacterial toxinosis. Therefore, we tested whether addition of exosomes can protect Vero E6 cells, which are highly susceptible to SARS-CoV-2 infection (Fig 3A). Exosomes were isolated from A549ACE2+ cells (Fig 3B) and mixed with human SARS-CoV-2 USA WA1/2020 isolate at an MOI of 0.01. We included a polyclonal neutralizing antibody targeting spike (nAb) as a positive control. Addition of ACE2+ exosomes led to a dose-dependent reduction in viral nucleoprotein (NP) staining by immunofluorescence microscopy at 24 h post-infection (Figs 3C and 3D and S2A), whereas ACE2− exosomes from untransduced control A549s failed to inhibit infection. Also, addition of the microvesicle fraction that does not contain exosomes (5,000xg) did not decrease infection levels (Fig 3C). We also tested neutralization in human airway epithelial cultures (HAECs), an air-liquid interfact (ALI) model that contains all major cell types found in the bronchial epithelium (basal, ciliated, secretory), organized in pseudostratified architecture [34]. Addition of ACE2+ exosomes mixed with SARS-CoV-2 to the apical side (“lumen”) blocked infection of HAECs, while ACE2− exosomes did not show any significant effect (Figs 3E and S2B). PPT PowerPoint slide

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TIFF original image Download: Fig 3. ACE2+ exosomes protect against SARS-CoV-2 infection. (A) Workflow for exosome neutralization assay used to assess protective effects of mixing SARS-CoV-2 and exosomes. Created with BioRender.com. (B) Representative histogram of cell-surface ACE2 on A549 and A549ACE2 cells. (C) Representative immunofluorescence images of Vero E6 cells infected with SARS-CoV-2, pellet from 5,000xg spin, approximately 20 K exosomes from ACE2− cells, approximately 20 K exosomes from ACE2+ cells, neutralizing antibody, or with media alone stained for viral N protein. Scale bar: 0.625 mm, (D) Percentage of infected Vero E6 cells based on positive staining for N protein after infection with SARS-CoV-2 mixed with exosomes isolated from A549ACE2 (teal) or untransduced A549 cells (black). Compiled means ± SEM from 5 experiments, values normalized to infection with SARS-CoV-2 alone. (E) Representative immunofluorescence images of HAECs infected with mock (media only control), SARS-CoV-2 alone, approximately 11 K (1) or approximately 22 K (2) ACE2+ exosomes with virus, approximately 150 K (3) ACE2− exosomes with virus, and neutralizing antibody with virus (nAb). Images were taken 72 hpi. Scale bar: 0.156 mm. (F) Representative flow cytometry plots of exosomes isolated from Vero E6 cells stimulated with CpG. FSC: forward scatter, SSC: side scatter. (G) Quantification of exosomes from Vero E6 cells and Vero E6 cells transduced with a non-targeting shRNA for ATG16L1 stimulated with PBS (n = 4) or 1 uM CpG-A (n = 4) for 16 h. (H) Quantification of infected cells after infection of Vero E6 cells with SARS-CoV-2 only, SARS-CoV-2 mixed with supernatants of cells pretreated overnight with CpG-A (induced), or SARS-CoV-2 added to CpG-A pretreated cells following removal of supernatant (wash). Exosomes: CD9, CD63, CD81+, PKH67+ events. Means ± SEM of at least 2 independent experiments. G Two-way ANOVA with Dunnett’s post-test compared to NT PBS. H One-way ANOVA with Dunnett’s post-test compared to PBS. Error bars represent the mean ± SEM of at least 2 independent experiments, with measurements taken from distinct samples. Underlying data can be found in S1 Data. ** P ≤ 0.01; **** P ≤ 0.0001. HAEC, human airway epithelial culture; hpi, hours post infection; ns, not significant; SARS‑CoV‑2, Severe Acute Respiratory Syndrome Coronavirus 2. https://doi.org/10.1371/journal.pbio.3001754.g003 We next tested whether inducing exosome production protects against viral infection without the need of adding exogenous exosomes from donor cells. We found that pretreatment with CpG-A, which we confirmed induces exosome production by Vero E6 cells in a manner dependent on ATG16L1 (Fig 3F and 3G), led to a reduction in cells infected with SARS-CoV-2 (Fig 3H). Removing the exosome-containing supernatant prior to infection led to similar levels of SARS-CoV-2 infection as unstimulated cells (Figs 3H and S2C). Further, knockdown of ATG16L1 in either A549 cells or Vero E6 cells did not impact viral replication levels in either cell type (S2D and S2E Fig). Vero E6 cells generally cannot produce IFNs [35,36], although hantavirus infection was shown to elicit IFNλ from these cells [37]. To formally demonstrate that the exosome fraction of the supernatant from CpG-A-treated Vero E6 cells contains the antiviral activity, we biochemically isolated exosomes and tested whether they inhibit SARS-CoV-2 infection of target cells. The enriched exosomes were sufficient to reduce viral infection, and the degree of inhibition was even greater than the unmanipulated supernatant. Together, these results show that defensosomes can inhibit SARS-CoV-2 infection.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001754

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