Autophagy-enhancing strategies to promote intestinal viral resistance and mucosal barrier function in SARS-CoV-2 infection

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of Coronavirus disease 19 (COVID-19), continues to circulate globally despite the widespread vaccination and therapeutics like Paxlovid, remdesivir, and molnupiravir. COVID-19 is associated with both respiratory and gastrointestinal manifestations, with persistent intestinal pathology contributing to the post-COVID-19 condition. We have previously demonstrated the antiviral activity of autophagy-blocking drugs, such as Berbamine dihydrochloride, against intestinal SARS-CoV-2 acquisition. In addition, the autophagy blockers restored the barrier function of infected intestinal epithelium. In this addendum, using human intestinal organoids, we present evidence for a protective role of intrinsic higher levels of autophagy flux in limiting intestinal SARS-CoV-2 infection. Pharmacological treatment with Akt inhibitor MK-2206 hydrochloride suppressed viral entry into the intestinal epithelium. This antiviral effect of MK-2206 was shown to be dependent on Synaptosomal-associated protein 29-dependent (SNAP-29)-mediated autophagy flux. Furthermore, extrinsically enhanced autophagy with MK-2206 also prevented SARS-CoV-2-induced intestinal barrier damage. Our findings thus underscore the intricate role of autophagy pathways in the dissemination and pathogenesis of intestinal SARS-CoV-2, highlighting the therapeutic potential of host-directed therapies targeting autophagy to intervene in COVID-19-associated sequelae and improve intestinal health.

Keywords: Antiviral immunity; SARS-CoV-2; autophagy; epithelial cells; gastrointestinal pathology; host-directed therapy; human intestinal organoids; post-COVID-19 condition.

Figure 1.

Intrinsic higher number of autophagosomes correlates with reduced intestinal SARS-CoV-2 infection. (A,B) viral infection of confluent intestinal epithelial monolayers exposed to SARS-CoV-2 pseudovirus for 5 days, determined by luciferase activity (relative light units, RLU). (A) Data are mean ± SD of n = 6 donors represented by open circles; *p < 0.05. (B) Stratification of viral infection levels in genotyped intestines: ATG16L1 rs6861(TT) donors (red colored circles) versus ATG16L1 rs6861 (CC) donors (blue-colored circles); *p < 0.05. (C) Flow-cytometric representation of autophagy flux as measured by saponin extraction of intracellular LC3-II accumulation (in fluorescent intensity, FI) upon incubation with 100 nM bafilomycin A1 for 4 h in ATG16L1 rs6861 (TT, red, versus CC, blue) genotyped intestinal epithelial cells. Data are representative of n=2 donors per genotype.

Figure 2.

Therapeutic enhancement of autophagy by MK-2206 suppresses intestinal SARS-CoV-2 infection via SNAP29-mediated autophagic degradation. (A) Viral infection of Caco-2 cells upon transfection with control siRNA or siATG13, followed by exposure to pseudotyped SARS-CoV-2 for 72 h, determined by luciferase activity (RLU). Open circles represent individual replicates, n = 3; *p < 0.05. (B) Schematic representation of the mechanism by which Akt inhibitor MK-2206 impacts its molecular target to pharmaceutically increase autophagy flux. (C,D) Percentage cell viability upon treatment of Caco-2 cell line (C) or ATG16L1 rs6861 (CC) genotyped intestinal epithelial monolayers (D) with MK-2206, determined by ATP-based CellTiter-Glo assay. Cells were treated with an optimized concentration of 5 μM MK-2206 or left untreated for 72 h (C) or 60 h (D); open circles represent individual replicates, n = 3. (E–G) Autophagy flux in U87.mCherry-GFP-LC3 autophagy reporter cell line upon incubation with 5 μM MK-2206 or 200 nM bafilomycin A1 as control, or left untreated for 24 or 48 h. Percentage GFP signal reduction is representative of autophagic flux, determined by imaging flow cytometry after 24 h (E) and flow cytometry after 48 h (F, G). (E) Representative imaging flow cytometry overlays for brightfield, mCherry, and GFP signals, three individual cells shown per condition are representative of n = 3 replicates (F) Representative flow cytometry plots, and (G) the quantification, data are mean ± SD of n = 3 replicates represented by open circles; ****p < 0.0001, ***p < 0.001. (H) Schematic representation of intestinal epithelial monolayer pre-treatment with MK-2206 and subsequent infection with SARS-CoV-2 pseudovirus. (I) Viral infection of confluent ATG16L1 rs6861 (CC) genotyped intestinal epithelial monolayers pre-treated with 5 μM MK-2206 or left untreated for 24 h, followed by infection with SARS-CoV-2 pseudovirus for 5 days, determined by luciferase activity (RLU). Data were normalized to untreated and are mean ± SD of n = 3 donors represented by open circles; *p < 0.05. (J) Viral infection of Caco-2 cells upon transfection with control siRNA or siSNAP29, followed by treatment with 5 μM MK-2206 and subsequently exposed to pseudotyped SARS-CoV-2 for 72 h, determined by luciferase activity (RLU). Circles represent individual replicates, n = 2 replicates.

Figure 3.

MK-2206 treatment hampers SARS-CoV-2-mediated disruption of the intestinal barrier. (A) Representative confocal microscopy imaging of the changes in morphology of ATG16L1 rs6861 (CC) genotyped intestinal epithelial monolayers upon pre-treatment with 5 μM MK-2206 or left untreated for 24 h, followed by exposure to SARS-CoV-2 pseudovirus for 5 days. Actin (Phalloidin) is visualized in green and nuclei (DAPI) in blue. Scale bar = 15 μm, images representative of n = 2 donors. (B) Quantitative analysis of fluorescence intensity of actin staining, as depicted in (A). Fluorescent intensity was determined at the original magnification by measuring the mean gray value with ImageJ software. Data are mean ± SD of 5 fields of view of a representative donor, represented by open circles, n = 2 donors; ***p < 0.0001.