Gastrointestinal symptoms and fecal shedding of SARS-CoV-2 RNA are frequently observed in COVID-19. However, it is unclear whether SARS-CoV-2 replicates in the human intestine and contributes to possible fecal-oral transmission. Here, we report productive infection of SARS-CoV-2 in ACE2 + mature enterocytes in human small intestinal enteroids. Expression of two mucosa-specific serine proteases, TMPRSS2 and TMPRSS4, facilitated SARS-CoV-2 spike fusogenic activity and promoted virus entry into host cells. We also demonstrate that viruses released into the intestinal lumen were inactivated by simulated human colonic fluid, and infectious virus was not recovered from the stool specimens of patients with COVID-19. Our results highlight the intestine as a potential site of SARS-CoV-2 replication, which may contribute to local and systemic illness and overall disease progression. RESULTS SARS-CoV-2 infects human intestinal enteroidsIn the intestine, ACE2 functions as a chaperone for the sodiumdependent neutral amino acid transporter B 0 AT1 (encoded by SLC6A19) on IECs and regulates microbial homeostasis (21,22). ACE2 expression is substantially higher in the small intestine than in all the other organs, including the lung, in both humans and mice (fig. S1, A and B). Given these data, we assessed whether SARS-CoV-2 can infect IECs as a first step for understanding its implications for fecal-oral transmission. We performed single-cell RNA sequencing (scRNA-seq) to capture the global transcriptomics in all IEC subsets in the mouse small intestinal epithelium (Fig. 1A, left). ACE2 mRNA was predominantly seen in Cd26 + Epcam + Cd44 − Cd45 − mature enterocytes (Fig. 1A, right) (23, 24). In addition, bulk RNA-seq revealed that primary human ileum enteroids had substantially higher mRNA levels of all known CoV receptors, including ACE2, than the of 10 that Ace2 high cells are also positive for Cd26 and Epcam but negative for Cd44 and Cd45. (B) Human duodenum enteroids were cultured in the Transwell monolayer system using maintenance (MAINT) or differentiation (DIFF) conditions for 3 days. Monolayers were stained for ACE2 (red) and actin (phalloidin, white). Scale bars, 32 m. (C) Human duodenum enteroids in monolayer, cultured in either maintenance (MAINT) or differentiation (DIFF) conditions, were apically infected with 1.5 × 10 5 plaque-forming units (PFU) of VSV-SARS-CoV-2 [multiplicity of infection (MOI) = 0.3] for 24 hours. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. p.i., post-infection. (D) Human duodenum enteroids in 3D Matrigel were cultured in maintenance (MAINT) medium or differentiation (DIFF) medium for 3 days and infected with 2.2 × 10 5 PFU of VSV-SARS-CoV-2 for 18 hours. Enteroids were stained for virus (green), actin (phalloidin, white), and nucleus (DAPI, blue). Scale bars, 50 m. (E) Same as (C) except that virus titers were measured using a TCID 50 assay instead of viral RNA levels by qPCR. (F) Same as (D) except that human ileum enteroids were used instead. Scale bars, ...

Cholesterol 25-hydroxylase (CH25H) is an interferon (IFN)-stimulated gene that shows broad antiviral activities against a wide range of enveloped viruses. Here, using an IFN-stimulated gene screen against vesicular stomatitis virus (VSV)-SARS-CoV and VSV-SARS-CoV-2 chimeric viruses, we identified CH25H and its enzymatic product 25-hydroxycholesterol (25HC) as potent inhibitors of SARS-CoV-2 replication. Internalized 25HC accumulates in the late endosomes and potentially restricts SARS-CoV-2 spike protein catalyzed membrane fusion via blockade of cholesterol export. Our results highlight one of the possible antiviral mechanisms of 25HC and provide the molecular basis for its therapeutic development.

Despite mounting evidence for SARS-CoV-2 engagement with immune cells, most express little, if any, of the canonical receptor of SARS-CoV-2, ACE2. Here, using a myeloid-cell receptor-focused ectopic expression screen, we identified several C-type lectins (DC-SIGN, L-SIGN, LSECtin, ASGR1, and CLEC10A) and Tweety family member 2 (TTYH2) as glycan-dependent binding partners of the SARS-CoV-2 spike. Except for TTYH2, these molecules primarily interacted with spike via regions outside of the receptor-binding domain. Single-cell RNA-sequencing analysis of pulmonary cells from COVID-19 patients indicated predominant expression of these molecules on myeloid cells. Although these receptors do not support active replication of SARS-CoV-2, their engagement with virus induced robust proinflammatory responses in myeloid cells that correlated with COVID-19 severity. We also generated a bispecific anti-spike nanobody that not only blocked ACE2-mediated infection but also the myeloid receptors-mediated proinflammatory responses. Our findings suggest SARS-CoV-2-myeloid receptor interactions promote immune hyper-activation, which represents potential targets for COVID-19 therapy.

Celiac disease (CD) is a chronic enteropathy elicited by a Th1 response to gluten peptides in the small intestine of genetically susceptible individuals. However, it remains unclear what drives the induction of inflammatory responses of this kind against harmless antigens in food. In a recent work, we have shown that the p31-43 peptide (p31-43) from α-gliadin can induce an innate immune response in the intestine and that this may initiate pathological adaptive immunity. The receptors and mechanisms responsible for the induction of innate immunity by p31-43 are unknown and here we present evidence that this may reflect conformational changes in the peptide that allow it to activate the NLRP3 inflammasome. Administration of p31-43, but not scrambled or inverted peptides, to normal mice induced enteropathy in the proximal small intestine, associated with increased production of type I interferon and mature IL-1β. P31-43 showed a sequence-specific spontaneous ability to form structured oligomers and aggregates in vitro and induced activation of the ASC speck complex. In parallel, the enteropathy induced by p31-43 in vivo did not occur in the absence of NLRP3 or caspase 1 and was inhibited by administration of the caspase 1 inhibitor Ac-YVAD-cmk. Collectively, these findings show that p31-43 gliadin has an intrinsic propensity to form oligomers which trigger the NLRP3 inflammasome and that this pathway is required for intestinal inflammation and pathology when p31-43 is administered orally to mice. This innate activation of the inflammasome may have important implications in the initial stages of CD pathogenesis.