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Variable susceptibility of intestinal organoid–derived monolayers to SARS-CoV-2 infection

['Kyung Ku Jang', 'Kimmel Center For Biology', 'Medicine At The Skirball Institute', 'New York University Grossman School Of Medicine', 'New York', 'United States Of America', 'Maria E. Kaczmarek', 'Department Of Microbiology', 'Simone Dallari', 'Ying-Han Chen']

Date: 2022-04

Gastrointestinal effects associated with Coronavirus Disease 2019 (COVID-19) are highly variable for reasons that are not understood. In this study, we used intestinal organoid–derived cultures differentiated from primary human specimens as a model to examine interindividual variability. Infection of intestinal organoids derived from different donors with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) resulted in orders of magnitude differences in virus replication in small intestinal and colonic organoid–derived monolayers. Susceptibility to infection correlated with angiotensin I converting enzyme 2 (ACE2) expression level and was independent of donor demographic or clinical features. ACE2 transcript levels in cell culture matched the amount of ACE2 in primary tissue, indicating that this feature of the intestinal epithelium is retained in the organoids. Longitudinal transcriptomics of organoid-derived monolayers identified a delayed yet robust interferon signature, the magnitude of which corresponded to the degree of SARS-CoV-2 infection. Interestingly, virus with the Omicron variant spike (S) protein infected the organoids with the highest infectivity, suggesting increased tropism of the virus for intestinal tissue. These results suggest that heterogeneity in SARS-CoV-2 replication in intestinal tissues results from differences in ACE2 levels, which may underlie variable patient outcomes.

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 an honoraria from Puretech Health, Genentech, and Abbvie. K.C. holds U.S. patent 10,722,600 and provisional patent 62/935,035 and 63/157,225. K.C. is a co-investigator on the Post-Acute Sequelae of SARS-CoV-2 Infection Initiative funded by the NIH (OT2HL161847). J.A. has received research support from BioFire Diagnostics. J.A. reports consultancy fees, honorarium, or advisory board fees from BioFire Diagnostics, Janssen, Abbvie, and Pfizer. J.A. holds U.S. patent 2012/0052124A1.

Introduction

Intestinal organoid cultures have transformed our ability to investigate properties of the human intestinal epithelium. Consisting of organized epithelial cell clusters differentiated from somatic stem cells, intestinal organoids generated from endoscopic pinch biopsies are capable of self-renewal and recreate many of the structural, functional, and molecular characteristics of the tissue of origin [1]. Investigators have exploited these versatile properties of intestinal organoids to study infectious agents that are otherwise difficult to examine, including viruses such as noroviruses [2–4]. Intestinal organoids can also inform our understanding of interindividual differences in disease susceptibility, such as elucidating the mechanisms by which mutations accumulate in patients with colorectal cancer [5,6]. Additionally, we and others have documented substantial heterogeneity in the growth, morphology, viability, and susceptibility to cytokine toxicity of human intestinal organoid lines [1,6–8]. However, how this heterogeneity relates to resistance of the intestinal epithelium to infectious agents remains unclear.

Although Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is primarily associated with dysfunction of the respiratory system, the gastrointestinal tract is also an established target organ in patients with Coronavirus Disease 2019 (COVID-19). As many as 60% of patients present with diarrhea, vomiting, abdominal pain, anorexia, and/or nausea [9–16]. Also, SARS-CoV-2 antigen in intestinal biopsies and viral RNA in the stool are readily detected, even after the virus is undetectable in respiratory samples [17–27]. To a limited extent, virions and infectious particles have been detected in patient intestinal and stool specimens [17,25,28,29]. Although the pathophysiological significance of these observations has not been resolved, the prolonged presence of viral antigens in the gut is likely to impact antibody evolution [17]. Consistent with a potential intestinal tropism, intestinal epithelial cells display robust expression of the SARS-CoV-2 receptor angiotensin I converting enzyme 2 (ACE2) and transmembrane proteases TMPRSS2 and TMPRSS4 that facilitate viral entry [30–32]. Further, human intestinal organoids derived from either somatic stem cells or inducible pluripotent stem cells support SARS-CoV-2 reproduction [32–35]. These studies have shown that ACE2 expression levels can differ based on the differentiation state and anatomical region from which the organoids are derived, but whether this affects the degree of SARS-CoV-2 infection is debated. A broader comparison of gene expression patterns and SARS-CoV-2 infection across organoids from different donors and culture conditions may help interpret the studies that have highlighted the extreme range of ACE2 expression in intestinal tissues associated with demographic and clinical features of individuals, which could have consequences for susceptibility to both viral infections and inflammatory conditions [36–41].

Only small numbers of independent small intestinal and colonic organoid lines were compared to one another in previous studies examining SARS-CoV-2 infection of intestinal epithelial cells. Thus, the importance of the anatomical origin of organoids and other variables remains unclear. We established and differentiated 3D organoid lines from small intestinal and colonic biopsies procured from 12 and 13 donors, respectively, from healthy donors and patients with inflammatory bowel disease (IBD) of both sexes (S1 Table). The expression of ACE2, TMPRSS2, the enterocyte marker of differentiation APOA1, and representative interferon-stimulated genes (ISGs) ISG15, OASL, and MX2 were significantly higher in small intestinal and colonic organoid lines cultured in 3D differentiation media (3DD) compared with those cultured in expansion media (3DE) that maintains organoids in an undifferentiated state (S1A, S1B, and S1D–S1G Fig). TMPRSS4 expression was similar in both conditions (S1C Fig). Donor-to-donor variability in expression of these genes may reflect the inflammatory environment from which the stem cells were procured. However, 3DE organoids derived from IBD and non-IBD donors displayed comparable gene expression patterns except the decreased OASL expression in IBD donor-derived colonic 3DE organoids (S1H Fig). Intestinal organoids can be grown as differentiated monolayers to expose the apical side and facilitate viral entry. We found that the level of ACE2, TMPRSS2, TMPRSS4, APOA1, ISG15, OASL, and MX2 expression in organoid-derived 2D monolayers correlated well with 3DD organoids generated from the same donor (Fig 1A–1D), suggesting that organoid lines retain their intrinsic gene expression patterns independent of these 2 culturing conditions. In addition, we found that monolayers exhibited the highest ACE2 and TMPRSS2 expression among the culture conditions we examined (Fig 1E and 1F), whereas 3DD organoids showed the highest TMPRSS4, APOA1, ISG15, OASL, and MX2 expression (Fig 1G–1K). Therefore, we used the monolayer model for subsequent analyses.

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TIFF original image Download: Fig 1. Heterogeneous gene expression patterns in small intestinal and colonic organoids. (A–D) Correlation analysis of ACE2 and TMPRSS2 (A), TMPRSS4 and APOA1 (B), ISG15 and OASL (C), or MX2 (D) expression in human intestinal (circle) and colonic (rectangle) organoids cultured as monolayers grown in differentiation media for 7 days with those cultured as human 3D organoids grown in differentiation media (3DD) for 7 days. (E–K) RT-PCR data comparing ACE2 (E), TMPRSS2 (F), TMPRSS4 (G), APOA1 (H), ISG15 (I), OASL (J), and MX2 (K) expression in human 3D organoids grown with expansion media (3DE), 3DD, and monolayers grown in differentiation media for 7 days. Data points are mean of at least 2 technical replicates of individual organoid lines. Bars represent mean ± SEM, and at least 2 independent experiments were performed. Underlying data can be found in S1 Data. P, P value; r, Pearson correlation coefficient. **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001 by simple regression analysis in A–D and paired t test, 2 tailed in E–K. ACE2, angiotensin I converting enzyme 2; RT-PCR, reverse transcription PCR. https://doi.org/10.1371/journal.pbio.3001592.g001

We investigated whether ACE2, TMPRSS2, or TMPRSS4 expression differed between small intestinal and colonic organoid–derived monolayers. Although ACE2 expression did not differ, TMPRSS2 and TMPRSS4 expression were higher, and APOA1 was decreased in colonic monolayers (S1I–S1L Fig). Five pairs of the small intestinal and colonic organoid lines were generated from the same individual. We found that gene expression patterns were generally the same when comparing small intestinal and colonic monolayers from the same donor (S2A Fig). The lack of correlation between ACE2 or TMPRSS2 expression and APOA1 expression suggested that heterogeneous ACE2 and TMPRSS2 levels were not an artifact caused by insufficient differentiation (S2B and S2C Fig). When we segregated the data based on disease status or sex, the only difference we observed was decreased ACE2 and APOA1 expression in colonic monolayers derived from IBD patients compared with non-IBD donors (S2D–S2F Fig). The expressions of ISG15, OASL, and MX2 were also not correlated with disease status or sex (S2E and S2F Fig), although we note that ISG15 and OASL transcripts were higher in colonic versus small intestinal monolayers (S3A and S3B Fig). These transcripts generally did not correlate with the age of participants (S2 Table).

ACE2 expression differed by as much as 5.9-fold when comparing monolayers with the highest and lowest expression of this gene (Fig 2A). To test whether such differences lead to heterogeneity in viral infection, we infected monolayers with SARS-CoV-2 at a multiplicity of infection (MOI) of 4 for 72 hours. Remarkably, the amount of virus detected in the supernatant of culture media by plaque assay differed by as much as 423-fold (Fig 2B). Immunofluorescence microscopy analyses of ACE2 and SARS-CoV-2 nucleoprotein (NP) in representative monolayers confirmed these findings—SI1 and C1 (susceptible small intestinal and colonic monolayers, respectively, with high ACE2 transcript levels) displayed higher degrees of ACE2 and NP staining compared with SI10 and C8 (resistant small intestinal and colonic monolayers, respectively) (Fig 2C and 2D). Indeed, SARS-CoV-2 infection correlated with ACE2 and TMPRSS2 expression, but not TMPRSS4 and APOA1 expression (Fig 2E–2H). We validated these findings based on relative ACE2 transcript levels by enumerating absolute RNA copy numbers. The strong correlation of high copy numbers of ACE2 (2.9 × 105 to 1.7 × 106 transcripts/μg of RNA) with SARS-CoV-2 infection supported the relationship between susceptibility to infection and ACE2 expression (S3D Fig). The amount of virus recovered from monolayers was comparable when the data were segregated by the tissue location, the disease status, or sex of the donors (S3E Fig). Similarly, virus production did not correlate with donor age (S3F Fig).

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TIFF original image Download: Fig 2. Differential susceptibility of intestinal organoid–derived monolayers correlates with ACE2 and TMPRSS2 expression. (A) RT-PCR analysis of ACE2 expression in small intestinal (SI1-SI12) and colonic (C1-C13) monolayers. (B) PFUs determined by virus titration on Vero E6 cells of supernatant from monolayers at 72 hours postinfection with SARS-CoV-2. (C) Representative immunofluorescence microscopy images showing co-staining of DAPI (blue), ACE2 (green), and SARS-CoV-2 NP (red) in SARS-CoV-2 infected SI1, SI10, C1, and C8 monolayer lines. An image of uninfected C1 is shown as a representative uninfected condition and a Matrigel-coated well without cells is shown as a control for background fluorescence. (D) Total intensity of NP and ACE2 normalized with cell counts of SI1, SI10, C1, and C8. (E–H) Correlation of SARS-CoV-2 PFU with ACE2 (E), TMPRSS2 (F), TMPRSS4 (G), or APOA1 (H) expression among monolayers. Data points in A, B, and D are each technical replicate, and data points in E–H are the mean of at least 2 technical replicates of individual organoid lines. Bars represent mean ± SEM, and at least 2 independent experiments were performed. Underlying data can be found in S1 Data. P, P value; r, Pearson correlation coefficient. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001 by simple regression analysis in E–H. ACE2, angiotensin I converting enzyme 2; NP, nucleoprotein; PFU, plaque-forming unit; RT-PCR, reverse transcription PCR; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; SI, small intestine. https://doi.org/10.1371/journal.pbio.3001592.g002

The association between susceptibility to SARS-CoV-2 infection and ACE2 expression was clear in most cases, but there were outliers for the colonic monolayers. For instance, C7 and C8 have moderate to high levels of ACE2 but low levels of virus production (Fig 2A and 2B). Thus, we investigated whether other factors may contribute to differential SARS-CoV-2 infectivity. Several polymorphisms are predicted to alter the stability of ACE2 or alter its affinity to the SARS-CoV-2 spike (S) protein [42–45]. However, the sequence of the ACE2 coding region of C7 and C8 were identical to other donors (S1 Table). Differences in interferon responses may also contribute to SARS-CoV-2 infectivity, where higher ISG levels are predicted to confer protection against viral infection [46–48]. Baseline ISG15, OASL, and MX2 expression in C7 and C8 did not explain the lower virus production (S3A–S3C Fig), and we did not observe correlations between ISG15, OASL, or MX2 and SARS-CoV-2 susceptibility (S3G–S3I Fig). Next, we examined ISG expression following stimulation with interferon beta (IFNβ) or interferon lambda 2 (IFNλ2). Although we observed varied levels of ISG induction, they were not associated with reduced viral infection (S4A Fig, S3 Table). Generally, we did not detect an association between the degree to which these ISGs were induced and properties of the donor tissue location, disease status, and age (S4A–S4C Fig, S3 Table). However, small intestinal monolayers from female donors displayed higher ISG expression than male donors following IFNβ or IFNλ2 stimulation (S4D and S4E Fig). ACE2 and TMPRSS2 expression were not altered by IFNβ or IFNλ2 (S5 Fig), indicating that ACE2 and TMPRSS2 are not ISGs in monolayers. We note that this limited survey of transcript level changes does not rule out a potential role for antiviral cytokines, and a comprehensive protein level analyses of immune mediators will be necessary to identify additional mechanisms of resistance.

Our results thus far are consistent with the possibility that ACE2 gene expression is a key determinant of the degree to which the intestinal epithelium of an individual is susceptible to SARS-CoV-2 infection. As organoids are differentiated from primary stem cells and expanded in culture [49,50], it was unclear whether interdonor differences reflect ACE2 levels in the primary tissues. Therefore, we measured ACE2 protein by immunofluorescence microscopy in small intestinal and colonic sections from the same donors corresponding to individual lines of organoid-derived monolayers (Fig 3, S6 Fig). ACE2 staining was restricted to the epithelium and most intense along the apical brush border (villi in the small intestine and top of the crypts in the colon; Fig 3A and 3B, S6A and S6B Fig), consistent with our data and previous studies showing that ACE2 expression is enriched in differentiated enterocytes [32,33]. Primary tissue specimens also displayed heterogeneous ACE2 protein levels (Fig 3C, S6 Fig). The ACE2 mean intensity was decreased in colonic sections of IBD patients, but did not differ when comparing tissue location or sex (Fig 3D). The mean intensity of ACE2 staining in intestinal tissue sections strongly correlated with ACE2 transcript and SARS-CoV-2 levels in monolayers derived from the same individual, but not with age (Fig 3E). Therefore, organoid-derived monolayers retain the variable ACE2 levels from its original tissue.

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TIFF original image Download: Fig 3. Differential ACE2 expression observed in individual intestinal organoid lines are conserved in primary tissue from the same donor. (A and B) Representative ACE2 staining images in primary tissues of terminal ileum (A) and ascending colon (B) from which SI1, SI10, C1, and C13 organoids were established. (C) Mean intensity of ACE2 per area in each field of view from the primary tissues from which small intestinal and colonic organoids were established. (D) Mean intensity of ACE2 by disease (left) or sex (right). (E) Correlation of ACE2 mean intensity with ACE2 expressions (left) and SARS-CoV-2 PFU (middle) among monolayers or participant age (right). Data points in C are the field of views, and data points in D and E are mean of at least 2 technical replicates of individual organoid lines. Bars represent mean ± SEM, and at least 2 independent experiments were performed. Underlying data can be found in S1 Data. Scale bars: 200 μm. P, P value; r, Pearson correlation coefficient. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001 by unpaired t test, 2 tailed in D and simple regression analysis in E. ACE2, angiotensin I converting enzyme 2; F, female; IBD, inflammatory bowel disease; M, male; PFU, plaque-forming unit; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; SI, small intestine. https://doi.org/10.1371/journal.pbio.3001592.g003

To further investigate how susceptible and resistant organoids differ from each other, we selected the 3 colonic monolayer lines that each displayed high infection (HI; C1, C2, and C3) or low infection (LI; C8, C12, and C13) for RNA sequencing (RNA-seq) analysis. We validated the transcriptional and microscopy analyses (Fig 2A, 2C and 2D) by western blot, which showed higher levels of ACE2 protein in HI compared with LI monolayers and comparable levels of TMPRSS2 protein (Fig 4A). We then infected the monolayers with SARS-CoV-2 for 24 and 72 hours (I24 and I72, respectively) and compared these samples with mock infected monolayers harvested at 0 and 72 hours (UI0 and UI72, respectively). SARS-CoV-2 continues to replicate in organoids after the initial 24 hours, likely due to the low proportion of cells that are initially infected [33]. We reasoned that sampling early and late time points may distinguish transcriptional changes that contribute to resistance and susceptibility to infection versus those that are a consequence. Because these 6 monolayer lines were prepared from independently thawed batches of frozen organoid stocks, we quantified virus and confirmed that higher amounts of SARS-CoV-2 were recovered from HI compared with LI monolayers at both time points (Fig 4B and 4C). Both HI and LI monolayers remained viable following SARS-CoV-2 infection (S7A Fig). Similar to our findings with IFN-stimulated monolayers (S5 Fig), ACE2 and TMPRSS2 transcripts in both HI and LI monolayers were stable during the course of infection (S7B and S7C Fig).

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TIFF original image Download: Fig 4. Transcriptome analysis reveals a heighted and delayed interferon response to SARS-CoV-2 infection in susceptible organoid-derived monolayers. (A) Western blot analysis of ACE2, TMPRSS2, and ACTB in high infection (HI; C1, C2, and C3) and low infection (LI; C8, C12, and C13) lines. Blots are representative of at least 3 independent repeats. (B) PFU determined by virus titration on Vero E6 cells of supernatant of HI and LI monolayers at 24 and 72 hours after infection with SARS-CoV-2. (C) RT-PCR analysis of SARS-CoV-2 expression in HI and LI monolayers upon 24 and 72 hours infection with SARS-CoV-2. (D) Venn diagram depicting the number and overlap of DEGs according to RNA-seq analysis (see S7 Fig) of HI or LI monolayers infected with SARS-CoV-2 for 72 hours. (E) Highly enriched biological process GO terms for the DEGs in HI and LI infected with SARS-CoV-2. (F and G) IPA of the transcriptome of SARS-CoV-2–infected HI or LI for upstream regulators. Interferon-related genes (F) or top 5 molecules within the classes cytokine, transcription regulator, transmembrane receptor, ligand-dependent nuclear receptor, and others (G) commonly associated with HI infected/uninfected and LI infected/uninfected conditions. (H) DEGs of LI infected/HI infected condition were analyzed by IPA for upstream regulators. Top 5 upstream regulators related to zinc ion homeostasis and interferons for the LI. Data points in B and C represent the mean of at least 2 technical replicates of individual organoids lines. Bars represent mean ± SEM, and at least 2 independent experiments were performed. Underlying data can be found in S1 Data. *P ≤ 0.05 and **P ≤ 0.01 by unpaired t test, 2 tailed in B and C. ACE2, angiotensin I converting enzyme 2; DEG, differentially expressed gene; GO, Gene Ontology; IFN, interferon; IFNα, interferon alpha; IFNAR, xxx; IFNβ, interferon beta; IFNλ, interferon lambda; IPA, ingenuity pathway analysis; PFU, plaque-forming unit; PRR, pattern recognition receptor; RNA-seq, RNA sequencing; RT-PCR, reverse transcription PCR; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2. https://doi.org/10.1371/journal.pbio.3001592.g004

The number of transcripts displaying >2-fold changes (adjusted P value < 0.05) in 12 pairwise comparisons are summarized in S7D Fig. The conditions that displayed the most differences from one another were those comparing early time points to 72 hours postinfection. Uninfected samples at 0 and 72 hours displayed no differences, indicating that the transcriptome of uninfected HI and LI monolayers remained stable over time. This result increased our confidence that monolayers were fully differentiated at the onset of our experiments. Also, few genes displayed differential expression when comparing uninfected HI and LI monolayers. Principal component analysis (PCA) showed that infection at 72 hours separated samples on PC1 and susceptibility to infection (HI versus LI) separated samples on PC2 (S7E Fig). PCA also confirmed observations from the pairwise comparison indicating that uninfected monolayers and those infected for 24 hours were transcriptionally similar.

At the 72-hour time point, where the largest transcriptional changes occurred between conditions, we found that the majority of the differentially expressed genes (DEGs) when comparing uninfected and infected conditions (55 of 71) were common to LI and HI monolayers, while most DEGs (65 of 81) when comparing infected HI and LI monolayers were unique to this comparison (Fig 4D). Gene ontology analyses and ingenuity pathway analysis (IPA) showed that SARS-CoV-2 infection impacts antiviral pathways, especially those related to the interferon response, and that this signature was more pronounced in HI monolayers compared with LI monolayers following infection (Fig 4E–4G, S7F Fig). Indeed, DEGs related to the response to type I IFN (SAMHD1, NLRC5, USP18, IFIT1, ZBP1, SHFL, STAT2, IRF7, SP100, MX1, OAS3, STAT1, ISG15, and OAS2) and JAK-STAT (STAT5A, STAT1, STAT2, CCL2, and NMI) included common ISGs, and, although these were up-regulated in both HI and LI monolayers infected by SARS-CoV-2, they were induced to a higher degree in HI monolayers (S7G and S7H Fig). Because these ISGs are more highly expressed in HI monolayers and not detected at 24 hours, they are likely a response to the increased degree of infection. These results are consistent with other studies suggesting that the interferon response to SARS-CoV-2 is delayed [33,48,51–53]. Unexpectedly, multiple genes associated with pathways related to zinc and copper homeostasis were specifically up-regulated in LI compared to HI monolayers after 72 hours of infection (Fig 4E and 4H, S7I Fig). The increased expression of MT1 genes encoding metallothioneins in LI monolayers was particularly striking and may be indicative of a stress response activated by viral perturbations in the epithelium [54,55]. Collectively, longitudinal transcriptome analyses identified robust yet late transcriptional changes induced by SARS-CoV-2, the magnitude of which corresponded to the levels of viral infection.

The transcriptome analysis did not provide additional insight into the difference in ACE2 expression displayed by HI and LI monolayers. The transcription factors BRG1, FOXM1, and FOXA2 mediate ACE2 expression [56,57] but consistent with the RNA-seq results, HI and LI monolayers displayed comparable BRG1, FOXM1, and FOXA2 expression by quantitative PCR (qPCR; S8A–S8C Fig). However, we detected increased protein levels of FOXA2 in HI monolayers by western blot, suggesting a probable mechanism for the high ACE2 expression observed in these donors (S8D Fig).

During the preparation of this manuscript, a new variant of concern designated as Omicron emerged with multiple amino acid substitutions in the S protein. Thus, we examined the ability of the Omicron S protein to mediate entry into intestinal epithelial cells using SARS-CoV-2 S protein–pseudotyped lentiviral reporter viruses [58]. Vesicular stomatitis virus G protein (VSV-G) pseudotyped control virus displayed high infectivity of organoid-derived monolayers demonstrating feasibility of this approach (S9A Fig). Although Omicron S protein has been observed to have weaker or comparable binding affinity to ACE2 [59,60], Omicron S protein pseudotyped virus displayed 2.5- and 5-fold higher infection than Delta and D614G pseudotypes, respectively (S9A Fig), suggesting that Omicron exploits different or additional cell entry pathways to replicate in human intestinal organoids. Consistent with our observation, a recent study showed efficient entry of Omicron using the endosomal route [61]. D614G and Omicron S protein pseudotyped viruses showed 1.2- to 1.3-fold higher infection of HI monolayers compared with LI monolayers, whereas the Delta S protein pseudotyped virus displayed comparable infectivity (S9B Fig). This marginal contribution of the differential ACE2 expression to infection of these pseudotyped viruses suggests that other factors may be involved in SARS-CoV-2 susceptibility of intestinal epithelial cells. For example, MT1 genes identified in our RNA-seq experiment (Fig 4E and 4H) are associated with resistance to hepatitis C virus and human cytomegalovirus, and zinc ion suppresses the SARS-CoV-2 replications by inhibiting its main proteases [62–65]. Although we caution against overinterpreting these results obtained with pseudotyped viruses, we believe that these preliminary results justify future studies using intestinal organoids and other donor-derived cell culture systems to examine differential susceptibility to intact viruses representing existing and future variants.

When taken together, our results show that human intestinal organoids reveal interindividual differences in responses to viral infection. Organoid-derived monolayers showed substantial differences in their susceptibility to SARS-CoV-2 infection, and ACE2 levels were the strongest correlate of susceptibility. Although transcriptome analysis identified many DEGs upon SARS-CoV-2 infection when comparing organoid lines, these differences were not apparent at 24 hours postinfection, a time point at which the degree of virus infection already diverged between resistant and susceptible monolayers. Therefore, these gene expression patterns are unlikely to account for differential susceptibility and instead, provide a glimpse as to how increased viral replication can affect properties of the intestinal epithelium. Although the presence of SARS-CoV-2 RNA in the gut has been associated with diarrhea in patients with COVID-19 [27], the consequence of intestinal epithelial infection remains largely unclear and an important area of investigation. Extensive experiments in animal models predict that activation of viral RNA sensors trigger immune responses including ISGs that impact the intestinal barrier [66–76]. Coculturing organoids with leukocytes may help our understanding of the downstream consequences of epithelial infections [7]. Additionally, loss of microbiome diversity is associated with COVID-19 severity [77–80]. It would be important to determine whether the microbiome is involved in infection of the epithelium or represents an independent variable of disease outcome.

Finally, it is notable that organoids retained the differential ACE2 levels observed in intact primary tissue sections from the same donor. These results indicate that at least some transcriptional properties of the original intestinal epithelium that are individual specific are retained following ex vivo differentiation. Consistent with this theme, a recent study demonstrates that gastric organoids can be used to investigate age-dependent features of the stomach and yield insight into differential infection and interferon responses of children versus adults to SARS-CoV-2 [53]. If these findings are generalizable, then organoids can be a powerful platform to investigate interindividual differences in infectious disease susceptibility.

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