Intestinal Host Response to SARS-CoV-2 Infection and COVID-19 Outcomes in Patients With Gastrointestinal Symptoms

Abstract

Background & aims: Given that gastrointestinal (GI) symptoms are a prominent extrapulmonary manifestation of COVID-19, we investigated intestinal infection with SARS-CoV-2, its effect on pathogenesis, and clinical significance.

Methods: Human intestinal biopsy tissues were obtained from patients with COVID-19 (n = 19) and uninfected control individuals (n = 10) for microscopic examination, cytometry by time of flight analyses, and RNA sequencing. Additionally, disease severity and mortality were examined in patients with and without GI symptoms in 2 large, independent cohorts of hospitalized patients in the United States (N = 634) and Europe (N = 287) using multivariate logistic regressions.

Results: COVID-19 case patients and control individuals in the biopsy cohort were comparable for age, sex, rates of hospitalization, and relevant comorbid conditions. SARS-CoV-2 was detected in small intestinal epithelial cells by immunofluorescence staining or electron microscopy in 15 of 17 patients studied. High-dimensional analyses of GI tissues showed low levels of inflammation, including down-regulation of key inflammatory genes including IFNG, CXCL8, CXCL2, and IL1B and reduced frequencies of proinflammatory dendritic cells compared with control individuals. Consistent with these findings, we found a significant reduction in disease severity and mortality in patients presenting with GI symptoms that was independent of sex, age, and comorbid illnesses and despite similar nasopharyngeal SARS-CoV-2 viral loads. Furthermore, there was reduced levels of key inflammatory proteins in circulation in patients with GI symptoms.

Conclusions: These data highlight the absence of a proinflammatory response in the GI tract despite detection of SARS-CoV-2. In parallel, reduced mortality in patients with COVID-19 presenting with GI symptoms was observed. A potential role of the GI tract in attenuating SARS-CoV-2-associated inflammation needs to be further examined.

Keywords: COVID-19; GI infection; GI symptoms; SARS-CoV-2; host immune response; outcomes.

Figure 1

Clinical timing, endoscopic findings, and histologic features in the small intestines of patients with COVID-19. (A) Timing of GI evaluation with respect to COVID-19 disease course. (B) Representative endoscopic images of the duodenum in patients with COVID-19 (left) and control individuals (right). (C) Histologically normal duodenal tissue in a patient with COVID-19. (D) Histologic signs of inflammation detected in duodenal biopsy samples of patients with COVID-19, including neutrophils (arrow) and increased intraepithelial lymphocytes (∗). Scale bar, 100 μm.

Figure 2

SARS-CoV-2 viral particles and protein are detectable in intestinal tissues of patients with COVID-19. (A–H) IF staining of (A, B) duodenal and (C, D) ileal biopsy samples of (B, D) patients COVID-19 and (A, C) controls with ACE2 (green), EPCAM (red), and DAPI (blue). (E–N) IF staining of (E–I) duodenal and (J–N) ileal biopsy samples from (E–H, J–M) patients and (I, N) control individuals with SARS-CoV-2 nucleocapsid (green), EPCAM (red), and DAPI (blue) including (G, L) isotype and (H, M) no primary controls. (O–Q) IF staining of (O, P) duodenal and (Q) ileal biopsy samples of patients with SARS-CoV-2 nucleocapsid (green), MUC2 (red), and DAPI (blue) showing SARS-CoV-2 nucleocapsid in goblet cells (∗MUC2⁺) and nongoblet epithelial cells (arrows, MUC2^–). (R–W) Electron tomography of a duodenal biopsy. (R) Montaged projection overview. (S) Tomographic reconstruction of the region indicated by the rectangle in R showing the goblet cell Golgi region. (V) Detail of the presumptive virion indicated by the red arrow in S. Note the dark nucleocapsid puncta and surface spikes (arrows). (T) Electron tomography of an ileal biopsy from a patient with COVID-19 , montaged tomographic reconstruction of a goblet cell Golgi region. (U) Detail of the region indicated by the rectangle in T, showing a presumptive exit compartment containing 5 presumptive SARS-CoV-2 virions. (W) Detail of a presumptive virion from U; membrane bilayer and surface spikes are evident. The virion structures in R–W are comparable with those from a SARS-CoV-2–infected cultured cell (Supplementary Figure 6 and Supplementary Movies 1 and 2). (X) Projection image of a presumptive SARS-CoV-2 virion within an intestinal epithelial cell from a biopsy obtained from a COVID-19 patient with labelled spike protein by Immuno-EM (arrows). Detail of the presumptive virion itself is not apparent in the projection image. (Y) A single slice (approximately 10 nm) from a tomographic reconstruction of the same area shown in X. The spherical shape and membrane bilayer of the presumptive SARS-CoV-2 virion (indicated by ∗) are discernible, with gold particles connoting anti-S labeling localized to the presumptive virion’s outer periphery. Scale bars: 100 μm (A–N), 10 μm (O–Q), 5 μm (R), 0.2 μm (S, U), 1 μm (T), 0.05 μm (V, W), and 0.025 μm (X, Y). DAPI, 4′,6-diamidino-2-phenylindole; EPCAM, epithelial cell adhesion molecule.

Figure 3

CyTOF-based analysis identified immune cell signatures in intestinal biopsy samples and blood from patients with COVID-19 and control individuals. Uniform manifold approximation and projection (UMAP) presentation of (A) the 8 clusters of LP immune populations based on 38 markers, (B, left) by infection status with COVID-19 patients (red) and controls (blue) and (B, right) by disease severity with control individuals (blue) and patients with severe (red) and asymptomatic/mild/moderate (green) COVID-19. (C) The heatmap depicting immune populations in the LP based on specific cell-type markers. (D) Representative histograms comparing CD206⁺ and CD123⁺ in DC subsets in patients (red) and control individuals (blue). (E) Relative frequencies of CD206⁺ cDC2 and plasmacytoid DCs in LP of patients and control individuals (unsupervised analysis). (F) Relative frequencies of PD-1⁺ CD38⁺ (effector) CD4⁺ and CD8⁺ T cells in the LP of control individuals and patients (supervised analysis). (G) UMAP presentation of the 8 clusters of immune populations based on 38 markers in the EC of intestinal biopsy samples. (H) Relative frequencies of CD206⁺ cDC2 and CD4^–CD8^– T cells in the EC of control individuals and patients (unsupervised analysis). (I) Relative frequencies of PD-1⁺CD38⁺ (effector) CD4⁺ and CD8⁺ T cells in blood of control individuals and patients (supervised analysis). Open red circles denote patients with asymptomatic/mild/moderate disease, and filled red circles denote patients with severe COVID-19. Bar plots represent median values. Freq., frequency.

Figure 4

Transcriptional changes in intestinal biopsy samples from patients with COVID-19 compared with control individuals. (A) Hierarchical clustering of average expression changes for 1063 genes (rows) with induced (red) or depleted (blue) expression (FDR, ≤0.05) in the EC and LP of intestinal biopsy samples from patients with COVID-19. The panel on the left indicates significant genes for each tissue fraction in yellow. The color bar indicates the average log₂ fold change (FC). (B) The top enriched pathways (KEGG) that are induced (red) or depleted (blue) in the LP of patients with COVID-19 are displayed. The dashed line indicates the P ≤ .05 cutoff. Gene names are indicated for main pathways. (C) Deconvolution of main gastrointestinal cell types enriched or depleted in the LP of patients with COVID-19 compared with control individuals. Reference single cell RNA–seq cell-type signatures were taken from Smillie et al (P ≤ .05, Fisher exact test). (D) Average expression changes for DC markers in the EC and LP. Reference small conditional RNA–seq cell-type signatures were taken from Martin et al. The color bar indicates the average log₂(FC). (E) Hierarchical clustering of average expression changes (columns) in the EC and LP for genes related to antiviral response to SARS-CoV-2 in post mortem lung tissue of patients with COVID-19, as described by Blanco-Mello et al (top) and for cytokines and chemokines (bottom). The color bar indicates the average log₂ FC. (F) The gene expression levels for the top 10 significant chemokines and cytokines in the LP of patients with COVID-19 and control individuals. ∗P < .05, ∗∗P < .01. moDC, monocyte-derived dendritic cell.

Figure 5

Patients with COVID-19 with GI symptoms had reduced severity and mortality despite similar NP viral loads compared to those without GI symptoms. (A) Kaplan-Meier curves for survival stratified by any GI symptoms (left) and diarrhea (right) for patients in the discovery cohort. P values from log rank test and 95% CIs of Kaplan-Meier curves are shown. The number of patients at risk are reported for the respective timepoints. (B) 95% CIs of ORs of GI symptoms based on 1000 bootstrap iterations in a multivariate logistic regression for severity (blue) and mortality (red). (C) Validation based on the external cohort. 95% CIs of ORs of the diarrhea covariate based on 1000 bootstrap iterations to capture mortality, ICU admission, and composite outcome of ICU admission or death. Results are based on multivariate models after accounting for confounders including BMI, age, sex, lung disease, heart disease, and hypertension. (D) Validation based on the internal cohort. Boxplot of AUC over 1000 bootstrap iterations to predict mortality and disease severity in the internal validation cohort. (E) 95% CI of the reduction in AUC based on 1000 bootstrap iterations for the model “age + BMI + any GI symptoms” after removing age (blue), GI symptoms (red), and BMI (green). (F) SARS-CoV-2 viral load copies per milliliter (log₁₀ transformed based on N2 primer with the addition of a constant) stratified by GI symptoms. The square corresponds to the average viral load, and the error bars show 1 standard deviation of uncertainty from the mean. P values from 2-tailed unpaired t tests are reported. CI, confidence interval.

Figure 6

Patients with COVID-19 with GI symptoms have reduced levels of circulating inflammatory cytokines. (A) Correlation matrix (Pearson) for 92 markers in the Olink panel across patients with any GI symptoms (top left) compared with no GI symptoms (top right) and patients with diarrhea (bottom left) compared with patients without diarrhea (bottom right). Cluster assignment is reported on the top of the heatmap. (B) Boxplot of Hallmark inflammatory response and KEGG JAK/STAT signaling pathway Z-scores stratified by GI symptoms that were significantly enriched at 10% FDR in cluster 4 and cluster 5, respectively. (C) Significant associations between proteomic clusters and GI symptoms at 10% (dark blue) and 15% (light blue) FDR based on unpaired 2-tailed t test. (D) Analytes associated with GI symptoms at 10% FDR based on unpaired t test. The intensity of the color is proportional to the –log₁₀P value. Negative associations are in blue, and positive associations are in red. On the right side of the heatmap, the cluster assignment for each marker is reported. (E) Boxplots represent the median and interquartile range of select differentially expressed markers stratified by GI symptoms. P values from the unpaired t test are reported.

Supplementary Figure 1

Sample allocation for different assays in patients with COVID-19 and control individuals. Venn diagrams showing blood and biopsy samples used for mass cytometry (#) and RNA sequencing (Δ) in patients with COVID-19 (red) and control individuals (blue). The numbers in the Venn diagrams refer to respective patient and control cases detailed Supplementary Table 2. The table summarizes the total number of blood and biopsy samples allocated for mass cytometry and RNA-seq. N/A, not applicable.

Supplementary Figure 2

Representative H&E staining of small intestinal biopsy specimens of patients with COVID-19. Patient number in the top left corner corresponds with the patient number in Supplementary Table 2. All biopsy specimens are duodenal with the exception of patient 12, which is from the terminal ileum. Scale bar: 100 μm.

Supplementary Figure 3

IELs are not increased in small intestinal biopsy samples from patients with COVID-19 compared to control individuals. (A) CD3⁺ and CD8⁺ IELs per millimeter of epithelium in patients with COVID-19 and uninfected control individuals in the duodenum (black) and ileum (gray). P values generated from unpaired t tests. (B) Representative IF images of small intestinal biopsy samples showing CD3 (green), CD8 (red), and DAPI (blue). Representative CD8⁺ IELs (arrowhead) and representative CD8^– IELs (arrow) are indicated. Scale bar: 100 μm. ns, not significant.

Supplementary Figure 4

Representative IF images of small intestinal biopsy specimens of patients with COVID-19. SARS-CoV-2 nucleocapsid (green), EPCAM (red), and DAPI (blue) in all patients with COVID-19 where tissue was available for IF. Patient number in the top right corner corresponds with the patient number in Supplementary Table 2. All biopsy specimens are duodenal with the exception of patient 12, which is from the terminal ileum. Patient 8 is missing because of technical difficulties during IF staining. Scale bar: 100 μm.

Supplementary Figure 5

Representative immunofluorescence images of small intestinal biopsy specimens of control individuals. SARS-CoV-2 nucleocapsid (green), EPCAM (red), and DAPI (blue) in duodenal biopsy specimens (upper) and ileal biopsy specimens (lower). Scale bar: 100 μm.

Supplementary Figure 6

Electron microscopy by high-pressure freezing/freeze substitution fixation of presumptive SARS-CoV-2 infection in culture Vero cells. (A) Montaged overview of an infected cell (150-nm section) (presented for comparison with analogous structures found in tissue samples (Figure 2 and Supplementary Movie 1), which could not be preserved under similar optimal conditions for electron microscopy). The cell exhibits large numbers of cytoplasmic vacuoles, surface blebbing, and general cytopathogenicity. (B) Montaged tomographic reconstruction of the central portion of the cell shown in A. Large numbers of presumptive SARS-CoV-2 virions are contained within cytoplasmic compartments; most are closely adjacent to the compartment’s peripheries. (C) Gallery of 30 individual presumptive SARS-CoV-2 virions taken from the tomogram shown in B. Each example is displayed as an equatorial view with a tomographic thickness of 4.7 nm.

Supplementary Figure 7

Altered immune populations in the EC of patients with COVID-19 compared to control individuals. (A) The heatmap shows the clustering and distribution of different cell types in the EC. Relative frequencies of (B) IELs and (C) plasma cells in the EC of control individuals and patients with COVID-19. Open red circles denote patients with asymptomatic/mild/moderate disease, and filled red circles denote patients with severe COVID-19. The bar plots show median frequencies. NK, natural killer; NKT, natural killer T.

Supplementary Figure 8

Altered immune populations in the blood of patients with COVID-19 compared to control individuals. (A) The heatmap shows the clustering and distribution of different immune cell types in the blood. Relative frequencies of (B) classical (dotted bars) and nonclassical monocytes (open bars), (C) CD4⁺ regulatory T cells, and (D) IgG⁺ plasma cells in the blood of control individuals and patients with COVID-19. Open red circles denote patients with asymptomatic/mild/moderate disease, and filled red circles denote patients with severe COVID-19. The bar plots show median frequencies. CM, central memory; EM, effector memory; PBMC, peripheral blood mononuclear cell.

Supplementary Figure 9

Altered immune populations in the LP of patients with COVID-19 compared to control individuals. (A) Relative frequencies of LP immune cells in control individuals and patients with COVID-19. Open red circles denote patients with asymptomatic/mild/moderate disease, and filled red circles denote patients with severe COVID-19. The bar plots show median frequencies. (B) The stacked bar graphs show the distribution of average frequencies of naive and memory CD4⁺ and CD8⁺ T cells in the LP of patients with COVID-19 and control individuals. EMRA, effector memory T cells that re-express CD45RA; Freq., frequency; T_REG, T regulatory.

Supplementary Figure 10

Altered T-cell populations in blood and intestinal biopsy samples of patients with COVID-19 compared to control individuals based on supervised analysis. Representative CyTOF plots and bar plots comparing the frequencies of CD29⁺CD38⁺CD4⁺ and CD29⁺CD38⁺CD8⁺ T cells in (A) the blood and (B) LP of control individuals (blue) and patients with COVID-19 (red). Open red circles denote patients with asymptomatic/mild/moderate disease, and filled red circles denote patients with severe COVID-19. The bar plots show median frequencies. CyTOF, cytometry by time of flight.

Supplementary Figure 11

Distinct expression profiles in the intestinal EC and LP. (A) Principal component analysis of EC and LP fractions of patients with COVID-19 and control individuals. The 2 tissue fractions separate on principal component 1 (x-axis). (B) Hierarchical clustering of the average expression changes for 6636 genes (rows) characterizing the EC (red) or LP (blue) fractions (FDR, ≤0.05) in the intestinal biopsy samples of patients with COVID-19 and control individuals. The left panel indicates significant genes in yellow for each tissue compartment. The color bar (right) indicates the average log₂(FC).

Supplementary Figure 12

Immune signatures in the EC of patients with COVID-19. GSEA was performed using a rank-ordered list of genes differentially expressed in the infected EC vs control EC. The metric for ranking was log(FC) × –log(P value). (A) GSEA was performed on the rank-ordered EC gene set using SARS-CoV-2–infected organoid data sets. The gene sets tested were molecular signatures curated from SARS-CoV-2–infected organoid experimental data sets using hSIOs grown in either (1) Wnt high expansion (EXP) medium (at adjP < .05) or (2) differentiation (DIF) medium (at adjP < .1). Only gene sets significantly enriched (at FDR of <0.05) are displayed. (B) GSEA was performed for the same rank-ordered EC gene set using the Hallmark pathway data sets. Two significantly enriched pathways were found to be associated with up-regulated genes in infected EC relative to control individuals (at FDR of <0.05). Normalized enrichment score (NES) and FDR values are as indicated.

Supplementary Figure 13

Flow diagram of the discovery cohort. The diagram shows the total number of patients admitted to the Mount Sinai Health System between April 1 and 15, 2020, and the selection process that was adopted to select patients in the discovery cohort. ED, emergency department.

Supplementary Figure 14

Nausea and vomiting were associated with reduced mortality and severity. Kaplan-Meier curves for mortality stratified by (A) nausea and (B) vomiting for patients in the discovery cohort. P values from log rank test and 95% CIs of Kaplan-Meier curves are shown. Below each Kaplan-Meier curve, the number of patients at risk for different timepoints are reported.

Supplementary Figure 15

Patients with COVID-19 with GI symptoms had reduced levels of circulating IL6 and IL8. (A) IL6, (B) IL8, (C) TNF-α, and (D) IL1β at the time of admission in patients with and without GI symptoms. Boxplots represent the median and interquartile range. P values were calculated using the unpaired 2-tailed t test.

Supplementary Figure 16

Correlation matrix (Pearson) for 92 markers contained in the Olink platform. (A) Correlation matrix across patients with nausea (left) compared to patients without nausea (right) and (B) patients with vomiting (left) compared to patients without vomiting (right). Cluster assignment derived using unsupervised consensus clustering is reported on the top of the heatmap.