Reduced circulating FABP2 in patients with moderate to severe COVID-19 may indicate enterocyte functional change rather than cell death.

The gut is of importance in the pathology of COVID-19 both as a route of infection, and gut dysfunction influencing the severity of disease. Systemic changes caused by SARS-CoV-2 gut infection include alterations in circulating levels of metabolites, nutrients and microbial products which alter immune and inflammatory responses. Circulating plasma markers for gut inflammation and damage such as zonulin, lipopolysaccharide and ß-glycan increase in plasma along with severity of disease. However, Intestinal Fatty Acid Binding Protein / Fatty Acid Binding Protein 2 (I-FABP/FABP2), a widely used biomarker for gut cell death, has paradoxically been shown to be reduced in moderate to severe COVID-19. We also found this pattern in a pilot cohort of mild (n = 18) and moderately severe (n = 19) COVID-19 patients in Milan from March to June 2020. These patients were part of the first phase of COVID-19 in Europe and were therefore all unvaccinated. After exclusion of outliers, patients with more severe vs milder disease showed reduced FABP2 levels (median [IQR]) (124 [368] vs. 274 [558] pg/mL, P < 0.01). A reduction in NMR measured plasma relative lipid-CH3 levels approached significance (median [IQR]) (0.081 [0.011] vs. 0.073 [0.024], P = 0.06). Changes in circulating lipid levels are another feature commonly observed in severe COVID-19 and a weak positive correlation was observed in the more severe group between reduced FABP2 and reduced relative lipid-CH3 and lipid-CH2 levels. FABP2 is a key regulator of enterocyte lipid import, a process which is inhibited by gut SARS-CoV-2 infection. We propose that the reduced circulating FABP2 in moderate to severe COVID-19 is a marker of infected enterocyte functional change rather than gut damage, which could also contribute to the development of hypolipidemia in patients with more severe disease.

Amino acid sensor GCN2 promotes SARS-CoV-2 receptor ACE2 expression in response to amino acid deprivation.

Angiotensin-converting enzyme 2 (ACE2) has been identified as a primary receptor for severe acute respiratory syndrome coronaviruses 2 (SARS-CoV-2). Here, we investigated the expression regulation of ACE2 in enterocytes under amino acid deprivation conditions. In this study, we found that ACE2 expression was upregulated upon all or single essential amino acid deprivation in human colonic epithelial CCD841 cells. Furthermore, we found that knockdown of general control nonderepressible 2 (GCN2) reduced intestinal ACE2 mRNA and protein levels in vitro and in vivo. Consistently, we revealed two GCN2 inhibitors, GCN2iB and GCN2-IN-1, downregulated ACE2 protein expression in CCD841 cells. Moreover, we found that increased ACE2 expression in response to leucine deprivation was GCN2 dependent. Through RNA-sequencing analysis, we identified two transcription factors, MAFB and MAFF, positively regulated ACE2 expression under leucine deprivation in CCD841 cells. These findings demonstrate that amino acid deficiency increases ACE2 expression and thereby likely aggravates intestinal SARS-CoV-2 infection.

SARS-CoV-2 spike spurs intestinal inflammation via VEGF production in enterocytes.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) can cause gastrointestinal (GI) symptoms that often correlate with the severity of COVID-19. Here, we explored the pathogenesis underlying the intestinal inflammation in COVID-19. Plasma VEGF level was particularly elevated in patients with GI symptoms and significantly correlated with intestinal edema and disease progression. Through an animal model mimicking intestinal inflammation upon stimulation with SARS-CoV-2 spike protein, we further revealed that VEGF was over-produced in the duodenum prior to its ascent in the circulation. Mechanistically, SARS-CoV-2 spike promoted VEGF production through activating the Ras-Raf-MEK-ERK signaling in enterocytes, but not in endothelium, and inducing permeability and inflammation. Blockage of the ERK/VEGF axis was able to rescue vascular permeability and alleviate intestinal inflammation in vivo. These findings provide a mechanistic explanation and therapeutic targets for the GI symptoms of COVID-19.

A New Butyrate Releaser Exerts a Protective Action against SARS-CoV-2 Infection in Human Intestine.

Butyrate is a major gut microbiome metabolite that regulates several defense mechanisms against infectious diseases. Alterations in the gut microbiome, leading to reduced butyrate production, have been reported in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. A new butyrate releaser, useful for all the known applications of butyrate, presenting physiochemical characteristics suitable for easy oral administration, (N-(1-carbamoyl-2-phenyl-ethyl) butyramide (FBA), has been recently developed. We investigated the protective action of FBA against SARS-CoV-2 infection in the human small intestine and enterocytes. Relevant aspects of SARS-CoV-2 infection were assessed: infectivity, host functional receptor angiotensin-converting enzyme-2 (ACE2), transmembrane protease serine 2 (TMPRSS2), neuropilin-1 (NRP1), pro-inflammatory cytokines expression, genes involved in the antiviral response and the activation of Nf-kB nuclear factor (erythroid-derived 2-like) 2 (Nfr2) pathways. We found that FBA positively modulates the crucial aspects of the infection in small intestinal biopsies and human enterocytes, reducing the expression of ACE2, TMPRSS2 and NRP1, pro-inflammatory cytokines interleukin (IL)-15, monocyte chemoattractant protein-1 (MCP-1) and TNF-α, and regulating several genes involved in antiviral pathways. FBA was also able to reduce the number of SARS-CoV-2-infected cells, and ACE2, TMPRSS2 and NRP1 expression. Lastly, through the inhibition of Nf-kB and the up-regulation of Nfr2, it was also able to reduce the expression of pro-inflammatory cytokines IL-15, MCP-1 and TNF-α in human enterocytes. The new butyrate releaser, FBA, exerts a preventive action against SARS-CoV-2 infection. It could be considered as an innovative strategy to limit COVID-19.

Diosmectite inhibits the interaction between SARS-CoV-2 and human enterocytes by trapping viral particles, thereby preventing NF-kappaB activation and CXCL10 secretion.

SARS-CoV-2 enters the intestine by the spike protein binding to angiotensin-converting enzyme 2 (ACE2) receptors in enterocyte apical membranes, leading to diarrhea in some patients. Early treatment of COVID-19-associated diarrhea could relieve symptoms and limit viral spread within the gastrointestinal (GI) tract. Diosmectite, an aluminomagnesium silicate adsorbent clay with antidiarrheal effects, is recommended in some COVID-19 management protocols. In rotavirus models, diosmectite prevents pathogenic effects by binding the virus and its enterotoxin. We tested the trapping and anti-inflammatory properties of diosmectite in a SARS-CoV-2 model. Trapping effects were tested in Caco-2 cells using spike protein receptor-binding domain (RBD) and heat-inactivated SARS-CoV-2 preparations. Trapping was assessed by immunofluorescence, alone or in the presence of cells. The effect of diosmectite on nuclear factor kappa B (NF-kappaB) activation and CXCL10 secretion induced by the spike protein RBD and heat-inactivated SARS-CoV-2 were analyzed by Western blot and ELISA, respectively. Diosmectite bound the spike protein RBD and SARS-CoV-2 preparation, and inhibited interaction of the spike protein RBD with ACE2 receptors on the Caco-2 cell surface. Diosmectite exposure also inhibited NF-kappaB activation and CXCL10 secretion. These data provide direct evidence that diosmectite can bind SARS-CoV-2 components and inhibit downstream inflammation, supporting a mechanistic rationale for consideration of diosmectite as a management option for COVID-19-associated diarrhea.

Molecular Insights into SARS-CoV2-Induced Alterations of the Gut/Brain Axis.

For a yet unknown reason, a substantial share of patients suffering from COVID-19 develop long-lasting neuropsychiatric symptoms ranging from cognitive deficits to mood disorders and/or an extreme fatigue. We previously reported that in non-neural cells, angiotensin-1 converting enzyme 2 (ACE2), the gene coding for the SARS-CoV2 host receptor, harbors tight co-expression links with dopa-decarboxylase (DDC), an enzyme involved in the metabolism of dopamine. Here, we mined and integrated data from distinct human expression atlases and found that, among a wide range of tissues and cells, enterocytes of the small intestine express the highest expression levels of ACE2, DDC and several key genes supporting the metabolism of neurotransmitters. Based on these results, we performed co-expression analyses on a recently published set of RNA-seq data obtained from SARS-CoV2-infected human intestinal organoids. We observed that in SARS-CoV2-infected enterocytes, ACE2 co-regulates not only with DDC but also with a specific group of genes involved in (i) the dopamine/trace amines metabolic pathway, (ii) the absorption of microbiota-derived L-DOPA and (iii) the absorption of neutral amino acids serving as precursors to neurotransmitters. We conclude that in patients with long COVID, a chronic infection and inflammation of small intestine enterocytes might be indirectly responsible for prolonged brain alterations.

Enterocytes, fibroblasts and myeloid cells synergize in anti-bacterial and anti-viral pathways with IL22 as the central cytokine.

IL22 is an important cytokine involved in the intestinal defense mechanisms against microbiome. By using ileum-derived organoids, we show that the expression of anti-microbial peptides (AMPs) and anti-viral peptides (AVPs) can be induced by IL22. In addition, we identified a bacterial and a viral route, both leading to IL22 production by T cells, but via different pathways. Bacterial products, such as LPS, induce enterocyte-secreted SAA1, which triggers the secretion of IL6 in fibroblasts, and subsequently IL22 in T cells. This IL22 induction can then be enhanced by macrophage-derived TNFα in two ways: by enhancing the responsiveness of T cells to IL6 and by increasing the expression of IL6 by fibroblasts. Viral infections of intestinal cells induce IFNß1 and subsequently IL7. IFNß1 can induce the expression of IL6 in fibroblasts and the combined activity of IL6 and IL7 can then induce IL22 expression in T cells. We also show that IL22 reduces the expression of viral entry receptors (e.g. ACE2, TMPRSS2, DPP4, CD46 and TNFRSF14), increases the expression of anti-viral proteins (e.g. RSAD2, AOS, ISG20 and Mx1) and, consequently, reduces the viral infection of neighboring cells. Overall, our data indicates that IL22 contributes to the innate responses against both bacteria and viruses.

Targeting the intestinal TMPRSS2 protease to prevent SARS-CoV-2 entry into enterocytes-prospects and challenges.

The transmembrane protease serine 2 (TMPRSS2) is a membrane anchored protease that primarily expressed by epithelial cells of respiratory and gastrointestinal systems and has been linked to multiple pathological processes in humans including tumor growth, metastasis and viral infections. Recent studies have shown that TMPRSS2 expressed on cell surface of host cells could play a crucial role in activation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein which facilitates the rapid early entry of the virus into host cells. In addition, direct suppression of TMPRSS2 using small drug inhibitors has been demonstrated to be effective in decreasing SARS-CoV-2 infection in vitro, which presents TMPRSS2 protease as a potential therapeutic strategy for SARS-CoV-2 infection. Recently, SARS-CoV-2 has been shown to be capable of infecting gastrointestinal enterocytes and to provoke gastrointestinal disorders in patients with COVID-19 disease, which is considered as a new transmission route and target organ of SARS-CoV-2. In this review, we highlight the biochemical properties of TMPRSS2 protease and discuss the potential targeting of TMPRSS2 by inhibitors to prevent the SARS-CoV-2 spreading through gastro-intestinal tract system as well as the hurdles that need to be overcome.

The differences in SARS-CoV and SARS-CoV-2 specific co-expression network mediated biological process in human gut enterocytes.

Novel coronavirus SARS-CoV-2 was recently outbreak worldwide causes severe acute respiratory syndrome along with gastrointestinal symptoms for some infected patients. Information on detail pathogenesis, host immune responses and responsible biological pathways are limited. Therefore, infection specific host gut responses and dietary supplements to neutralize immune inflammation demand extensive research. This study aimed to find differences in global co-expression protein-protein interaction sub-network and enriched biological processes in SARS-CoV and SARS-CoV-2 infected gut enterocytes cell line. Attempts have also been made to predict some dietary supplements to boost human health. The SARS-CoV and SARS-CoV-2 infected differential express proteins were integrated with the human protein interaction network and co-expression subnetworks were constructed. Common hubs of these sub-networks reshape central cellular pathways of metabolic processes, lipid localization, hypoxia response to decrease oxygen level and transport of bio-molecules. The major biological process enriched in the unique hub of SARS-CoV-2 significantly differ from SARS-CoV, related to interferon signaling, regulation of viral process and influenza-A enzymatic pathway. Predicted dietary supplements can improve SARS-CoV-2 infected person''s health by boosting the host immunity/reducing inflammation. To the best of our knowledge this is the first report on co-expression network mediated biological process in human gut enterocytes to predict dietary supplements/compounds.

The SARS-CoV-2 receptor and other key components of the Renin-Angiotensin-Aldosterone System related to COVID-19 are expressed in enterocytes in larval zebrafish.

People with underlying conditions, including hypertension, obesity, and diabetes, are especially susceptible to negative outcomes after infection with coronavirus SARS-CoV-2, which causes COVID-19. Hypertension and respiratory inflammation are exacerbated by the Renin-Angiotensin-Aldosterone System (RAAS), which normally protects from rapidly dropping blood pressure via Angiotensin II (Ang II) produced by the enzyme Ace. The Ace paralog Ace2 degrades Ang II, counteracting its chronic effects, and serves as the SARS-CoV-2 receptor. Ace, the coronavirus, and COVID-19 comorbidities all regulate Ace2, but we do not yet understand how. To exploit zebrafish (Danio rerio) to help understand the relationship of the RAAS to COVID-19, we must identify zebrafish orthologs and co-orthologs of human RAAS genes and understand their expression patterns. To achieve these goals, we conducted genomic and phylogenetic analyses and investigated single cell transcriptomes. Results showed that most human RAAS genes have one or more zebrafish orthologs or co-orthologs. Results identified a specific type of enterocyte as the specific site of expression of zebrafish orthologs of key RAAS components, including Ace, Ace2, Slc6a19 (SARS-CoV-2 co-receptor), and the Angiotensin-related peptide cleaving enzymes Anpep (receptor for the common cold coronavirus HCoV-229E), and Dpp4 (receptor for the Middle East Respiratory Syndrome virus, MERS-CoV). Results identified specific vascular cell subtypes expressing Ang II receptors, apelin, and apelin receptor genes. These results identify genes and cell types to exploit zebrafish as a disease model for understanding mechanisms of COVID-19.

Epithelial response to IFN-γ promotes SARS-CoV-2 infection.

SARS-CoV-2, the agent that causes COVID-19, invades epithelial cells, including those of the respiratory and gastrointestinal mucosa, using angiotensin-converting enzyme-2 (ACE2) as a receptor. Subsequent inflammation can promote rapid virus clearance, but severe cases of COVID-19 are characterized by an inefficient immune response that fails to clear the infection. Using primary epithelial organoids from human colon, we explored how the central antiviral mediator IFN-γ, which is elevated in COVID-19, affects epithelial cell differentiation, ACE2 expression, and susceptibility to infection with SARS-CoV-2. In mouse and human colon, ACE2 is mainly expressed by surface enterocytes. Inducing enterocyte differentiation in organoid culture resulted in increased ACE2 production. IFN-γ treatment promoted differentiation into mature KRT20+ enterocytes expressing high levels of ACE2, increased susceptibility to SARS-CoV-2 infection, and resulted in enhanced virus production in infected cells. Similarly, infection-induced epithelial interferon signaling promoted enterocyte maturation and enhanced ACE2 expression. We here reveal a mechanism by which IFN-γ-driven inflammatory responses induce a vulnerable epithelial state with robust replication of SARS-CoV-2, which may have an impact on disease outcome and virus transmission.

A Single-Cell RNA Expression Map of Human Coronavirus Entry Factors.

To predict the tropism of human coronaviruses, we profile 28 SARS-CoV-2 and coronavirus-associated receptors and factors (SCARFs) using single-cell transcriptomics across various healthy human tissues. SCARFs include cellular factors both facilitating and restricting viral entry. Intestinal goblet cells, enterocytes, and kidney proximal tubule cells appear highly permissive to SARS-CoV-2, consistent with clinical data. Our analysis also predicts non-canonical entry paths for lung and brain infections. Spermatogonial cells and prostate endocrine cells also appear to be permissive to SARS-CoV-2 infection, suggesting male-specific vulnerabilities. Both pro- and anti-viral factors are highly expressed within the nasal epithelium, with potential age-dependent variation, predicting an important battleground for coronavirus infection. Our analysis also suggests that early embryonic and placental development are at moderate risk of infection. Lastly, SCARF expression appears broadly conserved across a subset of primate organs examined. Our study establishes a resource for investigations of coronavirus biology and pathology.

Bioinformatic Analysis of Correlation between Immune Infiltration and COVID-19 in Cancer Patients.

In 2020, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused infections worldwide. However, the correlation between the immune infiltration and coronavirus disease 2019 (COVID-19) susceptibility or severity in cancer patients remains to be fully elucidated. ACE2 expressions in normal tissues, cancers and cell lines were comprehensively assessed. Furthermore, we compared ACE2 expression between cancers and matched normal tissues through Gene Expression Profiling Interactive Analysis (GEPIA). In addition, we performed gene set enrichment analysis (GSEA) to investigate the related signaling pathways. Finally, the correlations between ACE2 expression and immune infiltration were investigated via Tumor Immune Estimation Resource (TIMER) and GEPIA. We found that ACE2 was predominantly expressed in both adult and fetal tissues from the digestive, urinary and male reproductive tracts; moreover, ACE2 expressions in corresponding cancers were generally higher than that in matched healthy tissues. GSEA showed that various metabolic and immune-related pathways were significantly associated with ACE2 expression across multiple cancer types. Intriguingly, we found that ACE2 expression correlated significantly with immune cell infiltration in both normal and cancer tissues, especially in the stomach and colon. These findings proposed a possible fecal-oral and maternal-fetal transmission of SARS-CoV-2 and suggested that cancers of the respiratory, digestive or urinary tracts would be more vulnerable to SARS-CoV-2 infection.

The protein expression profile of ACE2 in human tissues.

The novel SARS-coronavirus 2 (SARS-CoV-2) poses a global challenge on healthcare and society. For understanding the susceptibility for SARS-CoV-2 infection, the cell type-specific expression of the host cell surface receptor is necessary. The key protein suggested to be involved in host cell entry is angiotensin I converting enzyme 2 (ACE2). Here, we report the expression pattern of ACE2 across > 150 different cell types corresponding to all major human tissues and organs based on stringent immunohistochemical analysis. The results were compared with several datasets both on the mRNA and protein level. ACE2 expression was mainly observed in enterocytes, renal tubules, gallbladder, cardiomyocytes, male reproductive cells, placental trophoblasts, ductal cells, eye, and vasculature. In the respiratory system, the expression was limited, with no or only low expression in a subset of cells in a few individuals, observed by one antibody only. Our data constitute an important resource for further studies on SARS-CoV-2 host cell entry, in order to understand the biology of the disease and to aid in the development of effective treatments to the viral infection.

Relative Abundance of SARS-CoV-2 Entry Genes in the Enterocytes of the Lower Gastrointestinal Tract.

There is increasing evidence of gastrointestinal (GI) infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We surveyed the co-expression of SARS-CoV-2 entry genes ACE2 and TMPRSS2 throughout the GI tract to assess potential sites of infection. Publicly available and in-house single-cell RNA-sequencing datasets from the GI tract were queried. Enterocytes from the small intestine and colonocytes showed the highest proportions of cells co-expressing ACE2 and TMPRSS2. Therefore, the lower GI tract represents the most likely site of SARS-CoV-2 entry leading to GI infection.

SARS-CoV-2 productively infects human gut enterocytes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can cause coronavirus disease 2019 (COVID-19), an influenza-like disease that is primarily thought to infect the lungs with transmission through the respiratory route. However, clinical evidence suggests that the intestine may present another viral target organ. Indeed, the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) is highly expressed on differentiated enterocytes. In human small intestinal organoids (hSIOs), enterocytes were readily infected by SARS-CoV and SARS-CoV-2, as demonstrated by confocal and electron microscopy. Enterocytes produced infectious viral particles, whereas messenger RNA expression analysis of hSIOs revealed induction of a generic viral response program. Therefore, the intestinal epithelium supports SARS-CoV-2 replication, and hSIOs serve as an experimental model for coronavirus infection and biology.

COVID-19, coronavirus, SARS-CoV-2 and the small bowel.

Although SARS-CoV-2 may primarily enter the cells of the lungs, the small bowel may also be an important entry or interaction site, as the enterocytes are rich in angiotensin converting enzyme (ACE)-2 receptors. The initial gastrointestinal symptoms that appear early during the course of Covid-19 support this hypothesis. Furthermore, SARS-CoV virions are preferentially released apically and not at the basement of the airway cells. Thus, in the setting of a productive infection of conducting airway epithelia, the apically released SARS-CoV may be removed by mucociliary clearance and gain access to the GI tract via a luminal exposure. In addition, post-mortem studies of mice infected by SARS-CoV have demonstrated diffuse damage to the GI tract, with the small bowel showing signs of enterocyte desquamation, edema, small vessel dilation and lymphocyte infiltration, as well as mesenteric nodes with severe hemorrhage and necrosis. Finally, the small bowel is rich in furin, a serine protease which can separate the S-spike of the coronavirus into two "pinchers" (S1 and 2). The separation of the S-spike into S1 and S2 is essential for the attachment of the virion to both the ACE receptor and the cell membrane. In this special review, we describe the interaction of SARS-CoV-2 with the cell and enterocyte and its potential clinical implications.