Résilience - COVID-19 - Résilience - Coronavirus disease 2019

Human intestine biomimetic model to test the impact of microbiota products during SARS-CoV-2 infection – COVIDMicroGutModel

Human intestine biomimetic model to test the impact of microbiota products during SARS-CoV-2 infection

Several lines of evidence reported that the gastrointestinal (GI) tract is targeted by SARS-CoV-2. GI symptoms occur in 15 to 60% of the cases and are associated with severe forms of the infection, and SARS-CoV-2 RNA is present in the stools of infected patients. However, the role played by the intestine during COVID-19 and whether this organ is important for the viral transmission and for downstream inflammatory response is not yet understood

Impact of microbiota derivatives on SARS-CoV-2 invasion, replication and consequences on inflammation

In this project we propose a human Intestinal model combining human primary cells (organoid derived) from colonic and small intestinal tissues with organ-on-chip technology to recapitulate the full hallmark of the small intestinal and colonic barrier (cells diversity, 3D topology, mechanical stimulation) and test the role of microbiota derived products during SARS-CoV-2 infection:<br />- To evaluate the impact of the gut microbiota, in particular of its bacterial components and metabolites, on the infection and replication of SARS-CoV-2.<br />- To determine the effects of the gut microbiota on induced inflammation during SARS-CoV-2 infection<br />- To evaluate the possible benefit of manipulating the gut microbiota to scale down COVID-19 severity.

We used a combination of human primary intestinal cells (derived from colonic an ileal organoids) with organ-on-chip technology to recapitulate the physiology of the human gut barrier (cell types, 3D architecture and mechanical stimulation, such as peristaltic motion and fluid flow). Primary human colonic/ileal cells in filter or in gut-on-chip are treated with microbial metabolites for several time and infected with SARS-CoV-2. Viral invasion and replication are assessed by qPCR and plaque assay. Impact of microbiota derivative on intestinal tissue is done by analysing gene expression of a subset of genes by qPCR and inflammation assess by cytokine gene expression (Nanostring) and FACS (Luminex).

1-Proportion of ACE2-positive cells in colon/ileum models
Our previous data showed that SARS-CoV-2 infection was more efficient in 3D chips than in 2D filters (10-fold increase), and more efficient in ileum than in colon. To understand whether this was linked to a differential expression of SARS-CoV-2’s cellular receptor, we tested the proportion of ACE2-positive cells in colon/ileum organoids grown in undifferentiated cells versus differentiated (DM) medium or grown in chips versus filters (Fig. 1A-B). We observed a two-fold increase of ACE2-positive cells when colonic/ileal cells were grown in DM, indicating a higher ACE2 productive cells upon differentiation (Fig. 1A). Moreover, maturating colonic or ileal cells in chips enhances by 5- and 3-fold respectively the proportion of ACE2-positive cells as compared to filter maturation (Fig. 1B). This indicates that organ-on-chip maturation increases ACE2-positive cells probably by enhancing cell differentiation. In addition, we always could observe a higher proportion of ACE2 positive cells in ileum than in colon. So altogether our data confirm that SARS-CoV-2 invasion efficiency strongly correlated with ACE2 expression.

2-Impact of microbiota derivatives on SARS-CoV-2 infection
Next, we first tested several microbial products on SARS-CoV-2 infection in colon/ileum maturated in 2D filters: Lipopolysaccharide (LPS), Peptidoglycan (PGN), Flagellin (Fla) or metabolites derivatives like Short Chain Fatty Acid (SCFA mix of acetate, proprionate and butyrate). We added these products 24h before SARS-CoV-2 infection and keep them all along the infection kinetics. Our results showed that only SCFA incubation reduced viral invasion in colonic cells. No clear effect was seen on ileal cells. Thus, we decided to go on our study with only colonic cells, also because these are the intestinal cells mostly interacting with the microbiota.
Pretreatment of colonic cells with SCFA during 4 days before the infection (at the moment of the differentiation) increases by almost 2-fold, its beneficial effect against viral invasion. Then, we compared the impact of SCFA on colonic cells grown either in 2D filter or in 3D chip (Fig.1C-E). Our data show that SCFA pretreatment reduces SARS-CoV-2 cellular invasion in both filter and chips by 20-fold and 50-fold respectively (Fig.1 C). However, while SCFA was also seen reducing the amount of secreted SARS-CoV-2 from the apex of colonic barrier in filter, no robust effect was seen on the secreted virus in chips (Fig. 1 D-E). However, it should be noticed that in organ-on-chips the viral excretion is stronger and faster and seem to reach a plateau after 24h post-infection that is not seen in filters. Altogether our data show a clear beneficial effect of gut microbial metabolites, such as SCFA during SARS-CoV-2 invasion and replication in the human intestine.

This very promising data encourage us to pursue this study to scale down COVID severity.
We are currently comparing the expression of several important players in coronavirus invasion (ACE2, TMPRSS2, BoAT1), as well as markers of several intestinal cell populations (colonocytes, enterocytes, goblet, enteroendocrine, Paneth cells and stem cells) between colonic/ileal cells grown in filter or chips and upon treatment with several microbiota derivatives. In addition, we will decipher the role of SCFA in gene expression, cell organization but also on viral-mediated inflammation by analyzing cytokines profile.

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Several lines of evidence reported that gut is targeted by SARS-CoV-2. Intestinal symptoms occur in 15 to 60% of the cases and are associated with severe forms of the infection. However, the role played by the intestine during COVID-19 and whether this organ is important for the viral transmission and for downstream inflammatory response is not yet understood. Alteration in the gut microbiota and consequently changes in microbiota-derived products in the gut lumen and host tissues, have been associated with many diseases. In particular, several examples are highlighting the role of the gut microbiota, in the susceptibility to viral lung infection. Thus, alteration of the gut microbiota in association with an impaired intestinal permeability may be involved in the second, inflammatory, phase of the SARS-CoV-2 infection, which is mainly responsible for mortality. Microbiota derived products, such as lipopolysaccharide (LPS), peptidoglycans (PG, MDP), short-chain fatty acids (SCFA), secondary bile acid, and tryptophan-derivative molecules, have been already reported to have positive or negative impact on the physiology of the intestinal epithelial barrier.
To obtain a physiological model of the human gut, we have been recently developing organ-on-chip (OOC) devices that combine tissue engineering and microfluidic technologies. These devices recapitulate the architecture and the mechanical forces observed in the gut. Thanks to this approach, we could reproduce the high infectivity of the human-restricted pathogen Shigella in the colon, as it was reported in human patients, hence revealing how useful this gut-on-chip model can be in preclinical studies. Furthermore, we already combined OOC technology with human organoid-derived gut primary cells to recapitulate the full hallmark of the small intestinal and colonic barrier (cells diversity, 3D topology, mechanical stimulation). We observed an efficient SARS-CoV-2 invasion, replication and propagation in these biomimetic human gut models.
Today our project aims at using these human colon/ileal surrogates to test the potential benefit of microbiota-derived products during SARS-CoV-2 infection.
We will use this colonic/ileal-on-chip model to interrogate the role of selected microbiota-derived products (LPS, MDP, SCFA, bile acids, tryptophan) on:
- The expression of host cell factors important for the virus invasion
- SARS-CoV-2 entry and replication
- The colonic/ileal barrier integrity and homeostasis upon infection
- Cytokine response and crosstalk with immune cells upon viral infection
Altogether our study will provide a better understanding of the infection and pathophysiology of SARS-CoV-2, and will evaluate the possible benefit of manipulating the gut microbiota to scale down COVID-19 severity. We expect significant benefits for patients and public health. We hope to identify microbiota-derived products that could be used exogenously (several of these molecules are already commercialized) or be increased by probiotic treatment that could allow the early treatment of patients with a high risk of severity. Overall, this project will evaluate the potential benefits of manipulating the intestinal microbiota to reduce the severity of COVID-19. Our innovative approach, sophisticated organ-on-a-chip technologies and organoid culture from human colon and ileum tissues, provide a new gut model that will Reduce, Replace and Refine the use of small animal experimentations and provide a preclinical model for finding therapeutics in COVID19.

Project coordination

Nathalie Sauvonnet (Institut Pasteur)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

IP Institut Pasteur
IP INSTITUT PASTEUR

Help of the ANR 79,744 euros
Beginning and duration of the scientific project: May 2021 - 12 Months

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