A single-cell survey of the small intestinal epithelium

da | Giu 1, 2021 | Biologia Molecolare

Figure 1. New cell types from the intestinal epithelium are identified. Created with BioRender.com.

Abstract

The small intestinal epithelium is a differentiated tissue, composed of heterogeneous cell populations. We do not know all types and subtypes of epithelial cells, their molecular characteristics and how they respond to different pathogens. A recent study (2017) published by Haber et al. attempted to overcome these limitations. Through the single-cell RNA sequencing (scRNA-seq) technique, researchers characterized the intestinal epithelial cells and investigated how they respond to pathogenic insults of Salmonella enterica and Heligmosomoides polygyrus. This work provides new insights to develop cell-specific approaches in case of pathological conditions.

We have not a transcriptional characterization of the intestinal epithelium at single-cell resolution, for this reason the researchers Haber et al. performed a scRNA-seq analysis of the small intestine. They profiled 53193 cells (extracted from the mouse gut) in homeostasis, they individuated new cell subsets and they characterized other subsets previously known. Afterwards, they have carried out the same experiment to analyze the way the intestinal epithelium responds to bacterial and helminth infections. This study shows how the epithelial cells respond in a specific way through antimicrobial programs and with more general mechanisms that involve all the cell population.

The intestinal epithelial cells were isolated from the mouse gut and selected with a specific epithelial marker EpCAM using FACS technique. Subsequently, 7216 cells were profiled with 3′ droplet-based scRNA-seq technique and 15 clusters were identified. For each cell they identified key transcription factors, gene signatures and specific G-protein coupled receptors. The results were validated by an independent analysis using full-length scRNA-seq on 1522 epithelial cells; these data were congruent with the droplet-based clusters, nevertheless the second technique detected only 8 clusters.

scRNA-seq technology made possible the analysis of a huge quantity of cells starting from a biological sample, in this way it is possible to create a survey of the intestinal epithelial cells based on the analysis of the transcriptomes at a single cell-level.

Droplet-based data allowed to identify 12 clusters of enteroendocrine (EE) cells according to a new taxonomy, which previously comprise 8 distinct subclasses. Each cluster has been now associated with a canonical hormone (if the cells expressed more than 50% of it). The obtained results highlighted how hormones cannot be actually used as specific marker for subtypes of EE cells. For example, hormone Cck, previously identified as a canonical marker of I cells, has been found expressed by five different EE subsets according to the new classification.  This study discovered that some EE subtypes have a specific regional localization, for example the subtype SIL-P is mostly localized in ileum, while the subtype SILA in duodenum.

Another cell type, that is revolutionized by this study, are the Tuft cells, which were previously identified (through the specific marker Trpm) as a unique cluster. Thanks to the scRNA-seq technique, researchers discovered two different subsets with two different gene programs: Tuft-1 (specialized in neuronal development) and Tuft-2 (related with inflammatory genes). Furthermore, the Ptprc gene has been identified in Tuft-2, it encodes the immune marker CD45. These data represent an unexpected result because it is the first time that CD45 is found expressed in cell not from the hematopoietic lineage.

In the first phase of the experiment the Microfold cells (another intestinal epithelial population) have not been detected. This cluster represents only the 10% of cells which reside in the rare follicle-associated epithelia of the small intestine. In order to study the Microfold cells, researchers treated Lgr5+ stem cells with the cytokine RANKL for 6 days to force the differentiation towards these cells. Once obtained, the Microfold cells were profiled, and new markers were identified.

Another subtype, represented by Paneth cells, was profiled and a new marker was discovered (Mptx2). This result was validated through immunofluorescence assay and single-molecule fluorescent in-situ hybridization (smFISH). Haber et al. identified two different subsets of Paneth based on their gene expression (Paneth-1 and Paneth-2). Moreover, they uncovered a specific regional localization: Paneth 1 are mostly localized in ileum, while Paneth 2 are mostly localized in duodenum and jejunum; these results have been validated by employing immunofluorescence assay.

The second part of the study analyses the immune response of the intestinal epithelia to two different pathogens: S. enterica (a group of mice were infected with 108 bacterial cells) and H. polygyrus (another group of mice were infected with 200 third-stage larvae).

Figure 2 Infected mice show different cells and different genes. Created with BioRender.com.

After Salmonella infection the research group observed an increase in enterocytes and Paneth cells. They highlighted how the infection induced the expression of specific genes as Saa (encoding A1 and A2) in the enterocytes. Moreover, some antimicrobial peptides (Reg3a, Reg3b and Reg3d), that are normally are expressed only in the enterocytes, were induced in all the intestinal epithelial cells.

During H. polygyrus infection the number of Goblet cells and Tuft-2 increased. Similar to the cellular response to the Salmonella infection, H. polygyrus also caused cell specific responses. Particularly, Goblet cells expressed RELM-β and other genes that are not expressed in physiological condition (Wars and Pnlipr2).

This research demonstrated how much the intestinal epithelium is a heterogenous tissue and its important role in inflammatory response. The authors provide novel gene markers to characterize intestinal cell populations and discover new cell subtypes. However, further experiments may be necessary to functionally validate these new subtypes. For example, the two Paneth cell subtypes, do not show different transcriptional programs regulating their main cell functions.  Rather, they show gene expression differences that could be caused by the different conditions present in the various intestinal regions (duodenum, jejunum and ileum) such as oxygen, pH, nutrients and gut flora. Remarkably, this article could be the starting point for new research in biomedical application as new therapy for intestinal bacteria and parasites. This survey will also lay the basis for future studies involving intestinal tissue.

References

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Francesco Delucchi

Master Industrial Biotechnology student

Chiara Giacoppo

Master Industrial Biotechnology student