Human small intestine: The mysterious security guard

The proximal small intestine, encompassing the duodenum and jejunum, is widely recognized as the primary site for nutrient digestion and absorption in humans.[1] Notably, its luminal environment exhibits diverse histological and physiological characteristics, including a longitudinal pH gradient, variations in oxygen pressure, and differing concentrations of antimicrobial peptides.[2] In addition, the small intestine is a highly organized structure composed of repeated crypt-villous units. In mice, epithelial, immune, and mesenchymal cells have been identified to exhibit significant spatial variability in the gene expression patterns along the crypt-villus axis. However, the spatial expression heterogeneity of the small intestine in humans remains largely unexplored. In a recent study published in Nature, Harnik et al[3] provided a more comprehensive picture of the detailed gene expression profiles of the human small intestinal mucosa and submucosa using surgical resection samples at the proximal intestine to the ligament of Treitz. They elegantly annotated the crypt points and calculated the distance between each point and the nearest crypt to pinpoint the stromal epithelial coordinates and accurately distinguish the architectures such as the lumen, submucosal layer, lamina propria, mucosal muscular layer, apical epithelium, and basal epithelium. Remarkably, they identified immune, stromal, and epithelial cells within the small intestine and characterized their spatial relationships and distributions along both the crypt-villus and stromal epithelial axes at single-cell resolution. Multiple intestinal cells in the small intestine appear signature genomic expression, defense genes such as Recombinant Human Regenerating Islet-Derived Protein 1-alpha (REG1A) and Deleted in Malignant Brain Tumors 1 (DMBT1) specifically expressed in the crypts, polymeric immunoglobulin receptor (PIGR) and glutathione-S-transferase A1 (GSTA1) expressed in the bottom of the villi, enzyme genes as diacylglycerol-acyltransferase 1 (DGAT1) and Retinol-binding protein 2 (RBP2) expressed in the middle of the villi, and transport and channel genes as apolipoprotein A1 (APOA1) and aquaporin 10 (AQP10) mainly expressed at the top of the villi.[3] The mucosa of small intestines forms many circular folds and numerous villi protruding to the intestinal lumen. The surface of each villus is composed of a layer of columnar epithelial cells; the cell membrane at the top of the columnar epithelial cells forms many small protrusions called microvilli. The presence of circular folds, villi, and microvilli increases the surface area to around 200 m2, giving it a huge absorption area. The diets stay in the small intestine for a relatively long time, usually 3–8 hours, providing ample time for full absorption. The intake food has been digested and broken down into small molecules.[4] The absorption process of fatty acids separates spatially along the villous axis. The key genes transmembrane protein 1 (TMEM1) and BSCL2 lipid droplet biogenesis associated seipin (BSCL2) involved in lipid droplet formation are highly expressed at the crypt villous bottom region, while triacylglycerol synthase (coded by monoacylglycerol O-acyltransferase 2 [MOGAT2], monoacylglycerol O-acyltransferase 3 [MOGAT3], and diacylglycerol O-acyltransferase 1 [DGAT1]) is expressed in the middle of the villous layer. Genes related to chyle granule formation (coded by microsomal triglyceride transfer protein [MTTP], apolipoprotein B [APOB], and diacylglycerol O-acyltransferase 2 [DGAT2]) are distributed at the villous tips. A similar spatial distribution also occurs in the absorption, storage, and output processes of iron. The absorption of iron is mainly localized at the crypts (transferrin receptor, TFRC) or the bottom of the villi (solute carrier family 11 member 2, SLC11A2), while the secretion of iron is partitioned toward the top of the villi, indicating that the crypt villous bottom plays an important role in absorbing iron, while the top of the villi is involved in the releases of iron.[3] The villi of the small intestine are composed of a single layer of columnar epithelia (including absorptive enterocytes, goblet cells, Paneth cells, tuft cells, and a small number of enteroendocrine cells), which is connected to the epithelium of the small intestinal gland (the invagination of the epithelium between adjacent villous roots, also known as the small intestinal crypt).[5] An in-depth analysis of the stratification and functional status of small intestinal villous stromal cells reveals distinct cellular distributions: CD4+ T cells and traditional dendritic cells are distributed at the bottom of the villi, whereas macrophages and CD8+ T cells are distributed in broader bands near the villi tips. In the layering of endothelial cells and fibroblasts spanning the matrix epithelial axis, lymphatic vessels are observed located at the matrix core, capillary cells at the matrix epithelial boundary, and endothelial cells between the two.[6] What is significantly different from the architecture of the mouse small intestine is the depth specificity of epithelial function along the villous axis. The homologous gene REG1B or other antimicrobial proteins are not upregulated in human intestinal cells at the villous bottom, indicating that these cells are not specifically engaged in antimicrobial defense and that genes related to defense responses are transferred to the crypts. In terms of morphology, the human small intestine appears as an additional circular fold formed by the epithelial layer being curled into a macroscopic shape, presenting morphological complexity, which is different from the smooth, finger-shaped fixed villi in mice.[3] In addition to the aforementioned functions, the small intestine also hosts a diverse and relatively sparse gut microbiota, with a biomass of approximately 105–107 microorganisms per milliliter of contents, significantly lower than the microbial density in the colon. Detections of mucosal biopsy samples obtained from endoscopies and operations enable the verification of the common compositions including Streptococcus, Bacteroides, Clostridium, Lactobacillus, and Enterobacteriaceae.[7] In our previous study, we discussed host–microbial interactions in Crohn’s disease. In total, 540 tissue samples from 30 Crohn’s disease patients and 50 tissue samples from 5 non-Crohn’s disease controls. These samples were derived from paired tissues including intestinal mucosal area, submucosa-muscularis serosa, mesenteric adipose tissue, mesentery, and mesenteric lymph nodes. The spatial distribution of proteins and gut microbiota was mapped. Host proteome and gut microbiome across different tissues are involved in the pathogenesis of the intestinal inflammation.[8] This underscores the essential interplay between the gut and its microbiota in maintaining immune homeostasis. In summary, a comprehensive spatial expression map of the human small intestine has been provided, highlighting the potential metabolic spatial specificity along the crypt-villous axis [Figure 1]. The bottom of the villi specifically absorbs trace elements such as heavy metals and ions, while the tip of the villi specifically absorbs large amounts of elements. The processes of lipid absorption, iron storage, and release are further distributed in different areas of the villi. The human small intestine contains abundant serrated and branched villi, which are enriched at the top of the circular folds. This provides valuable resources for better understanding the biology of the human small intestine and serves as a reference for studying changes in zoning patterns, especially on various metabolic issues and immune-mediated regulation. When the villi are damaged or atrophied, it can lead to a decline in absorption, which in turn can cause digestive problems such as malnutrition and diarrhea. This study will open an avenue for clarifying the pathogenesis of diseases in the small intestine. Clarifying the expression and functional status of specific genes in different structures has positive significance for the screening of specific pathogenic targets in subsequent disease models.Figure 1: Schematic representation of the specialized spatial organization of gene expression, immune cell distribution, and nutrient absorption processes along the crypt-villus axis in the human small intestine.Funding This research was funded by grants from the National Natural Science Foundation of China (Nos. 82070562, 82370532, and 82341219). Conflicts of interest None