
==== Front
BMC Med
BMC Med
BMC Medicine
1741-7015
BioMed Central London

39267041
3611
10.1186/s12916-024-03611-9
Research
Single-cell sequencing of the vermiform appendix during development identifies transcriptional relationships with appendicitis in preschool children
Meng Lingdu 1
Yang Yifan 1
He Shiwei 3
Chen Huifen 1
Zhan Yong 1
Yang Ran 1
Li Zifeng 1
Zhu Jiajie 1
Zhou Jin 1
Li Yi 1
Xie Lulu 1
Chen Gong 1
Zheng Shan szheng@shmu.edu.cn

1
Yao Xiaoying 13916738653@139.com

2
Dong Rui rdong@fudan.edu.cn

1
1 https://ror.org/05n13be63 grid.411333.7 0000 0004 0407 2968 Department of Pediatric Surgery, Shanghai Key Laboratory of Birth Defect, Children’s Hospital of Fudan University, 399 Wan Yuan Road, Shanghai, 201102 China
2 https://ror.org/04rhdtb47 grid.412312.7 0000 0004 1755 1415 Family Planning Department, Obstetrics and Gynecology Hospital of Fudan University, Shanghai, China
3 https://ror.org/050s6ns64 grid.256112.3 0000 0004 1797 9307 Department of Epidemiology and Health Statistics, School of Public Health, Fujian Medical University, Fujian, China
12 9 2024
12 9 2024
2024
22 38324 5 2024
4 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Background

The development of the human vermiform appendix at the cellular level, as well as its function, is not well understood. Appendicitis in preschool children, although uncommon, is associated with a high perforation rate and increased morbidity.

Methods

We performed single-cell RNA sequencing (scRNA-seq) on the human appendix during fetal and pediatric stages as well as preschool-age inflammatory appendices. Transcriptional features of each cell compartment were discussed in the developing appendix. Cellular interactions and differentiation trajectories were also investigated. We compared scRNA-seq profiles from preschool appendicitis to those of matched healthy controls to reveal disease-associated changes. Bulk transcriptomic data, immunohistochemistry, and real-time quantitative PCR were used to validate the findings.

Results

Our analysis identified 76 cell types in total and described the cellular atlas of the developing appendix. We discovered the potential role of the BMP signaling pathway in appendiceal epithelium development and identified HOXC8 and PITX2 as the specific regulons of appendix goblet cells. Higher pericyte coverage, endothelial angiogenesis, and goblet mucus scores together with lower epithelial and endothelial tight junction scores were found in the preschool appendix, which possibly contribute to the clinical features of preschool appendicitis. Preschool appendicitis scRNA-seq profiles revealed that the interleukin-17 signaling pathway may participate in the inflammation process.

Conclusions

Our study provides new insights into the development of the appendix and deepens the understanding of appendicitis in preschool children.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s12916-024-03611-9.

Keywords

Vermiform appendix
Development
Preschool children
Appendicitis
BMP pathway
IL-17 signaling pathway
issue-copyright-statement© BioMed Central Ltd., part of Springer Nature 2024
==== Body
pmcBackground

The appendix, or vermiform appendix, is a narrow, tube-shaped sac arising from the posteromedial cecal border near the ileocecal valve, originating from the midgut during embryonic development. By week 6 of gestation, the cecal diverticulum emerges as the precursor to the cecum and appendix [1]. By the eighth week, the appendix becomes visible [2]. As the colon elongates, the cecum and appendix rotate medially with the midgut and descend into the right lower abdomen.

The histological structure of the appendix closely resembles that of the colon but possesses a unique feature not found in the colon—a dense aggregation of lymphoid tissue. This lymphoid tissue, which begins to develop in the appendix mucosa during weeks 14 and 15 of gestation [2], is structurally reminiscent of lymph nodes, with B cells forming follicles and T cells residing in the interfollicular region [3]. Compared to other intestinal lymphoid tissues such as Peyer’s patches and colonic patches, appendix lymphoid tissue exhibits distinct differences in both structural and cellular composition [3–5]. Functionally, the appendix acts as a reservoir for beneficial gut bacteria, aiding in the repopulation of the gut microbiome following infection [3]. Additionally, the gut-associated lymphoid tissue in the appendix is believed to play a significant role in overall gut immunity, contributing to the maturation of B lymphocytes and the production of immunoglobulin (Ig)A antibodies [6, 7]. These features underscore the uniqueness of the appendiceal lymphoid tissue and the appendix itself. Despite these unique characteristics, research on the molecular and cellular development of the human appendix remains limited, adding to the organ’s mystery. Consequently, deciphering its structure, cellular composition, and function is crucial for a deeper understanding of its role in the immune system.

Acute appendicitis is a common surgical emergency in children, accounting for nearly 33% of pediatric admissions for abdominal pain [8–10]. However, it is relatively uncommon in preschool children, accounting for just 5% of cases [11]. Despite this low incidence, preschool patients often present with higher rates of complications such as perforation, abscess formation, peritonitis, and sepsis [12–14]. Perforation may already be present in younger children (affecting around 30%), at the time of diagnosis [15]. Some studies suggested that delayed diagnosis in preschool children leads to complications due to factors such as their inability to communicate, other associated illnesses, difficult physical examinations, and the lack of a specific clinical presentation [8, 10, 13, 16]. However, other studies propose that complicated appendicitis may have different pathogenesis compared to uncomplicated appendicitis [10, 17, 18]. These factors highlight the unique and severe nature of appendicitis in preschool-aged children.

Despite the availability of advanced diagnostic imaging, diagnosing acute appendicitis in young children remains challenging. In addition, in preschool-aged children, luminal obstruction, is considered to be a common feature in acute appendicitis, with fecalith being a less common cause [14]. These observations lead us to hypothesize that the unique anatomical and histological features of the preschool appendix during development may contribute to these clinical characteristics. Understanding the timeline of appendiceal maturation could provide valuable insights into the pathophysiology of acute appendicitis in this age group.

The developmental trajectory of the appendix at the transcriptomic level has not yet been fully elucidated. In this study, we performed single-cell RNA sequencing (scRNA-seq) on an early human embryo and fetal appendix samples (7–18 gestational weeks (GW)), as well as pediatric appendices (3 days to 13 years old). To explore the transcriptomic characteristics of preschool-age appendicitis, we additionally profiled inflammatory appendices and blood samples of this group. In total, 37 fetal and 31 pediatric specimens were included in this study. Using these data, as well as scRNA-seq profiles of pediatric colon data and bulk RNA-seq data of pediatric acute appendicitis from other studies, we aim to construct a comprehensive cellular atlas of the developing human appendix from the fetal stage to adolescence at the transcriptomic level. This study seeks to trace the differentiation dynamics and features of cells in the appendix, providing insights into the links between these cellular processes and preschool-age appendicitis.

Methods

Human specimens

A total of 37 fetal specimens (1 embryo and 36 fetal appendices), 31 pediatric samples (17 normal and 14 inflamed appendices), and 8 colonic mucosal biopsy samples (normal segments of Hirschsprung’s disease) were included in this study. Epithelial cell clusters were extracted from the embryo sequencing data for epithelium development. Colonic samples and data were utilized solely for comparative analyses with appendiceal epithelium. The fetal samples were obtained from elective terminations of pregnancy at different GW. Normal pediatric appendices were electively resected during the intra-abdominal surgery for a non-inflammatory condition, such as gastrointestinal perforation, intussusception, intraperitoneal foreign bodies, ileocecal mass, and ovarian cyst (Additional file 1: Table S1). The inflammatory samples were obtained from patients diagnosed with complicated acute appendicitis. The details are listed in Table S1.

Histology and immunostaining

After specimen collection, a portion was set aside for histological experiments. They were fixed in 10% buffered formalin for 48 h, dehydrated, embedded in paraffin, and sectioned at 5 μm. Hematoxylin and eosin (H&E) staining was performed with a staining kit (G1120; Solarbio) based on the manufacturer’s instructions. Normal immunohistochemistry (IHC) was performed using a DAB kit (GK500710, Gene Tech). Multi-color immunofluorescence was carried out using a fluorescence IHC kit (TSA-RM-275, Panovue) according to the protocol of the manufacturer. Briefly, antigen was retrieved by 10 × Tris–EDTA (pH9.0) and boiled in the microwave oven for 20 min. After blocking with goat serum, samples were incubated overnight at 4 °C with primary antibodies (Additional file 1: Table S2). A secondary horseradish peroxidase-conjugated antibody was added and incubated at room temperature for 1 h. Then samples were incubated with fluorescent dye at room temperature for 15 min. H&E and IHC images were acquired with a Leica light microscope (DM750). The immunofluorescent slides were imaged using a confocal microscope (Leica) and analyzed using Leica imaging software.

Single-cell RNA sequencing and analyses

Detailed methods for single-cell preparation, sequencing, and data analyses are provided in Additional file 1 (Supplementary Methods, Table S3).

Real-time quantitative PCR (Rt-qPCR)

Total RNA was extracted using TRIzol reagent (T9108, Takara); then, 1 µg RNA was transcribed into cDNA using a reverse-transcription kit (AK4601, Takara). PCR amplification kit (AKA606, Takara) was used to perform the real-time qPCR. Additional file 1 Table S4 lists primer sequences for each gene, and each sample was analyzed in duplicate. Relative RNA expression levels were calculated using the 2–ΔΔCt method, which normalized to GAPDH [19]. Wilcoxon tests were used to compare the significance between two groups. P < 0.05 was considered statistically significant.

Results

Depicting the cell atlas of the developing human vermiform appendix

Given the limited research on the development of the human appendix, we aimed to create a comprehensive cell atlas across different developmental stages. We obtained samples of human fetal and pediatric appendices and performed histological and molecular analyses. We divided the samples according to age, including fetal (before birth), neonatal (0–1 month), infant and toddler (< 3 years), preschool (3–6 years), and school-age (≥ 7 years) groups. Intuitively, the rapid growth in girth (and in length as well [1]), formation of villi and crypts, increased volume of lymphoid tissue, and incrassation of smooth muscle layers were the major physical changes (Fig. 1A, Additional file 1: Fig. S1A).Fig. 1 scRNA-seq profiling of fetal and pediatric appendix. A Representative H&E staining of fetal (n = 6) and pediatric (n = 4) appendices. B General workflow of experimental design for scRNA-seq. Lines on the arrow mark the age of the samples. Black lines represent normal samples (n = 13), and red lines represent the appendicitis samples (n = 2). GW gestational week, D day, Y year. C UMAP visualization of merged normal appendix profiles colored by cell types. D Dot plots displaying marker genes for each cell type. Color represents the average expression within each cell cluster, and size indicates the proportion of cells expressing each marker gene. E Bar plots showing the proportion of each cell type in the merged cells colored by cell types. Each bar represents an individual sample, with samples grouped by age into five categories. GW gestational week, D day, Y year. F Violin plot with embedded box plot representing the transcriptional similarities in different cell types of the pediatric dataset. The embedded box plot represents the median and interquartile range of the similarities scores

To further explore the whole cellular dynamics throughout the development of the human vermiform appendix, we analyzed the single-cell transcriptomic profiles of prenatal (8–18 GW) and pediatric (0–13 years) appendices, including gut endoderm cells from a 7 GW embryo (Fig. 1B). Over 91,000 high-quality cells were identified, revealing major clusters of T/NK (CD3D, NKG7), B (CD79A, MS4A1), myeloid (CD68, CD14), neural (HAND2, TUBB2B), epithelial (EPCAM, FABP1), mesenchymal (COL1A1, COL3A1), and endothelial (CLDN5, VWF) cells (Fig. 1C, D, Additional file 1: Fig. S1B, S1C). Fetal appendiceal samples were enriched in mesenchymal cells, while proportions of T/NK cells and B cells were highest in postnatal samples (Fig. 1E). Throughout development, the transcriptional similarities of B cells and T/NK cells were relatively greater than those of other cell types, rendering lymphoid cells had the least transcriptional diversity across the landscape of appendix development (Fig. 1F, Additional file 1: Fig. S1D). After further sub-clustering, we identified 76 cell types, and we discuss each compartment in the below section.

Deciphering mesenchymal components in appendix development

Mesenchymal cells proliferate and differentiate dramatically. For example, smooth muscle cells (SMCs) increased rapidly during development. SMCs were hard to distinguish in H&E sections at 8 GW but gradually thickened around 10 GW (Fig. 2A).Fig. 2 Development of mesenchymal compartment cell. A. Representative H&E staining of fetal appendices at 8 and 10 GW. GW gestational week. B UMAP visualization of merged mesenchymal cells in developing dataset colored by cell types. C Multicolor IHC identifying the location of S2 (PDGFRA+) and epithelial cells (CDH1+). D Enriched GO terms of the DEGs of ARID5B+ SMCs, TCF21+ fibroblasts, and PITX1+ fibroblasts. E Bar plots showing the proportion of each cell type in the mesenchymal compartment. F Multicolor IHC identifying C7+ S3 in appendices at the age of 8 GW and 8 years. Bar plots showing the relative number of C7+ S3 in postnatal (n = 3) and prenatal (n = 4) appendices. Error bars indicate the standard deviation (SD). Statistical analysis was performed using Student’s t-test. *p < 0.05; Y year. G Violin plots showing the scores of mesenchymal development and differentiation pathways among age groups. The embedded dot and stick represent the mean and standard deviation range, respectively. The dashed line showed the trend of change. H Intercellular communication between epithelial cells and other cell types. Numbers indicate the number of ligand–receptor pairs for each intercellular link. I Circos plot visualizing putative cross talk between epithelial cells and S2 cells. J Multicolor IHC identifying pericytes and endothelial cells in different age groups. Bar plots showing the ratio of pericytes to endothelial cells in infant and toddler (n = 3), preschool (n = 3), and school-age (n = 3) groups. Error bars indicate the standard deviation (SD). Statistical analysis was performed using Student’s t-test. *p < 0.05; **p < 0.01. K Violin plot with embedded box plot representing the vascular transport score in different age groups of endothelial cells. The embedded box plot represents the median and interquartile range of the scores. Wilcoxon test was performed. ns not significant; *p < 0.05; ***p < 0.001

We sub-grouped the cells into 13 subsets (Fig. 2B) based on canonical marker genes and differentially expressed genes (DEGs) (Additional file 1: Fig. S2A). Unlike the composition of mesenchymal cells in adult colons [20], stromal 3 (S3) (ASPN, OGN) cells constitute a larger proportion compared to stromal 1 (S1) (ADAMDEC1, ADAM28) cells in developing appendices. S1 cells were reported to be distributed throughout the lamina propria [20], where the appendix contains a large number of lymphoid follicles, probably resulting in a small quantity of S1 cells. We observed heterogeneity with S3 cells and categorized them into three clusters separately. HAND1+ S3 cells express embryonic development markers (HAND1, HAND2, DLK1, PITX2) [21], while C7+ S3 express mature adult hallmark S3 markers (C7, THY1) [22]. Meanwhile, the expression of FBLN1+ S3 cells was akin to the transitional S3 in the previous report [22], whose state was between S3 progenitors (NR2F1, FNDC1) and mature C7+ S3 cells. Stromal 2 (S2) cells, marked by F3, NPY, and CH25H, are important in intestinal epithelial barrier function [23, 24]. The expression of PDGFRA in S2 was similar to the villus-based telocytes which support the epithelium [25] (Fig. 2C) and contribute to intestinal development and renewal [26]. We also identified two other fibroblast clusters: PITX1+ fibroblasts and TCF21+ fibroblasts. According to the top DEGs, most of them are essential for cell differentiation and development in multiple organs [27–32], and similarly, ARID5B+ SMCs also have differentiation characteristics [33] (Fig. 2D), suggesting that these cells may play roles in mesenchyme development. Mesenchymal lymphoid tissue organizers (mLTo), defined by CCL19, CCL21, and CXCL13 as well as adhesion and NF-κB pathway molecules [34], were believed to be associated with the formation of lymphoid organs and observed at 10 GW in our dataset. Pericytes were identified by KCNJ8 and RGS5.

Fetal samples showed greater cell type diversity than pediatric samples (Fig. 2E, Additional file 1: Fig. S2B). The proportion of PITX1+ fibroblasts was almost decreasing with age and existed in the fetal period. Likewise, transitional (FBLN1+) and development-associated (HAND1+) S3 were also enriched before the neonatal period and so were the cycling fibroblasts and S2. On the contrary, the proportion of mature (C7+) S3 cells, which emerged at around 8 GW, increased after birth (Fig. 2F). Thus, it is not difficult to speculate that the development and differentiation of mesenchymal cells trends with age, which was confirmed by scoring related pathways (Fig. 2G).

S2 cells were reported to support epithelial homeostasis, which could be reflected in most receptors–ligands interactions with epithelial cells within the mesenchymal compartment (Fig. 2H) by ERBB3 and NRG1 (Fig. 2I). NRG1 was reported to enhance differentiation in developing intestine enteroid cultures [35]. Notch signaling was also found between the epithelial–mesenchymal interactions, which was considered as an essential role in intestinal development [36, 37]. However, the limited presence of S2 cells in preschoolers reduces epithelial barrier support, as they express key basement membrane components (Fig. 2E, Additional file 1: Fig. S2B). On the other hand, the proportion of pericytes was largest in the preschool group among all stages, and the ratio of pericytes to endothelial cells in the preschool group reached 1:2.4, which was the highest ratio recorded postnatally (Fig. 2J, Additional file 1: Fig. S2C). Greater pericyte coverage may accelerate the transportation of cytokines in the microcirculation during appendicitis, which probably accounts for the more severe inflammatory response in the preschool group [38] (Fig. 2K). SMCs and ARID5B+ SMCs compose the muscular layer of the appendix wall, and their proportions were limited in the preschool group (Additional file 1: Fig. S2D). Appendix wall is thinner in preschool than in school-age children, which may contribute to early perforation during appendicitis (Additional file 1: Fig. S2E).

Charting the map of endothelial cells in developing appendices

We divided the endothelial cells into nine groups (Fig. 3A) based on vessel types and size using reference marker genes (Additional file 1: Fig. S3A). The composition varied with age groups (Fig. 3B, Additional file 1: Fig. S3B). To be noticed, small-vessel endothelial cells transitioned to large-vessel types through development (Fig. 3B–C), with venous capillary endothelial cells decreasing and large-vessel cells increasing. We performed pseudotime analysis by RNA velocity and scored the cells with arteriovenous marker genes [39]. Then, we discovered the gradual changes of the arteriovenous features along the pseudotime, whose respective paths were almost opposite (Fig. 3D).Fig. 3 Endothelial and neural compartments in developing appendix. A UMAP visualization of merged endothelial cells in developing dataset colored by cell types. CP capillary, L large size, M medium size. B Bar plots showing the proportion of each cell type in the endothelial compartment. C Multicolor IHC identifying endothelial cells in appendices at the age of 18 GW and 8 years. GW gestational week, Y year. D Line plots showing the gam-smoothed fit curves of arterial (red) and venous (blue) scores for cells along the velocity-predicted pseudotime. E Bar plots showing the proportion of endothelial cells of all cells among different age groups. F Eclipse plots displaying the average (dot) and distribution range (oval shape) of arteriovenous scores for cells in each age group. G Violin plot with embedded box plot representing the scores of angiogenesis signature genes among age groups. The embedded box plot represents the median and interquartile range of the scores. Wilcoxon test was performed. ns not significant, *p < 0.05; ****p < 0.0001. H Box plots showing the scores of cellular junction signature genes among age groups. Wilcoxon test was performed. ns not significant; *p < 0.05; **p < 0.01; ****p < 0.0001. I IHC sections of S100B in fetal appendix samples at the age of 8 and 9 GW. J UMAP visualization of merged neural cells in developing dataset colored by cell types. K Bar plots showing the proportion of each cell type in the neural compartment. L Partition-based graph abstraction of neural cells development. M Dot plots showing the top highly expressed ligand-receptor interactions of Glial_3 cells and endothelial cells and myeloid cells

Generally, the preschool group had the smallest proportion of endothelial cells (Fig. 3E) and exhibited lower arterial and venous scores, suggesting a more naïve state (Fig. 3F). On the contrary, fetal groups not only possessed higher arteriovenous features but also showed a distinct ability of angiogenesis as well (Fig. 3G), which is considered to be beneficial for development. However, the preschool group received higher scores in angiogenesis. The vascular endothelium plays a critical role in the regulation of vessel walls, whose integrity maintains the fundamental barrier function. Among the elements involved in vascular structure and permeability, the intercellular junction is the most important one [40]. The scores of three types of junctions, especially tight junctions, in the preschool group, were the lowest among all groups (Fig. 3H). Downregulated genes in this group were also enriched in cell junctions (Additional file 1: Fig. S3C, S3D), including MARCKS [41], EFNB2 [42], CFL1 [43], and CDH5 [44]. Above all, these findings indicate the weakness of the endothelium (low proportion and vascular junction) in preschool-age appendices, whose relative dysfunction may accelerate the disease process when inflammation occurs.

Mapping the heterogeneity of neural cells in appendices

During early development (4–7 GW), neural crest–derived precursor cells have been found to colonize and migrate the human gut, then form into the neurons and glial cells [45]. We identified distinct neural cells at 10 GW (the earliest time point) in our data, and glial cells at 8–9 GW (Fig. 3I) on IHC sections. Neural cells were categorized into cycling (MKI67, CENPF, TOP2A), neuronal (ELAVL4, CHRNA3, GAP43), and glial (S100B, SOX10, PLP1) types (Fig. 3J). According to the DEGs, sub-populations were identified respectively (Additional file 1: Fig. S3E). Cycling neural cells took a small proportion in fetal samples and early postnatal appendices, while most postnatal neural cells were glial—specifically lymphoid-associated glial cells (Gial_3) (FGL2, GFRA3, RXRG) (Fig. 3K, Additional file 1: Fig. S3F). It is reasonable to speculate that the size of neuronal cells is too large to capture due to technical defects. In the meantime, the proportion of submucosal glial cells (Glial_2) (CEBPD, SOCS3, ZFP36) increased before birth, which may be associated with the development of epithelium. We delineated the relationships using partition-based graph abstraction (Fig. 3L) and found that neuronal and glial formed separate branches from cycling neural cells. On the other hand, lymphoid-associated glial cells shared the most receptor–ligands pairs with endothelial cells among all types of neural cells (Additional file 1: Fig. S3G). Among those pairs with high mean expression levels between endothelial cells and Glial_3, endothelial cells expressing Notch ligands activated Notch receptors on glial cells (Fig. 3M). The NOTCH signaling involved in interactions was reported to be a powerful means of determining cell fate in the nervous systems and gliogenesis [46]. Complementarily, glial cells interacted with endothelial cells by expressing PDGFC, VEGFB, and PGF to promote angiogenesis and vessel maturation (Fig. 3M). Especially, PDGFC can provide protection to neurons and blood vessels. Therefore, neurovascular cross talk was accomplished to maintain tissue homeostasis and function [47]. On the other hand, MDK binding with PTPRZ1 was reported to promote neuronal migration and embryonic neuron survival, which plays a vital role in enteric neuronal development [48]. We also found that Glial_3 had the most interactions with myeloid cells among immune cells by tumor necrosis factor-related pairs. Enteric glia can produce pro-inflammatory cytokines and influence disease processes by regulating either innate or adaptive immune responses [49, 50]. In addition, neural cells were reported to cause neurogenic appendicitis by overactivation of neuropeptides [51]. Thus, the increase of Glial_3 in the preschool group may have correlations with the appendicitis process.

Epithelial cell development in human appendices

At the very beginning, the appendix epithelium was thick and pseudostratified during 8–12 GW. At approximately 13 GW, it continuously warped and a single-cell layer of villus and crypt structures was generated, while in the meantime, the vacuole-like structure of goblet cells was observed (Fig. 4A).Fig. 4 Epithelial cell development in human appendices. A Representative H&E staining of fetal appendices at 8 and 13 GW. GW gestational week. B UMAP visualization of merged epithelial cells in developing dataset colored by cell types. C Violin plot with embedded box plot showing the scores of crypt–villus axis markers among different cell types. The embedded box plot represents the median and interquartile range of the scores. D scVelo graphs displaying epithelial cells with overlaid arrows colored by cell types. E Box plots showing the scores of cellular junction signature genes among age groups. Wilcoxon test was performed. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001. F Heatmap displaying the relative expression of mucus-related genes in different age groups. G Violin plot with embedded box plot showing the scores of mucus-related genes in goblet cells from the developing appendix dataset (left) and the dataset integrated with colonic cells (right). Wilcoxon test was performed. ns, not significant; **p < 0.01; ****p < 0.0001. H Box plots showing relative expression of the indicated genes between colon (n = 8) and appendix samples (n = 8) from qPCR analysis. Wilcoxon test was performed. *p < 0.05; ***p < 0.001. I DEGs analysis showing up- and down-regulated genes across all 14 clusters. J Multicolor IHC identifying SPINK4+ goblet cells in colon and appendix. K Violin plots showing the relative expression of the BMP-off gene (SPINK4) and BMP target gene (ID1) in the colon and appendix group. Wilcoxon test was performed. ****p < 0.0001. L Violin plots showing the scores of BMP pathway in colon and appendix group. Wilcoxon test was performed. ****p < 0.0001. M Rank for regulons in colon and appendix goblet cells based on the regulon specificity score (RSS). N Box plots showing relative expression of the indicated genes between colon (n = 8) and appendix samples (n = 8) from qPCR analysis. Wilcoxon test was performed. *p < 0.05; **p < 0.01; ***p < 0.001. O Differential regulons associated with types of goblet cells, ranked by correlation with appendiceal goblet scores using the appendix signature genes. P Violin plots showing the relative expression of DDIT3 and XBP1 in the colon and appendix group. Wilcoxon test was performed. ****p < 0.0001

We extracted appendiceal epithelial cells and combined the gut endoderm epithelial cells of an embryo (7 GW) for further investigation. We identified 10 cell types (Fig. 4B) according to the marker genes (Additional file 1: Fig. S4A). These clusters included cycling epithelial cells (MKI67, UBE2C), stem cells (LGR5, ASCL2), secretory (MAFB, HEXIM1), and absorptive progenitors (HMGCS2, FXYD3), goblet cells (MUC2, SPDEF), enterocytes (APOB, FABP6), enteroendocrine cells (CHGA, NEUROD1), tuft cells (LRMP, PTGS1), and microfold cells (CCL20, CCL23). Cycling and stem cells were dominant in the embryo gut epithelium (Additional file 1: Fig. S4B, S4C). With advancing age, the proportion of secretory cells increases, and that of absorptive cells decreases, proving the inferior absorptive function of appendices. Besides, no Paneth cells were detected in healthy appendices, and a cluster of BEST4+ enterocytes was observed in the first trimester of development (13 GW in our dataset), similar to in other reports, and they were considered pH-sensing cells [52, 53]. We scored the cells with selected crypt–villus axis markers to define the position of epithelial cell subsets along the axis [53]. Cycling cells and stem cells are localized to the crypt bottom, while differentiated enterocytes are localized to the top (Fig. 4C). Pseudotime analysis provided a two-direction trajectory, starting with stem cells and cycling cells, and then diverging to absorptive and secretory lineages (Fig. 4D). The pseudotime was generally consistent with the actual age groups, while epithelial cells were slightly less mature in the preschool group (Additional file 1: Fig. S4D).

The intestinal epithelial layers are the primary barrier between the body and the external environment, which are constituted by physical and biochemical barriers [54]. The physical barrier is constructed by the tight junctions between adjacent epithelial cells, which participate in regulating intestinal permeability and guarding against toxins or infections, and are crucial for epithelial barrier integrity [55]. Mucus secreted by goblet cells dilutes toxins and traps bacteria, then quickly washes away to prevent their penetration [56]. Moreover, mucins together with various other molecules (FCGBP, CLCA1, TFF3, ZG16, DMBT1, IgA, defensins, lysozyme) construct a thick chemical layer, which provides a diffusion barrier for a more effective immune response [57, 58]. To evaluate the functions of the barriers, we calculated the scores of tight junction- and mucus-related genes [57, 59] in all epithelial cells and goblet cells. The score of tight junctions gradually increased and reached the top in the infant and toddler group before decreasing in the preschool- and school-age groups (Fig. 4E), while the mucus score of goblet cells was slightly higher in the preschool group (Fig. 4 F, G). The scores of inflammasomes, reactive oxygen species, autophagy, endocytosis, and exocytosis, which are considered to modulate mucus secretions of goblet cells [59, 60], were similarly increased. Conversely, endoplasmic reticulum stress, which was proven to limit mucus secretion [61], exhibited lower scores in the preschool group (Additional file 1: Fig. S4E). Expressions of the mucus-related genes validated by rt-qPCR were also relatively higher (Additional file 1: Fig. S4F) in the preschool group. In the preschool group, the loss of tight junctions may provide more opportunities for pathogen invasion. Thicker mucus continuously secreted by epithelial cells may raise the possibility of luminal obstruction and result in increased intra-luminal pressure and appendix distension.

Since colonic and appendiceal epithelium are alike, we combined scRNA-seq data of colonic samples from He et al. [62] and epithelial cells from our data to compare differences (Additional file 1: Fig. S4G, S4H). The appendices had fewer secretory cell types, especially goblet cells (Additional file 1: Fig. S4I), such that they expressed lower mucus-related genes, as validated by qPCR (Fig. 4G, H). The position of the appendiceal epithelium along the crypt–villus axis was relatively greater, consistent with its higher BMP signaling score and lower WNT signaling score (Additional file 1: Fig. S4J). Focusing on all goblet cells, we noticed that cluster 7 was mostly in colonic epithelium (Additional file 1: Fig. S4K, S4L), which highly expresses SPINK4 (Fig. 4I). We performed immunofluorescent staining and confirmed that SPINK4+ goblet cells are mainly located in the colon (Fig. 4J). SPINK4+ goblet cells were documented when culturing intestinal organoids under BMP-off differentiation conditions (with BMP inhibitor), which is a standard differentiation condition in vitro, and colonic goblet cells expressed higher BMP-off genes [63] (Fig. 4K, Additional file 1: Fig. S4M). Meanwhile, BMP target genes (ID1, ID2, ID3, CREB3L1, KLF10, PRDX2) were significantly upregulated in appendiceal goblet cells [64] (Fig. 4K, Additional file 1: Fig. S4M). Thus, it is reasonable to presume that activation of BMP-related pathways may play roles in the development of appendiceal epithelium (Fig. 4L).

We next applied SCENIC analysis to further analyze their differences at the transcription factor (TF) level. In the appendix goblet cells, HOXC8 and PITX2 were identified as top specific regulons, while SMARCA4 and XBP1 were listed as top regulons of colonic goblet cells (Fig. 4M, Additional file 1: Fig. S4N). Rt-qPCR was performed to validate their expression, respectively (Fig. 4N). We next examined regulons correlated with appendix signatures that may be associated with organ development. We estimated the correlation between the top 50 signatures of the appendix and all identified regulons (Fig. 4O). Then, we found that appendiceal goblet cells upregulated TFs involved in stimulating BMPs (DDIT3 [65], EGR1 [66]) and inhibiting Wnt pathways (CEBPB [67]). On the other hand, in colonic goblet cells, the top regulons were relevant to epithelial cell proliferation (ARNT, XBP1, EHF) and development (SPDEF) (Additional file 1: Fig. S4O, S4P). Moreover, colonic epithelial signatures scored slightly higher in the preschool group (Additional file 1: Fig. S4Q).

Suppressed functioning of myeloid cells in the preschool group

We collected 2129 myeloid cells and divided them into 10 groups (Fig. 5A). Among dendritic cells (DCs), we obtained four different types: DC1 (CLEC9A), DC2 (CD1C), DC3 (LAMP3), and plasmacytoid DC (pDC) (JCHAIN). We designated two types of macrophages, which differed in their expressions of LYVE1 and SPP1 (Additional file 1: Fig. S5A). Through developmental time, we observed a decreased proportion of LYVE1+ macrophages (Fig. 5B, Additional file 1: Fig. S5B). Notably, we identified LYVE1+ macrophages in our earliest sample (8 GW) (Fig. 5C). It was reported in newborn mice that LYVE1hi macrophages may originate from embryonic progenitors [68]. LYVE1+ macrophages were considered as M2-like and may play a crucial part in angiogenesis and extracellular matrix remodeling [69, 70] during the fetal period. On the contrary, the proportion of LAMP3+ DCs, which are considered mature DCs [71], increased over time (Additional file 1: Fig. S5B). DC3 also expressed CCR7 and FSCN1, both of which positively directed the migration of mature DCs into secondary lymphoid nodes [72–74]. We scored the myeloid cells with several gene sets from MSIGDB. The developmental pathways scored higher in the fetal group (Fig. 5D), which also had the greatest proportion of cell-cycling myeloid cells (Additional file 1: Fig. S5B). The response to oxidative stress accumulated through pseudotime (Fig. 5E). Interestingly, the state of myeloid cells seemed to be relatively quiescent in the preschool group when comparing multiple activation signatures (Fig. 5F). Besides, we found that cytokine production, neutrophil chemotaxis, and migration were markedly suppressed in myeloid cells of the preschool group (Fig. 5G). In addition, myeloid cells from preschool group appendices exhibited significantly decreased expression of phagocytosis genes (MERTK, IL1B) and scavenger genes (MARCO, CD5L) (Fig. 5H). In total, the functions of activation, cytokine production, chemotaxis and migration, phagocytosis, and scavenger in myeloid cells from the preschool group were suppressed, which may attenuate the initial innate immune function in some ways, exacerbating the inflammation process during appendicitis.Fig. 5 Suppressed function of myeloid cells in developing dataset. A UMAP visualization of merged myeloid cells in the developing dataset colored by cell types. B Bar plot showing the proportion of each cell type in the myeloid compartment. C Multicolor IHC identifying LYVE1+ goblet cells in the fetal appendix at age of 8 GW. GW gestational week. D Violin plots showing the scores of myeloid development and differentiation pathways among age groups. The embedded dot and stick represent the mean and standard deviation range, respectively. The dashed line showed the trend of change. E Scatterplots displaying correlations of velocity-predicted pseudotime with myeloid development, differentiation, and oxidative stress response score. F Box plots showing the scores of myeloid activation-related pathways in different age groups. Wilcoxon test was performed. **p < 0.01; ***p < 0.001; ****p < 0.0001; ns not significant. G Box plots showing the scores of myeloid function-related pathways in different age groups. Wilcoxon test was performed. **p < 0.01; ***p < 0.001; ****p < 0.0001. H Heatmap displaying the relative expression of phagocytosis and scavenger-related genes in different age groups

Compositions of lymphoid cells in developing appendices

The cell composition evolves over time from fetal to pediatric appendices (Fig. 1E). Lymphoid cells were observed first at 10 GW from our dataset, composing 0.13% of all cells. With advancing age, the proportion of lymphoid cells increased to 5.7% at 18 GW, then sharply increased after birth (74.89%) and topped off (98.44%) at the preschool stage, indicating the maturity of the appendiceal immune structure. Oppositely, myeloid cells dominated in the early stage of gestation but gradually decreased (Additional file 1: Fig. S6A).

Histologically, T cells and B cells were first observed at around 12–13 GW, accumulating and congregating further at 16–17 GW (Fig. 6A). However, the lymphatic nodules with germinal centers were only observed in the postnatal appendix and not obvious in the 3-day-old appendix (Fig. 1B).Fig. 6 B cells and T cells in the appendix during development. A IHC sections of CD3 and CD79a in fetal appendix samples. B UMAP visualization of merged B cells in developing dataset colored by cell types. C Bar plots showing the proportion of each cell type in the B cell compartment. D Representative IHC staining of BCL6 of the fetal appendix at the age of 17 GW. GW gestational week. E Violin plots showing the scores of B cell development and differentiation pathways among age groups. The embedded dot and stick represent the mean and standard deviation range, respectively. The dashed line showed the trend of change. F Heatmap displaying the relative expression of genes from indicated pathways in different types of B cells. G UMAP visualization of merged T cells in developing dataset colored by cell types. H Bar plots showing the proportion of each cell type in the T cell compartment. I Box plots showing the scores of naïve, cytotoxic, resident, and exhaust-related signatures in different age groups. Wilcoxon test was performed. **p < 0.01; ****p < 0.0001. J scVelo graphs displaying T cells with overlaid arrows colored by cell types. K Scatterplots displaying correlations of velocity-predicted pseudotime with T-cell-mediated immunity, cytotoxicity, naïve signatures, and proliferation score. L Identification of the top 300 genes contributing to the development over pseudotime. M Circos plot visualizing the number of interactions between ILC3, ILC2, and LYVE1+ macrophages, monocytes, DC2, S2, and mLTo. N Dot plots showing the top highly expressed ligand–receptor interactions of ILC2 and monocytes, ILC3 and LYVE1+ macrophages, and ILC3 and mLTo

Focusing on B cells, we obtained 19,894 cells in total (Fig. 6B, Additional file 1: Fig. S6B). The pro-B cluster accounted for a relatively larger proportion of cells in fetal groups (Fig. 6C). Germinal-center B cells first appeared at 13 GW in our data, earlier than previously reported [2] (Fig. 6C), and were observed microscopically at 17 GW (Fig. 6D). The only plasma cell type that first existed in the fetal appendices was IgM plasma cells at 14 GW, which possibly suggests primary antigen stimulation at a very early fetal stage. After birth, IgA and IgG plasma cells emerged. Similarly, we scored B cells with several gene sets. The fetal group had higher scores in B cell developmental and differentiation pathways (Fig. 6E). DEGs of PLCG2+ B cells were associated with pathways like B cell differentiation (PTK2B, PLCL2, ITFG2), and regulation of B-cell–mediated immunity (IL4, FOXP3, TGFB1), as PLCG2 was required for many aspects of BCR-mediated signaling (VAV3, SYK, BLK) (Fig. 6F, Additional file 1: Fig. S6C), impacting B cell development and function [75, 76]. Additionally, the B cell activation score was lower in the preschool group (Additional file 1: Fig. S6D).

Re-clustering T/NK cells obtained 16 populations, including NK (KLRF1, KLRD1), gdT (TRDC, TRGC1, TRGC2), two innate lymphoid cells (ILCs) (IL9R, NCR2), cycling T cells (MKI67), regulatory T cells (Treg) (FOXP3), two naïve T cell subsets (CD4-C1-CCR7, CD8-C1-LEF1), four clusters of memory T cells (CD4-C2-ANXA1, CD4-C3-GPR183, CD8-C2-GPR183, CD8-C3-NR4A1), cytotoxic T cells (CD8-C4-GZMK), mucosal-associated invariant T cells (MAITs) (CD8-C5-SLC4A10), and two groups of exhausted T cells (CD4-C4-CXCL13, CD8-C6-CTLA4) (Fig. 6G, Additional file 1: Fig. S6E). In the fetal group, the composition of T cells was more balanced than that of after birth. ILCs, MAITs, and gdT were more abundant prenatally, while naïve and memory T cells were more dominant in postnatal groups (Fig. 6H, Additional file 1: Fig. S6F). We used signature marker genes to define the resident, cytotoxic, exhausted, and naïve scores of T cells [77, 78]. The naïve and cytotoxic signatures were downregulated after birth, while the resident score was upregulated. The exhausted score presented an ascending trend over time. Older groups had higher exhaustion scores. The preschool group had higher scores in resident and cytotoxic signatures compared to the infant and toddler group and the school-age group, respectively, suggesting that the function of T cells may be relatively mature in the preschool group (Fig. 6I), and reflecting the stronger immune reserve capacity in preschool appendices.

To uncover transcriptional dynamics during T cell maturation, we performed RNA velocity analysis on the T cell subsets (Fig. 6J), finding RNA velocity-predicted pseudotime positively correlated with T cell-mediated immunity and cytotoxic pathways and negatively correlated with naïve signatures and proliferation pathways (Fig. 6K, Additional file 1: Fig. S6G). Ordering the cells along the pseudotime revealed a group of driver genes related to the development (fit likelihood > 0.165) (Fig. 6L). These genes were generally enriched in the T cell activation, proliferation, and differentiation pathways (Additional file 1: Fig. S6H). At an early stage of the predicted timeline, T cells required the capacity to express genes of naiveness (SELL, CCR7) and differentiation (PLAC8 [79], IFITM1 [80]). At late time points, T cell exhaustion-associated genes (PDCD1, CTLA4, and TIGIT [81]) were listed as diver genes (Fig. 6L).

Using CellPhoneDB, we found that the ILC2 displayed the highest number of interactions with myeloid cells (monocytes, LYVE1+ macrophages, DC2) (Fig. 6M). ILC2 can produce the type 2 cytokines interleukin (IL)-4, IL-5, and IL-13 and assist in tissue remodeling and repair [82]. SAA1 was reported to induce type 2 immune responses via FPR2 [83], and CD99 negatively regulated monocyte adhesion and transmigration through PILRα [84]. ILC3 had strong cross talks with mesenchymal cells (s2, mLTo). The interactions between mLTo and lymphoid tissue inducers (LTi) initiated secondary lymphoid organogenesis [82, 85]. LTi-like ILC3 subsets decreased with increasing GW and drastically dropped after birth. mLTo secreted CCL19 and CCL21 and received by LTi-like ILC3 through CCR7 to further promote IL-7 and RANKL expression, which resulted in LTo activation and maturation [85] (Fig. 6N).

Transcriptional characteristics in preschool-age appendicitis

Multiple features of the preschool group stood out in each compartment of developing appendices and may contribute to the clinical characteristics of appendicitis in preschool children. To study the transcriptomic changes and further investigate the molecular alterations in preschool appendicitis, we combined the scRNA-seq data of two samples from preschool patients with complicated appendicitis, their peripheral blood mononuclear cells (PBMCs), two normal age-matched appendices, and published healthy PBMC data. In total, we obtained 41,008 cells and divided them into 12 sub-groups (Fig. 7A). The proportions of neutrophils and other myeloid cells were higher in appendicitis; most of these cells were sourced from the blood, while T and B cells were sourced mostly from the tissue (Fig. 7B, Additional file 1: Fig. S7A). The similarity within myeloid cells among these samples was less than that of T or B cells (Additional file 1: Fig. S7B). It was reported that appendectomy is related to inflammatory bowel disease (IBD) [86], so we evaluated the risk of IBD by its high-risk gene sets [87] and found a slightly elevated risk in the appendicitis group (Fig. 7C). We compared the DEGs and performed Gene Set Enrichment Analysis (GSEA) in the KEGG database, determining that top-scored pathways were infection-related (Fig. 7D, E). In particular, the IL-17 signaling pathway was at the top. Rt-qPCR analysis confirmed the upregulation of IL-17 pathway genes in preschool appendicitis (Fig. 7F).Fig. 7 Transcriptional characteristics in preschool-age appendicitis. A UMAP visualization of merged cells colored by cell types. B Bar plots showing the proportion of each cell type in merged cells and colored by disease group. C Violin plots showing the scores of IBD-risk genes among normal and appendicitis groups. Wilcoxon test was performed. ****p < 0.0001. D Volcano plot showing the top 10 DEGs between the appendicitis group (red) and normal group (blue). Gray dots represent insignificant genes. E Enriched KEGG terms of the DEGs in the appendicitis group. F Box plots showing relative expression of the indicated genes among normal (n = 8), preschool (n = 6), and school-age (n = 6) appendicitis samples from qPCR analysis. Wilcoxon test was performed. *p < 0.05; **p < 0.01; ***p < 0.001; ns not significant. G GSEA enrichment plot of expression signatures of IL-17 signaling pathway in myeloid cells, T cells, and B cells of appendicitis group, respectively. H–J Violin plots showing the scores of the IL-17 signaling pathway among different types of myeloid cells, T cells, and B cells, respectively. Wilcoxon test was performed. **p < 0.01; ****p < 0.0001. K Jaccard similarities of myeloid, T, and B cell clusters in developing dataset with the signatures of IL1B+ macrophages, CD8-C3-NR4A1, and CD69+ B cell, respectively. L Bar plots showing the proportion of Cluster_3 IL1B+ macrophages, CD8-C3-NR4A1, and Cluster_10 CD69+ B cells in the developing dataset among different age groups. M Box plots displaying the scores of IL-17 signaling pathway, CD69+ B cell signatures, CD8-C3-NR4A1 signatures, and IL1B+ macrophage signatures in the developing dataset among different age groups. Wilcoxon test was performed. ns not significant; ****p < 0.0001. N Scatterplots displaying correlations of IL-17 signaling scores with CD69+ B cell signatures, CD8-C3-NR4A1 signatures, and IL1B+ macrophage signatures scores. O Enriched KEGG terms of the DEGs in appendicitis group of bulk RNA-seq. P Heatmap depicting clinical information, IL-17 signaling score of the bulk RNA-seq. Q Violin plots showing the scores of the IL-17 signaling pathway between different groups. Wilcoxon test was performed. *p < 0.05. R Bar plots showing the scores in different age groups. S Scatterplots displaying correlations of IL-17 signaling scores, WBC counts, and histological scores

When inflammation happened, as the major effector cells, immune cells responded quickly. Neutrophils, CD4-C1-CCR7 T cells, and memory B cells took larger parts during appendicitis (Additional file 1: Fig. S7C–S7H). Firstly, DEGs of myeloid cells between the normal and appendicitis groups were enriched in the IL-17 signaling pathway and so were the DEGs of B cells and T cells (Fig. 7G, Additional file 1: Fig. S7I–S7K). After scoring myeloid cells with IL-17 pathway genes, IL1B + macrophages stood out with higher scores (Fig. 7H) and may be the responsible cells. To identify the IL1B+ macrophages in developing datasets, we extracted the signature genes of IL1B+ macrophages and found that cluster 3 of the developing datasets scored the highest (Fig. 7K, Additional file 1: Fig. S7L–S7N). In the T cell compartment, CD8-C3-NR4A1 T cells scored the highest in IL-17 signatures (Fig. 7I) and matched with CD8-C3-NR4A1 T cells from the developing datasets (Fig. 7K, Additional file 1: Fig. S7O). As for B cells, CD69+ B cells contributed the most to IL-17 scores (Fig. 7J). Likewise, it was cluster 10 that also had the greatest Jaccard similarities with CD69+ B cells and exhibited the greatest abundances of CD69+ B cells as predicted by CIBERSORTX (Fig. 7K, Additional file 1: Fig. S7P–S7R).

In total, IL1B+ macrophages, CD8-C3-NR4A1 T cells, and CD69+ B cells may be the main effector cells and promote inflammation during appendicitis. Interestingly, going through the developing datasets, cluster 3 as the IL1B+ macrophages, CD8-C3-NR4A1 T cells, and cluster 10 as the CD69+ B cells account for a significant percentage after birth in the preschool group (Fig. 7L). Moreover, the signatures of those cell types, along with IL-17 signaling pathways scored higher in the preschool group (Fig. 7M). Scores of IL1B+ macrophages signatures had the strongest correlation with IL-17 signaling pathways (Fig. 7N).

Next, we downloaded bulk RNA-seq data of appendicitis from Horwitz et al. [88] for external verification. According to the DEGs, the IL-17 pathway was also enriched at the top place (Fig. 7O, Additional file 1: Fig. S7S). We scored the samples with IL-17-related genes and found that the score was higher in patients with appendicitis, especially those without fecalith (Fig. 7P, Q). Comparing the clinical data, we found that histological scores from the article [88] (evaluated by H&E sections), WBC counts, and IL-17 scores were higher in the preschool group (Fig. 7R). IL-17 scores were also significantly positively correlated with WBC count and histological scores (Fig. 7S). To sum up, the IL-17 signaling pathway was upregulated in appendicitis, especially in preschool children.

Discussion

Our study is the first to construct a developing cell atlas of the normal human vermiform appendix at single-cell transcriptomic resolution, revealing dynamic changes in cellular components and differentiation state over time. Early prenatal samples showed various types of mesenchymal, endothelial, and neural cells, while postnatal samples showed a dominance of diverse immune cells.

Macroscopically, the structure of the appendix can be seen at 8 GW, and it grows rapidly in girth and length through time, mostly deriving benefits from the thickening muscularis layer and exploding lymphoid tissue. PITX1+ fibroblasts, a type of embryonic fibroblast that is mostly present in fetal appendices and declines with GW advancement, may be involved in muscle development of the fetal appendix. It was reported that PITX1+ in embryonic fibroblasts is an essential TF for inducing skeletal muscle progenitor cells [89] and plays a critical role in promoting chondrogenesis and myogenesis in the hindlimb [90]. The emergence of the lymphoid tissue started through the interaction between mLTo and ILCs during embryo development. It was reported that mLTo is derived from a group of perivascular myofibroblastic precursors. The precursors attracted the LTi cells and differentiated into mLTo by lymphotoxin LTα1β2 [91]. Then, activated mLTo secreted CCL2, CCL19, and CCL21 to further recruit and retain more LTi cells, thus forming a positive feed-forward loop. This interaction may initiate before 10 GW. Germinal-center B cells were observed at a very early stage, i.e., 13 GW in our data and 17 GW in the IHC section. Combining the observation of IgM plasma cells at 14 GW, we speculate that primary antigen stimulation in the appendix occurs at a very early fetal stage, consistent with the theory that the development of gut microbiota begins before birth [92]. The maternal microbiota travels to the fetus through the placenta in direct (microbes themselves) or indirect (microbiota-derived metabolites) manners [93].

When comparing the appendiceal and colonic epithelial cells, we found that BMP-related pathways may account for the differences. BMPs can control epithelial proliferation by blocking β-catenin activity, whose function is known to promote proliferation within intestinal crypts [94]. As daughter cells of stem cells move upwards from the base of the crypt, they encounter diminishing degrees of WNT signals and an increase in BMP signals [95]. We discovered an upregulated level of BMP target genes in appendiceal epithelial cells compared to colonic cells, indicating a likely poor state of proliferation in appendix epithelium. Regarding mucus secretion, appendiceal goblet cells exhibited lower scores than colonic goblet cells.

Appendicitis in preschool children is an uncommon surgical disease associated with a high perforation rate and increased morbidity. This was previously attributed to a delay in diagnosis owing to the atypical clinical symptoms. However, recent studies suggest that complicated appendicitis is likely a different disease from uncomplicated appendicitis. In other words, preschool children are more prone to developing complicated appendicitis. When analyzing the scRNA-seq profiles of the developing appendix by age group, we found various features of the preschool appendix that may explain the characteristics of preschool appendicitis. On the one hand, the rarity of preschool appendicitis could profit from its better microcirculation. Pericytes envelop the endothelial wall of the microcirculation. The ratio of pericytes to endothelial cells is the highest in the preschool group, which guarantees the blood vessel integrity and bloodstream [96], to avoid the colonization of pathogens and accumulation of inflammatory factors. The higher angiogenesis score of the endothelium also showed the capability to maintain general homeostasis, including in response to trauma [97]. Besides, the mucus secreted by the epithelium of preschool children was rich; to some extent, it can better prevent pathogen invasion.

On the other hand, preschool appendicitis progresses faster once the inflammation begins, possibly due to the thin-walled appendix. The lowest proportion of SMCs was present in this group, while school-age children exhibited higher proportions with less risk of perforation. Then, although higher pericyte coverage is good for vessels, it also appears to correlate positively with endothelial barrier selectivity and stringency [98]. During the inflammation process, pericytes may also potentiate inflammation by releasing inflammatory cytokines and chemokines and by overexpressing cell adhesion molecules [96]. Meanwhile, higher proportions of neural cells in this group may promote inflammation. It was reported that glia reactively proliferates and exhibits an increase in pro-inflammatory functions in acute colitis [49]. During acute inflammation, glial cells generate nitric oxide, IL-1β, tumor necrosis factor, and other pro-inflammatory mediators to recruit immune cells and enhance the response [50]. Moreover, the attenuated tight junctions within endothelial cells and epithelial cells facilitate greater permeability so that the inflammatory cytokines can more quickly enter the circulation and accelerate the response. Meanwhile, the thicker mucus and hyperplasic lymphoid tissue in the preschool group further lead to the obstruction of the appendiceal lumen and worsen the situation. Most importantly, preschool children exhibit the highest proportion of lymphoid cells, whose abundance may enhance immune surveillance and response [99, 100], and potentially explain many of the features we observed in this group. This lymphoid tissue grows rapidly during the toddler period, peaks in preschool-aged children, and plateaus during adolescence. During this period of rapid growth, other structures may respond correspondingly. For example, increased pericyte coverage and endothelial angiogenesis support vessel development and substance exchange, while goblet mucus secretion may be a response to the newly established gut flora. Conversely, the robust growth may weaken cell junctions as the appendix prepares for an increase in volume. To dig deeper, the gut microbiome may play a significant role in lymphoid tissue development, as bacterial translocation has been reported to parallel its growth [101]. However, these findings may also be influenced by heterogeneity between groups due to the small sample size. Further validation is needed to confirm our presumptions.

We discovered that the IL-17 signaling pathway was activated during the appendicitis process, especially in the preschool group. IL1B+ macrophages, CD8-C3-NR4A1 T cells, and CD69+ B cells, enriched in IL-17 signaling, maybe the responsible cell types and promote inflammation during appendicitis. Their higher proportion in normal preschool appendices may provide evidence for the quick inflammatory response. IL1B+ macrophage is one of the resident tissue macrophages with higher expression of IL1B and NLRP3, consistent with the role of NLRP3 inflammasome in IL-1β activation and regulation of intestinal homeostasis [102]. NR4A1 and CD69 were also tissue-resident genes, proving that those cell types were resident in the appendix and related to the development. Therefore, their larger proportion and greater devotion to IL-17 signaling probably are the characteristics of preschool appendicitis.

IL-17 signaling plays a vital role in protecting mammals against fungal and bacterial infections by enhancing host defense mechanisms [103]. It controls inflammation by regulating concerted actions of multiple inflammatory mediators and synergizing with other inflammatory signals [104]. IL-17 was also reported in embryonic development and tissue regeneration [105]. However, a disrupted IL-17 signaling pathway has been implicated in various inflammatory autoimmune disorders, including IBD, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, and asthma [103]. The IBD-risk gene set scored higher in appendicitis, and it is reported that undergoing appendectomy before developing ulcerative colitis can reduce the risk of colectomy as well as ulcerative colitis–related hospital admissions [106]. In an experimental appendicitis model, appendectomy substantially inhibits Th17 recruitment, differentiation, activation, and interleukin expression in the distal colon, thereby significantly suppressing Th17-pathway-mediated immunopathological damage in IBD or experimental colitis [107]. As a microbial reservoir, appendix has shown a positive correlation between Proteobacteria and IL-17A production by appendiceal epithelial CD4+ T cells in pediatric appendicitis [108], further validating that gut microbes can participate in Th17 responses [109, 110]. These findings suggest that appendix could function as an immune organ, interacting with gut microbiota and potentially contributing to autoimmune diseases by regulating the IL-17 signaling pathway. Researchers have targeted IL-17 for therapy and achieved significant efficacy in treating psoriasis, ankylosing spondylitis, and other IL-17-imbalanced autoimmune diseases [111], either by directly targeting the IL-17 pathway to inhibit inflammation or by indirectly inhibiting the IL-17-mediated inflammatory response through regulating Th17 cell differentiation [112]. Similarly, antibody therapy for preschool appendicitis patients might be a future treatment option. It could be administered preoperatively to alleviate symptoms and reduce the risk of complications or postoperatively to accelerate recovery.

Several limitations should be noted. Firstly, the sample size in this study is small, with a limited number of subjects in each age group and within the appendicitis group. There is heterogeneity both within and between groups, which has impacted the consistency of the developmental atlas. Secondly, we used normal segments of Hirschsprung’s disease as relatively healthy controls in the epithelium compartment. While this approach provided some comparative insights, using authentic healthy colonic specimens for comparison would have provided a more accurate baseline. Thirdly, most of the patients in the preschool group had complicated appendicitis, so our results did not classify between complicated and uncomplicated cases. Including more cases of uncomplicated appendicitis in preschool children would provide a more comprehensive understanding of preschool appendicitis. Lastly, most results are based on bioinformatic analyses and lack experimental validation. Further experiments and larger sample sizes are required in future studies.

Conclusions

In summary, we provide a detailed single-cell atlas of the human appendix during development and explore the transcriptional characteristics of preschool appendicitis. Higher pericyte coverage, endothelial angiogenesis, and goblet mucus scores in healthy preschool appendices may contribute to the rarity of preschool appendicitis. Meanwhile, the abundance of lymphoid-associated glial cells and lymphoid cells, suppressed function in myeloid cells, and activation of the IL-17 pathway likely contribute to the severe inflammation observed in preschool appendicitis. IL1B+ macrophages, CD8-C3-NR4A1 T cells, and CD69+ B cells may be the key cell types involved in preschool appendicitis. Overall, our study provides a developmental perspective on the transcriptional landscape of preschool appendicitis, offering new insights for future research in this area.

Supplementary Information

Additional file 1: Supplementary Methods, Table S1-S4, Fig. S1-S7. Table S1. Sample information; Table S2. Primary antibodies; Table S3. Gene sets used in cell scoring; Table S4. Primers used in qPCR assays. Fig. S1. Related to Fig. 1. Cell atlas of developing appendices; Fig. S2. Related to Fig. 2. Mesenchymal cells in different groups; Fig. S3. Related to Fig. 3. Endothelial and neural compartments in developing atlas; Fig. S4. Related to Fig. 4. Appendiceal epithelial cells in the development dataset and the differences between colonic epithelial cells; Fig. S5. Related to Fig. 5. Myeloid cells in developing appendices; Fig. S6. Related to Fig. 6. Lymphoid cells of appendices during the development; Fig. S7. Related to Fig. 7. Characteristics of preschool appendicitis.

Abbreviations

DCs Dendritic cells

DEGs Differentially expressed genes

GSEA Gene set enrichment analysis

GW Gestational weeks

H&E Hematoxylin and eosin

IBD Inflammatory bowel disease

Ig Immunoglobulin

IHC Immunohistochemistry

IL Interleukin

ILCs Innate lymphoid cells

LTi Lymphoid tissue inducers

MAITs Mucosal-associated invariant T cells

mLTo Mesenchymal lymphoid tissue organizers

PBMCs Peripheral blood mononuclear cells

pDC Plasmacytoid dendritic cell

Rt-qPCR Real-time quantitative PCR

scRNA-seq Single-cell RNA sequencing

SMCs Smooth muscle cells

TF Transcription factor

Treg Regulatory T cells

Acknowledgements

We wish to thank all the patients for their involvement in this study. Some of the icons and elements of diagrams were created with BioRender.com.

Authors’ contributions

LM, YY, and SH contributed to the study concept and design, data interpretation and analysis, drafting of the manuscript, and critical revision of the manuscript. HC participated in the study investigation. YZ, RY, and ZL contributed to data analysis. JZ1, JZ2, YL, LX, and GC collected the samples. SZ, XY, and RD supervised the study and critically revised the manuscript. All authors read and approved the final manuscript.

Funding

This study was supported by the Cyrus Tang Foundation, and Shanghai Hospital Development Center Foundation (SHDC 22022306).

Availability of data and materials

Healthy human PBMC data was downloaded from the 10 × Genomics websites (https://cf.10xgenomics.com/samples/cell/pbmc3k/pbmc3k_filtered_gene_bc_matrices.tar.gz). scRNA-seq data of the normal colon samples were gathered from the study of He et al. Bulk RNA-seq data of pediatric appendicitis were downloaded from the GSE9579 dataset. Raw sequencing data of fetal and pediatric appendices specimens in our study are available in the Genome Sequence Archive database under accession number HRA008187.

Declarations

Ethics approval and consent to participate

This study was reviewed and approved by the ethics committees of the Children’s Hospital of Fudan University (protocol no. 2020(420)) and the Obstetrics and Gynecology Hospital of Fudan University (protocol no. 2018–069). Informed consent was obtained from each patient or the legal guardians of each participant if younger than 18 years old before the surgery and the termination of pregnancy, and the anonymity of all samples was strictly maintained throughout the study.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Lingdu Meng, Yifan Yang, and Shiwei He contributed equally to this work.
==== Refs
References

1. Malas MA Sulak O Gökçimen A Sari A Development of the vermiform appendix during the fetal period Surg Radiol Anat 2004 26 3 202 207 10.1007/s00276-003-0208-9 15173960
Malas MA, Sulak O, Gökçimen A, Sari A. Development of the vermiform appendix during the fetal period. Surg Radiol Anat. 2004;26(3):202–7. 10.1007/s00276-003-0208-9.15173960 10.1007/s00276-003-0208-9
2. Schumpelick V Dreuw B Ophoff K Prescher A Appendix and cecum: embryology, anatomy, and surgical applications Surg Clin North Am 2000 80 1 295 318 10.1016/S0039-6109(05)70407-2 10685154
Schumpelick V, Dreuw B, Ophoff K, Prescher A. Appendix and cecum: embryology, anatomy, and surgical applications. Surg Clin North Am. 2000;80(1):295–318. 10.1016/S0039-6109(05)70407-2.10685154 10.1016/S0039-6109(05)70407-2
3. Girard-Madoux MJH Gomez de Agüero M Ganal-Vonarburg SC The immunological functions of the appendix: an example of redundancy? Semin Immunol. 2018 36 31 44 10.1016/j.smim.2018.02.005 29503124
Girard-Madoux MJH, Gomez de Agüero M, Ganal-Vonarburg SC, et al. The immunological functions of the appendix: an example of redundancy? Semin Immunol. 2018;36:31–44. 10.1016/j.smim.2018.02.005.29503124 10.1016/j.smim.2018.02.005
4. Spencer J Siu JHY Montorsi L Human intestinal lymphoid tissue in time and space Mucosal Immunol 2019 12 2 296 298 10.1038/s41385-018-0120-6 30542110
Spencer J, Siu JHY, Montorsi L. Human intestinal lymphoid tissue in time and space. Mucosal Immunol. 2019;12(2):296–8. 10.1038/s41385-018-0120-6.30542110 10.1038/s41385-018-0120-6
5. Kooij IA Sahami S Meijer SL Buskens CJ te Velde AA The immunology of the vermiform appendix: a review of the literature Clin Exp Immunol 2016 186 1 1 9 10.1111/cei.12821 27271818
Kooij IA, Sahami S, Meijer SL, Buskens CJ, te Velde AA. The immunology of the vermiform appendix: a review of the literature. Clin Exp Immunol. 2016;186(1):1–9. 10.1111/cei.12821.27271818 10.1111/cei.12821
6. Deshmukh S Verde F Johnson PT Fishman EK Macura KJ Anatomical variants and pathologies of the vermix Emerg Radiol 2014 21 5 543 552 10.1007/s10140-014-1206-4 24570122
Deshmukh S, Verde F, Johnson PT, Fishman EK, Macura KJ. Anatomical variants and pathologies of the vermix. Emerg Radiol. 2014;21(5):543–52. 10.1007/s10140-014-1206-4.24570122 10.1007/s10140-014-1206-4
7. Radenkovic G Petrovic V Zivanovic D Interstitial cells of cajal and neural structures in the human fetal appendix J Neurogastroenterol Motil 2021 27 1 127 133 10.5056/jnm20100 33380557
Radenkovic G, Petrovic V, Zivanovic D, et al. Interstitial cells of cajal and neural structures in the human fetal appendix. J Neurogastroenterol Motil. 2021;27(1):127–33. 10.5056/jnm20100.33380557 10.5056/jnm20100
8. Rentea RM St. Peter SD Pediatric appendicitis Surg Clin North America. 2017 97 1 93 112 10.1016/j.suc.2016.08.009
Rentea RM, St. Peter SD. Pediatric appendicitis. Surg Clin North America. 2017;97(1):93–112. 10.1016/j.suc.2016.08.009.10.1016/j.suc.2016.08.009
9. Glass CC Rangel SJ Overview and diagnosis of acute appendicitis in children Semin Pediatr Surg 2016 25 4 198 203 10.1053/j.sempedsurg.2016.05.001 27521708
Glass CC, Rangel SJ. Overview and diagnosis of acute appendicitis in children. Semin Pediatr Surg. 2016;25(4):198–203. 10.1053/j.sempedsurg.2016.05.001.27521708 10.1053/j.sempedsurg.2016.05.001
10. Almström M Svensson JF Svenningsson A Hagel E Wester T Population-based cohort study on the epidemiology of acute appendicitis in children in Sweden in 1987–2013 BJS Open 2018 2 3 142 150 10.1002/bjs5.52 29951638
Almström M, Svensson JF, Svenningsson A, Hagel E, Wester T. Population-based cohort study on the epidemiology of acute appendicitis in children in Sweden in 1987–2013. BJS Open. 2018;2(3):142–50. 10.1002/bjs5.52.29951638 10.1002/bjs5.52
11. Williams N Kapila L Acute appendicitis in the preschool child Arch Dis Child 1991 66 11 1270 1272 10.1136/adc.66.11.1270 1755634
Williams N, Kapila L. Acute appendicitis in the preschool child. Arch Dis Child. 1991;66(11):1270–2. 10.1136/adc.66.11.1270.1755634 10.1136/adc.66.11.1270
12. Mallick MS Appendicitis in pre-school children: a continuing clinical challenge. A retrospective study Int J Surg 2008 6 5 371 373 10.1016/j.ijsu.2008.06.003 18675602
Mallick MS. Appendicitis in pre-school children: a continuing clinical challenge. A retrospective study. Int J Surg. 2008;6(5):371–3. 10.1016/j.ijsu.2008.06.003.18675602 10.1016/j.ijsu.2008.06.003
13. Surana R Quinn F Puri P Appendicitis in preschool children Pediatr Surg Int 1995 10 2 68 70 10.1007/BF00171156
Surana R, Quinn F, Puri P. Appendicitis in preschool children. Pediatr Surg Int. 1995;10(2):68–70. 10.1007/BF00171156.10.1007/BF00171156
14. Almaramhy HH Acute appendicitis in young children less than 5 years: review article Ital J Pediatr 2017 43 1 15 10.1186/s13052-017-0335-2 28257658
Almaramhy HH. Acute appendicitis in young children less than 5 years: review article. Ital J Pediatr. 2017;43(1):15. 10.1186/s13052-017-0335-2.28257658 10.1186/s13052-017-0335-2
15. Howell EC Dubina ED Lee SL Perforation risk in pediatric appendicitis: assessment and management Pediatric Health Med Ther 2018 9 135 145 10.2147/phmt.S155302 30464677
Howell EC, Dubina ED, Lee SL. Perforation risk in pediatric appendicitis: assessment and management. Pediatric Health Med Ther. 2018;9:135–45. 10.2147/phmt.S155302.30464677 10.2147/phmt.S155302
16. Rothrock SG Pagane J Acute appendicitis in children: emergency department diagnosis and management Ann Emerg Med 2000 36 1 39 51 10.1067/mem.2000.105658 10874234
Rothrock SG, Pagane J. Acute appendicitis in children: emergency department diagnosis and management. Ann Emerg Med. 2000;36(1):39–51. 10.1067/mem.2000.105658.10874234 10.1067/mem.2000.105658
17. Livingston EH Woodward WA Sarosi GA Haley RW Disconnect between incidence of nonperforated and perforated appendicitis: implications for pathophysiology and management Ann Surg 2007 245 6 886 892 10.1097/01.sla.0000256391.05233.aa 17522514
Livingston EH, Woodward WA, Sarosi GA, Haley RW. Disconnect between incidence of nonperforated and perforated appendicitis: implications for pathophysiology and management. Ann Surg. 2007;245(6):886–92. 10.1097/01.sla.0000256391.05233.aa.17522514 10.1097/01.sla.0000256391.05233.aa
18. Andersson R Hugander A Thulin A Nyström PO Olaison G Indications for operation in suspected appendicitis and incidence of perforation BMJ 1994 308 6921 107 110 10.1136/bmj.308.6921.107 8298378
Andersson R, Hugander A, Thulin A, Nyström PO, Olaison G. Indications for operation in suspected appendicitis and incidence of perforation. BMJ. 1994;308(6921):107–10. 10.1136/bmj.308.6921.107.8298378 10.1136/bmj.308.6921.107
19. Livak KJ Schmittgen TD Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method Methods 2001 25 4 402 408 10.1006/meth.2001.1262 11846609
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402–8. 10.1006/meth.2001.1262.11846609 10.1006/meth.2001.1262
20. Kinchen J Chen HH Parikh K Structural remodeling of the human colonic mesenchyme in inflammatory bowel disease Cell 2018 175 2 372 86.e17 10.1016/j.cell.2018.08.067 30270042
Kinchen J, Chen HH, Parikh K, et al. Structural remodeling of the human colonic mesenchyme in inflammatory bowel disease. Cell. 2018;175(2):372-86.e17. 10.1016/j.cell.2018.08.067.30270042 10.1016/j.cell.2018.08.067
21. Falix FA Aronson DC Lamers WH Gaemers IC Possible roles of DLK1 in the Notch pathway during development and disease Biochimica et Biophysica Acta (BBA) – Mol Basis Dis. 2012 1822 6 988 95 10.1016/j.bbadis.2012.02.003
Falix FA, Aronson DC, Lamers WH, Gaemers IC. Possible roles of DLK1 in the Notch pathway during development and disease. Biochimica et Biophysica Acta (BBA) – Mol Basis Dis. 2012;1822(6):988–95. 10.1016/j.bbadis.2012.02.003.10.1016/j.bbadis.2012.02.003
22. Fawkner-Corbett D Antanaviciute A Parikh K Spatiotemporal analysis of human intestinal development at single-cell resolution Cell 2021 184 3 810 26.e23 10.1016/j.cell.2020.12.016 33406409
Fawkner-Corbett D, Antanaviciute A, Parikh K, et al. Spatiotemporal analysis of human intestinal development at single-cell resolution. Cell. 2021;184(3):810-26.e23. 10.1016/j.cell.2020.12.016.33406409 10.1016/j.cell.2020.12.016
23. Sheng N Ma Z Zhou Y Cholesterol 25-hydroxylase protects against experimental colitis in mice by modulating epithelial gut barrier function Sci Rep 2020 10 1 14246 10.1038/s41598-020-71198-1 32859970
Sheng N, Ma Z, Zhou Y, et al. Cholesterol 25-hydroxylase protects against experimental colitis in mice by modulating epithelial gut barrier function. Sci Rep. 2020;10(1):14246. 10.1038/s41598-020-71198-1.32859970 10.1038/s41598-020-71198-1
24. Abrams ME Johnson KA Perelman SS Oxysterols provide innate immunity to bacterial infection by mobilizing cell surface accessible cholesterol Nat Microbiol 2020 5 7 929 942 10.1038/s41564-020-0701-5 32284563
Abrams ME, Johnson KA, Perelman SS, et al. Oxysterols provide innate immunity to bacterial infection by mobilizing cell surface accessible cholesterol. Nat Microbiol. 2020;5(7):929–42. 10.1038/s41564-020-0701-5.32284563 10.1038/s41564-020-0701-5
25. Shoshkes-Carmel M Wang YJ Wangensteen KJ Subepithelial telocytes are an important source of Wnts that supports intestinal crypts Nature 2018 557 7704 242 246 10.1038/s41586-018-0084-4 29720649
Shoshkes-Carmel M, Wang YJ, Wangensteen KJ, et al. Subepithelial telocytes are an important source of Wnts that supports intestinal crypts. Nature. 2018;557(7704):242–6. 10.1038/s41586-018-0084-4.29720649 10.1038/s41586-018-0084-4
26. McCarthy N Manieri E Storm EE Distinct mesenchymal cell populations generate the essential intestinal bmp signaling gradient Cell Stem Cell 2020 26 3 391 402.e5 10.1016/j.stem.2020.01.008 32084389
McCarthy N, Manieri E, Storm EE, et al. Distinct mesenchymal cell populations generate the essential intestinal bmp signaling gradient. Cell Stem Cell. 2020;26(3):391-402.e5. 10.1016/j.stem.2020.01.008.32084389 10.1016/j.stem.2020.01.008
27. Nemec S Luxey M Jain D Pitx1 directly modulates the core limb development program to implement hindlimb identity Development 2017 144 18 3325 3335 10.1242/dev.154864 28807899
Nemec S, Luxey M, Jain D, et al. Pitx1 directly modulates the core limb development program to implement hindlimb identity. Development. 2017;144(18):3325–35. 10.1242/dev.154864.28807899 10.1242/dev.154864
28. Sadato D Ono T Gotoh-Saito S Eukaryotic translation initiation factor 3 (eIF3) subunit e is essential for embryonic development and cell proliferation FEBS Open Bio 2018 8 8 1188 1201 10.1002/2211-5463.12482 30087825
Sadato D, Ono T, Gotoh-Saito S, et al. Eukaryotic translation initiation factor 3 (eIF3) subunit e is essential for embryonic development and cell proliferation. FEBS Open Bio. 2018;8(8):1188–201. 10.1002/2211-5463.12482.30087825 10.1002/2211-5463.12482
29. El Amri M Fitzgerald U Schlosser G MARCKS and MARCKS-like proteins in development and regeneration J Biomed Sci 2018 25 1 43 10.1186/s12929-018-0445-1 29788979
El Amri M, Fitzgerald U, Schlosser G. MARCKS and MARCKS-like proteins in development and regeneration. J Biomed Sci. 2018;25(1):43. 10.1186/s12929-018-0445-1.29788979 10.1186/s12929-018-0445-1
30. Stryjewska A Dries R Pieters T Zeb2 regulates cell fate at the exit from epiblast state in mouse embryonic stem cells Stem Cells 2017 35 3 611 625 10.1002/stem.2521 27739137
Stryjewska A, Dries R, Pieters T, et al. Zeb2 regulates cell fate at the exit from epiblast state in mouse embryonic stem cells. Stem Cells. 2017;35(3):611–25. 10.1002/stem.2521.27739137 10.1002/stem.2521
31. Delfín DA DeAguero JL McKown EN The extracellular matrix protein ABI3BP in cardiovascular health and disease Front Cardiovasc Med 2019 6 23 10.3389/fcvm.2019.00023 30923710
Delfín DA, DeAguero JL, McKown EN. The extracellular matrix protein ABI3BP in cardiovascular health and disease. Front Cardiovasc Med. 2019;6:23. 10.3389/fcvm.2019.00023.30923710 10.3389/fcvm.2019.00023
32. Park J Ivey MJ Deana Y The Tcf21 lineage constitutes the lung lipofibroblast population Am J Physiol Lung Cell Mol Physiol 2019 316 5 L872 L885 10.1152/ajplung.00254.2018 30675802
Park J, Ivey MJ, Deana Y, et al. The Tcf21 lineage constitutes the lung lipofibroblast population. Am J Physiol Lung Cell Mol Physiol. 2019;316(5):L872–85. 10.1152/ajplung.00254.2018.30675802 10.1152/ajplung.00254.2018
33. Okazaki Y Murray J Ehsani A Increased glucose metabolism in Arid5b−/− skeletal muscle is associated with the down-regulation of TBC1 domain family member 1 (TBC1D1) Biol Res 2020 53 1 45 10.1186/s40659-020-00313-3 33023658
Okazaki Y, Murray J, Ehsani A, et al. Increased glucose metabolism in Arid5b−/− skeletal muscle is associated with the down-regulation of TBC1 domain family member 1 (TBC1D1). Biol Res. 2020;53(1):45. 10.1186/s40659-020-00313-3.33023658 10.1186/s40659-020-00313-3
34. Elmentaite R Kumasaka N Roberts K Cells of the human intestinal tract mapped across space and time Nature 2021 597 7875 250 255 10.1038/s41586-021-03852-1 34497389
Elmentaite R, Kumasaka N, Roberts K, et al. Cells of the human intestinal tract mapped across space and time. Nature. 2021;597(7875):250–5. 10.1038/s41586-021-03852-1.34497389 10.1038/s41586-021-03852-1
35. Holloway EM Czerwinski M Tsai YH Mapping development of the human intestinal niche at single-cell resolution Cell Stem Cell 2021 28 3 568 80.e4 10.1016/j.stem.2020.11.008 33278341
Holloway EM, Czerwinski M, Tsai YH, et al. Mapping development of the human intestinal niche at single-cell resolution. Cell Stem Cell. 2021;28(3):568-80.e4. 10.1016/j.stem.2020.11.008.33278341 10.1016/j.stem.2020.11.008
36. Le Guen L Marchal S Faure S de Santa BP Mesenchymal-epithelial interactions during digestive tract development and epithelial stem cell regeneration Cell Mol Life Sci 2015 72 20 3883 3896 10.1007/s00018-015-1975-2 26126787
Le Guen L, Marchal S, Faure S, de Santa BP. Mesenchymal-epithelial interactions during digestive tract development and epithelial stem cell regeneration. Cell Mol Life Sci. 2015;72(20):3883–96. 10.1007/s00018-015-1975-2.26126787 10.1007/s00018-015-1975-2
37. Faure S McKey J Sagnol S de Santa BP Enteric neural crest cells regulate vertebrate stomach patterning and differentiation Development 2015 142 2 331 342 10.1242/dev.118422 25519241
Faure S, McKey J, Sagnol S, de Santa BP. Enteric neural crest cells regulate vertebrate stomach patterning and differentiation. Development. 2015;142(2):331–42. 10.1242/dev.118422.25519241 10.1242/dev.118422
38. Spencer BG Estevez JJ Liu E Craig JE Finnie JW Pericytes, inflammation, and diabetic retinopathy Inflammopharmacology 2020 28 3 697 709 10.1007/s10787-019-00647-9 31612299
Spencer BG, Estevez JJ, Liu E, Craig JE, Finnie JW. Pericytes, inflammation, and diabetic retinopathy. Inflammopharmacology. 2020;28(3):697–709. 10.1007/s10787-019-00647-9.31612299 10.1007/s10787-019-00647-9
39. Hou S Li Z Dong J Heterogeneity in endothelial cells and widespread venous arterialization during early vascular development in mammals Cell Res 2022 32 4 333 348 10.1038/s41422-022-00615-z 35079138
Hou S, Li Z, Dong J, et al. Heterogeneity in endothelial cells and widespread venous arterialization during early vascular development in mammals. Cell Res. 2022;32(4):333–48. 10.1038/s41422-022-00615-z.35079138 10.1038/s41422-022-00615-z
40. Toborek M Kaiser S Endothelial cell functions. Relationship to atherogenesis Basic Res Cardiol. 1999 94 5 295 314 10.1007/s003950050156 10543305
Toborek M, Kaiser S. Endothelial cell functions. Relationship to atherogenesis. Basic Res Cardiol. 1999;94(5):295–314. 10.1007/s003950050156.10543305 10.1007/s003950050156
41. Chen Z Zhang W Selmi C The myristoylated alanine-rich C-kinase substrates (MARCKS): a membrane-anchored mediator of the cell function Autoimmun Rev 2021 20 11 102942 10.1016/j.autrev.2021.102942 34509657
Chen Z, Zhang W, Selmi C, et al. The myristoylated alanine-rich C-kinase substrates (MARCKS): a membrane-anchored mediator of the cell function. Autoimmun Rev. 2021;20(11):102942. 10.1016/j.autrev.2021.102942.34509657 10.1016/j.autrev.2021.102942
42. Frye M Stritt S Ortsäter H EphrinB2-EphB4 signalling provides Rho-mediated homeostatic control of lymphatic endothelial cell junction integrity eLife. 2020 9 e57732 10.7554/eLife.57732 32897857
Frye M, Stritt S, Ortsäter H, et al. EphrinB2-EphB4 signalling provides Rho-mediated homeostatic control of lymphatic endothelial cell junction integrity. eLife. 2020;9:e57732. 10.7554/eLife.57732.32897857 10.7554/eLife.57732
43. Indra I Troyanovsky RB Shapiro L Honig B Troyanovsky SM Sensing actin dynamics through adherens junctions Cell Rep 2020 30 8 2820 33.e3 10.1016/j.celrep.2020.01.106 32101754
Indra I, Troyanovsky RB, Shapiro L, Honig B, Troyanovsky SM. Sensing actin dynamics through adherens junctions. Cell Rep. 2020;30(8):2820-33.e3. 10.1016/j.celrep.2020.01.106.32101754 10.1016/j.celrep.2020.01.106
44. Sauteur L Krudewig A Herwig L Cdh5/VE-cadherin promotes endothelial cell interface elongation via cortical actin polymerization during angiogenic sprouting Cell Rep 2014 9 2 504 513 10.1016/j.celrep.2014.09.024 25373898
Sauteur L, Krudewig A, Herwig L, et al. Cdh5/VE-cadherin promotes endothelial cell interface elongation via cortical actin polymerization during angiogenic sprouting. Cell Rep. 2014;9(2):504–13. 10.1016/j.celrep.2014.09.024.25373898 10.1016/j.celrep.2014.09.024
45. Wallace AS Burns AJ Development of the enteric nervous system, smooth muscle and interstitial cells of Cajal in the human gastrointestinal tract Cell Tissue Res 2005 319 3 367 382 10.1007/s00441-004-1023-2 15672264
Wallace AS, Burns AJ. Development of the enteric nervous system, smooth muscle and interstitial cells of Cajal in the human gastrointestinal tract. Cell Tissue Res. 2005;319(3):367–82. 10.1007/s00441-004-1023-2.15672264 10.1007/s00441-004-1023-2
46. Gaiano N Fishell G The role of notch in promoting glial and neural stem cell fates Annu Rev Neurosci 2002 25 471 490 10.1146/annurev.neuro.25.030702.130823 12052917
Gaiano N, Fishell G. The role of notch in promoting glial and neural stem cell fates. Annu Rev Neurosci. 2002;25:471–90. 10.1146/annurev.neuro.25.030702.130823.12052917 10.1146/annurev.neuro.25.030702.130823
47. Lee C Zhang F Tang Z Liu Y Li X PDGF-C: a new performer in the neurovascular interplay Trends Mol Med 2013 19 8 474 486 10.1016/j.molmed.2013.04.006 23714575
Lee C, Zhang F, Tang Z, Liu Y, Li X. PDGF-C: a new performer in the neurovascular interplay. Trends Mol Med. 2013;19(8):474–86. 10.1016/j.molmed.2013.04.006.23714575 10.1016/j.molmed.2013.04.006
48. Maeda N Ichihara-Tanaka K Kimura T A receptor-like protein-tyrosine phosphatase PTPzeta/RPTPbeta binds a heparin-binding growth factor midkine. Involvement of arginine 78 of midkine in the high affinity binding to PTPzeta J Biol Chem. 1999 274 18 12474 9 10.1074/jbc.274.18.12474 10212223
Maeda N, Ichihara-Tanaka K, Kimura T, et al. A receptor-like protein-tyrosine phosphatase PTPzeta/RPTPbeta binds a heparin-binding growth factor midkine. Involvement of arginine 78 of midkine in the high affinity binding to PTPzeta. J Biol Chem. 1999;274(18):12474–9. 10.1074/jbc.274.18.12474.10212223 10.1074/jbc.274.18.12474
49. Delvalle NM Dharshika C Morales-Soto W Communication between enteric neurons, glia, and nociceptors underlies the effects of tachykinins on neuroinflammation Cell Mol Gastroenterol Hepatol 2018 6 3 321 344 10.1016/j.jcmgh.2018.05.009 30116771
Delvalle NM, Dharshika C, Morales-Soto W, et al. Communication between enteric neurons, glia, and nociceptors underlies the effects of tachykinins on neuroinflammation. Cell Mol Gastroenterol Hepatol. 2018;6(3):321–44. 10.1016/j.jcmgh.2018.05.009.30116771 10.1016/j.jcmgh.2018.05.009
50. Seguella L Gulbransen BD Enteric glial biology, intercellular signalling and roles in gastrointestinal disease Nat Rev Gastroenterol Hepatol 2021 18 8 571 587 10.1038/s41575-021-00423-7 33731961
Seguella L, Gulbransen BD. Enteric glial biology, intercellular signalling and roles in gastrointestinal disease. Nat Rev Gastroenterol Hepatol. 2021;18(8):571–87. 10.1038/s41575-021-00423-7.33731961 10.1038/s41575-021-00423-7
51. Sesia SB Mayr J Bruder E Haecker FM Neurogenic appendicopathy: clinical, macroscopic, and histopathological presentation in pediatric patients Eur J Pediatr Surg 2013 23 3 238 242 10.1055/s-0032-1333119 23444074
Sesia SB, Mayr J, Bruder E, Haecker FM. Neurogenic appendicopathy: clinical, macroscopic, and histopathological presentation in pediatric patients. Eur J Pediatr Surg. 2013;23(3):238–42. 10.1055/s-0032-1333119.23444074 10.1055/s-0032-1333119
52. Parikh K Antanaviciute A Fawkner-Corbett D Colonic epithelial cell diversity in health and inflammatory bowel disease Nature 2019 567 7746 49 55 10.1038/s41586-019-0992-y 30814735
Parikh K, Antanaviciute A, Fawkner-Corbett D, et al. Colonic epithelial cell diversity in health and inflammatory bowel disease. Nature. 2019;567(7746):49–55. 10.1038/s41586-019-0992-y.30814735 10.1038/s41586-019-0992-y
53. Elmentaite R Ross ADB Roberts K Single-cell sequencing of developing human gut reveals transcriptional links to childhood Crohn's disease Dev Cell 2020 55 6 771 83.e5 10.1016/j.devcel.2020.11.010 33290721
Elmentaite R, Ross ADB, Roberts K, et al. Single-cell sequencing of developing human gut reveals transcriptional links to childhood Crohn’s disease. Dev Cell. 2020;55(6):771-83.e5. 10.1016/j.devcel.2020.11.010.33290721 10.1016/j.devcel.2020.11.010
54. Odenwald MA Turner JR The intestinal epithelial barrier: a therapeutic target? Nat Rev Gastroenterol Hepatol 2017 14 1 9 21 10.1038/nrgastro.2016.169 27848962
Odenwald MA, Turner JR. The intestinal epithelial barrier: a therapeutic target? Nat Rev Gastroenterol Hepatol. 2017;14(1):9–21. 10.1038/nrgastro.2016.169.27848962 10.1038/nrgastro.2016.169
55. Peterson LW Artis D Intestinal epithelial cells: regulators of barrier function and immune homeostasis Nat Rev Immunol 2014 14 3 141 153 10.1038/nri3608 24566914
Peterson LW, Artis D. Intestinal epithelial cells: regulators of barrier function and immune homeostasis. Nat Rev Immunol. 2014;14(3):141–53. 10.1038/nri3608.24566914 10.1038/nri3608
56. Pelaseyed T Bergström JH Gustafsson JK The mucus and mucins of the goblet cells and enterocytes provide the first defense line of the gastrointestinal tract and interact with the immune system Immunol Rev 2014 260 1 8 20 10.1111/imr.12182 24942678
Pelaseyed T, Bergström JH, Gustafsson JK, et al. The mucus and mucins of the goblet cells and enterocytes provide the first defense line of the gastrointestinal tract and interact with the immune system. Immunol Rev. 2014;260(1):8–20. 10.1111/imr.12182.24942678 10.1111/imr.12182
57. Johansson MEV Hansson GC Immunological aspects of intestinal mucus and mucins Nat Rev Immunol 2016 16 10 639 649 10.1038/nri.2016.88 27498766
Johansson MEV, Hansson GC. Immunological aspects of intestinal mucus and mucins. Nat Rev Immunol. 2016;16(10):639–49. 10.1038/nri.2016.88.27498766 10.1038/nri.2016.88
58. Chelakkot C Ghim J Ryu SH Mechanisms regulating intestinal barrier integrity and its pathological implications Exp Mol Med 2018 50 8 1 9 10.1038/s12276-018-0126-x 30115904
Chelakkot C, Ghim J, Ryu SH. Mechanisms regulating intestinal barrier integrity and its pathological implications. Exp Mol Med. 2018;50(8):1–9. 10.1038/s12276-018-0126-x.30115904 10.1038/s12276-018-0126-x
59. Yang S Yu M Role of goblet cells in intestinal barrier and mucosal immunity J Inflamm Res 2021 14 3171 3183 10.2147/jir.S318327 34285541
Yang S, Yu M. Role of goblet cells in intestinal barrier and mucosal immunity. J Inflamm Res. 2021;14:3171–83. 10.2147/jir.S318327.34285541 10.2147/jir.S318327
60. Birchenough GM Johansson ME Gustafsson JK Bergström JH Hansson GC New developments in goblet cell mucus secretion and function Mucosal Immunol 2015 8 4 712 719 10.1038/mi.2015.32 25872481
Birchenough GM, Johansson ME, Gustafsson JK, Bergström JH, Hansson GC. New developments in goblet cell mucus secretion and function. Mucosal Immunol. 2015;8(4):712–9. 10.1038/mi.2015.32.25872481 10.1038/mi.2015.32
61. Naama M Telpaz S Awad A Autophagy controls mucus secretion from intestinal goblet cells by alleviating ER stress Cell Host Microbe 2023 31 3 433 46.e4 10.1016/j.chom.2023.01.006 36738733
Naama M, Telpaz S, Awad A, et al. Autophagy controls mucus secretion from intestinal goblet cells by alleviating ER stress. Cell Host Microbe. 2023;31(3):433-46.e4. 10.1016/j.chom.2023.01.006.36738733 10.1016/j.chom.2023.01.006
62. He S Wang J Huang Y Intestinal fibrosis in aganglionic segment of Hirschsprung's disease revealed by single-cell RNA sequencing Clin Transl Med 2023 13 2 e1193 10.1002/ctm2.1193 36738110
He S, Wang J, Huang Y, et al. Intestinal fibrosis in aganglionic segment of Hirschsprung’s disease revealed by single-cell RNA sequencing. Clin Transl Med. 2023;13(2):e1193. 10.1002/ctm2.1193.36738110 10.1002/ctm2.1193
63. Beumer J Puschhof J Yengej FY BMP gradient along the intestinal villus axis controls zonated enterocyte and goblet cell states Cell Rep 2022 38 9 110438 10.1016/j.celrep.2022.110438 35235783
Beumer J, Puschhof J, Yengej FY, et al. BMP gradient along the intestinal villus axis controls zonated enterocyte and goblet cell states. Cell Rep. 2022;38(9):110438. 10.1016/j.celrep.2022.110438.35235783 10.1016/j.celrep.2022.110438
64. Miyazono K Kamiya Y Morikawa M Bone morphogenetic protein receptors and signal transduction J Biochem 2010 147 1 35 51 10.1093/jb/mvp148 19762341
Miyazono K, Kamiya Y, Morikawa M. Bone morphogenetic protein receptors and signal transduction. J Biochem. 2010;147(1):35–51. 10.1093/jb/mvp148.19762341 10.1093/jb/mvp148
65. Chiba N Noguchi Y Seong CH Ohnishi T Matsuguchi T EGR1 plays an important role in BMP9-mediated osteoblast differentiation by promoting SMAD1/5 phosphorylation FEBS Lett 2022 596 13 1720 1732 10.1002/1873-3468.14407 35594155
Chiba N, Noguchi Y, Seong CH, Ohnishi T, Matsuguchi T. EGR1 plays an important role in BMP9-mediated osteoblast differentiation by promoting SMAD1/5 phosphorylation. FEBS Lett. 2022;596(13):1720–32. 10.1002/1873-3468.14407.35594155 10.1002/1873-3468.14407
66. Pereira RC Delany AM Canalis E CCAAT/enhancer binding protein homologous protein (DDIT3) induces osteoblastic cell differentiation Endocrinology 2004 145 4 1952 1960 10.1210/en.2003-0868 14684614
Pereira RC, Delany AM, Canalis E. CCAAT/enhancer binding protein homologous protein (DDIT3) induces osteoblastic cell differentiation. Endocrinology. 2004;145(4):1952–60. 10.1210/en.2003-0868.14684614 10.1210/en.2003-0868
67. Chung SS Lee JS Kim M Regulation of Wnt/β-catenin signaling by CCAAT/enhancer binding protein β during adipogenesis Obesity 2012 20 3 482 487 10.1038/oby.2011.212 21760632
Chung SS, Lee JS, Kim M, et al. Regulation of Wnt/β-catenin signaling by CCAAT/enhancer binding protein β during adipogenesis. Obesity. 2012;20(3):482–7. 10.1038/oby.2011.212.21760632 10.1038/oby.2011.212
68. Zhang N, Kim SH, Gainullina A, et al. LYVE1+ macrophages of murine peritoneal mesothelium promote omentum-independent ovarian tumor growth. J Exp Med. 2021;218(12). 10.1084/jem.20210924
69. Lim HY Lim SY Tan CK Hyaluronan receptor LYVE-1-expressing macrophages maintain arterial tone through hyaluronan-mediated regulation of smooth muscle cell collagen Immunity 2018 49 2 326 41.e7 10.1016/j.immuni.2018.06.008 30054204
Lim HY, Lim SY, Tan CK, et al. Hyaluronan receptor LYVE-1-expressing macrophages maintain arterial tone through hyaluronan-mediated regulation of smooth muscle cell collagen. Immunity. 2018;49(2):326-41.e7. 10.1016/j.immuni.2018.06.008.30054204 10.1016/j.immuni.2018.06.008
70. Kieu TQ Tazawa K Kawashima N Kinetics of LYVE-1-positive M2-like macrophages in developing and repairing dental pulp in vivo and their pro-angiogenic activity in vitro Sci Rep 2022 12 1 5176 10.1038/s41598-022-08987-3 35338195
Kieu TQ, Tazawa K, Kawashima N, et al. Kinetics of LYVE-1-positive M2-like macrophages in developing and repairing dental pulp in vivo and their pro-angiogenic activity in vitro. Sci Rep. 2022;12(1):5176. 10.1038/s41598-022-08987-3.35338195 10.1038/s41598-022-08987-3
71. Zhang Q He Y Luo N Landscape and dynamics of single immune cells in hepatocellular carcinoma Cell 2019 179 4 829 45.e20 10.1016/j.cell.2019.10.003 31675496
Zhang Q, He Y, Luo N, et al. Landscape and dynamics of single immune cells in hepatocellular carcinoma. Cell. 2019;179(4):829-45.e20. 10.1016/j.cell.2019.10.003.31675496 10.1016/j.cell.2019.10.003
72. Hong W, Yang B, He Q, Wang J, Weng Q. New insights of CCR7 signaling in dendritic cell migration and inflammatory diseases. Front Pharmacol. 2022;13
73. Riol-Blanco L Sánchez-Sánchez N Torres A The chemokine receptor CCR7 activates in dendritic cells two signaling modules that independently regulate chemotaxis and migratory speed J Immunol 2005 174 7 4070 4080 10.4049/jimmunol.174.7.4070 15778365
Riol-Blanco L, Sánchez-Sánchez N, Torres A, et al. The chemokine receptor CCR7 activates in dendritic cells two signaling modules that independently regulate chemotaxis and migratory speed. J Immunol. 2005;174(7):4070–80. 10.4049/jimmunol.174.7.4070.15778365 10.4049/jimmunol.174.7.4070
74. Yamakita Y Matsumura F Lipscomb MW Fascin1 promotes cell migration of mature dendritic cells J Immunol 2011 186 5 2850 2859 10.4049/jimmunol.1001667 21263068
Yamakita Y, Matsumura F, Lipscomb MW, et al. Fascin1 promotes cell migration of mature dendritic cells. J Immunol. 2011;186(5):2850–9. 10.4049/jimmunol.1001667.21263068 10.4049/jimmunol.1001667
75. Yu M Chen Y Zeng H PLCγ-dependent mTOR signalling controls IL-7-mediated early B cell development Nat Commun 2017 8 1 1457 10.1038/s41467-017-01388-5 29133930
Yu M, Chen Y, Zeng H, et al. PLCγ-dependent mTOR signalling controls IL-7-mediated early B cell development. Nat Commun. 2017;8(1):1457. 10.1038/s41467-017-01388-5.29133930 10.1038/s41467-017-01388-5
76. Wang D Feng J Wen R Phospholipase Cγ2 is essential in the functions of B cell and several Fc receptors Immunity 2000 13 1 25 35 10.1016/S1074-7613(00)00005-4 10933392
Wang D, Feng J, Wen R, et al. Phospholipase Cγ2 is essential in the functions of B cell and several Fc receptors. Immunity. 2000;13(1):25–35. 10.1016/S1074-7613(00)00005-4.10933392 10.1016/S1074-7613(00)00005-4
77. Guo X Zhang Y Zheng L Global characterization of T cells in non-small-cell lung cancer by single-cell sequencing Nat Med 2018 24 7 978 985 10.1038/s41591-018-0045-3 29942094
Guo X, Zhang Y, Zheng L, et al. Global characterization of T cells in non-small-cell lung cancer by single-cell sequencing. Nat Med. 2018;24(7):978–85. 10.1038/s41591-018-0045-3.29942094 10.1038/s41591-018-0045-3
78. Sun Y Wu L Zhong Y Single-cell landscape of the ecosystem in early-relapse hepatocellular carcinoma Cell 2021 184 2 404 21.e16 10.1016/j.cell.2020.11.041 33357445
Sun Y, Wu L, Zhong Y, et al. Single-cell landscape of the ecosystem in early-relapse hepatocellular carcinoma. Cell. 2021;184(2):404-21.e16. 10.1016/j.cell.2020.11.041.33357445 10.1016/j.cell.2020.11.041
79. Slade CD Reagin KL Lakshmanan HG Klonowski KD Watford WT Placenta-specific 8 limits IFNγ production by CD4 T cells in vitro and promotes establishment of influenza-specific CD8 T cells in vivo PLoS ONE 2020 15 7 e0235706 10.1371/journal.pone.0235706 32639988
Slade CD, Reagin KL, Lakshmanan HG, Klonowski KD, Watford WT. Placenta-specific 8 limits IFNγ production by CD4 T cells in vitro and promotes establishment of influenza-specific CD8 T cells in vivo. PLoS ONE. 2020;15(7):e0235706. 10.1371/journal.pone.0235706.32639988 10.1371/journal.pone.0235706
80. Yánez DC Ross S Crompton T The IFITM protein family in adaptive immunity Immunology 2020 159 4 365 372 10.1111/imm.13163 31792954
Yánez DC, Ross S, Crompton T. The IFITM protein family in adaptive immunity. Immunology. 2020;159(4):365–72. 10.1111/imm.13163.31792954 10.1111/imm.13163
81. Tirosh I Izar B Prakadan SM Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq Science 2016 352 6282 189 196 10.1126/science.aad0501 27124452
Tirosh I, Izar B, Prakadan SM, et al. Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq. Science. 2016;352(6282):189–96. 10.1126/science.aad0501.27124452 10.1126/science.aad0501
82. Vivier E Artis D Colonna M Innate lymphoid cells: 10 years on Cell 2018 174 5 1054 1066 10.1016/j.cell.2018.07.017 30142344
Vivier E, Artis D, Colonna M, et al. Innate lymphoid cells: 10 years on. Cell. 2018;174(5):1054–66. 10.1016/j.cell.2018.07.017.30142344 10.1016/j.cell.2018.07.017
83. Smole U Gour N Phelan J Serum amyloid A is a soluble pattern recognition receptor that drives type 2 immunity Nat Immunol 2020 21 7 756 765 10.1038/s41590-020-0698-1 32572240
Smole U, Gour N, Phelan J, et al. Serum amyloid A is a soluble pattern recognition receptor that drives type 2 immunity. Nat Immunol. 2020;21(7):756–65. 10.1038/s41590-020-0698-1.32572240 10.1038/s41590-020-0698-1
84. Kohyama M Matsuoka S Shida K Monocyte infiltration into obese and fibrilized tissues is regulated by PILRα Eur J Immunol 2016 46 5 1214 1223 10.1002/eji.201545897 26840635
Kohyama M, Matsuoka S, Shida K, et al. Monocyte infiltration into obese and fibrilized tissues is regulated by PILRα. Eur J Immunol. 2016;46(5):1214–23. 10.1002/eji.201545897.26840635 10.1002/eji.201545897
85. Krishnamurty AT Turley SJ Lymph node stromal cells: cartographers of the immune system Nat Immunol 2020 21 4 369 380 10.1038/s41590-020-0635-3 32205888
Krishnamurty AT, Turley SJ. Lymph node stromal cells: cartographers of the immune system. Nat Immunol. 2020;21(4):369–80. 10.1038/s41590-020-0635-3.32205888 10.1038/s41590-020-0635-3
86. Bhangu A Søreide K Di Saverio S Assarsson JH Drake FT Acute appendicitis: modern understanding of pathogenesis, diagnosis, and management Lancet 2015 386 10000 1278 1287 10.1016/s0140-6736(15)00275-5 26460662
Bhangu A, Søreide K, Di Saverio S, Assarsson JH, Drake FT. Acute appendicitis: modern understanding of pathogenesis, diagnosis, and management. Lancet. 2015;386(10000):1278–87. 10.1016/s0140-6736(15)00275-5.26460662 10.1016/s0140-6736(15)00275-5
87. Huang B Chen Z Geng L Mucosal profiling of pediatric-onset colitis and IBD reveals common pathogenics and therapeutic pathways Cell 2019 179 5 1160 76.e24 10.1016/j.cell.2019.10.027 31730855
Huang B, Chen Z, Geng L, et al. Mucosal profiling of pediatric-onset colitis and IBD reveals common pathogenics and therapeutic pathways. Cell. 2019;179(5):1160-76.e24. 10.1016/j.cell.2019.10.027.31730855 10.1016/j.cell.2019.10.027
88. Murphy CG Glickman JN Tomczak K Acute appendicitis is characterized by a uniform and highly selective pattern of inflammatory gene expression Mucosal Immunol 2008 1 4 297 308 10.1038/mi.2008.13 19079191
Murphy CG, Glickman JN, Tomczak K, et al. Acute appendicitis is characterized by a uniform and highly selective pattern of inflammatory gene expression. Mucosal Immunol. 2008;1(4):297–308. 10.1038/mi.2008.13.19079191 10.1038/mi.2008.13
89. Ito N Kii I Shimizu N Tanaka H Takeda S Direct reprogramming of fibroblasts into skeletal muscle progenitor cells by transcription factors enriched in undifferentiated subpopulation of satellite cells Sci Rep 2017 7 1 8097 10.1038/s41598-017-08232-2 28808339
Ito N, Kii I, Shimizu N, Tanaka H, Takeda S. Direct reprogramming of fibroblasts into skeletal muscle progenitor cells by transcription factors enriched in undifferentiated subpopulation of satellite cells. Sci Rep. 2017;7(1):8097. 10.1038/s41598-017-08232-2.28808339 10.1038/s41598-017-08232-2
90. Wang JS Infante CR Park S Menke DB PITX1 promotes chondrogenesis and myogenesis in mouse hindlimbs through conserved regulatory targets Dev Biol 2018 434 1 186 195 10.1016/j.ydbio.2017.12.013 29273440
Wang JS, Infante CR, Park S, Menke DB. PITX1 promotes chondrogenesis and myogenesis in mouse hindlimbs through conserved regulatory targets. Dev Biol. 2018;434(1):186–95. 10.1016/j.ydbio.2017.12.013.29273440 10.1016/j.ydbio.2017.12.013
91. Koliaraki V Prados A Armaka M Kollias G The mesenchymal context in inflammation, immunity and cancer Nat Immunol 2020 21 9 974 982 10.1038/s41590-020-0741-2 32747813
Koliaraki V, Prados A, Armaka M, Kollias G. The mesenchymal context in inflammation, immunity and cancer. Nat Immunol. 2020;21(9):974–82. 10.1038/s41590-020-0741-2.32747813 10.1038/s41590-020-0741-2
92. Stinson LF Boyce MC Payne MS Keelan JA The not-so-sterile womb: evidence that the human fetus is exposed to bacteria prior to birth Front Microbiol 2019 10 1124 10.3389/fmicb.2019.01124 31231319
Stinson LF, Boyce MC, Payne MS, Keelan JA. The not-so-sterile womb: evidence that the human fetus is exposed to bacteria prior to birth. Front Microbiol. 2019;10:1124. 10.3389/fmicb.2019.01124.31231319 10.3389/fmicb.2019.01124
93. Miko E, Csaszar A, Bodis J, Kovacs K. The maternal-fetal gut microbiota axis: physiological changes, dietary influence, and modulation possibilities. Life (Basel). 2022;12(3). 10.3390/life12030424
94. Shroyer NF Wong MH BMP Signaling in the intestine: cross-talk is key Gastroenterology 2007 133 3 1035 1038 10.1053/j.gastro.2007.07.018 17854606
Shroyer NF, Wong MH. BMP Signaling in the intestine: cross-talk is key. Gastroenterology. 2007;133(3):1035–8. 10.1053/j.gastro.2007.07.018.17854606 10.1053/j.gastro.2007.07.018
95. Beumer J Clevers H Cell fate specification and differentiation in the adult mammalian intestine Nat Rev Mol Cell Biol 2021 22 1 39 53 10.1038/s41580-020-0278-0 32958874
Beumer J, Clevers H. Cell fate specification and differentiation in the adult mammalian intestine. Nat Rev Mol Cell Biol. 2021;22(1):39–53. 10.1038/s41580-020-0278-0.32958874 10.1038/s41580-020-0278-0
96. Medina-Flores F Hurtado-Alvarado G Deli MA Gómez-González B The active role of pericytes during neuroinflammation in the adult brain Cell Mol Neurobiol 2022 10.1007/s10571-022-01208-5 35195811
Medina-Flores F, Hurtado-Alvarado G, Deli MA, Gómez-González B. The active role of pericytes during neuroinflammation in the adult brain. Cell Mol Neurobiol. 2022. 10.1007/s10571-022-01208-5.35195811 10.1007/s10571-022-01208-5
97. Thomas H, Cowin AJ, Mills SJ. The importance of pericytes in healing: wounds and other pathologies. Int J Mol Sci. 2017;18(6). 10.3390/ijms18061129
98. Zhang Z-S Zhou H-N He S-S Research advances in pericyte function and their roles in diseases Chin J Traumatol 2020 23 2 89 95 10.1016/j.cjtee.2020.02.006 32192909
Zhang Z-S, Zhou H-N, He S-S, et al. Research advances in pericyte function and their roles in diseases. Chin J Traumatol. 2020;23(2):89–95. 10.1016/j.cjtee.2020.02.006.32192909 10.1016/j.cjtee.2020.02.006
99. Mueller SN Mackay LK Tissue-resident memory T cells: local specialists in immune defence Nat Rev Immunol 2016 16 2 79 89 10.1038/nri.2015.3 26688350
Mueller SN, Mackay LK. Tissue-resident memory T cells: local specialists in immune defence. Nat Rev Immunol. 2016;16(2):79–89. 10.1038/nri.2015.3.26688350 10.1038/nri.2015.3
100. Hashimoto K Kouno T Ikawa T Single-cell transcriptomics reveals expansion of cytotoxic CD4 T cells in supercentenarians Proc Natl Acad Sci 2019 116 48 24242 24251 10.1073/pnas.1907883116 31719197
Hashimoto K, Kouno T, Ikawa T, et al. Single-cell transcriptomics reveals expansion of cytotoxic CD4 T cells in supercentenarians. Proc Natl Acad Sci. 2019;116(48):24242–51. 10.1073/pnas.1907883116.31719197 10.1073/pnas.1907883116
101. Gebbers J-O Laissue J-A Bacterial translocation in the normal human appendix parallels the development of the local immune system Ann N Y Acad Sci 2004 1029 1 337 343 10.1196/annals.1309.015 15681775
Gebbers J-O, Laissue J-A. Bacterial translocation in the normal human appendix parallels the development of the local immune system. Ann N Y Acad Sci. 2004;1029(1):337–43. 10.1196/annals.1309.015.15681775 10.1196/annals.1309.015
102. Zhang L Li Z Skrzypczynska KM Single-cell analyses inform mechanisms of myeloid-targeted therapies in colon cancer Cell 2020 181 2 442 59.e29 10.1016/j.cell.2020.03.048 32302573
Zhang L, Li Z, Skrzypczynska KM, et al. Single-cell analyses inform mechanisms of myeloid-targeted therapies in colon cancer. Cell. 2020;181(2):442-59.e29. 10.1016/j.cell.2020.03.048.32302573 10.1016/j.cell.2020.03.048
103. Kumar R Theiss AL Venuprasad K RORγt protein modifications and IL-17-mediated inflammation Trends Immunol 2021 42 11 1037 1050 10.1016/j.it.2021.09.005 34635393
Kumar R, Theiss AL, Venuprasad K. RORγt protein modifications and IL-17-mediated inflammation. Trends Immunol. 2021;42(11):1037–50. 10.1016/j.it.2021.09.005.34635393 10.1016/j.it.2021.09.005
104. Amatya N Garg AV Gaffen SL IL-17 Signaling: the yin and the yang Trends Immunol 2017 38 5 310 322 10.1016/j.it.2017.01.006 28254169
Amatya N, Garg AV, Gaffen SL. IL-17 Signaling: the yin and the yang. Trends Immunol. 2017;38(5):310–22. 10.1016/j.it.2017.01.006.28254169 10.1016/j.it.2017.01.006
105. Bie Q Jin C Zhang B Dong H IL-17B: a new area of study in the IL-17 family Mol Immunol 2017 90 50 56 10.1016/j.molimm.2017.07.004 28704706
Bie Q, Jin C, Zhang B, Dong H. IL-17B: a new area of study in the IL-17 family. Mol Immunol. 2017;90:50–6. 10.1016/j.molimm.2017.07.004.28704706 10.1016/j.molimm.2017.07.004
106. Myrelid P Landerholm K Nordenvall C Pinkney TD Andersson RE Appendectomy and the risk of colectomy in ulcerative colitis: a national cohort study Am J Gastroenterol 2017 112 8 1311 1319 10.1038/ajg.2017.183 28653667
Myrelid P, Landerholm K, Nordenvall C, Pinkney TD, Andersson RE. Appendectomy and the risk of colectomy in ulcerative colitis: a national cohort study. Am J Gastroenterol. 2017;112(8):1311–9. 10.1038/ajg.2017.183.28653667 10.1038/ajg.2017.183
107. Cheluvappa R Identification of new potential therapies for colitis amelioration using an appendicitis-appendectomy model Inflamm Bowel Dis 2019 25 3 436 444 10.1093/ibd/izy332 30329049
Cheluvappa R. Identification of new potential therapies for colitis amelioration using an appendicitis-appendectomy model. Inflamm Bowel Dis. 2019;25(3):436–44. 10.1093/ibd/izy332.30329049 10.1093/ibd/izy332
108. The SML Schreurs R Drewniak A Enhanced Th17 responses in the appendix of children with complex compared to simple appendicitis are associated with microbial dysbiosis Front Immunol 2023 14 1258363 10.3389/fimmu.2023.1258363 38239362
The SML, Schreurs R, Drewniak A, et al. Enhanced Th17 responses in the appendix of children with complex compared to simple appendicitis are associated with microbial dysbiosis. Front Immunol. 2023;14:1258363. 10.3389/fimmu.2023.1258363.38239362 10.3389/fimmu.2023.1258363
109. Di Gangi A Di Cicco ME Comberiati P Peroni DG Go with your gut: the shaping of t-cell response by gut microbiota in allergic asthma Front Immunol 2020 11 1485 10.3389/fimmu.2020.01485 32760404
Di Gangi A, Di Cicco ME, Comberiati P, Peroni DG. Go with your gut: the shaping of t-cell response by gut microbiota in allergic asthma. Front Immunol. 2020;11:1485. 10.3389/fimmu.2020.01485.32760404 10.3389/fimmu.2020.01485
110. Shi F Liu G Lin Y Altered gut microbiome composition by appendectomy contributes to colorectal cancer Oncogene 2023 42 7 530 540 10.1038/s41388-022-02569-3 36539569
Shi F, Liu G, Lin Y, et al. Altered gut microbiome composition by appendectomy contributes to colorectal cancer. Oncogene. 2023;42(7):530–40. 10.1038/s41388-022-02569-3.36539569 10.1038/s41388-022-02569-3
111. Huangfu L Li R Huang Y Wang S The IL-17 family in diseases: from bench to bedside Signal Transduct Target Ther 2023 8 1 402 10.1038/s41392-023-01620-3 37816755
Huangfu L, Li R, Huang Y, Wang S. The IL-17 family in diseases: from bench to bedside. Signal Transduct Target Ther. 2023;8(1):402. 10.1038/s41392-023-01620-3.37816755 10.1038/s41392-023-01620-3
112. Fletcher JM Moran B Petrasca A Smith CM IL-17 in inflammatory skin diseases psoriasis and hidradenitis suppurativa Clin Exp Immunol 2020 201 2 121 134 10.1111/cei.13449 32379344
Fletcher JM, Moran B, Petrasca A, Smith CM. IL-17 in inflammatory skin diseases psoriasis and hidradenitis suppurativa. Clin Exp Immunol. 2020;201(2):121–34. 10.1111/cei.13449.32379344 10.1111/cei.13449
