Retroviruses are unique in that they package their RNA genomes as non-covalently linked dimers. Failure to dimerize their genomes results in decreased infectivity and reduced packaging of genomic RNA into virus particles. Two models of retrovirus genome dimerization have been characterized: in murine leukemia virus (MLV), genomic RNA dimerization occurs co-transcriptionally in the nucleus, resulting in the preferential formation of genome homodimers; whereas in human immunodeficiency virus (HIV-1), genomic RNA dimerization occurs in the cytoplasm and at the plasma membrane, with a random distribution of heterodimers and homodimers. Although in vitro studies have identified the genomic RNA sequences that facilitate dimerization in Rous sarcoma virus (RSV), in vivo characterization of the location and preferences of genome dimerization has not been performed. In this study, we utilized three single molecule RNA imaging approaches to visualize genome dimers of RSV in cultured quail fibroblasts. The formation of genomic RNA heterodimers within cells was dependent on the presence of the dimerization initiation site (DIS) sequence in the L3 stem. Subcellular localization analysis revealed that heterodimers were present the nucleus, cytoplasm, and at the plasma membrane, indicating that genome dimers can form in the nucleus. Furthermore, single virion analysis revealed that RSV preferentially packages genome homodimers into virus particles. Therefore, the mechanism of RSV genomic RNA dimer formation appears more similar to MLV than HIV-1. Retroviruses are unique in that they package their single-stranded RNA genomes as non-covalently linked dimers. Genome dimerization is evolutionarily conserved in retroviruses; mutation of the genome dimerization site or failure of a genome to appropriately dimerize results in loss of infectivity of the resulting virus ,3,4,5,6.In vitro experiments have identified two regions of the genome that are important for retroviral genome dimerization: the dimer linkage structure (DLS) and the dimerization initiation site (DIS). The DLS was identified in Rous sarcoma virus (RSV) and characterized by electron microscopy as a physical region where the 5′ untranslated region (5′UTR) of two partially-denatured RSV genomes interacted to form a dimer structure ,15,16,17Previous studies in cell culture have characterized two models of genomic RNA dimerization in retroviruses. In murine leukemia virus (MLV), genome dimerization occurs in the nucleus in a co-transcriptional fashion, resulting in the preferential formation of genetically identical genome homodimers ,27,28,29To address these questions, we used single molecule RNA labeling methods to visualize RSV unspliced vRNAs in cells and virions. We were interested in determining the subcellular localization of unspliced vRNA heterodimers that were genetically distinct and therefore distinguishable using different fluorescence markers. Notably, vRNA homodimers could not be reliably differentiated from single vRNA molecules (monomers) using these methods because they both appeared as a single-colored focus within the cell.Using confocal microscopy, we detected RSV RNA heterodimers in the nucleus, cytoplasm, and at the plasma membrane, suggesting that genome dimerization may originate in the nucleus, similarly to MLV. Deletion of the DIS from the RSV leader sequence significantly reduced colocalization of genetically distinct unspliced vRNAs, indicating that these dual-colored foci were likely formed by the process of dimerization. Single virion analysis of genome composition demonstrated that RSV primarily packaged genomic RNA homodimers, once again similar to that of MLV. Lastly, we demonstrated that intracellular heterodimers were primarily composed of a pair of unspliced vRNAs rather than being influenced by the presence of a mixture of labeled unspliced and spliced vRNAs. Taken together, these findings define the genome dimerization preferences for RSV and suggest that dimers may initially arise in the nucleus, in an analogous fashion as MLV.pol gene and substitution of the pATV8 gag gene. The pRC.V8 plasmid only contains one splice acceptor site and generates one unspliced RNA and one spliced env RNA ,58mCherrem-loops , with a em-loops ,47 to vimCherry sequence does not interfere with virus assembly, as each of the proviral constructs used was budding competent and can generate virus particles detected by immunoblotting of cell lysates and supernatants  unspliced RNA heterodimers and single-color RNA foci (red or green) per cell were measured using each of the three imaging combinations . The totTo examine where genomic RNA dimerization occurs within the cell, the average number of genomic RNA heterodimers present in the nucleus, in the cytoplasm, and at the plasma membrane was quantitated . ColocalmCherry RNA sequence or a cassette of 24 copies of MS2 stem-loops was inserted into the intron to label unspliced vRNA   C. Two imTo determine whether RSV has a packaging preference for genome homo- or heterodimers, single virion imaging analysis was performed ,32. EquiAssuming random formation of genomic RNA dimers based on the Hardy–Weinberg equation, we would have expected to observe 50% homodimers and 50% heterodimers ,27,30,32env mRNA) [mCherry sequence to the env spliced subgenomic mRNA sequence, we selectively imaged only unspliced vRNAs versus both unspliced and spliced vRNAs  . Splicednv mRNA) , so we wed vRNAs A. Confoced vRNAs B.n = 19 cells and 24 cells, respectively; p = 0.2132; On a single cell basis, the average number of heterodimers per cell was 3.79 ± 0.49 for unspliced vRNA and 2.79 ± 1.10 for spliced plus unspliced vRNAs  contributed to genome dimerization, as the sequences have been used in previous studies for vRNA labeling and dimerization [This report is the first in vivo visualization of the role of the DIS sequence in genome dimerization using a full-length RSV genome. The sequences required for dimerization in the RSV leader sequence were previously identified using in vitro dimerization assays with truncated vRNAs that contained the 5′ UTR and minimal sequences in the g region ,60,61,62g region . This fiackaging . It is urization ,47,48,76As mentioned previously, the RSV psi packaging sequence is located upstream of the 5′ splice acceptor site and is thus present on both spliced and unspliced vRNAs. RSV unspliced vRNA is preferentially packaged into newly formed virions, and studies have suggested that the genome dimerization is linked to genome packaging ,5,8,36. We applied our imaging techniques to characterize the genome composition of genome heterodimers within cells when either only unspliced vRNAs or both spliced and unspliced vRNAs were labeled with different fluorescent markers . We founOur studies also demonstrated that each imaging technique has unique characteristics and caveats. As previously noted, the smFISH technique resulted in detection of brighter RNA foci in the nucleus, likely due to accumulation of multiple copies of genomic vRNAs, and fainter RNA foci in the cytoplasm and at the plasma membrane, likely due to the presence of fewer copies of genomic vRNAs. The difference in intensity resulted in uneven detection by the Imaris Spots algorithm, which uses an intensity threshold to identify foci. This is likely the explanation for the decreased number of total colocalized red-green foci detected by smFISH-smFISH as compared to MS2-YFP-smFISH and MS2-YFP-Bgl-YFP . The use of MS2 and Bgl aptamer labeling systems resulted in higher levels of nonspecific background fluorescence due to the presence of fluorophore-tagged coat proteins that were not bound to vRNA. We also noticed a labeling bias between the MS2 and Bgl aptamers, with more Bgl-labeled foci visualized as compared to MS2-labeled foci intracellularly and within virions. The bias was not due to the nature of the fluorophore fused to the coat protein because when the fluorophores were swapped, a Bgl labeling bias was still seen . All of In summary, the results of this study demonstrate that RSV genomic RNA dimer formation occurs in the nucleus, and dimers may also arise in the cytoplasm and at the plasma membrane. Genome heterodimer formation is dependent on the presence of the L3 stem-loop and palindromic DIS sequence and appear to be composed of a pair of unspliced vRNAs. Additionally, we demonstrated the use of three distinct imaging approaches to visualize distinct single molecule RNA genomes in cells. The presence of genome dimers in the nucleus is intriguing considering that RSV ribonucleoprotein complexes arise in the nucleus and nuclear Gag is important for efficient packaging of genomic RNA dimers ,64,65,67


Gallus gallus, GGA) chromosomes 1–9 and Z have been widely used to investigate avian chromosome homologies, GGA microchromosome probes are rarely used in these studies because most GGA microchromosome probes generated by flow sorting often contain multiple GGA microchromosomes. In contrast, the stone curlew  has an atypical low diploid chromosome number (42) karyotype and only 4 pairs of dot-like microchromosomes; a set of chromosome-specific painting probes that cover all BOE chromosomes has been generated. To get a genome-wide view of evolutionary chromosomal rearrangements in different lineages of Ciconiiformes, we used BOE painting probes instead of GGA painting probes to analyze the karyotypes of three ciconiiform species belonging to two different families: the eastern grey heron , the little egret  and the crested ibis .Previous cytogenetic studies show that the karyotypes of species in Ciconiiformes vary considerably, from 2n = 52 to 78. Their karyotypes include different numbers of small to minute bi-armed chromosomes that have evolved probably by fusions of two ancestral microchromosomes, besides macrochromosomes and dot-like microchromosomes. However, it is impossible to define the inter-species homologies of such small-sized bi-armed chromosomes based on chromosome morphology and banding characteristics. Although painting probes from the chicken  and EGA (EGA 5) or had fused with a part of the BOE 10 equivalent in NNI (NNI 5).BOE painting probes display the same hybridization pattern on chromosomes of ACI and EGA, while a different hybridization pattern is observed on chromosomes of NNI. BOE autosome probes detected 21 conserved homologous segments and 5 fusions on the sixteen pairs of recognizable chromosomes of ACI and EGA, while 16 conserved homologous segments and 4 fusions were found on the twelve pairs of recognizable chromosomes of NNI. Only a portion of smaller bi-armed chromosomes in the karyotypes of the ciconiiform species could have evolved from fusions of ancestral microchromosomes. In particular BOE 5, which is the result of a fusion between two segments homologous to Our painting results indicate that different chromosome rearrangements occur in different ciconiiform lineages. Some of the small-sized bi-armed chromosomes in ACI, EGA and NNI are derived from the fusions of two microchromosomes, indicating that microchromosome fusions play an important role in ciconiiform chromosome evolution. The fusion segment homologous to GGA 7 and 8 is a potential cytogenetic signature that unites Ardeidae and Threskiornithidae. According to traditional classification, the order Ciconiiformes comprises medium- to large-sized wading birds that have a worldwide distribution in temperate, subtropical, and tropical regions. There are 108 species in Ciconiiformes, belonging to three suborders, 5 families and 36 genera –17. OnlyGallus gallus, GGA) macrochromosomes 1–9 and Z, and four sets of probes from other bird species  macrochromosomes [Cross-species chromosome painting has been utilized to detect chromosomal homologies between different avian species for more than two decades. Up to 2019, 96 avian species from eighteen orders have been analyzed by chromosome painting mainly using probes from the chicken . Howeveiewed in ). For inomosomes , while sArdea cinerea, ACI), the little egret  and the crested ibis  are species from two different ciconiiform families: the former two belong to the Ardeidae, the last is a member of the Threskiornithidae. Previous comparative cytogenetic study indicated that both Ardeidae and Threskiornithidae constitute a clear-cut group based on changes in chromosome morphology; however, such chromosomal changes could not provide information on the relationships between these families due to their potential independent origins and the presence of undefined small chromosomes involved in the fusion with a segment of chromosome 1 [Burhinus oedicnemus, BOE, 2n = 42, Charadriiformes) were the first set of chromosome-specific painting probes from Neoaves species that were able to cover the entire genome of a given bird species [Gyps fulvus, GFU)  were used in hybridizations with ACI and NNI chromosomes. Our study provides some new insights into the karyotype evolution and phylogenetic relationships in Ciconiiformes.The eastern grey heron  to paint the chromosomes of ACI and NNI. Since ACI and EGA have similar karyotypes and identical hybridization patterns when painted with BOE probes, EGA is not included in this part of the study. Both ACI 3 (submetacentric) and NNI 3 (acrocentric) are painted by the probe of BOE 3 (corresponding to GGA 3) (see above). When we used the probe of GFU 16, which is homologous to part of BOE 3 , the disTaeniopygia guttata, Passeriformes) have been applied to investigate the chromosomal evolution of 41 species in 16 different orders [Previous studies have indicated that five pairs of BOE macrochromosomes  are highly conserved. As found in many other avian species, the five largest ancestral avian chromosomes, homologous to GGA 1–3, 4q and 5, are each retained as a single intact BOE chromosome (BOE 1–4 and 6), while the other 10 pairs of BOE bi-armed chromosomes  each represent a fusion product of two or three homologous GGA smaller macrochromosomes or microchromosomes . Therefot orders , 33–41. Up to now, the karyotypes of five species in three ciconiiform families have been studied by chromosome painting using probes from GGA, LAL and BOE , two species belonging to Charadrii which is one of the three major clades of Charadriiformes [V. chilensis, five species in Charadriiformes have been studied by chromosome painting; this association is not detected in the other three species of Charadriiformes [Among the fusions detected in Ardeidae and Threskiornithidae, two (corresponding to GGA 7 + 8 and 9 + 1 MIC) seem to be common for species in our study. BOE 5 (corresponding to GGA 7 + 8) as a conserved synteny was detected in ACI Fig. a, EGA Fb and NNIiiformes , 42. Inciiformes , 27, 43.iiformes , 31, 44.In Ciconiidae, three fusions  were observed in two Brazilian stork species, and one of the fusions (corresponding to GGA 8 + 9) was considered to be a shared character for storks . In our Although only five ciconiiform species have been studied by chromosome painting to date, these results demonstrate that storks differ from herons and ibises in chromosome rearrangements, and herons and ibises share a syntenic association that indicates a closer karyotype relationship. These chromosome painting data also provide support for the molecular phylogenomic studies that place herons and ibises in one clade and storks in another clade , 44–49. We have established genome-wide comparative chromosome maps between BOE and three ciconiiform species from two different families by chromosome painting using a set of BOE probes that cover the entire BOE genome. Our results indicate that chromosomal fusions play an important role in the karyotype evolution of species in Ciconiiformes. Besides fusions between macro- and microchromosome, fusions between microchromosomes are also found in species from Ardeidae and Threskiornithidae, leading to some small bi-armed chromosomes. A fusion of segments homologous to GGA 7 and 8 is shared by these three ciconiiform species, indicating that this fusion may be a cytogenetic signature linking Ardeidae and Threskiornithidae, and reinforcing the proposition that places herons and ibises in one clade as reported in previous molecular phylogenomic studies.A. cinerea, ACI), the little egret  and the crested ibis  were studied. The fibroblast cell lines of EGA  and NNI  were provided by Kunming Cell Bank, the Chinese Academy of Sciences. The cells were grown in DMEM (Dulbecco's Modified Eagle Medium) enriched with 15% fetal bovine serum. Metaphase preparation and slide preparation of NNI and EGA were carried out following conventional methods. Mitotic chromosomes of ACI were obtained from the bone marrow suspension of a male ACI prepared and stored in 1999 in Kunming Cell Bank, the Chinese Academy of Sciences.Three species belonging to two different families in Ciconiiformes, the eastern grey heron  were hybridized onto metaphases of ACI, EGA and NNI respectively to delineate the homologous segments between BOE, ACI, EGA and NNI. In addition, three painting probes from the chicken  and the griffon vulture  were used to verify results from BOE probes. The preparation of BOE, GGA and GFU painting probes, cross-species chromosome painting, image capture and processing were carried out as previously described [Painting probes from the stone curlew (escribed , 26. In escribed  were ind


Gastrointestinal cancer is currently one of the main causes of cancer death, with a large number of cases and a wide range of lesioned sites. A high fat diet, as a public health problem, has been shown to be correlated with various digestive system diseases and tumors, and can accelerate the occurrence of cancer due to inflammation and altered metabolism. The gut microbiome has been the focus of research in recent years, and associated with cell damage or tumor immune microenvironment changes via direct or extra-intestinal effects; this may facilitate the occurrence and development of gastrointestinal tumors. Based on research showing that both a high fat diet and gut microbes can promote the occurrence of gastrointestinal tumors, and that a high fat diet imbalances intestinal microbes, we propose that a high fat diet drives gastrointestinal tumors by changing the composition of intestinal microbes. According to 2020 statistics, 1,806,590 new cancer cases and 606,520 cancer deaths occurred in the United States, with gastrointestinal cancer being one of the leading causes of death With the development of the metagenome and macrotranscriptome in recent years, research into intestinal microorganisms has reached a new peak HFD can significantly promote the occurrence and development of gastrointestinal tumors, mainly involving metabolic reprogramming and the change of various carcinogenic molecules Figure .As early as in 1994, it was found that mice fed HFD had a higher incidence of esophageal cancer, suggesting a relationship between HFD and esophageal cancer In clinical epidemiological studies, Ibiebele et al. found that “meat and fat” were well correlated with esophageal adenocarcinoma, gastric esophagus adenocarcinoma and esophageal squamous cell carcinoma Many epidemiological studies have reported that dietary fat may be a risk factor for gastric cancer Arita and colleagues found that lipotoxicity associated with HFD induced precancerous lesions due to the disruption of gastric epithelial organelle homeostasis, tissue integrity, and stemness gene expression mediated by ObR signaling A study found that, during 8-20 weeks of HFD feeding, mitochondrial damage was found in gastric wall cells accompanied by increased mucosal thickness. The addition of free fatty acids (FFAs) can replicate this expression and promote metagenetic changes, indicating that the lipid toxicity of FFAs induces the death of parietal cells and the occurrence of precancerous lesions. Jiang et al. found that HFD could provide sufficient energy for metastasis and increase the level of O-Glc-N-acylation, which promoted the transcriptional activation of CD36. Upregulation of CD36 leads to increased fat uptake in gastric cancer cells, forming a vicious cycle that promotes gastric cancer metastasis Metabolic reprogramming, as a key intermediate, promotes the development of tumors by connecting many pro-oncogenic molecules. Studies have confirmed that HFD can cause significantly increased hepatic retention of hydrophobic bile acids, which are significantly associated with changes in intestinal microorganisms Reduced FGF21 is closely related to excessive proliferation, abnormal p53 and TGF-β/Smad signals IRE1α is linked to endoplasmic reticulum stress in liver cancer and drives pathogenesis .HFD can promote proliferation and inhibit abnormal cell clearance. A western diet induced excessive proliferation of pancreatic epithelial cells in mice and led to an increase in the frequency and possibility of mutations 38HFD can create both inflammatory and immunosuppressive tumor microenvironments. Philip et al. found that the pancreatic tissues of LSL-KRas/ela-CreERT mice fed with HFD had higher KRAS activity, fibrotic matrix, shorter survival time and higher degree of pancreatic intraepithelial neoplasms (PanIN) and pancreatic ductal adenocarcinoma (PDAC) G12D-expressing mice Metabolic reprogramming is also involved in PDAC. For example, HFD can promote the development of pancreatic tumors by over-activating oncogenic KRAS to induce aerobic glycolysis in vitro showed significant DNA damage and increased KRAS mutation, and they also found that KRAS mutant cells had growth advantages in both normal and high glucose environments.Hu et al. found that the mechanism of PDAC development may be related to DNA damage Epidemiological studies on CRC and western diets has confirmed the link between them There have also been many studies on the effects of HFD on cytokines or obesity factors. Zhu and colleagues found that elevated serum levels of insulin, leptin, TNF-α, IGF1, as well as increased levels of proliferating cell nuclear antigen, COX-2, cyclinD1, β-catenin and NFκB proteins in adenoma tissues indicate that HFD promotes the formation of colonic adenomas through inflammation and metabolic abnormalities, and influences cell cycle Many studies have demonstrated the key role of intestinal microorganisms in diseases induced by HFD, such as metabolic disorders Alistipes, Bilophila and Bacteroides) and reduce the level of Firmicutes that metabolize plant polysaccharides . Some metabolites, such as bile acid and metabolic enzymes (bisulfite reductase), are increased in the intestinal tract of whole-meat eaters. Bacteroidetes is highly correlated with animal proteins, various amino acids and saturated fats Bacteroides enterotypes, while children in Burkina Faso on a diet high in carbohydrates and low in animal protein were dominated by Privotella enterotypes David et al. have shown that short-term HFD can change the composition of gut microbes Firmicutes to BacteroidetesRecently, Bisanz et al. conducted a meta-analysis on the sequencing data of 27 animal and human studies on diet and flora and summarized the similarities between studies on the impact of HFD on intestinal flora composition. In most studies, the HFD raised the ratio of Previous studies have shown that intestinal microorganisms play an important pathogenic role in some gastrointestinal tumors. We hypothesized that intestinal microorganisms may have a causal effect on gastrointestinal tumors Figure .Helicobacter pylori infection has been shown to be negatively correlated with the occurrence of esophageal cancer H. pylori infection reduces the occurrence of gastroesophageal reflux and thus reduces the occurrence of Barrett's esophagus and esophageal cancer Fusobacterium nucleatum may lead to aggressive tumor behavior through the activation of chemokines such as CCL20 in esophageal tissues Microorganisms in the esophagus are affected by oral and stomach microbes H. pylori induces complex inflammatory and immune responses that lead to a series of morphological events, beginning with chronic gastritis in almost all infected individuals, from atrophy to intestinal metaplasia to dysplasia and eventually to gastric cancer HelicobacterH. pylori strains can stimulate the expression of interleukin by activating the transcription factor NFκB H. pylori-induced gastric cancer, and the transmembrane potential mediated by H2 oxidation catalyzed by H. pylori hydrogenase is involved in CagA transport and also affects DNA conversion efficiency H. pylori infection. Recently, Koch and Muller proposed that H. pylori can activate the dendritic cell (DC) inflammasomes to secrete IL-1β and IL-18 In addition to toxin proteins, increased secretion of pro-inflammatory cytokines, such as TNF and IL-1β, has also been shown to be closely related to tumors H. pylori bacteria in the stomach affects the development of GAC H. pylori but colonized with intestinal flora than in sterile mice H. pylori negative gastritis, H. pylori positive gastritis and H. pylori negative non-gastritis groups. Streptococcus spp., Haemophilus parainfluenzae and Treponema spp. were possible pathogens In addition, recent studies in humans have shown that the colonization of some non-H. pylori can also affect other microbiomes with an impact on gastric cancer. For example, a study showed that compared with the uninfected stomach, H. pylori infection increased Proteobacteria, Spirochetes and Acidobacteria, while it decreased the relative abundance of Actinobacteria, Bacteroidetes and FirmicutesClostridium and Lactobacillus) alone was sufficient to promote the gastric pathogenesis of INS-GAS mice, while H. pylori accelerated the occurrence and progression of gastrointestinal intraepithelial neoplasia in INS-GAS mice, and showed severe inflammation in serum and local gastric tissues H. pylori infection and gastric atrophy H. pylori infection that reduce the secretion of gastric acid enable the successful establishment of a new microbiome that promotes malignant transformation Veillonella, Prevotella, Fusobacterium and LeptotrichiaRecently, Inasco et al. performed studies on gastric cancer and proposed that lactic acid bacteria (LAB) can affect gastric cancer through various mechanisms There is increasing evidence that the pathophysiology and treatment of various liver diseases may be strongly influenced by the gut microbiota Animal model studies have shown that liver inflammation is correlated with intestinal mucosal barrier dysfunction, suggesting that bacterial translocation and imbalance caused by intestinal mucosal barrier dysfunction affect the occurrence of non-alcoholic fatty liver disease, non-alcoholic steatohepatitis (NASH) and progression to HCC Firmicutes/Bacteroidetes ratio was significantly reduced in the feces of pancreatic cancer patients Firmicutes, Deferribacteres, Actinobacteria and Proteobacteria decreased significantly. Moreover, the metabolism of the microbiota changed from dominant energy metabolism to enhanced polyamine and lipid metabolism  Fusobacterium negative group, the cancer-specific mortality observed in the positive group was significantly higher, and the presence of Clostridium was significantly correlated with a poor prognosis of pancreatic cancer In recent years, many studies have found that the occurrence and development of pancreatic cancer are correlated with intestinal microorganisms, and the microorganisms in the pancreas are affected by intestinal microorganisms. It was found that the Traditionally, the pancreas has been considered a sterile environment Enterococcus faecalis and GFP-labeled Escherichia coli into wild-type mice. They found that the bacteria could migrate to the pancreas Malassezia can promote the occurrence of pancreatic cancer. Meanwhile, Sethi et al. found that the removal of microorganisms significantly inhibited pancreatic tumor volume and reduced the risk of pancreatic cancer liver metastasis. However, in Rag1 knockout mice lacking mature T and B lymphocytes, the tumor inhibitory effect was significantly eliminated after the removal of microorganisms, suggesting that the tumor inhibition effect may be mediated by the immune system Regarding the mechanism of the influence of microorganisms on the occurrence of pancreatic cancer, Thomas and colleagues found that there is an extra-intestinal, long-distance interaction between intestinal microorganisms and the pancreas that can promote the development of pancreatic cancer and influence transcriptional changes in pancreatic cancer xenografts and the infiltration of innate immune cells F. nucleatum, Peptostreptococcus stomatis, Parvimonas micra and Solobacterium moorei were significantly correlated with the occurrence of CRC Ruminococcaceae, Clostridiaceae and Lachnospiracea in the feces of patients significantly decreased, while Bacillus and Gammaproteobacteria significantly increased. In addition to bacteria, Temperate bacteriophages have also been shown to be associated with the occurrence and progression of CRC, as they interact with the host bacterial community and accelerate the occurrence of CRC by changing its composition Mucispirillum schaedleri can promote the production of LPS and aggravate the inflammation. Lachnospiraceae bacterium A4 reduces butyrate generation and promotes the formation of cancers In clinical studies, gut microbes have been shown to be directly associated with the development of CRC. Yu et al. found that F. nucleatum, E. coli and Bacteroides fragilis. F. nucleatum is one of the common bacterial species in CRC F. nucleatum infections can activate the TLR4 and MyD88 signaling pathways, leading to the activation of NFκB and increased miR21 expression. It can also increase the expression of anoctamin-1 in CRC cells and prevent apoptosis Fusobacterium adhesin A (FadA) of F. nucleatum can bind to E-cadherin, promoting intracellular annexin A1 expression, forming a FadA/E-cadherin/annexin A1/β-catenin tetramer, thereby activating the β-catenin pathway and promoting CRC cell proliferation F. nucleatum was shown to up-regulate CARD3, activate the autophagy pathway in cells, and then promote the metastasis of CRC E. coli, researchers have known for years that it promotes CRC E. coli infection can induce the phosphorylation of H2AX (a marker of DNA double-strand break), transient DNA damage, incomplete DNA repair, chromosomal aberrations and increased cell division E. coli infection can also promote the occurrence and development of CRC by activating the P38 MAPK pathway in macrophages to up-regulate COX-2 expression  B2 E. coli isolated from CRC tissues was found to increase the invasion of multinucleated cells and the formation of crypt abscess and promote the expression of proliferative nuclear antigen in epithelial cells E. coli resulted in a distinct mutational signature to promote CRC Enterotoxigenic Bacteroides fragilis (ETBF)can activate the Stat3 pathway in colonic epithelial cells through Bacteroides fragilis toxin (BFT), combine with extracellular IL-17 to activate the NFκB signaling pathway via IL17R, and trigger the expression of chemokines such as CXCL1 which can lead to the accumulation of myeloid cells in the distal colon and lead to cancer hiMO-MDSCs E. coli, promoting colibactin-induced DNA damage to colonic epithelial cells Campylobacter jejuni 81-176 clinically isolated from humans could activate the cytolethal distending toxin (CDT) B gene, promote the production of the CDT subunit CDTB, and thereby play the role of gene toxin and promote the occurrence of CRC C. jejuni 81-176 also changes the expression of host genes, up-regulates the PPAR signaling and calcium signaling pathways and alters the intestinal microbial composition and gene transcription of the host. Lysinibacillus sphaericus in the gut degrades aspirin and reduces its chemopreventive effects regarding CRC in mice The role of some specific microbial communities in the development of CRC has been clarified, such as Proteobacteria increase in intestinal microorganisms. Flagellin is an important component of microorganisms, which can increase the secretion of IL-6 and CCL2/MCP-1 mRNA expression by C26 CRC cells, reduce caspase-1 activity and active oxygen production, thereby increasing cytotoxicity, leading to an increase in the inflammatory response, which plays a role in promoting cancer Intestinal microbial imbalance can promote the occurrence of colorectal inflammation and then lead to CRC. For example, under normal circumstances, symbiotic fungi in the intestine can be recognized by C-type lectin receptors, activating downstream SYK and CARD9, which in turn mediates the activation of inflammasomes and promotes the maturation of IL-18 and the anti-tumor T cell response, thereby limiting the development of CRC. Imbalanced changes in intestinal microorganisms may inhibit the anti-tumor effect of the SYK-CARD9 axis In addition, intestinal microbial metabolites can also promote CRC. For example, short-chain fatty acids (SCFAs) are products of microbial catabolism of dietary fiber in the colon and cecum, including acetate, propionate and butyrate 2S) has been shown to promote the occurrence of various cancers, which is a metabolite of intestinal microorganisms Desulfovibrio spp. and Clostridium lavalense to produce carcinogenic H2S 2S in the blood circulation is mainly microbial origin, and the amount of H2S in the blood of germ-free mice is very low 2S reaches the esophageal tissue through the blood circulation, by up-regulating HSP90, it promotes the proliferation, anti-apoptosis, angiogenesis and migration of esophageal cancer cells Changes in intestinal microbes due to HFD will cause an increase in immature myeloid cells and neutrophils of esophagus tissues. The progenitor cells, i.e. lgr5+ cells, expand into the esophagus accompanied by chronic inflammation, thereby causing metaplasia and dysplasia Meanwhile, the intestinal microbiota promotes the development of esophageal cancer by activating IL-8 via TLR signaling, which has an impact on the immune system and activates granulocytic myeloid cells Lactobacillales increased in the stomach of mice fed HFD, while Bifidobacteriales decreased. Lactobacillus converts lactose to lactic acid, acidifying the surface of the gastric mucosa Veillonella, Prevotella, Fusobacterium, Leptotrichia and promote tumorigenesis in various ways A recent study found that gastric leptin signal regulates gastric flora and intestinal metaplasia of gastric mucosa. Stomach flora transplantation in HFD group induces intestinal metaplasia in recipient mice, indicating that microbial changes promote intestinal metaplasia Enterobacteriaceae can increase endotoxin production, thereby further triggering chronic inflammation and accelerating obesity Desulfovibrionaceae may be an important group of endotoxin producers that can produce LPS and induce chronic inflammation and metabolic endotoxemia Desulfovibrionaceae reduces sulfate to H2S and destroys the intestinal barrier 2S upregulates the expression of the fatty acid receptor CD36 in gastric cancer cells and directly activates CD36, triggering lipid metabolism reprogramming and thereby promoting gastric cancer metastasis He et al. found that, after 12 weeks of HFD, the gastric flora was abnormal and the diversity of the flora decreased There exist some scholars who have done research on HFD promoting HCC through intestinal microorganisms. Xie et al. found in a mouse model of NASH-HCC induced by streptozotocin and HFD that hydrophobic bile acids significantly increased intrahepatic retention, and these are significantly associated with changes in intestinal microbes Atopobium spp., Bacteroides spp., Bacteroides vulgatus, Bacteroides acidifaciens, Bacteroides uniformis, Clostridium cocleatum, Clostridium xylanolyticum and Desulfovibrio spp. were significantly increased in model mice, positively correlated with LPS levels and the liver disease pathological progress Similarly, a study found that HFD can cause excessive growth of Gram-positive bacteria in the intestine and increase the level of deoxycholic acid in the liver. Moreover, the aging-related hepatic stellate cell secretory phenotype produces various inflammatory and tumor-promoting factors, thereby promoting the development of HCC in mice exposed to the chemical carcinogen DMBA Akkermansia muciniphilaIn addition to the pathways mentioned above, we have also investigated other pathways. For example, while HFD can cause abnormal accumulation of bile acids under the action of microorganisms, and Cui et al. found that the liver FGF15 (19)/FGFR4 signal is significantly enhanced by HFD, we infer that bile acids enhance FGF15 (19)/FGFR4 and then activate EMT and the Wnt/β-catenin pathway, leading to the cancerous conversion of liver cancer Lactobacillus in the upper small intestine. This flora is related to the expression of sodium glucose cotransporter-1 in the small intestine and the secretion of GLP 1 We propose several mechanisms to support the hypothesis that HFD promotes the development of pancreatic cancer by affecting the composition of intestinal microbes. A number of studies have shown that HFD can affect human blood glucose levels through the intestinal flora. For example, HFD can cause the decline of Bilophila wadsworthia, promotes the translocation of LPS to pancreatic tissues and accelerates the occurrence of pancreatic cancer through inflammation. We speculate that HFD exacerbates intestinal barrier dysfunction, resulting in the accumulation of flora and LPS, which may translocate into pancreatic tissue through the destroyed intestinal barrier and stimulate an inflammatory positive feedback loop to synergistically enhance Ras to promote pancreatic cancer. Since NFκB is a common targeting molecule downstream of Ras, we surmise that translocated LPS stimulates TLRs of pancreatic tissue and stimulates the downstream NFκB pathway, enables downstream COX-2 and promotes a positive inflammatory feedback loop through the production of PGE2 HFD proliferates pro-inflammatory flora such as Malassezia spp. to pancreatic tissue, which combines with mannose binding lectin (MBL) and stimulates the complement cascade by the lectin pathway, leading to the release of C3a, C5a and other substances. C3aR-mediated signal transduction triggers the release of neutrophil extracellular traps and the polarization of neutrophils to the tumorigenic phenotype F. nucleatum in the intestine, which may transfer from the intestine to the pancreas through mesenteric vein drainage, and promote the occurrence of pancreatic cancer. In HFD-fed mice, the abundance of Fusobacterium increased significantly F. nucleatum combines with TIGIT in the pancreas It has been pointed out that complement activation in the tumor microenvironment can promote tumor immune escape, proliferation, and metastasis by maintaining T cell immunosuppression and chronic inflammation Regarding the pathogenesis of CRC, some scholars have found that intestinal microbes can serve as a bridge in the process of HFD promoting CRC development, connecting the causal relationship between the two.Enterococcus faecalis, Proteus mirabilis and Candida parapsilosis. The colonization of these bacteria at the anastomosis destroys the healing intestine, resulting in increased permeability and transmigration of cancer cells, which promotes the recurrence of CRC. HFD increased collagenase-producing microbes Schulz and colleagues found that HFD feeding promoted the formation of tumors in the small intestine of mice, along with changes in the composition of intestinal microbes. This transformation of intestinal microbes is related to the reduction of host antibacterial defenses mediated by Paneth cells, which reduces the recruitment of DCs and the expression of MHC class II molecules in intestinal associated lymphoid tissues. In terms of microbial metabolites, HFD can cause a significant reduction of SCFAs in the small intestine and feces Clostridium in the intestine can convert primary bile acids synthesized by liver into secondary bile acids Parabacteroides in the intestine decreased Parabacteroides distasonis can reduce the activation of TLR4 signaling pathway and Akt, and inhibit the promoting role of HFD on CRC. Therefore, we speculate that HFD reduces the abundance of Parabacteroides distasonis and indirectly promotes the development of CRC Temperate phage and FusobacteriumF. nucleatum, it was found that the western diet has a strong correlation with F. nucleatum-positive CRC in an epidemiological survey, and it plays an important role in diet-mediated CRC, which further demonstrates the accuracy of our hypothesis Based on previous related studies, we propose other possible mechanisms. First, bile acids are one of the important metabolites of intestinal microbes In the preceding narrative, we demonstrated that HFD, intestinal microbes and gastrointestinal tumors are related to each other. There is a solid theoretical basis for the suggestion that the HFD can promote the occurrence of gastrointestinal cancer by changing intestinal microbes. It can be used as a direction for future research and experiments to verify whether this proposal is correct, but in the research process, we need to pay attention to distinguish whether HFD promotes tumors directly or requires microbial mediation. In addition, because the intestinal tract and various organs are linked in many ways, even if HFD unbalances intestinal microorganisms, its cancer-promoting effect is mediated through a long-distance method or when the pathogenic microorganisms directly enter the organs. These all need further research and elaboration.Gammaproteobacteria may make the tumor sensitive to gemcitabine In the treatment of tumors, the relationship between HFD, intestinal microbes and gastrointestinal tumors also provides us with new ideas. First, we suggest changing the lifestyle of HFD and reduce the amount of fat in the diet to prevent the occurrence of gastrointestinal tumors, and effectively control the incidence of tumors from food-borne pathways. Secondly, for patients already suffering from disease, it is also extremely important to take effective treatment measures to prevent or even interrupt the development of tumors, and intestinal microorganisms may be an effective target. Geller and colleagues found that removing cytidine deaminase or using antibiotics such as ciprofloxacin to remove Lactobacillus spp. and change the fecal contents of short chain fatty acids such as butyrate. This can induce proinflammatory genes such as Rac1, Map2k1, Map2k6 and Atf2, as well as many cytokines and chemokines , thereby affecting intestinal immunity and promoting inflammation Among many lifestyle factors, the diet is one of the most significant factors affecting the intestinal microbiome Mucispirillum is related with MHC-allele-dependent IgA response E. coliAlistipes (Rikenellaceae) and Bacteroides (Bacteroidaceae) genera were over-represented in Nod2 knock-out mice. Conversely, the Prevotella (Prevotellaceae) genus was reduced BifidobacteriumChristensenellaceae consortium To a certain extent, the composition of the gut microbiota may depend on the individual's genetics, although studies by scholars have shown that genes play a minor influence on the formation of gut microbes C. jejuni 81-176 infection will up-regulate two cancer-promoting signal pathways including Peroxisome proliferator-activated receptors signaling pathway and calcium signaling pathway, and promote the occurrence of CRC Molecular pathology epidemiology is also an important direction of microbial research in recent years. The tumorigenesis promoted by HFD has obvious molecular pathological characteristics. For example, we mentioned above that mice fed with HFD have higher activity of KRAS


The microenvironment (ME) plays a critical role in causing glioblastoma (GBM) to be a moving and incurable target. The main features governing the interaction between cancer cells and the ME include dependency, promotion, and in rare cases, even competition. In the original Stupp protocol, the alkylating agent temozolomide (TMZ) is the first-line chemotherapy drug to treat GBM, and it is broadly used together or after radiotherapy. Some studies have described TMZ as an adjuvant to other therapeutic approaches including immunotherapy because of its ability to induce an immunogenic death of cancer cells. TMZ also exerts immunomodulatory effects on the tumor and immune ME. These findings support the coexistence of two circuits, i.e., one that subverts local immunosuppressive mechanisms and another that exerts a harmful influence on the peripheral immune response. A bias toward the latter can drive the failure of treatments based on the combination of chemotherapy and immunotherapy approaches. In this review, we will reanalyze how intrinsic and acquired resistance to TMZ impacts the immunomodulatory effects previously described by way of inducing a functional alteration of local immune cells and promoting immunosuppression and how different components of the immune ME, with particular attention to tumor-associated macrophages and microglia, can cause TMZ resistance to circumvent potential local immunogenic mechanisms. Chemotherapy has historically been viewed as immunosuppressive until many studies have considered the immunomodulatory effects of conventional anticancer treatments . In 2012The treatment of glioblastoma (GBM), a devastating brain cancer with a dismal prognosis, is one of the most urgent unmet needs in oncology . HoweverIn evaluating the potential effect of TMZ on the tumor and immune ME, it is crucial to consider the heterogeneity of GBM and its resistance or sensitivity to TMZ and to evaluate the dynamics and genotype-dependent effects of the immune ME.Some data support the idea that the role of TMZ in influencing host immune responses depends on its dosage. For example, in mice and humans, transient lymphodepletion due to the administration of increasing doses of TMZ enhanced a specific antitumor immune response induced by immunotherapy while simultaneously increasing the frequency of regulatory T cells (Tregs) . FurtherUnlike the first evidence supporting the essential role of transient lymphodepletion in stimulating specific immune responses, recent studies have revealed that TMZ directly exerted a negative effect specifically on immune effector cells. In the preclinical model, the effect of anti-PD1 therapy was reversed by the systemic administration of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and TMZ . The immdim CD16+ NK cells under TMZ treatment were modulated by a specific single nucleotide polymorphism (-897DelC), which in turn correlated with a better clinical outcome  on their surface , in agre outcome . In anotAll these observations demonstrate that a considerable debate remains over the role of chemotherapy, such as TMZ, as an immunological adjuvant for immunotherapy treatments.Considering that GBM is sustained by a unique, heterogeneous, and highly immunosuppressive ME that is also responsible for therapy resistance, in this review, we will focus our attention on 1) how chemotherapy can impact the composition of the immune ME in relation to the genetic and molecular landscape of GBM; and 2) how the immune ME constructed by brain resident and infiltrating immune cells, such as microglia and macrophages, is involved in the chemoresistance of GBM.Malignant gliomas are typically characterized by an immunosuppressive ME in which macrophages and microglia are the most abundant components of innate immunity.Circulating monocyte-derived macrophages (Mo) and tissue-resident microglia (Mg) are often described as GAMs (glioma-associated microglia/macrophages) that significantly contribute to tumor growth and invasion and immunosuppression due to their inadequate antigen-presenting capacity and suppression of T-cell proliferation. The percentage of GAMs present in GBM is positively correlated with tumor aggressiveness, as shown by their higher abundance in the mesenchymal (MES) molecular subtype –16.IDH (Isocitrate DeHydrogenase) mutational status also impacts GAM subpopulation prevalence, resulting in a higher proportion of Mo-GAMs in more aggressive IDH wild-type gliomas , which have been well characterized by single-cell RNA sequencing . GAMs lo gliomas , 16, 21.MGMT), whose expression is silenced by promoter methylation in 30%–50% of patients. Suppression of MGMT confers a greater sensitivity to TMZ and consequently improves survival , VDR (Vitamin D Receptor), TNFSF9 (TNF Superfamily Member 9), TNFRSF9 (TNF Receptor Superfamily Member 9), and LILRA5 (Leukocyte Immunoglobulin Like Receptor A5), were implicated in remodeling an immunosuppressive ME favoring M2 macrophage polarization. The expression of corresponding proteins was verified in GBM specimens by immunohistochemistry and the authors showed that they were significantly upregulated in GBM specimens with unmethylated MGMT compared with MGMT-methylated specimens , which regulates ROS levels and protects tumor cells against oxidative stress . The samPhagocytosis of cancer cell by macrophages can restrict tumor growth and progression. Tumors adopt several immune escape mechanisms to avoid engulfment; one of these involves the Toll-like receptor 4 (TLR4). TLRs are key components of the innate immune system and, in the tumor ME, are expressed by both tumor and immune cells . TLR4 exIn GBM, the heterogeneity of the immune contexture within the ME can impact the effects of TMZ.in vitro and in vivo tumorigenicity. Furthermore, human microglia can promote TMZ resistance by the augmented expression of inflammatory IL-11, which activates the STAT3 -MYC pathway in tumor cells -γ inhibition reduces IL-11 accumulation in the tumor ME, which mimics the TMZ response of the so-called “exceptional responders” whose tumors show a reduced trafficking and infiltration of GAMs  II chaperone CD74 is a cell-surface receptor for the cytokine macrophage migration inhibitory factor (MIF). Some data suggest that CD74 has a pro-tumorigenic role. In particular, gliomas may escape the pro-inflammatory M1 conversion of macrophages/microglia e of GBM –40. MIF reatment . CD74 waotherapy , 43. KitThe tight association between GBM cells and GAMs in chemoresistance was confirmed by Kazantseva et al. in a study on the TP53 isoform Δ133p53β, which has pro-tumorigenic functions such as the induction of tumor growth and migration, protection from apoptosis, promotion of angiogenesis, and reduction of chemotherapy responsiveness. In addition, this isoform reduces sensitivity to TMZ treatment and promotes cell survival by reducing oxidative stress in an immunosuppressive ME characterized by increasing the recruitment of CD163 and colony stimulating factor 1-receptor (CSF1-R) positive macrophages mediated by CCL2 .GBM is an urgent unmet medical need. Genetic driver mutations of tumor cells, biological and molecular heterogeneity of the tumor, and immune ME can impact the response to chemotherapy administered as a standard or adjuvant therapy.Despite the promising role of TMZ in treating newly diagnosed GBM, it has not proven to be effective against recurrent GBM. The dynamic interplay between GBM cells and immune ME influences many aspects of tumor evolution: survival, proliferation, recurrence, and therapeutic response. Therapy sensitivity is not governed only by a few molecular pathways but is modulated by intrinsic and acquired factors that involve and/or depend on ME. In a significantly immunosuppressive context, macrophages can play a crucial role in TMZ resistance and GBM relapse Figure 1MGMT status -like tumors, which are considered as the more aggressive molecular subtype of GBM . In thisT status .The wealth of information on microglia and macrophages can be exploited to overcome the challenges of combinatorial targeted therapies. Looking forward, standard therapeutic protocols, including TMZ, will be harmonized and combined with innovative (immuno)therapeutic approaches that take dynamic heterogeneity of tumor cells and macrophage/microglia into consideration. In addition, immune modulation of ME by reprogramming macrophages/microglia can reverse chemoresistance and improve TMZ efficacy.Writing—original draft preparation: NI, MM, and SP. Review and editing: ME and SP. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Increasing life expectancies are unfortunately accompanied by increased prevalence of Alzheimer's disease (AD). Regrettably, there are no current therapeutic options capable of preventing or treating AD. We review here data indicating that AD is accompanied by gut dysbiosis and impaired renin angiotensin system (RAS) function. Therefore, we propose the potential utility of an intervention targeting both the gut microbiome and RAS as both are heavily involved in proper CNS function. One potential approach which our group is currently exploring is the use of genetically-modified probiotics (GMPs) to deliver therapeutic compounds. In this review, we specifically highlight the potential utility of utilizing a GMP to deliver Angiotensin (1–7), a beneficial component of the renin-angiotensin system with relevant functions in circulation as well as locally in the gut and brain. Life expectancy continues to increase in developed countries worldwide, with persons aged >65 years representing the fastest growing segment of the population . In line with the need for fresh approaches, it is possible that alternative strategies are needed that target other physiologic systems beyond the CNS. One prominent possibility is targeting the gut microbiota—the microorganisms which compose the complex microenvironment of the human intestinal tract—given their known roles in neural communication  to deliver therapeutic compounds. GMPs allow for the oral delivery of therapeutic compounds by embedding the compound of interest within a bacterial  strain safe for human consumption. Indeed, several authors have purported the potential benefits and therapeutic applications of GMPs—particularly those from lactic acid bacteria  modified to express Ang (1–7); Ang (1–7) GMP] not only increased circulating levels of Ang , it significantly altered several components of the gut-brain-axis in ways that direct systemic Ang  administration did not  release of pro-inflammatory cytokines to activate the hypothalamic pituitary adrenal (HPA) axis or directly impact CNS immune activity, (2) production of short chain fatty-acids, (3) release of neurotransmitters, or (4) by modulating tryptophan metabolism and downstream metabolites , left S. In summDrosophila demonstrated that indeed gut dysbiosis accelerates the progression of AD in the fly model of AD . While patients with MCI demonstrate impaired cognitive processes beyond those of normal aging, they are not as severe as individuals with more advanced neurodegenerative disease states. While individuals with MCI are more likely to develop Alzheimer's disease (AD) and other forms of more advanced dementia than age matched controls , one of the most prevalent forms of dementia, currently affects 5.8 million people in the United States and is the sixth overall leading cause of death. Typical AD pathology includes extra cellular deposits of amyloid β-protein (Aβ) and neurofibrillary tangles of hyperphosphorylated tau Selkoe, , though Indeed, emerging evidence indicates individuals with AD demonstrate an altered microbiome profile with decreased diversity beyond that of age-matched controls  is another neurodegenerative disease with potential cognitive impacts and affects ~1-million individuals in the United States alone , but also by the more recently discovered Ang (1–7) axis whereby the binding of Ang (1–7) to the Mas (AT7) receptor promotes several beneficial actions (In recent years a number of pleiotropic effects have been ascribed to the renin-angiotensin system (RAS) that extend beyond lowering blood pressure Sica, . As we r actions , left. H actions .Among the purported benefits of the Ang (1–7) axis is improved brain health. Components of the axis are found throughout the central nervous system (CNS) including within neurons, astrocytes, cerebral arteries, and various brain regulatory centers including the paraventricular nucleus (PVN) and hypothalamus  axis modulation benefits multiple aspects of cognition in several models of cognitive dysfunction, including AD . However, systemic delivery of Ang (1–7) typically requires burdensome infusions and/or repeated injections as well as the expensive and cumbersome production process for the drug. Thus, a different formulation—particularly one which could be administered orally–could provide practical benefits to patients from both a cost and ease of use standpoint. In line with this reasoning, our previous work has demonstrated that orally delivered Ang  GMP influences far beyond the gut, including altering levels of neurotransmitters and components of the RAS  is being utilized in clinical trials for clinically-urgent conditions including inoperable tumors, breast cancer, mitigation of chemotherapy induced bone marrow toxicity, and wound healing (Source: Components of the Ang (1–7) axis are also present throughout the gut including the small intestinal brush border, muscularis mucosa, and propria, as well as microvascular endothelium and vascular smooth muscle cells  to CNS function is in the metabolism of the essential amino acid tryptophan . Under nLactobacilli promote the production of Indole-3-carboxaldehyde (I3A) which promotes the production of interleukin 22 and the maintenance of mucosal reactivity  hold promise for altering these neuro-regulatory pathways.The remaining (~95%) tryptophan is then metabolized by the human host and absorbed in the intestines for transport to the liver and release into the circulation. Once into the circulation, a minority of tryptophan is broken to support protein synthesis and the production of the neurotransmitter serotonin. Meanwhile the vast majority of tryptophan is metabolized to the neuro-regulatory kynurenine pathway—which has been proposed a key link between gut dysbiosis and various neurologic conditions including AD  GMP reduced neuroinflammatory gene expression in the pre-frontal cortex while also increasing circulating concentrations of neuroregulatory compounds picolinic acid and serotonin in older F344/BN rats , may provide a better way to target AD and age-related dementia. Moreover, the delivery of therapeutic compounds through probiotics is particularly useful due to their ability to survive gastric acids and bile, allowing them to reach the intestinal target. The specific GMP discussed here utilized LP as the live vector for oral delivery. The LP was then modified to express Ang  as a secreted fusion protein utilizing cholera toxin subunit B , which functions as a trans-epithelial carrier, allowing for uptake into circulation -expressing First and foremost, the vascular effects of any RAS-affecting compound should be considered. Direct application of the Ang  GMP into the gut is able to get into circulation  GMP work to improve CNS function through the targeting other peripheral tissues, rather than through targeting the brain directly. Impaired gut function can impair brain health and cognitive functioning, and may result in neurodegenerative conditions , with two-thirds of diagnosed patients being female. Notably, this gender discrepancy is not explained by increased life expectancy as the incidence, cognitive decline, and amyloid pathology are all greater in women compared to age-matched men with AD  and other non-classical components of the RAS  and aldosterone levels  is one of the most prevalent age-related inflictions among the elderly population, but there are currently no available therapeutics of any kind capable of preventing or curing this disease state. One potential approach is through the gut, but significant work is required to determine how such interventions can be implemented and the degree to which they can be efficacious. Probiotic-based interventions may be a good choice, provided there is an appropriate vessel through which systemic alterations can be administered. Though there is limited clinical evidence for the effectiveness of probiotics in dementia, there is a strong link between gut microbiome and brain function/dysfunction, including distinct microbial profiles in major depressive disorder  may hold promise for AD-related impairments via oral delivery of therapeutic compounds. Specifically, the utilization of GMPs from lactic acid bacteria which itself has inherent benefits and these GMPs can be further utilized to deliver compounds such as Angiotensin (1–7) as there is strong evidence for the role of the RAS in AD (Wright and Harding, The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.AH, AB, CC, and TB contributed to conceptual framework and writing of this manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The intestinal immune system represents the largest collection of immune cells in the body and is continually exposed to antigens from food and the microbiota. Here we discuss the contribution of single-cell transcriptomics in shaping our understanding of this complex system. We consider the impact on resolving early intestine development, engagement with the neighbouring microbiota, diversity of intestinal immune cells, compartmentalisation within the intestines and interactions with non-immune cells. Finally, we offer a perspective on open questions about gut immunity that evolving single-cell technologies are well placed to address. Since the recognition of a localised intestinal immune system in 19192 has revolutionized the field of immunology, revealing an unappreciated complexity of immune cell subsets, identifying new cell types and states, redefining cellular ontogeny and enabling inference of cell fate trajectories and function4. ScRNAseq is able to piece together existing knowledge of cell markers, ontology and interactions into an integrative picture of the building blocks of human tissues. Applied to human mucosal immunity, scRNAseq is particularly powerful as it allows for systematic analysis of cells within these complex and highly-immunologically active tissues, thereby making the most of small and often difficult to obtain clinical samples. Although transcriptional expression is not a perfect readout of protein expression5, scRNAseq allows for the hypothesis-generating phase of research to begin with and be guided by tissue-specific clues. Targeted experiments in model systems can then be used to support findings and test biological mechanisms. In this way and spurred on by the conception of the Human Cell Atlas (HCA) initiative in 2016, scRNAseq has been applied with great effect to several human barrier tissues including skin6, reproductive organs8 and mouth9, and recently in the context of SARS-CoV-2 infection14.The invention of single-cell RNA sequencing (scRNAseq) in 2009In this review, we focus on the immune system of the intestinal tract and specifically discuss how single-cell transcriptomics has advanced knowledge in this field. We provide an introduction to scRNAseq methods and analysis tools with particular use in this area and highlight studies that have shed light on the origins of intestinal immunity, cell diversity and plasticity, interactions with non-immune cells and compartmentalisation within the tissue architecture.18, focus on regional differences21 or investigate intestinal development24 applying either in-depth or high-throughput methods, and increasingly combining other technologies such as V(D)J sequencing and spatial transcriptomics to better understand cell heterogeneity, lineage relationships and spatial locations in tissue24. Below we outline the current and emerging technologies and analysis tools for studying intestinal immunity.The scRNAseq field is rapidly evolving, with the number of cells captured per experiment now in the millions. Approaches to single-cell profiling intestinal tissues vary between studies and depend on tissue availability and biological questions being asked. Current studies on intestinal immunity compare cells of healthy or IBD patients28. Platforms relying on the isolation of dissociated cells by fluorescence-activated cell sorting (FACS) or microfluidic devices include STRT-seq29, CEL2-seq30, MARS-seq31 and SMART-seq2-334. Use of FACS provides auxiliary information of proteins targeted by a select panel of fluorophore-tagged antibodies and can help unite transcriptional profiles to traditional cell type identities. These methods are lower in throughput due to limited capture sites, ranging from 100–1000’s of cells per experiment  or in microwells (Seq-Well S3 37 and STRT-seq-2i38). These methods tag either the 3′ or 5′ end of mRNA, incorporating a unique molecular identifier and applying a cell-specific barcode early after cell capture. Total mRNA can then be pooled for downstream library preparation allowing for processing of 1000’s-millions of cells per experiment  and neural network based algorithms without a prior reference   and within the Seurat framework.Pre-processing and analysis of scRNAseq data from low- and high-throughput platforms follows the same general workflow and is detailed in a number of review articles and online tutorials64.Intestinal immunity is established during development in utero. Prior to the single-cell genomics era, survey of immune populations during human development has been challenging, owing to tissue access, and there has been limited knowledge about the populations and markers expressed at this stage. Single-cell transcriptomics have been instrumental in understanding the diversity of immune and non-immune populations in these precious human intestinal developmental tissues65, our understanding of the development of these structures has previously relied on experiments in animals66. In mouse studies, early GALT formation has been described to involve interactions between mesenchymal lymphoid tissue organising (mLTo), endothelial LTo (eLTo) and lymphoid tissue inducer ) cells66. Interactions between these cell types are critical for recruiting and retaining immune cells at the sites of developing lymphoid structures. Through scRNAseq of human fetal gut samples, central players in secondary lymphoid organ formation have been resolved in humans, and their communication programs in initiating PP formation are defined from as early as 12 weeks post-conception , suggesting reactivation of developmental programs to support intestinal inflammation24. Fawkner-Corbett et al. described a population of mLTo-like stromal cells  with similarities to a subtype of stromal cells expanded in ulcerative colitis (UC)24. Taking advantage of recent spatial transcriptomics technology, the authors showed localisation of these cells and confirmed the likelihood of relevant cell-cell interactions in lymphoid follicle formation in situ24. This shows that the formation of secondary organs is not restricted to development and is required for proper maturation and response of the immune system.The intestinal immune system is supported and regulated by gut-associated lymphoid tissues (GALTs), including mesenteric lymph nodes, Peyer’s Patches (PPs) and cryptopatches. Despite obvious differences in gut lymphoid tissue size and location between species68. Microbial dysbiosis is well recognised in diseases such as IBD, colorectal cancer, metabolic disorders and in conditions including pregnancy although mechanistic insight into the host:microbial relationship remains in its infancy69. Whether the interaction is between the host and its resident microbiota or a direct response to a specific infectious entity, microbes communicate with the host through attachment to mucosal surfaces, binding specifically to host receptors, production of metabolites such as short chain fatty acids and bile acids or adapting their growth and metabolism based on changes we make to their local environment. In parallel host immune responses attempt to continuously decipher between microbial friend or foe.We live in an era where the relationship between our immune system and microbes has never received such unprecedented attention. Characterising human-associated microbiotas and their role in health and disease has become the holy grail of current medicine. It is well-established that the human-associated microbiota contains a wide and complex community of microorganisms that is unique to individuals and constantly evolves in response to its environment70. Conversely, microscopic images of bacterial-like structures with mucin threads within the gut lumen during the second gestational trimester provide compelling evidence for in utero seeding as well as aligning with other studies that detail in utero antigenic priming of the fetal immune system71. However, these studies are caveated with the potential of contamination with environmental sources of microbes (reviewed in ref. 72) making their physiological relevance questionable.The question of when host–microbe interactions become established has become a topic of intense investigation, with the presence of microbiota during in utero development still highly debated. A recent study of the meconium microbiota in human neonates (at term) before birth, controlling for process/delivery mode-induced contamination indicated that microbial colonization most likely occurs either during birth via maternal seeding or post-birth via environmental seeding73. In particular, multiple scRNAseq studies have shown that memory CD4+ and CD8+ T cells are present and clonally expanded in the intestines in the first and second trimester of development  in combination with BCR sequencing showed that B cells are immature during second-trimester human development compared to those found in infants75. Our scRNAseq of human fetal intestines up to 17 weeks post conception also showed no evidence of B cell clonal expansion, class-switching or germinal centre formation21. Prenatal B cells may similarly be involved in development of lymphoid structures and have no need for class switching, while postnatal B cells undergo these events due to the presence of microbiome75. Through more precise analysis of cell phenotypes, these single cell studies promote the emerging concept that immune cell activation at least until second-trimester development is a product of their support of a highly controlled process of tissue generation rather than due to microbial seeding. Whether this is also the case in the third trimester of human development remains to be determined.Nevertheless, priming of the immune system and unexpected activation of immune cells have been suggested to be linked to the early microbial colonisation in fetal organs, especially the gutent Fig. 74–76. Ho78. Studies have consistently highlighted differences in bacterial gut communities associated with NEC that result in an imbalance between pro- and anti-inflammatory gut immune mediators80. Work by Cho et al. used mouse models to highlight an imbalance within the adaptive immune system in the NEC intestinal environment typified by type 3/T helper (Th)17 polarization, with reduced Th1, Th2, and Treg responses81. These findings were further supported by scRNAseq studies showing preferential presence of TNF-α-producing CD4+ T cells in early intestinal development and an enrichment for these cells in the intestines of preterm infants with NEC  is a devastating intestinal disease that occurs primarily in premature infants, resulting in impairment of the epithelial barrier and in extreme cases causing intestinal perforation and tissue necrosis84. A recent study analysed microbial features predictive of NEC and identified Enterobacteriaceae overgrowth; including specifically Klebsiella—known to possess secondary metabolite gene clusters related to quorum sensing and bacteriocin production to be replicating more rapidly in the days prior to NEC diagnosis85. The transcriptional and proportional cell changes are likely reflected in these preterm infants and future single-cell studies will be instrumental in defining these changes.Bacterial dysbalance in the premature intestines is considered one of the key factors contributing to NEC. No single microbe has been identified as the mea culpa for NEC although increased abundance of Proteobacteria are frequently reported in NEC infants4. In this way scRNAseq has refined classical immune cell type labels, defined new populations and predicted the role of cell types and states in the intestinal immune system of both mice86 and humans89.The immune system must exhibit diversity and plasticity to respond to the countless challenges incurred throughout life. The conventional approach to studying diversity in immune cells has been top down—focussing on a cell type and iteratively subdividing it into more distinct subsets based on marker gene expression. This approach has been essential in understanding the intestinal immune system, but relies on pre-selection of markers and is limited in resolving heterogeneity within distinct cell groups. A strength of scRNAseq is its ability to explore heterogeneity from the bottom up—dividing cells into distinct groups and then defining molecular profiles that best describe each population90. While they do not possess a functional TCR, they draw parallels in function and subtype classification with Th cells. ILCs are typically divided into 5 types- ILC1, ILC2, ILC3, natural killer and LTi cells91. A study by Muzzurana et al. compared sorted CD127+ ILCs from human blood, lung, colon and a past tonsil dataset92 using the Smartseq2 platform87. Adopting a bottom-up approach to classifying ILC subtypes, they performed unbiased clustering followed by differential expression analysis and correlation analysis on the pooled data. ILCs subdivided into 20 subsets, clustering largely by tissue origin and FACS phenotype. Highest ILC3-associated gene expression was detected in the colon as expected90, but also the highest degree of diversity covering a spectrum of signatures ranging from migratory  to activation and tissue residence 87. ILC heterogeneity has similarly been shown with scRNAseq of mouse intestines86. To show the influence of the neighbouring microbiota on ILC signatures, the authors of this study treated mice with antibiotics prior to scRNAseq analysis. In antibody-treated mice, profiles of ILC1 and ILC2 more closely resembled ILC3 cells  compared to mice with an intact microbiota86. This points towards ILC3 representing the “default” phenotype with environmental factors driving diversification.ILCs are innate immune cells that defend against both intra- and extracellular infections and are particularly abundant in mucosal tissues93. A scRNAseq study by Kiner et al. observed extensive heterogeneity and blended signatures of colonic T cells in specific pathogen-free mice88. In an attempt to drive Th differentiation they infected mice with Citrobacter rodentium, an inducer of Th17 cells (determined by IL-17 expression), Heligmosomoides polygyrus and Nippostrongylus brasiliensis, both inducers of Th2 (IL-5 and IL-13) responses or Salmonella enterica, a bacterial infection inducing Th1 (IFNγ) responses. While FACS of T cells from infected mice confirmed the expected skewing of Th differentiation, scRNAseq analysis and unbiased clustering separated cells by infection system rather than characteristic Th genes. Expression of canonical Th cytokines dominated opposing sides of the same clusters in their data, arguing against discrete subsets and in favour of a polarised continuum of Th phenotypes driven by the infection setting88 . However, plasticity or merging between these subsets has been a frequent observation in mice and humansCD4+ CD8+ cells expanded in UC16. Given the known association between Th17 cells and IBD99, the authors hypothesised the role of the Th17-like cells in driving inflammation, although this remains to be confirmed16. In contrast, an independent UC study showed expansion of a IL26-expressing subset of Th17-like CD8+ T cells with an immunoregulatory signature89. Trajectory and TCR sequencing analysis of the single cell profiles further characterised this population as clonally expanded and arising from tissue-resident T cells or representing a post-effector state. To understand the significance of IL26 expression by these cells during inflammation the authors compared pathology of dextran sodium sulfate (DSS)-induced acute colitis in wild-type mice to humanised IL26-expressing transgenic mice - the Il26 gene does not naturally exist in rodents89. The IL26 transgenic mice experienced less severe disease, a phenotype that could be reversed with the administration of an IL26-antibody89. This suggests a possible role for IL26 in protecting against inflammation. In the context of colorectal carcinoma, paired TCR and transcriptome sequencing identified 8 distinct populations of CD8+ T cells, with signatures ranging from naive, central and effector memory cells, recently activated effector memory/effector cells  to dysfunctional exhausted cells (expressing PDCD1 and HAVCR2)100. Distinct TCR clonal populations and trajectory mapping supported two possible differentiation paths for T effector memory cells- either towards TEMRA or exhausted T cell states. The authors suggest that skewing differentiation towards beneficial TEMRA and away from a state of exhaustion could represent a possible avenue for therapeutic intervention100. Furthermore, this study showed tumour-specific T cell responses, with enrichment and clonal expansion of pro-inflammatory CXCL13+ BHLHE40+ TH1-like cells in tumours with microsatellite instability, but moderate enrichment for Th17 cells in those with microsatellite stability100. The enrichment of CXCL13+ T cells in tumours with high mutational burden was supported by a second scRNAseq study and offers a possible explanation for why this patient cohort responds better to checkpoint blockade therapy101. Together these studies highlight how scRNAseq can assist in understanding the complexity of intestinal diseases and the nuanced involvement of cell types and states in disease progression and control.ScRNAseq studies of human intestinal disease have similarly added to our extensive understanding of the diversity in T cell phenotypes and highlighted specific enriched populations of likely significance to pathology. One such study observed a Th17-like population of Immune cells do not act in isolation, rather their phenotype and response is shaped by their local environment. The intestinal tract in particular comprises unique microenvironments at the macro-anatomical level in terms of distinct tissue regions and at the microanatomical level within the cross-sectional layers of the intestinal wall. Single-cell transcriptomics studies have built upon knowledge of zonation of cells within the human and mouse intestinal tract through providing the full breadth of molecular profiles of cell states, suggesting distinct roles for cells between different zones and how these contribute to the physiological functions of the intestines.102, the epithelial barrier cells have notable variability between small and large intestines. For example, small intestinal epithelium forms villi and crypts while large intestines form only crypts, and Paneth cells that secrete antimicrobial peptides are only present in the small intestine103, while mucus secreting goblet cells are more abundant in the large intestine where they maintain a thicker mucus layer104. Through unbiased analysis of gene expression, scRNAseq has shown variability in the expression of nutrient absorption and antimicrobial defence genes by the epithelia between small and large intestines, leading to identification of a Paneth-like cell in the latter19. ScRNAseq has also resolved further rare subtypes based on distinct gene expression and shown that these change by gut regions. BEST2+ goblet cells that are restricted to the colon106, have been deeply profiled at single cell level in humans. This analysis revealed their specific expression of Kallikreins KLK15 and KLK3, and protease inhibitors WFDC2 and WFDC3 compared to other colonocytes21. Similarly BEST4+ epithelial cells, first identified in the intestinal tract by Ito et al.105, have been shown to be transcriptionally distinct from other epithelial cells in the human intestines16. Building on previous work in which a rare subset of small intestinal epithelial cells was reported to highly express CFTR, encoding a key channel mutated in cystic fibrosis108, further scRNAseq studies showed that BEST4+ epithelial cells of the human small, but not large, intestine co-expressed CFTR  to permit communication with the epithelium and support its remodelling in response to diet . Th17 cells and their cytokines are known to be key mediators of the pathogenesis of CD119. ScRNAseq has further shown that Th17 cells accumulate within the intraepithelial space at the expense of CD8+ T, γδ T, Tfh and T regulatory cells during active CD compared to controls120. Studies of pediatric colitis reported a decreased abundance of CD8-ENTPD1 (expressing the gene encoding CD39) and γδT-ENTPD1 cells in the intraepithelial compartment116. The transcriptional profile of these specific cell subsets led the authors to hypothesise that a defective cAMP pathway was at play and contributing to disease pathogenesis. To test this theory, the phosphodiesterase inhibitor and anti-platelet drug, dipyridamole, was used to drive the cAMP pathway in a mouse model of colitis and in patients, resulting in a dose-dependent increase in T cell CD39 expression and improved epithelial integrity and decreased colitis severity116.ScRNAseq has similarly been applied to better resolve the compartmentalisation of T cells and expression of known risk factors121. Bulk RNA sequencing (RNAseq) analysis of macrophages from these physically separated compartments showed separate expression profiles122. However, while fluorescence microscopy of mouse intestinal tissue highlighted at least two morphologically distinct populations of muscularis macrophages122, the nature and origins of further subsets of macrophages within each compartment remained a mystery. An unpublished study by Domanska et al. implemented scRNASeq of adjacent normal colorectal cancer tissues to address these questions113. They showed that lamina propria macrophages comprise 13 transcriptionally distinct subsets with a spectrum of proinflammatory signature  or high antigen presenting and phagocytic capacity (high levels of HLA class II genes and gene ontology pathways enrichment for endocytosis). Trajectory analysis predicted the majority of these subtypes arise from bone marrow-derived monocytes113. In the submucosal space, the majority of macrophages expressed LYVE1 (associated with vasculature) and COLEC12 (associated with neurons) and had low antigen presenting capacity, but high chemotactic and tissue-protective properties  and homeostatic properties . Homeostatic muscularis macrophages were also positive for PMP22 and EMP1, genes expressed by Schwann cells, suggesting these macrophages phagocytose Schwann cells and are in close contact with neurons113. Macrophages in both compartments showed ligand/receptor expression enabling them to interact extensively with tissue resident cells indicating that their expression profile is heavily influenced by their local microenvironment113.Separate populations of macrophages exist within the lamina propria, submucosa and muscularis propria. A wealth of earlier research has described diverse roles for these populations appropriate to their microenvironment- lamina propria macrophages phagocytose bacterial antigens and produce mediators that drive epithelial cell renewal and muscularis macrophages interact closely with the enteric nervous system123, exposure to morphogen gradients124, and hypoxia125, low-resolution approaches were unable to determine the positional effects on enterocyte function in mice127 or humans128. A study by Moor et al. applied laser capture microdissection of mouse enterocytes followed by scRNAseq to elegantly resolve a continuous gradient of transdifferentiation along the villus axis114. The villus tip enterocytes expressed an immune-modulatory program with the capacity to modulate immune reaction to the microbiota in the gut lumen  possess characteristics distinct from villus epithelium131. Microdissection and RNAseq of mouse intestinal epithelium followed by single-cell validation of gene expression with single molecule fluorescence in situ hybridization showed that follicle-associated epithelium expresses lower levels of antimicrobial and nutrient absorption genes132. This suggests that epithelium at these sites is tuned for the optimal and efficient sampling of bacterial antigens by M cells and immune cells, rather than nutrient absorption and antimicrobial activity  consisting of IgG plasma cells, inflammatory mononuclear phagocytes, activated T cells, and stromal cells that corresponded with failure to achieve durable corticosteroid-free remission upon anti-TNF therapy in a fraction of patients. A real strength of scRNAseq here was the capacity to compare ligand receptor pair expression between equivalent cells of patient groups. In this way, the authors showed that enriched cellular interactions between myeloid cells, activated endothelial cells and activated CCL2+ CCL7+ stromal cells were generating a positive inflammatory feedback loop in the GIMATS samples18. Last, Smillie et al. identified a population of inflammation associated fibroblasts (IAFs) that were expanded 189-fold in biopsies from UC patients versus controls16. The profile of IAFs was comparable to cancer-associated fibroblasts, a key player in creating an immune tolerant tumour environment. IAFs also highly express OSMR, a predictor of resistance to anti-TNF therapy in UC patients, and ranked high for a resistance gene signature determined from bulk RNAseq data. The gene encoding the ligand for OSMR, OSM was most strongly expressed by inflammatory monocytes and cDC2s in the scRNAseq data, implicating interactions between these cells in resistance to treatment16.Mesenchymal or stromal cells of the intestine reside in the subepithelial layers and contribute largely to structural integrity. Three recent studies comparing healthy and IBD intestinal tissue have applied scRNAseq to not only map the diversity of intestinal stromal subtypes, but also pinpoint which cell subtypes and interactions are at play during inflammation135. Previous bulk RNAseq had identified neuronal and inflammation gene signatures from these cells136, but was unable to resolve whether these programs were from one population of cells or distinct subtypes. Using scRNAseq Haber et al. carried out unbiased clustering of Tuft cells from the small intestines of mice and identified two distinct subsets contributing these profiles137. Tuft-2 cells were enriched for immune-related genes particularly those supporting Th2 responses (Il4ra and Il13ra1 and Il17rb). Incredibly, this population also expressed Ptprc (encoding the pan-immune marker CD45), the first recording of this in non-hematopoietic cells and blurring the lines of the traditional definition of immune cells137. While equivalent findings have not been made from single cell analysis of human intestinal tuft cells, a fraction of human and mouse Tuft cells were shown to express immune signalling machinery, specifically activating and inhibitory Fc gamma receptors and downstream mediators for IgG signalling21. This could facilitate direct activation of Tuft cells in response to signals from plasma cells. These findings formed the basis of experiments in mouse models of intestinal colitis in which Tuft cells upregulated the inhibitory receptor suggested their potential as a rheostat of intestinal inflammation21.Tuft cells are chemosensors of the gut epithelium, transmitting messages in the form of a spectrum of biological effector molecules to immune and neuronal cells138. Recent scRNAseq experiments have taken this further to pinpoint exact subpopulations. Work from the Xavier and Regev laboratories combined scRNAseq, flow cytometry and immunofluorescence assays to define three novel subtypes of Lgr5+ intestinal stem cells in the mouse small intestine82. Although not as high as DCs, two of these populations expressed MHC-II at significant levels and were capable of presenting antigen to antigen-specific T cells in co-culture experiments. While the exact role of antigen presentation by these cells is unknown, the authors speculated that it could be a non-essential means for the epithelial layer to respond to infection or be a means by which T helper cells can interact with ISCs and shape their appropriate differentiation into mature epithelial cell types. The latter explanation is particularly interesting in light of further results showing Tregs cells promote ISC renewal while Th1 and Th17 cells promote differentiation82.Antigen presentation is a critical step in the transmission of immune activation to the adaptive immune system with primary antigen presenters regarded as conventional DCs, macrophages and naive B cells. A body of prior work has extended this role to various epithelial cell types via MHC-II expression, with particular roles for microfold cells localised to PPsSingle-cell studies have provided a wealth of knowledge about the complex cellular landscape of the intestinal immune system. In their short history, they have detailed the spectrum of cell phenotypes, provided resolution of zonation of immune cells and shown the impact of their engagement with the neighbouring microbiota in health and disease. As these methods continue to evolve, there is little doubt that they will continue to provide insights into the field of intestinal immunity Box .24 and will be a key feature of future studies. The application of scRNAseq to in vitro systems and experimental models will offer the ability to look in detail at the mechanism of therapeutic and biological agents  on intestinal immune cells. ScRNAseq has already been adapted for capture of bacterial RNA in pioneering studies139, opening the possibility to study the function of specific bacteria. Integration of modalities for example combining scRNAseq of host cells with single-cell metatranscriptomics and patient genotype data will also provide the opportunity to study the interaction between these factors in shaping intestinal immune environments. Last, as the first chapter of the HCA approaches completion140, studies of individual organ systems will be combined to provide a global picture of human biology. We anticipate that this will bring with it studies of the contribution of gut immune cells to human biology and disease at a systems-wide level.Spatial transcriptomics, while not yet at the single-cell with whole transcriptome level, has already placed gut immune signatures in their tissue contextBox 1 Areas of open investigation