Potential role of the intratumoral microbiota in colorectal cancer immunotherapy.

Colorectal cancer (CRC) has been one of the most common malignancies worldwide. Despite the advances in current therapies, the mortality rate of CRC remains high. Among them, immunotherapy has achieved satisfactory results in some CRC patients, however, how to expand the use of immunotherapy in CRC patients remains an urgent challenge. Surprisingly, the intratumoral microbiota has been found in multiple tumor tissues, including CRC. It has been demonstrated that the intratumoral microbiota is associated with the progression and treatment of CRC, and is able to enhance or decrease anti-tumor immune responses via different mechanisms as well as influence the immunotherapy efficacy, providing new potential therapeutic targets for CRC immunotherapy. In this review, we focus on the characteristics of the intratumoral microbiota, its roles in the genesis and development of CRC, its modulation of anti-tumor immune responses and immunotherapy, and propose potential applications of the intratumoral microbiota in CRC immunotherapy. Additionally, we propose possible directions for future research on the intratumoral microbiota related to CRC immunotherapy.

W-GA nanodots restore intestinal barrier functions by regulating flora disturbance and relieving excessive oxidative stress to alleviate colitis.

Inflammatory bowel disease (IBD) may arise due to disruption of mucosal barriers as a result of dysregulation of the intestinal flora and excessive oxidative stress. The creation of nanomaterials with only microbiota-regulating effects often leads to inadequate therapeutic outcomes caused by the disruption of a healthy microbial balance and the emergence of tissue harm caused by excessive oxidative stress. This report describes the multifunctional activity of ultrasmall W-GA nanodots, which can precisely regulate the intestinal microbiome by inhibiting the abnormal expansion of Enterobacteriaceae during colitis and alleviating the damage caused by oxidative stress to the reconstructive microflora, ultimately restoring intestinal barrier function. W-GA nanodots have been synthesized through a simple coordination reaction and can be dispersed in various solvents in vitro, demonstrating favorable safety profiles in cells, significant clearance of reactive oxygen and nitrogen species (RONS), and increased cell survival in models of oxidative stress induced by hydrogen peroxide (H2O2). Through oral or intravenous administration, the W-GA nanodots were shown to be highly safe when tested in vivo, and they effectively reduced colon damage in mice with DSS-induced colitis by restoring the integrity of the intestinal barrier. W-GA nanodots have enabled the integration of microflora reprogramming and RONS clearance, creating a potent therapeutic strategy for treating gut inflammation. Consequently, the development of W-GA nanodots represents a promising strategy for enhancing the formation and preservation of the intestinal barrier to treat IBD by suppressing the growth of Enterobacteriaceae, a type of facultative anaerobic bacterium, and facilitating the effective removal of RONS. Ultimately, this leads to the restoration of the intestinal barrier's functionality. STATEMENT OF SIGNIFICANCE: An increasing number of nanoparticles are under development for treating inflammatory bowel disease. Although they can alleviate inflammation symptoms by regulating reactive oxygen and nitrogen species (RONS) and microbiota, their understanding of the mechanism behind microbiota regulation is limited. This study synthesized W-GA nanodots using a straightforward one-pot synthesis method. Simple synthesis holds significant promise for clinical applications, as it encompasses multiple nanoenzyme functions and also exhibits Enterobacteriaceae inhibitory properties.Thus, it contributes to ameliorating the current medical landscape of inflammatory bowel disease.

Updated Progress on Polysaccharides with Anti-Diabetic Effects through the Regulation of Gut Microbiota: Sources, Mechanisms, and Structure-Activity Relationships.

Diabetes mellitus (DM) is a common chronic metabolic disease worldwide. The disturbance of the gut microbiota has a complex influence on the development of DM. Polysaccharides are one type of the most important natural components with anti-diabetic effects. Gut microbiota can participate in the fermentation of polysaccharides, and through this, polysaccharides regulate the gut microbiota and improve DM. This review begins by a summary of the sources, anti-diabetic effects and the gut microbiota regulation functions of natural polysaccharides. Then, the mechanisms of polysaccharides in regulating the gut microbiota to exert anti-diabetic effects and the structure-activity relationship are summarized. It is found that polysaccharides from plants, fungi, and marine organisms show great hypoglycemic activities and the gut microbiota regulation functions. The mechanisms mainly include repairing the gut burrier, reshaping gut microbiota composition, changing the metabolites, regulating anti-inflammatory activity and immune function, and regulating the signal pathways. Structural characteristics of polysaccharides, such as monosaccharide composition, molecular weight, and type of glycosidic linkage, show great influence on the anti-diabetic activity of polysaccharides. This review provides a reference for the exploration and development of the anti-diabetic effects of polysaccharides.

A human milk oligosaccharide prevents intestinal inflammation in adulthood via modulating gut microbial metabolism.

Observational evidence suggests that human milk oligosaccharides (HMOs) promote the growth of commensal bacteria in early life and adulthood. However, the mechanisms by which HMOs benefit health through modulation of gut microbial homeostasis remain largely unknown. 2'-fucosyllactose (2'-FL) is the most abundant oligosaccharide in human milk and contributes to the essential health benefits associated with human milk consumption. Here, we investigated how 2'-FL prevents colitis in adulthood through its effects on the gut microbial community. We found that the gut microbiota from adult mice that consumed 2'-FL exhibited an increase in abundance of several health-associated genera, including Bifidobacterium and Lactobacillus. The 2'-FL-modulated gut microbial community exerted preventive effects on colitis in adult mice. By using Bifidobacterium infantis as a 2'-FL-consuming bacterial model, exploratory metabolomics revealed novel 2'-FL-enriched secretory metabolites by Bifidobacterium infantis, including pantothenol. Importantly, pantothenate significantly protected the intestinal barrier against oxidative stress and mitigated colitis in adult mice. Furthermore, microbial metabolic pathway analysis identified 26 dysregulated metabolic pathways in fecal microbiota from patients with ulcerative colitis, which were significantly regulated by 2'-FL treatment in adult mice, indicating that 2'-FL has the potential to rectify dysregulated microbial metabolism in colitis. These findings support the contribution of the 2'-FL-shaped gut microbial community and bacterial metabolite production to the protection of intestinal integrity and prevention of intestinal inflammation in adulthood.IMPORTANCEAt present, neither basic research nor clinical studies have revealed the exact biological functions or mechanisms of action of individual oligosaccharides during development or in adulthood. Thus, it remains largely unknown whether human milk oligosaccharides could serve as effective therapeutics for gastrointestinal-related diseases. Results from the present study uncover 2'-FL-driven alterations in bacterial metabolism and identify novel B. infantis-secreted metabolites following the consumption of 2'-FL, including pantothenol. This work further demonstrates a previously unrecognized role of pantothenate in significantly protecting the intestinal barrier against oxidative stress and mitigating colitis in adult mice. Remarkably, 2'-FL-enhanced bacterial metabolic pathways are found to be dysregulated in the fecal microbiota of ulcerative colitis patients. These novel metabolic pathways underlying the bioactivities of 2'-FL may lay a foundation for applying individual oligosaccharides for prophylactic intervention for diseases associated with impaired intestinal homeostasis.

New Awareness of the Interplay Between the Gut Microbiota and Circadian Rhythms.

Circadian rhythms influence various aspects of the biology and physiology of the host, such as food intake and sleep/wake cycles. In recent years, an increasing amount of genetic and epidemiological data has shown that the light/dark cycle is the main cue that regulates circadian rhythms. Other factors, including sleep/wake cycles and food intake, have necessary effects on the composition and rhythms of the gut microbiota. Interestingly, the gut microbiota can affect the circadian rhythm of hosts in turn through contact-dependent and contact-independent mechanisms. Furthermore, the gut microbiota has been shown to regulate the sleep/wake cycles through gut-brain-microbiota interaction. In addition to diabetes, the gut microbiota can also intervene in the progression of neuro- degenerative diseases through the gut-brain-microbiota interaction, and also in other diseases such as hypertension and rheumatoid arthritis, where it is thought to have a spare therapeutic potential. Even though fecal microbiota transplantation has good potential for treating many diseases, the risk of spreading intestinal pathogens should not be ignored.

Clostridium butyricum Reduces Obesity in a Butyrate-Independent Way.

Accumulating evidence from recent studies links the gut microbiota to obesity, and microbiome therapy has been examined as a treatment. Clostridium butyricum (C. butyricum), an intestinal symbiont, protects the host from a range of diseases. Studies have shown a negative correlation between the relative abundance of C. butyricum and a predisposition for obesity. However, the physiological function and material basis of C. butyricum for obesity are unclear. Here, five C. butyricum isolates were administered to mice on a high-fat diet (HFD) to determine their anti-obesity effects. All isolates suppressed the formation and inflammation of subcutaneous fat, and the two effective strains considerably reduced weight gain and ameliorated dyslipidemia, hepatic steatosis, and inflammation. These positive effects were not achieved by increasing the concentration of intestinal butyrate, and the effective strains could not be replaced by sodium butyrate (NaB). We also discovered that oral supplementation with the two most effective strains changed the metabolism of tryptophan and purine and altered the composition of the gut microbiota. In summary, C. butyricum improved the metabolic phenotypes under the HFD by controlling the composition of the gut microbiota and modulating intestinal metabolites, thereby demonstrating its ability to fight obesity and providing a theoretical foundation for microbial preparations production.

Timing matters: age-dependent impacts of the social environment and host selection on the avian gut microbiota.

BACKGROUND: The establishment of the gut microbiota in early life is a critical process that influences the development and fitness of vertebrates. However, the relative influence of transmission from the early social environment and host selection throughout host ontogeny remains understudied, particularly in avian species. We conducted conspecific and heterospecific cross-fostering experiments in zebra finches (Taeniopygia guttata) and Bengalese finches (Lonchura striata domestica) under controlled conditions and repeatedly sampled the faecal microbiota of these birds over the first 3 months of life. We thus documented the development of the gut microbiota and characterised the relative impacts of the early social environment and host selection due to species-specific characteristics and individual genetic backgrounds across ontogeny by using 16S ribosomal RNA gene sequencing. RESULTS: The taxonomic composition and community structure of the gut microbiota changed across ontogenetic stages; juvenile zebra finches exhibited higher alpha diversity than adults at the post-breeding stage. Furthermore, in early development, the microbial communities of juveniles raised by conspecific and heterospecific foster parents resembled those of their foster family, emphasising the importance of the social environment. In later stages, the social environment continued to influence the gut microbiota, but host selection increased in importance. CONCLUSIONS: We provided a baseline description of the developmental succession of gut microbiota in zebra finches and Bengalese finches, which is a necessary first step for understanding the impact of the early gut microbiota on host fitness. Furthermore, for the first time in avian species, we showed that the relative strengths of the two forces that shape the establishment and maintenance of the gut microbiota (i.e. host selection and dispersal from the social environment) change during development, with host selection increasing in importance. This finding should be considered when experimentally manipulating the early-life gut microbiota. Our findings also provide new insights into the mechanisms of host selection. Video Abstract.

Crosstalk between traditional Chinese medicine-derived polysaccharides and the gut microbiota: A new perspective to understand traditional Chinese medicine.

Polysaccharide is a kind of macromolecule polymer composed of monosaccharides connected by glycosidic bonds. Traditional Chinese medicine (TCM), composed of various bioactive ingredients, is usually rich in polysaccharides. In recent years, extensive research on TCM polysaccharides has demonstrated their pharmacological effects. Polysaccharides can hardly be catabolized by enzymes encoded by the human genome but can be degraded to absorbable metabolites by bacteria inhabiting the colon. Hence, the gut microbiota plays a vital role in degrading TCM polysaccharides into short-chain fatty acids (SCFAs) which exert physiological functions locally and systemically. Besides, TCM polysaccharides can also modulate the composition and activities of the gut microbiota by promoting the growth of beneficial bacteria and inhibiting the colonization of pathogenic bacteria, ultimately restoring gut homeostasis and improving human health. In this review, we discuss the extraction and pharmacological effects of TCM polysaccharides, various functions of the gut microbiota, and the interactions between TCM polysaccharides and the gut microbiota, illuminating the mechanisms of TCM polysaccharides modulating host physiology via the gut microbiota. To firmly establish the clinical efficacy of TCM polysaccharides, further high-quality studies especially clinical trials are needed. Generally, discussion on the interplay between TCM polysaccharides and the gut microbiota is expected to elucidate their application prospects and inspire new thoughts in the development of TCM.

Gut Microbiota and Immunotherapy.

The gut microbiota is the largest microbiota in the body, which is closely related to the immune state of the body. A number of studies have shown that gut microbiota and its metabolites are involved in host immune regulation. Immune checkpoint inhibitors have become an important drug for the treatment of many malignant tumors, which can significantly improve the prognosis of tumor patients. However, a considerable number of patients cannot benefit from immune checkpoint inhibitors. At present, the known treatment methods of microbiota manipulation mainly include fecal microbiota transplantation, dietary regulation, prebiotics and so on. Therefore, this paper will discuss the possibility of improving the anti-tumor efficacy of immunotherapy from the perspectives of the gut microbiota and immunotherapy.

The improvement of parturition duration by high intake of dietary fibre in late gestation is associated with gut microbiota and metabolome in sows.

Prolonged parturition duration has been widely demonstrated to be a risk factor for incidence of stillbirth. This study evaluated the supply of dietary fibre on the parturition duration, gut microbiota and metabolome using sows as a model. A total of 40 Yorkshire sows were randomly given diet containing normal level of dietary fibre (NDF, 17·5 % dietary fibre) or high level of dietary fibre (HDF, 33·5 % dietary fibre). Faecal microbiota profiled with 16S rRNA amplicon sequencing, SCFA and metabolome in the faeces and plasma around parturition were compared between the dietary groups. Correlation analysis was conducted to further explore the potential associations between specific bacterial taxa and metabolites. Results showed that HDF diet significantly improved the parturition process as presented by the shorter parturition duration. HDF diet increased the abundance of the phyla Bacteroidetes and Synergistetes and multiple genera. Except for butyrate, SCFA levels in the faeces and plasma of sows at parturition were elevated in HDF group. The abundances of fifteen and twelve metabolites in the faeces and plasma, respectively, markedly differ between HDF and NDF sows. These metabolites are involved in energy metabolism and bacterial metabolism. Correlation analysis also showed associations between specific bacteria taxa and metabolites. Collectively, our study indicates that the improvement of parturition duration by high fibre intake in late gestation is associated with gut microbiota, production of SCFA and other metabolites, potentially serving for energy metabolism.

Hematopoietic Cell Transplantation Rescues Inflammatory Bowel Disease and Dysbiosis of Gut Microbiota in XIAP Deficiency.

BACKGROUND: X-linked inhibitor of apoptosis protein (XIAP) deficiency is an infrequent inborn error of immunity that is often associated with refractory inflammatory bowel disease (IBD). The natural course of XIAP deficiency is typically associated with poor prognosis, and hematopoietic cell transplantation (HCT) is the only curative treatment. OBJECTIVE: To study (1) the effect of HCT on patients with XIAP deficiency undergoing HCT, (2) the status of XIAP deficiency-associated IBD after HCT, and (3) the gut microbiota of XIAP deficiency-associated IBD before and after HCT. METHODS: A nationwide survey of patients with XIAP deficiency was conducted. A spreadsheet questionnaire was collected from the physicians. Feces samples collected from the patients before and after HCT and their healthy family members were analyzed. RESULTS: Twenty-six patients with XIAP deficiency underwent HCT by the end of March 2020, and 22 patients (84.6%) survived. All the survivors underwent a fludarabine-based reduced-intensity condition regimen. Acute graft-versus-host disease was observed in 17 patients (65.4%). Nineteen patients experienced refractory IBD before undergoing HCT. IBD improved remarkably after HCT. After HCT, the colonoscopic and pathological symptoms were restored to normal, and the pediatric ulcerative colitis activity index improved significantly. Gut microbiota indicated dysbiosis before HCT; however, it was improved to resemble that of the healthy family members after HCT. CONCLUSIONS: This study revealed that HCT has a favorable outcome for XIAP deficiency. HCT rescues gut inflammation and dysbiosis in patients with XIAP deficiency.

Bacillus subtilis-Based Probiotic Improves Skeletal Health and Immunity in Broiler Chickens Exposed to Heat Stress.

The elevation of ambient temperature beyond the thermoneutral zone leads to heat stress, which is a growing health and welfare issue for homeothermic animals aiming to maintain relatively constant reproducibility and survivability. Particularly, global warming over the past decades has resulted in more hot days with more intense, frequent, and long-lasting heat waves, resulting in a global surge in animals suffering from heat stress. Heat stress causes pathophysiological changes in animals, increasing stress sensitivity and immunosuppression, consequently leading to increased intestinal permeability (leaky gut) and related neuroinflammation. Probiotics, as well as prebiotics and synbiotics, have been used to prevent or reduce stress-induced negative effects on physiological and behavioral homeostasis in humans and various animals. The current data indicate dietary supplementation with a Bacillus subtilis-based probiotic has similar functions in poultry. This review highlights the recent findings on the effects of the probiotic Bacillus subtilis on skeletal health of broiler chickens exposed to heat stress. It provides insights to aid in the development of practical strategies for improving health and performance in poultry.

Gut Microbiota Perturbation in IgA Deficiency Is Influenced by IgA-Autoantibody Status.

BACKGROUND & AIMS: IgA exerts its primary function at mucosal surfaces, where it binds microbial antigens to regulate bacterial growth and epithelial attachment. One third of individuals with IgA deficiency (IgAD) suffers from recurrent mucosal infections, possibly related to an altered microbiota. We aimed to delineate the impact of IgAD and the IgA-autoantibody status on the composition and functional capacity of the gut microbiota. METHODS: We performed a paired, lifestyle-balanced analysis of the effect of IgA on the gut microbiota composition and functionality based on fecal samples from individuals with IgAD and IgA-sufficient household members (n = 100), involving quantitative shotgun metagenomics, species-centric functional annotation of gut bacteria, and strain-level analyses. We supplemented the data set with 32 individuals with IgAD and examined the influence of IgA-autoantibody status on the composition and functionality of the gut microbiota. RESULTS: The gut microbiota of individuals with IgAD exhibited decreased richness and diversity and was enriched for bacterial species encoding pathogen-related functions including multidrug and antimicrobial peptide resistance, virulence factors, and type III and VI secretion systems. These functional changes were largely attributed to Escherichia coli but were independent of E coli strain variations and most prominent in individuals with IgAD with IgA-specific autoreactive antibodies. CONCLUSIONS: The microbiota of individuals with IgAD is enriched for species holding increased proinflammatory potential, thereby potentially decreasing the resistance to gut barrier-perturbing events. This phenotype is especially pronounced in individuals with IgAD with IgA-specific autoreactive antibodies, thus warranting a screening for IgA-specific autoreactive antibodies in IgAD to identify patients with IgAD with increased risk for gastrointestinal implications.

A behavioral model for mapping the genetic architecture of gut-microbiota networks.

The gut microbiota may play an important role in affecting human health. To explore the genetic mechanisms underlying microbiota-host relationships, many genome-wide association studies have begun to identify host genes that shape the microbial composition of the gut. It is becoming increasingly clear that the gut microbiota impacts host processes not only through the action of individual microbes but also their interaction networks. However, a systematic characterization of microbial interactions that occur in densely packed aggregates of the gut bacteria has proven to be extremely difficult. We develop a computational rule of thumb for addressing this issue by integrating ecological behavioral theory and genetic mapping theory. We introduce behavioral ecology theory to derive mathematical descriptors of how each microbe interacts with every other microbe through a web of cooperation and competition. We estimate the emergent properties of gut-microbiota networks reconstructed from these descriptors and map host-driven mutualism, antagonism, aggression, and altruism QTLs. We further integrate path analysis and mapping theory to detect and visualize how host genetic variants affect human diseases by perturbing the internal workings of the gut microbiota. As the proof of concept, we apply our model to analyze a published dataset of the gut microbiota, showing its usefulness and potential to gain new insight into how microbes are organized in human guts. The new model provides an analytical tool for revealing the "endophenotype" role of microbial networks in linking genotype to end-point phenotypes.

Dysbiosis of the gut microbiota maybe exacerbate orf pathology by promoting inflammatory immune responses.

Orf is a contagious disease caused by the epitheliotropic orf virus (ORFV) that mainly affects goats and sheep. Orf occurs worldwide and can cause great losses to livestock production. Mounting evidence has shown that gut microbiota plays a pivotal role in shaping the immune responses of the host and thus affecting the infection process of a wide range of pathogens. However, it is unclear whether gut microbiota plays a role during orf development. In this study, we exploited asymptomatic ORFV-carrier goats to explore the potential effects of gut microbiota on orf pathogenesis. The results showed that antibiotics-induced gut microbiota disruption significantly aggravated orf, as indicated by the greater disease severity and higher percentage of animals manifesting clinical orf symptoms. Further analysis suggested IL-17-induced excessive neutrophil accumulation in the diseased lips was potentially responsible for the tissue pathology. In addition, skin γδT cells may be an important source of IL-17. In conclusion, our study showed that the gut microbiota of ORFV-carrier goats plays a central role in controlling inflammatory pathology during ORFV infection, partly through suppressing IL-17-mediated local proinflammatory immune responses. This finding can provide help for elucidating the pathogenesis of orf and also suggests an efficient strategy to minimize the inflammatory pathology by maintaining a healthy gut microbiota during orf development.

Effects of products designed to modulate the gut microbiota on hyperlipidaemia.

PURPOSE: Fatalities due to heart and cerebrovascular diseases caused by uncontrolled hyperlipidaemia increase every year; on the other hand, lipid-lowering drugs are known to cause side effects. The gut microbiota has been thoroughly investigated by researchers and consumers, because they have unique functional properties and littler side effects. However, the effects of the gut microbiota remain controversial. We conducted a meta-analysis to assess the effects of products designed to modulate the gut microbiota on various hyperlipidaemias. METHODS: We systematically searched PubMed, Embase, Cochrane Library (Central), and Web of Science for randomized controlled trials (published before June 2017, and those only in English) to compare treatment (products designed to modulate the gut microbiota) versus placebo. Our main endpoints were total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in serum. We assessed pooled data using a fixed effects model. RESULTS: Of 1337 identified studies, 21 were eligible and included in our analysis (n = 1436 participants). The combined estimate of effect size for the impact of products designed to modulate the gut microbiota on serum TC (WMD - 11.07 mg/dL, 95% CI - 13.72 to - 8.43, p < 0.001), LDL-C (WMD - 10.96 mg/dL, 95% CI - 13.37 to - 8.56, p < 0.001), and HDL-C (WMD 0.72 mg/dL, 95% CI 0.06-1.38, p = 0.032) were statistically significant, while no significant effect was found on TG concentrations (WMD - 0.56 mg/dL, 95% CI - 5.59 to 4.47, p = 0.828). Subgroup analysis showed parallel trials, probiotics, and long-term intervention had better effects on lowering blood lipid levels. CONCLUSION: Products designed to modulate the gut microbiota results in changes of the plasma lipid concentrations and these changes may protect against cardiovascular disease.

Colonization and Gut Flora Modulation of Lactobacillus kefiranofaciens ZW3 in the Intestinal Tract of Mice.

This study evaluated the distribution and colonization of Lactobacillus kefiranofaciens ZW3 and determined its capacity to modulate the gut microbiota in an animal model. Based on (1) fluorescence imaging, (2) flow cytometry, and (3) qPCR, we found that ZW3 successfully adhered to mouse mucous tissue and colonized the mouse ileum. Gut microbiota profiling was performed using high-throughput sequencing. After continuous intubation with ZW3 for 1 week, the proportion of Lachnospiraceae, a family of butyric acid-producing bacteria, increased at day 7 (11.9% at day 0 versus 18.4% at day 7). In addition, Lactobacillaceae showed an increasing trend (4% at day 0 versus 13% at day 7) that was accompanied by an observable decline in the Rikenellaceae family (1.58% at day 7, 0.14% at day 14, and 0.75% at day 21) in the tested mouse. The results demonstrate that ZW3 could successfully adhere to and colonize the mouse gut throughout the course of the experiment. The profiling analysis of the gut microbiota also provided evidence supporting the function of ZW3 in improving the intestinal flora of mice.

Targeted deletion of RANKL in M cell inducer cells by the Col6a1-Cre driver.

The gut-associated lymphoid tissues (GALTs), including Peyer's patches (PPs), cryptopatches (CPs) and isolated lymphoid follicles (ILFs), establish a host-microbe symbiosis by the promotion of immune reactions against gut microbes. Microfold cell inducer (MCi) cells in GALTs are the recently identified mesenchymal cells that express the cytokine RANKL and initiate bacteria-specific immunoglobulin A (IgA) production via induction of microfold (M) cell differentiation. In the previous study, the Twist2-Cre driver was utilized for gene deletion in mesenchymal cells including MCi cells. In order to investigate MCi cells more extensively, it will be necessary to develop experimental tools in addition to the Twist2-Cre driver mice and characterize such drivers in specificity and efficiency. Here we show that M cell differentiation and IgA production are impaired in the targeted deletion of RANKL by the Col6a1-Cre driver. We compared Col6a1-Cre with Twist2-Cre in terms of the specificity for mesenchymal cells in GALTs. Col6a1-Cre CAG-CAT-EGFP mice exhibited EGFP expression in podoplanin+CD31- cells including MCi cells, while Twist2-Cre mice were shown to target endothelial cells and podoplanin+CD31- cells. Tnfsf11fl/ΔCol6a1-Cre mice exhibited the absence of M cells and severe IgA reduction together with an alteration in gut microbial composition. Moreover, we analyzed germ free mice to test whether changes in the microbiota are the cause of M cell deficiency. M cell differentiation was normal in the CPs/ILFs of germ free mice, indicating that MCi cells induce M cells independently of microbial colonization. This study demonstrates that Col6a1-Cre driver mice are as useful as Twist2-Cre driver mice for functional analyses of GALT-resident mesenchymal cells, including MCi cells.