M cell maturation and cDC activation determine the onset of adaptive immune priming in the neonatal Peyer's patch.

Early-life immune development is critical to long-term host health. However, the mechanisms that determine the pace of postnatal immune maturation are not fully resolved. Here, we analyzed mononuclear phagocytes (MNPs) in small intestinal Peyer's patches (PPs), the primary inductive site of intestinal immunity. Conventional type 1 and 2 dendritic cells (cDC1 and cDC2) and RORgt+ antigen-presenting cells (RORgt+ APC) exhibited significant age-dependent changes in subset composition, tissue distribution, and reduced cell maturation, subsequently resulting in a lack in CD4+ T cell priming during the postnatal period. Microbial cues contributed but could not fully explain the discrepancies in MNP maturation. Type I interferon (IFN) accelerated MNP maturation but IFN signaling did not represent the physiological stimulus. Instead, follicle-associated epithelium (FAE) M cell differentiation was required and sufficient to drive postweaning PP MNP maturation. Together, our results highlight the role of FAE M cell differentiation and MNP maturation in postnatal immune development.

ONECUT2 regulates RANKL-dependent enterocyte and microfold cell differentiation in the small intestine; a multi-omics study.

Microfold (M) cells reside in the intestinal epithelium of Peyer's patches (PP). Their unique ability to take up and transport antigens from the intestinal lumen to the underlying lymphoid tissue is key in the regulation of the gut-associated immune response. Here, we applied a multi-omics approach to investigate the molecular mechanisms that drive M cell differentiation in mouse small intestinal organoids. We generated a comprehensive profile of chromatin accessibility changes and transcription factor dynamics during in vitro M cell differentiation, allowing us to uncover numerous cell type-specific regulatory elements and associated transcription factors. By using single-cell RNA sequencing, we identified an enterocyte and M cell precursor population. We used our newly developed computational tool SCEPIA to link precursor cell-specific gene expression to transcription factor motif activity in cis-regulatory elements, uncovering high expression of and motif activity for the transcription factor ONECUT2. Subsequent in vitro and in vivo perturbation experiments revealed that ONECUT2 acts downstream of the RANK/RANKL signalling axis to support enterocyte differentiation, thereby restricting M cell lineage specification. This study sheds new light on the mechanism regulating cell fate balance in the PP, and it provides a powerful blueprint for investigation of cell fate switches in the intestinal epithelium.

Intersecting pathways: The role of hybrid E/M cells and circulating tumor cells in cancer metastasis and drug resistance.

Cancer metastasis and therapy resistance are intricately linked with the dynamics of Epithelial-Mesenchymal Transition (EMT) and Circulating Tumor Cells (CTCs). EMT hybrid cells, characterized by a blend of epithelial and mesenchymal traits, have emerged as pivotal in metastasis and demonstrate remarkable plasticity, enabling transitions across cellular states crucial for intravasation, survival in circulation, and extravasation at distal sites. Concurrently, CTCs, which are detached from primary tumors and travel through the bloodstream, are crucial as potential biomarkers for cancer prognosis and therapeutic response. There is a significant interplay between EMT hybrid cells and CTCs, revealing a complex, bidirectional relationship that significantly influences metastatic progression and has a critical role in cancer drug resistance. This resistance is further influenced by the tumor microenvironment, with factors such as tumor-associated macrophages, cancer-associated fibroblasts, and hypoxic conditions driving EMT and contributing to therapeutic resistance. It is important to understand the molecular mechanisms of EMT, characteristics of EMT hybrid cells and CTCs, and their roles in both metastasis and drug resistance. This comprehensive understanding sheds light on the complexities of cancer metastasis and opens avenues for novel diagnostic approaches and targeted therapies and has significant advancements in combating cancer metastasis and overcoming drug resistance.

An M cell-targeting recombinant L. lactis vaccine against four H. pylori adhesins.

The acidic environment and enzyme degradation lead to oral vaccines often having little immune effect. Therefore, it is an attractive strategy to study an effective and safe oral vaccine delivery system that can promote gastrointestinal mucosal immune responses and inhibit antigen degradation. Moreover, the antigens uptake by microfold cells (M cells) is the determining step in initiating efficient immune responses. Therefore, M cell-targeting is one promising approach for enhancing oral vaccine potency. In the present study, an M cell-targeting L. lactis surface display system (plSAM) was built to favor the multivalent epitope vaccine antigen (FAdE) to achieve effective gastrointestinal mucosal immunity against Helicobacter pylori. Therefore, a recombinant Lactococcus lactic acid vaccine (LL-plSAM-FAdE) was successfully prepared, and its immunological properties and protective efficacy were analyzed. The results showed that LL-plSAM-FAdE can secretively express the recombinant proteins SAM-FAdE and display the SAM-FAdE on the bacterial cell surface. More importantly, LL-plSAM-FAdE effectively promoted the phagocytosis and transport of vaccine antigen by M cells in the gastrointestinal tract of mice, and simulated high levels of cellular and humoral immune responses against four key H. pylori adhesins (Urease, CagL, HpaA, and Lpp20) in the gastrointestinal tract, thus enabling effective prevention of H. pylori infection and to some extent eliminating H. pylori already present in the gastrointestinal tract. KEY POINTS: • M-cell-targeting L. lactis surface display system LL- plSAM was designed • This system displays H. pylori vaccine-promoted phagocytosis and transport of M cell • A promising vaccine candidate for controlling H. pylori infection was verified.

Sorbitol Destroyed Intestinal Microfold Cells (M Cells) Development through Inhibition of PDE4-Mediated RANKL Expression.

Objective: Microfold cells (M cells) are specific intestinal epithelial cells for monitoring and transcytosis of antigens, microorganisms, and pathogens in the intestine. However, the mechanism for M-cell development remained elusive. Materials and Methods: Real-time polymerase chain reaction, immunofluorescence, and western blotting were performed to analyze the effect of sorbitol-regulated M-cell differentiation in vivo and in vitro, and luciferase and chromatin Immunoprecipitation were used to reveal the mechanism through which sorbitol-modulated M-cell differentiation. Results: Herein, in comparison to the mannitol group (control group), we found that intestinal M-cell development was inhibited in response to sorbitol treatment as evidenced by impaired enteroids accompanying with decreased early differentiation marker Annexin 5, Marcksl1, Spib, sox8, and mature M-cell marker glycoprotein 2 expression, which was attributed to downregulation of receptor activator of nuclear factor kappa-В ligand (RANKL) expression in vivo and in vitro. Mechanically, in the M-cell model, sorbitol stimulation caused a significant upregulation of phosphodiesterase 4 (PDE4) phosphorylation, leading to decreased protein kinase A (PKA)/cAMP-response element binding protein (CREB) activation, which further resulted in CREB retention in cytosolic and attenuated CREB binds to RANKL promoter to inhibit RANKL expression. Interestingly, endogenous PKA interacted with CREB, and this interaction was destroyed by sorbitol stimulation. Most importantly, inhibition of PDE4 by dipyridamole could rescue the inhibitory effect of sorbitol on intestinal enteroids and M-cell differentiation and mature in vivo and in vitro. Conclusion: These findings suggested that sorbitol suppressed intestinal enteroids and M-cell differentiation and matured through PDE4-mediated RANKL expression; targeting to inhibit PDE4 was sufficient to induce M-cell development.

NOX1 is essential for TNFα-induced intestinal epithelial ROS secretion and inhibits M cell signatures.

OBJECTIVE: Loss-of-function mutations in genes generating reactive oxygen species (ROS), such as NOX1, are associated with IBD. Mechanisms whereby loss of ROS drive IBD are incompletely defined. DESIGN: ROS measurements and single-cell transcriptomics were performed on colonoids stratified by NOX1 genotype and TNFα stimulation. Clustering of epithelial cells from human UC (inflamed and uninflamed) scRNASeq was performed. Validation of M cell induction was performed by immunohistochemistry using UEA1 (ulex europaeus agglutin-1 lectin) and in vivo with DSS injury. RESULTS: TNFα induces ROS production more in NOX1-WT versus NOX1-deficient murine colonoids under a range of Wnt-mediated and Notch-mediated conditions. scRNASeq from inflamed and uninflamed human colitis versus TNFα stimulated, in vitro colonoids defines substantially shared, induced transcription factors; NOX1-deficient colonoids express substantially lower levels of STAT3 (signal transducer and activator of transcription 3), CEBPD (CCAAT enhancer-binding protein delta), DNMT1 (DNA methyltransferase) and HIF1A (hypoxia-inducible factor) baseline. Subclustering unexpectedly showed marked TNFα-mediated induction of M cells (sentinel cells overlying lymphoid aggregates) in NOX1-deficient colonoids. M cell induction by UEA1 staining is rescued with H2O2 and paraquat, defining extra- and intracellular ROS roles in maintenance of LGR5+ stem cells. DSS injury demonstrated GP2 (glycoprotein-2), basal lymphoplasmacytosis and UEA1 induction in NOX1-deficiency. Principal components analyses of M cell genes and decreased DNMT1 RNA velocity correlate with UC inflammation. CONCLUSIONS: NOX1 deficiency plus TNFα stimulation contribute to colitis through dysregulation of the stem cell niche and altered cell differentiation, enhancing basal lymphoplasmacytosis. Our findings prioritise ROS modulation for future therapies.

Inhibition of microfold cells ameliorates early pathological phenotypes by modulating microglial functions in Alzheimer's disease mouse model.

BACKGROUND: The gut microbiota has recently attracted attention as a pathogenic factor in Alzheimer's disease (AD). Microfold (M) cells, which play a crucial role in the gut immune response against external antigens, are also exploited for the entry of pathogenic bacteria and proteins into the body. However, whether changes in M cells can affect the gut environments and consequently change brain pathologies in AD remains unknown. METHODS: Five familial AD (5xFAD) and 5xFAD-derived fecal microbiota transplanted (5xFAD-FMT) naïve mice were used to investigate the changes of M cells in the AD environment. Next, to establish the effect of M cell depletion on AD environments, 5xFAD mice and Spib knockout mice were bred, and behavioral and histological analyses were performed when M cell-depleted 5xFAD mice were six or nine months of age. RESULTS: In this study, we found that M cell numbers were increased in the colons of 5xFAD and 5xFAD-FMT mice compared to those of wild-type (WT) and WT-FMT mice. Moreover, the level of total bacteria infiltrating the colons increased in the AD-mimicked mice. The levels of M cell-related genes and that of infiltrating bacteria showed a significant correlation. The genetic inhibition of M cells (Spib knockout) in 5xFAD mice changed the composition of the gut microbiota, along with decreasing proinflammatory cytokine levels in the colons. M cell depletion ameliorated AD symptoms including amyloid-ß accumulation, microglial dysfunction, neuroinflammation, and memory impairment. Similarly, 5xFAD-FMT did not induce AD-like pathologies, such as memory impairment and excessive neuroinflammation in Spib-/- mice. CONCLUSION: Therefore, our findings provide evidence that the inhibiting M cells can prevent AD progression, with therapeutic implications.

Biological effects of polystyrene micro- and nano-plastics on human intestinal organoid-derived epithelial tissue models without and with M cells.

Micro- and nano-plastics (MPs and NPs) released from plastics in the environment can enter the food chain and target the human intestine. However, knowledge about the effects of these particles on the human intestine is still limited due to the lack of relevant human intestinal models to validate data obtained from animal studies or tissue models employing cancer cells. In this study, human intestinal organoids were used to develop epithelia to mimic the cell complexity and functions of native tissue. Microfold cells (M cells) were induced to distinguish their role when exposure to MPs and NPs. During the exposure, the M cells acted as sensors, capturers and transporters of larger sized particles. The epithelial cells internalized the particles in a size-, concentration-, and time-dependent manner. Importantly, high concentrations of particles significantly triggered the secretion of a panel of inflammatory cytokines linked to human inflammatory bowel disease (IBD).

Plumbagin Exhibits Genotoxicity and Induces G2/M Cell Cycle Arrest via ROS-Mediated Oxidative Stress and Activation of ATM-p53 Signaling Pathway in Hepatocellular Cells.

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone, PLB), a naturally occurring naphthoquinone mainly isolated from the plant Plumbago zeylanica L., has been proven to possess anticancer activities towards multiple types of cancer. Although there has been an increasing amount of research regarding its anticancer effects, the association between oxidative stress, genotoxicity and the cell cycle arrest induced by PLB still remains unclear. Therefore, it is important to investigate their potential connections and the involvement of DNA damage and the ataxia telangiectasia mutated protein (ATM)-p53 signaling pathway in PLB's anticancer mechanism. The present study showed that PLB exposure significantly reduced HCC cell viability and colony formation. In addition, PLB-induced G2/M cell cycle arrest, oxidative stress, and DNA damage was detected, which could be almost blocked by NAC pretreatment. PLB could trigger a DNA damage response by activating cell cycle checkpoints such as ATM, checkpoint kinase 1 (Chk1), checkpoint kinase 2 (Chk2) and p53. Meanwhile, the key modulator of the G2/M transition factor, Cell Division Cycle 25C (cdc25C), was significantly downregulated in an ROS-dependent manner. Furthermore, pretreatment with ATM and p53 inhibitors (KU55933 and Pifithrin-α) could reduce the occurrence of G2/M cell cycle arrest by inhibiting the activation of the ATM-p53 pathway. Taken together, these results indicate that ROS-mediated oxidative stress plays a key role in PLB-induced G2/M cell cycle arrest mediated by the ATM-p53 pathway.

Baicalein Induces G2/M Cell Cycle Arrest Associated with ROS Generation and CHK2 Activation in Highly Invasive Human Ovarian Cancer Cells.

Ovarian cancer is a lethal gynecological cancer because drug resistance often results in treatment failure. The CHK2, a tumor suppressor, is considered to be an important molecular target in ovarian cancer due to its role in DNA repair. Dysfunctional CHK2 impairs DNA damage-induced checkpoints, reduces apoptosis, and confers resistance to chemotherapeutic drugs and radiation therapy in ovarian cancer cells. This provides a basis for finding new effective agents targeting CHK2 upregulation or activation to treat or prevent the progression of advanced ovarian cancer. Here, the results show that baicalein (5,6,7-trihydroxyflavone) treatment inhibits the growth of highly invasive ovarian cancer cells, and that baicalein-induced growth inhibition is mediated by the cell cycle arrest in the G2/M phase. Baicalein-induced G2/M phase arrest is associated with an increased reactive oxygen species (ROS) production, DNA damage, and CHK2 upregulation and activation. Thus, baicalein modulates the expression of DNA damage response proteins and G2/M phase regulatory molecules. Blockade of CHK2 activation by CHK2 inhibitors protects cells from baicalein-mediated G2/M cell cycle arrest. All the results suggest that baicalein has another novel growth inhibitory effect on highly invasive ovarian cancer cells, which is partly related to G2/M cell cycle arrest through the ROS-mediated DNA breakage damage and CHK2 activation. Collectively, our findings provide a molecular basis for the potential of baicalein as an adjuvant therapeutic agent in the treatment of metastatic ovarian cancer.

Structural changes of layer-by-layer self-assembled starch-based nanocapsules in the gastrointestinal tract: Implications for their M cell-targeting delivery and transport efficiency.

Characterizing the structural changes of cell-targeting delivery carriers in gastrointestinal tract (GIT) is crucial for understanding their effectiveness in cell targeting and transport. Herein, RGD peptide-grafted carboxymethyl starch (CMS) and cationic quaternary ammonium starch (QAS) were utilized to fabricate quintet-layered nanocapsules loaded with ovalbumin (OVA). The aim was to improve delivery and transportation efficiency, specifically targeting M cells. The research analyzed the impact of pH and enzyme variations in GIT on the structure of nanocapsules, interactions between carriers and the release behavior of OVA. Results showed that the size of nanocapsules increased from 229.2 to 479.8 nm and the zeta potential decreased from -1.08 to -33.33 mV during oral delivery. This was evident in TEM images, showing a more relaxed core-shell structure. Isothermal titration calorimetry and molecular dynamic simulation indicated that pH changes primarily affected the electrostatic interaction between carriers. Increasing pH led to reduced affinity constants, and around 84.42 % of OVA was successfully delivered to M cells. Moreover, the transport efficiency of nanocapsules to M cells was five times greater than that of Caco-2 cells. This suggests the feasibility of developing a nanocapsules delivery system capable of adapting to pH changes in GIT by regulating electrostatic interactions between carriers.

Differential role of M cells in enteroid infection by Mycobacterium avium subsp. paratuberculosis and Salmonella enterica serovar Typhimurium.

Infection of ruminants such as cattle with Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease, a disease characterized by chronic inflammation of the small intestine and diarrhoea. Infection with MAP is acquired via the faecal-to-oral route and the pathogen initially invades the epithelial lining of the small intestine. In this study we used an in vitro 3D mouse enteroid model to determine the influence of M cells in infection of the gut epithelia by MAP, in comparison with another bacterial intestinal pathogen of veterinary importance, Salmonella enterica serovar Typhimurium. The differentiation of M cells in the enteroid cultures was induced by stimulation with the cytokine receptor activator of nuclear factor-κB ligand (RANKL), and the effects on MAP and Salmonella uptake and intracellular survival were determined. The presence of M cells in the cultures correlated with increased uptake and intracellular survival of Salmonella, but had no effect on MAP. Interestingly neither pathogen was observed to preferentially accumulate within GP2-positive M cells.

A novel pectin polysaccharide from vinegar-baked Radix Bupleuri absorbed by microfold cells in the form of nanoparticles.

Polysaccharides of vinegar-baked Radix Bupleuri (VBCP) have been reported to exhibit liver-targeting and immunomodulatory activities through oral administration, but the absorption behavior and mechanism of VBCPs have not been extensively studied. In this study, a novel HG type pectin polysaccharide, VBCP1-4, with a high molecular weight of 2.94 × 106 Da, was separated from VBCP. VBCP1-4 backbone was contained 1,4-α-D-GalpA, 1,4-α-D-GalpA6OMe, 1,3,4-α-D-GalpA and 1,2,4-α-D-Rhap. The branches were mainly contained 1,5-α-L-Araf, 1,3,5-α-L-Araf, t-α-L-Araf and t-α-D-Galp, which linked to the 3 position of 1,3,4-α-D-GalpA and the 4 position of 1,2,4-α-D-Rhap. VBCP1-4 could self-assemble to nanoparticles in water, with CMC values of 106.41 µg/mL, particle sizes of 178.20 ± 2.82 nm and zeta potentials of -23.19 ± 1.44 mV. The pharmacokinetic study of VBCP1-4, which detected by marking with FITC, revealed that it could be partially absorbed into the body through Peyer's patches of the ileum. In vitro absorption study demonstrated that VBCP1-4 was difficult to be absorbed by Caco-2 cell monolayer, but could be absorbed by M cells in a time and concentration dependent manner. The absorption mechanism was elucidated that VBCP1-4 entered M cells through clathrin-mediated endocytosis in the form of nanoparticles. These findings provide valuable insights into the absorption behavior of VBCP and contribute to its further development.

Deciphering the M-cell niche: insights from mouse models on how microfold cells "know" where they are needed.

Known for their distinct antigen-sampling abilities, microfold cells, or M cells, have been well characterized in the gut and other mucosa including the lungs and nasal-associated lymphoid tissue (NALT). More recently, however, they have been identified in tissues where they were not initially suspected to reside, which raises the following question: what external and internal factors dictate differentiation toward this specific role? In this discussion, we will focus on murine studies to determine how these cells are identified (e.g., markers and function) and ask the broader question of factors triggering M-cell localization and patterning. Then, through the consideration of unconventional M cells, which include villous M cells, Type II taste cells, and medullary thymic epithelial M cells (microfold mTECs), we will establish the M cell as not just a player in mucosal immunity but as a versatile niche cell that adapts to its home tissue. To this end, we will consider the lymphoid structure relationship and apical stimuli to better discuss how the differing cellular programming and the physical environment within each tissue yield these cells and their unique organization. Thus, by exploring this constellation of M cells, we hope to better understand the multifaceted nature of this cell in its different anatomical locales.

Discrimination of distinct chicken M cell subsets based on CSF1R expression.

In mammals, a subset of follicle-associated epithelial (FAE) cells, known as M cells, conduct the transcytosis of antigens across the epithelium into the underlying lymphoid tissues. We previously revealed that M cells in the FAE of the chicken lung, bursa of Fabricius (bursa), and caecum based on the expression of CSF1R. Here, we applied RNA-seq analysis on highly enriched CSF1R-expressing bursal M cells to investigate their transcriptome and identify novel chicken M cell-associated genes. Our data show that, like mammalian M cells, those in the FAE of the chicken bursa also express SOX8, MARCKSL1, TNFAIP2 and PRNP. Immunohistochemical analysis also confirmed the expression of SOX8 in CSF1R-expressing cells in the lung, bursa, and caecum. However, we found that many other mammalian M cell-associated genes such as SPIB and GP2 were not expressed by chicken M cells or represented in the chicken genome. Instead, we show bursal M cells express high levels of related genes such as SPI1. Whereas our data show that bursal M cells expressed CSF1R-highly, the M cells in the small intestine lacked CSF1R and both expressed SOX8. This study offers insights into the transcriptome of chicken M cells, revealing the expression of CSF1R in M cells is tissue-specific.

Unlocking the potential of microfold cells for enhanced permeation of nanocarriers in oral drug delivery.

The therapeutic effects of orally administered nanocarriers depend on their ability to effectively permeate the intestinal mucosa, which is one of the major challenges in oral drug delivery. Microfold cells are specialized enterocytes in the intestinal epithelium known for their high transcytosis abilities. This study aimed to compare and evaluate two targeting approaches using surface modifications of polymer-based nanocarriers, whereas one generally addresses enterocytes, and one is directed explicitly to microfold cells via targeting the sialyl LewisA motif on their surface. We characterized the resulting carriers in terms of size and charge, supplemented by scanning electron microscopy to confirm their structural properties. For predictive biological testing and to assess the intended targeting effect, we implemented two human intestinal in vitro models containing microfold-like cells. Both models were thoroughly characterized prior to permeation studies with the different nanocarriers. Our results demonstrated improved transport for both targeted formulations compared to undecorated carriers in the in vitro models. Notably, there was an enhanced uptake in the presence of microfold-like cells, particularly for the nanocarriers directed by the anti-sialyl LewisA antibody. These findings highlight the potential of microfold cell targeting to improve oral administration of drugs and emphasize the importance of using suitable and well-characterized in vitro models for testing novel drug delivery strategies.

Molecular weight-mediated interaction changes for enhancing structural stability, release behavior and M cells-targeting transport efficacy of starch-based nanoparticles.

Molecular weight (Mw) of ligand-mediated nanocarriers plays a pivotal role in their architecture and properties. In this study, self-assembled ovalbumin (OVA)-loaded nanoparticles were meticulously engineered by starch polyelectrolytes with different Mw. Results unveiled that, tailoring Mw of GRGDS pentapeptides-grafted carboxymethyl starch (G-CMS) displayed strong binding-affinity and transport efficiency through microfold cells (M cells) pathway in the simulated intestinal epithelial cell monolayer in which M cells were randomly located in the Caco-2 cells monolayer. Notably, nanoparticles assembled from G-CMS with relatively higher Mw exhibited more compact structures due to the stronger interactions between layers compared to that with relatively lower Mw, which rendered remarkably stable and only 19.01 % in vitro OVA leakage under conditions of the upper gastrointestinal tract. Subsequently, more intact nanoparticles reached M cells after in vitro digestion and exhibited higher transport efficiency through the M cells pathways (apparent permeability: 9.38 × 10-5 cm/s) than Caco-2 cells, attributing to specific- and non-specific binding affinity towards M cells. Therefore, optimal Mw tailoring of starch polyelectrolytes can mediate the molecular interactions among their assembled layers and the interactions with M cells to balance the structural compactness, release and transport efficacy of nanoparticles, holding promise for advancing M cells-targeting oral delivery technologies.