PRC2 Regulated Atoh8 Is a Regulator of Intestinal Microfold Cell (M Cell) Differentiation

Intestinal microfold cells (M cells) are a dynamic lineage of epithelial cells that initiate mucosal immunity in the intestine. They are responsible for the uptake and transcytosis of microorganisms, pathogens, and other antigens in the gastrointestinal tract. A mature M cell expresses a receptor Gp2 which binds to pathogens and aids in the uptake. Due to the rarity of these cells in the intestine, their development and differentiation remain yet to be fully understood. We recently demonstrated that polycomb repressive complex 2 (PRC2) is an epigenetic regulator of M cell development, and 12 novel transcription factors including Atoh8 were revealed to be regulated by the PRC2. Here, we show that Atoh8 acts as a regulator of M cell differentiation; the absence of Atoh8 led to a significant increase in the number of Gp2+ mature M cells and other M cell-associated markers such as Spi-B and Sox8. In vitro organoid analysis of RankL treated organoid showed an increase of mature marker GP2 expression and other M cell-associated markers. Atoh8 null mice showed an increase in transcytosis capacity of luminal antigens. An increase in M cell population has been previously reported to be detrimental to mucosal immunity because some pathogens like orally acquired prions have been able to exploit the transcytosis capacity of M cells to infect the host; mice with an increased population of M cells are also susceptible to Salmonella infections. Our study here demonstrates that PRC2 regulated Atoh8 is one of the factors that regulate the population density of intestinal M cell in the Peyer’s patch.

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Atoh8 is a regulator of intestinal microfold cell (M cell) differentiation

10.1101/2021.05.10.443378 ◽

2021 ◽

Author(s):

Joel Johnson George ◽

Laura Martin Diaz ◽

Markus Ojanen ◽

Keijo Viiri

Keyword(s):

Cell Differentiation ◽

Mucosal Immunity ◽

M Cells ◽

M Cell ◽

Polycomb Repressive Complex 2 ◽

Salmonella Infections ◽

Epigenetic Regulator ◽

Development And Differentiation ◽

Host Mouse ◽

Microfold Cells

Intestinal microfold cells (M cells) are a dynamic lineage of epithelial cells that initiate mucosal immunity in the intestine. They are responsible for the uptake and transcytosis of microorganisms, pathogens and other antigens in the gastrointestinal tract. A mature M cell expresses a receptor Gp2 which binds to pathogens and aids in the uptake. Due to the rarity of these cells in the intestine, its development and differentiation remains yet to be fully understood. We recently demonstrated that polycomb repressive complex 2 (PRC2) is an epigenetic regulator of M cell development and 12 novel transcription factors including Atoh8 were revealed to be regulated by the PRC2. Here, we show that Atoh8 acts as a regulator of M cell differentiation; absence of Atoh8 led to a significant increase in the number of Gp2+ mature M cells and other M cell associated markers. Atoh8 null mice showed an increase in transcytosis capacity of luminal antigens. Increase in M cell population has been previously reported to be detrimental to mucosal immunity because some pathogens like orally acquired prions have been able to exploit the transcytosis capacity of M cells to infect the host; mouse with increased population of M cells are also susceptible to Salmonella infections. Our study here demonstrates that the population density of intestinal M-cell in the Peyer's patch is regulated by the PRC2 regulated Atoh8.

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Polycomb Repressive Complex 2-controlled Essrg regulates intestinal Microfold cell differentiation

10.1101/2020.11.13.379610 ◽

2020 ◽

Author(s):

Joel Johnson George ◽

Mikko Oittinen ◽

Laura Martin-Diaz ◽

Veronika Zapilko ◽

Sharif Iqbal ◽

...

Keyword(s):

Transcription Factors ◽

Cell Differentiation ◽

Cell Development ◽

Polycomb Repressive Complex ◽

M Cell ◽

Polycomb Repressive Complex 2 ◽

Epigenetic Regulator ◽

Microfold Cells ◽

Estrogen Related Receptor

AbstractMicrofold cells (M cells) are immunosurveillance epithelial cells located in the Peyer’s patches in the intestine responsible for monitoring and transcytosis of antigens, microorganisms and pathogens. Many transcription factors, e.g., Spi-B and Sox8, necessary to M cell differentiation have been described but the exhaustive set of factors sufficient for differentiation and development of a mature M cell remains elusive. Moreover, the role of polycomb repressive complex 2 (PRC2) as an epigenetic regulator of M cell development has not yet been interrogated. Here, we show that PRC2 regulates a significant set of genes during the M cell differentiation including many transcription factors. Estrogen related receptor gamma (Esrrg) is a novel M cell specific transcription factor acting on a RankL-Rank induced NF-kB pathway, upstream of Sox8 and necessary but not sufficient for a mature M cell marker Gp2 expression. To conclude, with the aid of PRC2 target survey we identified the list of developmental genes specifically implicated in M cell development and Essrg as a necessary factor for Sox8-mediated M cell differentiation.

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Development of intestinal M cells and follicle-associated epithelium is regulated by TRAF6-mediated NF-κB signaling

Journal of Experimental Medicine ◽

10.1084/jem.20160659 ◽

2018 ◽

Vol 215 (2) ◽

pp. 501-519 ◽

Cited By ~ 28

Author(s):

Takashi Kanaya ◽

Sayuri Sakakibara ◽

Toshi Jinnohara ◽

Masami Hachisuka ◽

Naoko Tachibana ◽

...

Keyword(s):

Cell Differentiation ◽

Tumor Necrosis Factor Receptor ◽

M Cells ◽

M Cell ◽

Antigen Uptake ◽

Conditional Deletion ◽

Follicle Associated Epithelium ◽

Necrosis Factor

M cells are located in the follicle-associated epithelium (FAE) that covers Peyer’s patches (PPs) and are responsible for the uptake of intestinal antigens. The differentiation of M cells is initiated by receptor activator of NF-κB. However, the intracellular pathways involved in M cell differentiation are still elusive. In this study, we demonstrate that the NF-κB pathway activated by RANK is essential for M cell differentiation using in vitro organoid culture. Overexpression of NF-κB transcription factors enhances the expression of M cell–associated molecules but is not sufficient to complete M cell differentiation. Furthermore, we evaluated the requirement for tumor necrosis factor receptor–associated factor 6 (TRAF6). Conditional deletion of TRAF6 in the intestinal epithelium causes a complete loss of M cells in PPs, resulting in impaired antigen uptake into PPs. In addition, the expression of FAE-associated genes is almost silenced in TRAF6-deficient mice. This study thus demonstrates the crucial role of TRAF6-mediated NF-κB signaling in the development of M cells and FAE.

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Role of M Cells in Human Mucosal Immunity to Mycobacterium tuberculosis

Open Forum Infectious Diseases ◽

10.1093/ofid/ofx162.111 ◽

2017 ◽

Vol 4 (suppl_1) ◽

pp. S48-S48

Author(s):

Vidhya Nair ◽

Haaris Khan ◽

Ron Mitchell ◽

Michael U Shiloh

Keyword(s):

Mycobacterium Tuberculosis ◽

Virulence Factor ◽

Mucosal Immunity ◽

Cell Biology ◽

Infection Model ◽

M Cells ◽

Dependent Manner ◽

M Cell ◽

Wide Range

Abstract Background Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a bacterial pathogen that infects roughly one-third of the worldÕs population and causes 1–2 million deaths per year. The current paradigm is that phagocytosis of Mtb by patrolling alveolar macrophages initiates Mtb infection. While this model can account for pulmonary TB, it does not adequately explain the occurrence of extrapulmonary forms of TB that manifest in the absence of obvious lung involvement, such as tuberculous cervical lymphadenitis, also known as scrofula. We hypothesized that specialized epithelial cells called microfold cells (M cells) may be an alternate portal of entry for Mtb. Previously we demonstrated that Mtb is able to transcytose across an epithelial barrier in an M cell dependent manner and that M cell mediated transcytosis is vital for Mtb pathogenesis in a mouse model of tuberculosis. Methods We used an in vitro M-cell mediated translocation assay and a Mtb mutant lacking a key virulence factor, ESAT6. We used biochemistry and genetics to identify a novel receptor for ESAT6. We also developed a novel explanted human adenoid Mtb infection model to study mucosal immunity. Results We now demonstrate that the Mtb virulence factor ESAT6 is necessary and sufficient to mediate binding and transcytosis by M cells in vitro and in vivo, and that uptake of Mtb by M cells requires a unique cell surface ESAT6 receptor. We developed a novel explanted human adenoid model of M cell biology and demonstrate rapid Mtb transcytosis by primary human tissue within 60–120 minutes. Using flow cytometry we find that Mtb is first ingested by M cells and then after transcytosis, by tissue resident antigen-presenting cells. Explanted adenoids from 10 independent donors display a wide range of Mtb uptake. Conclusion We conclude that Mtb ESAT6 is necessary for Mtb uptake by M-cells and that binding and transcytosis require a host receptor. Because explanted adenoids display a wide range of Mtb uptake, M cell mediated transcytosis may confer differential susceptibility to scrofula and disseminated disease. These findings are significant as M cells could potentially serve as the basis for novel therapeutic targets against primary Mtb infection. Disclosures All authors: No reported disclosures.

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A201 UNDERSTANDING THE INFECTION DYNAMICS OF MYCOBACTERIUM PARATUBERCULOSIS (MAP)

Journal of the Canadian Association of Gastroenterology ◽

10.1093/jcag/gwab002.199 ◽

2021 ◽

Vol 4 (Supplement_1) ◽

pp. 225-226

Author(s):

G M Baruta ◽

H Zhang ◽

L Alston ◽

S A Hirota

Keyword(s):

Cell Differentiation ◽

Cell Model ◽

Ex Vivo ◽

Gastrointestinal Disease ◽

Mycobacterium Avium Subspecies Paratuberculosis ◽

Experimental System ◽

M Cells ◽

M Cell ◽

Cellular Tropism

Abstract Background Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne’s disease (JD) in ruminants. Following infection, JD may present as enteritis, leading to wasting, often causing premature culling of livestock. Beyond veterinary medicine, several mycobacterium species, including MAP, have been implicated in human gastrointestinal disease. While MAP has been incriminated in causing Crohn’s disease (a claim that has yet to be substantiated), there are confirmed cases of MAP infection in immunocompromised individuals, causing profuse diarrhea, fever, and drastic weight loss. Given the economic burden associated with MAP infection, considerable efforts have sought to understand its dynamics. However, these processes have not been completely characterized, hindering our ability to generate novel anti-infective agents. While the current paradigm suggests that MAP travels to the small intestine, gaining entry through the epithelium, the exact cellular tropism and the mechanism(s) of entry are not well defined. Therefore, we have developed an ex vivo enteroid-based system to visualize invasion of MAP in distinct cells of the intestinal epithelium using a GFP-expressing MAP strain. With this, we sought to test the hypothesis that MAP invasion occurs via M cells through receptor-mediated transcytosis. Aims 1) Characterize experimental system and visualize MAP invasion 2) Determine cellular tropism 3) Uncover mechanisms underlying MAP invasion Methods Enteroids (2D and 3D) were generated and M cell differentiation induced via addition of RANKL. Confluent ileal monolayers were exposed to GFP-expressing MAP strain (K10 pWES4). Confocal microscopy was performed, and barrier function was measured via transepithelial electrical resistance (TEER). Results We generated 3D enteroids and confluent enteroid-derived monolayers with functional M cells capable of transcytosis. MAP was detected mainly within M cells. We further confirmed this finding using a human in vitro M cell model, the Caco-2/Raji-B co-culture system. Furthermore, alterations in TEER following MAP exposure in monolayers cultured with RANKL, triggering M cell differentiation, suggest the existence of a novel mechanism by which MAP disrupts the barrier to invade the mucosa. Conclusions Our results suggest that MAP translocates across the epithelium predominantly via M cells, as shown both in a human and murine model. This newly optimized approach provides an experimental system that will enable us to better characterize M cell-MAP interactions, with the hopes of identifying new therapeutic targets to prevent the spread of MAP and reduce economic impact of JD. Beyond MAP infection, this novel ex vivo system has potential to elucidate other host-pathogen interactions. Funding Agencies Natural Sciences and Engineering Research Council of Canada (NSERC)

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Comparative Analysis of EspF Variants in Inhibition of Escherichia coli Phagocytosis by Macrophages and Inhibition of E. coli Translocation through Human- and Bovine-Derived M Cells

Infection and Immunity ◽

10.1128/iai.00023-11 ◽

2011 ◽

Vol 79 (11) ◽

pp. 4716-4729 ◽

Cited By ~ 19

Author(s):

Amin Tahoun ◽

Gabriella Siszler ◽

Kevin Spears ◽

Sean McAteer ◽

Jai Tree ◽

...

Keyword(s):

Escherichia Coli ◽

Epithelial Cells ◽

Cellular Localization ◽

M Cells ◽

Binding Assays ◽

M Cell ◽

Content Type ◽

E Coli ◽

Coculture System

ABSTRACTThe EspF protein is secreted by the type III secretion system of enteropathogenic and enterohemorrhagicEscherichia coli(EPEC and EHEC, respectively). EspF sequences differ between EHEC O157:H7, EHEC O26:H11, and EPEC O127:H6 in terms of the number of SH3-binding polyproline-rich repeats and specific residues in these regions, as well as residues in the amino domain involved in cellular localization. EspFO127is important for the inhibition of phagocytosis by EPEC and also limits EPEC translocation through antigen-sampling cells (M cells). EspFO127has been shown to have effects on cellular organelle function and interacts with several host proteins, including N-WASP and sorting nexin 9 (SNX9). In this study, we compared the capacities of differentespFalleles to inhibit (i) bacterial phagocytosis by macrophages, (ii) translocation through an M-cell coculture system, and (iii) uptake by and translocation through cultured bovine epithelial cells. TheespFgene fromE. coliserotype O157 (espFO157) allele was significantly less effective at inhibiting phagocytosis and also had reduced capacity to inhibitE. colitranslocation through a human-derivedin vitroM-cell coculture system in comparison toespFO127andespFO26. In contrast,espFO157was the most effective allele at restricting bacterial uptake into and translocation through primary epithelial cells cultured from the bovine terminal rectum, the predominant colonization site of EHEC O157 in cattle and a site containing M-like cells. Although LUMIER binding assays demonstrated differences in the interactions of the EspF variants with SNX9 and N-WASP, we propose that other, as-yet-uncharacterized interactions contribute to the host-based variation in EspF activity demonstrated here.

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Identifying human and murine M cells in vitro

Experimental Biology and Medicine ◽

10.1177/1535370219838674 ◽

2019 ◽

Vol 244 (7) ◽

pp. 554-564 ◽

Cited By ~ 1

Author(s):

Ana Klisuric ◽

Benjamin Thierry ◽

Ludivine Delon ◽

Clive A Prestidge ◽

Rachel J Gibson

Keyword(s):

Epithelial Cell ◽

Epithelial Cells ◽

In Vitro Models ◽

Oral Drug ◽

M Cells ◽

Cell Marker ◽

M Cell ◽

Follicle Associated Epithelium

M cells are an epithelial cell population found in the follicle-associated epithelium overlying gut-associated lymphoid tissues. They are specialized in the transcytosis of luminal antigens. Their transcytotic capacity and location in an immunocompetent environment has prompted the study of these cells as possible targets for oral drug delivery systems. Currently, the models most commonly used to study M cells are restricted to in vivo experiments conducted in mice, and in vitro studies conducted in models comprised either of primary epithelial cells or established cell lines of murine or human origin. In vitro models of the follicle-associated epithelium can be constructed in several ways. Small intestinal Lgr5+ stem cells can be cultured into a 3D organoid structure where M cells are induced with RANKL administration. Additionally, in vitro models containing an “M cell-like” population can be obtained through co-culturing intestinal epithelial cells with cells of lymphocytic origin to induce the M cell phenotype. The evaluation of the efficiency of the variations of these models and their relevance to the in vivo human system is hampered by the lack of a universal M cell marker. This issue has also hindered the advancement of M cell-specific targeting approaches aimed at improving the bioavailability of orally administered compounds. This critical review discusses the different approaches utilized in the literature to identify M cells, their efficiency, reliability and relevance, in the context of commonly used models of the follicle-associated epithelium. The outcome of this review is a clearly defined and universally recognized criteria for the assessment of the relevance of models of the follicle-associated models currently used. Impact statement The study of M cells, a specialized epithelial cell type found in the follicle-associated epithelium, is hampered by the lack of a universal M cell marker. As such, many studies lack reliable and universally recognized methods to identify M cells in their proposed models. As a result of this it is difficult to ascertain whether the effects observed are due to the presence of M cells or an unaccounted variable. The outcome of this review is the thorough evaluation of the many M cell markers that have been used in the literature thus far and a proposed criterion for the identification of M cells for future publications. This will hopefully lead to an improvement in the quality of future publications in this field.

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Comparative study of transcriptomics-based scoring metrics for the epithelial-hybrid-mesenchymal spectrum

10.1101/2020.01.02.892604 ◽

2020 ◽

Author(s):

Priyanka Chakraborty ◽

Jason T George ◽

Shubham Tripathi ◽

Herbert Levine ◽

Mohit Kumar Jolly

Keyword(s):

Cancer Cells ◽

Cancer Metastasis ◽

Epithelial Mesenchymal Transition ◽

Multinomial Logistic Regression ◽

M Cells ◽

M Cell ◽

Mesenchymal Transition ◽

Tumor Types

AbstractThe Epithelial-mesenchymal transition (EMT) is a cellular process implicated in embryonic development, wound healing, and pathological conditions such as cancer metastasis and fibrosis. Cancer cells undergoing EMT exhibit enhanced aggressive behavior characterized by drug resistance, tumor-initiation potential, and the ability to evade immune system. Recent in silico, in vitro, and in vivo evidence indicates that EMT is not an all-or-none process; instead, cells stably acquire one or more hybrid epithelial/mesenchymal (E/M) phenotypes which often can be more aggressive than purely epithelial or mesenchymal cell populations. Thus, the EMT status of cancer cells can prove to be a critical estimate of patient prognosis. Recent attempts have employed different transcriptomics signatures to quantify EMT status in cell lines and patient tumors. However, a comprehensive comparison of these methods, including their accuracy in identifying cells in the hybrid E/M phenotype(s), is lacking. Here, we compare three distinct metrics that score EMT on a continuum, based on the transcriptomics signature of individual samples. Our results demonstrate that these methods exhibit good concordance among themselves in quantifying the extent of EMT in a given sample. Moreover, scoring EMT using any of the three methods discerned that cells undergo varying extents of EMT across tumor types. Separately, our analysis also identified tumor types with maximum variability in terms of EMT and associated an enrichment of hybrid E/M signatures in these samples. Moreover, we also found that the multinomial logistic regression (MLR) based metric was capable of distinguishing between ‘pure’ individual hybrid E/M vs. mixtures of epithelial (E) and mesenchymal (M) cells. Our results, thus, suggest that while any of the three methods can indicate a generic trend in the EMT status of a given cell, the MLR method has two additional advantages: a) it uses a small number of predictors to calculate the EMT score, and b) it can predict from the transcriptomic signature of a population whether it is comprised of ‘pure’ hybrid E/M cells at the single-cell level or is instead an ensemble of E and M cell subpopulations.

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Aging-Related Impairments to M Cells in Peyer’s Patches Coincide With Disturbances to Paneth Cells

Frontiers in Immunology ◽

10.3389/fimmu.2021.761949 ◽

2021 ◽

Vol 12 ◽

Author(s):

David S. Donaldson ◽

Barbara B. Shih ◽

Neil A. Mabbott

Keyword(s):

Cell Differentiation ◽

Paneth Cell ◽

The Elderly ◽

Paneth Cells ◽

Peyer's Patches ◽

Peyer’S Patches ◽

M Cells ◽

Aging Effects ◽

M Cell ◽

Aged Mice

The decline in mucosal immunity during aging increases susceptibility, morbidity and mortality to infections acquired via the gastrointestinal and respiratory tracts in the elderly. We previously showed that this immunosenescence includes a reduction in the functional maturation of M cells in the follicle-associated epithelia (FAE) covering the Peyer’s patches, diminishing the ability to sample of antigens and pathogens from the gut lumen. Here, co-expression analysis of mRNA-seq data sets revealed a general down-regulation of most FAE- and M cell-related genes in Peyer’s patches from aged mice, including key transcription factors known to be essential for M cell differentiation. Conversely, expression of ACE2, the cellular receptor for SARS-Cov-2 virus, was increased in the aged FAE. This raises the possibility that the susceptibility of aged Peyer’s patches to infection with the SARS-Cov-2 virus is increased. Expression of key Paneth cell-related genes was also reduced in the ileum of aged mice, consistent with the adverse effects of aging on their function. However, the increased expression of these genes in the villous epithelium of aged mice suggested a disturbed distribution of Paneth cells in the aged intestine. Aging effects on Paneth cells negatively impact on the regenerative ability of the gut epithelium and could indirectly impede M cell differentiation. Thus, restoring Paneth cell function may represent a novel means to improve M cell differentiation in the aging intestine and increase mucosal vaccination efficacy in the elderly.

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Identification of scavenger receptor B1 as the airway microfold cell receptor for Mycobacterium tuberculosis

eLife ◽

10.7554/elife.52551 ◽

2020 ◽

Vol 9 ◽

Author(s):

Haaris S Khan ◽

Vidhya R Nair ◽

Cody R Ruhl ◽

Samuel Alvarez-Arguedas ◽

Jorge L Galvan Rendiz ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Scavenger Receptor ◽

Cell Receptor ◽

The Body ◽

M Cells ◽

M Cell ◽

Type Vii Secretion ◽

Multiple Routes ◽

Mechanistic Basis ◽

Microfold Cells

Mycobacterium tuberculosis (Mtb) can enter the body through multiple routes, including via specialized transcytotic cells called microfold cells (M cell). However, the mechanistic basis for M cell entry remains undefined. Here, we show that M cell transcytosis depends on the Mtb Type VII secretion machine and its major virulence factor EsxA. We identify scavenger receptor B1 (SR-B1) as an EsxA receptor on airway M cells. SR-B1 is required for Mtb binding to and translocation across M cells in mouse and human tissue. Together, our data demonstrate a previously undescribed role for Mtb EsxA in mucosal invasion and identify SR-B1 as the airway M cell receptor for Mtb.

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