scholarly journals A201 UNDERSTANDING THE INFECTION DYNAMICS OF MYCOBACTERIUM PARATUBERCULOSIS (MAP)

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)

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

2018 ◽  
Vol 215 (2) ◽  
pp. 501-519 ◽  
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.

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

2021 ◽  
Vol 22 (17) ◽  
pp. 9355
Author(s):  
Joel Johnson George ◽  
Laura Martin-Diaz ◽  
Markus J. T. Ojanen ◽  
Rosa Gasa ◽  
Marko Pesu ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Mucosal Immunity ◽  
M Cells ◽  
M Cell ◽  
Polycomb Repressive Complex 2 ◽  
Salmonella Infections ◽  
Epigenetic Regulator ◽  
Development And Differentiation ◽  
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, 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.

scholarly journals Transcytosis of Murine-Adapted Bovine Spongiform Encephalopathy Agents in an In Vitro Bovine M Cell Model

2010 ◽  
Vol 84 (23) ◽  
pp. 12285-12291 ◽  
Author(s):  
Kohtaro Miyazawa ◽  
Takashi Kanaya ◽  
Ikuro Takakura ◽  
Sachi Tanaka ◽  
Tetsuya Hondo ◽  
...  
Keyword(s):  
Bovine Spongiform Encephalopathy ◽  
Cell Model ◽  
Transmissible Spongiform Encephalopathies ◽  
Epithelial Cell Line ◽  
Spongiform Encephalopathy ◽  
M Cells ◽  
Intestinal Epithelial ◽  
M Cell ◽  
Spongiform Encephalopathies

ABSTRACT Transmissible spongiform encephalopathies (TSE), including bovine spongiform encephalopathy (BSE), are fatal neurodegenerative disorders in humans and animals. BSE appears to have spread to cattle through the consumption of feed contaminated with BSE/scrapie agents. In the case of an oral infection, the agents have to cross the gut-epithelial barrier. We recently established a bovine intestinal epithelial cell line (BIE cells) that can differentiate into the M cell type in vitro after lymphocytic stimulation (K. Miyazawa, T. Hondo, T. Kanaya, S. Tanaka, I. Takakura, W. Itani, M. T. Rose, H. Kitazawa, T. Yamaguchi, and H. Aso, Histochem. Cell Biol. 133:125-134, 2010). In this study, we evaluated the role of M cells in the intestinal invasion of the murine-adapted BSE (mBSE) agent using our in vitro bovine intestinal epithelial model. We demonstrate here that M cell-differentiated BIE cells are able to transport the mBSE agent without inactivation at least 30-fold more efficiently than undifferentiated BIE cells in our in vitro model. As M cells in the follicle-associated epithelium are known to have a high ability to transport a variety of macromolecules, viruses, and bacteria from gut lumen to mucosal immune cells, our results indicate the possibility that bovine M cells are able to deliver agents of TSE, not just the mBSE agent.

scholarly journals BHLHE40 Regulates IL-10 and IFN-γ Production in T Cells but Does Not Interfere With Human Type 1 Regulatory T Cell Differentiation

2021 ◽  
Vol 12 ◽  
Author(s):  
Molly Javier Uyeda ◽  
Robert A. Freeborn ◽  
Brandon Cieniewicz ◽  
Rosa Romano ◽  
Ping (Pauline) Chen ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
Ex Vivo ◽  
Surface Expression ◽  
Surface Molecules ◽  
Ifn Γ ◽  
And Function ◽  
Tr1 Cells

Type 1 regulatory T (Tr1) cells are subset of peripherally induced antigen-specific regulatory T cells. IL-10 signaling has been shown to be indispensable for polarization and function of Tr1 cells. However, the transcriptional machinery underlying human Tr1 cell differentiation and function is not yet elucidated. To this end, we performed RNA sequencing on ex vivo human CD49b+LAG3+ Tr1 cells. We identified the transcription factor, BHLHE40, to be highly expressed in Tr1 cells. Even though Tr1 cells characteristically produce high levels of IL-10, we found that BHLHE40 represses IL-10 and increases IFN-γ secretion in naïve CD4+ T cells. Through CRISPR/Cas9-mediated knockout, we determined that IL10 significantly increased in the sgBHLHE40-edited cells and BHLHE40 is dispensable for naïve CD4+ T cells to differentiate into Tr1 cells in vitro. Interestingly, BHLHE40 overexpression induces the surface expression of CD49b and LAG3, co-expressed surface molecules attributed to Tr1 cells, but promotes IFN-γ production. Our findings uncover a novel mechanism whereby BHLHE40 acts as a regulator of IL-10 and IFN-γ in human CD4+ T cells.

scholarly journals Treatment with Exogenous Trypsin Expands In Vitro Cellular Tropism of the Avian Coronavirus Infectious Bronchitis Virus

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1102
Author(s):  
Phoebe Stevenson-Leggett ◽  
Sarah Keep ◽  
Erica Bickerton
Keyword(s):  
Cell Culture ◽  
Infectious Bronchitis Virus ◽  
Ex Vivo ◽  
Serial Passage ◽  
In Ovo ◽  
Infectious Bronchitis ◽  
Protease Treatment ◽  
Cellular Tropism ◽  
Passage Cell

The Gammacoronavirus infectious bronchitis virus (IBV) causes a highly contagious and economically important respiratory disease in poultry. In the laboratory, most IBV strains are restricted to replication in ex vivo organ cultures or in ovo and do not replicate in cell culture, making the study of their basic virology difficult. Entry of IBV into cells is facilitated by the large glycoprotein on the surface of the virion, the spike (S) protein, comprised of S1 and S2 subunits. Previous research showed that the S2′ cleavage site is responsible for the extended tropism of the IBV Beaudette strain. This study aims to investigate whether protease treatment can extend the tropism of other IBV strains. Here we demonstrate that the addition of exogenous trypsin during IBV propagation in cell culture results in significantly increased viral titres. Using a panel of IBV strains, exhibiting varied tropisms, the effects of spike cleavage on entry and replication were assessed by serial passage cell culture in the presence of trypsin. Replication could be maintained over serial passages, indicating that the addition of exogenous protease is sufficient to overcome the barrier to infection. Mutations were identified in both S1 and S2 subunits following serial passage in cell culture. This work provides a proof of concept that exogenous proteases can remove the barrier to IBV replication in otherwise non-permissive cells, providing a platform for further study of elusive field strains and enabling sustainable vaccine production in vitro.

scholarly journals 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

2011 ◽  
Vol 79 (11) ◽  
pp. 4716-4729 ◽  
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.

scholarly journals Identifying human and murine M cells in vitro

2019 ◽  
Vol 244 (7) ◽  
pp. 554-564 ◽  
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.

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

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.

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

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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