M-like Cells Research Articles

Alginate-Based Oral Delivery Systems to Enhance Protection, Release, and Absorption of Catalase.

Oxidative stress, overproduction of reactive oxygen species (ROS), plays an important role in the development of inflammatory bowel diseases. Catalase has great therapeutic potential by scavenging hydrogen peroxide, one of the ROSs produced in cellular metabolisms. However, in vivo application to scavenge ROS is currently limited especially in oral administrations. Here, we introduced an alginate-based oral drug delivery system that effectively protected catalase from the simulated harsh conditions of the gastrointestinal (GI) tract, released it in the small intestine mimicked condition, and enhanced its absorption via M cells, highly specialized epithelium cells in the small intestine. First of all, catalase was encapsulated in alginate-based microparticles with different amounts of polygalacturonic acid or pectin, which achieved an encapsulation efficiency of more than 90%. It was further shown that catalase was released from alginate-based microparticles in a pH-dependent manner. Results indicated that alginate-polygalacturonic acid microparticles (60 wt % Alg:40 wt % Gal) released 79.5 ± 2.4% of encapsulated catalase at pH 9.1 in 3 h, while they only released 9.2 ± 1.5% of encapsulated catalase at pH 2.0. Even when catalase was encapsulated in microparticles (60 wt % Alg:40 wt % Gal) and exposed to pH 2.0 followed by pH 9.1, it still retained 81.0 ± 11.3% enzyme activity compared to that in microparticles prior to the pH treatment. We then investigated the efficiency of RGD conjugation to catalase on the catalase uptake by M-like cells, the coculturing of human epithelial colorectal adenocarcinoma; Caco-2 cells and B lymphocyte; Raji cells. RGD-catalase protected M-cells more efficiently from the cytotoxicity of H2O2, a typical ROS. RGD conjugation to catalase enhanced the uptake by M-cells with 87.6 ± 0.8% RGD-catalase, whereas 11.5 ± 9.2% of RGD-free catalase passed across M-cells. From the results of protection, release, and absorption of model therapeutic proteins from the harsh pH conditions, alginate-based oral drug delivery systems will have numerous applications for the controlled release of drugs that are easily degradable in the GI tract.

Effect of Lentinan on Peyer's patch structure and function in an immunosuppressed mouse model

Lentinan (LNT), a polysaccharide isolated from Lentinus edodes, has been shown to stimulate immune response. Despite its widespread use owing to its health benefits, epidemiologic and experimental studies that address the biological activities of LNT after oral administration to animals or humans are rare. In this study, the effects of LNT on the intestinal mucosal immune system of immunosuppressed mice were investigated. The number and size of the Peyer's patches (PPs), proliferation ability of PP lymphocytes, T and B lymphocyte percentage, and T-cell activation proportion, as well as the number of M-like cells in PPs, were determined. The antigen transfer ability of M-like cells and intestinal secretory immunoglobulin A (IgA) levels were also detected. After oral administration of LNT for 7 days, the number of PPs, lymphocytes in PPs, and the level of intestinal soluble IgA in immunosuppressed mice were increased. LNT maintained the percentage of T and B lymphocytes and upregulated the proportion of activated T cells in PPs. Furthermore, the number of M-like cells, which were differentiated from intestinal epithelial cells, and their antigen transfer ability were enhanced. These results indicate that orally administered LNT can improve the immune status of the intestinal mucosa in immunosuppressed mice.

A comparison of three Peyer's patch "M-like" cell culture models: particle uptake, bacterial interaction, and epithelial histology.

Intestinal Peyer's patch (PP) microfold (M) cells transport microbes and particulates across the follicle-associated epithelium (FAE) as part of the mucosal immune surveillance system. In vitro human M-like cell co-culture models are used as screens to investigate uptake of antigens-in-nanoparticles, but the models are labour-intensive and there is inter-laboratory variability. We compared the three most established filter-grown Caco-2/Raji B cell co-culture systems. These were Model A (Kernéis et al., 1997), Model B (Gullberg et al., 2000), and Model C (Des Rieux et al. 2007). The criteria used were transepithelial resistance (TEER), the apparent permeability coefficient (Papp) of [14C]-mannitol, M cell-like histology, as well as latex particle and Salmonella typhimurium translocation. Each co-culture model displayed substantial increases in particle translocation. Truncated microvilli compared to mono-cultures was their most consistent feature. The inverted model developed by des Rieux et al. (2007) displayed reductions in TEER and an increased (Papp), accompanied by the largest increase in particle translocation compared to the other two models. The normally-oriented model developed by Gullberg et al. (2000) was the only one to consistently display an increased translocation of Salmonella typhimurium. By applying a double Matrigel™ coating on filters, altering the medium feeding regime for Raji B cells, and restricting the passage number of B cells, improvements to the Gullberg model B were achieved, as reflected by increased particle translocation and improved histology. In conclusion, this is the first time all three designs have been compared in one study and each displays phenotypic features of M-like cells. While Model C was the most robust co-culture, the Model B protocol could be improved by optimizing several variables and is less complicated to establish than the two inverted models.

Escherichia albertii, a novel human enteropathogen, colonizes rat enterocytes and translocates to extra-intestinal sites.

Diarrhea is the second leading cause of death of children up to five years old in the developing countries. Among the etiological diarrheal agents are atypical enteropathogenic Escherichia coli (aEPEC), one of the diarrheagenic E. coli pathotypes that affects children and adults, even in developed countries. Currently, genotypic and biochemical approaches have helped to demonstrate that some strains classified as aEPEC are actually E. albertii, a recently recognized human enteropathogen. Studies on particular strains are necessary to explore their virulence potential in order to further understand the underlying mechanisms of E. albertii infections. Here we demonstrated for the first time that infection of fragments of rat intestinal mucosa is a useful tool to study the initial steps of E. albertii colonization. We also observed that an E. albertii strain can translocate from the intestinal lumen to Mesenteric Lymph Nodes and liver in a rat model. Based on our finding of bacterial translocation, we investigated how E. albertii might cross the intestinal epithelium by performing infections of M-like cells in vitro to identify the potential in vivo translocation route. Altogether, our approaches allowed us to draft a general E. albertii infection route from the colonization till the bacterial spreading in vivo.

Abstract 2320: CD70 immune checkpoint ligand is associated with the epithelial-to-mesenchymal transition in non-small cell lung cancer

Abstract Non-small-cell lung cancers (NSCLC) account for 85% of lung cancers and are mostly diagnosed at advanced stage. Drugs interrupting immune checkpoints, such as anti-CTLA-4, anti-PD-1, anti-PD-L1, have proven to unleash anti-tumor immunity and mediate durable cancer regressions in a portion of patients with NSCLC. Epithelial-to-mesenchymal transition (EMT) is a developmental process that enables reprogramming of polarized epithelial cells towards a mesenchymal phenotype with migratory and invasive properties. EMT has been involved in escape from oncogenic-induced senescence, resistance to chemotherapy and development of metastatic disease. Moreover, EMT promotes cancer cell plasticity and favors their adaptability to selective pressures. Here we made use of in silico approaches and further in vivo validation to evaluate the potential role of EMT as a major escape mechanism to tumor immunosurveillance in NSCLC. We performed an initial in silico analysis to assess the expression of immune checkpoint ligands (ICPLs) in 129 NSCLC cell lines (CCLE), in relation to their EMT status defined by a 72-gene EMT signature that was previously reported in NSCLC. Unsupervised hierarchical clustering analysis revealed that the expression of selected ICPLs was associated with the mesenchymal or epithelial phenotype. In particular, CD70, a member of the tumor necrosis factor superfamily, was significantly associated with the mesenchymal status (p < 0.001). We identified an E-box sequence (CANNTG) in CD70 gene promoter thus suggesting the possible regulation of CD70 by ZEB1 and/or SNAI. We extended the in silico analysis to a set of 488 adenocarcinomas (ADC) and 501 squamous cell carcinomas (SCC) from the TCGA and confirmed that CD70 expression was associated with a mesenchymal status (q-value <0.001). In a set of E-like (H441 and H1650) and M-like (H23 and HCC44) NSCLC cell lines we validated the association between an increased level of CD70 by flow cytometry and the mesenchymal status. Expression of CD70 by immunohistochemistry was observed in 5/51 lung ADC (10%), 6/45 (13%) lung SCC, 16/26 (63%) pulmonary sarcomatoid carcinomas and 2/10 (20%) small cell lung carcinomas. Interestingly, in pulmonary sarcomatoid carcinomas, CD70 expression was closely associated with the mesenchymal component. Our results suggest an association between the expression of CD70 and the mesenchymal phenotype, thus shedding light on the potential role of CD70 in immune escape of a subset of NSCLC. Citation Format: Sandra Ortiz-Cuaran, Aurélie Swalduz, Maria A. Albaret, Christine Menetrier-Caux, Véronique Haddad, Arnaud Paré, Geneviève De Souza, Anne P. Morel, Maurice Pérol, Christophe Caux, Sylvie Lantuejoul, Alain Puisieux, Pierre Saintigny. CD70 immune checkpoint ligand is associated with the epithelial-to-mesenchymal transition in non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2320.

A 3D in vitro model to explore the inter-conversion between epithelial and mesenchymal states during EMT and its reversion.

Epithelial-to-mesenchymal transitions (EMT) are strongly implicated in cancer dissemination. Intermediate states, arising from inter-conversion between epithelial (E) and mesenchymal (M) states, are characterized by phenotypic heterogeneity combining E and M features and increased plasticity. Hybrid EMT states are highly relevant in metastatic contexts, but have been largely neglected, partially due to the lack of physiologically-relevant 3D platforms to study them. Here we propose a new in vitro model, combining mammary E cells with a bioengineered 3D matrix, to explore phenotypic and functional properties of cells in transition between E and M states. Optimized alginate-based 3D matrices provided adequate 3D microenvironments, where normal epithelial morphogenesis was recapitulated, with formation of acini-like structures, similar to those found in native mammary tissue. TGFβ1-driven EMT in 3D could be successfully promoted, generating M-like cells. TGFβ1 removal resulted in phenotypic switching to an intermediate state (RE cells), a hybrid cell population expressing both E and M markers at gene/protein levels. RE cells exhibited increased proliferative/clonogenic activity, as compared to M cells, being able to form large colonies containing cells with front-back polarity, suggesting a more aggressive phenotype. Our 3D model provides a powerful tool to investigate the role of the microenvironment on metastable EMT stages.

Surface-modified yeast cells: A novel eukaryotic carrier for oral application

The effective targeting and subsequent binding of particulate carriers to M cells in Peyer's patches of the gut is a prerequisite for the development of oral delivery systems. We have established a novel carrier system based on cell surface expression of the β1-integrin binding domain of invasins derived from Yersinia enterocolitica and Yersinia pseudotuberculosis on the yeast Saccharomyces cerevisiae. All invasin derivatives were shown to be effectively expressed on the cell surface and recombinant yeast cells showed improved binding to both human HEp-2 cells and M-like cells in vitro. Among the different derivatives tested, the integrin-binding domain of Y. enterocolitica invasin proved to be the most effective and was able to target Peyer's patches in vivo. In conclusion, cell surface-modified yeasts might provide a novel bioadhesive, eukaryotic carrier system for efficient and targeted delivery of either antigens or drugs via the oral route.

In vitro M-like cells genesis through a tissue-engineered triple-culture intestinal model.

Although fewer in number, M-cells are considered antigen sampling cells, acting as a gateway for antigens from the gut lumen and presenting an impressive aptitude for particle transcytosis. These features make M-cells attractive targets for oral drug delivery studies, but this has been poorly explored. New and reproducible tissue-like in vitro models for studying intestinal sampling and permeability mechanisms are needed. The combination of different cell players in such models offers improved microenvironments with higher physiologic relevance. Here, a tissue-engineered model was established, by co-culturing Caco-2 absorptive cells, HT29-MTX mucus-producing cells and Raji B lymphocytes. After 3 weeks of cell co-culture, the presence of M-like cells was evidenced by the loss of brush-border organization, detected by the lack of microvilli. The triple-culture model showed to be efficient for insulin transport, a process that was influenced by the tightness of junctions between epithelial cells and the presence of mucus and M-like cells. Ultimately, the proposed tissue-engineered model provides a more complete and reliable tool to perform drug permeability tests, as compared to traditional models, and may also find applicability as an in vitro system to study transdifferentiation mechanisms of M cells.

30 Role of Long Polar Fimbriae in the Interactions of Enterohemorrhagic Escherichia coli With Peyer's Patches and Inhibition by a Probiotic Yeast

Enterohemorrhagic Escherichia coli (EHEC) are food-borne pathogens associated with diarrhea, hemorrhagic colitis and life-threatening complications such as hemolytic-uremic syndrome. EHEC interact with the Follicule-Associated Epithelium (FAE) of Peyer's patches (PPs) of the distal ileum in humans and translocate across the intestinal epithelium via M cells. Molecular mechanisms are still unknown but Long Polar Fimbriae (LPF), which contribute to intestinal colonization, may be involved. Currently no specific treatment is available in EHEC infections and use of antibiotics remains controversial. Probiotic could be an alternative strategy. The objectives of the study were to investigate the role of LPF in EHEC tropism to PPs, and to explore the influence of a probiotic yeast, Saccharomyces cerevisiae, on EHEC interactions with intestinal mucosa. The expression of lpf genes (encoded by two lpf operons) of EHEC O157:H7 strain EDL933 was analyzed using in vitro models of the human upper GI tract and large intestine. To investigate the involvement of LPF in the ability of EDL933 to target PPs, we generated the D lpfA1,DlpfA2, DlpfA1-DlpfA2 isogenic mutants and trans-complemented them with lpf genes. LPF interaction with M-like cells was investigated using an in vitro model of specialized M cells. In vivo interactions of EHEC with murine PPs were analyzed in ileal loop assays. Mice were infected with a mixture of two bacterial strains, and the numbers of PPs-interacting bacteria were determined using a competitive index analysis. To investigate the effect of S. cerevisiae, mice were given the probiotic for 7 days before ileal loops assays were conducted with O157:H7 wild type. Lpf isogenic mutants (i) were not able to interact with ileal biopsies containing PPs compared to the wild type strain in competitive colonization assays and (ii) translocated across M cells at levels significantly lower than those observed for the wild type strain. Trans-complementation of the mutants with the cloned lpf genes restored their ability to interact with PPs and M cells, indicating that expression of lpfA1 or/and lpfA2 genes is required for interactions with PPs. Regarding probiotic strategy, S. cerevisiae exerts a trophic effect on the intestinal mucosa. Bloodshot PPs were macroscopically observed following EHEC infection of murine ileal loops. We showed that pre-treatment with yeast significantly inhibited O157:H7 interactions with PPs and reduced the number of hemorrhagic PPs in murine ileal loops. Yeast cell surface are rich in mannose and the role of such carbohydrates in inhibiting EHEC interactions with PPs will be investigated. We conclude that LPF are involved in the interactions of EHEC with murine PPs and are needed for an active translocation across M cell monolayer. Tropism of EHEC to PPs can be limited by a probiotic S. cerevisiae strain.

The IbeA invasin of adherent-invasive Escherichia coli mediates interaction with intestinal epithelia and macrophages.

Adherent-invasive Escherichia coli (AIEC) pathogroup isolates are a group of isolates from the intestinal mucosa of Crohn's disease patients that can invade intestinal epithelial cells (IECs) or macrophages and survive and/or replicate within. We have identified the ibeA gene in the genome of AIEC strain NRG857c and report the contribution of IbeA to the interaction of AIEC with IECs and macrophages and colonization of the mouse intestine. An ibeA deletion mutant strain (NRG857cΔibeA) was constructed, and the in vitro effect on AIEC adhesion and invasion of nonpolarized and polarized Caco-2 cells, the adhesion and transcytosis of M-like cells, the intracellular survival in THP-1 macrophages, and the contribution to intestinal colonization of the CD-1 murine model of infection were evaluated. A significant reduction in invasion was observed with the ibeA mutant in Caco-2 and M-like cells, whereas adhesion was not affected. Complementation of the mutant reestablished Caco-2 invasive phenotype to wild-type levels. Reduction in invasion did not significantly affect transcytosis through M-like cells at early time points. The absence of ibeA significantly affected AIEC intramacrophage survival up to 24 h postinfection. No significant changes associated with IbeA were found in AIEC colonization across the murine gastrointestinal tract, but a slight reduction of gamma interferon was observed in the ceca of mice infected with the ibeA mutant. In addition, a decrease in the pathology scores was observed in the ilea and ceca of mice infected with the ibeA mutant. Our data support the function of IbeA in the AIEC invasion process, macrophage survival, and inflammatory response in the murine intestine.

Oral and Anal Vaccination Confers Full Protection against Enteric Redmouth Disease (ERM) in Rainbow Trout

The effect of oral vaccines against bacterial fish diseases has been a topic for debate for decades. Recently both M-like cells and dendritic cells have been discovered in the intestine of rainbow trout. It is therefore likely that antigens reaching the intestine can be taken up and thereby induce immunity in orally vaccinated fish. The objective of this project was to investigate whether oral and anal vaccination of rainbow trout induces protection against an experimental waterborne infection with the pathogenic enterobacteria Yersinia ruckeri O1 biotype 1 the causative agent of enteric redmouth disease (ERM). Rainbow trout were orally vaccinated with AquaVac ERM Oral (MERCK Animal Health) or an experimental vaccine bacterin of Y. ruckeri O1. Both vaccines were tested with and without a booster vaccination four months post the primary vaccination. Furthermore, two groups of positive controls were included, one group receiving the experimental oral vaccine in a 50 times higher dose, and the other group receiving a single dose administered anally in order to bypass the stomach. Each group was bath challenged with 6.3×108 CFU/ml Y. ruckeri, six months post the primary vaccination. The challenge induced significant mortality in all the infected groups except for the groups vaccinated anally with a single dose or orally with the high dose of bacterin. Both of these groups had 100% survival. These results show that a low dose of Y. ruckeri bacterin induces full protection when the bacterin is administered anally. Oral vaccination also induces full protection, however, at a dose 50 times higher than if the fish were to be vaccinated anally. This indicates that much of the orally fed antigen is digested in the stomach before it reaches the second segment of the intestine where it can be taken up as immunogenic antigens and presented to lymphocytes.

Murine Norovirus Transcytosis across anIn VitroPolarized Murine Intestinal Epithelial Monolayer Is Mediated by M-Like Cells

Noroviruses (NoVs) are the causative agent of the vast majority of nonbacterial gastroenteritis worldwide. Due to the inability to culture human NoVs and the inability to orally infect a small animal model, little is known about the initial steps of viral entry. One particular step that is not understood is how NoVs breach the intestinal epithelial barrier. Murine NoV (MNV) is the only NoV that can be propagated in vitro by infecting murine macrophages and dendritic cells, making this virus an attractive model for studies of different aspects of NoV biology. Polarized murine intestinal epithelial mICcl2 cells were used to investigate how MNV interacts with and crosses the intestinal epithelium. In this in vitro model of the follicle-associated epithelium (FAE), MNV is transported across the polarized cell monolayer in the absence of viral replication or disruption of tight junctions by a distinct epithelial cell with microfold (M) cell properties. In addition to transporting MNV, these M-like cells also transcytose microbeads and express an IgA receptor. Interestingly, B myeloma cells cultured in the basolateral compartment underlying the epithelial monolayer did not alter the number of M-like cells but increased their transcytotic activity. Our data demonstrate that MNV can cross an intact intestinal epithelial monolayer in vitro by hijacking the M-like cells' intrinsic transcytotic pathway and suggest a potential mechanism for MNV entry into the host.

Pathogenesis of Human Enterovirulent Bacteria: Lessons from Cultured, Fully Differentiated Human Colon Cancer Cell Lines

Hosts are protected from attack by potentially harmful enteric microorganisms, viruses, and parasites by the polarized fully differentiated epithelial cells that make up the epithelium, providing a physical and functional barrier. Enterovirulent bacteria interact with the epithelial polarized cells lining the intestinal barrier, and some invade the cells. A better understanding of the cross talk between enterovirulent bacteria and the polarized intestinal cells has resulted in the identification of essential enterovirulent bacterial structures and virulence gene products playing pivotal roles in pathogenesis. Cultured animal cell lines and cultured human nonintestinal, undifferentiated epithelial cells have been extensively used for understanding the mechanisms by which some human enterovirulent bacteria induce intestinal disorders. Human colon carcinoma cell lines which are able to express in culture the functional and structural characteristics of mature enterocytes and goblet cells have been established, mimicking structurally and functionally an intestinal epithelial barrier. Moreover, Caco-2-derived M-like cells have been established, mimicking the bacterial capture property of M cells of Peyer's patches. This review intends to analyze the cellular and molecular mechanisms of pathogenesis of human enterovirulent bacteria observed in infected cultured human colon carcinoma enterocyte-like HT-29 subpopulations, enterocyte-like Caco-2 and clone cells, the colonic T84 cell line, HT-29 mucus-secreting cell subpopulations, and Caco-2-derived M-like cells, including cell association, cell entry, intracellular lifestyle, structural lesions at the brush border, functional lesions in enterocytes and goblet cells, functional and structural lesions at the junctional domain, and host cellular defense responses.

C5a receptor-targeting ligand-mediated delivery of dengue virus antigen to M cells evokes antigen-specific systemic and mucosal immune responses in oral immunization

Oral mucosal immunization is a feasible and economic vaccination strategy. In order to achieve a successful oral mucosal vaccination, antigen delivery to gut immune inductive site and avoidance of oral tolerance induction should be secured. One promising approach is exploring the specific molecules expressed on the apical surfaces of M cells that have potential for antigen uptake and immune stimulation. We previously identified complement 5a receptor (C5aR) expression on human M-like cells and mouse M cells and confirmed its non-redundant role as a target receptor for antigen delivery to M cells using a model antigen. Here, we applied the OmpH ligand, which is capable of targeting the ligand-conjugated antigen to M cells to induce specific mucosal and systemic immunities against the EDIII of dengue virus (DENV). Oral immunization with the EDIII–OmpH efficiently targeted the EDIII to M cells and induced EDIII-specific immune responses comparable to those induced by co-administration of EDIII with cholera toxin (CT). Also, the enhanced responses by OmpH were characterized as Th2-skewed responses. Moreover, oral immunization using EDIII–OmpH did not induce systemic tolerance against EDIII. Collectively, we suggest that OmpH-mediated targeting of antigens to M cells could be used for an efficient oral vaccination against DENV infection.

Mechanism for exploiting the complement 5a anaphylatoxin receptor on M cells by mucosal pathogen is closely related with that of mucosal immune induction (P3212)

Abstract The mucosal epithelium is exposed to enormous amount of microorganisms containing commensal and pathogenic bacteria. Although many pathogens initiate their infection in mucosal epithelium and/or M cells of Peyer’s patch, detailed mechanism for the infection is poorly understood. We explored the possible survival mechanism of Yersinia enterocolitica in M cells in relation to crosstalk between C5aR and TLRs based on the observation that outermembrane protein H (OmpH) of Y. enterocolitica is one of the ligands for C5aR. Localization of C5aR and TLRs in human M-like cells and mouse M cells was confirmed during Y. enterocolitica infection. Also, regulation of cAMP- and/or autophagy-associated gene expression by Y. enterocolitica infection was characterized. Moreover, C5aR antagonist treatment reduced the survival of Y. enterocolitica in human M-like cells and the bacteria showed low infection efficiency in C5aR knock-out mouse. It is suggested that the OmpH of Y. enterocolitica could be used as M cell-targeting ligand in oral mucosal vaccination model. Collectively, we assume that the inhibition of cAMP expression by interaction between Y. enterocolitica and both C5aR and TLRs is closely associated with the bacterial survival in M cells and this interaction could be used as targeting of oral mucosal vaccine.

In vivo murine and in vitro M-like cell models of gastrointestinal anthrax

Bacillus anthracis is the causative agent of anthrax and is acquired by three routes of infection: inhalational, gastrointestinal and cutaneous. Gastrointestinal (GI) anthrax is rare, but can rapidly result in severe, systemic disease that is fatal in 25%–60% of cases. Disease mechanisms of GI anthrax remain unclear due to limited numbers of clinical cases and the lack of experimental animal models. Here, we developed an in vivo murine model of GI anthrax where spore survival was maximized through the neutralization of stomach acid followed by an intragastric administration of a thiabendazole paste spore formulation. Infected mice showed a dose-dependent mortality rate and pathological features closely mimicking human GI anthrax. Since Peyer's patches in the murine intestine are the primary sites of B. anthracis growth, we developed a human M (microfold)-like-cell model using a Caco-2/Raji B-cell co-culturing system to study invasive mechanisms of GI anthrax across the intestinal epithelium. Translocation of B. anthracis spores was higher in M-like cells than Caco-2 monolayers, suggesting that M-like cells may serve as an initial entry site for spores. Here, we developed an in vivo murine model of GI anthrax and an in vitro M-like cell model that could be used to further our knowledge of GI anthrax pathogenesis.

Increased Translocation of Atypical Enteropathogenic Escherichia coli Through M-like cells cultured in vitro.

Event Abstract Back to Event Increased Translocation of Atypical Enteropathogenic Escherichia coli Through M-like cells cultured in vitro. Denise Yamamoto1* 1 University of Sao Paulo, UNIFESP (DMIP), Brazil Yamamoto, D.1; Hernandes, R.T.1, Abe, C. M.2; Gomes, T.A.T.1 1. UNIFESP (DMIP); Universidade Federal de São Paulo; Rua Botucatu, 862 cep 04023-062 - São Paulo, SP. 2. Instituto Butantan, Laboratório de Biologia Celular. Avenida Vital Brazil, 1.500 cep 05503-900 - São Paulo, SP - Brasil Enteropathogenic Escherichia coli (EPEC) is sub-grouped into typical (tEPEC) and atypical (aEPEC), based on the presence of the EPEC adherence factor plasmid in the former group. aEPEC strains are widely spread in children and adults in developed and developing countries. Previously, we showed that some aEPEC strains invade HeLa and polarized T84 intestinal cells cultivated in vitro, more efficiently than tEPEC prototype strain E2348/69. We also showed that pre-treatment of polarized T84 cell monolayers with Ethylenediamine tetraacetic acid significantly increased aEPEC strain 1551-2 (serotype O non-typable, non-motile) invasion, suggesting preferential penetration through the basolateral surface. Other pathogens, such as Shigella spp, can reach the enterocyte basolateral surface after crossing M cells. Furthermore, it has been shown that tEPEC E2348/69 adheres to but does not translocate M-like cells. In this study, we evaluated the ability of aEPEC 1551-2 strain to translocate through M-like cells obtained in vitro. To induce M-like differentiation, Caco-2 cells (105) were cultured on the upper chamber of Transwell membranes for 10 days; afterwards, Raji-B cells (106) were inoculated into the lower chamber and kept on culture for 6 days. Either Caco-2 or M-like cells were infected separately (107 UFC/filter) with aEPEC 1551-2 and tEPEC E2348/69 strains and incubated for 6 or 3 h, respectively. Aliquots of the media at the lower chamber were then collected and CFU were quantified by serial dilutions. Our data demonstrated that the presence of M-like cells promoted a significant increase of aEPEC 1551-2 translocation (from 119.6±75.7 to 2869±762.1, p=0.03), but not of tEPEC E2348/69 (from 2.3±2.3 to 27.8±16.5, p=0.18) as compared in Caco-2 and M-like cells, respectively. These data suggest that aEPEC 1551-2 might reach the basolateral surface of the enterocytes in vivo, after M cells translocation. Keywords: aEPEC 1551-2 strain, in vitro, M-like cells, translocation Conference: ECMIS - E. coli and the Mucosal Immune System : Interaction, Modulation and Vaccination, Ghent, Belgium, 2 Jul - 5 Jul, 2011. Presentation Type: Oral Presentation Topic: Infection and innate immunity, immunosupression and/or immunostimulation Citation: Yamamoto D (2012). Increased Translocation of Atypical Enteropathogenic Escherichia coli Through M-like cells cultured in vitro.. Front. Immunol. Conference Abstract: ECMIS - E. coli and the Mucosal Immune System : Interaction, Modulation and Vaccination. doi: 10.3389/conf.fimmu.2012.01.00001 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 07 Oct 2011; Published Online: 09 Jan 2012. * Correspondence: Dr. Denise Yamamoto, University of Sao Paulo, UNIFESP (DMIP), Sao Paulo, 04827-130, Brazil, dyamamoto@unifesp.br Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Denise Yamamoto Google Denise Yamamoto Google Scholar Denise Yamamoto PubMed Denise Yamamoto Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

M cells expressing the complement C5a receptor are efficient targets for mucosal vaccine delivery

In the mucosal immune system, M cells are known as specialized epithelial cells that take up luminal antigens, although the receptors on M cells and the mechanism of antigen uptake into M cells are not well-understood. Here, we report the expression of the complement C5a receptor (C5aR) on the apical surface of M cells. C5ar mRNA expression in co-cultured Caco-2 human M-like cells was six-fold higher than in mono-cultured cells. C5aR expression was detected together with glycoprotein 2, an M-cell-specific protein, on the apical surface of M-like cells and mouse Peyer's patch M cells. Interestingly, after oral administration of Yersinia enterocolitica which expresses outer membrane protein H (OmpH) that is homologous to the Skp α1 domain of Escherichia coli, a ligand of C5aR, dense clustering and phosphorylation of C5aR were detected in M cells. Finally, targeted antigen delivery to M cells using C5aR as a receptor was achieved using the OmpH α1 of Y. enterocolitica such that the induction of ligand-conjugated antigen-specific immune responses was confirmed in mice after oral immunization of the OmpH β1α1-conjugated antigen. Collectively, we identified C5aR expression on M cells and suggest that C5aR could be used as a target receptor for mucosal antigen delivery.

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

The EspF protein is secreted by the type III secretion system of enteropathogenic and enterohemorrhagic Escherichia 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. EspF(O127) is important for the inhibition of phagocytosis by EPEC and also limits EPEC translocation through antigen-sampling cells (M cells). EspF(O127) has 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 different espF alleles 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. The espF gene from E. coli serotype O157 (espF(O157)) allele was significantly less effective at inhibiting phagocytosis and also had reduced capacity to inhibit E. coli translocation through a human-derived in vitro M-cell coculture system in comparison to espF(O127) and espF(O26). In contrast, espF(O157) was 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.

Use of a gene expression signature related to epithelial-to-mesenchymal transition (EMT) to predict for overall survival (OS) in cohorts of lung and head and neck cancer (HNSCC) patients.

7010 Background: Improved predictions for survival and response to therapy will help to refine clinical care and potentially improve outcomes for patients with cancers of the upper aerodigestive tract. Methods: Gene expression analysis using three mesenchymal (M) and three epithelial (E) non-small cell lung cancer (NSCLC) cell lines identified 574 differentially expressed probes based on a false discovery rate (FDR) of 30%, which were then used to categorize a panel of 50 cell lines on the Illumina gene expression platform into 30 E-like and 20 M-like cell lines. 46 of these cell lines were analyzed with Affymetrix arrays, corresponding to 27 E-like and 19 M-like cell lines, and the top 4054 differentially expressed probes based on a FDR of 20% were filtered to retain probes with at least 2-fold average difference in expression. Overlap with the Director’s Consortium (DC) of mostly early stage lung adenocarcinoma patients treated with surgery and adjuvant therapy as appropriate resulted in 991 probes, which were then ranked using a survival-based Cox regression model with patients combined from the UM and HLM cohorts in the DC. The top 112 probes based on a FDR of 25% were then used in a supervised PCA analysis and two-evenly divided groups of patients were compared using Kaplan-Meier survival analysis. The 112 probe signature was independently tested in the DC MSK cohort and a cohort of surgical-pathologic high-risk HNSCC patients treated with surgery and post-operative radiotherapy. Results: Gene expression related to EMT is able to identify two highly different E-like and M-like groups of cell lines. This cell-line derived and clinically refined EMT-related gene expression signature predicted for OS in an independent cohort in the DC (p = 0.0016) as well as in HNSCC patients (p = 0.003). Conclusions: A gene expression signature related to EMT predicts for OS in lung cancer and HNSCC patients. Further prospective validation will be needed to verify these results. In addition, this signature distinguishes between unique biological phenotypes and therefore may also be significant in predicting response to specific therapeutic interventions.