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Perry SS, Brice DC, Sakr AA, Kandeil A, DeBeauchamp J, Ghonim M, Jones J, Miller L, Vegesana K, Crawford JC, Langfitt DM, Kercher L, Abdelsamed HA, Webster RG, Thomas PG, Webby RJ, Okda FA. Modulation of cytokeratin and cytokine/chemokine expression following influenza virus infection of differentiated human tonsillar epithelial cells. J Virol 2025; 99:e0146024. [PMID: 39791909 PMCID: PMC11852761 DOI: 10.1128/jvi.01460-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2024] [Accepted: 12/08/2024] [Indexed: 01/12/2025] Open
Abstract
The tonsils have been identified as a site of replication for Epstein-Barr virus, adenovirus, human papillomavirus, and other respiratory viruses. Human tonsil epithelial cells (HTECs) are a heterogeneous group of actively differentiating cells. Here, we investigated the cellular features and susceptibility of differentiated HTECs to specific influenza viruses, including expression of avian-type and mammalian-type sialic acid (SA) receptors, viral replication dynamics, and the associated cytokine secretion profiles. We found that differentiated HTECs possess more abundant α2,3-linked SA (preferentially bound by avian influenza viruses) than α2,6-linked SA (preferentially bound by mammalian strains). This dual receptor expression suggests a role in influenza virus adaptation and tropism within the tonsils by facilitating the binding and entry of multiple influenza virus strains. Our results indicated the susceptibility of differentiated HTECs to a wide range of influenza viruses from human, swine, and avian hosts. Virus production for most strains was detected as early as 1 day post-infection (dpi), and typically peaked by 3 dpi. However, pandemic H1N1 virus showed remarkably delayed replication kinetics that did not peak until at least 7 dpi. Notably, influenza virus infection impacted the expression of cytokeratins in HTEC cultures, which correlated with altered cytokine secretion patterns. These patterns varied within the strains but were most distinct in swine H3N2 infection. In conclusion, differentiated HTECs exhibited a strain-specific pattern of influenza virus replication and innate immune responses that included changes in cytokeratin and cytokine expression. These studies shed light on the complex interplay between influenza viruses and host cells in the tonsils. IMPORTANCE To develop effective interventions against influenza, it is important to identify host factors affecting pathogenesis and immune responses. Tonsils are lymphoepithelial organs characterized by infiltration of B and T lymphocytes into the squamous epithelium of tonsillar crypts, beneath which germinal centers play key roles in antigen processing and the immune response. Influenza virus tropism in the human upper respiratory tract is a key determinant of host-range, pathogenesis, and transmission. Accordingly, experimental models using primary cells from the human respiratory tract are relevant for assessing virus tropism and replication competence. Our study addresses the dynamics of influenza virus replication in HTECs, including cellular tropism, infectivity, and cytokeratin and cytokine expression. The results of this study highlight the complex interplay between structural proteins and immune signaling pathways, all of which provide valuable insights into host-virus interactions.
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Affiliation(s)
- S. Scott Perry
- Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA
| | - David C. Brice
- Department of Host-Microbe Interactions, St Jude Children's Research Hospital, Memphis, Tennessee, USA
| | - Ahmed Atef Sakr
- Cornell Veterinary Biobank, Cornell University College of Veterinary Medicine, Ithaca, New York, USA
| | - Ahmed Kandeil
- Department of Host-Microbe Interactions, St Jude Children's Research Hospital, Memphis, Tennessee, USA
- National Research Center, Giza, Egypt
| | - Jennifer DeBeauchamp
- Department of Host-Microbe Interactions, St Jude Children's Research Hospital, Memphis, Tennessee, USA
| | - Mohamed Ghonim
- Department of Host-Microbe Interactions, St Jude Children's Research Hospital, Memphis, Tennessee, USA
| | - Jeremy Jones
- Department of Host-Microbe Interactions, St Jude Children's Research Hospital, Memphis, Tennessee, USA
| | - Lance Miller
- Department of Host-Microbe Interactions, St Jude Children's Research Hospital, Memphis, Tennessee, USA
| | - Kasi Vegesana
- Department of Host-Microbe Interactions, St Jude Children's Research Hospital, Memphis, Tennessee, USA
| | - Jeremy Chase Crawford
- Department of Host-Microbe Interactions, St Jude Children's Research Hospital, Memphis, Tennessee, USA
| | - Deanna M. Langfitt
- Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA
| | - Lisa Kercher
- Department of Host-Microbe Interactions, St Jude Children's Research Hospital, Memphis, Tennessee, USA
| | - Hossam A. Abdelsamed
- Immunology Center of Georgia (IMMCG), Department of Physiology, Medical College of Georgia (MCG), Augusta University, Augusta, Georgia, USA
| | - Robert G. Webster
- Department of Host-Microbe Interactions, St Jude Children's Research Hospital, Memphis, Tennessee, USA
| | - Paul G. Thomas
- Department of Host-Microbe Interactions, St Jude Children's Research Hospital, Memphis, Tennessee, USA
| | - Richard J. Webby
- Department of Host-Microbe Interactions, St Jude Children's Research Hospital, Memphis, Tennessee, USA
| | - Faten A. Okda
- Department of Host-Microbe Interactions, St Jude Children's Research Hospital, Memphis, Tennessee, USA
- National Research Center, Giza, Egypt
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Miguelena Chamorro B, Hameed SA, Dechelette M, Claude JB, Piney L, Chapat L, Swaminathan G, Poulet H, Longet S, De Luca K, Mundt E, Paul S. Characterization of Canine Peyer's Patches by Multidimensional Analysis: Insights from Immunofluorescence, Flow Cytometry, and Single-Cell RNA Sequencing. Immunohorizons 2023; 7:788-805. [PMID: 38015460 PMCID: PMC10696420 DOI: 10.4049/immunohorizons.2300091] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2023] [Accepted: 11/06/2023] [Indexed: 11/29/2023] Open
Abstract
The oral route is effective and convenient for vaccine administration to stimulate a protective immune response. GALT plays a crucial role in mucosal immune responses, with Peyer's patches (PPs) serving as the primary site of induction. A comprehensive understanding of the structures and functions of these structures is crucial for enhancing vaccination strategies and comprehending disease mechanisms; nonetheless, our current knowledge of these structures in dogs remains incomplete. We performed immunofluorescence and flow cytometry studies on canine PPs to identify cell populations and structures. We also performed single-cell RNA sequencing (scRNA-seq) to investigate the immune cell subpopulations present in PPs at steady state in dogs. We generated and validated an Ab specifically targeting canine M cells, which will be a valuable tool for elucidating Ag trafficking into the GALT of dogs. Our findings will pave the way for future studies of canine mucosal immune responses to oral vaccination and enteropathies. Moreover, they add to the growing body of knowledge in canine immunology, further expanding our understanding of the complex immune system of dogs.
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Affiliation(s)
- Beatriz Miguelena Chamorro
- Centre International de Recherche en Infectiologie, Team GIMAP (Saint-Etienne), Université Claude Bernard Lyon 1, Inserm, U1111, CNRS, UMR5308, ENS Lyon, UJM, F69007 Lyon, France
- Global Innovation, Boehringer Ingelheim, Saint-Priest, France
| | | | | | | | - Lauriane Piney
- Global Innovation, Boehringer Ingelheim, Saint-Priest, France
| | - Ludivine Chapat
- Global Innovation, Boehringer Ingelheim, Saint-Priest, France
| | | | - Hervé Poulet
- Global Innovation, Boehringer Ingelheim, Saint-Priest, France
| | - Stéphanie Longet
- Centre International de Recherche en Infectiologie, Team GIMAP (Saint-Etienne), Université Claude Bernard Lyon 1, Inserm, U1111, CNRS, UMR5308, ENS Lyon, UJM, F69007 Lyon, France
| | - Karelle De Luca
- Global Innovation, Boehringer Ingelheim, Saint-Priest, France
| | - Egbert Mundt
- Global Innovation, Boehringer Ingelheim, Saint-Priest, France
| | - Stéphane Paul
- Centre International de Recherche en Infectiologie, Team GIMAP (Saint-Etienne), Université Claude Bernard Lyon 1, Inserm, U1111, CNRS, UMR5308, ENS Lyon, UJM, F69007 Lyon, France
- International Center for Infectiology Research, INSERM 1408 Vaccinology, Saint-Etienne, France
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Wiarda JE, Becker SR, Sivasankaran SK, Loving CL. Regional epithelial cell diversity in the small intestine of pigs. J Anim Sci 2023; 101:skac318. [PMID: 36183288 PMCID: PMC9831138 DOI: 10.1093/jas/skac318] [Citation(s) in RCA: 13] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2022] [Accepted: 09/28/2022] [Indexed: 01/13/2023] Open
Abstract
Understanding regional distribution and specialization of small intestinal epithelial cells is crucial for developing methods to control appetite, stress, and nutrient uptake in swine. To establish a better understanding of specific epithelial cells found across different regions of the small intestine in pigs, we utilized single-cell RNA sequencing (scRNA-seq) to recover and analyze epithelial cells from duodenum, jejunum, and ileum. Cells identified included crypt cells, enterocytes, BEST4 enterocytes, goblet cells, and enteroendocrine (EE) cells. EE cells were divided into two subsets based on the level of expression of the EE lineage commitment gene, NEUROD1. NEUROD1hi EE cells had minimal expression of hormone-encoding genes and were dissimilar to EE cells in humans and mice, indicating a subset of EE cells unique to pigs. Recently discovered BEST4 enterocytes were detected in both crypts and villi throughout the small intestine via in situ staining, unlike in humans, where BEST4 enterocytes are found only in small intestinal villi. Proximal-to-distal gradients of expression were noted for hormone-encoding genes in EE cells and nutrient transport genes in enterocytes via scRNA-seq, demonstrating regional specialization. Regional gene expression in EE cells and enterocytes was validated via quantitative PCR (qPCR) analysis of RNA isolated from epithelial cells of different small intestinal locations. Though many genes had similar patterns of regional expression when assessed by qPCR of total epithelial cells, some regional expression was only detected via scRNA-seq, highlighting advantages of scRNA-seq to deconvolute cell type-specific regional gene expression when compared to analysis of bulk samples. Overall, results provide new information on regional localization and transcriptional profiles of epithelial cells in the pig small intestine.
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Affiliation(s)
- Jayne E Wiarda
- Food Safety and Enteric Pathogens Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, IA, USA
- Immunobiology Graduate Program, Iowa State University, Ames, IA, USA
- Department of Veterinary Microbiology and Preventative Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA, USA
- Oak Ridge Institute for Science and Education, Agricultural Research Service Participation Program, Oak Ridge, TN, USA
| | - Sage R Becker
- Food Safety and Enteric Pathogens Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, IA, USA
- Immunobiology Graduate Program, Iowa State University, Ames, IA, USA
- Department of Veterinary Microbiology and Preventative Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA, USA
| | - Sathesh K Sivasankaran
- Food Safety and Enteric Pathogens Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, IA, USA
- Genome Informatics Facility, Iowa State University, Ames, IA, USA
| | - Crystal L Loving
- Food Safety and Enteric Pathogens Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, IA, USA
- Immunobiology Graduate Program, Iowa State University, Ames, IA, USA
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Niazi AM, ZiHeng Z, Fuke N, Toyama K, Habibi WA, Kawaguchi N, Yamaguchi R, Hirai T. Detection of Swine Influenza A and Porcine Reproductive and Respiratory Syndrome Viruses in Nasopharynx-Associated Lymphoid Tissue. J Comp Pathol 2022; 197:23-34. [DOI: 10.1016/j.jcpa.2022.06.006] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2022] [Revised: 05/23/2022] [Accepted: 06/28/2022] [Indexed: 10/15/2022]
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Sutton KM, Orr B, Hope J, Jensen SR, Vervelde L. Establishment of bovine 3D enteroid-derived 2D monolayers. Vet Res 2022; 53:15. [PMID: 35236416 PMCID: PMC8889782 DOI: 10.1186/s13567-022-01033-0] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2021] [Accepted: 02/03/2022] [Indexed: 12/28/2022] Open
Abstract
Three-dimensional (3D) intestinal enteroids are powerful in vitro models for studying intestinal biology. However, due to their closed structure direct access to the apical surface is impeded, limiting high-throughput applications of exogenous compounds and pathogens. In this study, we describe a method for generating confluent 2D enteroids from single-cell suspensions of enzymatically-dissociated ileum-derived bovine 3D enteroids. Confluent monolayers were first achieved using IntestiCult media but to establish a defined, cost-effective culture media, we also developed a bovine enteroid monolayer (BEM) medium. The monolayers cultured in BEM media proliferated extensively and formed confluent cell layers on both Matrigel-coated plastic plates and transwell inserts by day 3 of culture. The 2D enteroids maintained the epithelial cell lineages found in 3D enteroids and ileum tissue. In addition, the monolayers formed a functional epithelial barrier based on the presence of the adherens and tight junction proteins, E-cadherin and ZO-1, and electrical resistance across the monolayer was measured from day 3 and maintained for up to 7 days in culture. The method described here will provide a useful model to study bovine epithelial cell biology with ease of access to the apical surface of epithelial cells and has potential to investigate host-pathogen interactions and screen bioactive compounds.
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Affiliation(s)
- Kate M Sutton
- Division of Infection and Immunity, The Roslin Institute & R(D)SVS, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, UK.
| | - Brigid Orr
- Division of Infection and Immunity, The Roslin Institute & R(D)SVS, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, UK
| | - Jayne Hope
- Division of Infection and Immunity, The Roslin Institute & R(D)SVS, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, UK
| | - Stina R Jensen
- Novozymes A/S, Animal Health and Nutrition, 2800, Lyngby, Denmark
| | - Lonneke Vervelde
- Division of Infection and Immunity, The Roslin Institute & R(D)SVS, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, UK.
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Correa F, Luise D, Bosi P, Trevisi P. Weaning differentially affects the maturation of piglet peripheral blood and jejunal Peyer's patches. Sci Rep 2022; 12:1604. [PMID: 35102264 PMCID: PMC8803882 DOI: 10.1038/s41598-022-05707-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2021] [Accepted: 01/03/2022] [Indexed: 11/09/2022] Open
Abstract
The study aimed to assess how the post-weaning condition changes piglet peripheral blood (PB) and jejunal Peyer's patches (JPPs) as compared to the suckling period, and how these changes are associated with intestinal microbiota evolution. Sixteen pigs were slaughtered and sampled for PB, JPPs and jejunal content (JC) at weaning (26 days) or at 12 days fed on a pre-starter diet. The PB and JPP transcriptomes were analysed using mRNA-seq. The Gene Set Enrichment Analysis was used to demonstrate enriched gene clusters, depending on sampling time. Jejunal microbiota was profiled using 16S rRNA gene sequencing. Post-weaning JPPs were enriched for processes related to the activation of IFN-γ and major histocompatibility complex (MHC) class I antigen processing which clustered with the reduced abundance of the Weisella genus and Faecalibacterium prausnitzii in JC. The post-weaning microbiome differed from that seen in just-weaned pigs. For just-weaned PB, the enrichment of genes related to hemoglobin and the iron metabolism indicated the greater presence of reticulocytes and immature erythrocytes. The JPP genes involved in the I MHC and IFN-γ activations were markers of the post-weaning phase. Several genes attributable to reticulocyte and erythrocyte maturation could be interesting for testing the iron nutrition of piglets.
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Affiliation(s)
- Federico Correa
- Department of Agricultural and Food Sciences (DISTAL), University of Bologna, Viale G. Fanin 46, 40127, Bologna, Italy
| | - Diana Luise
- Department of Agricultural and Food Sciences (DISTAL), University of Bologna, Viale G. Fanin 46, 40127, Bologna, Italy
| | - Paolo Bosi
- Department of Agricultural and Food Sciences (DISTAL), University of Bologna, Viale G. Fanin 46, 40127, Bologna, Italy.
| | - Paolo Trevisi
- Department of Agricultural and Food Sciences (DISTAL), University of Bologna, Viale G. Fanin 46, 40127, Bologna, Italy
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Investigation of Early Supplementation of Nucleotides on the Intestinal Maturation of Weaned Piglets. Animals (Basel) 2021; 11:ani11061489. [PMID: 34064055 PMCID: PMC8223990 DOI: 10.3390/ani11061489] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2021] [Revised: 05/19/2021] [Accepted: 05/20/2021] [Indexed: 01/04/2023] Open
Abstract
Simple Summary Nucleotides represent a group of bioactive compounds essential for the development of the gastrointestinal tract and immune function. This study aimed to evaluate the short-term effect of oral administration of nucleotides before and after weaning on growth performance, health, development of the intestinal immunity and microbiome of piglet. A nucleotide-based product (NU) was orally given four times before weaning and once after to one group of piglets, while a second group was used as a control (CO). The NU pigs did not grow more than the control until 12 days post-weaning but had increased hemoglobin and hematocrit values. At weaning, feces of NU piglets had a microbial profile more typical of growing pigs, while those of CO were more representative of suckling pigs. The upregulation of genes in the blood of control pigs at weaning was indicative of more activation towards an inflammatory response, while genes of erythropoiesis were more active in NU pigs post-weaning. NU supplementation stimulated genes for proliferative activity in the intestinal immune system, a sign of possible anticipated maturation. NU supplementation did not influence the growth performance of piglets but may have expressed a positive effect on pig microbiota anticipating its maturation at weaning, with possible immunostimulant activity on the intestinal immune system. Abstract Nucleotides are essential for the development of the gastrointestinal tract and immune function, but their intake with milk by piglets could be insufficient. The effect of nucleotides on growth and health was tested on 98 piglets divided into two groups: NU, orally administrated with 4 mL of a nucleotide-based product (SwineMOD®) at 10, 15, 18, 21, 27 days, or not (CO). Blood and feces were sampled at weaning (26 d, T1), and at 38 d (T2). Per each group and time-point, eight piglets were slaughtered and jejunal Peyer’s patches (JPPs) were collected. NU increased hemoglobin content and hematocrit, but not growth. At weaning, the NU fecal microbiota was characterized by the abundance of Campylobacteraceae, more typical of the growing phase, compared to CO, with a greater abundance of Streptococcaceae. For the blood transcriptome, an initial greater inflammatory activation was seen in CO, while at T2, NU enriched gene sets related to erythropoiesis. The activation of gene groups ranging from epigenetic response to transcriptional regulation evidenced an intense proliferative activity in NU JPPs. NU supplementation did not influence the growth performance of piglets but could have expressed a positive effect on pig microbiota anticipating its maturation at weaning. This immunostimulant activity in the JPPs could moderate the inflammation in the immediate pre-weaning.
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Furukawa M, Ito S, Suzuki S, Fuchimoto D, Onishi A, Niimi K, Usami K, Wu G, Bazer FW, Ogasawara K, Watanabe K, Aso H, Nochi T. Organogenesis of Ileal Peyer's Patches Is Initiated Prenatally and Accelerated Postnatally With Comprehensive Proliferation of B Cells in Pigs. Front Immunol 2020; 11:604674. [PMID: 33424851 PMCID: PMC7793923 DOI: 10.3389/fimmu.2020.604674] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2020] [Accepted: 11/05/2020] [Indexed: 11/21/2022] Open
Abstract
Morphogenesis and differentiation of organs is required for subsequent functional maturation. The morphological features of Peyer's patches vary among species. In pigs, they develop extensively in the ileum as ileal Peyer's patches (IPPs). However, the role of IPPs in the porcine immune system remains to be elucidated because of a lack of complete understanding of IPP organogenesis. Results of the present study revealed that development of porcine IPPs is initiated prenatally between embryonic days 76 and 91. The process of IPP organogenesis is concomitant with increased transcriptional patterns of CXCL13 and CCL19. IPPs undergo further development postnatally by forming central, marginal, and subepithelial zones. Importantly, a large number of proliferating B cells and apoptotic cells are found in porcine IPPs postnatally, but not prenatally. The expression level of IgM in proliferating B cells depends on the zone in which distinct B cells are separately localized after birth. Specifically, IgM+ cells are predominantly found in the central zone, whereas IgM-/low cells are abundant in the marginal zone. Importantly, the cellular feature of IPPs differs from that of mesenteric lymph nodes (MLNs) where such distinct zones are not formed both prenatally and postnatally. Our findings suggest that IPPs (not MLNs) in postnatal pigs are involved in complementing functions of the primary lymphoid tissue that promotes the differentiation and maturation of B cells.
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Affiliation(s)
- Mutsumi Furukawa
- International Education and Research Center for Food and Agricultural Immunology, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan
| | - Shun Ito
- International Education and Research Center for Food and Agricultural Immunology, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan
| | - Shunichi Suzuki
- Division of Animal Science, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Tsukuba, Japan
| | - Daiichiro Fuchimoto
- Division of Animal Science, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Tsukuba, Japan
| | - Akira Onishi
- Department of Animal Science and Resources, Nihon University College of Bioresource Sciences, Fujisawa, Japan
| | - Kanae Niimi
- International Education and Research Center for Food and Agricultural Immunology, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan
| | - Katsuki Usami
- International Education and Research Center for Food and Agricultural Immunology, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan
| | - Guoyao Wu
- Department of Animal Science, Texas A&M University, College Station, TX, United States
| | - Fuller W Bazer
- Department of Animal Science, Texas A&M University, College Station, TX, United States
| | - Kouetsu Ogasawara
- Department of Immunobiology, Tohoku University Institute of Development, Aging and Cancer, Sendai, Japan
| | - Kouichi Watanabe
- International Education and Research Center for Food and Agricultural Immunology, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan
| | - Hisashi Aso
- International Education and Research Center for Food and Agricultural Immunology, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan
| | - Tomonori Nochi
- International Education and Research Center for Food and Agricultural Immunology, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan.,International Research and Development Center for Mucosal Vaccines, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
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Chen YM, Helm ET, Gabler N, Hostetter JM, Burrough ER. Alterations in Intestinal Innate Mucosal Immunity of Weaned Pigs During Porcine Epidemic Diarrhea Virus Infection. Vet Pathol 2020; 57:642-652. [PMID: 32880235 DOI: 10.1177/0300985820932140] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
In the small intestine, localized innate mucosal immunity is critical for intestinal homeostasis. Porcine epidemic diarrhea virus (PEDV) infection induces villus injury and impairs digestive function. Moreover, the infection might comprise localized innate mucosal immunity. This study investigated specific enterocyte subtypes and innate immune components of weaned pigs during PEDV infection. Four-week-old pigs were orally inoculated with PEDV IN19338 strain (n = 40) or sham-inoculated (n = 24). At day post inoculation (DPI) 2, 4, and 6, lysozyme expression in Paneth cells, cellular density of villous and Peyer's patch microfold (M) cells, and the expression of polymeric immunoglobulin receptor (pIgR) were assessed in the jejunum and ileum by immunohistochemistry, and interleukin (IL)-1β and tumor necrosis factor (TNF)-α were measured in the jejunum by ELISA. PEDV infection led to a decrease in the ratios of villus height to crypt depth (VH-CD) in jejunum at DPI 2, 4, and 6 and in ileum at DPI 4. The number of villous M cells was reduced in jejunum at DPI 4 and 6 and in ileum at DPI 6, while the number of Peyer's patch M cells in ileum increased at DPI 2 and then decreased at DPI 6. PEDV-infected pigs also had reduced lysozyme expression in ileal Paneth cells at DPI 2 and increased ileal pIgR expression at DPI 4. There were no significant changes in IL-1β and TNF-α expression in PEDV-infected pigs compared to controls. In conclusion, PEDV infection affected innate mucosal immunity of weaned pigs through alterations in Paneth cells, villous and Peyer's patch M cells, and pIgR expression.
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Abstract
The pig is an omnivorous, monogastric species with many advantages to serve as an animal model for human diseases. There are very high similarities to humans in anatomy and functions of the immune system, e g., the presence of tonsils, which are absent in rodents. The porcine immune system resembles man for more than 80% of analyzed parameters in contrast to the mouse with only about 10%. The pig can easily be bred, and there are less emotional problems to use them as experimental animals than dogs or monkeys. Indwelling cannulas in a vein or lymphatic vessel enable repetitive stress-free sampling. Meanwhile, there are many markers available to characterize immune cells. Lymphoid organs, their function, and their role in lymphocyte kinetics (proliferation and migration) are reviewed. For long-term experiments, minipigs (e.g., Göttingen minipig) are available. Pigs can be kept under gnotobiotic (germfree) conditions for some time after birth to study the effects of microbiota. The effects of probiotics can be tested on the gut immune system. The lung has been used for extracorporeal preservation and immune engineering. After genetic modifications are established, the pig is the best animal model for future xenotransplantation to reduce the problem of organ shortage for organ transplantation. Autotransplantation of particles of lymphnodes regenerates in the subcutaneous tissue. This is a model to treat secondary lymphedema patients. There are pigs with cystic fibrosis and severe combined immune deficiency available.
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Affiliation(s)
- Reinhard Pabst
- Institute of Immunomorphology, Centre of Anatomy, Medical School Hannover, Hanover, Germany.
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11
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Özbek M, Bayraktaroğlu AG. Developmental study on the ileal Peyer's patches of sheep, and cytokeratin-18 as a possible marker for M cells in follicle associated epithelium. Acta Histochem 2019; 121:311-322. [PMID: 30745250 DOI: 10.1016/j.acthis.2019.01.005] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2018] [Revised: 01/15/2019] [Accepted: 01/17/2019] [Indexed: 01/08/2023]
Abstract
Peyer's patches are known as the immune sensors of the intestine because of their ability to transport luminal antigens. The objective of this study was both to assess the prenatal and postnatal development of sheep ileal Peyer's patches with respect to histomorphology, distribution of CD4+ and CD8+ cells, and localization of proliferating and apoptotic cells, and to examine the morphology of M cells and expression of CK18 in follicle associated epithelium (FAE). We also hypothesized that CK18 could be a potential marker for M cell. Peyer's patches completed their histomorphological development in prenatal period and involuted in the postnatal period. The distribution of the CD4+ and CD8+ cells was similar in the last trimester of pregnancy (days 120-150) and the postnatal period, but differed in the early stages of foetal development (days 70-120). In the prenatal period, the follicular area displayed high levels of proliferation and apoptosis. We observed CK18 immunoreaction only in FAE. While M cells were devoid of microfolds in the early stages of the prenatal period, these cells acquired a prismatic shape and bore distinct apical microfolds in the late prenatal period and postnatal period. As a result, it was determined that, in sheep, the development of the ileal Peyer's patches occurred in the prenatal period, independent of exogenous antigenic stimulation, and in association with high levels of lymphopoiesis and apoptosis in the follicles. We found, for the first time, that CK18 is a novel and reliable marker for FAE in sheep ileal Peyer's patches. We suggest that CK18 positive cells in FAE are M cells.
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Affiliation(s)
- Mehmet Özbek
- Mehmet Akif Ersoy University, Faculty of Veterinary Medicine, Department of Histology and Embryology, Burdur, Turkey.
| | - Alev Gürol Bayraktaroğlu
- Ankara University, Faculty of Veterinary Medicine, Department of Histology and Embryology, Ankara, Turkey
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Klisuric A, Thierry B, Delon L, Prestidge CA, Gibson RJ. Identifying human and murine M cells in vitro. Exp Biol Med (Maywood) 2019; 244:554-564. [PMID: 30907132 DOI: 10.1177/1535370219838674] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
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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Affiliation(s)
- Ana Klisuric
- 1 Division of Health Sciences, University of South Australia, Adelaide 5000, Australia.,2 ARC Centre of Excellence in Convergent Bio and Nano Science and Technology, University of South Australia, Frome Road, Adelaide 5000, Australia.,3 School of Pharmacy and Medical Science, University of South Australia, Adelaide 5000, Australia
| | - Benjamin Thierry
- 2 ARC Centre of Excellence in Convergent Bio and Nano Science and Technology, University of South Australia, Frome Road, Adelaide 5000, Australia.,4 Future Industries Institute, University of South Australia, Mawson Lakes 5095, Australia
| | - Ludivine Delon
- 1 Division of Health Sciences, University of South Australia, Adelaide 5000, Australia.,2 ARC Centre of Excellence in Convergent Bio and Nano Science and Technology, University of South Australia, Frome Road, Adelaide 5000, Australia.,4 Future Industries Institute, University of South Australia, Mawson Lakes 5095, Australia
| | - Clive A Prestidge
- 1 Division of Health Sciences, University of South Australia, Adelaide 5000, Australia.,2 ARC Centre of Excellence in Convergent Bio and Nano Science and Technology, University of South Australia, Frome Road, Adelaide 5000, Australia
| | - Rachel J Gibson
- 1 Division of Health Sciences, University of South Australia, Adelaide 5000, Australia
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Xia L, Yang Y, Wang J, Jing Y, Yang Q. Impact of TGEV infection on the pig small intestine. Virol J 2018; 15:102. [PMID: 29914507 PMCID: PMC6006930 DOI: 10.1186/s12985-018-1012-9] [Citation(s) in RCA: 75] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2018] [Accepted: 06/03/2018] [Indexed: 02/07/2023] Open
Abstract
Background Pig diarrhea causes high mortality and large economic losses in the swine industry. Transmissible gastroenteritis virus (TGEV) causes pig diarrhea, with 100% mortality in piglets less than 2 weeks old. No investigation has yet been made of the small intestine of piglets that survived infection by TGEV. Methods In this study, we evaluated the impact of TGEV infection on the small intestine of recovered pigs. Results Histological analyses showed that TGEV infection led to villi atrophy, and reduced villous height and crypt depth. The number of SIgA positive cells, CD3+T cells, and dendritic cells (DCs) in jejunum decreased after TGEV infection in vivo. In contrast, microfold cell (M cell) numbers and cell proliferation increased in infected pigs. TGEV infection also significantly enhanced the mRNA expression levels of cytokine IL-1β, IL-6, TNF-α, IL-10, and TGF-β. Additionally, lower gene copy numbers of Lactobacillus, and higher numbers of Enterobacteriaceae, were detected in mucosal scraping samples from TGEV-infected pigs. Conclusions TGEV infection damages the small intestine, impairs immune functions, and increases pathogenic bacterial loading, all of which may facilitate secondary infections by other pathogens. These findings help quantify the impact of TGEV infection and clarify the pathogenic mechanisms underlying its effects in pigs.
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Affiliation(s)
- Lu Xia
- MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Weigang 1, Nanjing, Jiangsu, 210095, People's Republic of China
| | - Yunhan Yang
- MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Weigang 1, Nanjing, Jiangsu, 210095, People's Republic of China
| | - Jialu Wang
- MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Weigang 1, Nanjing, Jiangsu, 210095, People's Republic of China
| | - Yuchao Jing
- MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Weigang 1, Nanjing, Jiangsu, 210095, People's Republic of China
| | - Qian Yang
- MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Weigang 1, Nanjing, Jiangsu, 210095, People's Republic of China.
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Early feed restriction of lambs modifies ileal epimural microbiota and affects immunity parameters during the fattening period. Animal 2018; 12:2115-2122. [PMID: 29679995 DOI: 10.1017/s1751731118000836] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
Bacteria firmly attached to the gastrointestinal epithelium during the pre-weaning phase may show a significant impact on nutrient processing, immunity parameters, health and feed efficiency of lambs during post-weaning phases. Thus, the aim of this study was to describe the differences in the ileal epimural microbiota (e.g. total bacteria, Prevotella spp., Bifidobacterium spp. and Lactobacillus spp.) of fattening lambs promoted by early feed restriction during the suckling phase trying to elucidate some of the underlying mechanisms behind changes in feed efficiency during the fattening period. A total of 24 Merino lambs (average BW 4.81±0.256 kg) were used, 12 of them (ad libitum, ADL) kept permanently in individual pens with their mothers, whereas the other 12 lambs were separated from their dams for 9 h each day to be exposed to milk restriction (RES). After weaning (BW=15 kg) all the animals were penned individually, offered the same complete pelleted diet (35 g/kg BW per day) and slaughtered at a BW of 27 kg. During the fattening period, reduced gain : feed ratio (0.320 v. 0.261, P<0.001) was observed for the RES group. Moreover, increments of Prevotella spp. were detected in the ileal epimural microbiota of RES lambs (P<0.05). There were also higher numbers of infiltrated lymphocytes (T and B cells) in the ileal lamina propria (P<0.05), a higher M-cell labelling intensity in ileal Peyer's patches domes (P<0.05) and a trend towards a thickening of the submucosa layer when compared with the ADL group (P=0.057). Some other immunological parameters, such as an increased immunoglobulin A (IgA) production (pg IgA/µg total protein) and increments in CD45+ cells were also observed in the ileum of RES group (P<0.05), whereas transforming growth factor β and toll-like receptor gene expression was reduced (P<0.05). In conclusion, early feed restriction during the suckling phase promoted changes in ileal epimural microbiota and several immunity parameters that could be related to differences in feed efficiency traits during the fattening period of Merino lambs.
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A new cell-to-cell interaction model for epithelial microfold cell formation and the enhancing effect of epidermal growth factor. Eur J Pharm Sci 2017; 106:49-61. [DOI: 10.1016/j.ejps.2017.05.054] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2017] [Revised: 04/12/2017] [Accepted: 05/23/2017] [Indexed: 12/22/2022]
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16
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Prims S, Pintens N, Vergauwen H, Van Cruchten S, Van Ginneken C, Casteleyn C. Effect of artificial rearing of piglets on the volume densities of M cells in the tonsils of the soft palate and ileal Peyer’s patches. Vet Immunol Immunopathol 2017; 184:1-7. [DOI: 10.1016/j.vetimm.2016.12.009] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2016] [Revised: 12/12/2016] [Accepted: 12/22/2016] [Indexed: 10/20/2022]
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17
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Geisler F, Leube RE. Epithelial Intermediate Filaments: Guardians against Microbial Infection? Cells 2016; 5:cells5030029. [PMID: 27355965 PMCID: PMC5040971 DOI: 10.3390/cells5030029] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2016] [Revised: 06/15/2016] [Accepted: 06/21/2016] [Indexed: 12/21/2022] Open
Abstract
Intermediate filaments are abundant cytoskeletal components of epithelial tissues. They have been implicated in overall stress protection. A hitherto poorly investigated area of research is the function of intermediate filaments as a barrier to microbial infection. This review summarizes the accumulating knowledge about this interaction. It first emphasizes the unique spatial organization of the keratin intermediate filament cytoskeleton in different epithelial tissues to protect the organism against microbial insults. We then present examples of direct interaction between viral, bacterial, and parasitic proteins and the intermediate filament system and describe how this affects the microbe-host interaction by modulating the epithelial cytoskeleton, the progression of infection, and host response. These observations not only provide novel insights into the dynamics and function of intermediate filaments but also indicate future avenues to combat microbial infection.
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Affiliation(s)
- Florian Geisler
- Institute of Molecular and Cellular Anatomy, RWTH Aachen University, Wendlingweg 2, 52074 Aachen, Germany.
| | - Rudolf E Leube
- Institute of Molecular and Cellular Anatomy, RWTH Aachen University, Wendlingweg 2, 52074 Aachen, Germany.
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Hondo T, Someya S, Nagasawa Y, Terada S, Watanabe H, Chen X, Watanabe K, Ohwada S, Kitazawa H, Rose MT, Nochi T, Aso H. Cyclophilin A is a new M cell marker of bovine intestinal epithelium. Cell Tissue Res 2016; 364:585-597. [DOI: 10.1007/s00441-015-2342-1] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2015] [Accepted: 12/03/2015] [Indexed: 12/20/2022]
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19
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Wang M, Gao Z, Zhang Z, Pan L, Zhang Y. Roles of M cells in infection and mucosal vaccines. Hum Vaccin Immunother 2015; 10:3544-51. [PMID: 25483705 DOI: 10.4161/hv.36174] [Citation(s) in RCA: 51] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023] Open
Abstract
The mucosal immune system plays a crucial part in the control of infection. Exposure of humans and animals to potential pathogens generally occurs through mucosal surfaces, thus, strategies that target the mucosa seem rational and efficient vaccination measures. Vaccination through the mucosal immune system can induce effective systemic immune responses simultaneously with mucosal immunity compared with parenteral vaccination. M cells are capable of transporting luminal antigens to the underlying lymphoid tissues and can be exploited by pathogens as an entry portal to invade the host. Therefore, targeting M-cell-specific molecules might enhance antigen entry, initiate the immune response, and induce protection against mucosal pathogens. Here, we outline our understanding of the distribution and function of M cells, and summarize the advances in mucosal vaccine strategies that target M cells.
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Key Words
- ANX, Annexin; BALT, bronchus-associated lymphoid tissue
- C5aR, C5a receptor
- DCs, dendritic cells
- DENV, dengue virus
- EDIII, envelope domain III
- FAE, follicle-associated epithelium
- GALT, gut-associated lymphoid tissue
- GENALT, genital-associated lymphoid tissue
- GP2, Glycoprotein 2
- Hsp60, heat shock protein 60
- LPS, lipopolysaccharide
- M cells
- M cells, microfold cells
- MALT, mucosa-associated lymphoid tissue
- NALT, nasopharynx- or nose-associated lymphoid tissue
- OVA, ovalbumin
- OmpH, outer membrane protein H
- PP, Peyer's patches
- PRRs, pathogen recognition receptors
- PrPC, cellular prion protein
- SELEX, Systematic Evolution of Ligands by EXponential enrichment
- SIgA secretory IgA
- TLR-4, Toll-like receptor-4
- UEA-1,Ulex europaeus agglutinin-1
- antigen
- infection
- mucosal immunity
- pσ1, reovirus surface protein σ1
- vaccine
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Affiliation(s)
- Miao Wang
- a State Key Laboratory of Veterinary Etiological Biology; National Foot-and-Mouse Disease Reference Laboratory; Lanzhou Veterinary Research Institute; CAAS ; Lanzhou , Gansu , China
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Early events in the pathogenesis of foot-and-mouth disease in pigs; identification of oropharyngeal tonsils as sites of primary and sustained viral replication. PLoS One 2014; 9:e106859. [PMID: 25184288 PMCID: PMC4153717 DOI: 10.1371/journal.pone.0106859] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2014] [Accepted: 08/02/2014] [Indexed: 11/30/2022] Open
Abstract
A time-course study was performed to elucidate the early events of foot-and-mouth disease virus (FMDV) infection in pigs subsequent to simulated natural, intra-oropharyngeal, inoculation. The earliest detectable event was primary infection in the lingual and paraepiglottic tonsils at 6 hours post inoculation (hpi) characterized by regional localization of viral RNA, viral antigen, and infectious virus. At this time FMDV antigen was localized in cytokeratin-positive epithelial cells and CD172a-expressing leukocytes of the crypt epithelium of the paraepiglottic tonsils. De novo replication of FMDV was first detected in oropharyngeal swab samples at 12 hpi and viremia occurred at 18–24 hpi, approximately 24 hours prior to the appearance of vesicular lesions. From 12 through 78 hpi, microscopic detection of FMDV was consistently localized to cytokeratin-positive cells within morphologically characteristic segments of oropharyngeal tonsil crypt epithelium. During this period, leukocyte populations expressing CD172a, SLA-DQ class II and/or CD8 were found in close proximity to infected epithelial cells, but with little or no co-localization with viral proteins. Similarly, M-cells expressing cytokeratin-18 did not co-localize with FMDV proteins. Intra-epithelial micro-vesicles composed of acantholytic epithelial cells expressing large amounts of structural and non-structural FMDV proteins were present within crypts of the tonsil of the soft palate during peak clinical infection. These findings inculpate the paraepiglottic tonsils as the primary site of FMDV infection in pigs exposed via the gastrointestinal tract. Furthermore, the continuing replication of FMDV in the oropharyngeal tonsils during viremia and peak clinical infection with no concurrent amplification of virus occurring in the lower respiratory tract indicates that these sites are the major source of shedding of FMDV from pigs.
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21
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Mair KH, Sedlak C, Käser T, Pasternak A, Levast B, Gerner W, Saalmüller A, Summerfield A, Gerdts V, Wilson HL, Meurens F. The porcine innate immune system: an update. DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY 2014; 45:321-43. [PMID: 24709051 PMCID: PMC7103209 DOI: 10.1016/j.dci.2014.03.022] [Citation(s) in RCA: 195] [Impact Index Per Article: 17.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/19/2014] [Revised: 03/30/2014] [Accepted: 03/31/2014] [Indexed: 05/21/2023]
Abstract
Over the last few years, we have seen an increasing interest and demand for pigs in biomedical research. Domestic pigs (Sus scrofa domesticus) are closely related to humans in terms of their anatomy, genetics, and physiology, and often are the model of choice for the assessment of novel vaccines and therapeutics in a preclinical stage. However, the pig as a model has much more to offer, and can serve as a model for many biomedical applications including aging research, medical imaging, and pharmaceutical studies to name a few. In this review, we will provide an overview of the innate immune system in pigs, describe its anatomical and physiological key features, and discuss the key players involved. In particular, we compare the porcine innate immune system to that of humans, and emphasize on the importance of the pig as model for human disease.
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Affiliation(s)
- K H Mair
- Institute of Immunology, Department of Pathobiology, University of Veterinary Medicine Vienna, Veterinärplatz 1, 1210 Vienna, Austria
| | - C Sedlak
- Institute of Immunology, Department of Pathobiology, University of Veterinary Medicine Vienna, Veterinärplatz 1, 1210 Vienna, Austria
| | - T Käser
- Vaccine and Infectious Disease Organization-International Vaccine Centre (VIDO-InterVac), University of Saskatchewan, 120 Veterinary Road, S7N 5E3 Saskatoon, Saskatchewan, Canada
| | - A Pasternak
- Vaccine and Infectious Disease Organization-International Vaccine Centre (VIDO-InterVac), University of Saskatchewan, 120 Veterinary Road, S7N 5E3 Saskatoon, Saskatchewan, Canada
| | - B Levast
- Vaccine and Infectious Disease Organization-International Vaccine Centre (VIDO-InterVac), University of Saskatchewan, 120 Veterinary Road, S7N 5E3 Saskatoon, Saskatchewan, Canada
| | - W Gerner
- Institute of Immunology, Department of Pathobiology, University of Veterinary Medicine Vienna, Veterinärplatz 1, 1210 Vienna, Austria
| | - A Saalmüller
- Institute of Immunology, Department of Pathobiology, University of Veterinary Medicine Vienna, Veterinärplatz 1, 1210 Vienna, Austria
| | - A Summerfield
- Institute of Virology and Immunoprophylaxis (IVI), Sensemattstrasse 293, 3147 Mittelhäusern, Switzerland
| | - V Gerdts
- Vaccine and Infectious Disease Organization-International Vaccine Centre (VIDO-InterVac), University of Saskatchewan, 120 Veterinary Road, S7N 5E3 Saskatoon, Saskatchewan, Canada
| | - H L Wilson
- Vaccine and Infectious Disease Organization-International Vaccine Centre (VIDO-InterVac), University of Saskatchewan, 120 Veterinary Road, S7N 5E3 Saskatoon, Saskatchewan, Canada
| | - F Meurens
- Vaccine and Infectious Disease Organization-International Vaccine Centre (VIDO-InterVac), University of Saskatchewan, 120 Veterinary Road, S7N 5E3 Saskatoon, Saskatchewan, Canada.
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22
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Infection dynamics of foot-and-mouth disease virus in pigs using two novel simulated-natural inoculation methods. Res Vet Sci 2014; 96:396-405. [DOI: 10.1016/j.rvsc.2014.01.009] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2013] [Revised: 12/03/2013] [Accepted: 01/26/2014] [Indexed: 11/23/2022]
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Casteleyn C, Van den Broeck W, Gebert A, Tambuyzer BR, Van Cruchten S, Van Ginneken C. M cell specific markers in man and domestic animals: Valuable tools in vaccine development. Comp Immunol Microbiol Infect Dis 2013; 36:353-64. [DOI: 10.1016/j.cimid.2013.03.002] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2012] [Revised: 03/01/2013] [Accepted: 03/21/2013] [Indexed: 12/13/2022]
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Erysipelothrix rhusiopathiae exploits cytokeratin 18-positive epithelial cells of porcine tonsillar crypts as an invasion gateway. Vet Immunol Immunopathol 2013; 153:260-6. [DOI: 10.1016/j.vetimm.2013.03.013] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2012] [Revised: 03/15/2013] [Accepted: 03/20/2013] [Indexed: 11/20/2022]
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25
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Tozaki K, Kimura J, Yasuda M, Ryu N, Nasu T, Pernthaner A, Hein WR. C6, a new monoclonal antibody, reacts with the follicle-associated epithelium of calf ileal Peyer's patches. J Vet Sci 2013; 14:1-6. [PMID: 23388432 PMCID: PMC3615225 DOI: 10.4142/jvs.2013.14.1.1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2012] [Accepted: 08/08/2012] [Indexed: 11/20/2022] Open
Abstract
The follicle-associated epithelium (FAE) of Peyer's patches (PPs) contains M cells that are important for reducing mucosal immune responses by transporting antigens into the underlying lymphoid tissue. We generated a monoclonal antibody (C6) that reacted with the FAE of calf ileal PPs, and analyzed the characteristics of C6 using immunohistochemistry and Western blotting. FAE of the ileal PP was stained with C6 during both late fetal developmental and postnatal stages. Neither the villous epithelial cell nor intestinal crypt basal cells were stained at any developmental stage. During the prenatal stages, FAE of the jejunal PP was C6-negative. However, a few C6-positive cells were distributed diffusely in some FAE of the jejunal PPs during the postnatal stages. The protein molecular weight of the antigen recognized by C6 was approximately 45 kDa. These data show that C6 is useful for identifying the FAE in ileal PPs and further suggest that differentiation of the FAE in these areas is independent of external antigens.
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Affiliation(s)
- Kana Tozaki
- Department of Veterinary Anatomy, Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan
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26
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Natural alternatives to in-feed antibiotics in pig production: can immunomodulators play a role? Animal 2012; 3:1644-61. [PMID: 22443549 DOI: 10.1017/s1751731109004236] [Citation(s) in RCA: 52] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
As a result of the European ban of in-feed growth-promoting antibiotics, new strategies are being developed to increase the resistance to disease in farm animals. In pig production, this is of particular importance during the weaning transition when piglets are subjected to major stressful events, making them highly sensitive to digestive disorders. At this time, the development of both innate and adaptive immunity at the mucosal surface is critical in preventing the potential harmful effects of intestinal pathogenic agents. Strategies aiming at stimulating natural host defences through the use of substances able to modulate immune functions have gained increasing interest in animal research, and different bioactive components a priori sharing those properties have been the subject of in vivo nutritional investigations in pig. Among these, yeast derivates (β-glucans and mannans) are able to interact with immune cells, particularly phagocytic cells. However, studies where they have been fed to pigs have shown inconsistent results, suggesting that their ability to target the sensitive immune cells through the oral route is questionable. The plant extracts, which would benefit from a positive image in the public opinion, have also been tested. However, due to a lack of data on the bioactive components of particular plants and the large diversity of species, it has proved difficult to prepare extracts of equivalent potency and thus, the literature on their influence on pig immunity remains inconclusive. In considering piglet immunity and health benefits, the most promising results to date have been obtained with spray-dried animal plasma, whose positive effects would be provided by specific antibodies and non-specific competition of some plasma components with bacteria for intestinal receptors. The major positive effect of spray-dried animal plasma is in reducing the infiltration of gut-associated lymphoid tissue by immune cells, which is likely to be the result of a decreased colonisation by potentially harmful bacteria. This review also highlights the limitations of some of the published in vivo studies on the immunomodulatory activity of certain feed additives. Among those, the lack of standardisation of extracts and the heterogeneity of piglet-rearing conditions (e.g. exposure to pathogens) are likely the most limiting.
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27
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Mucosal vaccines to prevent porcine reproductive and respiratory syndrome: a new perspective. Anim Health Res Rev 2012; 13:21-37. [PMID: 22717576 DOI: 10.1017/s1466252312000023] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Porcine reproductive and respiratory syndrome (PRRS) is an economically important infectious disease of swine. Constant emergence of variant strains of PRRS virus (PPRSV) and virus-mediated immune evasion followed by viral persistence result in increased incidence and recurrence of PRRS in swine herds. Current live and killed PRRSV vaccines administered by a parenteral route are ineffective in inducing complete protection. Thus, new approaches in design and delivery of PRRSV vaccines are needed to reduce the disease burden of the swine industry. Induction of an effective mucosal immunity to several respiratory pathogens by direct delivery of a vaccine to mucosal sites has proven to be effective in a mouse model. However, there are challenges in eliciting mucosal immunity to PRRS due to our limited understanding of safe and potent mucosal adjuvants, which could potentiate the mucosal immune response to PRRSV. The purpose of this review is to discuss methods for induction of protective mucosal immune responses in the respiratory tract of pigs. The manuscript also discusses how PRRSV modulates innate, adaptive and immunoregulatory responses at both mucosal and systemic sites of infected and/or vaccinated pigs. This information may help in the design of innovative mucosal vaccines to elicit superior cross-protective immunity against divergent field strains of PRRSV.
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Valpotić H, Kovšca Janjatović A, Lacković G, Božić F, Dobranić V, Svoboda D, Valpotić I, Popović M. Increased number of intestinal villous M cells in levamisole -pretreated weaned pigs experimentally infected with F4ac⁺ enterotoxigenic Escherichia coli strain. Eur J Histochem 2012; 54:e18. [PMID: 22073366 PMCID: PMC3167307 DOI: 10.4081/ejh.2010.e18] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2009] [Accepted: 03/05/2010] [Indexed: 12/01/2022] Open
Abstract
Immunoprophylaxis of porcine postweaning colibacillosis (PWC) caused by enterotoxigenic Escherichia coli (ETEC) expressing F4 fimbriae is an unsolved problem. Just as ETEC strains can exploit intestinal microfold (M) cells as the entry portal for infection, their high transcytotic ability make them an attractive target for mucosally delivered vaccines, adjuvants and therapeutics. We have developed a model of parenteral/oral immunization of 4-weeks-old pigs with either levamisole or vaccine candidate F4ac+ non-ETEC strain to study their effects on de novo differentiation of antigen-sampling M cells. Identification, localization and morphometric quantification of cytokeratin 18 positive M cells in the ileal mucosa of 6-weeks-old pigs revealed that they were: 1) exclusively located within villous epithelial layer, 2) significantly numerous (P< 0.01) in levamisole pretreated/challenged pigs, and 3) only slightly, but not significantly numerous in vaccinated/challenged pigs compared with non-pretreated/challenged control pigs. The fact that levamisole may affect the M cells frequency by increasing their numbers, makes it an interesting adjuvant to study development of an effective M cell-targeted vaccine against porcine PWC.
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Affiliation(s)
- H Valpotić
- Department of Animal Nutrition, Veterinary Faculty, University of Zagreb, Croatia
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Mazzoni M, Bosi P, De Sordi N, Lalatta-Costerbosa G. Distribution, organization and innervation of gastric MALT in conventional piglet. J Anat 2011; 219:611-21. [PMID: 21781093 DOI: 10.1111/j.1469-7580.2011.01415.x] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022] Open
Abstract
Mucosa-associated lymphoid tissue (MALT) is the initial inductive site for mucosal immunity. It is present in the different layers of the mucosal wall and consists of organized lymphoid tissue which may occur as isolated or aggregated lymphoid follicles (LFs) and interfollicular areas. It is present in many organs, including the pig stomach. Gastric MALT has been intensely studied in experimentally infected pigs but few data are available in healthy, non-gnotobiotic or germ-free animals. In the present study we described the gastric MALT in conventional piglets in the cardiac mucosa of the gastric diverticulum, in the pyloric mucosa, and in the sites of transition from cardiac to oxyntic and from cardiac to pyloric mucosa by means of histological and immunohistochemical stains. The majority of LFs were located in the cardiac mucosa and in the transition from the cardiac to the oxyntic mucosa. Here the LFs were mainly located in the submucosa and reached the mucosa; we called these submucosal lymphoid follicles (SLFs). In the pyloric mucosa and in the transition sites from the cardiac to the pyloric mucosa, LFs were located in the mucosa; we called these mucosal lymphoid follicles (MLFs). In SLFs, a compartmental organization of T and B lymphocytes was present; by contrast, in the MLFs, the T and B cells were intermingled, suggesting the possibility of different roles for the two types of follicles. In the epithelium overlying the lymphoid tissue, numerous T lymphocytes and some cells immunoreactive to cytokeratin-18 were observed. Following the application of the fluorescent tracer DiI into the SLFs of the diverticulum, enteric neurones located in the submucosal plexus were labelled, confirming the interplay between the immune and the enteric nervous system.
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Affiliation(s)
- Maurizio Mazzoni
- Department of Veterinary Medical Science, University of Bologna, Bologna, Italy.
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Hondo T, Kanaya T, Takakura I, Watanabe H, Takahashi Y, Nagasawa Y, Terada S, Ohwada S, Watanabe K, Kitazawa H, Rose MT, Yamaguchi T, Aso H. Cytokeratin 18 is a specific marker of bovine intestinal M cell. Am J Physiol Gastrointest Liver Physiol 2011; 300:G442-53. [PMID: 21193527 DOI: 10.1152/ajpgi.00345.2010] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Microfold (M) cells in the follicle-associated epithelium (FAE) of Peyer's patches have an important role in mucosal immune responses. A primary difficulty for investigations of bovine M cells is the lack of a specific molecular marker. To identify such a marker, we investigated the expression of several kinds of intermediate filament proteins using calf Peyer's patches. The expression patterns of cytokeratin (CK) 18 in jejunal and ileal FAE were very similar to the localization pattern of M cells recognized by scanning electron microscopy. Mirror sections revealed that jejunal CK18-positive cells had irregular and sparse microvilli, as well as pocket-like structures containing lymphocytes, typical morphological characteristic of M cells. However, CK18-negative cells had regular and dense microvilli on their surface, typical of the morphology of enterocytes. In contrast, CK20 immunoreactivity was detected in almost all villous epithelial cells and CK18-negative cells in the FAE. CK18-positive proliferating transit-amplifying cells in the crypt exchanged CK18 for CK20 above the mouth of the crypt and after moving to the villi; however, CK18-positive M cells in the crypt continued their expression of CK18 during movement to the FAE region. Terminal deoxynucleotidyl-transferase-mediated deoxyuridine-triphosphate-biotin nick-end labeling-positive apoptotic cells were specifically detected at the apical region of villi and FAE in the jejunum and ileum, and all were also stained for CK20. These data indicate that CK18 may be a molecular marker for bovine M cells in FAE and that M cells may transdifferentiate to CK20-positive enterocytes and die by apoptosis in the apex of the FAE.
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Affiliation(s)
- Tetsuya Hondo
- Cellular Biology Laboratory, Graduate School of Agricultural Science, Tohoku Univ., Sendai, Miyag, Japan
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Beyaz F, Ergün E, Bayraktaroğlu AG, Ergün L. Identification of intestinal M cells in isolated lymphoid follicles and Peyer’s patches of the Angora rabbit. Cell Tissue Res 2010; 341:417-27. [DOI: 10.1007/s00441-010-1005-5] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2010] [Accepted: 06/08/2010] [Indexed: 02/08/2023]
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The identification of intestinal M cells in the sacculus rotundus and appendix of the Angora rabbit. Vet Res Commun 2010; 34:255-65. [PMID: 20217227 DOI: 10.1007/s11259-010-9349-6] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 02/25/2010] [Indexed: 12/18/2022]
Abstract
The present study was aimed at the immunohistochemical demonstration of M cells, found in the follicle-associated epithelium (FAE) of the sacculus rotundus (SR) and appendix of the Angora rabbit, using anti-vimentin primary antibodies, and at the determination of certain fine structural characteristics. Ten adult Angora rabbits constituted the material of the study. Immunohistochemical staining revealed that many cells composing the FAE, which covered the dome regions of the SR and appendix, reacted positively with vimentin. FAE contained two different types of vimentin-positive cells. The first type surrounded intraepithelial lymphocytes (IEL) with a basolateral invagination in the apex and periphery of the dome epithelium, whilst the second type consisted of columnar cells found in the FAE near crypts. The immunoreactivity of the cells found in the FAE covering the apex and periphery of the domes was observed particularly in the perinuclear cytoplasm and the cytoplasm surrounding the IEL. Electron microscopic examination demonstrated that the M cells found in the FAE covering the apex and periphery of the dome regions of the SR and appendix did not exhibit any microvilli on their apical surface. The FAE near crypts contained columnar cells, which resembled enterocytes. The apical membrane of these cells exhibited shorter and irregular microvilli, in contrast to neighbouring enterocytes. It was determined that M cells, found in the FAE of the SR and appendix in the Angora rabbit, displayed similarities in terms of localization and fine structure. This situation may be indicative of the two lymphoid structures with different localization having similar functional properties.
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Rothkötter HJ. Anatomical particularities of the porcine immune system--a physician's view. DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY 2009; 33:267-272. [PMID: 18775744 DOI: 10.1016/j.dci.2008.06.016] [Citation(s) in RCA: 84] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/10/2008] [Revised: 06/30/2008] [Accepted: 06/30/2008] [Indexed: 05/26/2023]
Abstract
In this article the anatomical structure of the porcine immune organs is described. The focus is on their particularities that are related to the use of pigs as an animal model. Key issues of the intrauterine development of the lymphoid organs are presented, such as the specific epithelio-chorial placenta, the appearance of the thymic tissue and the initial development of B cells. The role of the thymus for the development of alpha/beta and gamma/delta T cells and the location of tonsillar tissue in the naso-pharynx, in the oral cavity and at the basis of the tongue are described. The porcine spleen is of interest for surgical techniques to treat splenic trauma adequately. The observation of the inverted lymph node structure of pigs is puzzling and it remains unclear why only few species have this distinct morphological organisation. Based on the functional differences in lymphocyte recirculation observed in pigs, specific lymph cannulation experiments are possible in the porcine immune system. The porcine intestinal lymphoid tissue and the lymphocytes in the mucosal epithelium and lamina propria are of interest for studying the gut immune responses. For use as a model the fact that the pig is a monogastric omnivorous animal represents an advantage, although the porcine ileal Peyer's patch has no obvious anatomical equivalent in man. Based on the detailed knowledge of porcine immune morphology the pig is suitable as model animal for immunology--in addition to the various experimental approaches in physiology, pharmacology, surgery, etc. that are applicable to human medicine.
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Affiliation(s)
- Hermann-Josef Rothkötter
- Institute of Anatomy, Medical Faculty, Otto-von-Guericke-University Magdeburg, Leipziger Strasse 44, 39120 Magdeburg, Germany.
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Kanaya T, Miyazawa K, Takakura I, Itani W, Watanabe K, Ohwada S, Kitazawa H, Rose MT, McConochie HR, Okano H, Yamaguchi T, Aso H. Differentiation of a murine intestinal epithelial cell line (MIE) toward the M cell lineage. Am J Physiol Gastrointest Liver Physiol 2008; 295:G273-84. [PMID: 18556421 DOI: 10.1152/ajpgi.00378.2007] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
M cells are a kind of intestinal epithelial cell in the follicle-associated epithelium of Peyer's patches. These cells can transport antigens and microorganisms into underlying lymphoid tissues. Despite the important role of M cells in mucosal immune responses, the origin and mechanisms of differentiation as well as cell death of M cells remain unclear. To clarify the mechanism of M cell differentiation, we established a novel murine intestinal epithelial cell line (MIE) from the C57BL/6 mouse. MIE cells grow rapidly and have a cobblestone morphology, which is a typical feature of intestinal epithelial cells. Additionally, they express cytokeratin, villin, cell-cell junctional proteins, and alkaline phosphatase activity and can form microvilli. Their expression of Musashi-1 antigen indicates that they may be close to intestinal stem cells or transit-amplifying cells. MIE cells are able to differentiate into the M cell lineage following coculture with intestinal lymphocytes, but not with Peyer's patch lymphocytes (PPL). However, PPL costimulated with anti-CD3/CD28 MAbs caused MIE cells to display typical features of M cells, such as transcytosis activity, the disorganization of microvilli, and the expression of M cell markers. This transcytosis activity of MIE cells was not induced by T cells isolated from PPL costimulated with the same MAbs and was reduced by the depletion of the T cell population from PPL. A mixture of T cells treated with MAbs and B cells both from PPL led MIE cells to differentiate into M cells. We report here that MIE cells have the potential ability to differentiate into M cells and that this differentiation required activated T cells and B cells.
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Affiliation(s)
- Takashi Kanaya
- Cellular Biology Laboratory, Graduate School of Agricultural Science, Tohoku Univ., 1-1 Tsutsumidori Amamiyamachi, Aoba-ku, 981-8555 Sendai, Japan
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Transcytosis of F4 fimbriae by villous and dome epithelia in F4-receptor positive pigs supports importance of receptor-dependent endocytosis in oral immunization strategies. Vet Immunol Immunopathol 2008; 124:29-40. [DOI: 10.1016/j.vetimm.2006.10.014] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2006] [Revised: 09/26/2006] [Accepted: 10/11/2006] [Indexed: 11/24/2022]
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Chin K, Onishi S, Yuji M, Inamoto T, Qi WM, Yamamoto K, Warita K, Yokoyama T, Hoshi N, Kitagawa H. Special sugar expression on apoptotic epithelial cells of Peyer's patches and intestinal villi in rat small intestine. J Vet Med Sci 2007; 69:193-9. [PMID: 17339765 DOI: 10.1292/jvms.69.193] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Our previous study clarified that the apical regions of both the follicle-associated epithelium (FAE) of Peyer's patches and the intestinal villi are the only adhesion sites of indigenous bacteria in rat jejuno-ileum. To survey the ligands against bacterial lectins, sugar expression patterns on epithelial cells were lectin-histochemically investigated using 21 lectins in the jejuno-ileal Peyer's patches of rats. As a result, (D-glcNAc)(2-4), detected by Solanum tuberosum (STL) and by Lycopersicon esculentum (LEL), and beta-D-gal(1-3)-D-galNAc detected by Peanut agglutinin (PNA), were strongly expressed on the brush borders of the apical regions of the FAE and the intestinal villi. On the other hand, neither sugar was expressed on the brush borders of the basal regions of both FAE and intestinal villi. The positive intensities for the lectins correlated with the progression of epithelial apoptosis in the FAE and in the intestinal villi. Moreover, the double staining with lectin histochemical method and the in situ nick end-labeling method could simultaneously detect the strong expression of both sugars and nuclear DNA fragmentation in epithelial cells at the late apoptotic stage. Other sugar expression patterns in the intestinal villi were similar with those in the FAE. There were no lectins specific for M cells in the FAE. From these findings, the possible sugars of ligands against some indigenous bacterial lectins, expressing specially on the apoptotic epithelial cells, might be narrowed down in rat jejuno-ileum.
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Affiliation(s)
- Keigi Chin
- Department of Bioresource and Agrobiosciences, Graduate School of Science and Technology, Kobe University, Japan
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Kanaya T, Aso H, Miyazawa K, Kido T, Minashima T, Watanabe K, Ohwada S, Kitazawa H, Rose MT, Yamaguchi T. Staining patterns for actin and villin distinguish M cells in bovine follicle-associated epithelium. Res Vet Sci 2007; 82:141-9. [PMID: 16949627 DOI: 10.1016/j.rvsc.2006.05.009] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2005] [Revised: 04/19/2006] [Accepted: 05/30/2006] [Indexed: 01/16/2023]
Abstract
M cells play a central role in the initiation of mucosal immune responses. However, a primary source of difficulty for investigations of this is the lack of an available specific marker for bovine M cells. As M cells possess irregular and short microvilli, we investigated the distribution and localization of the microvillar proteins actin and villin by immunohistochemistry of the gut of calves. In ileum of the calf, actin and villin were clearly and continuously immunostained in the brush border of the villous epithelia, however, discontinuous immunostaining with patches of no staining were observed in follicle-associated epithelium (FAE). Electron microscopy revealed that M cells had irregular microvilli and lacked the typical brush border, and it was inferred that these patches of no staining might be the intercellular crevices of M cells. As the microvilli of M cells were very sparse, there were several areas of weak immunostaining in calf jejunal FAE. These results suggest that M cells in calf FAE are detectable by the absence of staining for actin and villin.
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Affiliation(s)
- Takashi Kanaya
- Laboratory of Functional Morphology, Tohoku University, 1-1 Tsutsumidori, Amamiyamachi, Aoba-ku, 981-8555 Sendai, Japan
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Abstract
The gastrointestinal tract represents the largest mucosal membrane surface in the human body. The immune system in the gut is the first line of host defense against mucosal microbial pathogens and it plays a crucial role in maintaining mucosal homeostasis. Membranous or microfold cells, commonly referred to as microfold cells, are specialized epithelial cells of the gut-associated lymphoid tissues (GALT) and they play a sentinel role for the intestinal immune system by delivering luminal antigens through the follicle-associated epithelium to the underlying immune cells. M cells sample and uptake antigens at their apical membrane, encase them in vesicles to transport them to the basolateral membrane of M cells, and from there deliver antigens to the nearby lymphocytes. On the flip side, some intestinal pathogens exploit M cells as their portal of entry to invade the host and cause infections. In this article, we briefly review our current knowledge on the morphology, development, and function of M cells, with an emphasis on their dual role in the pathogenesis of gut infection and in the development of host mucosal immunity.
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des Rieux A, Fievez V, Théate I, Mast J, Préat V, Schneider YJ. An improved in vitro model of human intestinal follicle-associated epithelium to study nanoparticle transport by M cells. Eur J Pharm Sci 2007; 30:380-91. [PMID: 17291730 DOI: 10.1016/j.ejps.2006.12.006] [Citation(s) in RCA: 223] [Impact Index Per Article: 12.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2006] [Revised: 12/13/2006] [Accepted: 12/29/2006] [Indexed: 11/28/2022]
Abstract
An alternative in vitro model of human follicle-associated epithelium (FAE) to study nanoparticle transport mechanisms by M cells was developed and characterized. The previous in vitro model of human FAE has been improved by inverting inserts after Caco-2 cell seeding. Raji and M cells were identified only in inverted co-culture cell monolayers by immunohistochemistry, confocal microscopy, and electron microscopy. The M cell conversion rate evaluated by scanning electron microscopy ranged between 15 and 30% of cells. Transport of 200 nm carboxylated polystyrene nanoparticles was higher and more reproducible in the inverted model. Nanoparticle transport was temperature-dependent, not affected by the presence of EGTA or by potassium depletion, but inhibited by EIPA or nystatin, suggesting that it occurs most likely by macropinocytosis. The inverted model appears more physiologic, functional and reproducible than the normally oriented model.
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Affiliation(s)
- Anne des Rieux
- Université catholique de Louvain, Unité de Pharmacie Galénique, Avenue E. Mounier, 73-20, 1200 Brussels, Belgium
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Bimczok D, Post A, Tschernig T, Rothkötter HJ. Phenotype and distribution of dendritic cells in the porcine small intestinal and tracheal mucosa and their spatial relationship to epithelial cells. Cell Tissue Res 2006; 325:461-8. [PMID: 16673104 DOI: 10.1007/s00441-006-0195-3] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2005] [Accepted: 02/15/2006] [Indexed: 01/22/2023]
Abstract
Dendritic cells (DC) as key mediators of tolerance and immunity perform crucial immunosurveillance functions at epithelial surfaces. In order to induce an immune response, the DC have to gain access to antigens present at the luminal surface of mucosal epithelia. The mechanisms of this process are still largely unclear. We have therefore analysed the distribution of DC in the porcine intestinal and respiratory mucosa and their spatial relationship to epithelial cells by immunohistology. Immunofluorescence analysis of cryosections taken from jejunal Peyer's patches and double-stained for DC and M cells (specialised for antigen uptake) have revealed that 35.2+/-3.9% of M cells are located directly adjacent to DC in the subepithelial domes, representing possible antigen transfer sites. In normal jejunal villi, a rare population of lamina propria DC extending cytoplasmic processes between enterocytes has been identified as a possible correlate for direct luminal antigen uptake. Like small intestinal DC, DC in the porcine trachea mostly co-express CD16 with MHC-II. Tracheal DC have been found at high densities both above and below the basement membrane (BM) of the tracheal epithelium, with 32.4 DC/mm BM and 23.0 DC/mm BM, respectively. The intraepithealial DC population forms a dense network, with many of the cytoplasmic processes being directed towards the tracheal lumen. Our morphological analyses indicate that DC at mucosal epithelial sites are ideally positioned for the uptake of luminal antigens.
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Affiliation(s)
- Diane Bimczok
- Institute of Anatomy, Medical Faculty, Otto von Guericke University, 39120 Magdeburg, Germany.
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Snoeck V, Verfaillie T, Verdonck F, Goddeeris BM, Cox E. The jejunal Peyer's patches are the major inductive sites of the F4-specific immune response following intestinal immunisation of pigs with F4 (K88) fimbriae. Vaccine 2006; 24:3812-20. [PMID: 16099554 DOI: 10.1016/j.vaccine.2005.07.025] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/01/2022]
Abstract
A recently developed oral immunisation model in pigs in which F4 (K88) fimbriae of enterotoxigenic Escherichia coli are administered to induce a protective intestinal immunity, was used to determine the optimal inductive sites of the F4-specific intestinal immune response. Hereto, pigs were immunised with F4 orally, in the lumen of the mid-jejunum, ileum or mid-colon. Throughout the small intestine, the highest number of ASC was found following jejunal immunisation, followed by ileal, oral and colonic immunisation. To determine the signifance of Peyer's patches in the induced immune response, F4 was injected into the jejunal Peyer's patches (JPP), lamina propria (LP) and ileal Peyer's patches (IPP). Immunisation in the JPP induced the highest number ASC in the small intestine, whereas immunisation in the LP and IPP resulted in lower intestinal antibody responses. In conclusion, we have shown that the JPP are the major inductive sites of the F4-specific intestinal antibody response. This knowledge could be important when using the pig as an animal model for vaccination studies.
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Affiliation(s)
- V Snoeck
- Laboratory of Veterinary Immunology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium.
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Shakweh M, Ponchel G, Fattal E. Particle uptake by Peyer's patches: a pathway for drug and vaccine delivery. Expert Opin Drug Deliv 2005; 1:141-63. [PMID: 16296726 DOI: 10.1517/17425247.1.1.141] [Citation(s) in RCA: 106] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
Particle uptake by Peyer's patches offers the possibility of tailoring vaccines that can be delivered orally. However, particle uptake by the follicle-associated epithelium in the gastrointestinal tract depends on several different factors that are the physicochemical properties of the particles, the physiopathological state of the animal, the analytical method used to evaluate the uptake and finally the experimental model. These parameters do not allow a clear idea about the optimal conditions to target the Peyer's patches. The goal of this review is to clarify the role of each factor in this uptake.
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Affiliation(s)
- Monjed Shakweh
- University of Paris-South, Faculty of Pharmacy, UMR CNRS 8612, 5 rue Jean-Batiste Clement, 92290 Chatenay-Malabry Cedex, France
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Miyazawa K, Aso H, Kanaya T, Kido T, Minashima T, Watanabe K, Ohwada S, Kitazawa H, Rose MT, Tahara K, Yamasaki T, Yamaguchi T. Apoptotic process of porcine intestinal M cells. Cell Tissue Res 2005; 323:425-32. [PMID: 16283391 DOI: 10.1007/s00441-005-0086-z] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2005] [Accepted: 09/12/2005] [Indexed: 12/20/2022]
Abstract
Membranous (M) cells of the follicle-associated epithelium (FAE) are believed to sample antigens from the gut lumen. However, the origin, differentiation mechanism, and cell death of M cells are still a matter of controversy. Therefore, we investigated the process of M cell differentiation and determined their fate in the intestine of three-way crossbred female pigs. We used anti-cytokeratin 18 and anti-PCNA antibodies to distinguish M cells and proliferative cells and performed immunohistochemistry, enzyme histochemistry, and scanning electron microscopy on fresh ileal Peyer's patches. Cell migration and apoptotic cells were detected by BrdU labeling and the TUNEL method, respectively. The turnover of the FAE was similar to that of the villi. M cells were mostly observed from the FAE crypt to the FAE periphery, but not in the FAE apex. As proliferative M cells (cytokeratin 18(+)/PCNA(+) cells) have previously been detected in the FAE crypt, porcine M cells may be directly derived from intestinal epithelial stem cells and committed as a distinct cell lineage in the crypts. M cells from the FAE periphery were unstained or only weakly stained for alkaline phosphatase, whereas cytokeratin 18(+)/alkaline phosphatase(+) cells lying near to the FAE apex showed a columnar shape similar to that of adjacent enterocytes. These data suggest that the committed M cells differentiate to mature M cells by contact with lymphocytes at the FAE periphery, and that they trans-differentiate to enterocytes and are finally excluded near the FAE apex.
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Affiliation(s)
- Kohtaro Miyazawa
- Laboratory of Functional Morphology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, 981-8555, Sendai, Japan
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Schierack P, Nordhoff M, Pollmann M, Weyrauch KD, Amasheh S, Lodemann U, Jores J, Tachu B, Kleta S, Blikslager A, Tedin K, Wieler LH. Characterization of a porcine intestinal epithelial cell line for in vitro studies of microbial pathogenesis in swine. Histochem Cell Biol 2005; 125:293-305. [PMID: 16215741 DOI: 10.1007/s00418-005-0067-z] [Citation(s) in RCA: 304] [Impact Index Per Article: 15.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 08/17/2005] [Indexed: 11/25/2022]
Abstract
In vitro studies on the pathogenesis in swine have been hampered by the lack of relevant porcine cell lines. Since many bacterial infections are swine-specific, studies on pathogenic mechanisms require appropriate cell lines of porcine origin. We have characterized the permanent porcine intestinal epithelial cell line, IPEC-J2, using a variety of methods in order to assess the usefulness of this cell line as an in vitro infection model. Electron microscopic analyses and histochemical staining revealed the cells to be enterocyte-like with microvilli, tight junctions and glycocalyx-bound mucin. The functional integrity of monolayers was determined by transepithelial electrical resistance (TEER) measurements. Both commensal bacteria and important bacterial pathogens were chosen for study based on their principally different infection mechanisms: obligate extracellular Escherichia coli, facultative intracellular Salmonella and obligate intracellular Chlamydia. We determined the colonization and proliferation of the bacteria on and within the host cells and monitored the host cell response. We verified the expression of mRNAs encoding the cytokines IL-1alpha, -6, -7, -8, -18, TNF-alpha and GM-CSF, but not TGF-beta or MCP-1. IL-8 protein expression was enhanced by Salmonella invasion. We conclude that the IPEC-J2 cell line provides a relevant in vitro model system for porcine intestinal pathogen-host cell interactions.
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Affiliation(s)
- Peter Schierack
- Institut für Mikrobiologie und Tierseuchen, Freie Universität Berlin, Berlin, Germany.
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Tohno M, Shimosato T, Kitazawa H, Katoh S, Iliev ID, Kimura T, Kawai Y, Watanabe K, Aso H, Yamaguchi T, Saito T. Toll-like receptor 2 is expressed on the intestinal M cells in swine. Biochem Biophys Res Commun 2005; 330:547-54. [PMID: 15796917 DOI: 10.1016/j.bbrc.2005.03.017] [Citation(s) in RCA: 55] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2005] [Indexed: 12/13/2022]
Abstract
The Toll-like receptor (TLR) 2 binds a wide variety of microbial cell wall components. In this study, we investigated the expression pattern of TLR2 in adult swine gut-associated lymphoid tissues using real-time quantitative PCR, Western blotting, immunohistochemistry, and flow cytometric analysis. The mRNA for TLR2 was preferentially expressed in the mesenteric lymph nodes (MLNs) and Peyer's patches (Pps) of adult swine. Expression in these two tissues was approximately 15- and 9-fold higher than that of spleen, respectively. Western blotting further confirmed that the TLR2 protein was highly expressed in the MLNs and Pps. Interestingly, TLR2-expressing cells were found not only in immune cells, such as T cells and B cells, but also in membranous (M) cells. In addition, double immunostaining for TLR2 and cytokeratin 18 revealed that TLR2 was strongly expressed not only in the cytoplasm but also in the apical membrane of the pocket-like M cells. These results indicate that TLR2 on the MLNs and Pps enable the host defense to respond to a variety of cell wall components. Furthermore, the potential function of TLR2 as a pattern recognition receptor and its cellular distribution suggest that TLR2 plays an important role in ligand-specific transcytosis and transport in M cells.
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Affiliation(s)
- Masanori Tohno
- Laboratory of Animal Products Chemistry, Graduate School of Agricultural Science, Tohoku University, Aobaku, Sendai 981-8555, Japan
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Shimosato T, Tohno M, Kitazawa H, Katoh S, Watanabe K, Kawai Y, Aso H, Yamaguchi T, Saito T. Toll-like receptor 9 is expressed on follicle-associated epithelia containing M cells in swine Peyer's patches. Immunol Lett 2004; 98:83-9. [PMID: 15790512 DOI: 10.1016/j.imlet.2004.10.026] [Citation(s) in RCA: 61] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2004] [Revised: 10/27/2004] [Accepted: 10/27/2004] [Indexed: 12/13/2022]
Abstract
The precise distribution and expression of Toll-like receptor (TLR) 9 in gut-associated lymphoid tissues (GALTs) has not been elucidated. In this study, we investigated the expression pattern of TLR9 in adult and neonatal swine GALTs by real-time quantitative PCR, western blot, confocal laser microscopy and flow cytometric analysis. The swine TLR9 gene was preferentially expressed in adult Peyer's patches (Pps) and mesenteric lymph nodes (MLNs), which contained approximately three times higher TLR9 than the spleen. Other tissues exhibited only weak expression of TLR9. In neonatal swine, elevated expression of TLR9 was detected only in MLNs. We firstly showed that highly expressive (TLR9(+)) cells were formed in Pps and MLNs. In addition, TLR9(+) cells were present not only in immune cells such as dendritic cells and B cells but also in follicle-associated epithelia (FAE) including membranous cells (M cells) in Pps. These results suggest that Pps and MLNs provide the host defense with the ability to respond to a variety of bioactive oligonucleotides (ODNs) from bacteria at a conductive site of initial immune responses.
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Affiliation(s)
- Takeshi Shimosato
- Laboratory of Animal Products Chemistry, Graduate School of Agricultural Science, Tohoku University, Japan
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Carapelli A, Regoli M, Nicoletti C, Ermini L, Fonzi L, Bertelli E. Rabbit tonsil-associated M-cells express cytokeratin 20 and take up particulate antigen. J Histochem Cytochem 2004; 52:1323-32. [PMID: 15385578 DOI: 10.1177/002215540405201008] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
Abstract
M-cells are believed to play a pivotal role in initiation of the immune response. These cells, located in the epithelia that overlie mucosal lymphoid follicles, are responsible for the active uptake of particulate antigens and for their translocation to the underlying lymphoid tissue. The identification of reliable markers for M-cells is therefore extremely important for the study of the initial steps that lead to the immune response. For this purpose, we studied cytokeratin 20 (CK20) expression in the epithelium of rabbit palatine tonsils by immunofluorescence, confocal microscopy, and Western blotting. CK20+ cells were observed in all rabbit palatine tonsils examined. By Western blotting, one CK20-immunoreactive band was identified at 46 kD on samples of proteins from the intermediate filament-enriched cytoskeletal fraction of tonsil epithelium. Double labeling of CK20+ cells with cell-specific markers confirmed that such cells were actually M-cells. Moreover, CK20+ M-cells displayed a mature phenotype (they formed pockets harboring lymphoid cells) and were functionally competent because they could take up particulate antigens from the pharyngeal lumen. We conclude that CK20 is an M-cell marker for rabbit palatine tonsils. Moreover, we can hypothesize the use of M-cells as a possible site for antigen delivery of particle-based vaccines.
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Affiliation(s)
- Alessandro Carapelli
- Dept. of Pharmacology "Giorgio Segre," Section of Morphology, Via Aldo Moro 4, University of Siena, Siena, Italy.
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Schauser K, Olsen JE, Larsson LI. Immunocytochemical studies of Salmonella Typhimurium invasion of porcine jejunal epithelial cells. J Med Microbiol 2004; 53:691-695. [PMID: 15184542 DOI: 10.1099/jmm.0.45582-0] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Although infection of pigs with Salmonella Typhimurium represents a serious problem, most studies on Salmonella infection have been carried out in other species. The purpose of the current study was to examine the route(s) of entry of Salmonella Typhimurium in pigs, using a jejunal loop model. The infection process was followed over 240 min using single to triple immunocytochemical detection of Salmonella and intestinal cell markers. Salmonella invasion was observed in both cytokeratin-18-positive and -negative cylindrical absorptive cells within 5-10 min. Subepithelial invasion of ordinary villi was consistently less marked than invasion of the subepithelial layer of Peyer's patches. Our results show that several epithelial cell types were invaded by Salmonella, and that Peyer's patches represent the main portal of entry in early Salmonella infection. Additionally, infection was associated with alterations in the keratin and F-actin cytoskeleton of intestinal epithelial cells, probably reflecting toxin-mediated actions. Such changes were confined to the proximal region of the jejunum, demonstrating a regional heterogeneity of intestinal epithelial cell responses to Salmonella infection.
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Affiliation(s)
- Kirsten Schauser
- Department of Anatomy and Physiology1 and Department of Veterinary Microbiology2, The Royal Veterinary and Agricultural University, Gronnegaardsvej 7, DK-1870 Frederiksberg C, Denmark
| | - John Elmerdahl Olsen
- Department of Anatomy and Physiology1 and Department of Veterinary Microbiology2, The Royal Veterinary and Agricultural University, Gronnegaardsvej 7, DK-1870 Frederiksberg C, Denmark
| | - Lars-Inge Larsson
- Department of Anatomy and Physiology1 and Department of Veterinary Microbiology2, The Royal Veterinary and Agricultural University, Gronnegaardsvej 7, DK-1870 Frederiksberg C, Denmark
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Gebert A, Steinmetz I, Fassbender S, Wendlandt KH. Antigen transport into Peyer's patches: increased uptake by constant numbers of M cells. THE AMERICAN JOURNAL OF PATHOLOGY 2004; 164:65-72. [PMID: 14695320 PMCID: PMC1602236 DOI: 10.1016/s0002-9440(10)63097-0] [Citation(s) in RCA: 66] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Membranous (M) cells are specialized epithelial cells of the Peyer's patches that sample antigens from the gut lumen, thereby enabling the host to respond immunologically. Recent studies suggest that this transport can be up-regulated within hours by de novo formation of M cells from enterocytes. To test this hypothesis, we used an in vivo model and induced the transcytosis of tracers in Peyer's patches by application of Streptococcus pneumoniae R36a into the gut lumen. Using cell-type-specific markers, we quantified M cells in the Peyer's patch domes, lymphocytes associated with M cells, and the transport rate for experimentally applied microbeads after 3 hours of exposure to R36a. The transport of latex microbeads was significantly increased by +131% in the R36a-treated patches as compared to buffer controls (P < 0.001). While in controls, each M cell was associated with 2.05 +/- 0.64 lymphocytes, a significant increase (+55.1%; P < 0.001) was determined in the R36a-treated patches. However, no statistical difference was detected in the percentage of M cells in the dome epithelia (46.0 +/- 4.6% versus 45.5 +/- 3.8%). It is concluded that bacteria-induced up-regulation of particle transport in Peyer's patch domes is due to an increased transport rate of the M cells, but not to a de novo formation of M cells. The data support the hypothesis that M cells represent a separate cell lineage that does not derive from enterocytes on the domes.
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Affiliation(s)
- Andreas Gebert
- Institute of Anatomy, University of Lübeck, Lübeck, Germany.
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