M-cells

1. M-cells Not your average mucosal epithelial cell Iahn Cajigas (iahn@mit.edu) HST.120 Gastrointestinal Pathophysiology Group 4 Dr. Rufo, Rm 425 Session Presentation

2. Outline • Overview and Introduction (5 min) • What are M-cells? • Where are they located? • What is their role in normal physiology? • M-cells as a route for host invasion • Medical Use of Normal Physiologic Role of M-cells for Vaccine Development • Presentation of one key research article (10 min) • Efficacy of a food plant-based oral cholera toxin B subunit vaccine • Critical analysis of the findings and implications of the article (10 min) • Question and Answer (5 min)

3. What are M-cells? • Specialized epithelial cells located in the follicle-associated epithelium (FAE). • Deliver intact antigens through active trans-epithelial vesicular transport from the GI lumen to intraepithelial lymphoid cells and to organized mucosal lymphoid tissues that are designed to process antigens and initiate mucosal immune response. • Restricted to sites directly over lymphoid follicles. • Hypothesis: Reduces the inherent risk of transporting intact foreign material and microbes across the epithelial barrier by assuring immediate exposure to phagocytes and antigen-presenting cells. • M cells are well suited for efficient endocytosis and transcytosis. • Lack the rigid brush border cytoskeleton. • Apical surfaces have broad membrane microdomains from which endocytosis occurs. Lack villous structure of enterocytes. • Basolateral membrane forms large intraepithelial “pocket” containing T lymphocytes ,B lymphocytes, and macrophages (Neutra et al., 1996). • Intraepithelial “pocket” brings the basolateral cell surface to within a few microns of the apical surface and greatly shortens the distance that transcytotic vesicles must travel to cross the epithelial barrier. • Endocytic or phagocytic uptake of foreign antigens or particles is followed by rapid transcytosis directly to the intraepithelial pocket, with little or no retention in M cell lysosomes. • Contain apical receptors for IgA whereas enterocytes have IgA-specific receptors in their basolateral membranes. • enterocytes transport newly synthesized IgA into the GI lumen and M-cells take up antigens that have been recognized by secreted IgA and reintroduce them in the subepithelial pocket for antigen processing and presentation by APCs.

4. Where are they? Lymphoid Follicles • Located in the small intestine, colon, rectum, tonsils, and adenoids. • Luminal contents sampled by M-cells and “periscoping” DCs. • “Periscoping” DCs typically seen in muscosal surfaces that lack lymphoid follicles (eg. vaginal epithelium), but also present in follicles. • Consist of M-cells, DCs, T-cells, and B-cells Mucosal vaccines: the promise and the challenge. Neutra MR, Kozlowski PA. Nat Rev Immunol. 2006 Feb;6(2):148-58.

5. Normal Physiological Role Antigen uptake: Enterocytes vs. M-cells • M-cells “constrained” to locations with underlying follicles. • Thought to limit the transport of intact antigens to locations prepared to process and present potential pathogens. • Lack think apical glycocalyx and villous structure of enterocytes. • Sample luminal contents. • Clathrin-mediated endocytosis observed to occur. Other methods like receptor-mediated endocytosis, pinocytosis, etc. have not been ruled out. • Enterocytes do not transport intact antigens. • Have MHC II on basolateral membrane. • Upregulate co-stimulatory molecules upon binding of TLR to luminal antigens. • Release pro-inflammatory or anti-inflammatory cytokines. Mucosal vaccines: the promise and the challenge. Neutra MR, Kozlowski PA. Nat Rev Immunol. 2006 Feb;6(2):148-58.

6. M-cell: Apical Surface Glycocalyx Glycocalyx Enterocyte M-cell Short apical-to-basolateral distance. Subepithelial pocket Epithelial M Cells: Gateways for Mucosal Infections and Immunization Neutra et al. Cell, Vol. 86, 345–348, August 9, 1996

7. M-cell: Luminal Antigen Transport • Intact antigen presentation is a double-edged sword for the immune system. • Can sample intact luminal antigens so that neutralizing antibodies can be made. • Mechanism can be hijacked by various pathogens • Shigella, Salmonella, Listeria, Prions, and HIV are known to primarily enter hosts through M-cells. • pathogenic strains of Shigella are selectively transcytosed by M cells; followed by local invasion of adjacent epithelial cells via basolateral cell surfaces, rapid influx of neutrophils and macrophages, and cytokine-mediated disruption of the epithelial barrier (Perdomo et al., 1994; Figure 2C). Reovirus M-cell basolateral membrane Epithelial M Cells: Differentiation and Function, Kraehenbuhl, J., Neutra, M.R., Annu. Rev. Cell Dev. Biol. 2000. 16:301–32.

8. Research Article Discussion

9. Experimental Design • Goal: Investigate trangenic food plants for the production and delivery of oral vaccine against microbial enteropathogens and their toxins, using CT (the prototypical diarrhea-inducing toxin), as a model system. • Immunization of CD-1 mice by transgenic potato plants transformed with the CTB gene. • 4 Group • 30ug CTB in Sodium Bicarbabonate immunization group (pH 8.5) by oral feeding (Positive control) • 1g of untransformed-potato tissues (5 mice) (Negative Control) • 1g of transformed-potato tissue (10 mice) • 3g of transformed-potato tissue (8 mice)

10. Experimental Design (cont.) CTB Pentamer present with boiling

11. Anti-CTB Antibody Titers Fecal IgA on Day 38 Serum anti-CTB Antibodies on days 35 and 70 Mucosal anti-CTB antibody titers

12. Cholera Toxin Neutralization Assay in Cell Culture Vero cells + CT + serum of immunized mice Vero cells + CT + serum of unimmunized mice Not shown: Vero cells + serum of unimmunized moue looks like A. Conclusion: Serum of immunized mouse contain protective factor (i.e. neutralizing antibodies) to CT.

13. Neutralization Titers of CT vary depending on method of vaccination • Vaccination with CT directly lead to a much higher concentration of serum neutralizing antibody as demonstrated by the ability of higher dilutions of CTB-vaccinated mice serum to still neutralize CTB in cell cultures. • Could be due to a multitude of factors including: differing effective concentrations of antigen delivered to M-cells, decreased antigenicity of transgenic CTB pentamer (due to ER localizing sequence), or decreased expression of CTB protein within the transgenic plant (e.g. promoter effects)

14. Reduction of CT-induced diarrhea in immunized mice Ileal loop of unimmunized mouse exposed to CTB • Significant difference between negative-control group and orally immunized mice. • Injection of saline to ileal loops of both immunized and non-immunized mice did not result in accumulation of fluid suggesting that mechanical disturbance during loop ligation did not result in inflammatory response. Ileal loop of Immunized mouse exposed to CTB

15. Gm1-ELISA assay of CT binding to Gm1-ganglioside. • CTB preferentially binds to GM1-gangliosides (mostly on M-cells in vivo) as demonstrated by the GM1-ELISA result on the left. • Addition of Immune serum prevents CTB from binding to GM1-gangliosides and results in reduced intensity on the ELISA. • The negative control indicates that in the absence of GM1-gangliosides neither immune serum or non-immune serum bind to the wells.

16. Analysis and Implications • Oral immunization against cholera toxin was achieved by a transgenic potato plant. • Achieves both a serum and mucosal immunity by administration of only oral vaccine. • Demonstrated by neutralization of cholera toxin using serum of immunized mice both visually with a VERO (African green monkey kidney cells) cell assay. Indicates presence of serum neutralizing antibodies. • Both IgA and IgG present in feces of immunized mice • The immunity is not as strong as the that observed with just the cholera toxin. • Change in sequence affects the conformation of the protein. • Decreased affinity to the GM1-ganglioside. • Although CTB binds to Gm1-gangliosides (expressed on all epithelial cells), it preferentially binds M-cells due to the decreased size of the glycocalyx and lack of villous structure these cells. CTB can thus function as an effective carrier for induction of a mucosal immune response to polypeptides to which CTB is chemically or genetically conjugated.

17. Analysis and Implications (cont.) • Experiment demonstrates that antibody titers can be boosted by oral administration of additional vaccine food plant tissues when protective titer declines. • Application of the CTB pentamer as an effective carrier for conjugated peptides in food plants will move us closer to achievement of low cost, convenient, effective, and safe strategy for prevention of infections enteric diseases as well as autoimmune diseases in animals and in man, especially in regions of the world where conventional vaccines are unaffordable as well as unavailable. • Zero risk of transformation of attenuated vaccine strains into pathogenic strains! • Need to keep carrier-conjugated protein complex within specific size and charge to allow proper interaction with M-cells. Protein complex must maintain its conformation through the GI-tract until it binds to the M-cells so that the full immune response can be achieved.

18. Questions? • Please be gentle.

19. Oral Tolerance • A) Generation of an immune response requires ligation of the T-cell receptor with peptide-MHC complexes in the presence of appropriate costimulatory molecules (CD80 and CD86) and cytokines. • B) With high doses of oral antigen, T-cell receptor cross-linking can occur in the absence of costimulation or in the presence of inhibitory ligands (CD95 and CD95 ligand), leading to anergy or deletion, respectively. • C) Low doses of oral antigen lead to the activation of regulatory T cells, which suppress immune responses through soluble or cell surface–associated suppressive cytokines (IL-10 and TGF-b).