Roles of M cells in infection and mucosal vaccines

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.

Keywords:

• antigen
• infection
• M cells
• mucosal immunity
• vaccine

Abbreviations:

• 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
• MALT, mucosa-associated lymphoid tissue
• M cells, microfold cells
• NALT, nasopharynx- or nose-associated lymphoid tissue
• OmpH, outer membrane protein H
• OVA, ovalbumin
• PP, Peyer's patches
• PrPC, cellular prion protein
• PRRs, pathogen recognition receptors
• pσ1, reovirus surface protein σ1
• SIgA secretory IgA
• SELEX, Systematic Evolution of Ligands by EXponential enrichment
• TLR-4, Toll-like receptor-4
• UEA-1,Ulex europaeus agglutinin-1

Introduction

Despite continuous exposure to a broad range of biological stimuli, the mucosal surfaces provide a first line of defense against potential pathogens and nonself antigens, because most infections are initiated at mucosal sites. The mucosal immune system plays a vital role in maintaining mucosal homeostasis and consistently surveilling potential harmful agents. The mucosal immune system is essentially divided into 2 compartments known as inductive and effector sites, which work together to maintain the mucosal barrier.¹ The inductive sites mainly consist of mucosa-associated lymphoid tissue (MALT), including gut-associated lymphoid tissue (GALT), nasopharynx- or nose-associated lymphoid tissue (NALT), bronchus-associated lymphoid tissue (BALT), and genital-associated lymphoid tissue (GENALT), where antigen sampling occurs and stimulates cognate naive T and B lymphocytes. Although the effector sites are primarily constituted by the lamina propria of various mucosae, stroma of exocrine glands, and surface epithelia, at which antigen-specific antibodies and immune cells perform their specific action.²

M cells (membranous epithelial, microfold or microvillous cells), first observed in 1965,³ are functional units constituting inductive sites of the intestinal mucosal immune system. In the human gastrointestinal tract, M cells are predominantly present on the follicle-associated epithelium (FAE) which overlies the dome structure of Peyer's patches (PPs) in the small intestine,⁴ and have been identified in the crypt epithelium of human tonsils and adenoids⁵ and colon and rectum of mice.⁶ They are specialized epithelial cells of GALT, playing a crucial role in the intestinal immune system by sampling and uptake of antigens, and then transporting them from the gut lumen to the underlying immune system.⁷ Gastrointestinal contents transported via M cells can be captured by dendritic cells (DCs) resident beneath M cells to initiate mucosal immune responses, leading to the production of antigen specific IgA by B cells.⁸ M cells are among the most important epithelial cell types involved in the adhesion, uptake and sampling of antigens at mucosal surfaces, which facilitates M and adjacent cells working together to maintain mucosal homeostasis.

Distribution and function of M cells

Distribution of M cells

M cells exist principally in the FAE overlying both isolated (i.e. solitary nodules) and aggregated (i.e., PP) lymphoid follicles⁹ in GALT, and as well in NALT¹⁰ and BALT.¹¹ They can be easily recognized and distinguished from neighboring enterocytes and goblet cells by their unique morphological characters, which include irregular brush border, high endocytic activity, reduced microvilli, thin glycocalyx, sparse lysosomes, and basolateral pockets containing mononuclear phagocytes and lymphocytes. They possess tight junctions and desmosomes that contact adjacent columnar cells and interdigitating latacoria (Fig. 1). These features are adaptions allowing materials (macromolecules and microorganisms) in the lumen to have easy access to the internalized apical surface of M cells and subsequently to be transported to the underlying tissues. However, the ultrastructure of M cells shows some variation between species and anatomical regions.⁷ Therefore, identifying M cells needs morphological features at the light microscopic level and specific M cells markers to isolate or target them. Cellular morphology requires ultrastructural examination using scanning and transmission electron microscopy, which are labor intensive and not available to many researchers. Specific M cell markers are essential, whereas the expression of cellular antigens by M cells varies widely among species and anatomical sites.⁷ Despite no universal M cell markers to date, many potential markers have been studied in humans and experimental animals in the past few years, using species- and tissue-specific M cell markers (Table 1).¹²

Table 1. M cell markers in various species and MALT sites

M cell markers	Species	MALT sites	References
Clusterin	Human	Tonsils, adenoids, PPs, appendix, colon	^75
Cathepsin E	Human	Tonsils, adenoids, PPs, appendix, colon (not specific, also found in FAE and crypt epithelium)	^59, 76
GP2	Mouse	PPs	^38, 45
Vimentin	Rabbit, horse	Tonsils, PPs, adenoids, appendix and cecal patches.	^77–79
β1 integrin	Mouse	PPs	^80
Annexin V	Mouse	PPs	^54
Polymerized actin	Mouse	Cecal patches	^81
Cytokeratin 8	Rat	PPs	^82
Cytokeratin 18	Swine, bovine	PPs and cecal patches	^83, 84
Fucose	Mouse, rabbit	PPs and cecal patches	^85–88

PPs, Peyer's patches; GP2, Glycoprotein 2; FAE, follicle-associated epithelium; MALT, mucosa-associated lymphoid tissue.

Figure 1. Schematic diagram of intestinal epithelium showing M cells, PPs, intestinal epithelial cells and translocation mechanisms of several kinds of antigen. Gut-associated mucosal immune system has 2 parts, including inductive sites and effector sites. The inductive sites are mainly composed of MALT, and the effector sites are primarily constituted by the lamina propria of various mucosae, stroma of exocrine glands, and surface epithelia. In the FAE, luminal antigens are transported across the epithelium by M cells, where primary immune responses can be induced, and then phagocytosed by DCs present in the dome of the PPs, delivered to the mesenteric lymph nodes, which can directly prime T-cell responses to antigens. DCs also sample antigens through enterocytes. sIgA, secreted by mature plasma cells in the lamina propria, from epithelial cells into the gut lumen, may have a controlling role in bacterial persistence and uptake. M cells can transport bacteria, viruses, parasites and non-infectious particles through the apical membrane to the basolateral surface, exploiting many molecules and proteins to recognize and transport antigen. M cells lack microvilli and possess basolateral pockets containing mononuclear phagocytes and lymphocytes, which contribute antigens transported to the underlying tissues. PP, Peyer's patches; DC, dendritic cell; GP2, Glycoprotein 2; PrP^C, cellular prion protein; C5aR, C5a receptor; FAE, follicle-associated epithelium; MALT, mucosa-associated lymphoid tissue.

Recently, the presence of intestinal villous M cells has challenged the notion of an exclusive location for M cells within the FAE region.¹³ Intestinal villous M cells are located at the top of intestinal villous epithelium, forming small dense clusters or with diffuse distribution. They have a function similar to the PP M cells, and evidence from several studies shows that antigen-specific mucosal immune responses can be induced in GALT- and PP-deficient mice.^13,14

Function of M cells

M cells have the ability to transport bacteria,^15-18 viruses,^19-24 parasites²⁵ and non-infectious particles²⁶ through the apical membrane to the basolateral surface. When bacteria and large particles get into the mucosal lumen, M cells undergo apical membrane ruffling, actin cytoskeleton rearrangements,²⁷ and interdigitation, and then phagocytose these large particles. Viruses and small particles with adhesion properties undergo phagocytosis in clathrin-coated vesicles²⁸ and nonadherent antigens undergo fluid-phase pinocytosis.²⁹ After transporting endocytotic vesicles to the endosomal compartment, internalization occurs quickly, and the vesicles are secreted by exocytosis to the basolateral membrane.³⁰

In contrast, M cells also provide an entry for many pathogens to invade the host and establish infection. Brucella abortus is internalized only in M cells among intestinal epithelial cells, demonstrating a role for M cells as an entry portal for B. abortus after oral infection. Researchers have also detected co-localization of cellular prion protein (PrP^C) and the bacterium on the apical surface of M cells, indicating that PrP^C on M cells serves as a major uptake receptor for B. abortus during oral infection.³¹ As a result, the capacity of M cells for antigen sampling can accelerate invasion by potentially harmful enteric microorganisms.³² An in vitro study in an M-like cell model that expressed caveolin-1 elucidated the role of caveolin-1 in the entry of Salmonella. Downregulation of caveolin-1 expression by siRNA reduced the level of Salmonella transcytosis across the M-like cells.³³ However, M cells deliver these pathogens directly to regions of the immune system fully equipped for coping with an emergency. The best evidence supporting this condition above is provided by studies carried on Shigella spp.,¹⁸ Salmonella spp,³⁴ Yersinia spp.,³⁵ Vibrio cholera, reovirus type-1, poliovirus and HIV-1.³⁶ It has been shown that M cells or antigen sampling by M cells can result in significant attenuation of the antigen-specific immune response in PPs in mice with oral administration of Salmonella enterica serovar Typhimurium.^37,38 In addition, M cells have the ability to distinguish among different commensal bacteria and alter subsequent immune responses.³⁹

It is particularly worth mentioning that M cells also have the capacity to transport proteins,⁴⁰ especially secretory IgA (SIgA). SIgA selectively interacts with M cells, targets DCs located in the sub-epithelial dome region of PP, resulting in limited mucosal and systemic immune responses against a non-self-associated protein antigen,⁴¹ and exploits M cells as the primary route for delivery to the GALT. A recent study has demonstrated the mechanism by which SIgA is selectively bound and taken up. Both the Cɑ1 region and glycosylation, more particularly sialic acid residues, take part in M-cell-mediated reverse transcytosis, and M cells take up SIgA via the Dectin-1 receptor, in which Siglec-5 may act as a co-receptor.⁴² There are several mechanisms of targeting M cells for induction of mucosal SIgA responses in animal models. For instance, it has been demonstrated that M cell targeting mediated by a Claudin-4-specific targeting peptide enhances mucosal IgA responses above the response to nontargeted mucosal antigen, which have promise in delivery of mucosal vaccination.⁴³

M cell-targeted mucosal immunization

For many years, researchers have been exploiting the potential of M-cell-specific mechanisms for drug and vaccine delivery to the mucosal immune system.⁴⁴ Many M-cell-targeted molecules have been used for development of mucosal vaccines (Table 2).

Table 2. M-cell-targeting molecules for mucosal vaccination

Receptors on M cells/M cell-specific molecules	Ligand	Reference
α(1,2)-fucose-containing carbohydrate	Antibody NKM 16–2–4	^61
α-l-fucose (murine PP M cells)	UEA-1	^69, 70, 87, 89, 90
C5aR	Peptide Co1, OmpH (Yersinia)	^51, 52, 64
α2,3 sialic acid	σ1 protein (reovirus)	^71, 91
β1 integrin	Invasion (Yersinia)	^80
GP2	FimH (E. coli and Salmonella)	^38, 45–47
TLR-4	LPS	^92
Cellular prion protein	Hsp60 of B. abortus	^31, 49, 50
Annexin A5	LPS	^55
bacterial peptidoglycan	Peptidoglycan recognition protein-1	^53

UEA-1,Ulex europaeus agglutinin-1; PP, Peyer's patches; C5aR, C5a receptor; GP2, Glycoprotein 2; TLR-4, Toll-like receptor-4; OmpH, outer membrane protein H; LPS, lipopolysaccharide; Hsp60, heat shock protein 60.

M-cell-specific molecules in mucosal vaccine development

M cells express a large amount of immune-surveillance receptors on the apical surfaces, contributing to the variety of microbial pathogens and antigens. They are provided with an array of molecules to present luminal antigens to the underlying mucosal lymphoid tissues. Therefore, identifying M-cell-specific targeting molecules has been a focus, by recognizing molecules exploited by pathogens to invade M cells.

Glycoprotein 2 (GP2)

GP2 is specially expressed on M cells.⁴⁵ This protein is highly expressed on the apical membranes of PP M cells, but not highly expressed on other enterocyte populations. Recent studies have revealed that GP2 acts as a transcytotic receptor, bound to FimH⁺ bacteria such as Escherichia coli and S. Typhmurium, by recognizing FimH, a major component of the type 1 pilus on the outer membrane of a subset of Gram-negative enterobacilli.³⁸ Thus, GP2 on M cells can act as a transcytotic receptor for bacterial antigens, and worthy of note, participate in the mucosal immune responses to these particular bacteria; a subset of commensal and pathogenic enterobacteria (E. coli and S. Typhmurium).^46,47 Other research has shown that a murine GP2 (mGP2)-specific aptamer, isolated using Systematic Evolution of Ligands by EXponential enrichment (SELEX), with a loop structure and the nucleotide sequence, AAAUA (both important for binding to mGP2), binds to mGP2 expressed on the cell surface, indicating that the aptamer serves as a promising tool for testing M-cell-targeted vaccine delivery in murine model systems.⁴⁸

PrP^C

PrP^C is highly expressed on the luminal side of the apical plasma membrane of murine M cells and co-localized with GP2, suggesting that it is an antigen receptor candidate on M cells.⁴⁹ PrP^C interacts with heat shock protein (Hsp)60 of B. abortus, which had been recognized as an immunodominant antigen of many microbes.⁵⁰ Accumulated evidence suggests that PrP^C on M cells is well placed to contribute to mucosal immunosurveillance by enhancing transcytosis of B. abortus or other exogenous antigens.⁴⁹

C5a receptor (C5aR)

The expression and nonredundant role of C5aR in human M-like cells and mouse M cells have been demonstrated, indicating the role of C5aR as a target receptor to induce the immune response. Sae-Hae Kim et al. verified phosphorylation of C5aR in vivo after oral infection of mice by Yersinia enterocolitica. They confirmed the expression of C5aR in the apical area of mouse M cells and human M-like cells by measuring the expression levels of mRNA and protein.⁵¹ Sae-Hae Kim et al. also used the outer membrane protein H (OmpH) ligand of Y. enterocolitica, which acts as a targeting ligand to C5aR in M cells, to induce specific mucosal and systemic immunity against envelope domain III (EDIII) of dengue virus (DENV), suggesting OmpH-mediated targeting of antigens to M cells as an efficient oral vaccination against DENV infection.⁵²

Other specific molecules

There are other M-cell-specific molecules that may specifically bind to components of potential pathogenic organisms. Peptidoglycan recognition protein 1 is an innate recognition protein binding to bacterial peptidoglycan, and is also expressed highly in M cells.⁵³ Annexin (ANX) A5 expressed by M cells⁵⁴ can bind to lipopolysaccharide (LPS) of Gram-negative bacteria and block endotoxin activity, suggesting that ANXA5 on M cells acts as an uptake receptor for Gram-negative bacteria.⁵⁵ The discovery of M-cell-targeting receptors using pathogen-exploited molecules could be a promising approach in the development of effective mucosal vaccines. Clusterin, fatty acid binding protein, cathepsin E, secretogranin V and other M-cell-expressed proteins may have potential roles in M cell functions, but these are less clearly understood.^56-59 The increasing evidences have demonstrated that M-cell-specific molecular antibody, which is conjugated with antigen protein or liver vector, can transport the antigen to mucosal tissues, leading to produce efficient immune responses. However, some molecules, selected as M-cell-specific molecules, are not uniquely expressed on M cells, resulting in producing a non-ideal oral delivery system for targeting M cells. With the development research on the mechanism of M cell differentiation, we can regulate the immune processes by means of artificial mediation of the M-cell-specific molecules gene expression. For instance, we can increase the efficiency of mucosal vaccination, through booting the expression of certain M-cell-specific molecules. Meanwhile, we can even inhibit the viral infection, by reducing the expression of some molecules, which are necessary for the entry of some virus particles.

M cell ligands as novel and effective mucosal vaccine targets

Many researchers have studied M cell ligands, in order to take advantage of the fact that targeting specific receptors on the apical membrane of M cells could specifically increase antigen uptake and presentation, evoking immune responses and providing protection against infection.⁶⁰

NKM 16-2-4

NKM 16–2–4 can be used as monoclonal antibody to target vaccine antigens to the M-cell-specific carbohydrate moiety.⁶¹ It can distinguish α(1, 2)-fucosylated M cells from goblet cells containing abundant sialic acids neighboring the α(1, 2) fucose moiety and from non-α(1, 2)-fucosylated epithelial cells.⁶¹ The use of NKM 16–2–4 targeting vaccine antigens to M cells could be effective for vaccine delivery into the intestinal mucosa. Oral vaccination using antigen-encapsulated liposomes coupled with UEA-1 could lead to increased uptake by PP M cells and the induction of higher antigen specific sIgA responses.⁶²

Co1 ligand

Many studies have investigated the M-cell-targeting ligand, Co1, selected from a phage display library against differentiated M-like cells, and have produced recombinant antigen fused to the selected ligands using the model antigen.^63-65 Co1 ligand promotes the uptake of fused antigen and enhances the immune response against the fused antigen, indicating that Co1 could be used as an adjuvant for targeted antigen delivery into the mucosal immune system to enhance immune induction.⁶⁴ Another promising approach used Co1 ligand to induce specific immune responses against a pathogenic viral antigen, EDIII of DENV. Efficient antigen delivery into PPs was observed and the antibodies induced by the Co1-ligand-conjugated EDIII antigen showed effective virus-neutralizing activity.⁶⁵ Taken together, these results reveal that M-cell-targeting ligands with adjuvant activity can be designed to exploit our knowledge of receptors expressed on the apical surface of M cells involved in pathogen invasion.^66,67

Caveolin-1

Caveolin-1 is the major structural component of caveolae. It was examined its expression in Caco-2-driven M-like cells, and was verified that co-culturing with B lymphocytes, caveolin-1 could increase the susceptibility of M cells to Salmonella infection.⁶⁸ Some recent studies have shown that caveolin-1 is not only a good marker of human M cells, but also a potent candidate for understanding M cell transcytosis as a novel target for mucosal immunity.

Ulex europaeus agglutinin (UEA)-1

UEA-1 has been confirmed as a specific ligand for α-l-fucose present on the apical membrane of M cells, anchored for selective delivery of antigen to GALT. Some researchers have used nanoparticles coated by UEA-1-conjugated alginate to induce immunological response in BALB/c mice, and compared them with aluminum hydroxide gel-based conventional vaccine. The results demonstrated that immunization with the former induced efficient systemic as well as mucosal immune responses against BSA compared to other formulations, which indicated the potential of UEA–alginate-coated nanoparticles as an effective oral delivery system.⁶⁹ However, UEA-1 lectin also reacted strongly with other issues, such as goblet cells and the mucus layer covering the intestinal epithelium.⁷⁰

Reovirus surface protein σ 1 (pσ1)

pσ1 has the ability to bind M cells, which facilitates reovirus infection via pσ1. A genetic fusion between ovalbumin (OVA) and pσ1 was applied nasally, to enhance tolerogen uptake. Studies showed that OVA– pσ1-mediated tolerance was lost in the absence of interleukin-10, demonstrating that the feasibility of using pσ1 as a mucosal delivery platform specifically for low-dose tolerance induction.⁷¹ Another targeted transgene vaccination using pσ1 conjugated to polylysine through intranasal immunization, could induce mucosal immunity and enhance cell-mediated immunity, leading to prolong mucosal IgA and produce antigen-specific serum IgG.⁷²

The number of M cell receptors and their ligands that have been identified so far is limited, and most of them are not just expressed in M cells, but in neighboring enterocytes as well.⁶⁰ Toll-like receptor (TLR)-4 and α5β1 integrin, belonging to pathogen recognition receptors (PRRs), are expressed on the surface of human and mouse M cells. Interaction between these innate immune system molecules with pathogen-associated molecular patterns is essential for bacteria translocation across the lumen. Nevertheless, PRRs are also expressed in other enterocytes and not merely in M cells. For example, α5β1 integrin is both dispersed on the lateral and basolateral surfaces of enterocytes and on the apical surface of M cells, which is a challenge in targeting M cells alone.⁶⁰

M-cell-targeting ligands can enhance the uptake of oral vaccines by M cells and improve antigen-specific immune responses in both mucosal and systemic immunity. It seems that targeting ligand to antigen is a very promising approach in the development of efficient mucosal vaccine. However, simple targeting of antigen to M cells does not ensure the production of efficient protective immunity. We should pay more attention to the ligand study and find out the “optimal transporter," presenting antigens to M cells, leading to efficient immune responses.

Concluding Remarks

Compared with the parenteral vaccines, mucosal vaccines have many advantages, such as safety, low cost, convenient administration, being innocuous, and effectiveness due to induction of both local and systemic protective immune responses. Nevertheless, there are still some limits not to be ignored in mucosal vaccines. Mucosal tolerance can be induced by repeated oral administration of large doses of antigen in experimental animals.⁷³ Mucosal vaccines also should overcome 2 major obstacles, effectiveness and safety, because of the uniqueness of the mucosal environment.⁷⁴ The appearance of a new potent mucosal vaccine also brings other challenges, such as antigen degradation by proteolytic activities, low dose of antigen absorbed, and lack of potent mucosal adjuvants.⁶⁰ We need to probe oral vehicle delivery systems more deeply to overcome these issues. The capacity of M cells to sample antigens, transfer luminal bacteria, and stimulate mucosal immune responses, makes them fascinating targets for mucosal vaccine delivery. Results of previous studies imply that we require safe and effective oral mucosal vaccines with M-cell-targeting strategies, because the M-cell-targeting vaccines now available do not actively direct antigens to M cells. Recent studies have been exploited to search for specific M cell molecules to develop M-cell-targeted strategies, which will likely promote progress in mucosal vaccines. However, many mechanisms of M cells are still unclear and not known as well-known as those of DCs. Therefore, to develop M-cell-targeting strategies in mucosal vaccines, further understanding of mucosal immunology and M cell biology is required, and M cell function, and identification of more M-cell-specific molecules and effective mucosal adjuvants are crucial for further development of M-cell-targeted vaccines.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

This study was financially supported by the Chinese “863” National Programs for High Technology Research and Development (Grant No.: 2011AA10A211), the National Pig Industrial System (CARS-36–06B), and the Special Fund for Agro-scientific Research in the Public Interest (201203039).