Abstract

Intestinal M (microfold or membranous) cells are an enigmatic lineage of intestinalepithelial cells that initiate mucosal immune responses through the uptake andtranscytosis of luminal antigens. Due to their rarity, the mechanisms of M-cell functionand differentiation are poorly understood. To overcome this problem, experimentalstrategies to enrich for M-cells have been established. Transcriptome analyses haveprovided valuable insight, especially on the receptors for antigen uptake, and suchstudies have broadened our knowledge of M-cell function. In another line of investigation,we and others have begun to dissect the molecular pathways of M-cell differentiation.Among them, receptor activator of NF-κB ligand (RANKL) has been identified as an essentialfactor for M-cell differentiation. We have focused on the M-cell inducible activity ofRANKL and have been able to observe temporal transitions during M-cell differentiation byusingin vivo ectopic M-cell differentiation induced by exogenous RANKLtreatment. We have found that the ets-family transcription factor Spi-B is essential forfunctional maturation of M cells. In the absence of Spi-B, the immune response toSalmonella Typhimurium is severely impaired, suggesting that M cellsare important for maintaining intestinal homeostasis.

INTRODUCTION

The mucosal surface of the mammalian gut is continuously exposed to a variety of foreignproteins and microorganisms, some of which are potentially harmful to the host. To protectitself from these dangers, the host has evolved specialized organized lymphoid tissue,gut-associated lymphoid tissue (GALT), which includes Peyer’s patches (PPs) and isolatedlymphoid follicles. GALT is the inductive site for intestinal immunity but is different fromother peripheral lymphoid tissues; it lacks afferent lymphatics and instead directly samplesmucosal antigens across the epithelial barrier to initiate immune responses. This task isthought to be mainly accomplished by specialized epithelial cells known as M cells(microfold or membranous), which are found in the follicle-associated epithelium (FAE)covering the lymphoid follicles of GALT [1]. M cellshave a tremendous capacity for antigen uptake and transcytosis, functions that allow therapid transport of antigens to underlying lymphoid tissue, especially to antigen-presentingcells. Processed antigens are then presented to T cells, which support cognate B-cellactivation, ultimately leading to the generation of plasma cells that produce polymericimmunoglobulin A (IgA) [2]. Thus, M-cell-mediatedtransport of antigens is an important step in the initiation of mucosal immuneresponses.

To better understand the biological significance of M cells in host defense, the importanceof an M-cell deficient model cannot be overestimated. One plausible approach to achieve sucha model would be to inactivate genes critically involved in M-cell differentiation. Althoughthe functional and morphological features of M cells were initially described nearly 40years ago, many basic questions about M-cell differentiation and function remain unsolved[3,4,5]. In this review, we introduce the basics of M-cellbiology and introduce recent findings concerning M-cell differentiation.

DETECTION OF M CELLS AND IDENTIFICATION OF M-CELL SPECIFIC MOLECULES

For a long time, the detection of M cells depended on morphological analysis using electronmicroscopy. M cells have characteristic morphological features that set them apart fromother subsets of intestinal epithelial cells. They have shorter and irregular microvilli ontheir apical surface and a pocket-like basolateral invagination of the plasma membrane thathouses lymphocytes and antigen-presenting cells (Fig.1). These morphological features enable the recognition of M cells by electronmicroscopy and are likely to be conserved in M cells throughout GALT in the small and largeintestine, such as isolated lymphoid follicles and colonic patches [6,7]. In some species, cytoskeletalproteins such as actin, villin, cytokeratin and vimentin are used for the detection of Mcells [8,9,10,11,12]. In addition,Ulex europaeusagglutinin-I (UEA-I), which binds α-1,2 fucosylated residues, has become a classical markerfor murine M cells [13,14]. Although useful as M-cell markers, these molecules are not uniformlyapplicable among different species, thus hindering a global understanding of M-cell biology.Several approaches for identifying molecules highly and specifically expressed in M cellshave been used to overcome this problem and find universal M-cell markers. We summarized theM-cell markers identified so far inTable1 [2,15,16,17,18,19,20,21,22]. Hase et al. established a method toisolate FAE from murine PPs and compared the gene expression profile of these cells withthat of the much more abundant villous epithelial cells. Based on this analysis, they foundthat secretory granule, neuroendocrine protein 1 (sgne-1), encoded by theScg5 gene, is selectively expressed in M cells [18]. Verbrugghe et al. also performed a similar type of analysis andreported that annexin A5 is abundantly expressed by murine M cells [15]. Terahara et al. developed an M-cell recognizing monoclonal antibody(NKM 16-2-4) by immunizing a rat with UEA-I⁺ cells from the murine smallintestine and used it to sort out a putative M-cell fraction from murine PPs for geneexpression profiling [17,23]. The identification of such M-cell-specific molecules has enabled usto detect M cells relatively easily by immunohistochemical analysis.

ANTIGEN-UPTAKE RECEPTORS EXPRESSED ON THE M-CELL SURFACE

M cells provide an efficient portal through which gut luminal antigens can be transportedinto the underlying lymphoid tissue. In keeping with this function, M cells have a highcapacity for phagocytosis and transcytosis. To identify the molecules involved in thesefunctions, gene expression profiling of M cells as described above has been utilized. Tworesearch groups have independently identified glycoprotein-2 (GP2) as an M-cell-specificmolecule because of its prominent expression on M cells [2,17]. GP2 is aglycosylphosphatidylinositol (GPI)-anchored protein and selectively binds to the type I pilion the outer membrane of certain bacteria, such asEscherichia coli andSalmonella Typhimurium. In the absence of GP2, uptake and transcytosis ofthese bacteria and the subsequent immune response are severely impaired, suggesting that GP2is an important immunosurveillance receptor for luminal antigens [2].

Cellular prion protein (PrP^c), another GPI-anchored protein highly expressed onM cells, was also identified as an antigen-uptake receptor. PrP-deficient mice have a defectin uptake of the gram-negative bacteriumBrucella abortus into PPs. Antigenuptake in this case seems to depend on the affinity of PrP for heat-shock protein 60 (Hsp60)expressed byB. abortus [24]. Manyother proteins in addition to GP2 and PrP have been identified on the M-cell surface. Toclarify the molecular mechanisms of antigen uptake, the interaction between these moleculesand luminal antigens should be investigated.

M CELLS DERIVE FROM LGR5+ INTESTINAL EPITHELIAL STEM CELLS IN THE CRYPTS

All intestinal epithelial lineage cells, including absorptive enterocytes, goblet cells,Paneth cells (only in the small intestine) and enteroendocrine cells originate fromintestinal epithelial stem cells located at the bottom of the intestinal crypts ofLieberkühn. These intestinal epithelial stem cells express the leucine-richrepeat-containing G protein-coupled receptor 5 (Lgr5) [25]. Lineage-tracing studies using transgenic mice expressing a reporter gene(LacZ) under control of theLgr5 promoter have confirmedthat all epithelial cells within the FAE, including M cells, are also derived from Lgr5+intestinal epithelial stem cells [26]. Different fromother epithelial cell lineages, specification into the M-cell lineage from stem cells ortransit-amplifying cells, which directly derive from stem cells and rapidly proliferate, hasbeen mainly examined with respect to extrinsic factors from the adjacent microenvironment,as described below.

THE EXTRINSIC FACTORS FOR M-CELL DIFFERENTIATION

TRACING M-CELL DIFFERENTIATION AFTER EXOGENOUS RANKL TREATMENT

As there are normally no M cells in the VE, we thought that RANKL-induced ectopic M-celldifferentiation would enable us to observe the transition stages in the process of M-celldifferentiation. As expected, the expression kinetics of M-cell markers after treatment withRANKL are distinct; the expressions of myristoylated alanine-rich C kinase substrate like 1(Marcksl1), CCL9 and GP2 peak after 1, 2 and 3 days, respectively. Furthermore, thelocalization of these M-cell markers moves from the crypt zones toward the tips of the villiwith time after RANKL treatment, with Marcksl1 limited to the crypt-villus junction at day 1and GP2 expression restricted to the upper part of the villi on day 3. Given that theposition of the cells along the intestinal crypt-villus axis reflects their degree ofmaturation, these observations indicate that Marcksl1 expression is initiated at an earlystage of M-cell differentiation, whereas expression of CCL9 and GP2 requires furthermaturation of the RANKL-induced M cells [16]. Ourfindings parallel the tempo of expression of these M-cell marker molecules during thephysiological development of M cells in ontogeny and provide novel insight with respect toM-cell differentiation.

In terms of the distribution of RANKL-induced M cells in the VE, these cells seem to besimilar to villous M-like cells. Actually, it has been shown that villous M-like cellsharbor the typical M-cell morphologies and capacity for antigen uptake [38]. To define the features of villous M-like cells, thegene expression profile of villous M-like cells was compared with those of VE or PP M cells.Although villous M-like cells share traits with PP M cells in terms of chemokine expression,typical M-cell markers such as GP2 were not expressed in villous M-like cells [17]. Considering that RANKL-induced M cells mimic thegene expression profile of PP M cells, villous M-like cells are distinct from RANKL-inducedM cells.

EXPRESSION OF THE ETS-FAMILY TRANSCRIPTION FACTOR SPI-B DURING M-CELLDIFFERENTIATION

Identification of lineage-specific transcription factors expressed during M-celldifferentiation is a key to elucidating the molecular mechanisms of the process. Manytranscription factors are known to be involved in the cell-fate decisions made as intestinalepithelial stem cells in the crypt differentiate into one of the recognized types ofterminally differentiated intestinal epithelial cells. Atoh1, which is repressed by theNotch effector transcriptional repressor Hes1 [39] isessential for commitment of epithelial progenitor cells into the secretory lineages,including goblet cells, Paneth cells, enteroendocrine cells and tuft cells [40]. Downstream of Atoh1, the specification of theindividual secretory cell lineages requires at least one additional transcription factor:KLF4 is required for the maturation of goblet cells [41]; Sox9 is required for the maturation of Paneth cells [42,43]; and neurogenin 3 isrequired for the maturation of enteroendocrine cells [44]. We hypothesized that M-cell differentiation also requires regulation by oneor more distinct transcription factors. To identify the transcription factor (s) involved inM-cell differentiation, we performed gene expression profiling during RANKL-induced M-celldifferentiation. We focused on transcription factors upregulated early during M-celldifferentiation and found that the ets-family transcription factor Spi-B is highly expressedin M cells [16]. The expression of Spi-B is conservedin multiple species, including human M cells and in anin vitro bovineM-cell model [35], indicating its crucial role inM-cell differentiation and function.

SPI-B IS ESSENTIAL FOR THE MATURATION OF M CELLS

To clarify the role of Spi-B in M-cell development, we examined Spi-B-deficient mice andfound that in the PP, GP2+ M cells were completely absent from the FAE, whereas Marcksl1 andannexin A5 were not silenced. Based on the kinetics of appearance of these markers duringM-cell differentiation, it seems likely that expression of GP2 requires terminal maturationof M cells. We have therefore hypothesized that the Marcksl1+ annexin A5-positive cellsbelong to a functionally immature stage in the M-cell lineage. Consistent with thishypothesis, these cells lack typical features of M cells, such as the disorganizedmicrovilli on their apical surface and the pocket-like structure harboring immune cells ontheir basolateral side. In addition to these morphological abnormalities, M cells inSpi-B-deficient mice exhibited functional defects in the transport of antigens, such as asignificant reduction in transport of orally administeredS. TyphimuriumandYersinia enterocolitica to PPs [16].

We also found that Spi-B was expressed in M cells of other organized lymphoid tissues inthe gut, such as isolated lymphoid follicles in the small intestine and colonic patches inthe colon [16]. To investigate the contribution ofSpi-B in non-gut M-cell differentiation, we assessed Spi-B expression in nasal-associatedlymphoid tissues (NALT). As we expected, M cells within the epithelium covering (NALT) alsoexpressed Spi-B (unpublished data). These observations indicate that the expression of Spi-Bis also conserved throughout mucosal surfaces.

THE LACK OF FUNCTIONALLY MATURE M CELLS IMPAIRS MUCOSAL IMMUNE RESPONSES

The severe reduction in transport of pathogenic bacteria into PPs in Spi-B-deficient miceraises the possibility that bacteria-specific immune responses are impaired in the absenceof functionally mature M cells. To examine this possibility, we evaluated theS. Typhimurium-specific immune response by using adoptive transfer of Tcells from anS. Typhimurium-specific T-cell receptor transgenic mouse,SM1. In the absence of Spi-B, T-cell activation by orally administeredS.Typhimurium and subsequent proliferation were significantly impaired [16]. These findings indicate that the transport of antigen via M cells isessential for inducing normal mucosal immune responses (Fig. 2), thus possibly resolving the issue that has been debated for many years.

SPI-B-INDEPENDENT REGULATION OF M-CELL DIFFERENTIATION

Apart from our study, Sato et al. more recently reported that there is a population of PP Mcells that arises independently from Spi-B, as they found thatAlcaligenesspp., which are Gram-negative bacteria, were taken up into PPs in the absence of Spi-B[22]. Another research group demonstrated thatSpi-B transduction is insufficient to give rise to M cells in the intestinal epithelialcells [26]. In addition, we have identified Marcksl1+annexin A5-positive cells in Spi-B-deficient mice. Taken together, other factors are likelyrequired for the development of M cells. In support of this hypothesis, it has been reportedthat the noncanonical NF-κB pathway evokes Spi-B in response to RANKL [35]. This pathway may induce other transcription factors as well as Spi-Bfor M-cell differentiation.

PERSPECTIVES OF FUTURE M-CELL STUDIES

The main functions of M cells are antigen uptake and transcytosis. As described above, afew receptors for specific antigen uptake have been identified, and the molecular basis forantigen uptake has been partially clarified. On the other hand, there is much lessinformation with respect to the transcytosis of antigens. Recently, Asai et al. used thein vitro M-like cell culture derived from the Caco-2 cell system todemonstrate that the SRC family tyrosine kinase HCK is involved in transcytosis of antigens[45]. They also suggest that Spi-B regulatesHCK-dependent transcytosis. Other than HCK, it seems likely that multiple moleculesregulated by Spi-B are involved in transcytosis. These initial studies suggest thatdetermining Spi-B targets in M cells may provide important new insight into the mechanismsof transcytosis. Recently, a novelin vitro M-cell differentiation modelhas been established by using organoid cultures from intestinal crypts [26]. Thesein vitro-derived M cellsexhibited similar gene expression profiles asin vivo M cells and may allowfor advanced analyses of M-cell biology.

References