Enteroendocrine Cells Are Specifically Marked by Cell Surface Expression of Claudin-4 in Mouse Small Intestine

Enteroendocrine cells are solitary epithelial cells scattered throughout the gastrointestinal tract and produce various types of hormones, constituting one of the largest endocrine systems in the body. The study of these rare epithelial cells has been hampered by the difficulty in isolating them because of the lack of specific cell surface markers. Here, we report that enteroendocrine cells selectively express a tight junction membrane protein, claudin-4 (Cld4), and are efficiently isolated with the use of an antibody specific for the Cld4 extracellular domain and flow cytometry. Sorted Cld4+ epithelial cells in the small intestine exclusively expressed a chromogranin A gene (Chga) and other enteroendocrine cell–related genes (Ffar1, Ffar4, Gpr119), and the population was divided into two subpopulations based on the activity of binding to Ulex europaeus agglutinin-1 (UEA-1). A Cld4+UEA-1− cell population almost exclusively expressed glucose-dependent insulinotropic polypeptide gene (Gip), thus representing K cells, whereas a Cld4+UEA-1+ cell population expressed other gut hormone genes, including glucagon-like peptide 1 (Gcg), pancreatic polypeptide–like peptide with N-terminal tyrosine amide (Pyy), cholecystokinin (Cck), secretin (Sct), and tryptophan hydroxylase 1 (Tph1). In addition, we found that orally administered luminal antigens were taken up by the solitary Cld4+ cells in the small intestinal villi, raising the possibility that enteroendocrine cells might also play a role in initiation of mucosal immunity. Our results provide a useful tool for the cellular and functional characterization of enteroendocrine cells.


Introduction
The intestinal epithelial cell layer consists of functionally heterogeneous cell populations, including absorptive epithelial cells, goblet cells, paneth cells, M cells, cup cells, tuft cells, and enteroendocrine cells, all of which are derived from Lgr5 + crypt base columnar stem cells [1][2][3]. Among these, enteroendocrine cells include more than 10 different cell types producing distinct hormones or hormone-like substances, such as serotonin, secretin, glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide 1 (GLP-1), pancreatic polypeptide-like peptide with Nterminal tyrosine amide, and cholecystokinin [4]. Those producing GIP and GLP-1 are called K cells and L cells [5,6], respectively, and are responsible for ''incretin effects'' regulating postprandial insulin secretion [7][8][9][10][11]. Although enteroendocrine cells comprise only a small population of the intestinal epithelial cells, they function as essential regulators of digestion, appetite, gut motility, and metabolism, making the gut one of the largest endocrine organ in the body [12]. Several key transcriptional factors for the differentiation of enteroendocrine cells have been identified [13,14]. However, how the cell lineages diverge and inter-relate remain poorly understood, in part because of the lack of specific cell markers for purifying live enteroendocrine cells. It has been reported that Ulex europaeus agglutinin-1 (UEA-1) binds some enteroendocrine cells, but it also reacts with other epithelial cell components, such as M cells in the follicle-associated epithelium (FAE) and exocrine goblet cells [15]. More recently, transgenic mice carrying fluorescent reporters under the control of Gcg and Gip promoters have enabled the identification and isolation of L cells and K cells, respectively [16,17]. Nonetheless, general cell surface markers for the enteroendocrine cell population have not been identified.
Claudins (Clds), integral membrane proteins with four transmembrane domains, are crucial components of tight junctions (TJs) that function as a primary barrier to solutes and water as well as charge-selective channels between the apical and basal sides of epithelial cellular sheets [18,19]. The Cld gene family comprises at least 24 members in mice and in humans [19][20][21]. Typically, multiple Clds are expressed in most types of epithelial cells, and the combination and ratio of different types of Clds in TJ strands may determine the permeability of each epithelial cellular sheet [20,22]. Recent studies have revealed that Clds may also be involved in nonbarrier functions such as the regulation of cell proliferation and cell signaling [23][24][25][26][27][28][29]. A Cld family member, Cld4, may be one of these unique types of Clds. We previously reported that Cld4 is expressed in various TJ-deficient cells, such as thymic epithelial cells and developing T cells [28,30]. In the intestinal mucosa, Cld4 is expressed in a portion of the tips of villi and FAE of the Peyer's patches [31][32][33], providing a molecular target for drug delivery of the efficient mucosal vaccine [34][35][36]. In the current study, we demonstrate that Cld4 is selectively and abundantly expressed on the cell surface of enteroendocrine cells and serves as an effective molecular marker for their identification and isolation.

Selective Expression of Cld4 in Intestinal Solitary Epithelial Cells Displaying Chromogranin A
It was reported that several types of Clds are expressed in epithelial cells of mouse small intestine, including Cld3, Cld4, and Cld10 [24]. The expression of Cld10 was sharply concentrated at cell-cell contact sites of an entire epithelial cell sheet at the most apical region of the plasma membrane, colocalizing with ZO-1 ( Figure 1A), suggesting that Cld10 expression is confined to TJs. Although Cld3 was also localized at cell-cell borders of the epithelial cellular sheet, the expression was much broader, covering entire basolateral regions ( Figure 1A). In contrast, Cld4 expression was detected in rare and solitary cells scattered within the epithelial cellular sheet of the intestinal villi ( Figure 1A). In these cells, Cld4 was localized diffusely and strongly throughout the entire cell surface in addition to the concentrated localization at ZO-1 + TJs formed with neighboring epithelial cells ( Figure 1B). The characteristic immunostaining pattern was confirmed with the use of an independent rat monoclonal antibody that recognizes the extracellular domain of Cld4 (HKH-189) [28] ( Figure S1). The signal with either antibody was completely absent in the intestine of Cldn4 2/2 mice [29] (Figure S1), confirming the specificity of the signal. The solitary Cld4 + cells showed a spindle-shape morphology ( Figure 1A and B) reminiscent of enteroendocrine cells; therefore, we performed coimmunostaining of Cld4 with chromogranin A (CgA), a general marker of enteroendocrine cells [4] and UEA-1, which binds to goblet cells and some enteroendocrine cells [15]. We found that almost all of the solitary Cld4 + cells coexpressed CgA, irrespective of UEA-1 binding ( Figure 2). On the other hand, UEA-1 + CgA 2 cells, most likely representing goblet cells, did not exhibit Cld4 expression ( Figure 2). The results suggest that Cld4 is selectively expressed in enteroendocrine cells of small intestinal villi.

The Expression of Enteroendocrine Cell-Associated Genes Is Confined to Isolated Cld4 + Intestinal Epithelial Cells
To confirm the features of Cld4 + solitary epithelial cells, we isolated them with a cell sorter using the rat anti-Cld4 mAb (HKH-189). Fluorescence-activated cell sorting (FACS) analysis revealed that approximately 8% of the total viable epithelial cell fraction (PI 2 CD45 2 Ter119 2 EpCAM + ) of the intestinal single-cell preparation from wild-type (WT) mice showed positive staining with anti-Cld4 mAb (HKH-189), whereas virtually no positive signal was detected in that from Cldn4 2/2 mice ( Figure 3A). We then separately sorted Cld4 + and Cld4 2 fractions with a cell sorter. A Cld4 + fraction showed three-times more Cldn4 transcripts than a Cld4 2 fraction, whereas both cell fractions contained comparable levels of ZO-1(Tjp1) and Cld3 (Cldn3) transcripts ( Figure 3B). In agreement with the immunostaining analysis, the transcripts of the chromogranin A gene (Chga) were confined exclusively to the Cld4 + cell fraction ( Figure 3B). It has been reported that Gprotein-coupled receptors, such as GPR40, GPR119, and GPR120, are specifically expressed in enteroendocrine cells and are involved in sensing free fatty acids, leading to the release of incretin hormones [37][38][39]. The transcripts of all these G-proteincoupled receptor genes (Ffar1, Gpr119, Ffar4) were also expressed preferentially in the Cld4 + cell fraction ( Figure 3B). The results indicate that Cld4 + cells isolated with a cell sorter are highly enriched with all the enteroendocrine cells in the small intestine.

Transepithelial Passage of Luminal Antigen by Cld4 + Enteroendocrine Cells
Exocrine goblet cells are reported to be capable of capturing luminal antigens and delivering them to lamina propria [42], and therefore we investigated the antigen-capturing capacity of enteroendocrine cells with the use of rhodamine-conjugated dextran (10 kDa) as a model antigen. In 30 minutes following oral administration, the luminal surface of the intestinal epithelial cells was coated with dextran without diffuse invasion into the epithelial cellular sheets; however, characteristic cylindrical dextran columns projecting through the villus epithelium into the lamina propria were observed sporadically ( Figure 6A). Some of the dextran columns coincided with Cld4 2 CgA 2 epithelial cells, most likely representing goblet cells ( Figure 6A). However, we also observed that a portion of the dextran columns coincided with spindle-shape CgA + Cld4 + epithelial cells ( Figure 6A), although not all Cld4 + cells were associated with dextran columns at a given time (30 minutes) after dextran administration. FACS analysis confirmed that about 23% of Cld4and 32% of Cld4 + epithelial cells were labeled with fluorescein after fluorescein-dextran administration, whereas no staining was detected in the epithelial cells from untreated mice ( Figure 6B). In the Cld4cell population, the strong fluorescein staining was confined to UEA1 + cells representing goblet cells and M cells, with only marginal staining in UEA1 2 absorptive epithelial cells ( Figure 6C). Within the Cld4 + enteroendocrine cell population, the vast majority (88.5%) of UEA-1 + cells revealed a strong fluorescein staining, although only a minor proportion (17.4%) of UEA-1cells representing K cells showed a marginal staining, if any ( Figure 6C). These results collectively suggest that a proportion of enteroendocrine cells are capable of efficiently capturing luminal antigens, with the exception of K cells.

Discussion
Enteroendocrine cells comprise a very small population of gut epithelial cells that are scattered solitarily throughout the gastrointestinal tract, and the physical isolation of these cells has been hampered by the lack of specific cell surface markers. In the current study, we found that CgA + enteroendocrine cells in small intestine rather specifically express a Cld family member, Cld4, diffusely and strongly on the cell surface as revealed by immunostaining. Flow cytometric analysis using the rat anti-Cld4 monoclonal antibody (HKH-189) that recognizes the extracellular portion indicated that approximately 8% of total small intestinal epithelial cells expressed Cld4, and that the isolated Cld4 + cells exclusively exhibited the expression of the genes associated with enteroendocrine cells, such as Chga, Ffar1, Gpr119, and Ffar4. The proportion could be an overestimate for enteroendocrine cells, because the Cld4 + cell population may also include other minor epithelial cells such as FAE in Peyer's patches and villous tip enterocytes [31][32][33]. We confirmed that a portion of these cells expressed Cld4 locally at TJs as well as laterally. However, we presume that such epithelial cells expressing a relatively low level of Cld4 may be hardly detected with FACS analysis because low-residual Cldn4 transcripts could be detected in the Cld4 2 cell population. Thus, in practice, it is strongly suggested that Cld4 serves as a specific cell surface marker covering entire enteroendocrine cells, providing a useful means for the isolation of this minor epithelial cell population as viable cells.
It has been reported that UEA-1 binds to a portion of enteroendocrine cells [15] and, in agreement, FACS analysis revealed that the Cld4 + epithelial cells consisted of UEA-1 + and UEA-1 2 populations, both of which comparably expressed Chga. The Cld4 + UEA-1 2 cell population almost exclusively expressed Gip, with undetectable level of Cck or Sct, thus representing K cells. The population expressed lower levels of Gcg and Pyy, and it remains to be determined whether this was attributable to a minor contamination of UEA-1 low cells, or to overlapping hormone expression in a single cell type as suggested previously [17,40,41]. On the other hand, the Cld4 + UEA-1 + cell population contained abundant transcripts of a series of hormones, including Gcg, Pyy, Cck, Sct, and Tph1, but no detectable Gip. Thus, although the cell population was much smaller than the Cld4 + UEA-1 2 cell population, it was suggested that the Cld4 + UEA-1 + cell population comprises functionally heterogeneous enteroendocrine cells, including L cells. The results of gene expression analysis were confirmed at the protein level with immunostaining analysis; GIP expression was associated with Cld4 + UEA-1 2 cells, whereas GLP-1 expression was detected with Cld4 + UEA-1 + cells with a minor  Although M cells present in the FAE of Peyer's patches are specialized for capturing luminal antigens and delivering them to lamina propria to initiate mucosal immunity [43], a recent report indicated that exocrine goblet cells are also capable of doing so [42]. Our current study indicates that Cld4 + CgA + enteroendocrine cells have an activity of taking up luminal materials as well. The antigencapturing activity was found mainly in the minor Cld4 + UEA-1 + cells population, with little activity in the Cld4 + UEA-1 2 K cells. Although enteroendocrine cells apparently form ZO-1 + Cld4 + TJs with adjacent absorptive epithelial cells, it is unclear whether the luminal antigen is delivered through a transcellular route like M cells [44], or via a paracellular route due to the permissive TJs as implicated in goblet cells [42,45]. Because our current results indicate that the fluorescein-labeled dextran could be detected in both goblet and enteroendocrine cells by FACS analysis, we presume that luminal antigens may be transferred into basolateral sites through a transcellular route of these cells. It also remains to be investigated whether such an activity of enteroendocrine cells is linked to the initiation of antigen-specific mucosal immunity, or related to any other biological functions. Nonetheless, Cld4 may be a potential target for the delivery of mucosal vaccines or peptide drugs [34][35][36]. In conclusion, our current results indicate that Cld4 is a new marker of enteroendocrine cells and provides a useful means for the study of their development and function.

Ethics
This study was conducted according to the principles expressed in the Declaration of Helsinki. The study was approved by Animal Research Committee, Graduate School of Medicine, Kyoto University (MedKyo13045).

Mice
8-week-old to 10-week-old C57BL/6 WT female mice were purchased from Japan SLC and kept for at least 1 week before experiments in our animal room. Cldn4 2/2 mice were generated as described [29]. Mice were maintained in specific pathogen-free conditions at the Kyoto University Laboratory Animal Center in accordance with university guidelines. Mice were euthanized by cervical dislocation and used for experiments.

Histological Analysis
Histological analysis was performed as described, with some modifications [46,47]. For intestinal villi, tissue samples were fixed with 10 % formalin (Wako) for 16 hours at 4uC, followed by dehydration with 10% and 20% (wt/vol) sucrose solution before being frozen with liquid nitrogen in Tissue-Tek OCT compound (Sakura). Unless otherwise noted, cryostat sections (6 mm) were blocked with 2% (vol/vol) fetal calf serum in phosphate-buffered saline (PBS) for 30 minutes at room temperature, followed by being incubated with primary antibodies or reagents for 16 hours at 4uC. Samples were then washed with 0.1% PBS-Tween and PBS, each for 5 minutes, followed by staining with secondary reagents for 30 minutes at room temperature. Samples were washed with PBS twice and then counterstained with DAPI (Sigma-Aldrich) for 10 minutes at room temperature to visualize the nucleus. Finally, samples were washed with PBS twice, mounted in Mowiol (Calbiochem), and examined under a fluorescence photo microscope (Axiovert 200M; Carl Zeiss).

Flow Cytometry Analysis and Cell Sorting
FACS analysis and cell sorting were performed as described previously [48]. In brief, the small intestine was cut longitudinally and washed in cold PBS. Then, a 2-cm piece of intestine was cut and incubated in 2% (vol/vol) fetal calf serum in RPMI 1640 (Wako) containing 0.5 mM EDTA (Nacalai Tesque) for 15 minutes at 37uC. Cells were filtered with a 70-mm cell strainer (Becton Dickinson) and blocked with Fc Block (2.4G2; Becton Dickinson) for 5 minutes at room temperature, followed by staining antibodies and reagents for 30 minutes at 4uC. Dead cells were excluded by propidium iodide (PI) staining. Epithelial cell fraction was defined by PI 2 CD45 2 -TER119 2 EpCAM + gating and biotinylated HKH-189 was used for Cld4 staining. Samples were analyzed by FACSCanto (Becton Dickinson) or sorted by FACSAria (Becton Dickinson).

Statistical Analysis
Data are presented as mean values 6 standard deviation. Statistical significance was determined with the Student t test or ANOVA.