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DOI: 10.1002/eji.201343782

Yosuke Kurashima et al.

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Immunity at the Barrier Review Series

Mucosal innate immune cells regulate both gut homeostasis and intestinal inflammation Yosuke Kurashima1,2,3 , Yoshiyuki Goto4 and Hiroshi Kiyono1,2,5 1

Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan 2 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Tokyo, Japan 3 Division of Infectious Genetics, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan 4 Department of Microbiology and Immunology, Columbia University Medical Center, New York, NY, USA 5 International Research and Development Center for Mucosal Vaccine, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan Continuous exposure of intestinal mucosal surfaces to diverse microorganisms and their metabolites reflects the biological necessity for a multifaceted, integrated epithelial and immune cell-mediated regulatory system. The development and function of the host cells responsible for the barrier function of the intestinal surface (e.g., M cells, Paneth cells, goblet cells, and columnar epithelial cells) are strictly regulated through both positive and negative stimulation by the luminal microbiota. Stimulation by damage-associated molecular patterns and commensal bacteria-derived microbe-associated molecular patterns provokes the assembly of inflammasomes, which are involved in maintaining the integrity of the intestinal epithelium. Mucosal immune cells located beneath the epithelium play critical roles in regulating both the mucosal barrier and the relative composition of the luminal microbiota. Innate lymphoid cells and mast cells, in particular, orchestrate the mucosal regulatory system to create a mutually beneficial environment for both the host and the microbiota. Disruption of mucosal homeostasis causes intestinal inflammation such as that seen in inflammatory bowel disease. Here, we review the recent research on the biological interplay among the luminal microbiota, epithelial cells, and mucosal innate immune cells in both healthy and pathological conditions.

Keywords: Epithelial cells

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Homeostasis

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Inflammation r Intestinal immunity r Mast cells

Introduction to the function and homeostasis of the innate barrier The intestinal mucosa is constantly being exposed to a wide range of biological stimuli, including commensal and pathogenic microorganisms and food materials. More than 1014 microorganisms

Correspondence: Prof. Hiroshi Kiyono e-mail: [email protected]

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reside in the intestinal lumen and provide nutrients for their host via enzymatic synthesis from food materials (reviewed in [1]). For example, microbial metabolites produced in the large intestine, such as short-chain fatty acids produced through polysaccharide fermentation, regulate the body’s energy supply, the integrity of the epithelium, and the function of immune cells via the G protein-coupled receptors GPR41 and GPR43 (Fig. 1A, reviewed in [2]). In addition, the use of recent technologies, such as 16S ribosomal RNA gene sequencing, has shown the connection between the variation and composition of the commensal

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Figure 1. Interactions among the commensal microbiota, epithelial barrier, and innate immune cells for the maintenance of homeostasis in the mucosa. (A) Regulation of immune homeostasis by commensal microbiota. Bacterial components and bacteria-derived food metabolites, such as short-chain fatty acids, affect the epithelial layer and thereby support the production of antimicrobial peptides (AMPs). Epithelial cells respond by producing immunomodulatory factors such as IL-25, which control the functions of myeloid cell subsets. These myeloid cell subsets then regulate IL-22 production by group 3 innate lymphoid cells (ILC3) through cell–cell interactions. (B) Regulation of bacterial homeostasis by mucosal immune cells. DC–ILC interaction directly affects epithelial integrity and antimicrobial peptide production though the production of IL-22. This pathway maintains the relative proportions of commensal bacteria by regulating the production of antimicrobial peptides such as RegIII-β and RegIII-γ, which prevent the dissemination of Alcaligenes spp. to the periphery and control the expansion of segmented filamentous bacteria (SFB). Mast cells (MCs) stimulate IgA production by B cells/plasma cells via IL-6 and CD40–CD40L pathways, also contributing to bacterial homeostasis at the mucosa.

microbiota and various pathophysiological conditions, such as intestinal inflammation, obesity, diabetes, and even behavior (reviewed in [3, 4]). Furthermore, a deficiency in Toll-like receptor 5 (TLR5), a receptor for the bacterial component flagellin, has been shown to cause spontaneous colitis and obesity in mice [5]. Bacterial components from the luminal microbiota also modify and strengthen the barrier function and integrity of the intestinal epithelium by stimulating pathogen recognition sensors such as TLRs that detect bacteria, viruses, fungi, and protozoa; nucleotidebinding oligomerization domain (NOD)-like receptors (NLRs) that detect bacteria; and RIG-I-like receptors (RLRs) that detect viral components [2]. Interactions between TLRs and NLRs lead to the production of proinflammatory cytokines (e.g., IL-1-β and IL-18). Stimulation of TLRs and NLRs induces the assembly of protein complexes called inflammasomes (reviewed in [6]). Inflammasomes are composed of a bacterial sensor, an adaptor protein, and a caspase, and they detect danger signals, such as goutassociated uric acid crystals, calcium pyrophosphate dihydrate crystals, extracellular ATP, cytosolic DNA, and bacterial products, that are released from dying cells. The inflammasomes are complexes that can be formed by members of the NLR (NLRP1, 2, 3, 6, 7, or NLRC4) or the PYHIN (AIM2 or IFII6) protein family (reviewed in [6, 7]). Deficiencies in any of the inflammasome components reduce the integrity of the epithelial barrier and increase susceptibility to intestinal inflammation [7]. This constant exposure of the epithelial surface to microbial components and products therefore requires sufficient epithelial regen C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

eration and integrity to maintain a healthy mucosal barrier in the intestine. Directly below the epithelium, numerous innate and acquired immune cells create a homeostasis between the commensal flora of the intestine and the host immune system [8]. Among these immune cells, innate lymphoid cells (ILCs) and mast cells (MCs) are key players, which have multiple roles in maintaining homeostasis and innate immune surveillance to protect the host against invading parasitic worms (e.g., Nippostrongylus brasiliensis and Trichinella spiralis) and enteric pathogens (e.g., Citrobacter rodentium), and which also act as gatekeepers to the mucosal compartment (reviewed in [9–11]).

Homeostasis of the mucosal barrier by epithelial cells The intestinal epithelium is constructed of a monolayer of epithelial cells that includes M cells, goblet cells, Paneth cells, and columnar epithelial cells, which are all cells that differentiate from crypt stem cells. These cells cover the mucosa and play critical roles in the regulation of the ecology of the commensal microbiota and in the host immune response (reviewed in [12]). A mucus layer contains antimicrobial peptides and mucins, which are produced by Paneth cells and goblet cells, respectively, as well as secretory immunoglobulin A (SIgA), which is transcytosed by columnar epithelial cells. These molecules limit bacterial access to the www.eji-journal.eu

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Figure 2. Crosstalk between the commensal microbiota and the mucus layer. Bacterial components such as LPS and flagellin stimulate bacterial sensors such as TLRs, nucleotide-binding oligomerization domain-containing protein 2 (NOD2), and inflammasomes expressed by various intestinal immune and epithelial cells. This stimulation increases the production of mucus components such as antimicrobial peptides, trefoil factor 3 (TFF3), and secretory IgA (SIgA), which optimize immunological resistance against invading pathogens and the relative proportions of commensal bacterial at the intestinal surface.

epithelial surface and create a suitable cohabitation niche for the commensal microbiota, and can be induced by bacterial stimulation (e.g., via TLR-dependent pathways; Fig. 2) [12]. Indeed, depletion of MyD88, the central adaptor molecule for nearly all TLRs, in the intestinal epithelium decreases the expression of polymeric immunoglobulin receptor (a major component for the formation and transportation of SIgA), mucin 2, and antibacterial peptides [13]. These observations indicate the importance of the crosstalk between the commensal microbiota and epithelial cells in optimizing resistance to infectious diseases and maintaining epithelial integrity. Paneth cells reside near the stem cell crypt in the villi of the small intestine and directly sense commensal microbiota through a TLR–MyD88-dependent pathway [14, 15]. MyD88 expressed in Paneth cells regulates RegIII-γ production [16]. This cascade induces the expression of antimicrobial factors such as RegIII-γ, CRP-ductin, and RELM-β, which possibly contribute to the creation of a protective environment for intestinal crypt stem cells [14, 15] (Fig. 1B and 2). In addition, defensin production by Paneth cells is induced via stimulation of TLRs and intracellular bacterial sensors such as NODs and NLRs (Fig. 2) [6]. Defective Paneth cells and susceptibility to intestinal inflammation have been revealed in mice deficient in several Crohn’s disease-associated genes, such as Nod2 and Atg16l1. A frameshift mutation in NOD2 reduces antimicrobial peptide production by Paneth cells, creating a predisposition for the development of Crohn’s disease [17]. Indeed, in patients with Crohn’s disease, NOD2 mutation is associated with diminished α-defensin expression [17]. Furthermore, patients with Crohn’s disease carrying the ATG16L1 mutation are associated with abnormal Paneth cell granules.  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

The development and function of goblet cells are also regulated in part via TLRs [16, 18]. For instance, goblet-cell development is negatively regulated via the TLR4 pathway [18]. Goblet cells produce mucus that blocks the direct attachment of commensal bacteria to the epithelial layer, thereby spatially regulating the colonization of commensal bacteria [19]. The importance of goblet cells in the protection of the intestinal mucosa is evident in the roles of goblet-cell-derived mucin 2, a major colonic gel-forming mucin, and the small peptide trefoil factor 3 (TFF3). In mucin 2-deficient mice, commensal bacteria are able to directly contact and alter the morphology of epithelial cells, which leads to the spontaneous development of colitis and colonic cancer [20]. TFF3 synergizes with mucin and enhances the protective barrier properties of the mucus layer by promoting the physiological migration of intestinal epithelium to the mucosal surface and the healing of the epithelium (Fig. 2) [15]. TFF3 expression is regulated by TLR2-mediated signals [21]. Indeed, a colitisassociated variant of TLR2 results in impaired mucosal repair because of a lack of TFF3 expression [21]. It is noteworthy that mice lacking either mucin 2 or TFF3 are highly susceptible to experimental colitis [15, 20]. Different types of epithelial cells induce different immune responses. It has been recently reported that goblet cells are involved in oral tolerance, where they preferentially deliver low molecular weight Ags to tolerogenic CD103+ DCs to induce immunological unresponsiveness to Ags that would otherwise provoke an immune response [22]. Unlike tolerance-inducing goblet cells, M cells are involved in the initiation of acquired Ag-specific immune responses, such as IgA induction, via the uptake of luminal Ags [23]. M cells specialize in taking up Ag www.eji-journal.eu

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from the intestinal lumen and are therefore also referred to as gateway or Ag-sampling cells. M cells reside in the follicleassociated epithelia of Peyer’s patches and isolated lymphoid follicles, which are organized lymphoid tissues involved in immune surveillance and the induction of Ag-specific immune responses within the mucosa [23]. Our recent study indicates that M cells are the point of entry for Alcaligenes spp., a newly identified intratissue commensal flora that is involved in the induction of IgA in Peyer’s patches [23]. M cells are also the point of entry for infectious pathogens (e.g., Salmonella enterica serovar Typhimurium) [24]. Importantly, M cells express glycoprotein 2 (GP2), a receptor for FimH, which is a component of the type I pili of gram-negative bacteria [24,25]. Thus, GP2-deficient mice show reduced immune responses against infected S. enterica serovar Typhimurium [24]. Because the translocation of Ags by M cells through DCs located beneath M cells induces strong acquired Ag-specific immune responses, Ag delivery to M cells via M-cell-specific molecules promotes effective Ag-specific mucosal immune responses; this may therefore be a suitable route for the delivery of mucosal vaccines and adjuvants [23]. Columnar epithelial cells form a monolayer surface barrier by firmly attaching to each other via tight junctions and their function and integrity is regulated by the commensal microbiota. Both antibiotic-treated mice and MyD88-deficient mice showed severe inflammatory responses in a dextran sodium sulfate-induced colitis model [26]. Stimulation of TLRs and NLRs by commensal bacteria activates NF-κB, which is required for the homeostasis of the epithelial layer. It has been revealed that epithelial-cell-specific deletion of NF-κB essential modulator (also known either as NEMO or inhibitor of NF-κB kinase [IKK-γ]), one of the regulatory subunits of the IKK complex, results in the spontaneous induction of intestinal inflammation [27]. It has been proposed that cellular death resulting from NEMO deficiency allows commensal bacteria to penetrate the epithelial layer and leads to excessive TNF production by mucosal myeloid lineage cells and subsequent epithelial apoptosis [27], because the absence of TNF receptors and MyD88 rescues the development of colitis in epithelial-specific NEMO-deficient mice [27]. In addition, mice lacking inflammasome components such as NLRP3 or caspase-1, or lacking the production of IL-1β and IL-18, show severe inflammation in an experimental colitis model [7]. Under these conditions, exogenous administration of IL-18 reverses the pathological phenotypes resulting from the defective inflammasomes [28]. NLRP6-deficient mice also show reduced IL-18 expression together with spontaneous intestinal hyperplasia and exacerbation of colitis as well as an aberrant fecal microbiota composition [7]. Additionally, NLRP6 inflammasome-deficient mice show a different microbiota composition that includes increased levels of the bacterial phyla Bacteroidetes (Prevotellaceae) and TM7 compared with WT mice [7] (Fig. 2). This indicates that the composition of the commensal microbiota is regulated by NLRP6 inflammasome-IL-18 pathways [7]. In addition to their primary role as a physical protective barrier separating the inside and outside environments, intestinal epithelial cells create a harmonized interface that avoids unneces C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

HIGHLIGHTS

sary reactions to innocuous Ags. Disruption of the epithelial barrier results in invasion by commensal bacteria and the excessive production of inflammatory cytokines by mucosal immune cells, eventually resulting in pathological inflammation that may subsequently develop into chronic intestinal inflammation [12]. Thus, epithelial cells tightly mediate the crosstalk between the mucosal immune system and the commensal microbiota.

Innate mucosal conductors of physiological inflammation: ILCs ILCs, which have recently been identified as important innate cell populations in mouse and human, are involved in the maintenance of the intestinal barrier, the induction of inflammation, and the regulation of tissue repair [6, 8]. ILCs are categorized into three groups [11, 29, 30]: Group 1 ILCs (ILC1 cells and NK cells) express T-bet and produce IFN-γ; Group 2 ILCs (ILC2 (natural helper cells), nuocytes, and innate helper 2 cells) express retinoic acid receptor (RAR)-related orphan receptor (ROR)α and GATA3, and produce IL-5 and IL-13; and Group 3 ILCs (fetal lymphoid tissue-inducer cells, NKp46-positive NK22 cells, and NKp46-negative ILC3 cells) express RORγt and produce either IL-17A, IL-17F, or IL-22 (reviewed in [29, 30]). In addition to the different transcription factor and cytokine expressions among the three groups of ILC cells, the cytokine receptor expression patterns also differ: ILC1 cells express IL-12R-β2, ILC2 cells express IL-33R and IL-25R, and ILC3 cells express IL-1R and IL-23R [30]. Of these ILC populations, ILC3 cells, together with intestinal CD4+ T cells (Th17 and Th22 cells), are the main source of IL-22, which is one of the homeostatic cytokines critical for epithelial-cell-mediated immune responses under physiological conditions [6]. IL-22 receptors are specifically expressed on intestinal epithelial cells in the steady state [31], and epithelial cells are in turn activated via IL-22 receptors leading to the production of the antimicrobial peptides RegIII-β and RegIII-γ, which eliminate bacterial pathogens [32, 33]. IL-22-deficient mice are susceptible to radiation-induced tissue damage, suggesting the importance of IL-22 produced by ILC3 cells in the maintenance of epithelial homeostasis [14]. It has been reported that Th22 cells develop via an IL-6-dependent pathway, whereas IL-22-producing ILCs develop via an IL-23-dependent pathway [34]. The production of IL-22 by ILC3 cells is regulated by various means. For instance, IL-23 produced by LTβR+ intestinal DCs regulates IL-22 production in the steady state (Fig. 1B) [8]. In addition, TLR5 expressed on CD103+ CD11b+ intestinal DCs plays an important role in the production of IL-23 in response to bacterial flagellin, leading to IL-22 release from ILC3 cells [32]. In this context, IL-22 production by ILC3 cells is influenced by the commensal microbiota [16,31,35]. In addition, IL-22 production from ILCs is tightly inhibited by IL-25 produced by epithelial cells stimulated by the commensal microbiota (Fig. 1A) [35]. Because ILC3 cells do not express receptors for IL-25, it is likely that the involvement of the IL-25 receptor IL-17BR, which is expressed by DCs, plays a role in the inhibition of IL-22 production [30,35]. The commensal microbiota plays a central role in the regulation of the epithelial cell and www.eji-journal.eu

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ILC axis mucosal barrier, which controls the production of IL-22 and IL-25 as promoting and inhibitory cytokines, respectively. Further investigations are needed to understand the interaction between IL-22-producing ILC3 cells and intestinal epithelial cells, and the contribution of intestinal DCs to the homeostasis of the mucosal barrier. Another way in which ILCs regulate intestinal inflammation is by the production of antimicrobial peptides. RegIII-γ induced by IL-22-producing ILC3 cells provides a defensive platform against C. rodentium infection by promoting bacterial clearance through direct antimicrobial effects [36]. RegIII-γ also regulates the spatial colonization of commensal bacteria and RegIII-γ-deficient mice show high numbers of gram-positive Eubacterium rectale and segmented filamentous bacteria (SFB) attached to the epithelium (Fig. 1B) [16]. RegIII-γ-deficient mice also have increased levels of SIgA and CD4+ T cells in the intestinal compartment [16]. Furthermore, LT-β receptor-deficient mice lacking ILC3-cell-mediated antimicrobial peptide production (e.g., RegIII-γ) show a different microbiota composition and increased bacterial colonization (e.g., increased numbers of SFB) compared with their WT littermates [37]. These studies indicate that loss of the spatial harmonization between the commensal microbiota and the intestinal epithelial layer causes enhanced activation of intestinal adaptive immunity (e.g., expansion of the numbers of Th1 cells and IgA-producing cells) [16]. In addition to the central role of ILCs in the induction of IL-22 and antimicrobial molecules, ILCs also play a critical role in the induction of acquired mucosal immune responses such as the IgA induction pathway [38]. Intestinal IgA is involved in the development and maintenance of the homeostasis between the commensal microbiota and the host immune system [39]. An aberrant commensal microbiota is found in activation-induced (cytidine) deaminase-deficient mice, which results in a defect in IgA classswitching, leading to reduced IgA production in the intestine [40]. Colonies of anaerobic commensal bacteria, especially SFB, are expanded in activation-induced (cytidine) deaminase-deficient mice, subsequently causing hyperplasia of isolated lymphoid follicles. It is therefore possible that ILCs orchestrate both innate (e.g., epithelial cell-derived RegIII-γ) and acquired (e.g., IgA) mucosal immunity for the maintenance of the homeostasis of both the luminal commensal microbiota and the host mucosal immune system. A recent study has indicated the regulatory function of ILCs in the anatomical containment of commensal bacteria [41]. Alcaligenes is an opportunistic gram-negative bacterial species frequently detected in hospitalized patients with HIV, cancer, or cystic fibrosis [41]. In na¨ıve mice, Alcaligenes spp. are detected in GALTs, such as Peyer’s patches, isolated lymphoid follicles, and MLNs, but not in systemic lymphoid organs such as spleen [41]. Alcaligenes spp. have also been detected in the GALT of nonhuman primates and humans [41]. This anatomical compartmentalization of Alcaligenes spp. is presumably mediated by IL-22regulating antimicrobial molecules such as RegIII-β, RegIII-γ, S100a8, and S100a9 (Fig. 1B). In this context, the elimination of ILCs by administration of ILC-depleting monoclonal antibodies or neutralization of IL-22 induces dissemination of Alcaligenes spp.  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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from the Peyer’s patches into the systemic compartments resulting in pathological evidence of inflammation in mice (e.g., enlargement of the spleen) [41]. Thus, ILCs and IL-22 play major roles in the maintenance of a homeostatic symbiotic relationship between intestinal commensal bacteria and the host mucosal immune system via, for example, the containment of intratissue habituated commensal microorganisms. This study was the first to show the importance of the innate immune system in maintaining anatomical niches for opportunistic commensal bacteria, and therewith, intestinal homeostasis. In addition to the anatomical containment of commensal bacteria, another recent study has indicated that Ag presentation by MHC class II-expressing ILC3 cells is required to limit the expansion of pathological CD4+ T cells against commensal bacteria in the intestinal compartment [42]. Selective deletion of ILC3 cells in mice results in spontaneous colitis with dysregulation of commensal bacteria-dependent CD4+ T cells [42]. These observations indicate the importance of ILCs for the regulation of both commensal bacteria and host adaptive immune responses.

A state of acrimonious inflammation: Mucosal barrier disruption and innate immune cell deregulation Homeostatic interactions between innate immune cell populations and the intestinal epithelium are strictly regulated; however, once the regulatory axis between the innate mucosal and epithelial cells is disrupted, intestinal inflammation is inevitable. Notwithstanding the importance of ILCs in the maintenance of epithelial integrity, as well as in the regulation of the composition and localization of the commensal microbiota, these cells are also involved in intestinal inflammation. IL-17- and IFN-γ-producing IL-23-responsive ILCs have been identified as inducers of intestinal inflammation in murine colitis models (e.g., Helicobacter hepaticus-infected mice or dextran sodium sulfate-treated mice with a human immune system) [9, 29]. Indeed, treatment of mice with a neutralizing Ab against the IFN-γ produced by ILCs ameliorates the progression of intestinal inflammation in a murine colitis model [43]. Recently, ILC3 cells have been shown to differentiate into IFN-γ-producing ILC1 cells (NKp44+ CD103+ ) in response to IL-12 and IL-15 [44]. In human Crohn’s disease patients, the frequencies of ILC1 and ILC3 cells are higher in the intestinal compartment compared with those in healthy subjects [43, 44]. These observations indicate that ILCs, in addition to Th1 and Th17 cells, are a promising target for the treatment of intestinal inflammation. MCs are versatile cells that only terminally mature once they reach peripheral tissues [45]. It has been suggested that local conditions, such as the presence/absence of inflammation, within the peripheral tissues influence MC phenotype (e.g., the expression patterns of MC proteases) [45]. MCs promote the clearance of pathogens (e.g., T. spiralis and N. brasiliensis) by producing proteases (e.g., MC protease-1) and recruiting neutrophils and eosinophils to the intestine by increasing vascular permeability [10]. MCs also contribute to the maintenance of mucosal homeostasis by supporting IgA production and inducing epithelial www.eji-journal.eu

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turnover [46, 47]. MCs promote the differentiation of B cells to IgA-producing plasma cells in the intestinal compartment through the production of IL-6 as well as through cell–cell interactions, especially through the CD40L–CD40 pathway (Fig. 1B) [46]. At the basal level, paracellular and transcellular epithelial permeability is tightly regulated by proteases released by MCs (e.g., MC protease-4). Thus, MCs maintain the architecture of the intestinal epithelium by regulating epithelial migration [47]. Furthermore, MC-deficient mice, as well as chymase-deficient mice, show decreased epithelial migration and permeability that subsequently alters villus morphology (Fig. 1B) [47]. Even though MCs also play important roles in the maintenance of mucosal homeostasis in the intestine, such as in optimizing epithelial turnover, excessive activation of MCs is positively correlated with the severity of allergic and inflammatory conditions in the gut such as food allergies and inflammatory bowel disease (IBD) [48, 49]. Accumulation of activated or degranulated MCs and increased levels of tryptase derived from MCs are found in the intestines of patients with IBD [47–50]. Recent analyses indicate that MC-derived tryptase is involved in the induction of inflammation in mice with experimental colitis [50]. These results imply that activation of MCs is required for the maintenance of a basal level of mucosal homeostasis, but that abnormal or excess activation induces intestinal inflammation. The factors responsible for MC activation during intestinal inflammation might be considered as possible targets for treating IBD. Our recent study indicates that MCs promote inflammation after activation by extracellular adenosine triphosphate (ATP), which is considered to be a damage signal [50]. Extracellular ATP, which is released from both necrotic cells and activated monocytes during inflammation, promotes a wide range of pathophysiological responses via the activation of cell-surface purinergic P2 receptors [50]. For instance, increased levels of ATP are released in the peritoneal cavity of mice showing inflammation during graft-versushost disease [51]. In addition, a colorectal biopsy of mice with experimental colitis showed a significant increase in ATP release compared with that of healthy mice [49]. The P2X receptor family currently includes seven unique receptor subtypes (P2X1–7). P2X1–7 are receptors for extracellular ATP that act as ATP-gated ion channels [52]. It has been demonstrated that P2X7 is involved in various inflammatory symptoms, such as contact hypersensitivity and asthma [53, 54]. MCs in the colon express high levels of P2X1, 4, and 7 receptors [49]. Extracellular ATP stimulation induces the release of inflammatory cytokines (e.g., IL-1β, IL-6, TNF-α), chemokines (e.g., CCL2, CXCL2), and lipid mediators (e.g., leukotriene B4) from MCs [49]. The molecular cascade of ATP and P2X7 plays a critical role in mucosal MC-mediated inflammation, and inhibition of the ATP–P2X7 pathway reduces colonic inflammatory responses [49]. Furthermore, P2X7-expressing MCs accumulate at inflammatory sites in the colon of patients with Crohn’s disease [49]. It has also been reported that commensal microbiota are involved in the migration of colonic MCs. Stimulation by commensal bacteria via TLRs induces CXCR2 ligand production by the epithelium, which in turn induces the migration of MCs [55]. The function of MCs in inflammatory disorders  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

may also be related to defects in the development and functions of non-MC cells, especially those cells expressing c-kit [56]. ILC2 and ILC3 cells express c-kit; however, the importance of c-kit deficiency in the development and function of ILCs remains to be clarified [29, 30]. These studies clearly demonstrate that excess and topical activation of innate immune cells such as ILCs and MCs, together with modulation via stimulation by the commensal microbiota, initiates and advances intestinal inflammation. It is important to further understand the regulatory and inhibitory mechanisms of the abnormal activation of these mucosal innate cell populations.

Conclusion Our understanding of the immunobiological characteristics of the intestinal surface barrier in both healthy and disease states has dramatically progressed in the past few years. Research has shown the roles of innate cells and sensors in the regulation of both physiological and pathological inflammation. The context is complicated, but the interplay and crosstalk among the luminal microbiota, epithelial cells, and innate immune cells such as ILCs and MCs possesses a dual nature in that it is capable of both protecting the mucosal barrier and initiating and advancing inflammation. The contribution of the crosstalk in innate–innate and innate–acquired cell interactions in the mucosal barrier remains to be elucidated. Recently, it was demonstrated that MCs and ILCs dynamically interact in the skin compartment and that this interaction plays an important role in the induction of skin inflammation in mice [57]. In the context of innate–innate cell interactions, it could be possible that this line of crosstalk also occurs in the mucosal compartments. Understanding the molecular and cellular spatiotemporal regulatory mechanisms for the maintenance of homeostasis and initiation of pathological conditions in mucosal compartments such as the intestinal tract will uncover immunobiologically unique host–parasite interaction machineries and new targets for the development of novel therapeutic and preventive approaches for various diseases.

Acknowledgements: This work is supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Grant-in-Aid for JSPS Fellows [023-04859 to Y.K.], Scientific Research S [23229004 to H.K.], and the Young Researcher Overseas Visits Program for Vitalizing Brain Circulation [to H.K., Y.K., and Y.G.]); the Global Center of Excellence Program of the Center of Education and Research for Advanced Genome-based Medicine (to H.K.); and the Core Research for Evolutional Science and Technology Program of the Japan Science and Technology Agency (to H.K.). We are grateful for helpful comments from Aayam Lamichhane and Anne M. O’Connor. www.eji-journal.eu

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Conflict of interest: The authors declare no financial or commercial conflict of interest.

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18 Sodhi, C. P., Neal, M. D., Siggers, R., Sho, S., Ma, C., Branca, M. F., Prindle, T. et al., Intestinal epithelial Toll-like receptor 4 regulates goblet cell development and is required for necrotizing enterocolitis in mice. Gastroenterology 2012. 143: 708–718, e701–705.

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factor 3 Full correspondence: Prof. Hiroshi Kiyono, Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan Fax: +81-3-5449-5411 e-mail: [email protected] See all articles in the Immunity at the Barrier Review Series at http://onlinelibrary.wiley.com/doi/10.1002/eji.v43.12/issuetoc

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Received: 7/6/2013 Revised: 25/9/2013 Accepted: 30/9/2013

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