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Intestinal microbiota and its effects on the immune system.1 Maria Rescigno Department of Experimental Oncology, European Institute of Oncology Milan, Italy

Running title: The immune system and the microbiota

Correspondence to: Maria Rescigno, PhD Department of Experimental Oncology. European Institute of Oncology Via Adamello, 16 20139 Milan, Italy Dir.: +39-0257489925 Fax: +39-0294375990 [email protected]

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/cmi.12301

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The microbiota colonizes every surface exposed to the external world and in the gut, it plays important roles in physiological functions such as the maturation of the immune system, the degradation of complex food macromolecules and also behavior. As such, the immune system has developed tools to cohabit with the microbiota, but also to keep it under control. When this control is lost, dysbiosis, i.e. deregulation in bacterial communities, can occur and this can lead to inflammatory disorders, including inflammatory bowel disease (IBD), obesity, diabetes and autism. For these reasons, the analysis of the microbiota, its interactions with the host and its composition in disease, have been intensively investigated in the last few years. In this review, we summarize the major findings in the interaction of the microbiota with the host immune system.

Introduction The analysis of microbiota composition and its role on our health have recently exploded due to the advent of new techniques that allow to clearly identify the composing species. It is emerging that we are made of ten times more microbial than mammalian cells which contribute to a hundred times more genes. Hence, the microbiota provides a set of new functions that over the years our organism has learned to exploit (Qin et al., 2010). This enormously enhances the genetic variation

among individuals that is provided by the human genome (Li et al., 2008; Mueller et al., 2006; Qin et al., 2010). One important function is the maturation of the immune

system and protection against some infectious agents (Hooper et al., 2012; Khoruts et al., 2010; Reid et al., 2011; Swiatczak et al., 2011). It is becoming clear that especially in the early phases of life the microbiota ‘educate’ our immune system to

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deal with both innocuous and harmful bacteria and to establish a balance among the two that is characteristic of a healthy gut. Indeed, the microbiota is composed by symbiotic innocuous bacteria and potential pathogens also called pathobionts (Chow et al., 2011) . In this review we will revise recent findings and new challenges of this exciting field.

Establishment of the microbiota. Children’s delivery mode has an impact on early microbiota composition (Dominguez-Bello et al., 2010). Vaginally delivered children display a microbiota,

which reminds that of the vaginal microbiota and includes Lactobacillus, Prevotella,

Atopobium, or Sneathia spp., while babies delivered with caesarian section have more skin taxa that include Staphylococcus spp (Dominguez-Bello et al., 2010). This suggests that the microbiota derives at least in part from the mothers during the delivery. The generation of diversity of the microbiota occurs in the first three years of life and then stabilizes (Yatsunenko et al., 2012). Interpersonal variations are

higher among children than among adults. This is partly inherited by the parents as siblings and twins not only have a microbiota that is more similar among them than with unrelated children, but their microbiota is closer to that of their mother or father than unrelated parents (Yatsunenko et al., 2012). Further, individuals coming from different geographic areas also display different microbiota. In particular, individuals from Malawi or Amerindians have a more diversified microbiota than those from the US (Yatsunenko et al., 2012). This may reflect a different diet, but also the exposure

to diverse environmental challenges that require a more heterogeneous phylogenetic composition of the fecal microbiota.

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Microbiota and innate immunity The direct interaction of the microbiota with epithelial cells and innate immune cells can be prevented by the presence of a mucous layer. In the proximal mouse small intestine the mucous is more patchy (Shan et al., 2013), while in the terminal ileum and colon (Linden et al., 2008) the mucous physically separates the intestinal lumen

from the epithelium (Johansson et al., 2008). The dense mucous layer (Johansson

et al., 2008) sanitizes around a 50-m region closest to the epithelium thanks also to the production of antimicrobial peptides (Vaishnava et al., 2011). Early in life when this region is not yet formed, or when the microbiota can breach it due to specific molecular characteristics or when there is a deregulation in its formation, then the microbiota can interact with the underlining epithelial cells and immune cells.

Microbiota and innate lymphoid cells Recently, a new class of immune cells has been described that are called innate lymphoid cells (ILCs) (reviewed in (Spits et al., 2013)). These cells have been studied mostly in the mouse system and very little is known in humans. These cells can be grouped in three subgroups based on the expression of transcription factors, surface markers and cytokines and continuously interact with the microbiota (Chen and Kasper, 2013; Sonnenberg and Artis, 2012). Groups 1 and 2 remind T helper cells 1 and 2, respectively as they express either T-bet and IFN- (group 1) or Gata-3, IL-5 and IL-13 (group 2). A more heterogeneous group expresses Rort and is called group 3. This comprises at least other three subgroups: one expressing T-bet, AhR, NCR and secretes IL-22 (classical ILC3); another one expressing only Rorγt and not NCR, but producing IL-17, IL-22 and IFN- (colitogenic ILC3); and the last one also called

LTi cells as they are involved in secondary lymphoid organ generation, that expresses

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AhR and the cytokines IL-17 and IL-22 (Spits et al., 2013). NCR+ ILC3 seem to be involved in the homeostasis of the gut. These cells express NKP46 but are not natural killer cells and are induced by the microbiota (Sanos et al., 2009). They produce IL-

22, a cytokine involved in epithelial cell repair and antibacterial activity (Cella et al., 2009; Luci et al., 2009; Sanos et al., 2009; Satoh-Takayama et al., 2008; Vivier et al., 2009; Zheng et al., 2008). IL-22 can be induced in ILC group 3 by several means. Aryl hydrocarbon receptor (AhR) engagement by ILCs induces IL-22 (Lee et al., 2012; Qiu et al., 2013). Ligands of the AhR can be produced as metabolic products of tryptophan by the microbiota, and in particular by some lactobacilli such as L. reuterii

(Zelante et al., 2013). IL-22 production is controlled by the microbiota also via the induction of IL-25 by intestinal epithelial cells (Sawa et al., 2011). IL-22 is also induced by lamina propria dendritic cells (expressing the marker CD103) in response to bacterial flagellin, after its binding to TLR5, and transient production of IL-23 (Kinnebrew et al., 2012). Altogether, these findings suggest that the induction of IL-

22 by ILCs can be a result of a multi party control among different compartments, microbiota, epithelial cells and dendritic cells. ILC3 can also control the proliferation of T cells in response to the microbiota independently of IL-22, IL-23 or IL-17 production (Hepworth et al., 2013). NKp46-Tbet- ILC3 can process and present antigens in the context of MHC class II molecules but do not induce T cell proliferation, rather they limit CD4+ T cell responses (Hepworth et al., 2013). Mice lacking MHC II on ILC3 display spontaneous colitis, indicating an important role of these cells in controlling T cell expansion and inflammation. Group 3 ILCs are also involved in Immunoglobulin (Ig)A induction via the release of soluble lymphotoxin (LT), presumably via regulation of T cell homing to the lamina propria (Kruglov et al., 2013). Expression of membrane bound lymphotoxin by ILCs, by contrast,

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regulates T cell-independent IgA induction, presumably via the production of iNOS by lamina propria phagocytes (Kruglov et al., 2013). As a consequence, lack of expression of LT by ILCs results in deregulation of microbiota composition, also called dysbiosis, and increase in segmented filamentous bacteria.

Microbiota and intraepithelial T cells. Together with ILCs, intraepithelial lymphocytes are the first line of defense against invading pathogens and participate to the shaping of the microbiota. The most abundant intestinal intraepithelial lymphocytes bear the  T cell receptor (Kunisawa

et al., 2007). Mouse  T cells promote repair of injured gut epithelia (Komano et al., 1995).  T cells play a major role in limiting the entrance of commensal bacteria after epithelial injury via the release of antimicrobial factors (Ismail et al., 2009). This response is mostly induced by the microbiota as in its absence (germ-free mice) the induction of the majority of genes related to inflammation and to the antimicrobial response is drastically reduced after intestinal epithelial disruption (Ismail et al., 2009).  T cells release anti-microbial peptides in response to invading pathobionts,

however, this is not due to a direct interaction between the microbiota and  T cells but is mediated by epithelial cell signaling via MyD88, an adaptor of the Toll-like receptor (TLR) signaling pathway (Ismail et al., 2011). A subset of mouse  T cells characterized by the expression of IL-1R1 are expanded by the microbiota and produce IL-17 in response to IL-23 and IL-1 (Duan et al., 2010), presumably

produced by other innate immune cells such as the ILC 3. Intraepithelial lymphocytes are equipped with peptidoglycan recognition protein 2 (PGLYRP-2) an enzyme involved in the degradation of peptidoglycan that destroys the nucleotide binding oligomerization domain containing protein (Nod)2-detected

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muramyl dipeptide structure, thus decreasing its availability and probably limiting an excessive activation of the innate response (Duerr et al., 2011).

Microbiota and granulocytes Selective depletion of the microbiota through antibiotic treatment results in uncontrolled proliferation of basophils, the increase of serum IgEs and the development of allergic reactions, suggesting that the microbiota plays a role also in the regulation of mouse basophil homeostasis (Hill et al., 2012). The increase in basophil numbers and serum IgE is dependent on signaling via MyD88 in B cells, suggesting a direct control of IgE in basophil homeostasis. Hence, the effect of the microbiota is not direct on basophils, but is dependent on B cells via control of IgE synthesis (Hill et al., 2012). Increased IgE levels in germ free mice can also cause

activation of IgE-bound mast cells and oral-induced systemic anaphylaxis (Cahenzli

et al., 2013). Peptidoglycan is one of the major constituents of the bacterial cell wall. Immune cells can sense degradation products of peptidoglycan via intracellular receptors NOD1 and NOD2. NOD1 recognizes primarily meso-diamino pimelic acid that derives from the degradation of peptidoglycan from gram negative bacteria. It has been recently shown that peptidoglycan translocated across the epithelial barrier can reach the blood stream and can activate neutrophils via NOD1 (Clarke et al., 2010). PGN-activated neutrophils have better bacterial killing properties, suggesting that the microbiota can directly influence neutrophil activity and prepares them for possible invading pathobionts (Clarke et al., 2010). Recognition by pattern recognition receptors hence does not only have a role in protecting against pathogens, but also that of ‘educating’ the innate immune system to preserve intestinal homeostasis.

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Microbiota and NKT cells Cd1d is an MHC class 1-like molecule expressed on natural killer T cells (NKT cells). CD1d recognizes self and foreign lipids and glycolipids. It has been shown that mice lacking CD1d display increased translocation of the microbiota as well as a different microbiota composition (Nieuwenhuis et al., 2009). This may be due to the capacity of NKT cells to regulate the function of Paneth cells, the major producers of antimicrobial peptides. The microbiota is involved in the phenotypic maturation of NKT cells (Wingender et al., 2012). However, the correct number and function of NKT cells is regulated by the microbiota only early in life. Microbiota colonization of adult germ-free mice does not correct the higher number of NKT cells observed in germ free mice and this correlates with increased severity of oxazolone-mediated colitis (Olszak et al., 2012).

Microbiota, dendritic cells and macrophages Dendritic cells and macrophages in the gut play important roles both for tolerance and for immunity induction. The distinction between macrophages and DCs in the gut is not obvious based on surface markers (Pabst and Bernhardt, 2010). This confusion rises because besides classical macrophages, in the gut there is an atypical population of macrophages that expresses CD11c, a marker previously associated to DCs. Among the cells that express CD11c, four different subsets are found in the mouse having different origin and function (Coombes and Powrie, 2008; Farache et al.,

2013b; Pabst and Bernhardt, 2010). Not much is known on what are the human counterpart gut APCs. Peripheral blood monocytes give rise to either CD11c+ macrophages or inflammatory DCs depending on the milieu found in the colon at the time of recruitment (Rivollier et al., 2012; Zigmond et al., 2012). These cells are

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characterized by the expression of high or intermediate levels of the chemokine receptor CX3CR1. CD11c+CX3CR1highF4/80+ resident macrophages display antiinflammatory properties (Rivollier et al., 2012): they express interleukin-10 (IL-10)

that is required to restimulate T regulatory cells (Treg) in situ (Hadis et al., 2011) and inhibit T cell proliferation via a contact dependent mechanism (Kayama et al., 2012). These macrophage-like cells are mostly sessile (Schulz et al., 2009), a characteristic conferred by the microbiota, as they can migrate out of the intestine after antibiotic

treatment (Diehl et al., 2013). By contrast, inflammatory DCs (CD11c+CX3CR1int) develop under intestinal inflammation, produce large quantities of inflammatory mediators (interleukin-12 (IL-12), IL-23, inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF)), and are capable of migrating to the draining lymph node and activate T helper-1 (Th1) T cells (Rivollier et al., 2012). A population of ‘classical’ migratory DCs is characterized by the expression of the αE integrin CD103. These cells can be further subdivided based on the expression of CD11b. CD11b-CD103+ DCs are most likely associated to isolated lymphoid follicles

that contaminate the lamina propria (LP) cell preparation (Bogunovic et al., 2009). Conversely, CD11b+CD103+ cells are present in the LP of the small and large

intestine, and migrate to the draining lymph nodes for the induction of T reg cells via the release of retinoic acid (RA), a metabolite of vitamin A, and transforming growth factor-

-β) (Coombes et al., 2007; Sun et al., 2007) and the activity of

Indoleamine 2,3-dioxygenase, IDO (Matteoli et al., 2010), an enzyme involved in

tryptophan catabolism. Furthermore, Interestingly, mice lacking both CD103+ DCs still retain the capacity to drive Foxp3+ T reg cells in the mesenteric lymph nodes, suggesting that there may be mechanisms of compensation, but the number of T regs is strongly reduced in the LP (Welty et al., 2013). This is due to the inability in these

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mice to induce CCR9 expression on T regs which confers them gut homing properties, suggesting that CD103+ DCs are absolutely required to drive the

development of gut homing T regs. Interestingly, the absence of these DC populations does not impact on mouse microbiota composition (Welty et al., 2013). The tolerogenic function of CD103+ DCs are imparted by factors released by

intestinal epithelial cells both in the mouse and human systems (Iliev et al., 2009a; Iliev et al., 2009b). Mouse CD11b+CD103+ DCs have been shown to be also required for the maintenance of mucosal Th17 T cells (Persson et al., 2013; Schlitzer et al., 2013). This function is a property of a subpopulation that does not express ALDH, the enzyme involved in retinoic acid generation from retinal (Janelsins et al., 2014). These cells also express acyloxyacyl hydrolase (AOAH), an enzyme that inactivates LPS. Colonic DCs deficient for AOAH have reduced ability to secrete IL-6 and to drive Th17 cell polarization (Janelsins et al., 2014). It would be interesting to know whether these mice have reduced ability to control infections or increased susceptibility to colitis. Mouse mononuclear phagocytes are actively involved in sampling the gut luminal content, via the extension of dendrites between epithelial cells (Rescigno et al., 2001) especially, under steady state, in the upper part of the small intestine (Chieppa et al., 2006). CX3CR1+ macrophages are most efficient in sending protrusions out into the

lumen and capture bacteria (Niess et al., 2005), soluble proteins (Mazzini et al., 2014) and fungi (Vallon-Eberhard et al., 2006). By contrast, CD103+ DCs are less frequent

at extending protrusions in the unperturbed state and capture primarily bacteria (Farache et al., 2013a). Interestingly, in the mouse small intestine, macrophages and DCs are in close physical interaction and can exchange antigens via a Connexin 43and gap junction-dependent mechanism (Mazzini et al., 2014).

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Some of the anti-inflammatory properties of DCs or macrophages may be due to their interaction with the microbiota or their metabolic products. Cd11c+ and CD11b+ phagocytes express GPR109A (encoded by Niacr1) a receptor for butyrate and niacin that are produced by the gut microbiota. GPR109A signaling leads to the production of IL-10 and the differentiation of T reg cells (Singh et al., 2014). The microbiota may control the activity of intestinal phagocytes also via epigenetic mechanisms such as gene expression regulation by microRNA. The expression of miR-107, a miRNA involved in controlling the expression of IL-23, for instance, is downregulated in CD11c+ intestinal colonic phagocytes during colitis (Xue et al., 2013). miR-107 is downregulated in DCs by the inflammatory cytokines IFN-γ, IL-6, and TNF-α, while it is promoted by TGF-β (Xue et al., 2013). Hence, the expression of miR-107 may

depend on the capacity of the microbiota to promote inflammatory or antiinflammatory cytokines. Indeed, the expression of miR107 is much higher in germ free (GF) than conventional specific pathogen free (SPF) mice (Xue et al., 2013). Mice in which the Fas-associated death domain (FADD) is deleted only in DCs have reduced numbers of DCs in gut associated lymphoid tissue such as the Peyer’s Patches (PP) and the mesenteric lymph nodes (MLN) (Young et al., 2013). This

correlates with systemic inflammation and increased sensitivity to endotoxin shock. DCs lacking FADD die by necroptosis which is responsible for the systemic inflammation and is dependent on the presence of the microbiota and the expression of MyD88, not by the DCs (Young et al., 2013). This is consistent with a maturation rather than a dying property of Fas in DCs (Rescigno et al., 2000). Hence, DCs are protected from microbiota-driven cell death and this ensures the maintenance of immune homeostasis.

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Mice in which DCs lack a signaling molecule (TRAF6) involved in TLR signaling, display loss of mucosal tolerance characterized by development of spontaneous colitis in the small intestine, reduced numbers of T regs and increased Th2 expansion. This susceptibility is dependent on the microbiota, but does not occur in mice that lack MyD88 in DCs. This suggests that the microbiota may activate a pathway leading to mucosal tolerance that is independent of MyD88, but dependent on TRAF6 (maybe a different pattern recognition receptor, or a different downstream signaling pathway after TLR ligation).

Microbiota and Adaptive immunity. Microbiota and B cells B cell ontogeny is not affected by the microbiota, however the microbiota can impact on the amount and isotypes of the produced immunoglobulins (Cahenzli et al., 2013; Hansson et al., 2011; Macpherson et al., 2000). In addition, the maturation of B cells

is dependent on the encounter with different bacterial species, with E. coli and bifidobacteria being among the major players in the infant human gut (Lundell et al., 2012). In the gut, the major function of B cells is to respond to the microbiota and eventual pathogens and to produce immunoglobulins of the A subtype that are protective against bacteria (Cerutti and Rescigno, 2008). IgA ontogeny and function has been extensively reviewed in (Cerutti and Rescigno, 2008; Mantis et al., 2011; Pabst, 2012; Sutherland and Fagarasan, 2012). IgA can serve several purposes: they allow the microbiota to anchor to the mucus for their colonization (Corthesy, 2007); they contrast the inflammatory activity of the bacteria through opsonization; they reduce the expression of inflammatory epitopes on the microbiota (Peterson et al., 2007). IgA are fundamental to preserve a correct balance of the microbiota, and in

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their absence there is a selected expansion of pathobionts, such as segmented filamentous bacteria (SFB) (Suzuki et al., 2004). SFB is present only in the infant

human gut and is not known whether it may be present under pathological conditions (Yin et al., 2013). Mouse IgA producing plasma cells in the gut have been shown to also express antimicrobial mediators such as TNF-a and iNOS and to have characteristics of monocyte/granulocyte lineages, suggesting that they may serve also other innate functions than just IgA production (Fritz et al., 2012). Interestingly, the genetic background is not the only determinant of IgA diversity, and in mice genetically identical, only a minority of IgA clones are expanded in young mice, while the diversity increases over age (Lindner et al., 2012). This suggests that early in life the exposure to the microbiota drives an initial diversity that is then further acquired with maturity. The correct production of IgA in response to the microbiota is ensured by the expression of the inhibitory co-receptor programmed cell death-1 (PD1) that controls the activity of T follicular helper cells. In the absence of PD-1 the selection of IgA precursor cells in the Peyer’s Patches is aberrant and this results in the alteration of microbial communities in the gut (Kawamoto et al., 2012). As mentioned above, the activity of B cells is controlled by the microbiota. Mouse microbiota alterations due to large spectrum antibiotics or germ free conditions can lead to a switch to IgE rather than IgA and subsequent activation of basophils (Hill et al., 2012) and mast cells (Cahenzli et al., 2013). This results in increased allergic

reactions. Hence, B cells are strongly controlled in their function by the microbiota, either directly or indirectly, but the outcome has profound effects on both microbiota diversity and the correct immune response. No surprise if in mice lacking B cells or IgAs, the microbiota drives a shift in epithelial cells towards reduced metabolic functions and increased IFN-related response (Shulzhenko et al., 2011).

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Microbiota and T cells As mentioned also for other immune cells, also in the case of T cell activation and differentiation, metabolic products of the microbiota can play a role either directly, or indirectly through the activity of other cell types. ATP, for instance, is released by aerobic bacteria during oxygen-dependent pyruvic acid catabolism. ATP acts on lamina propria CX3CR1+ macrophages to promote the differentiation of Th17 cells (Atarashi et al., 2008). SFB that is expanded when there is a defect in IgA production,

can induce a full activation of the mouse immune response (Gaboriau-Routhiau et al., 2009) and in particular of Th 17 cells (Ivanov et al., 2009). Given the ability of SFB

to contact epithelial cells, it might directly activate immune cells. Other components of the microbiota like B. fragilis can protect mice from experimental colitis via the induction of IL-10 producing Treg cells (Mazmanian et al., 2008). In the colon, both in the mouse and human system, Foxp3+ T reg differentiation is promoted by a combination of several strains of clostridia (Atarashi et al., 2013; Atarashi et al., 2011) or by a selected flora called altered Schaedler’s flora (ASF) that also contains

some clostridia species (Geuking et al., 2011). Microbiota driven T reg cells seems to

be specific for the microbiota (Lathrop et al., 2011). The mechanisms through which the microbiota impacts on T reg differentiation have recently started to be unraveled. B. fragilis releases outer membrane vesicles that contain the capsular polysaccharide

(PSA) that is involved in T reg differentiation (Shen et al., 2012). PSA targets TLR2 on DCs to allow their differentiation in T reg promoting cells. Three independent reports have shown that bacteria-derived short chain fatty acids, including butyrate, that are produced during starch degradation are involved in T reg differentiation

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(Arpaia et al., 2013; Furusawa et al., 2013; Smith et al., 2013). The involvement of GPR41, GPR43 or GPR109a in the observed effect has been proposed. Hence, the microbiota plays a major role in extrathymic T reg development either via microbe associated molecular patterns (such as PSA) or their metabolites.

Conclusions In conclusion, in this review we discussed primarily the role of the microbiota in controlling the maturation and activity of immune cells at steady-state. The immune system is profoundly regulated by the microbiota either directly or indirectly through the effect of barrier cells. Dendritic cells and macrophages respond to the microbiota and their metabolic products either directly or via the intervention of epithelial cells, and their activity can be regulated by microbiota-driven epigenetic mechanisms. T regulatory cells can be induced by metabolic products of the microbiota or by their cellular components. Microbiota-immune cell communication can be a two-way interaction. For instance, B cells are induced to mature by the microbiota. This has an impact on their capacity to drive isotype switching. A preference of IgE rather than IgA can drive the activation of basophils and mast cells and this results in a modified microbiota. This suggests that the action of the microbiota can result in complex immune interactions whose outcome is the control of the composition of the microbiota and the maintenance of immune homeostasis. We mainly focused on microbiota-immune cell interactions under physiological conditions and only touched upon what happens or would happen when these interactions are deregulated. Another review would be required for a full analysis on how these deregulations may promote immune disorders, particularly in humans.

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Acknowledgements This work is supported by grants of the European Commission (7th Framework programme: ERC-Dendroworld and HomeoGUT); by the Association for International Cancer Research (AICR); by the Associazione Italiana per la Ricerca sul Cancro (AICR); by the Italian ministry of health (Ricerca finalizzata). I declare no conflict of interest.

Figure legend The microbiota has profound effects on the immune system The microbiota can affect the inflammatory properties of DCs via an effect on the TLR signaling molecule TRAF6 or via epigenetic modifications through microRNA expression, such as MiR107; it can also control the viability of DCs via Fas signaling (FADD). CD103+ DCs can induce Treg development after ‘education’ by epithelial cell-derived factors (TSLP, TGF-b and retinoic acid) that in turn have been exposed to food, microbiota or its metabolites. IL-10 producing T regs can be induced by B. fragilis derived PSA via TLR2 engagement on DCs. Colonic Foxp3+ Tregs can be induced by microbiota-derived metabolic products, such as short chain fatty acids (SCFA). Bacteria-derived peptidoglycan (PGN) can be translocated across the epithelium and be sensed by NOD1for the activation of the antibacterial systemic activity of neutrophils (Nf). The microbiota or its components can be sensed via MyD88 by B cells that produce IgA rather than IgE. In the absence of this control, IgEs are elevated in the serum and this drives activation of basophils (Bf) and mast cells (MC) leading to allergic reactions. The microbiota can control the activity of ILC3 cells for the production of IL-22, epithelial cell protection and production of

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antimicrobial peptides (AMP). NKp46- ILC3 expressing MHC II can regulate the proliferation of T cells. ILC3 can also induce IgA production via a mechanism mediated by LTa.

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19

Accepted Article

Furusawa, Y., Obata, Y., Fukuda, S., Endo, T.A., Nakato, G., Takahashi, D., Nakanishi, Y., et al. (2013). Commensal microbe-derived butyrate induces the differentiation of colonic regulatory T cells. Nature 504: 446-450.

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20

Accepted Article

Iliev, I.D., Mileti, E., Matteoli, G., Chieppa, M., and Rescigno, M. (2009a). Intestinal epithelial cells promote colitis-protective regulatory T-cell differentiation through dendritic cell conditioning. Mucosal Immunol 2: 340-350.

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21

Accepted Article

Kawamoto, S., Tran, T.H., Maruya, M., Suzuki, K., Doi, Y., Tsutsui, Y., Kato, L.M., et al. (2012). The inhibitory receptor PD-1 regulates IgA selection and bacterial composition in the gut. Science 336: 485-489.

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22

Accepted Article

Lathrop, S.K., Bloom, S.M., Rao, S.M., Nutsch, K., Lio, C.W., Santacruz, N., Peterson, D.A., et al. (2011). Peripheral education of the immune system by colonic commensal microbiota. Nature 478: 250-254.

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23

Accepted Article

Matteoli, G., Mazzini, E., Iliev, I.D., Mileti, E., Fallarino, F., Puccetti, P., Chieppa, M., et al. (2010). Gut CD103+ dendritic cells express indoleamine 2,3-dioxygenase which influences T regulatory/T effector cell balance and oral tolerance induction. Gut 59: 595-604.

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24

Accepted Article

Pabst, O., and Bernhardt, G. (2010). The puzzle of intestinal lamina propria dendritic cells and macrophages. Eur J Immunol 40: 2107-2111.

Persson, E.K., Uronen-Hansson, H., Semmrich, M., Rivollier, A., Hagerbrand, K., Marsal, J., Gudjonsson, S., et al. (2013). IRF4 transcription-factor-dependent CD103(+)CD11b(+) dendritic cells drive mucosal T helper 17 cell differentiation. Immunity 38: 958-969.

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25

Accepted Article

Rescigno, M., Urbano, M., Valzasina, B., Francolini, M., Rotta, G., Bonasio, R., Granucci, F., et al. (2001). Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nat Immunol 2: 361-367.

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26

Accepted Article

Schulz, O., Jaensson, E., Persson, E.K., Liu, X., Worbs, T., Agace, W.W., and Pabst, O. (2009). Intestinal CD103+, but not CX3CR1+, antigen sampling cells migrate in lymph and serve classical dendritic cell functions. J Exp Med 206: 3101-3114.

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27

Accepted Article

Spits, H., Artis, D., Colonna, M., Diefenbach, A., Di Santo, J.P., Eberl, G., Koyasu, S., et al. (2013). Innate lymphoid cells--a proposal for uniform nomenclature. Nature reviews. Immunology 13: 145-149.

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28

Accepted Article

Welty, N.E., Staley, C., Ghilardi, N., Sadowsky, M.J., Igyarto, B.Z., and Kaplan, D.H. (2013). Intestinal lamina propria dendritic cells maintain T cell homeostasis but do not affect commensalism. The Journal of experimental medicine 210: 2011-2024.

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Accepted Article

Zheng, Y., Valdez, P.A., Danilenko, D.M., Hu, Y., Sa, S.M., Gong, Q., Abbas, A.R., et al. (2008). Interleukin-22 mediates early host defense against attaching and effacing bacterial pathogens. Nat Med 14: 282-289.

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30

SCFA

PSA

IgA

AMP

EC

Mir107 TRAF6 FADD

TSLP RA TGF-b DC

GPRs

DC

Foxp3+ Treg Protection against colitis/ inflammation

TLR2

colonic Foxp3+ Treg

PGN NOD1

MyD88

IL-22 IgA

LTa

B

Nf

IgA

IgE Bf

ILC3 MHC II

MC

T

T IL-10 Treg Control of pathogen systemic spreading

Control of allergy

Control of expansion of commensal specific T cells