Scandinavian Journal of Gastroenterology. 2015; 50: 24–33

REVIEW

The innate immune system and inflammatory bowel disease

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JULIE M. DAVIES & MARIA T. ABREU Miller School of Medicine, University of Miami, Miami Fl, USA

Abstract The innate immune system is a key factor in understanding the pathogenesis of inflammatory bowel disease (IBD) and in the hopes of improving its treatment. NOD2, a pattern recognition receptor, was one of the first major susceptibility genes identified in Crohn’s disease (CD). This discovery has been followed by genome-wide association studies that have identified other genes involved in innate immune responses. Most notably, polymorphisms in the interleukin (IL)-23 receptor have also been linked to IBD – both CD and ulcerative colitis. At the core of the innate immune defects associated with IBD is a lack of generating a robust response to control invasive commensal or pathogenic bacteria. The defect sometimes lies in a failure of the epithelium to express antimicrobial peptides or in defective control of intracellular bacteria by phagocytic cells such as dendritic cells, macrophages, or neutrophils. The recent identification of innate lymphoid cells that express the IL-23 receptor and generate both proinflammatory and protective or regulatory responses to commensal or pathogenic bacteria provides another layer of complexity to the interplay of host protection and dysregulated inflammation. Although inhibition of tumor necrosis factor has been highly successful as a strategy in treating IBD, we must better understand the nuanced role of other innate cytokines before we may incorporate these in the treatment of IBD.

Key Words: Crohn’s disease, dendritic cells, interleukin-17, interleukin-23, inflammatory bowel disease, innate lymphoid cells, NOD-like receptors, pattern recognition receptors, Toll-like receptors, ulcerative colitis

Introduction The intestine is unique, even among mucosal surfaces, for its interaction with potentially pathogenic and non-self entities, including bacteria, fungi, viruses, and the often forgotten category of food antigens. The ability of the intestine to navigate this complex environment and function to absorb nutrients and water, at the same time as excluding the microbiota, is the delicate balance of intestinal mutualism. The microbiota is required for effective extraction of nutrients from food but also carries within it the potential for host invasion and pathological infection. The intestinal immune system must walk the fine line between defense against invasion and inflammation-associated tissue damage to maintain homeostasis and optimal energy absorption.

Due to the incredible balancing act between the intestinal immune system and the microbiota, it is not surprising that this balance can be toppled resulting in inflammation and systemic release of the luminal microbiota. Inflammatory bowel disease (IBD) is a result of this imbalance. IBD is an immune-mediated disease comprising two distinct conditions with varying clinical presentations. Ulcerative colitis (UC) is an ulcerating inflammation of the mucosal layer generally restricted to the colon. Crohn’s disease (CD) is a deep transmural inflammation that mostly occurs in the terminal ileum but can occur anywhere from the mouth to the anus. UC and CD share many immunological features in common including increased cytokine secretion and accumulation of mononuclear cells in the affected areas. However, adaptive T-cell responses are thought to differ between the two diseases. T-helper (Th)1/Th17 responses dominate

Correspondence: Maria T. Abreu, MD, Department of Medicine, Division of Gastroenterology, Miller School of Medicine, University of Miami, Miami, Fl 33136, USA. Tel: +1 305 243 6404. Fax: +1 305 243 6125. E-mail: [email protected]

(Received 9 September 2014; accepted 13 September 2014) ISSN 0036-5521 print/ISSN 1502-7708 online
2015 Informa Healthcare DOI: 10.3109/00365521.2014.966321

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Innate immunity in IBD CD, whereas aberrant Th2 responses have been suggested to drive UC [1]. The innate immune system comprises pattern recognition receptors (PRRs) and all the immune cells that do not undergo somatic rearrangement, whose responses are germ-line encoded. In this review, we will focus on innate immune responses in IBD, as there is ample human evidence that deregulation in this arm of the immune system drives inflammation. A recent worldwide effort to elucidate the genetic associations in IBD was performed on 75,000 cases and controls. This work implicated a number of genes regulating innate immunity in susceptibility to IBD, including an enrichment of genes involved in the Kyoto Encyclopedia of Genes and Genomes terms: regulation of cytokine production, lymphocyte activation, and response to molecules of bacterial origin. Dendritic cell (DC)-associated genes showed the strongest association with susceptibility genes in IBD [2]. The preponderance of data suggesting altered innate regulation in IBD is further underlined by the appearance of IBD-like symptoms in patients with certain monogenic diseases. These include IBD-like disease in patients with common variable immune deficiency (CVID) which presents with a variety of genetic immunological defects. In a historical review of gastrointestinal samples from CVID patients, colon samples usually demonstrated lymphoid aggregates and increased apoptosis [3]. Defects in lipopolysaccharide (LPS)-responsive and beige-like anchor protein, which shuttles protein to signaling scaffolds following LPS recognition, a type 8 CVID, lead to deficits in autophagy which is one of the pathways that is highly significantly associated with IBD disease [4]. Chronic granulomatous disease patients have a genetic defect in parts of the nicotinamide adenine dinucleotide phosphate complex responsible for oxidative burst killing of intracellular bacteria. These patients are also prone to severe CDlike disease [5,6]. Defective bacterial killing in these patients may lead to increased cytokine production in the face of unproductive bacterial clearance. For a complete review of 40 monogenic diseases that have an increased risk of IBD-like disease, please see [4]. In this review, we will focus on PRRs and the innate effector cells that have been implicated with pathology in IBD. Our focus is on the hematopoietic compartment, as epithelial cells will be reviewed elsewhere in this issue. The gastrointestinal tissue microenvironment alters PRR signaling compared to systemic responses. How intestinal innate immune cells decode PRR signals regulates the balance between tolerance and inflammation that is required for proper function in the intestine. How these signals

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are deranged in IBD leading to disease is the focus of this review. Patten recognition receptors Toll-like receptors Toll-like receptors (TLRs) are a family of integral membrane proteins with an N-terminal ligand binding domain and a C-terminal Toll-interleukin (IL) 1 receptor (TIR) domain used for signal transduction. TLRs are situated either on the plasma membrane (TLR1, 2, 4, 5, 6) or intracellularly in endosomes (TLR3, 7, 8, 9). TLRs are responsive to pathogenassociated molecular patterns (PAMPs) found on bacteria and viruses. These patterns are generally conserved across the different phylum allowing for broad recognition of both pathogens and commensals by the same signaling molecules (for comprehensive reviews of TLR signaling, see [7–9]). Activation of TLRs initiates a signaling cascade that culminates in nuclear factor kappa-light-chain-enhancer of activated B cell (NF-kB)-dependent gene expression of inflammatory cytokines. Additionally, activation of bacterial sensors TLR4 and TLR9 and the viral sensors TLR3, TLR7, and TLR8 activate interferon (IFN) regulatory factor-3/7-induced, type I IFN gene expression [7]. In the intestine, TLRs are found on both epithelial cells and immune cells to varying degrees. Colonic epithelial cells express low levels of TLR2 and TLR4, and this is found basolaterally in healthy controls [10–12]. This spatial organization of the TLRs at the interface with the microbiota is the key to preserving hyporesponsiveness to the luminal microbiota, while maintaining the capacity to respond to invasion and breaches of the epithelial layer. As TLR signaling induces potent inflammatory cytokine secretion, its signaling is tightly regulated and several negative regulators exist to limit and attenuate TLRmediated signals. These include: soluble decoys (sTLR2, sTLR4), transmembrane proteins that sequester receptors from co-receptors (ST2, single immunoglobulin and TIR domain [SIGIRR], RP105), and several intracellular inhibitors of signal transduction (TOLLIP, IL-1 receptor-associated kinase 3 [IRAKM], SOCS1) (for a comprehensive review of negative regulators of TLR signaling, please see [13]). TLR expression in IBD is increased in both the epithelial and lamina propria compartments [10,11,14,15]. Upregulation of TLRs is likely to drive pathological cytokine secretion in the affected tissues in CD and UC patients. Interestingly, efficient TLR signaling is also protective in human IBD. Human polymorphisms (Asp299Gly and Thr399Ile) that

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result in impaired TLR signaling in vitro [16] are also associated with CD and UC risk in certain populations [17–19]. However, in the recent genome-wide association studies conducted across numerous populations, this association was no longer found [2]. Additionally, in CD patients having the Asp299Gly polymorphism of TLR4 in combination with NOD2 polymorphisms also have increased susceptibility compared to having one or the other [17]. This demonstrates the hypothesis of multiple genetic hits significantly increasing susceptibility due to the cross-regulation of many innate immune pathways. TLR5 sensitivity is also upregulated in CD patients. TLR5 recognizes bacterial flagellin, and flagellin reactivity and antibodies are both increased in CD patients [20,21] and high antibody titers are associated with complicated disease [22]. In experimental colitis models, TLR signaling can be protective or pathogenic, depending on the cell type involved and the model. Mice deficient in the TLR adaptor molecule MyD88 [23], TLR2 [24], and TLR4 [25], have worse colitis in response to the chemical colitigenic agent dextran sodium sulfate (DSS), which induces more severe epithelial injury compared to control wild-type mice. In these models, severe colitis is associated with defects in epithelial resistance to injury and lack of productive healing. Conversely, Rag2 / mice (lack T and B cells) infected with Helicobacter hepaticus are protected from inflammation when they lack MyD88 in their hematopoietic compartment. Bone marrow chimeras reconstituted with MyD88 / cells were unable to mount an inflammatory response to H. hepaticus infection and thus were protected from developing chronic inflammation in this model [26]. The authors postulated that DC recognition of H. hepaticus drove inflammatory production of the IL-23 receptor and the cytokine IL-17A, which were pathological in this model. Combined, these studies reveal alternate functions for innate immune response in different cell types. TLR activation is important for epithelial restitution following injury and thus prevents inflammation. But TLR signaling in DCs and other innate immune cells in the colonic environment can drive pathological inflammation. Worse colitis also occurs when negative regulators of TLRs are lost. SIGIRR deletion leads to increased colitis in a chronic DSS model [27,28] and Campylobacter jejuni and Citrobacter rodentium infection models [29,30]. In the DSS model, SIGIRR function was most important in regulating inflammation in epithelial cells but was not completely rescued by SIGIRR expression in epithelial cells indicating at least some role for SIGIRR downregulation of TLR signaling in hematopoietic cells. Interestingly, in the C. rodentium

infection model, loss of SIGIRR resulted in excess inflammatory responses when TLR signaling enhanced the production of antimicrobial proteins by epithelial cells which depleted the commensal microbiota to such an extent that the pathogenic bacteria were able to colonize the intestine [30]. Similarly, loss of the negative TLR regulatory protein TOLLIP increased DSS-induced colitis severity even when the microbiota was depleted by antibiotic treatment [31]. These studies highlight the importance of the fine balance in TLR signaling required for optimal tolerance to, and protection from, the intestinal flora mediated by both the epithelial and the hematopoietic compartments. C-type lectin receptors C-type lectin receptors (CLRs) are a diverse family of receptors that share a carbohydrate recognition domain and are able to recognize all classes of pathogens including yeast, helminths, fungi, viruses, and bacteria. CLRs can either signal directly or enhance other PRR signaling pathways. Caspase recruitment domain-containing protein (CARD9), a part of the dectin-1 signaling scaffold, is the candidate gene in an IBD susceptibility locus on chromosome 9 [2]. Dectin-1 (CLEC7) recognizes b 1–3 glucan structures in fungi including Candida albicans and activates NF-kB through a scaffold composed of CARD9, B-cell chronic lymphocytic leukemia (CLL)/lymphoma 10 (Bcl-10) and mucosa-associated lymphoid tissue lymphoma translocation protein 1. Upstream of the scaffold, spleen tyrosine kinase (SYK) is the adaptor molecule for dectin-1 [32]. SYK is required in DCs for fungal protection as it initiates a series of events culminating in the production of granulocyte macrophage colony-stimulating factor by natural killer (NK) cells and activation of phagocytic neutrophils to contain fungal spread [33]. Loss of dectin-1 resulted in increased colitis severity and increased pathogenic invasive fungal loads over non-pathogenic fungus in the lumen [34]. NOD-like receptors NOD-like receptors (NLRs) are a family of intracellular PRRs sharing similar structural domains, including CARDs, which facilitate binding to the other scaffold proteins; leucine-rich repeat domains, which are the PAMP-binding domains; and the eponymous nucleotide binding oligomerization domain (NOD also known as NACHT domain). There are 22 identified NLRs in humans and 33 in mice [35]. Generally, stimulation of NLRs results in activation of the caspase-1 signaling complex (known as the

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Innate immunity in IBD inflammasome) mediating cleavage of pro-IL-18 and pro-IL-1b or activation of NF-kB signaling. There are five classes of NLR, but here we will only discuss those which have been shown to be important in IBD. Mutations in the NOD2 gene are very significantly associated with CD but are actually protective from UC [2]. This kind of dichotomy is unusual in IBD and only one other susceptibility locus, encompassing the protein tyrosine phosphatase, nonreceptor type 22 (lymphoid) (PTPN22) candidate gene, demonstrates this kind of inverse risk pattern between CD and UC [2]. Receptor-interacting serine-threonine kinase 2 (RIPK2), an accessory protein in the NOD2 signaling cascade, is also in a susceptibility locus for CD, indicating the importance of proper functioning of this pathway in prevention of intestinal inflammation. NOD2. Activation of NOD2 by recognition of Grampositive and -negative bacteria-derived muramyl dipeptide (MDP) leads to NF-kB signaling in both epithelial cells and immune cells. NOD2 signaling is intimately entwined with TLR signaling such that NOD2 recognition of MDP tolerizes cells to further TLR2 or TLR4 activation. This ability to modulate TLR signaling is lost in CD patients with NOD2 mutations (Leu1007insC) [36] or (3020insC) [37] and may contribute to overactivation of TLR signaling in these patients. The negative TLR regulators IRAK-M [36] and activating transcription factor 3 [38] are upregulated with long term MDP stimulation, which mediates the decrease in TLR responsiveness. Additionally, new evidence has implicated another unexpected function of the CD-associated autophagy protein ATG16L1 in regulation of NOD1/ 2 signaling. Whereas NOD2 activation induces autophagy [39], ATG16L1 was found to function independently of autophagy and sequester RIPK2 away from NOD1/2 and prevent formation of the downstream signaling complex. In the CD allelic variant, this function was abolished [40]. This provides more evidence that deregulation of NOD2 signaling significantly affects inflammatory signaling in CD patients. Inflammasome activating NLRPs. The inflammasome is a signaling complex comprising the specific NLR with apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) and caspase-1. Activation of the inflammasome complex induces IL-1b and IL-18 transcription and caspase-1 cleaves the pro-IL-1b and pro-IL-18 proteins to their active forms. The NLRP3 protein is induced downstream of NF-kB [41] or reactive

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oxygen species (ROS) activation [42]. As NLRP3 activation is secondary to prior PRR activation by PAMPS or danger-associated molecular patterns (DAMPs), numerous stimuli have been implicated in upregulation of NLRP3 [43]. Due to the importance of IL-1b signaling in colitis, and the fact that it is upregulated in both CD and UC tissues [44], it is unsurprising that loss of NLRP3 protects from DSS colitis [42]. Alternately, loss of NLRP6 led to worse DSS-induced colitis, and this was postulated to be due to IL-18 regulation of the composition of the colonic microbiota. Loss of NLRP6 increased the abundance of Prevotellaceae and TM7. The altered microbiota, when transferred to wild-type hosts, led to worse colitis in those mice underlining the role of the microbiota in driving the colitis [45]. Follow-up studies from this report identified a crucial role for the interaction of autophagy with inflammasome signaling. NLRP6 / mice had reduced autophagic capacity which prevented mature mucus granules from being released by goblet cells. This nearly completely destroyed the mucus layer overlaying the epithelium and allowed pathogens to contact deep into the crypts driving inflammation [46]. So the link between the inflammasome and autophagy has been extended to include control of proper cellular secretory function in specialized epithelial cells. The abovementioned functional studies of TLRs, CLRs, and NLRs in mouse models demonstrate that the loss of important regulators of microbial defense leads to changes in innate immune cells, epithelial cells, and ultimately the composition of the microbiota that can trigger inflammation. Classical innate immune cells in IBD Dendritic cells DCs and macrophages are phagocytic, professional antigen-presenting cells that bridge innate and adaptive immunities. Recognition of PAMPs by PRRs on DCs determines their activation status; thus, antigenspecific T-cell responses are sculpted by the PRR pathway activated in DCs. Tissue-specific signals set the tone for the responsiveness of DCs [47,48]. Under normal colon homeostatic conditions, antigen presentation by DCs induces tolerance. Currently, there is some ambiguity about the expression of TLRs on intestinal DCs with some studies finding lower levels [49,50], whereas others found high levels [51]. However, the cytokine response of intestinal DCs to TLR4 stimulation is consistently reported to be attenuated compared to spleen DCs with a decrease in IL-12p70 and increased IL-10 secretion

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[49–52]. As DCs are very important in the development of antigen-specific responses, their deregulation in IBD has far-reaching effects. Two distinct subsets of DCs have been reported with opposing functions. The CD103+ DC subset has been implicated in driving regulatory T-cell (Treg) responses, whereas the CX3CR1+ subset is more inflammatory. Human studies have demonstrated accumulation of DCs in IBD with higher expression levels of TLR2, TLR4, and costimulatory molecules, which secreted increased cytokines compared to healthy controls. Treatment with anti-tumor necrosis factor (TNF) antibodies in these patients reduced DC costimulatory molecule expression [53], demonstrating their activated phenotype. In preclinical models, DCs play opposing roles depending on the timing of the inflammation. At early stages of inflammation, DCs are protective, and their loss leads to exacerbated inflammation [54,55]. This may be due to the role of CD103+ DCs in promoting the accumulation of Tregs [56]. Later stages of inflammation in the colon suffer from the lack of tissue-induced tolerance imprinted on the newly recruited DC precursors. These cells have trafficked from the blood as monocytic precursors and arrive into the inflamed colonic tissue. Under these conditions, the DCs are not tolerized by tissue-specific signals and instead induce pathogenic inflammatory T-cell responses, including enhancing Th17 responses by sensing invading bacteria through the NOD/RIPK2 pathway [57]. Neutrophils Neutrophils are short-lived innate immune cells that have an arsenal of weapons located in preformed granules at their disposal to destroy extracellular and intracellular invading pathogens. Neutrophils are also the most abundant white blood cell in blood and produce significant quantities of cytokines [58]. As an abundant effector cell, neutrophils play an important role in IBD. Activated neutrophils accumulate in the blood of UC patients at roughly three times the number in healthy patients [59]. Their effector enzyme neutrophil elastase [60] is increased in colonic tissue of UC patients. Neutrophils are the major source of ROS and blood neutrophils in CD patients were found to have increased ROS production in response to N-formyl-L-methionyl-L-leucylphenylalanine [61]. Recruitment of neutrophils (Ly6G+) into the inflamed colon occurs early following DSS injury; a significant increase in their number is observed in colons 24 h following administration, beating both CD11c+ DCs and F480+ macrophages to the colons by 4 days [62]. Additionally, blockade of neutrophil recruitment to the colon using a neutrophil

selective [63] or ICAM-1 blocking antibodies [64] attenuates DSS colitis, demonstrating the importance of this cell type in colonic inflammation. Classical innate immune cells like DCs and neutrophils utilize TLR, CLR, and NLR signals to not only choreograph first-line effector functions but also fine-tune later adaptive response. As such, they are critical innate cellular mediators of the development of inflammation in IBD patients. Innate lymphoid cells and IBD Innate lymphoid cells (ILCs) are a heterogeneous population of cells that secrete significant quantities of cytokines and are mainly located at barrier surfaces. The role of ILCs in maintaining epithelial integrity and in defense against invading pathogens is currently being unraveled. ILCs have been subdivided into three groups – ILC1, 2, and 3 – and are functionally distinct. ILCs derive from a common inhibitor of DNA binding 2 (ID2)-dependent lymphoid precursor but do not undergo somatic rearrangement like T and B cells. ILC1s include NK cells and are dependent on T-bet driven transcription factor function to secrete TNF-a and IFN-g. ILC2s are driven by the transcription factor GATA-binding factor 3 (GATA3) and secrete similar cytokines as Th2 cells, including IL-5 and IL-13, and are responsive to IL-33. ILC3s are RAR-related orphan receptor gamma (RORgt)+ cells and contain two subsets that secrete IL-17A or IL-22, respectively, which make them of particular interest in IBD. ILC3s are responsive to IL-23 and IL-1b for their own cytokine secretion. A further subset of ILC3s is the lymphoid tissue inducer cells that function early during development to generate isolated lymphoid follicles in the intestine. ILC3s have heterogeneous expression of NK cytotoxic receptors such as NKp44 and NKp46. ILCs are implicated in IBD as they respond to, and secrete large quantities of IBD-assoicated cytokines. Large quantities of IL-17A and IL-22 have been observed in the non–T-cell (CD3 ) population of peripheral blood and intestinal tissue of IBD patients [65–67]. Recent investigations of ILC populations in CD patients revealed increased numbers of a CD56+ ILC subset in involved tissue, which secreted more IL-22 and IL-26 than control patient tissues, while a second CD56 ILC subset produced more IL-17A and IL-17F in CD tissues than those from controls in response to IL-23. Interestingly, there were differences in the accumulation of these populations between CD and UC patients. The CD56 , IL-17A, and IL-17F-producing subset was increased in CD, but not UC patients [65]. Another study using

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Innate immunity in IBD slightly different markers found that CD3-CD56+ population could be further subdivided into NKp56+ or NKp44+ cells. In CD patients an imbalance toward the NKp46+ population and higher IFN-g accumulation was found, compared to UC patients [68]. Thus, the human studies to date have demonstrated a role of ILCs in the pathology of IBD. ILCs have been shown to produce large quantities of IL-17 and IL-22 [69] and have been implicated in the pathogenesis of murine models of innate-driven bacteria-induced colitis. Rag1 / mice that lack functional T or B cells develop colitis when infected with H. hepaticus, and this is dependent on RORgt+ and IL-23 responsive ILCs. This colitis is driven by high levels of IFN-g and IL-17A from ILCs [70]. In another model of innate-driven colitis, administration of an activating CD40 antibody to Rag / mice induces robust colitis and colonic IL-17A [71] expression along with a systemic wasting disease. IL-23 specifically drives the intestinal pathology in this model but is not involved in the systemic wasting disease. When the CD40 antibody was given to Rag / mice depleted of Thy1+ ILCs or given to Rorc / Rag / mice which do not have ILC3 cells and do not produce significant amounts of IL-23, colitis was prevented, but the systemic wasting disease was unchanged [70]. This result demonstrates the importance of IL-23 and ILCs specifically in the colon as the wasting disease induced in this model was not impacted by loss of either IL-23 or ILCs. Polymorphisms in the receptor of IL-23 are highly significant genetic susceptibility factors in IBD [2]. Indeed, inhibition of IL-12p40 (ustekinumab), the

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common receptor subunit of IL-12 and IL-23, is effective in treating CD [72]. ILCs can directly sense bacteria (at least through TLR2 [73]) to induce their effector functions; however, their most potent effector functions are stimulated by sensing the production of IL-23 by macrophages and DCs activated in response to microbial stimulation [57]. An essential function of ILCs is the production of IL-22. Early in life, ILCs produce abundant IL-22 in response to microbial colonization, but this tapers off with time and is repressed by epithelial production of IL-25 in response to the microbiota [74]. Destruction of the epithelial layer by administration of DSS induces IL-23 secretion from DCs and macrophages and decreases epithelial production of IL-25. This removes the break from ILC IL-22 secretion [74]. IL-22 drives activation of signal transducer and activation of transcription 3 in epithelial cells [75], which in turn, induces the upregulation of antimicrobial proteins including S100A8, S100A9, Reg3b, Reg3g, Muc1, Muc13, and bdefensins [74,76]. Neutrophils [77] and NK cells [78] infiltrating the colonic tissue after DSS-induced injury can also produce IL-22 in an IL-23-dependent manner and contribute to tissue repair. Thus, it is a tripartite system of cellular crosstalk, where PRR recognition of PAMPs and DAMPs induces IL-23 from CD103+ DCs that, in turn, induces IL-22 secretion from RORgt+ ILCs, which subsequently enhances epithelial production of antimicrobial proteins and promotes epithelial protection from bacterial invasion (Figure 1) [79].

Luminal bacteria S100A8, Reg3γ, Reg3β, Muc1, β-defensins Epithelial layer

Goblet cells

IL-17/IL22 IL-25

TLR

NFKB

NLR

TLR

Neutrophil

Dendritic cell

IL-12p40 IL-23p19

IL-23

MHCII

TCR

T cell

RORγt

ILC3

Figure 1. Orchestration of the innate immune system in the intestine. Recognition of bacteria or bacterial products by lamina propria DCs in the intestine initiates a cascade of events involving several different innate immune cell types. IL-23 secretion in response to PRR stimulation activates ILCs to produce IL-17 and IL-22. This cytokine is sensed by epithelial cells, which induce antimicrobial protein secretion to promote epithelial defense against invasion. Cytokine secretion from DCs also recruits neutrophils from the periphery to aid in phagocytosis, and participate in secretion of effector cytokines. DC = Dendritic cell; IL = Interleukin; PRR = Pattern recognition receptor.

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ILCs also play an active role in preventing commensal bacteria that reside in the gut-associated lymphoid tissue (GALT) from systemic escape. Loss of IL-22 secreting RORgt+ ILCs allowed the GALTresident commensal species Alcaligenes xylosoxidans to escape to the spleen and liver causing systemic inflammation. Secretion of IL-22 from the ILCs normally induces S100A8/S100A9 which contains the Alcaligenes, but this function was lost with the loss of the ILCs [80]. The ability of ILCs to produce IL-22 and participate in reinforcing epithelial defenses makes them important mediators of defense in the intestine. A further role for a subset of RORgt+ ILCs has lately been suggested. ILCs express major histocompatibility complex (MHC)-II and have been shown to present antigen to T cells and modulate adaptive responses. ILC-mediated antigen presentation in the intestine induces T-cell tolerance responses to commensal bacteria because they do not express costimulatory molecules. Mice lacking RORgt have increased peripheral T-cell numbers and increased commensal reactive immunoglobulin G, suggesting activated adaptive immunity in the absence of ILCs. Elegant studies investigating the specific role of MHC-II in ILCs showed that antigen presentation by ILCs in the steady state contributed to control of inflammatory responses to the commensal microbiota, and loss of the ability of ILCs to present antigen lead to significant inflammation [81]. ILCs, like other innate immune cells, have a myriad of roles to play in both maintenance of epithelial integrity as well as in defense against pathogen invasion. They are of considerable interest due to the high quantities of cytokines that they produce, especially as they are significant sources of and responders to IL-23, and IL-23R is one of the most significant genes in IBD susceptibility. Their additional roles as antigen-presenting cells and of gatekeepers for tissue-resident bacteria will be of continued interest in future studies. Dual nature of IL-17 in IBD Several studies have implicated increased IL-17 family members with active CD and UC [82,83]. The Th17 subset of Th cells produces abundant IL-17A and IL-17F and the pathological effects of these cells and effector cytokines has recently been extensively studied. In preclinical models of T-cell-mediated colitis, transfer of Il23r / T cells to Rag / (T- and B-cell deficient) recipients prevented intestinal inflammation and reduced accumulation of IL-17 and IL-22 in intestinal tissue. This demonstrates a role for Th17 cell production of pathogenic cytokines in promoting inflammation.

Based on these studies, investigations began to examine the potential for blockade of IL-17 as a treatment for IBD. Neutralizing antibodies were generated against IL-17A and despite some preclinical modeling that suggested a protective role for IL-17A in colitis models [84–86], secukinumab (anti-IL17A -AIN457) [87] and brodalumab (anti-IL-17RA-AMG827) [88] were tested in human clinical trials. The secukinumab trial has revealed that not only was there no clinical benefit from blockade of IL-17A, there was actually a worsening of symptoms in treated patients. Indeed, four patients on secukinumab developed mucocutaneous candidiasis, an infection of mucosal tissues with Candida albicans. It is noteworthy that humans with autoimmune polyendocrine syndrome type I have neutralizing autoantibodies to IL-17A, IL-17F, and IL-22 and also often develop chronic mucocutaneous candidiasis. These data suggest that although IL-17A has the potential to drive damaging chronic inflammation, it is also essential for the control of the commensal and pathogenic microbiota. Despite the accumulation of IL-17 in the periphery and tissue of CD and UC patients, blocking this inflammatory cytokine did not ameliorate disease but actually worsened it. The dependence of the epithelium on IL-17 for stimulation of IL-22 and promotion of antimicrobial proteins highlights the complexity of intestinal cytokine networks. The interplay between inflammation and protection is a very tight line and is filled with moving players which makes predicting outcomes of interventions challenging.

Concluding remarks All aspects of the innate immune system are on full alert in the intestine. Orchestration of tissue-specific responses to PRR stimulation by DCs impacts the resident ILC population as well as the epithelial layer. Cytokine and chemokine secretion then recruits effector cells like neutrophils, but also enhances the antimicrobial response of epithelial cells – sometimes to their detriment. It is thus understandable that the balance of these factors is deranged in IBD. Current efforts to block the effector cytokines and cells involved in the pathogenesis of IBD must take into consideration that not all activities of inflammatory cytokines are pathological and that there is a fine balance in the cytokine network, especially in the intestine. Future studies of the innate immune system in IBD will need to consider the plurality of effects of each receptor and cell type in the context of the intestinal tissue to more fully understand the outcome of intervention.

Innate immunity in IBD Acknowledgments

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Funding for this review was provided by grants to support the laboratory of MTA (National Institute of Health, National Cancer Institute 5R01CA137869-05, National Institute of Diabetes and Digestive and Kidney Diseases 2R01DK099076-06A1, Crohn’s and Colitis Foundation of America Senior Investigator Award). Declaration of interest: The authors have read the journal’s policy and have the following competing interests: MT Abreu has served as a consultant to Abbott Laboratories, Bristol-Myers Squibb, Hospira, Merck, Pfizer, Sanofi-Aventis, Janssen, GSK Holding Americas, Inc., and UCB. References [1] Sartor RB. Mechanisms of disease: pathogenesis of Crohn’s disease and ulcerative colitis. Nat Clin Pract Gastroenterol Hepatol 2006;3:390–407. [2] Jostins L, Ripke S, Weersma RK, Duerr RH, McGovern DP, et al. Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease. Nature 2012;491:119–24. [3] Daniels JA, Lederman HM, Maitra A, Montgomery EA. Gastrointestinal tract pathology in patients with common variable immunodeficiency (CVID): a clinicopathologic study and review. Am J Surg Pathol 2007;31:1800–12. [4] Uhlig HH. Monogenic diseases associated with intestinal inflammation: implications for the understanding of inflammatory bowel disease. Gut 2013;62:1795–805. [5] Marks DJ, Miyagi K, Rahman FZ, Novelli M, Bloom SL, Segal AW. Inflammatory bowel disease in CGD reproduces the clinicopathological features of Crohn’s disease. Am J Gastroenterol 2009;104:117–24. [6] Damen GM, van Krieken JH, Hoppenreijs E, van Os E, Tolboom JJ, Warris A, et al. Overlap, common features, and essential differences in pediatric granulomatous inflammatory bowel disease. J Pediatr Gastroenterol Nutr 2010;51:690–7. [7] Kumar H, Kawai T, Akira S. Toll-like receptors and innate immunity. Biochem Biophys Res Commun 2009;388:621–5. [8] Takeda K, Kaisho T, Akira S. Toll-like receptors. Annu Rev Immunol 2003;21:335–76. [9] McGettrick AF, O’Neill LA. Localisation and trafficking of Toll-like receptors: an important mode of regulation. Curr Opin Immunol 2010;22:20–7. [10] Vamadevan AS, Fukata M, Arnold ET, Thomas LS, Hsu D, Abreu MT. Regulation of Toll-like receptor 4-associated MD-2 in intestinal epithelial cells: a comprehensive analysis. Innate Immun 2010;16:93–103. [11] Cario E, Podolsky DK. Differential alteration in intestinal epithelial cell expression of toll-like receptor 3 (TLR3) and TLR4 in inflammatory bowel disease. Infect Immun 2000; 68:7010–17. [12] Abreu MT. Toll-like receptor signalling in the intestinal epithelium: how bacterial recognition shapes intestinal function. Nat Rev Immunol 2010;10:131–44. [13] Liew FY, Xu D, Brint EK, O’Neill LA. Negative regulation of toll-like receptor-mediated immune responses. Nat Rev Immunol 2005;5:446–58.

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