27.1

Introduction

The gastrointestinal barrier is more than a tissue with physiological function. As reviewed by others, it is now recognized as a complex ecosystem that requires a delicate homeostatic balance in order for the tissue to remain healthy and functional. The niche involves a multifaceted and dynamic mixture of host cells and molecules; microbes and their metabolites; as well as environmental factors that may reflect diet or contamination with toxins. Furthermore, gene expression in the host can modulate transcription in microbes resulting in changes in the metabolic profile and local immune responses that resonate throughout the body. Thus, these biological spheres are highly interconnected. A more recent and important concept is that the microbial-host interactions that begin in the digestive tract not only impact gastrointestinal inflammation and risks of local cancer, but they also affect the hosťs susceptibility to conditions ranging from diabetes, obesity to various neurological conditions.

Part of the role of the barrier is to confer innate immunity to protect the host from “danger” such as that threatened by a microbial challenge. This immunity entails a physical barrier as well as host responses that are either produced constitutively or induced in response to changes in the local ecosystem. While innate immunity may have evolved to protect the host from infection, its responses transition to adaptive immunity that enhances the health of the gastrointestinal tissues. However, sometimes protection is misguided to immune-mediated disease.

27.2

Composition of the Barrier

We propose a broader definition of the “barrier” than the single epithelial cell layer on the luminal surface of the gut. Overlying the epithelium is a biofilm—a mixed community of microbes that live in a complex relationship with the host and each other. This film is found adjacent to the apical surface and presumably thrives there due to the nutrients provided, in part, by the host and neighboring organisms. Protective microbes within the biofilm can occupy the niche and block colonization by pathogens or inhibit the growth of other organisms through the production of various antibiosis molecules. The important role for commensal organisms is illustrated by the increase in disease in mice treated with streptomycin and infected with Salmonella typhimurium. Further, the chronic diarrhea caused by C. difficile in humans after prolonged antimicrobial therapy is often effectively treated by restoring the microbiota with a fecal transplant while gastric microbiota in children have been suggested to limit the inflammation induced by infection with H. pylori. Mucus is another important element that contributes to the barrier. It provides a physical impediment to particle entry as well as serving as an “antiseptic paint” containing antimicrobial factors produced by the host. For example, mucus secretions contain antimicrobial peptides (defensins) from Paneth cells (PC) ; secretory IgA transported by epithelial cells ; and other protective molecules including lactoferrin and lysozyme.

Some microbes negotiate their way past the biofilm and mucus to become juxtaposed next to the epithelial layer. Indeed, this migration allows H. pylori to escape the harsh acidity in the lumen of the stomach by colonizing the more comfortable pH sanctuary under the mucus. In this niche, the gastric epithelium can alter gene expression in H. pylori to favor their colonization and subsequent molecular interactions affecting the sensing and the bi-directional interactions that define the relationship.

An important consideration, as covered elsewhere in this text, is the multiple lineages of epithelial cells including absorptive and secretory cells, goblet cells, enterochrommaffin cells, as well as more regionally limited, and highly specialized cells such as Paneth and parietal cells. All of these subsets contribute to the barrier and have been implicated, in one way or another, in innate immunity. This heterogeneity, along with the microbial diversity and complex metabolic profile in the lumen and adjacent tissue, contributes to the breadth of the innate responses that can be induced in the gut barrier.

27.3

Sensing Provocations to the GI Barrier

The epithelial layer provides a relevant “wall” that serves as a comprehensive, three-dimensional barrier that cannot be climbed over or walked around. In addition to sensing the presence of microbes, this barrier can be penetrated allowing adjacent leukocytes to sample and respond to microbes. Dietary or environmental antigens have been reported to be preferentially sampled by M cells overlying Peyer’s patches. While enterocytes and M cells may be the best-known sites in which microbes can invade and antigens are sampled, goblet cells are proficient in sampling antigen and passing immunogenic material on to underlying dendritic cells or mast cells (Simon Hogan, Personal communication). Sampling of material that reflects danger can lead to adaptive immunity while persistent exposure to benign antigens, including dietary molecules, can induce oral tolerance. Subsequent to the transition to adaptive immunity, leukocytes, such as mast cells, can be triggered, inducing secretions (mucus, electrolytes, water) that enhance the barrier and through catharsis, purge microbial communities from the host.

As described by Polly Matzinger, the immune system exists to protect us from danger. This is often, although not always, a microbial pathogen. There are hundreds of species of bacteria found in the digestive tract, plus a vast array of viruses, parasites, and fungi. Traditionally, we have considered these microbes as pathogens or commensals; however, as our understanding of host-microbial interactions develops, we now appreciate that benign bacteria can also cause disease when the conditions are appropriate. These organisms are referred to as pathobionts and illustrate the plasticity of the ecosystem as a single species can transition from benign to pathogenic. One example is an E. coli (NC101) that can be relatively nonthreatening but appears to contribute to colon cancer in the context of inflammation. The contribution of many of the members of this complex community to health and disease remains to be elucidated.

There are too many microbes to describe but an important concept to consider is the means by which an aggressive pathogen is distinct from a commensal and represents a danger signal to the host. Lipopolysaccharide (LPS) is lethal when the most pro-inflammatory forms find their way in sufficient concentration into the blood. Yet, both commensals and pathogens can produce LPS. Other factors define virulence and some of the most significant properties of pathogens include their ability to invade the barrier and/or to produce other potentially toxic molecules.

Salmonella enterica serovar Typhimurium is a well-studied model that illustrates the case. Salmonella have a set of genes clustered in a region referred to as Spi-1. These gene products encode molecules for a type III secretion system that translocates microbial virulence factors into epithelial cells where they activate GTPases, induce actin rearrangements, and facilitate bacterial internalization. This process of entry is referred to as invasion. As discussed below, metabolites have a dramatic impact on microbes including their virulence. For example, the expression of Spi-1 by Salmonella typhimurium is differentially regulated in the small and large intestine by short chain fatty acids. Bacterial LPS can also be recognized by host pattern-recognition receptors including TLR4 and BAI1, the latter enhancing the internalization into target cells independent of invasion.

In addition to microbial stimulation, other danger-associated molecular patterns (DAMPs), including DNA, RNA, and purines, can be recognized within the GI barrier. Dead cells can confer a danger signal through the chromatin-associated protein high-mobility group box 1 (HMGB1), which is recognized by TLR2, TLR4, and RAGE (receptor for advanced glycation end products). Phosphatidyl serine expressed on the surface of apoptotic cells is a molecular cue that enables the recognition of dead cells. Like LPS, it is recognized by BAI1 but it differs by imparting an anti-inflammatory signal. Engulfment of sterile apoptotic epithelial cells by adjacent enterocytes has been implicated in conferring the anergic tone that is characteristic of gut mucosal immune cells. Genetically engineering mice so they lack BAI1 expression in the epithelium makes them more susceptible to DSS-induced colitis.

DAMPs overlap somewhat with metabolic influences in the gut. For example, purines are a heterogeneous family of molecules with multiple functions mediated through several purinergic receptors. Some intestinal microbes or apoptotic cells release ATP that serves as a chemoattractant for macrophages as well as a signal to induce the differentiation of Th17 cells. Adding to the complexity of barrier function is the fact that ATP is also a neurotransmitter. ATP can be metabolized to ADP, AMP, and finally adenosine—the latter being a potent anti-inflammatory molecule. In addition, bacteria can produce adenosine, which is necessary for their colonization. Presumably this occurs due to the ability of adenosine to suppress neutrophil-mediated anti-bacterial responses. Adenosine has also been implicated in enhancing the survival of microbes within a host niche. As illustrated by this single metabolic pathway, there are multiple known, and likely many more unknown, molecular interactions that govern homeostasis within the ecosystem of the gastrointestinal barrier.

A new understanding of the complexity of the barrier lies in the study of the metabolome. The metabolic machinery of the host and microbes is connected very closely. For example, the antigenic mass in the gut would stimulate a life-threatening degree of inflammation if it were not regulated. This can be achieved in part by “protective” bacteria producing metabolites such as short chain fatty acids that themselves favor an anti-inflammatory phenotype. In addition, bacterial molecules can be recognized by Toll-like receptors and modify responses. One provocative example is polysaccharide A (PSA) that induces protective regulatory helper T-cell (Treg) responses capable of preventing excessive inflammation in models of inflammatory bowel disease. This is sensed by TLR2 on the cell surface and subsequently, intracellular Nod2 drives the protective responses. When mice lack Nod2, they lose their ability to generate protective Treg in response to PSA.

Further complicating the host-microbial interaction is the fact that host responses can shape microbial communities and the expression of virulence factors by bacteria. Dietary molecules have profound effects on innate responses in the gastrointestinal barrier. For example, they serve as substrates for microbes as evidenced by the ability of bacteria to digest cellulose to yield short chain fatty acids. These compounds have potent effects on host reactivity as they lead to the activation of AMP kinase and shift energy metabolism in host cells toward oxidative phosphorylation, which in turn, favors anergy. A well-studied short chain fatty acid is butyrate, which has been implicated in the inhibition of pro-inflammatory signaling and promotes barrier function. Amino acid metabolism has also been shown to have significant effects on innate immunity and homeostasis. Tryptophan is metabolized by either microbes or the host to yield kynurenine, a compound that favors the development of Treg through its effects on aryl hydrocarbon receptors (AhR). AhR have ambiguous effects on homeostasis as, depending on the nature of the ligand, they can favor the differentiation of either Treg or pro-inflammatory T-cell responses. In the context of diet and anergy, vitamin A is metabolized to retinoic acid (RA) by epithelial cells and dendritic cell subsets. RA promotes anergic responses by innate and adaptive immune cells and can contribute to the development of Tregs. Thus, the impact of metabolism on innate responses in the barrier and the transition to adaptive immunity has to be considered in the context of multiple, and sometimes competing, pathways.

While much remains to be learned, there are several points that emerge. First, all host immunity emanates from innate responses that are initiated in the barrier mediated by epithelial cells or adjacent leukocytes. Secondly, the metabolic profile contributed by the diet, host, and microbes modulate these host responses which, in turn, regulates the microbe. The luminal content, biofilm, mucus layer, epithelial barrier, and adjacent cells provide a steady stream of molecules that range from nutrients associated with digestion to bacterial growth factors to small molecules that regulate host responses. These events are highly interconnected and they are complex.

27.3.1

ROS Signaling

There are other metabolic responses that impact innate immunity in the epithelial barrier including purines, polyamines, reactive nitrogen species, hydrogen sulfide, and many more. One of the first metabolic responses that is induced subsequent to provocation of innate cells is the accumulation of reactive oxygen species (ROS, Fig. 27.1 ). ROS accumulation begins with the generation of superoxides by NADPH oxidases (NOX) or dual oxidases (DUOX) and exists in several forms including superoxide, hydrogen peroxide, and hydroxyl radicals. The accumulation of ROS is historically associated with antimicrobial responses in phagocytes. For example, deficiency in the activation of NOX2 (gene symbol, CYBB) due to mutations in CYBB or the related components causes chronic granulomatous disease, characterized by recurrent infections. Excessive amounts of ROS can lead to cell death and tissue damage while at lower concentrations, they are an important aspect of cell signaling.

ROS accumulation. Infections are often immediately followed by the accumulation of various metabolites including ROS. These play an important role in the reductive activation of transcription factors that lead to the production of cytokines and chemokines. In turn, neutrophils and monocytes are recruited to the site, activated, and contribute to innate immunity in the epithelial barrier and subepithelial region. ROS and RNS also confer antimicrobial effects. Persistence of the infection can lead to chronic inflammation as well as adaptive immunity which, in many cases, cumulates in a chronic, active inflammation.

Two isoforms of DUOX exist, DUOX 1 which is mainly expressed in the airway epithelia, and DUOX2 which is expressed in the gastric and colonic epithelium. Expression of DUOX2 is promoted by commensal bacteria through TIR-domain-containing adaptor protein including interferon-β (TRIF) and NF-κB signaling or MyD88 and p38 in the ileum and colon, respectively. The expression of DUOX2 is associated with gastrointestinal infection and inflammation.

Of the various oxidases, NOX1 and DUOX2 are the principle sources of ROS in the gastrointestinal epithelium in response to infection. The NOX1 isoform plays a key role in generating low levels of ROS that regulate signaling events essential for innate immunity. In contrast to DUOX, NOX1 function requires Rac1, which has been shown to be inhibited by the ROS-sensitive molecule, AP endonuclease 1 (APE1), which mediates a negative feedback on ROS production, presumably by inhibiting the activation of Rac1.

While NOX2 is the major source of ROS within myeloid cells, NOX1 and DUOX2 can produce levels of ROS that play a role in regulating gene expression and cell differentiation, although they also have been associated with oxidative stress. These levels of ROS can be induced by commensal organisms or pathogens but the inter-relationship of the responses that follow infections of microbes with such different effects on human health is largely unknown.

27.3.3

Swarming of Innate Immunity

Part of the complexity reflected in innate responses is that virtually all cells in the body have the ability to produce and/or respond to innate mediators. We have defined the “barrier” as multiple epithelial lineages as well as fibroblasts, nerves, endocrine cells and juxtaposed immune and inflammatory cells that are so closely interconnected that innate signals must necessarily reverberate throughout the barrier.

Most host responses to a microbial challenge are composed of a mixed inflammatory cell infiltrate. Beginning with resident cells, such as intraepithelial leukocytes, antigen presenting cells, mast cells, innate lymphoid cells (ILC), and some polymorphonuclear cells, provocation leads to their activation and the recruitment of other innate cells to the site of infection. Key to this response is the production of chemokines as well as microbial products that engage receptors on various lineages and through these signals, attract cells (initially neutrophils and monocytes) from the blood to migrate across the endothelium and into the tissue. The recruited cells are not “educated” or conditioned to the healthy immunological environment and thus, usually differentiate into more reactive cells in the context of an infection or chronic inflammation.

ILC are a relatively new population of leukocytes to consider during the innate response to infection. ILC phenotypically resemble T cells in several ways and their responses to microbial challenges are predicted to be highly mimicked by T lymphocytes. They are distinct in that they lack T-cell receptors and recognize their microbial ligands through poorly understood mechanisms. ILC are categorized into subsets much like Th cell subsets. In many cases, the ligands, cytokine receptors, signaling pathways, and transcription factors in ILC and Th cells are similar and thus, they become activated together as an insult persists. Thus, ILC1 resemble Th1 cells; ILC2 resemble Th2 cells; and ILC3 have properties resembling Th17 cells.

ILC3 contribute to immunological homeostasis by modulating Th cells including Treg. ILC also regulate the composition of microbes in intestinal tissues and the associated inflammatory responses, making them inextricably interrelated to the impact of Th cells on homeostasis. Intestinal ILC have been shown to contribute to immunity to Citrobacter rodentium and Clostridium difficile.

The swarming of innate responses could reflect a sequential activation of cells or a somewhat haphazard effect that reflects the access of the offending stimulus to the innate cells. We propose that innate responses in the gastrointestinal tract entail both sequential and haphazard responses. The epithelium plays an important role in the initial colonization and sensing of an infection such that it transduces the signal to other innate cells that, in turn, amplify the response. For example, chemokine production by epithelial cells will recruit and activate other innate cells that mediate a response that is broader and of higher magnitude than epithelial cells alone. However, the variation in epithelial barrier function can greatly alter the access of the microbe or its PAMPs to the underlying myofibroblasts and myeloid cells. This, along with the effects of existing immunity, other microbes, the biofilm, and the metabolic milieu are all modifiers that have the potential to add a degree of randomness (or what often feels like chaos experimentally) that affects the rate at which PAMPs engage molecular sensors on different cells as well as the scope and the magnitude of the response.

The amplification of the innate response can have adverse effects on the host. For example, the production of TNF-α from the epithelial or underlying layers is important for protecting the host but when produced in high quantities, or over extended periods of time, it contributes to tissue damage including DNA damage ( Fig. 27.2 ). As receptors for TNF-α are found on most cells, its presence can lead to an enhanced accumulation of ROS, increased levels of several chemokines, and other pro-inflammatory responses. Its promiscuous effects render it an effective therapeutic target in many patients with inflammatory bowel disease.

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