Intestinal epithelial cells lining the small and large bowel are an integral part of the gastrointestinal innate immune system, involved in responses to pathogens and antigen sampling for delivery to the adaptive immune system in the gut. There are several different types of intestinal epithelial cells, all derived from a common precursor found in the crypts of intestinal glands. Among these are the mucus-secreting goblet cells, which reside at the top of the small intestinal villi and the top of crypts of the colon; cytokine-secreting tuft cells dispersed in small and large intestinal epithelium; antigen-sampling microfold (M) cells, found in specialized dome structures overlying lymphoid tissues; and antibacterial peptide–secreting cells, including Paneth cells found at the bottom of the small intestinal crypts or deep crypt secretory cells in the colon (see Fig. 1). All these cell types contribute in different ways to the barrier function of the mucosa, as we will discuss later.
Fig1. The gastrointestinal immune system. (A) Schematic diagram of the cellular components of the mucosal immune system in the intestine. The main features include an epithelial barrier covered by secreted mucus, dendritic cells (DCs) and microfold (M) cells that sample antigens, Tuft cells that respond to helminths by secreting cytokines, various innate sentinel cells, and lymphocytes in the lamina propria beneath the epithelial layer, organized mucosal-associated lymphoid tissues beneath the epithelial barrier, such as Peyer’s patches, draining mesenteric lymph nodes, and plasma cells beneath the epithelium that secrete immunoglobulin A (IgA), which is transported into the lumen. (B) Photomicrograph of mucosal lymphoid tissue in the human intestine. Similar aggregates of lymphoid tissue are found throughout the gastrointestinal tract. ILC, Innate lymphoid cell.
Innate immune protection in the gut is mediated in part by the physical and chemical barrier provided by the mucosal epithelial cells and their mucus secretions. Adjacent intestinal epithelial cells are held together by proteins that form tight junctions, which block the movement of microbes between the cells into the lamina propria. In addition, mucosal epithelial cells produce antimicrobial substances, including defensins. Several cell types located in the mucosa, including epithelial cells, DCs, macrophages, and ILCs, are capable of mounting inflammatory and antiviral responses. Most of these responses are induced by pattern recognition receptor engagement by microbial ligands.
Several different extensively glycosylated proteins, called mucins, are secreted by goblet cells and form a viscous physical barrier that blocks microbes from contacting the epithelial lining of the gastrointestinal tract. Mucins contain many different O-linked oligosaccharides and include secreted and cell surface glycoproteins. Most of the intestinal mucus layer is composed of MUC2, a mucin that forms a hydrated gel ranging from 300 to 700 μm in thickness. In the lumen of the small bowel, the mucus forms a single layer, and most of the bacteria are in the outer portion (away from the epithelial surface) of the mucus. Therefore, bacteria rarely make direct contact with small intestine epithelial cells except at the tips of villi that extend toward the top of the mucus layer. In contrast, colonic mucosa has two layers: an outer less-dense layer that is colonized by bacteria and an inner denser layer that is attached to the epithelium and is bacteria free. These mucus layers also serve as a matrix for dis play of antimicrobial substances produced by the epithelial cells. Some mucins act as decoy molecules that can be shed from the epithelial cells and bind to the adhesin proteins that pathogenic bacteria use to attach to host cell membranes. In addition to the secreted mucus, the apical surface of gastrointestinal epithelial cells is coated with membrane-bound mucin proteins, which combine with various glycolipids to form the glycocalyx. This is a dense macromolecular layer, which ranges from 30 to 500 nm in thickness in different locations in the gut. The glycocalyx, like the secreted mucus, serves as a physical barrier to prevent microbial contact.
The mucus barrier of the intestine undergoes turnover and chemical changes in response to various environmental and immune signals, which allow rapid increases in mucosal barrier function. Mucins are constitutively produced by the goblet cells in the gastrointestinal epithelium and by the submucosal glands. They are replaced by newly synthesized molecules every 6 to 12 hours, and many liters of mucus are secreted each day in the adult gut. Several different environmental and immune stimuli can induce dramatic increases in mucin production. These stimuli include bacterial PAMPs binding multiple TLRs, cytokines (interleukin-1 [IL-1], IL-4, IL-6, IL-9, IL-13, tumor necrosis factor [TNF], and type I interferons), neutrophil products (such as elastase), and microbial adhesive proteins. These stimuli not only increase mucin gene expression but also alter the glycosylation of the mucins because of induced changes in the expression of glycosyltransferase enzymes. The changes in quantity and glycosylation of mucins are thought to increase barrier function against pathogens.
Defensins produced by intestinal epithelial cells provide innate immune protection against luminal bacteria. Defensins are peptides produced by various cell types in the body that exert lethal toxic effects on microbes by inserting into and causing loss of integrity of their outer phospholipid membranes. In the small bowel, the major defensins are the α-defensins, including human defensin 5 (HD5) and HD6, produced constitutively as inactive precursor proteins by Paneth cells located at the base of crypts between microvilli. Trypsin, also produced by Paneth cells, proteolytically cleaves the defensin precursors to generate active HD5 and HD6 peptides. In the colon, β-defensins are produced by absorptive epithelial cells in the intestinal crypts, some constitutively and others in response to IL-1 or invasive bacteria. In addition, neutrophil granules are rich in α-defensins, which likely contribute to their antimicrobial functions in the setting of infections or ischemic injury of the bowel wall.
Paneth cells and other epithelial cells of the intestine also secrete C-type lectins called regenerating islet-derived proteins III (REGIIIs). There are five different human REGIIIs, which act as antimicrobial peptides that bind to bacterial membranes, have bactericidal effects, and thereby block bacterial colonization of the epithelial surface. IL-22 produced by ILC3s stimulates the intestinal epithelia cell production of some of the REGIIIs.
Toll-like receptors (TLRs) are required to maintain a strong epithelial barrier in the gut, which keeps abundant commensal organisms and rare pathogens from invading. As we discussed in Chapter 4, TLRs are membrane-bound cellular recep tors that recognize pathogen-associated molecular patterns (PAMPs) produced by microbes and generate signals that promote inflammatory and antiviral responses by the cells. Most luminal bacteria of the gut are nonpathogenic if they are retained outside the epithelial barrier, yet they may express the same array of PAMPs that pathogenic bacteria express, such as lipopolysaccharide, peptidoglycan, CpG DNA, and flagellin. Intestinal epithelial cells express a wide range of TLRs, including TLRs 1, 2, 4, 5, 6, 7, and 9. Various gene-knockout studies in mice have demonstrated that these TLRs, stimulated by their ligands expressed by commensal bacteria, are required for homeostatic maintenance of a protective mucosal intestinal barrier. For example, TLR1 and TLR2 signaling induces the translocation of zona occludens 1 and occludin to the tight junctions between intestinal epithelial cells, thereby tightening the junctions. Furthermore, ligand-induced signaling by TLR1-TLR2, TLR4, and TLR5 stimulates goblet cells to secrete MUC2, needed to maintain a protective mucus layer. TLR signaling also stimulates the secretion of defensins, REGIII proteins, and IgA, all of which will prevent bacterial transgression of the barrier.
TLR expression patterns and functions are regulated to limit immune responses and inflammation in the absence of pathogens. Because inflammatory responses that involve the intestinal epithelial cells can impair barrier function and absorptive functions of the gut, it is not surprising that the expression of TLRs that recognize bacterial product are different in different parts of the gut, as are the responses to TLR signaling. The expression of TLRs by intestinal epithelial cells is lower in the small bowel than in the colon. TLR signaling by intestinal epithelial cell TLRs may also be dependent on cell polarization. For example, secretion of the proinflammatory chemokine IL-8 by intestinal epithelial cells is induced by flagellin binding to basolateral TLR5 but not TLR5 on the apical surface; therefore, only invading bacteria will stimulate an inflammatory response. Similarly, NLR family receptors for flagellins (e.g., NAIP) are expressed in the cytosol of intestinal epithelial cells and will activate inflammatory responses only when pathogenic bacteria or their products gain access to the cytosol.
In healthy individuals, DCs and macrophages in the gut inhibit inflammation and maintain homeostasis. Some intestinal macrophages have a unique phenotype that enables them to phagocytose and kill microbes while secreting antiinflammatory cytokines, such as IL-10. This phenotype is apparently induced in the local mucosal environment by transforming growth factor-β (TGF-β). Some gut DCs migrate into the epithelial layer and in response to retinoic acid and mucus and differentiate into a noninflammatory phenotype, while other DCs in the lamina propria maintain an inflammatory phenotype.
ILCs in the intestinal mucosa contribute to immune defense against bacteria and parasites, promote epithelial barrier function, and may suppress responses to commensal bacteria. ILCs do not express T-cell antigen receptors, but rather respond to local cytokine cues by secreting effector cytokines, and subsets of ILCs exist that secrete cytokines typical of helper T-cell subsets. Some of the cytokines that activate ILCs are referred to as alarmins because they are released by epithelial cells in response to injury or infection and serve as an alarm for the immune system. Most of the ILC3s in the body are found in the gut. In response to IL-1 and IL-23 made by gut epithelial cells, ILC3s secrete IL-17 and IL-22. IL-17 promotes acute inflammatory response to the microbes, and both IL-17 and IL-22 enhance intestinal mucosal barrier function by stimulating production of defensins and REGIII proteins, and by enhancing epithelial tight junction function. Studies in mice show that ILC2s play an important role in intestinal innate immunity against helminths. In response to the alarmin cytokine IL-33 released by stressed or damaged epithelial cells and the epithelium-derived cytokine IL-25, ILC2s secrete IL-5 and IL-13. IL-5 activates eosinophils, which secrete enzymes that degrade the outer integument of helminths, and IL-13 increases mucus production, contributing to expulsion of the worms. A specialized intestinal epithelial cell type called the tuft cell (also called brush cell), so named because of its characteristic apical tuft of short microvilli, plays a role in innate responses to helminths. Tuft cells are activated by helminths and other microbes to secrete abundant IL-25, which stimulates ILC2s to secrete IL-13, which in turn stimulates the differentiation of mucus-secreting goblet cells and more tuft cells from intestinal crypt stem cells. Tuft cells also secrete thymic stromal lymphopoietin (TSLP) and prostaglandins, which likely contribute to their immunomodulatory functions. Related tuft cells are found in various tissues, including the lung.
ILC function and inflammation in the intestinal mucosa are regulated in part by enteric neurons and external vagal sympathetic innervation. Studies in rodents have revealed that ILCs in the gut are stimulated by neuropeptides made by enteric neurons in the gut wall and inhibited by neurotransmitters produced by sympathetic neurons innervating the gut. Much of the evidence for neuronal regulation of ILCs has focused on ILC2s. Many enteric neurons produce neuropeptides, including neuromedin U, and mucosa innervating neurons secrete vasointestinal peptide. Production of these molecules increases after intestinal helminth infections, and these peptides can strongly and rapidly stimulate ILC2s to secrete IL-5 and IL-13, thereby enhancing antihelminth immunity. Conversely, norepinephrine produced by intestinal sympathetic neurons inhibits ILC2 production of IL-5 and IL-13, which may serve to downregulate the inflammatory response after parasite elimination. Enteric glial cells, which are closely associated with enteric neurons, sense PAMPs from microbes in the gut lumen and secrete ligands for RET, a signaling receptor expressed on intestinal ILC3s. The RET ligands derived from the glial cells stimulate IL-22 production by ILC3s, which promotes integrity of the intestinal epithelial barrier.
Mucosa-associated invariant T (MAIT) cells are specific for vitamin B metabolites generated by intestinal bacteria and fungi. Most human MAIT cells are in the liver and thus are in a position to respond to microbes or microbial metabolites delivered there from the gut via the portal circulation.
