Chapter 13 Barrier Immunity: The Immunology of Mucosa and Skin in Part I Introduction

The Gut Immune System Maintains a Barrier between the Microbiome and the Epithelium

The intestinal immune system employs three major strategies to prevent microbes from penetrating the gut epithelium without permission, and we touched on these earlier when describing the epithelial cell types. First, specialized epithelial cells produce mucus that inhibits bacterial mobility. In the large intestine, goblet cells are particularly abundant and produce a very thick layer of protective mucus that microbes have difficulty penetrating. In the small intestine, both goblet cells and Paneth cells are responsible for mucus production, but create a much thinner layer (see Figure 13-6).

Second, epithelial cells also generate molecules that have direct effects on the microbial communities. Paneth cells in the small intestine produce a wide range of antimicrobial peptides (AMPs) and other proteins, including defensins, which generate pores in bacterial membranes; lysozyme, which digests bacterial cell walls; and members of the regenerating islet-derived protein (REG3) family, which are especially lethal for gram-positive bacteria. Enterocytes throughout the intestine also have the ability to produce AMPs, which are concentrated in the mucus layer just above the epithelial cells.

Finally, plasma cells in the healthy lamina propria secrete large amounts of IgA, which binds to commensal organisms and dietary antigens (see Figure 13-7b). Secreted IgA (sIgA) antibodies are typically dimers joined by a small J-chain protein. IgA dimers are transcytosed across the epithelial barrier by binding polyIgRs expressed on the basolateral surfaces of multiple cell types in the intestinal epithelium. After transcytosis, the IgA dimer leaves with a portion of the polyIgR called the secretory component (SC), which helps to stabilize the dimer in the intestinal lumen. The protective role of antibody transcytosis is underscored by studies showing that mice lacking polyIgR are more susceptible to Salmonella typhimurium and Helicobacter pylori infections.

Some IgA antibodies are broadly specific for microbe-associated molecular patterns (MAMPs) and others bind very specifically to antigens encountered by the adaptive immune system at an earlier time. Regardless of specificity, IgA binding has several effects. It inhibits microbes from penetrating the epithelial cell, and at the same time discourages inflammatory responses to any of the antigens it binds to. Secretory IgA also plays a role in transporting antigen from the lumen to the subepithelial space and delivering it to antigen-presenting cells in a manner that promotes tolerance. We will discuss the source of the IgA antibodies shortly, when we describe the activities of the adaptive immune response.

Antigen Is Delivered from the Intestinal Lumen to Antigen-Presenting Cells in Multiple Ways

In order to maintain a healthy balance between immune tolerance and responsiveness, our intestinal immune system must find a way to communicate the presence and identity of microbial and other antigens in the lumen. Our intestinal immune system has, indeed, evolved an elaborate system of sampling the microbiome and transferring microbes to antigen-presenting cells, so that they can be further evaluated by the adaptive immune system.

Several intestinal epithelial cells (IECs) are involved in antigen sampling and transfer from lumen to lamina propria (Figure 13-8). As we have discussed previously, M cells are highly specialized for this purpose. Although they are found throughout the intestine, they are most abundant in the small intestine and are typically associated with underlying secondary lymphoid tissue, such as isolated lymphoid follicles (ILFs) and Peyer’s patches. Although best known for their ability to transcytose antigens and microbes nonspecifically, M cells also express receptors that allow them to convey specific classes of microbes to the lamina propria. They and other intestinal epithelial cells express receptors for antibody that allow them to carry IgA-antigen complexes back from the lumen into the lamina propria. Goblet cells are able to convey small, soluble antigens from lumen to lamina propria. And finally, some resident antigen-presenting cells extend processes between epithelial cells and sample antigen directly from the lumen.

A sectional view of a part of the small intestine describes the four types of movements of antigen from lumen to antigen-presenting cells. — FIGURE 13-8 How antigen is delivered from the lumen to antigen-presenting cells. (a) Antigen and whole microorganisms can be delivered to antigen-presenting cells (macrophages and dendritic cells) by M cells, which transcytose antigen and sometimes microbes from the lumen. (b) Antigen-antibody complexes can be carried across epithelial cells by Fc receptors (FcR). (c) Small antigens can also be transcytosed by goblet cells and delivered to underlying dendritic cells. Finally (d) antigen can interact directly with antigen-presenting cells that have extended their processes into the intestinal lumen.

Immune Homeostasis in the Intestine Is Promoted by Several Innate and Adaptive Cell Types

Epithelial cells and antigen-presenting cells both produce molecules that influence the outcome of antigen presentation in the intestine (Figure 13-9; see also Table 13-1). Under healthy conditions, epithelial cells are stimulated by commensal microbes that interact with PRRs, often Toll-like receptors (TLRs). These interactions result in the production of TGF-β, the vitamin A metabolite retinoic acid (RA), and thymic stromal lymphopoietin (TSLP). This trio of molecules maintains a tolerogenic environment in part by programming antigen-presenting cells to polarize T-cell differentiation toward the regulatory T cell (T_REG) lineage, an event that also depends on IL-10.

A sectional view describes the process of maintaining homeostasis and microbe tolerance on the surface of the intestine. — FIGURE 13-9 Maintaining homeostasis and tolerance to the microbiome at the intestinal surface. Innate and adaptive immune cells coordinate a response to gut commensal microorganisms that results in the generation of tolerogenic regulatory T cells and protective IgA-secreting B cells. See text for details.

The sectional view shows the epithelial layer in the small intestine. The region above the epithelial layer is labeled Lumen, the region below the epithelial layer is labeled Lamina propria, and the region that lies below Lamina propria is labeled Peyer’s patch or lymph node. Antigenic wall fragments, commensal microorganisms, and SIgA are found in the Lumen region. TLR (Toll like receptors) are anchored to the upper portion of the epithelial layer.

First process

On T L R activation, T S L P, T G F- beta, and R A are produced. They activate C D 1 0 3 plus D Cs, which then travels in two pathways. The M H C receptor on the C D 1 0 3 plus D C binds to the T C R receptor of the naïve T Cell to trigger the release of R A and T G F- beta, leading to the formation of T R E G cell. In the second pathway, the C D 1 0 3 plus D Cs trigger the B cell, which in turn triggers the release of I L-10, R A, and T G F-beta that travel to the I G a plus plasma cell. Text corresponding to this arrow reads, Returning cells express homing receptors C C R 9 and alpha subscript 4 beta subscript 7.

Second process

Antigenic wall fragments travel through the epithelial layer to bind to the T L R 5 receptor on the D C cell, which then triggers I L-23, and activates I L C that has R O R gamma t¬ plus to release I L – 22 that travels back to the epithelial layer.

Third process

A Commensal microbe binds to the C X 3 C R 1¬ Macrophage to trigger the release of I L-10, which activates T R E G cell.

Fourth process

TLR activation leads to the release of APRIL and BAFF, which activate T R E G cell.

S I g A proteins are seen both in the Lumen and the Lamina propria.

Some epithelial cells and antigen-presenting cells also produce BAFF (B-cell activating factor) and APRIL (a proliferation-inducing ligand), which promote IgA production by lamina propria B cells in both T-independent and T-dependent manners. IL-10 is also involved in IgA production by B cells.

IL-10, a potent anti-inflammatory cytokine, is prevalent in the healthy mucosa. Once thought to be made only by T cells, it is now known to be produced by many different cell types, including macrophages, dendritic cells, and B cells. Its importance is underscored by the observation that individuals with lower levels of IL-10 are susceptible to inflammatory bowel disease (IBD).

Intestinal antigen-presenting cells that produce or stimulate the production of IL-10 are particularly important in maintaining immune tolerance in the gut. Which innate immune cells play this role during homeostasis remains a topic of controversy and active study. It is fair to say that the extent of phenotypic diversity among gut APCs has surprised investigators (see Advances Box 13-1). Figure 13-9 shows examples of APCs that play important roles in maintaining gut tolerance: CX₃CR1⁺ resident macrophages, which extend processes between epithelial cells and sample microbial antigen directly; and CD103⁺ migratory dendritic cells, which receive signals from epithelial cells and travel to local secondary lymphoid tissue to interact with naïve T and B cells. Both of these cell types are likely sources of IL-10, as well as other molecules that influence homeostasis.

Epithelial cells and antigen-presenting cells communicate what they learn from sampling the microbiome to both innate lymphoid cells ^{ILCs as well as conventional T and B lymphocytes. The cell subsets that play the most prominent role in maintaining gut homeostasis are T_REG cells, IgA-secreting B cells, T follicular helper (T_FH) cells, T_H17 cells, ILC3s, and intraepithelial lymphocytes (IELs) (see Advances Box 13-1). Conventional B and T lymphocytes are activated in the secondary lymphoid tissues associated with the gut, while ILCs are activated in the lamina propria.}

Generating T_REG cells

Activated dendritic cells (DCs) travel via the lymphatics to mesenteric lymph nodes or Peyer’s patches, where they meet circulating naïve T cells. If DCs first meet their antigen in the tolerizing cytokine environment described earlier, they skew T-cell differentiation toward the FoxP3⁺ T_REG lineage. Whereas only 10% of circulating T cells are of the T_REG subtype, T_REG cells represent 30% of all T cells in the healthy gut lamina propria. Individuals with conditions that reduce T_REG populations are, in fact, susceptible to a variety of immune-mediated diseases, including colitis.

Regulatory T cells have two distinct sources. Thymic T_REGs commit to this lineage during development in the thymus. Peripheral T_REGs are generated from naïve T cells activated by tolerogenic antigen-presenting cells, including gut CD103⁺ dendritic cells. Peripheral T_REGs activated by gut APCs are often induced to express gut homing receptors and travel back to the intestinal mucosa where they release cytokines, such as IL-10, that reinforce tolerance. Whether the origin of T_REGs makes a difference in their function in the gut remains a question.

Generating IgA-Secreting B Cells

IgA-producing B cells are uniquely abundant in intestinal tissues and produce a hefty 3 grams of IgA every day. As we have discussed, IgA antibody is central to the intestine’s strategy to maintain a healthy distance from commensal luminal microbes. It is a first line of defense against pathogenic microbes and toxins and also is involved in transporting antigen from lumen to lamina propria for further evaluation. Where do they come from?

Some of our IgA comes from conventional B cells activated in B-cell follicles in the Peyer’s patches of the small intestine. Much also comes from B cells in isolated lymphoid follicles as well as B cells activated in draining mesenteric lymph nodes. What favors class switching to IgA? Interestingly, IgA class switching occurs in both T-dependent and T-independent manners and depends on the unique cytokine milieu generated by intestinal immune cells (Figure 13-10).

A two-part illustration describes T cell–dependent and T cell–independent IgA induction. — FIGURE 13-10 Generating IgA-producing B cells. IgA-secreting B cells can be generated in a T cell–dependent (a) and T cell–independent (b) manner (see text). (a) Briefly, antigen delivered to migratory dendritic cells travels to lymphoid tissue and stimulates naïve T cells to become T_FH cells, which provide cognate help to B cells that have also received signals from cytokines, such as TGF-β, that encourage their development into IgA-secreting plasma cells. These express integrins (α₄β₇) and chemokine receptors (CCR9) that direct them back to the mucosa. (b) Intestinal epithelial cells and several different APCs in the mucosa can also be induced by interaction with the microbiome to generate cytokines, including BAFF and APRIL, which stimulate lamina propria B cells to class switch to IgA.

The first part of the illustration titled T cell- dependent IgA induction shows an epithelial cell barrier layer with an M- cell. Antigens penetrate the epithelial layer through the M cell activating D C and follicular D C. The activated D C moves to the T-Cell zone and binds to a T cell. The MHC class II receptor of D C binds to the T C R of T cell and C D 80 or C D 86 of D C binds to the C D 28 receptor of the T cell, converting the T cell to T F H cell. The T F H cell moves to the Germinal center. The CD40L receptor and TCR receptors of the T F H cell bind to the C D 40 and M H C class II receptor of the B cell respectively. Simultaneously, the activated Follicular dendritic cell binds to the B C R receptor of the B cell. At the same time, T G F-beta particles bind to the T G F-Beta R receptor of the B cell. The T-cell B cell complex activates I g AThe second part of the illustration titled T cell-independent IgA induction shows an epithelial cell layer with a deep crypt. A T L R anchored to the epithelial cell is activated, and triggers production of APRIL and BAFF, which travel toward B cell. T L R activation simultaneously triggers release of T S L P that activates D C to release APRIL and BAFF, which in turn activates B cell. P D C cell also releases BAFF and APRIL, which activate B Cell. B cell with T C R receptor and antigen-bonded B C R activate the I g A¬ plus plasma cell to release I g A. plasmablast cell to express C C R 9 and alpha subscript 4 beta subscript 7 integrins.

The second part of the illustration titled T cell-independent IgA induction shows an epithelial cell layer with a deep crypt. A T L R anchored to the epithelial cell is activated, and triggers production of APRIL and BAFF, which travel toward B cell. T L R activation simultaneously triggers release of T S L P that activates D C to release APRIL and BAFF, which in turn activates B cell. P D C cell also releases BAFF and APRIL, which activate B Cell. B cell with T C R receptor and antigen-bonded B C R activate the I g AThe second part of the illustration titled T cell-independent IgA induction shows an epithelial cell layer with a deep crypt. A T L R anchored to the epithelial cell is activated, and triggers production of APRIL and BAFF, which travel toward B cell. T L R activation simultaneously triggers release of T S L P that activates D C to release APRIL and BAFF, which in turn activates B cell. P D C cell also releases BAFF and APRIL, which activate B Cell. B cell with T C R receptor and antigen-bonded B C R activate the I g A¬ plus plasma cell to release I g A. plus plasma cell to release I g A.

T-dependent class switching to IgA occurs in the traditional manner described in Chapter 11. Briefly, B cells are activated by antigen in follicles of mesenteric lymph nodes or Peyer’s patches. They generate germinal centers, where they undergo somatic hypermutation and class switching. T_FH cells are particularly important at this stage and provide the B cells with the combination of signals that favor IgA switching, including CD40L and the key cytokine TGF-β. Interestingly, intestinal T_H17 and T_REG cells both have the capacity to convert into T_FH cells that induce IgA class switching. B cells that successfully mature in lymphoid tissue generate plasma cells that express the adhesion molecule α₄β₇ and chemokine receptor CCR9, which cause them to home to the mucosa (see Figure 13-10). It takes about 7 days to produce IgA antibodies in this manner. They also undergo somatic hypermutation and tend to have high affinities for their antigens.

IgA antibodies can also be produced in a less traditional, but quicker, T-independent manner. This route is dependent on the cytokines BAFF and APRIL, which are produced by stimulated epithelial cells, as well as mucosal dendritic cells. It occurs not only in ILFs, but even in the lamina propria itself. IgA antibodies produced in this way tend to be of lower affinity.

Role of ILC3 and T_H17 Cells in Intestinal Immune Homeostasis

The ILC3 subset plays a particularly important role in maintaining tolerance in the gut mucosa. In response to a variety of signals produced by innate immune cells, including IL-23 and RA, ILC3 cells produce IL-17 and IL-22 (see Figure 13-9). In fact, ILC3s are now thought to be the major source of intestinal IL-22, which stimulates epithelial cells to secrete antimicrobial peptides, principally REG3. ILC3s also directly enhance B-cell production of IgA, by producing molecules that stimulate B-cell differentiation, and indirectly by inducing the development of isolated lymphoid follicles. In fact, a subset of ILC3 cells (called lymphoid tissue inducer or LTi cells) are required for the development of isolated lymphoid follicles and their cryptopatch precursors.

ILC1s and ILC2s are also found in small numbers in the healthy gut, but their role in intestinal homeostasis is unclear. They appear to play more important roles in the gut response to pathogens.

T_H17 cells were initially recognized for the detrimental role they play in inflammatory and autoimmune disorders (see Chapter 10). However, like ILC3 cells, they are also abundant in the healthy gut lamina propria. They, too, help maintain barrier immunity by secreting IL-22 and enhancing the health and activity of epithelial cells. An unexpected series of observations, described in Advances Box 13-3, led to the recognition that their development is encouraged by specific commensal microbes, including segmented filamentous bacteria (SFB). The beneficial effects of gut TH17 cells are underscored by the observation that, in their absence, mice are more susceptible to invasion by bacteria, including Citrobacter.

ADVANCES BOX 13-3

Germ-Free Animal Model Systems

Since Louis Pasteur advanced the germ theory of disease, humankind has been working to obliterate germs from our bodies and environments. Some of these efforts are appropriate, particularly when trying to prevent and treat disease-causing infection. However, as we have learned in this chapter, a germ-free existence is not necessarily a healthy one. The relationship we cultivate with our microbiome, probably from the earliest time in our life, shapes our entire immune system and contributes to both our physical and mental health. Even antibiotics, which have had a profoundly positive influence on animal health, also have a darker side in their counter-effects on our commensal microbiome.

Germ-free animal models, also called axenic models, have been an invaluable resource for studying the influence of the microbiome on animal health. Developed in the early 1900s as part of an effort to address diseases caused by pathogens, germ-free rodents have provided some of the strongest evidence for the association of specific microbial species with beneficial biological outcomes.

How do you raise a germ-free mouse? This is not trivial and requires a facility that isolates animals and prevents them from coming in contact with all microorganisms in the air, on surfaces, in food, on handlers, and on other animals (Figure 1a). A combination of barriers, radiation, high-temperature treatment, air filters, and very careful handling has made this possible. In addition, founder animals must be delivered sterilely, via cesarean section, to avoid contact with maternal microbes in the vagina/birth canal. Ideally, these founder animals are subsequently fostered by germ-free mothers. As one can imagine, food, which is naturally rich in microbes, presents a unique challenge. Early efforts to sterilize food reduced its nutritional value dramatically. New technologies that stabilize important nutrients and supplement with vitamins that are made by microbes have helped. Although germ-free mouse facilities are now common, vigilance is always important. Microbes are opportunists and can too easily find ways through barriers. Differences in experimental results from laboratory to laboratory always raise the possibility that some facilities are more germ-free than others.

FIGURE 1 Maintaining germ-free mice. (a) Example of an isolation chamber that maintains a sterile barrier between the external environment, the handler, and germ-free mice. (b) Multiple approaches used to assess the impact of microorganisms on health. Single microorganisms or communities of microorganisms from the laboratory, from other mice, or even from other donors, including humans, can be introduced. Diet can also be controlled and manipulated.

The photo shows an isolation chamber used in a laboratory. Various apparatus placed in sterile packs are arranged on the two shelves of the portable chamber. The illustration shows two mice labeled colonization of germ-free mice. Two arrows from donor microbiota and synthetic microbiota point toward each mouse. Shown below are nuts, cheese, mouse food pellets, and a strand of DNA. An arrow from the mouse food points toward two graphs labeled physiological characterization. The bar graph shows a pair of short grey and high blue columns. In the first set, difference between the heights of the two bars is less. In the second set, the difference between the two bars is high. A line graph shows two lines. Line one (grey), starting from the vertical axis, rises steeply and then gradually decreases. Line two (blue) seen below the first line, also starts from the same point, rises steeply and then gradually decreases.

What is the difference between a germ-free and a normally raised mouse? First, they simply look different. Littermates raised apart in germ-free versus normal conditions differ in weight gain; those in germ-free environments tend to be skinnier and, in fact, need 30% more calories to maintain their weight. This is likely due to the contributions the microbiome makes to an organism’s nutritional status, by digesting food that the host organism cannot and generating vitamins and other predigested nutrients.

The differences in the mucosal immune systems of the two types of animal are also striking. Mucosal immune cells of germ-free mice are dramatically reduced in number and in secondary lymphoid tissues, such as Peyer’s patches, which are dramatically reduced in size. IgA secretion is diminished, as are the numbers of T_H17 cells. Although one might expect germ-free mice to have fewer regulatory T cells, too, results have been conflicting and may reflect variations in diet, as antigens provided in the food may also induce regulatory T-cell development. Interestingly, although germ-free mice appear less anxious, they also do not seem to have as high a cognitive or memory capacity.

Germ-free mice have been very useful in understanding the effects of the microbiome on multiple body systems including the immune system. By repopulating these mice with selected and characterized populations of microorganisms, investigators gain understanding of the influence of specific microbial species and diet on many aspects of animal health (Figure 1b).

One of the first experiments to definitively reveal a specific effect of a microbial species on mucosal immune functions came out of an intriguing observation that laboratory mice purchased from two different vendors had different mucosal immune responses. Specifically, the investigators noticed that inbred C57BL/6 mice from the Jackson Laboratory had fewer gut T_H17 cells than the same mouse strain from Taconic Biosciences. Quantification of 16S RNA sequences specific for bacterial species revealed a difference in the gut microbiome (Figure 2). Strikingly, the Jackson Laboratory mice had little or no segmented filamentous bacteria (SFB). These investigators hypothesized that this microbe played a specific role in maintaining gut T_H17 cells and tested their idea using germ-free animals. Before reading on, see if you can devise a well-controlled experiment that would test their hypothesis.

FIGURE 2 Mice from different laboratories harbor different microorganisms. (a) A section of the small intestine was evaluated for the presence of segmented filamentous bacteria (SFB) by performing quantitative PCR of 16S rRNA sequences. The difference in amount of SFB-specific 16S rRNA sequences between mice from Taconic Biosciences (Tac) versus the Jackson Laboratory (Jax) are shown on the left (nd, not detectable). Right: The amount of total bacteria in the intestine of both sets of mice (a control). (b) Scanning electron microscopy of the small intestine shows that the Taconic mice have a much higher concentration of filaments on the epithelial surfaces.

Part A shows two bar graphs. Graph 1 compares the Relative fold change of segmented filamentous bacteria in mice from two laboratories. The vertical axis is labeled relative fold change with markings from 0 to 180 with increments of 20. The column representing Taconic Biosciences Lab reads 140 and no data is shown for Jackson Laboratory. Graph 2 shows the amount of total bacteria in the intestine of mice from the two different laboratories. The markings on the vertical axis range from 0 to 1.5 with increments of 0.5. The column representing Taconic Biosciences Lab reads 0.5 and the column representing Jackson Laboratory reads 1.5. Vertical error bars are shown on all the three columns. Two micrographs, labeled Taconic Biosciences Lab and Jackson Laboratory, show the scanning electron microscopic image of bacteria in the small intestine of mice from two different laboratories. Taconic Biosciences Lab mice have a higher concentration of filaments on the epithelial surface compared to the bacteria cultured in Jackson Laboratory.

The results of one of their experiments are shown in Figure 3. In this experiment, they introduced segmented filamentous bacteria (SFB) into germ-free (GF) mice and measured the production of IL-17 by small intestinal CD4^⁺ T cells. They also assessed IL-17 production by CD4^⁺ T cells from the intestines of specific pathogen–free (SPF) mice as a control. (These mice are raised so that they are not exposed to a subset of potential pathogens; however, they have a fairly healthy commensal microbiome.) What do you conclude, and what other controls may be helpful in drawing a definitive conclusion?

FIGURE 3 SFB colonization affects IL-17 production by intestinal helper T cells. Briefly, CD4^⁺ T cells were isolated from the intestine of germ-free (GF) mice, GF mice colonized specifically with SFB (GF + SFB), and specific pathogen–free (SPF) mice that have a healthy commensal microbiome. Each set of cells was stimulated under conditions that promote IL-17 production, and the percentage of T cells making IL-17 was measured by flow cytometry. See text for full explanation.

These results were the first to establish the importance of SFB as commensal bacteria and their influence on the health of one of our gut T-cell subsets. See Clinical Focus Box 16-1 for another example of the use of germ-free systems in understanding the immune response.

The cytokines produced by ILC3s and T_H17 cells can also enhance inflammation and cause disease. Chronic production of IL-17A and IL-17F, for instance, contribute to colitis and psoriasis, respectively. How ILC3s and T_H17 cells balance their health-promoting and inflammation-promoting functions is still not fully understood. The cytokine milieu probably plays a key role. Whereas the tolerogenic IL-10 and TGF-β cytokines promote the development of anti-inflammatory T_H17 cells, the presence of the proinflammatory cytokine IL-6, which is produced by multiple cell types after infection, can induce T_H17 cells to take on more inflammatory roles. It is, indeed, possible that these two subsets offer the gut a uniquely flexible defense mechanism with the ability to quickly convert from quiet inhabitants to proinflammatory defenders.

Role of Intraepithelial Lymphocytes (IELs) in Intestinal Immune Homeostasis

TCR γδ and TCR αβ intraepithelial lymphocytes (IELs) also play important roles in intestinal immunity and typically insert themselves between epithelial cells, below their tight junctions (see Advances Box 13-1). They can be recruited in a conventional immune response to an invading pathogen; however, they also participate in homeostasis by maintaining the integrity of the epithelial layer, producing antimicrobial peptides and immunosuppressive cytokines. Some, the natural IELs, come directly from the thymus and take up residence in the intestine; these tend to be CD4⁻CD8⁻. Induced IELs, on the other hand, develop from naïve T cells that have been activated in gut-associated lymphoid tissue. These are typically CD4⁺ TCR αβ⁺ cells that express the unusual CD8αα homodimer, which interacts with MHC class I. CD4⁺ IELs appear to repress inflammatory T-cell activity and may develop from mucosal FoxP3⁺ T_REG cells.

Key Concepts:

Tolerance to the intestinal commensal microbiome is achieved through interactions between the microbiome, epithelial cells, and immune cells in the lamina propria.
The homeostatic environment of the gut is dominated by anti-inflammatory cytokines including IL-10, TGF-β, and RA, as well as cells that maintain barrier health, including T_REG cells, IgA-producing B cells, ILC3 and T_H17 cells, and intraepithelial lymphocytes.
Antigen-presenting cells in the healthy intestine sample antigen, travel to secondary lymphoid tissue, and polarize naïve T cells to the T_REG lineage. Many of these cells take up residence in the lamina propria and help maintain tolerance to the microbiome.
IgA-producing B cells are generated in both T cell–dependent and T cell–independent manners. The latter depends on the presence of several molecules, including BAFF and APRIL, which are generated by epithelial cells and DCs in response to direct interaction with the microbiome.
ILC3 and T_H17 cells are also abundant in the intestinal lamina propria and play dual roles. They help maintain homeostasis by producing IL-22, which stimulates epithelial cells to produce protective AMPs. However, they can also contribute to inflammatory responses.

The Immune Systems in the Small and Large Intestines Differ

Although the small and large intestines share general strategies for managing their relationship with the microbiome, it is important to emphasize that the physiology of each segment differs, as do the communities of microbes and responding immune cells that reside in them (see Figures 13-5 and 13-6).

The small intestine harbors a smaller and less diverse commensal community than the large intestine, which is the site of the most abundant and diverse microbial community in the body. Whereas the lumen of the small intestine has fewer than a million commensal bacteria per milliliter of fluid, the lumen of the large intestine can contain between a billion and a trillion bacteria per milliliter.

The small and large intestines are also vulnerable to different pathogens. For example, Clostridium difficile, a bacterium associated with diarrhea outbreaks in hospitals and care centers, is found in the large intestine. Norovirus, which is associated with outbreaks of diarrhea on cruise ships, infects the small intestine. The large intestine is the site of infection for whipworm (Trichuris trichiura), whereas the common roundworm (Ascaris lumbricoides) does its damage in the small intestine. Similarly, the small and large intestines are susceptible to different inflammatory disorders and cancers. Celiac disease results from inflammatory reactions in the small intestine, and ulcerative colitis is a damaging inflammation of the large intestine. Finally, tumors are also more common in the large versus the small intestine. These are only a few of the biological distinctions between the two locations, which suggest parallel distinctions in the immune strategies adopted by each intestinal site.

As we discussed earlier, the relative concentrations of particular epithelial cell types also vary from small to large intestine. The small intestine has many Paneth cells, the large intestine has very few. The large intestine has many goblet cells that generate a much thicker and more effectively protective mucus layer. Peyer’s patches are present only in the small intestine, and their associated M cells are more prevalent there, too. Isolated lymphoid follicles are more common in the large intestine, which has no villi and, unlike the small intestine, does not have to occupy itself with the absorption and digestion of food. More distinctions continue to be identified and understanding the differences and their relationship to disease remains a topic of great interest.

Commensal Microbes Help Maintain Tolerogenic Tone in the Intestine

The research community is still working to understand all the criteria the gut immune system uses to decide whether to generate a tolerogenic or an immune and/or inflammatory response to the microbes and antigens it encounters. However, it is clear that both evolutionary and developmental events are at play. Microbes that co-evolved with us influenced the evolution of our immune receptors, including invariant T-cell receptors that have fixed specificities for common microbial antigens. Microbes that colonize our guts in early life (“old friends”) tune and tolerize our immune system by inducing the development of regulatory T cells and the production of IgA specific for commensal bacteria.

The availability of germ-free animal models (see Advances Box 13-3) has allowed investigators to more precisely determine the influence of specific microbial species. Investigators have found that several species of commensal bacteria are particularly good at stimulating T_REG accumulation and tolerance (Figure 13-11). These include bacteria in the phylum Firmicutes, such as Clostridium; and bacteria in the phyla Actinobacteria and Bacteroidetes, all of which are prominent members of the healthy human microbiome. Firmicutes bacteria and others produce short-chain fatty acids (SCFAs) by fermenting dietary fiber. SCFAs directly influence dendritic cells in the gut and enhance the development of regulatory T cells. Segmented filamentous bacteria (SFB), another member of the Firmicutes phylum, colonize the small intestine and alone enhance IgA production and T_H17 development. Remarkably, recent data show that even a single molecule of polysaccharide A (PSA) produced by a single intestinal bacterial species (Bacteroides fragilis) helps to maintain the systemic regulatory T-cell pool.

An illustration shows various types of commensal bacteria such as microbiota, Firmicutes, and Bacteroides Fragilis, and their effect on intestinal immune response — FIGURE 13-11 Effect of commensal bacteria on intestinal immune responses. Shown are several bacterial species that are common members of our commensal microbiome. These express and/or produce molecules (e.g., PSA, SCFA) that interact with epithelial cells (and APCs) and stimulate the development and activity of ILCs and helper T-cell subsets, each of which contributes differently to intestinal immune homeostasis (see text).

Although we still have much to learn about the viral communities that inhabit our gut, recent work shows that virus exposure also has beneficial effects on intestinal development and systemic immune function. Remarkably, when germ-free mice, whose intestinal epithelial barrier is more porous and whose mucosa is depleted of immune cells, are exposed to a single viral species (e.g., norovirus), immune cell populations are restored, epithelial cell connections are strengthened, and immune homeostasis in the gut is re-established. This is, in part, due to the interaction of the virus with pattern recognition receptors, which triggers the cascade of events responsible for maintaining healthy, tolerogenic immune activity in the intestine.

Finally, and importantly, it turns out that what happens in the gut does not simply stay in the gut. Not only does our commensal gut microbiome help tolerize our gut immune system, but it influences the entire immune system. When tolerogenic responses are impaired in the gut, autoimmune responses are more common elsewhere. For instance, patients with systemic lupus erythematosus (SLE) appear to have a microbiome that promotes inflammatory responses, and may be helped by supplementation with a bacterium, Bifidobacterium, from the phylum Actinobacteria. Bifidobacterium is now a relatively common probiotic and may act in part by promoting regulatory T-cell development.

Fecal transplantation, otherwise known as bacteriotherapy, was inspired by studies that show the beneficial influence of the microbiome. It has been performed by large-animal veterinarians for over 100 years and was first used in humans over 50 years ago. Most often used today in patients with C. difficile infections and the diarrhea it produces, fecal transplantation involves the introduction of a healthy donor’s colonic microbiome to an infected individual. The re-introduced commensal bacteria can outcompete disease-causing bacteria and restore epithelial integrity, although more work needs to be done to standardize therapy and to understand which specific microbes are beneficial.