Springing into Action: Intestinal Immune System Response to Invasion

Most of our commensal organisms are not pathogenic and contribute to the tolerogenic environment of the intestine at steady state. Nevertheless, the gut immune system maintains a healthy distance between epithelial surfaces and even beneficial commensal bacteria, by secreting IgA antibodies and generating mucus. These strategies help us fend off the few commensal bacteria, termed pathobionts, that do have the capacity to cause disease in compromised hosts.

Most dangerous microorganisms, however, come from the outside. Our gut has evolved several different strategies to recognize, respond to, and expel them. We describe in this section some of the current thinking behind inflammatory immune responses of the intestine, recognizing that our understanding is still evolving rapidly.

The Gut Immune System Recognizes and Responds to Harmful Pathogens

While we still do not fully understand all the variables that influence the intestinal decision to initiate an inflammatory rather than a tolerogenic response, infection of, or damage to, the epithelial barrier by pathogens, toxins, or trauma often is the distinguishing factor (Figure 13-12). Inflammation can cause discomfort, of course, but most inflammatory responses are ultimately protective and designed to clear pathogen and repair epithelial integrity. These successful inflammatory responses should be distinguished from immune responses that result in chronic inflammation, such as inflammatory bowel disease (IBD), which has increased in incidence and media presence.

FIGURE 13-12 Conditions that cause switch from homeostatic (a) to inflammatory (b) immune responses. Environmental, physiological, and genetic factors can compromise the integrity of the epithelium and/or alter the balance between beneficial and virulent microorganisms (see text). Antibiotics, diet, and ingestion of infectious organisms can alter the commensal microbiome directly. Pollutants and invading microorganisms can trigger inflammatory responses by the epithelium and antigen-presenting cells. Hormones produced by stress can have direct and indirect effects on the microbiome and epithelial integrity. Inherited mutations in immune molecules (e.g., IL-10 and IL-23 receptors) can also compromise our ability to maintain homeostasis.

Illustration a shows four layers, Gut lumen, Mucus, Healthy epithelial barrier, and Lamina propria. The first layer, gut lumen has physiological microbiota and the second layer, mucus has peace-keeping bacterium and pathobiont. The third layer is healthy epithelial barrier and below it is Lamina Propria, which has T H 1 cell, I G A plus plasma cell, T REG cell, and T H 17 cell. Homeostasis or physiological inflammation takes place in Lamina Propria. Illustration b shows the altered gut environment in which antibiotics, diet, hygiene, pollutants, stress, infection, and genetic changes decrease peace-keeping bacteria and increase pathobionts and invaders. This process is labeled Dysbiosis. Pollutants and infectious microorganisms penetrate the epithelial barrier by damaging them and cause pathological inflammation. The text corresponding to the epithelial barrier reads, damaged epithelial barrier, increased bacterial adherence, and penetration.

To successfully invade the gut, pathogens first need to battle commensal bacteria for space and nutrients. Some pathogens feed off of metabolites released by commensal bacteria and metabolize molecules that commensals cannot, thus exploiting niches not available to commensal organisms. Ironically, antibiotics, themselves, also provide invading bacteria with a competitive advantage and better access to the intestinal epithelium.

The response to pathogens that invade the gut is divided into two phases—the inductive phase, when the response is initiated and shaped, and the effector phase, when mature immune cells actively work to clear the pathogen and repair damage (Figure 13-13).

FIGURE 13-13 Intestinal immune system response to Salmonella bacterial infection: an example of a type 1 response. (a) The inductive phase; (b) the effector phase. See text for details.

Illustration a shows the inductive response to Salmonella. The illustration shows an epithelial layer with goblet cell, paneth cell, and M cells labeled. Mucins and anti-microbial peptides are seen in the mucus layer above the epithelial layer. The Peyer's patch below the epithelial barrier is labeled. Transcytosis of salmonella occurs through M cells resulting in the infection of epithelial cells. The bacteria are absorbed by C X subscript 3 C R plus macrophages and DCs.

Illustration b shows the effector response to salmonella. Salmonella enters the intestine through the M Cells. Salmonella enter the T L Rs in the intestine. T L Rs with salmonella produce N L R C 4 and N L R P 3, which activate I L – 18 that, in turn activates C D 8, N K, T H 1 cell. The C D 8, N K, T H 1 cells releases I F N – gamma, which goes back to the T L Rs. Chemokines activate Neutrophil. I L-1beta moves toward the epithelial layer. T L Rs with salmonella also produce I L-23 that stimulates T H 17, gamma delta T, N K T cells to release I L- 17, which moves toward the epithelial layer. T H 17, gamma delta T, N K T cells also release I L-22 that moves toward the epithelial layer. The effector response results in increased production of mucins and antimicrobial peptides in the mucus layer.

The inductive phase shares many features with other immune responses. It is initiated by epithelial cells and antigen-presenting cells that have been alerted to the presence of microbes via pattern recognition receptors that generate proinflammatory rather than tolerogenic signals. For instance, interactions with TLR5, a receptor for bacterial flagellin, tend to induce inflammation, whereas interactions with TLR2 can be tolerizing and important for resolving infection and epithelial integrity. Pathogens that activate NOD-like receptors (NLRs; see Chapter 4) and those that trigger inflammasome pathways in macrophages and epithelial cells also shift the immune system away from tolerogenic responses and trigger protective inflammatory responses (the inflammasome is described in Advances Box 4-2).

The intracellular location of PRRs also provides immune cells with information that allows them to distinguish between healthy and unhealthy interactions with microbes. For instance, TLR5 is located only on the basolateral and not the apical surface of epithelial cells in the large intestine. Therefore, bacteria will only trigger this PRR if the mucosal layer is damaged or the bacteria is invasive.

Activated APCs alert naïve T cells to the presence and general identity of the antigen. Rather than polarize naïve T cells to the regulatory lineage, APCs activated by pathogens polarize naïve T cells to the T_H1 and inflammatory T_H17 lineages or T_H2 and T_H9 lineages, depending on the microorganism encountered (see Advances Box 13-1, Figure 2, and specific examples discussed here). Rather than induce IgA class switching, the T cells and APCs generated after an encounter with pathogens encourage class switching of B cells to more proinflammatory IgG classes, which protect the body from the spread of the infection.

The effector phase involves the recruitment of cells and strategies that clear or expel the invading organism, and varies depending on the identity of the invading organism. For example, some bacterial infections are thwarted by the activation of a type 1 response, where inflammatory ILC3 and T_H17 cells recruit phagocytic neutrophils. Bacterial infections can also induce ILCs to secrete IL-22, which stimulates epithelial cells to produce antimicrobial peptides that kill bacteria. Worm invasion stimulates type 2 responses that include ILC2 and T_H2 activation, which lead, in turn, to the recruitment of eosinophils and enhanced mucus production and peristalsis, the muscle activity that ripples through intestines during digestion and helps expel worms. Resolution of the infection also requires the repair of epithelial damage.

Key Concepts:

• The intestinal immune response to infection includes an inductive phase, when cells are alerted to the presence of a pathogen and assemble their cytokine and cell arsenal, and an effector phase when these cells work to clear the infection.
• In order to successfully invade the intestine, pathogens must compete successfully with commensal organisms and evade or withstand the inflammatory response induced by pattern recognition receptors.

The Intestinal Immune System Can Mount Both Type 1 and Type 2 Responses

The intestinal immune responses to specific pathogens follow these general themes. However, they also reveal additional complexities, some of which we illustrate below, where we describe the response to two classes of pathogens: the single-cell prokaryote Salmonella, which causes diarrhea, and the multicellular worm Ascaris.

A Type 1 Response to Salmonella Bacteria

Bacterial species that cause severe diarrhea include Salmonella species, Clostridium difficile, Citrobacter, and enterohemorrhagic Escherichia coli (EHEC). We will focus on the immune response to Salmonella typhimurium, which is a common and highly contagious bacterium that causes fever, cramps, and diarrhea in all vertebrates. The infection is usually cleared after a week or so, but some individuals, particularly those who have compromised immune systems, may suffer for much longer.

Salmonella is transferred by food, water, or feces from infected individuals. It spreads easily and rapidly and, once swallowed, successfully finds its way to the small and large intestines, where it can invade the epithelium. As you know, the gut has established several barriers that must be breached for infection to occur. Salmonella must first compete with the healthy commensal bacteria community and find a way around the defensins, the antimicrobial proteins made by the gut epithelium. Some, but not all, antimicrobial peptides (e.g., defensins) are particularly good at killing Salmonella, and these can sometimes eliminate an intestinal infection all by themselves. Pre-existing IgA with broad specificity also limits the access of Salmonella to the epithelial barrier.

However, Salmonella has multiple other means of entry into intestinal tissue (see Figure 13-13a). It is often transcytosed by M cells, it can use its own secretory machinery to enter epithelial cells, and it can be engulfed by resident macrophages. During infection, Salmonella meets the next line of defense—our pattern recognition receptors (PRRs). Surface Toll-like receptors (e.g., TLR4 and TLR5) and internal TLR9 engage multiple antigens on this gram-negative pathogen, including lipopolysaccharide (LPS), flagellin, and CpG. Internal NOD-like receptors (NLRs) bind Salmonella products, too, and trigger inflammasome activity.

Several innate immune cells in the intestine express TLRs and NLRs and contribute to the inductive response to Salmonella. These include epithelial cells, antigen-presenting cells, and ILCs, which cooperate in developing a cytokine milieu that encourages a type 1 response (see Figure 13-13b; see also Figure 4-16).

Pattern recognition receptor engagement results in the expression of IL-23, a particularly potent cytokine that enhances the production of IL-17 and IL-22 by T_H17 and ILC3 cells. IL-22 is protective and up-regulates the production of antimicrobial proteins, including but not limited to REG3. IL-17 also enhances the production of chemokines that attract neutrophils. Neutrophils are particularly effective at clearing Salmonella and also produce additional IL-22 and IL-17 cytokines. This series of reactions, known as the IL-23–T_H17 cell axis, works within 2 to 3 days of a Salmonella infection to restore epithelial integrity.

Pathogen interactions with NLRs induce inflammasome activity in macrophages and activate caspase-1. Caspase-1, in turn, activates the cytokines IL-1β and IL-18, which contribute to the type 1 response by inducing T-cell and ILC subsets to produce IFN-γ (see Figure 13-12b). Finally, antibodies are also critical in controlling Salmonella infection and dissemination. Although IgA antibodies may play a role in the initial defense against Salmonella, antibodies of the IgG class are more potent in managing an active infection. Antigen sampled by M cells is relayed to the B-cell follicles where it activates antigen-specific B cells. These are induced to class switch by appropriate T helper cells. The IgG2a antibody class seems particularly effective against Salmonella infection. Unlike IgA, IgGs are not secreted into the lumen of the intestine unless the epithelial layer is physically damaged.

Salmonella has also evolved very clever defenses against these protective responses. It actually thrives on some of the carbohydrates that decorate proteins on epithelial surfaces, including sialic acid. If internalized by a macrophage, it takes advantage of the acidic pH of endosomal vesicles to activate its own virulence genes. It seems more resistant to some antimicrobial proteins than some commensal bacteria, and may even enjoy a growth advantage in the presence of these proteins. Hence, a successful immune response is a battle of time and evolutionary wits between host and microbe.

A Type 2 Response to Worm Infection

Billions of people are infected with parasitic worms. Intestinal worms include hookworms, roundworms, whipworms, tapeworms, and Trichinella, and can cause disorders and diseases that range from the very mild to the completely devastating. Ironically, a growing group of individuals in industrialized nations are gaining interest in therapeutic worm infection as a way to avoid autoimmune diseases and asthma. Although skepticism is warranted, there is also experimental evidence for the health benefits of some worm infections.

Just as our immune system has co-evolved with our commensal bacteria, it has also adapted over millions of years to a co-existence with intestinal worms. Some worms are treated as commensal organisms and generate tolerogenic responses that enhance the production of regulatory T cells and the generation of IL-10 and TGF-β cytokines (Figure 13-14a). Worms that invade our gut, however, meet a well-organized defense that reflects our long evolutionary history with these organisms.

FIGURE 13-14 Intestinal immune system response to worm infection: an example of a type 2 response. (a) The major inductive events that occur in the intestine after contact of epithelial tuft cells with a worm. (b) The effects of cytokines IL-4 and IL-5 on the effector response to the worm. See text for details. See also Videos 13-14v1 and 13-14v2. These two videos, both taken by Michael Patnode, show human eosinophils coating a Caenorhabditis elegans larval nematode.

Illustration a shows epithelial cell layer with tuft cell and goblet cell labeled. Mucin is seen near the goblet cell in the lumen region. A worm comes in contact with the epithelial cell. This contact activates D Cs that in turn activate T REG cell, which releases I L-10 and T G F- beta. The damage to the epithelial cell stimulates the cytokines, I L-25, I L-33, and T S L P, which activate I L C 2 cell. I L-13 and I L-5 are produced. An arrow from IL-5 points to eosinophil. Simultaneously, I L-25, I L-33, and T S L P activate Basophil. Both eosinophil and basophil release I L-4 that activates T H 2 cell, leading to release of I L-4, I L-5, and I L-13. Illustration b shows I L-4 stimulate worm-specific I g E antibodies to bind to the worm through the antigen–binding sites. I L- 5 stimulates I g E antibodies to bind with eosinophils at High affinity-F c epsilon R I site and release granule mediators that kill worms.

Infectious worms trigger a type 2 immune response, which is characterized by the activities of ILC2 and CD4⁺ T_H2 cells and the generation of the classic type 2 cytokine trio IL-4, IL-5, and IL-13. In fact, investigators speculate that our type 2 immune response evolved specifically as a strategy to manage helminth infections (see Figure 13-14a).

Type 2 responses in the gut are initiated by a specific subset of cytokines called alarmins. These include TSLP, IL-25, and IL-33, which are released by gut epithelial cells. These act on a variety of type 2 immune cells, including ILC2s, mast cells, basophils, and ultimately CD4⁺ T_H2 cells, to induce the release of IL-4, IL-5, and IL-13.

How are intestinal cells alerted to the presence of worms? What stimulates them to secrete alarmins? Until recently, we did not know. However, investigators have now shown that tuft cells play a key role (see Advances Box 13-1). Tuft cells, also called brush cells, were identified in the 1960s but their function was not well understood. They are rare cells and share intriguing features with the sensory cells in the tongue that respond to umami and bitter tastes.

In fact, using sensory receptors, tuft cells seem to “taste” products of multiple parasites, including worms and some protozoa. This induces the production of the alarmin IL-25. IL-25 stimulates lamina propria ILC2 cells to secrete the key type 2 cytokine IL-13. IL-13 has many effects, including a positive feedback influence on tuft cell development. It acts on stem cells in the intestinal epithelium, inducing the development and proliferation of more tuft cells. Mice without tuft cells fail to produce enough IL-25 and fail to generate an optimal type 2 response to worms.

Type 2 cytokines have multiple additional effects on the intestinal immune system. IL-13 enhances goblet cell production and secretion of mucus, which helps to clear worms physically from the gut. IL-4 produced by eosinophils and basophils helps polarize naïve T cells to the T_H2 subtype. T_H2 cells enhance B-cell class switching to IgE. Worm-specific IgE antibodies bind via their antigen-binding sites to worms and via their constant regions to granulocytes such as eosinophils, and induce the release of granule mediators; these granule mediators have potent toxic effects that can be fatal to worms (Figure 13-14b and associated videos).

Some of these mediators, including histamines, are well known to those suffering from allergy. The allergic response is thought to be a consequence of inappropriate activation of this ancient system, which evolved to manage worms, protozoa, and other complex parasites.

The recognition that the incidence of allergy and autoimmunity was increased in industrialized areas, where people were less exposed to the full array of microbes available to our ancestors, led David Strachan and others to advance the hygiene hypothesis (see Chapter 1, Clinical Focus Box 1-3). Briefly, this is a proposal that exposure to microbes, including worms, tunes the immune system toward a more tolerogenic, less inflammatory state. The hypothesis now has experimental support, although perspectives on the biological and cellular bases for the phenomenon are evolving.

Intriguingly and somewhat paradoxically, worm exposure also has a powerful dampening effect on the immune system. Not only do parasitic worms inspire type 2 immune responses, but they also inspire tolerogenic responses characterized by the development of T_REG cells and the activities of IL-10 and TGF-β (see Figure 13-14a). How these responses are regulated and coregulated is an active area of investigation. However, it is clear that the tolerogenic state has systemic consequences. Anecdotal evidence that worm exposure helps to quell autoimmune and allergic states has inspired some individuals to lobby for worm therapy to ameliorate autoimmune symptoms. While some research supports these observations, some highly publicized results remain anecdotal and much more needs to be done to validate such a therapeutic approach. Even if the notion gains more support, it is also important to remember that regulatory T cells can interfere with our immune response, particularly type 1 responses to intracellular pathogens.

It is also important to recognize that this description of a worm infection is an oversimplification. Worms are very diverse metazoans with complex life cycles and the ability to traverse different parts of our bodies. Different worms affect our immune system in unique ways. They inspire multilayered immune responses that promise to yield deeper insights into our intimate immune relationship with microbes.

Key Concepts:

• Pattern recognition receptors and NLRs expressed by epithelial cells, APCs, and ILCs trigger a type 1 response to the gram-negative bacterium Salmonella, which is coordinated by several cytokines, including IL-23 and IL-17, and several cell types, including T_H1 cells and ILCs. It culminates in the recruitment of phagocytic neutrophils.
• Worms are sensed by tuft cells in the epithelium, which generate alarmins and cytokines, including IL-25, that trigger a type 2 effector response coordinated by ILC2 and T_H2 cells, which produce the trio of type 2 cytokines, IL-4, IL-5, and IL-13. The response culminates in the production of IgE and the degranulation of eosinophils, which kills the worms directly.