The Superorganism

Of the microbes that live on the skin and mucosal surfaces, the largest such community is found in the intestine. The intestinal microflora consists of more than 1000 bacterial species in humans, with each individual housing about 160 species and about a hundred trillion (10¹⁴) bacterial cells. (Thus nine out of ten cells in the human body are bacterial. The figure for domestic mammals is unlikely to be very different!) Given the large number of bacterial species involved, they contain about 100 times more genes than the human genome. Because of the enormous number of microorganisms involved, the concept has emerged that animals together with their microflora form “superorganisms” in which enormous numbers of genes act together to promote survival and the whole is regulated by the immune system. In addition to considering the bacteria as part of the animal microbiome, it is also apparent that many viruses live within the body, collectively constituting a virome of unknown significance.

The presence of large stable populations of symbiotic bacteria implies that their elimination is neither feasible nor desirable. The immune system must therefore adapt to the presence of this microflora while at the same time retaining its ability to fight pathogens. The first bacteria to colonize body surfaces are acquired by a newborn animal from its mother at the time of birth. Within days, these are followed by many different environmentally acquired bacteria, generally as a result of chance microbial encounters. These organisms compete and adapt to their environment so that over the first months of life, their composition gradually changes to the stable adult-like microflora. The size and complexity of this microflora is greatest in the gastrointestinal tract. Most intestinal microbes are bacteria (Gram-positive and Gram-negative, facultative aerobic and anaerobic), but there are some archaea, a few eukaryotes, and many viruses and bacteriophages. The composition of the microflora varies among surfaces and even among locations on those surfaces. Additionally, the composition of the intestinal microflora differs among individuals and depends on their diet. The bacterial density is relatively low in the stomach but rises in the rumen, the distal small intestine, and especially the colon (Figure 22-2). The rumen mainly contains anaerobic bacteria, with some protozoa and fungi. In the large intestine, the microflora mainly consists of strict anaerobes. It has been estimated that this microbial population can eventually reach a density of 10¹² organisms per gram of intestinal contents.

As commensals, the intestinal microflora helps to break down plant cell walls and digest complex carbohydrates such as cellulose and other plant polysaccharides. These commensals synthesize some essential vitamins and, by maintaining an appropriate balance within the microbial population, restrict the growth of potential pathogens. In addition, they play a central role in the development and regulation of the immune system.

Although essential for the health and proper functioning of the animal body, it must not be assumed that the normal microflora does not present a threat to an animal. Microbes can only be excluded by continual vigilance. Many members of the microflora can be considered pathobionts, which retain the potential to cause disease. Remember too that the decomposition of a dead animal is initiated by its own microflora around the time of death immediately after the failure of its immune defenses.

Controlling the Microflora

Animals and their microflora have coevolved as a result of the benefits gained by each partner, but the huge mass of commensal bacteria within the intestine is a potential threat to host integrity and must be closely watched by the immune system. There are two obvious threats facing any animal. The first is the critical importance of ensuring that the normal microflora is kept in its place and prevented from any attempt at invasion. The second is that the microflora and the molecules it releases might trigger innate responses such as inflammation. If these organisms can invade, and especially if they cause inappropriate inflammation, bad things will happen.

Exclusion of Commensals

The first cellular barrier to microbial invasion consists of intestinal epithelial cells (enterocytes). These cells maintain barrier integrity by having tight junctions between cells and a coating of attached mucins that form a glycocalyx, and they produce multiple antimicrobial peptides. Specialized intestinal epithelial cells called Paneth cells express toll-like receptors (TLRs) (Figure 22-3). When triggered by the microflora, they secrete multiple antimicrobial peptides. These enteric defensins, known as cryptdins, accumulate within intestinal crypts and achieve very high local concentrations. They prevent commensals from entering the crypt space and so protect enterocytes from invasion. In cattle, expression of cryptdin genes occurs throughout the small intestine and colon. These cryptdins are secreted as active molecules, as opposed to the human and mouse molecules that are secreted as inactive precursors and subsequently activated by trypsin within the intestine. Parasitic or other intestinal infections may increase production of α- and β-defensins. Cryptdins serve a barrier function since they are found in highest concentrations in the fluid layer closest to the epithelium and therefore reduce microbial contact with enterocytes. The cryptdin mixture selectively kills some bacterial species and as a result regulates the precise composition of the microflora.

The gastrointestinal mucus layer is also critical to the exclusion of both commensals and pathogens. The mucus layer consists of a gel made of mucins, glycoproteins, and lipids that prevents bacteria from contacting the epithelium. This mucus acts as a lubricant, blocks chemical insults, and can capture and then expel pathogens. The mucus forms an inner and outer layer. The inner layer next to the enterocytes consists of firm mucus rich in defensins and lysozyme and contains few bacteria. The looser mucus in the intestinal lumen is composed primarily of mucins produced by intestinal goblet cells. Many bacteria are embedded in this mucus, where it prevents their washout. Its thickness and composition vary, but it tends to be thickest where the microflora is abundant.

The epithelial cell brush border is also covered by a glycocalyx—a layer of acidic polysaccharides and glycoproteins that binds to the apical surface of the cells and serves as a protective barrier while still permitting the absorption of nutrients.

The intestinal microflora acts competitively against potential invaders and supplements the physical defenses of this system (Figure 22-4). By occupying and exploiting the intestinal microenvironment, commensals block subsequent colonization by pathogenic bacteria. (It is, for example, possible to block Salmonella species colonization of the chicken intestine by administering an appropriate mixture of commensal bacteria to birds.) The microflora determines local environmental conditions by, for example, keeping the pH and oxygen tension low. The microflora is also influenced by the diet; as a result, the intestine of milk-fed animals is colonized largely by lactobacilli, which produce bacteriostatic lactic and butyric acids. These acids inhibit colonization by potential pathogens such as Escherichia coli, so young animals suckled naturally tend to have fewer digestive disturbances than animals weaned early in life. Genetically identical pigs housed in an outdoor environment, an indoor environment, or experimental isolators have had their intestinal flora studied. It has been found that 90% of the bacteria within the intestine of the outdoor group were Firmicutes, especially lactobacilli. In contrast, Firmicutes constituted less than 70% of the intestinal flora in the indoor group and about 50% in the isolated pigs. Pigs from cleaner environments had smaller proportions of lactobacilli. There is a strong negative correlation between the level of lactobacilli and the level of pathogens in the intestine. These differences also influence the expression of immune system genes. For example, animals raised in isolation express more genes involved in inflammatory immune responses such as type I interferons. In contrast, genes associated with T cell function are expressed more highly in the pigs housed outdoors.

Regulation of T Cell Function

Molecules produced by commensal bacteria can influence the functions of all the major T cell subsets. For example, Treg cells producing IL-10 are present in the intestine. Several commensal bacteria such as Bacteroides fragilis and some clostridia seem especially effective in inducing these FoxP3+, IL-10-producing T cells. The bacterial molecule polysaccharide A (PSA) is key to this process. PSA suppresses proinflammatory cytokine responses in the intestine and inhibits lymphocyte infiltration. Treg populations are reduced in germ-free mice.

Th17 cell development in the intestine is also affected by the microflora (Figure 22-5). Germ-free mice are deficient in IL-17. Segmented filamentous bacteria drive T cell development and stimulate Th17 production. SFBs also promote germinal center development, IgA production, and recruitment of intraepithelial lymphocytes. Because it is believed that Th17 cells may originate from T reg precursors, it is suggested that the microflora regulates the Th17-Treg switch.

Dysbiosis and the Hygiene Hypothesis

Changes in the composition of the intestinal microflora have been implicated in the development of allergies. The hygiene hypothesis suggests that a lack of exposure to certain commensal bacteria early in life affects the development of the immune system and increases an individual’s chances of developing allergic diseases. In support of this, it has been shown that children who develop allergic disease have different intestinal microflora than children who do not. Studies on piglets exposed to differing levels of intestinal colonization have shown that the complexity of the intestinal flora influences gene expression in the immune system. Animals with a limited intestinal flora expressed more genes involved in inflammation. Conversely, piglets with a more diverse intestinal flora expressed more genes related to T cell function. Alterations in the intestinal microflora also influence the development of autoimmune diseases such as rheumatoid arthritis, some mouse models of autoimmune arthritis, ankylosing spondylitis, insulin-dependent diabetes mellitus, and experimental autoimmune encephalitis (Chapter 35).

Inductive Sites

The mucosa-associated lymphoid tissues (MALTs) possess the three cell types required to initiate immune responses: T cells, B cells, and dendritic cells. These tissues include lymphoid tissues in the eyelids, nasal mucosa, tonsils, pharynx, tongue, and palate (collectively called Waldeyer’s ring); Peyer’s patches; solitary lymphoid nodules; the appendix in the intestine; and numerous lymphoid nodules in the lung. These lymphoid tissues are known by their acronyms. Thus GALT (gut-associated lymphoid tissue) is the collective term for all the lymphoid nodules, Peyer’s patches, and individual lymphocytes found in the intestinal walls. Similarly, BALT is the acronym used for the bronchus-associated lymphoid tissue in the lungs. These organized lymphoid tissues, unlike lymph nodes, do not react to foreign antigens delivered through afferent lymph but rather sample them directly from the surface.

The tonsils are especially important in inducing immunity on mucosal surfaces. Some organisms, however, can overcome the defenses of the tonsils and use them as a portal of entry into the body. For example, pathogens such as bovine herpesvirus-1, Mannheimia hemolytica, Streptococcus suis, and Mycobacterium tuberculosis can persist indefinitely within the tonsils.

The surface of the intestine is covered by a layer of enterocytes that form intercellular tight junctions and which thus form an effective barrier to both microbes and macromolecules (Figure 22-6). (Molecules larger than about 2 kDa are excluded.) Obviously, some aggressive invasive bacteria may damage this barrier and trigger local inflammatory and immune responses. There are two alternative routes by which organisms and macromolecules can penetrate the intact intestinal wall and be directed toward the intestinal lymphoid tissues. One route involves penetrating specialized epithelial cells (M cells) found in the epithelium directly over aggregates of lymphoid tissue or Peyer’s patches; the other involves dendritic cells that reside in the submucosa but extend their cytoplasmic processes between the enterocytes into the intestinal lumen. The tight junctions remain intact, but antigen samples can enter within the dendritic cell cytosol. This route provides a mechanism whereby noninvasive bacteria and macromolecules can be sampled and presented to nearby T cells.

Peyer’s patches are the largest of the mucosal lymphoid tissues. A newborn calf normally has about 100 Peyer’s patches, and these may cover as much as half of the ileal surface. Collectively, therefore, the intestine contains more lymphocytes than the spleen. In ruminants and pigs, there are two types of Peyer’s patch that differ in location, structure, and functions. The ileocecal Peyer’s patches of ruminants are primary lymphoid organs, whereas the jejunal Peyer’s patches are secondary lymphoid organs (Figure 12-8). In lambs, the ileocecal patches increase in size from birth to 6 months of age and then regress, leaving only a small scar. In contrast, the jejunal patches persist throughout adult life and continue to play a major role in intestinal defense. Both types of Peyer’s patch consist of masses of lymphocytes arranged in follicles and covered with an epithelium that contains M cells. M cells are specialized epithelial cells involved in antigen transportation. They have microfolds (M) rather than microvilli on their surface (Figure 22-7). The mucus layer tends to thin out over Peyer’s patches so that the M cells protrude into the lumen. M cells phagocytose the macromolecules and microbes they encounter, but rather than destroy them, they transport the antigens to their underlying lymphoid tissue. M cells may transport soluble macromolecules such as IgA, small particles, and even whole organisms. (Some pathogens, such as salmonellae, Yersinia and Listeria species, M. tuberculosis, and the reoviruses may take advantage of the M cells and use them to gain access to the body.) The proportion of M cells in the follicle-associated epithelium varies from 10% in humans and mice to 50% in rabbits and to 100% in the terminal ileum of pigs and calves.