Abstract
The postnatal period represents a crucial time for the developing mucosal immune system that coincides with early life colonization of the host by microbial pioneers and initial exposure to an infant’s surrounding environment. Immediately after birth, opportunistic commensal communities disseminate throughout the gastrointestinal tract and fundamentally influence the maturation, instruction, and function of immune regulatory and effector subsets in order to facilitate tolerance to environmental antigens and guide the symbiotic relationship between the host and its emerging microbiome. In certain contexts, deviation from optimal host-commensal interactions during infancy can have durable and potentially deleterious consequences on the microbial training of specific immune subsets that can manifest as chronic inflammation and/or autoimmune disease in later life. Here we discuss the role of early life microbial education of the immune system during this “window of opportunity” when the ecological succession of commensal populations has a potentially critical impact on human health and disease.
Keywords
Microbiota, Neonate, Host-commensal interactions, Innate immune response, Adaptive immune response, Inflammatory bowel disease, Window of opportunity
34.1
Introduction
The gastrointestinal tract harbors the largest population of commensal organisms in the human body, whose homeostasis requires immunoregulatory mechanisms to prevent unnecessary activation of the immune system against antigens generated by environmental exposures including host-associated microbes. The establishment of immunological tolerance is a result of proper education of resident and newly immigrated immune populations suggesting a dynamic and active relationship between the nascent and naive immune system and pioneering microbial communities that make up the host-commensal microbiome. Exposure to microbes begins in utero after which opportunistic commensal populations disseminate the gastrointestinal tract, skin, and oral cavities. Metagenomic surveys of fecal matter as a measure of the composition of the commensal population suggest higher levels of diversity in the gut microbiota of newborns that reach relatively stable proportions through early childhood and adolescence. The identity and relative diversity of commensal species are influenced by environmental factors, including the mode of delivery, diet, antibiotic exposure, and infection that affect the access of microbial communities to nutrients and suitable habitat. Given the relative instability of the microbiome during infancy, and particularly during the first year of life, studies suggest external factors, such as diet or exposure to antibiotics, can contribute to the volatility of newly emerging and opportunistic microbial populations. At the same time, recent animal studies have suggested that many regulatory and effector immune subsets are permissive to microbial instruction during a restrictive period of early life, we term the “window of opportunity” that contributes to proper (or improper) immune responses to local and systemic endogenous and environmental stimuli throughout life. In this chapter, we discuss the role of the microbiota during immune development throughout early life and consider how disruption of these interactions alters host susceptibility to disease in later life.
34.2
Microbiota Influences on the Structural Development and Function of the Immune System Within the Gastrointestinal Tract
The establishment of the field of gnotobiology , which is the selective colonization of germ-free (GF) (sterile) animals, supports the notion that there is a dynamic interaction between the host immune system and the microbiota. Initial comparisons between animals raised under GF or specific pathogen-free (SPF) environments revealed that virtually the entire ultra-structural development of the gastrointestinal tract is actively influenced by the presence of bacteria. GF animals harbor an enlarged cecum, principally due to an accumulation of nondegraded mucus and aberrant intestinal epithelial cell (IEC) morphology characterized by longer villi and shorter crypts in comparison to genetically identical animals raised under conventional (SPF) settings. In addition, GF animals exhibit extensive deficiencies in the development of gut-associated lymphoid tissues (GALTs), harbor fewer and smaller Peyer’s patches and mesenteric lymph nodes (mLNs), and exhibit impaired development and maturation of isolated lymphoid follicles (ILFs) versus their SPF counterparts. Significantly, introducing gut bacteria to formerly GF animals normalize many of these associated structural abnormalities. These latter studies not only emphasize the influence of the microbiota on proper lymphoid tissue development, but also intimate that specific microbial communities can interact with the immune system of the gut, particular at the mucosal interface.
34.2.1
A Role for Commensals During Colonization and Infection
GF animals are more susceptible to infection by certain bacterial, viral, and parasitic invaders suggesting a general role for the endogenous microbiota to provide protective immunity against pathogens. One mode of defense against exogenous pathogens by commensals relates to the competition between these two classes of organisms for the same ecological niche within the host, through a phenomenon known collectively as colonization resistance. One strategy that favors the indigenous microbial community is the preferential consumption of nutrients required for growth of competing pathogenic bacteria. Thus, by consuming common limited resources, Escherichia coli ( E. coli ) can outcompete enterohaemmorrhagic E. coli (EHEC) for organic acids, amino acids, and other nutrients. Commensal metabolism can also restrict the virulence of invading pathogens. Production of the short chain fatty acid (SCFA) butyrate by commensals specifically suppresses expression of virulence genes encoding the Type 3 secretion system (T3SS) machinery of Salmonella entera , including Serovar Enteritidis, and Typhimurium. In an analogous fashion, mucin- derived fucose, generated by fucosidase-bearing commensal bacteria, like Bacteroides thetaiotaomicron , modulates the expression of the virulence factor ler limiting the growth of EHEC. Finally, commensals can also produce specific antimicrobial peptides that directly affect pathogen growth or survival; for instance, E. coli produce bacteriocins, proteinaceous toxins that specifically inhibit the grown of the same or similar bacterial species, including EHEC. A consequence of the disruption of commensal-mediated colonization resistance is that susceptibility to enteric infection may increase. Administration of antibiotics to SPF animals or introduction of low-complexity microbiota to GF animals leads to increased accumulation and dissemination of infectious bacteria in the context of infection or during a breach in epithelial barrier function.
34.2.2
Modulation of the Mucosal Immune System by Commensal Microbes Throughout Life
During infancy, microbial communities exist in a volatile state suggesting colonization resistance has not yet been established and as such, the ability of the host to accept pioneering microbes can in part be explained by the relatively poor immune surveillance capacity of the neonatal immune system at birth including the presence of immune cells that promote a characteristic tolererogenic environment within mucosal sites throughout the body. The developing immune system is characterized by low inflammatory cytokine production and the enrichment of immune subsets of T and B cells with regulatory rather than effector capacity. Consistent with this, the neonatal system responds uniquely to conserved microbial-associated molecular patterns (MAMPs) to promote healthy microbial colonization through impaired production of inflammatory mediators, such as radical oxygen species, and elevated production of antiinflammatory cytokines such as interleukin 10 (IL-10) compared to adults. Although the underlying mechanisms responsible for these observations remain elusive, comparisons of GF and SPF animals demonstrate that signals derived from the microbiota can modulate many aspects of immune regulatory and effector function within both the innate and adaptive immune system. Introducing commensals to previously GF animals can restore or enhance many host defense mechanisms ; however, more recent studies suggest that optimal development of the intestinal immune system can only be achieved if the host is exposed and responds to these microbes during a critical and restricted period of time during neonatal development. Failure to “correct” deficiencies associated with appropriate microbial exposure during this “window of opportunity” in early life results in suboptimal maturation of the developing immune system with potentially deleterious ramifications for the host that can extend into adulthood. Below, we discuss specific regulatory and effector cells of the mucosal immune system subject to microbial regulation in the context of intestinal development during health and disease.
34.2.2.1
T-Cell Subsets
GF animals exhibit gross abnormalities in the development of primary and lymphoid architecture—including the GALT, spleen, and thymus—and harbor decreased frequencies of CD4 + and CD8 + intestinal T-cell subsets as well as reduced numbers of intraepithelial lymphocytes. The T-cell subsets present are characterized by altered cytokine polarization and a lack of the oligoclonal diversity of αβ T-cell receptor (TCR) that is otherwise typical of the SPF environment. In each case, introduction of either murine or human microbiota from healthy individuals to GF hosts at any time during development normalizes these gross morphological and cellular defects. The human symbiont, Bacteroides fragilis (B. fragilis) , expresses polysaccharide A (PSA) with the capacity to induce and expand IL-10-producing regulatory T (Treg) cells while concomitantly limiting Th17 responses. Gnotobiotic mice colonized with B. fragilis , via PSA expression, protect mice from experimental colitis by Helicobacter hepaticus . Given that Tregs are both a major source of IL-10 and capable of recognizing commensal-derived antigens to support tolerance to intestinal microbes it is not surprising that many commensal species, such as Clostridium spp. and others, promote regulatory T-cell development and function in gnotobiotic conditions when introduced at various times during early life and adulthood.
Segmented filamentous bacteria (SFB), a Gram-positive anaerobic species that colonizes the terminal ileum of mice, exerts robust immunomodulatory effects when introduced in GF animals of any age by promoting the accumulation of both Th17 and Th1 cells in the small intestine and drives the production of Immunoglobuin A (IgA). Consistent with enhanced production of Interleukin 17 (IL-17), gnotobiotic mice colonized with SFB confer enhanced protection compared to GF animals during infection with Citrobacter rodentium infection.
34.2.2.2
B Cells
GF animals experience normal B-cell maturation but exhibit general defects in the production of IgA and IgG1 antibodies both systemically and within mucosal compartments that can be normalized by conventionalization with healthy microbiota. IgA specific for commensals plays an important role in the compartmentalization of intestinal bacteria and are produced with the help of intestinal dendritic cells that sample commensals associated with the epithelium and interact with T and B cells in the Peyer’s patches. IgA + B cells migrate to the intestinal lamina propria to secrete IgA that is subsequently transcytosed across epithelial cells preventing adhesion of commensal bacteria to epithelial surfaces. The induction of IgA production by B cells in the small intestine during the course of microbial conventionalization is thought to reflect the restoration of ILFs in the small intestine.
34.2.2.3
Innate Lymphoid Cells
Innate lymphoid cells (ILC) are a newly emergent lymphoid subclass characterized by the lack of either B- or T-cell receptors but retain cytotoxic and immunomodulatory capacity. Group 1 ILCs (which includes Natural Killer cells) are indirectly hyperactivated in GF animals through the release of inflammatory signals derived from nonmucosal mononuclear phagocytes that are normally restricted by the presence of commensal bacteria. In addition, GF animals exhibit decreased expression of interleukin 22 (IL-22), a multifunctional cytokine that promotes production of antimicrobial peptides, enhances epithelial regeneration, increases mucus production, and regulates wound repair and is produced by several immune lineages, most prominently Group 3 ILCs, phenotypically characterized as Nkp46 + and RAR-related orphan receptor-γt (RORγt) + . Dysregulation of IL-22 expression can be normalized by conventionalization with healthy microbiota at any time during development, though the means by which this happens remains controversial.
34.2.2.4
Epithelial Cells
IECs, which line the gut and form a physical barrier between the luminal contents and the underlying cells of the immune system, have altered patterns of microvilli formation and decreased rates of cell turnover in GF animals compared to those raised under conventional settings. Epithelial barrier function is enhanced by the presence of commensal bacteria based on the observation that GF animals exhibit impaired production of antimicrobial peptides, particularly by Paneth cells in the small intestine compared to SPF animals. Colonization of GF neonates and adults with healthy microbiota restores expression of the antimicrobial peptide, RegIIIγ, a secreted C-type lectin through both direct mechanisms and indirectly through increased IL-22 production by Group 3 ILCs. Given RegIIIγ specifically targets Gram-positive bacteria, it is intriguing that gnotobiotic mice harboring the Gram-negative commensal organism B. thetaiotaomicron but not the Gram-positive microbe Bifidobacterium longum that induces RegIIIγ expression, it is tempting to speculate that symbiotic bacteria can tailor host immune responses to promote preferentially their survival. Commensal populations can indirectly influence epithelial cell immune function by inducing resistin-like molecule β (RELMβ) in goblet cells via the modulation of intestinal macrophage responses and major histocompatibility complex (MHC) class II expression through the induction of interferon γ (IFNγ). Transcriptome analyses of GF and SPF animals also reveal substantial gene expression differences within IEC populations including altered expression of Interleukin 25 (IL-25), transforming growth factor-β (TGF-β), B-cell activating factors (BAFFs), the proliferative factor APRIL, thymic stromal lymphopoietin (TSLP), and the chemokine CXCL16. Many of these changes that are under the influence of the microbiota occur through epigenetic modifications. The functional consequence of this dysregulation needs to be further explored.
34.2.3
Temporally Restricted Modulation of the Immune System by Commensal Microbes: Defining the “Window of Opportunity”
At birth, the infant gut exists largely as an aerobic environment, after which facultative aerobes including Escherichia and Enterococcus colonize and promote an anaerobic environment enabling an ecological succession of obligate anaerobes including Firmicutes and Bacteroides spp. This unique and dynamic environment is associated with developmental events occurring during early infancy and presumably can exert temporally constrained effects on immune surveillance capacity of the emergent mucosal immune system. In recent years, experimental evidence has supported the existence of specific periods of life in which specific immune subsets are permissive to microbial training, but after which mechanisms arise that prevent the microbial influence on these same subsets. It follows that improper education of these immune populations may persist to adulthood altering host response to environmental stimuli encountered later in life with potentially deleterious consequences.
34.2.3.1
Invariant Natural Killer T Cells
Invariant natural killer T (iNKT) cells are characterized by an invariant TCR-α chain coupled with a limited repertoire of TCR-β chains that recognize both endogeneous and exogeneous (including bacterial) lipid antigens presented by the nonpolymorphic MHC class I-like molecule, CD1d. In the absence of microbes, iNKT cell frequency is mildly decreased in most peripheral tissues including the spleen and liver and iNKT cells exhibit hyporesponsiveness to lipid antigen stimulation that can be restored by introducing specific bacteria bearing CD1d-restricted lipid antigens. In contrast, specific mucosal sites including the colon and lung, but not the small intestine, contain elevated numbers of iNKT cells in GF animals versus SPF animals regardless of genetic background, correlating with exaggerated inflammatory responses in iNKT-dependent experimental models of inflammatory bowel disease (IBD) and allergic asthma, respectively. Colonization of GF animals with healthy microbes prior to birth but not after weaning restores iNKT cell frequency in both the colon and lung to that of SPF animals. Furthermore, this resolution of iNKT cell frequency by the formerly GF host in response to colonization also reverses their susceptibility to colitis and allergic asthma, whereas the inability of these same commensal populations to resolve iNKT frequency by introducing them too late during development results in host responses similar to naïve GF animals despite harboring microbial levels equivalent to that of an SPF animal. This study represents the first documented evidence of a temporal window during which iNKT expansion within two mucosal organs (colon, lung) are permissive to microbial signals but after which these same populations are no longer modulated by commensals. The accumulation of iNKT cells in the colon of GF animals is associated with elevated levels of the chemokine, CXCL16, in the serum and mucosal tissues whose expression is normalized by conventionalization at birth but not after weaning. Neutralization of CXCL16 in GF animals during early life restores iNKT cell frequency to that of SPF animals. Interestingly, the absence of microbes alters the epigenetic state of the cxcl16 locus in bulk colonic IEC populations suggesting that commensal populations can modulate the chromatin architecture of these populations in a temporally restricted manner. In a separate study, the introduction of a human commensal B. fragilis in GF animals also restored iNKT frequency in the colon, but not the lung, to that of SPF animals corresponding with its colonization restriction to the intestinal tract. The ability of B. fragilis to resolve iNKT frequency is dependent on the production of microbial lipids as mutant strains harboring a deletion of a rate-limiting enzyme within the glycosphingolipid pathway fail to resolve iNKT cell frequencies in the colon using gnotobiotic mice harboring a B. fragilis mutant that lacks the ability to generate CD1d-activating sphingolipids. Mice that are colonized with mutant B. fragilis exhibit elevated responses to the experiment IBD model oxazolone colitis compared to cohorts colonized with wild type B. fragilis. Taken together, these studies reveal two nonmutually exclusive mechanisms by which modulation of host chemokine production to commensals or CD1d-restricted responses to bacterial lipid antigens can limit iNKT cell frequency during a restricted period of life during development that, if not corrected, leads to poor clinical outcomes associated with IBD and asthma in response to environmental triggers of inflammation during adulthood.
34.2.3.2
Immunoglobulin E Production
Whereas IgA and IgG1 levels are decreased in GF animals, in contrast, GF animals generate elevated levels of serum IgE once they reach postweaning age (~ 30 days) due to enhanced rates of B cell IgE isotype class switching in Peyer’s patches (PP) and MLNs. Hyperproduction of IgE is associated with exaggerated responses to orally induced systemic anaphylaxis partially mediated by interleukin 4 (IL-4) production by CD4 + T cells in PPs and MLNs. Colonization of GF animals with healthy microbiota at various times from birth to 4 weeks but not thereafter resolves IgE levels in adults to that of SPF animals defining a “window of opportunity” in which IgE can be modulated by commensals but after which IgE levels are no longer responsive to the microbiota. Interestingly, IgE production in adult mice is not only modulated by the time of initial exposure to microbes but also to the relative diversity of bacterial composition as gnotobiotic mice harboring 1 or 2 bacterial species retain hyper-IgE production compared to SPF counterparts.
34.3
Durable Consequences of Early Life Perturbation of the Microbiota in the Context of Human Disease
The nature of the interactions of the microbiota and the host immune system during early life development is increasingly being recognized as an important developmental window that, when closed, may result in potentially persistent immune abnormalities. In addition to the examples discussed above, evidence is emerging that suggest a prevalent but temporally restricted role of the microbiota on immune maturation in the context of lung disease, psoriasis, infection, and cancer. Collectively, these studies provide mechanistic explanations for the physiology underlying a phenomenon termed the “hygiene hypothesis” or “microbial hypothesis,” which proposes that allergic diseases may be “prevented by infection in early childhood, transmitted by unhygienic contact with older siblings”.
In support of this concept, children exposed to agricultural environments have a decreased risk for the development of allergic disease. Moreover, mechanisms exist to allow vertical transmission of these protective mechanisms as exposure to farms during pregnancy can confer protection against asthma in a mother’s offspring. It has been hypothesized that these protective responses that are enriched in farming communities may be related to the increased microbial diversity within these individuals compared to the population as a whole. Conversely, decreasing the complexity of the microbiota, particularly during early life, can be associated with poor clinical outcomes during later adolescence and adulthood. Perturbing the microbiota, such as exposure to antibiotics, is associated with persistent changes in microbial composition in both adults and infants. The use of antibiotics during the first 6 months of life is associated with increased susceptibility to allergy and asthma at 6 years of age and increased incidence of wheezing and eczema at 8 years of age, controlling for respiratory infections during infancy. Altering the composition or diversity of the microbiota through early life antibiotic usage or cesarean section has also be implicated in the risk for obesity and type 2 diabetes in later life though the mechanistic basis for these observations require further study.
The causes of complex disorders like IBD have increasingly been recognized to involve interactions between underlying genetic susceptibility and environmental and lifestyle factors. Genetic risk factors for both Crohn’s disease (CD) and ulcerative colitis (UC) include loci associated with genes involved in epithelial barrier maintenance as well as responses by both the innate and adaptive immune system. Furthermore, numerous studies have shown that both CD and UC are associated with reduced complexity of the commensal microbiota and shifts to a dysbiotic state though which of these changes are causative or reflective of a disease state remain elusive.
Studies like these discussed above support an indirect association between perturbation of microbial composition during early life and development of disease in later life and epidemiological evidence exist suggesting exposure to antibiotics in childhood, and especially during the first year of life, is associated with an increased risk for development of IBD. However, there remains a need to identify specific tractable markers that measure how these early life events impact immune development in order facilitate better monitoring and tailored therapy to a complex disease like IBD. A good example of this strategy is investigated in a recent clinical study of children with eosinophilic esophagitis (EoE), which mouse models suggest is an iNKT cell- mediated disease. Retrospective data suggest that patients with EoE display increased markers of iNKT cell frequency and activity in serum when exposed to antibiotics in early life. Moreover, dietary intervention to treat EoE resolved expression of iNKT-associated markers only in cohorts of patients that clinically responded successfully to therapy.
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