Recent advances in immune-mediated disease research have provided a considerable body of proof revealing the importance of the early gut microbiome for neonatal immune system development and disease pathogenesis [see Ref. (1) for a review]. The drastic increase of allergies and other immune-mediated diseases in industrialized countries has been hypothesized to be a result of deficiencies in the exposure to microbial organisms and their products, resulting in impaired immune system development, a concept first introduced as the hygiene hypothesis (2, 3). Pioneer work has identified the first 6 months after birth as a “window of opportunity” (4–7) during which contact with specific microbe-associated molecular patterns (MAMPs) triggers a cascade of reactions crucial for infant gut maturation (8–10). Disrupting early gut community succession may lead to dysbiosis, a state of ecological imbalance ensuing when the community loses key taxa, diversity, and/or metabolic capacity. This state can lead to a reduction of colonization resistance, allowing for a subsequent bloom in opportunistic pathogens [(11); for a definition of relevant ecological concepts refer to Table 1]. Concomitantly, microbial dysbiosis during infancy may also lead to health-related consequences in the neonatal stage or later in life. Preterm neonates can develop necrotizing colitis (NEC), a life-threatening disease strongly associated with microbial dysbiosis (12). Infants may also experience an elevated risk of developing inflammatory diseases such as asthma and allergies (13, 14), type 1 diabetes (15, 16), celiac disease (17), inflammatory bowel disease (18, 19), and obesity (20, 21) when exposed to a microbial dysbiosis early in life. Thus, studying the patterns of microbiome assembly and how disturbances to this process reflect in the developing immune system is of utmost importance to understand the origin of human diseases responsible for enormous health and economic burden to societies.

The infant gut microbiome is a complex ecosystem involving a great number and diversity of members (e.g. bacteria, phages, fungi, viruses, protozoans) that interact in a spatially and temporally structured environment (26–28). The neonatal gut microbiota can be considered a complex adaptive system in which both low-level local interactions and selection mechanisms combine to create high-level patterns (22). Complex adaptive systems are non-linear (output not proportional to the input, thus impeding predictability) in that they are heavily influenced by stochastic temporal events that result in a plethora of variable outcomes (22). The infant gut microbiome supports a set of emergent properties contributing to host physiology, including nervous, metabolic, and immune development (29–31), as well as tissue differentiation (32, 33). The emergent properties of a complex adaptive system are considered to be supported by combinations of taxonomic and/or functional diversity, as well as key taxonomic and/or functional groups, both of which insure community resilience (22), and increase the difficulty of attributing a cause–effect relationship to unique features or groups. Therefore, including community ecology theory to study the temporal dynamics of the infant gut microbiome has the potential to provide key information about its influence on the host immune system maturation.

Until 2 years of age, the human infant microbiome remains highly heterogeneous and lacks stability (34), being influenced by temporally structured environmental factors such as (1) maternal factors (35–37), (2) birth (38–41), (3) neonatal nutrition (27, 42, 43), and (4) other non-temporally structured factors, such as antibiotic treatments (41, 44, 45). The initial intestinal bacterial community composition of vaginally born infants involves higher levels of a multitude of bacterial groups (e.g., Atopobium, Bacteroides, Clostridium, Escherichia coli, Streptococcus spp. and Prevotella), while the community of infants born by C-section is dominated by skin-related taxa including Staphylococcus spp. (38). Key bacterial groups are also transferred to the infant by breastfeeding: Bifidobacterium and Lactobacillus (46–48). The multiple studies that have shown how intestinal dysbiosis can lead to detrimental immune-mediated outcomes (e.g., asthma, allergies, NEC, etc.) [see Ref. (30) for a review] suggest that the human immune system relies on an evolutionary conserved temporally structured succession of microbiome assembly. Unraveling the links between the temporal mosaics of the gut microbiome (structured succession patterns) with the emergent properties of this ecosystem (e.g., taxonomic and functional diversity, resilience, etc.) is key to improve our understanding of the importance of the infant microbiome for the development of the immune system.

The successful identification of the mechanisms linking the infant gut microbiome and immune development depends on our capacity to disentangle the relative effects of multiple factors (host genetics, environmental factors), key actors (e.g., Bacteroidetes, Bifidobacterium, etc.) and their interactions. Resilience, the ability of a system to adjust its activity to retain its basic functionality after a disturbance, is a crucial property of complex adaptive systems (49) and could be a key characteristic protecting the infant gut microbiome from reaching a dysbiotic state. Here, we review the recent findings on the links between infant gut microbiota and immune system maturation. Our review highlights the reliance of the neonate immune system development on a complex set of host-specific, environmental, temporal, and self-organizing characteristics of the infant gut microbiome. We propose that future studies should consider multi-level dynamics of the infant gut microbial community by disentangling the ecosystem reliance on (1) temporally structured patterns of alpha- and beta-diversity, both taxonomic and functional; (2) keystone species or microbial groups; and (3) inter-kingdom interactions. This will require a conceptual framework based on the understanding that the infant gut harbors a complex and diverse set of microbial species interacting in a temporally structured, multi-level, and non-linear network. Rightfully recognizing these structural characteristics has the potential to enable the identification of disturbance thresholds threatening the healthy development of the infant gut microbiome and its role in immune system training.

Multiple studies and comprehensive reviews discuss how the maturation of the immune system relies on the exposure to MAMPs (50–52). Here, we discuss the recent findings demonstrating that the efficiency of microbial exposure in immune system training can be age dependent, suggesting the importance of microbial composition and infant gut microbiome temporal succession patterns.

The gastrointestinal tract is already anatomically and functionally developed at birth in full-term infants, yet important aspects of its maturation occur postnatally and depend on exogenous stimulations with microbial cells, metabolites, hormones, growth factors, and antigens (53, 54). Recent studies in murine models have revealed that several aspects of immune development are more permissive to microbial-mediated changes during early life, and that certain microbial taxa are crucial in these interactions. For instance, oral administration of Bifidobacterium breve was effective in inducing proliferation of FoxP3-positive regulatory T cells (FoxP3⁺ Tregs) only if administered during the pre-weaning stage in mice (55). This age-dependent promotion of an important tolerogenic immune cell was also shown to be species specific, thereby suggesting that the tolerogenic gut immune response may have adapted to respond to specific—and important—bacterial taxa. Bifidobacterium species and subspecies are dominant members of the infant gut microbiome (56) and are strong modulators of the immune response (57). Their role as keystone taxa of the infant gut is proposed later in this review. Another microbial species that cause an age-dependent immune effect is the Helicobacter pylori, which ameliorated airway hyperresponsiveness more effectively when administered before weaning in two relevant mouse models (58), although it remains unclear if and when this bacterium colonizes the infant gastrointestinal tract.

While age-dependent modulation of the host’s immune response can be attributed to specific microbial taxa, most studies point to global changes in the microbial community (diversity shifts, and metabolites of poly-microbial origin) as drivers of immune development. Cahenzli et al. (59) showed that regulation of IgE responses and amelioration of antigen-induced oral anaphylaxis is dependent upon increased microbial diversity during early life. Their work thus suggests that there may be a diversity threshold necessary for proper maturation of these Th2 immune mechanisms. Furthermore, several other studies have demonstrated the immune consequences of the disruption of the early-life gut microbial community using antibiotics. Antibiotics induce drastic compositional and diversity shifts that lead to changes in crucial immune functions, including Treg proliferation (60, 61), IgE response (60, 62), Th-17 response (61, 63), and basophil-mediated Th2-cell responses (62). Given the influence exerted by these immune functions on widespread tissues and systems, it is not surprising that antibiotic-induced immune alterations during early life in animal models aggravate autoimmune diabetes (61, 64), allergic lung inflammation (60, 62, 63), inflammatory chronic colitis (65), and obesity (20, 21).

Early-life immune development is also reliant on the actions of a group of bacterial metabolites known as short-chain fatty acids (SCFAs). These compounds are direct by-products of bacterial colonic fermentation and are produced at very high rates (66). Acetate, propionate, and butyrate are the SCFAs produced in highest concentrations in the human gut, and are rapidly taken up by the gut epithelium through passive and active transport mechanisms (67). SCFAs are essential energy sources for colonocytes cells in the mammalian gut, and are precursors for gluconeogenesis, liponeogenesis, and protein and cholesterol synthesis (68). Among many of their immune functions [reviewed in Ref. (66)], SCFAs have been shown to induce extrathymic proliferation of Foxp3⁺ T cells (68–70), which orchestrate peripheral tolerance in mucosal tissues. This critical immune function of SCFA has been shown to be relevant for the offspring even if exposure occurred before birth. Oral administration of acetate during pregnancy was sufficient for the priming of FoxP3⁺ Treg cells and preventing allergic airway inflammation in the adult offspring (36), suggesting that in utero exposure to maternal gut microbial metabolites contributes to the development of immune functions in the airways of the offspring.

In addition to interactions with the developing immune system, a recent study by Kim et al. (71) suggests that the early gut microbiome confers colonization resistance through the production of bacterial metabolites resulting from age-dependent colonization with key bacterial taxa. Clostridial species from Clostridium clusters IV and XIVa, which increase in abundance with age, induced colonization resistance to intestinal mouse pathogens Salmonella enterica subsp. typhimurium and Citrobacter rodentium. Interestingly, the conferred mechanism of resistance is unrelated to immune adaptors MyD88 and TRIF, and independent of B and T cell function. The settlement of Clostridia in the gut of GF mice was also greatly reduced by the absence of neonatal bacteria, which may help explain the increased susceptibility of newborns and young infants to these GI infections.

Collectively, these studies constitute compelling evidence that key taxa, microbial community diversity, and bacterial metabolites constitute modulatory triggers of host immune function maturation. Although considerable research effort has been made, a great deal of the age-dependent processes through which microbial exposure drives immune system development remains to be identified. The infant gut microbiome temporal succession patterns, driven by birth, weaning, and introduction of solid foods, match marked changes in host immune function (72, 73). Therefore, future studies designed during these events, such as human longitudinal cohorts, hold great potential to improve our understanding of the dynamics at play.

Succession in ecology is defined as the pattern of changes in a community after a disturbance or after the opening of a new patch to colonization (74). Correspondingly, succession in the infant gut microbiome starts with the arrival of pioneer species that transform the gut habitat and enable the settlement of first succession species. The identity of the infant gut pioneer and first succession species is influenced by factors such as maternal factors (e.g., body weight and stress) (35–37), delivery mode (38–41), and type of milk consumption [(27, 42); Figure 1]. The temporal structure of these environmental factors contributes to the identity and dynamics of the infant gut microbiome and plays a role in the immune system training.

The impact of early-life dysbiosis on the risk of developing several human diseases has led to the hypothesis that there is a critical window during which changes in the gut microbiome are most influential in immune development. During this “window of opportunity,” the infant gut harbors a highly variable and increasingly diverse microbial community of low resilience, which renders it easily disrupted by disturbances such as antibiotic treatments (41). During this period of time, a loss of diversity or change in community composition has the potential to disrupt the development of certain aspects of neonate immune system and to promote a bloom of pathogens, thus increasing the risk of developing immune-mediated and infectious diseases. However, it remains unclear if community diversity per se represents a robust indicator of infant gut microbiome disruption, especially since (1) there could a threshold to be crossed for the gut ecosystem to suffer a significant loss of function; and (2) diversity as a diagnostic tool provides no information on the gut microbial community composition or functional properties.

Many studies have argued that a loss of community diversity could indicate a disruption of the natural infant gut microbiome community. After birth, both the taxonomic and functional diversity of the infant bacterial microbiome have been shown to increase (88). Life-threatening diseases such as NEC have been suggested to occur as an effect of disruption of the natural succession in the infant gut microbiome after antibiotic treatment (120, 121), lowering community diversity and creating an opportunity for other bacterial groups (e.g., Gammaproteobacteria) to dominate the normal bacterial community (122, 123). At that stage, a loss of community diversity can also hinder the training of the immune system by reducing its ability to recognize commensal bacteria [see Ref. (52) for a review]. Recent studies have confirmed that a significant loss in gut microbial diversity is indicative of an increased risk of developing autoimmune diseases (80, 124). In addition, a loss of diversity can promote a long-term increase in IgE levels, which has been suggested to trigger immune-mediated disorders in mice (59).

However, it remains to be determined if the link between the development of immune diseases and the loss of microbial diversity is caused by a reduction of microbial species alone or, more precisely, by a loss of key taxonomic or functional microbial groups essential to the development of the infant immune system. The work of Arrieta et al. (13) on 319 infants in a longitudinal cohort, showed no significant relationship between fecal microbial alpha-diversity and the risk of developing asthma. Yet, four bacterial taxa (Faecalibacterium, Lachnospira, Rothia, and Veillonella), fecal acetate and deconjugated bile acids were significantly altered in babies at risk of asthma. By contrast, Kostic et al. (15) identified that a significant reduction in infant gut community alpha-diversity is a characteristic condition of the T1D state in a cohort of 33 infants predisposed to type 1 diabetes. This loss in alpha-diversity was combined with an alteration of the metabolic pathways and microbial community phylogenetic structure (15). These studies suggest that both subtle and global changes in community composition may lead to immune impairment and disease development, and that functional dysbiosis can occur independently of significant changes in community alpha-diversity.

Community alpha-diversity may also not be a reliable indicator across all human populations given its geographic variability (27). In a study comparing European to Burkina Faso children, De Filippo et al. (125) showed that the latter group had a greater gut microbial diversity and shift in community composition, potentially associated with their high fiber diet. However, other lifestyle factors and environmental exposures may also explain these differences. In addition, bacterial alpha-diversity fluctuates significantly during the first year of life, making it an unreliable ecosystem measurement unless studies are strictly age- and population matched. Further, an opposite relationship between alpha-diversity and health status occurs during the first weeks of life, where lower alpha-diversity and a predominance of a few subspecies of Bifidobacterium longum is associated with better growth (126).

Another factor that is rarely taken into account when assessing microbiome alpha-diversity is the impact of other non-bacterial microbes. In a unique study targeting both infant gut bacterial and fungal communities, Fujimura et al. (14) showed that infant gut bacterial alpha-diversity increased with time while the fungal alpha-diversity decreased in reciprocal correlation. This finding suggests that microbial diversity per se might naturally fluctuate depending on the targeted organism and that currently unexplored inter-kingdom gut microbial associations may influence these dynamics. Most interestingly, their work demonstrated that the fungal beta-diversity better predicted atopy risk than bacterial beta-diversity. Therefore, fluctuations in infant gut fungal community composition could play a role in influencing infant’s susceptibility to childhood allergies and asthma.

The increase in both taxonomic and functional diversity of the infant bacterial gut microbiome in the few months after birth appears to be associated with multiple aspects of the immune system development, providing further evidence that the immune system relies on a temporally structured succession of the gut microbiome. However, the infant gut microbial diversity per se might not be an indicator conveying enough information to be considered as a diagnostic tool. Notwithstanding, studies to date do suggest that the training of the immune system relies on a particular pattern of microbial diversity increasing from birth until 3 years old, and that disrupting this pattern can increase the risk of developing immune-mediated disorders. Future research disentangling the relative impact of species richness, community taxonomic, and functional composition on the retention of infant gut ecosystem emergent properties (e.g., infant immune system development) will provide key information for the development of diagnostic tools.

In community ecology, the concept of a keystone species or group of species is described as an actor of a community that is so important to its organization and diversity that losing it provokes a massive cascade of extinctions and loss of ecosystem function (23, 24, 127). In other words, a keystone species has a remarkable impact in relation to its abundance (128). In an ecosystem, keystone species can belong to any trophic levels, from low-level species providing the resources on which a plethora of other species depends, to high-level species applying top-down regulation on the community. Keystone taxa of the infant gut microbiome contribute significantly to the ecosystem by (1) contributing to the establishment of other species; (2) by producing important metabolites including SCFAs (e.g., butyrate) that trigger local trophic cascades; (3) by improving ecosystem resistance against invading pathogenic species; and (4) by aiding in sustaining a balanced symbiosis with the host, which will in turn favor the stability of the microbial ecosystem. Because of the high inter-individual [i.e., Ref. (7)] and temporal (27, 34, 39, 40) variability of the infant gut ecosystem, identifying keystone taxa is a great challenge. Here, we discuss the potential for Bifidobacterium and Bacteroides to be keystone taxa and their role on infant immune system training.

At its beginning, complexity theory suggested that ecosystems exhibiting a higher complexity were more stable when sustaining disturbances such as species loss (167, 168). However, mathematical model simulations of food webs led to the proposal that instead of focusing on the stability of individual populations within an ecosystem, a better comprehension of complex systems could be gained from studying emergent properties such as productivity, resilience, and biomass (169, 170). From this point, studies have employed multiple properties to characterize ecosystems including species richness, taxonomic composition, functional profile, the level of interactions between species of the ecosystem, and the strength of these interactions. This transition in community ecology theory mirrors the improvement of our comprehension of complex ecosystems shifting from a singular to a multi-level perspective.

In this review, we advocate that the infant gut microbiome should be considered as a complex adaptive system crucial to the maintenance of various emergent properties (e.g., infant immune system training). These ecosystem properties are hardly attributable to a single group, instead they seem to rely on a temporally structured pattern of bacterial diversity increase after birth and the succession of particular keystone groups. The properties of complex adaptive systems highlight the great challenges faced by studies of the infant gut microbiome: a system far-from equilibrium dynamics, characterized by permanent novelty and incessant adaptation, dispersed multi-level interactions, and the absence of a global controller (171). The emergent properties of this ecosystem highlight the necessity of prospective, longitudinal infant gut microbiome studies, both taxonomic and functional, which will eventually allow us to identify the critical points at which this system loses its emergent properties and reaches a state of dysbiosis, impeding adequate immune system development. In addition, there is a need to disentangle the influence of loss of taxonomic and functional diversity, as well as of shifts in keystone taxa on immune system training and subsequent disease development. From past studies, we now understand that the maturation of the immune system relies on a temporally structured dynamic, starting in utero with maternal effects, influenced by environmental factors (delivery mode, type of milk consumption, and solid foods) and host biology, and depending heavily on auto-correlated local interactions between microbial groups. Further understanding of this complex adaptive system will also require (1) sampling a variety of geographically distinct human populations, (2) carrying out longitudinal cohorts that sample numerous times during the first 12 months, and (3) combining amplicon-based surveys with functional assays, such as metagenomics and metabolomics.

Another important influence in gut microbiome composition that remains vastly unexplored is the role of non-bacterial microorganisms. The role of the virome, the collection of viruses colonizing the host, has been previously explored in adult animals. Similarly to the bacteriome, the virome strongly interacts with the host immune system, with both positive and negative consequences for host health [see Ref. (172–174) for reviews]. However, it remains unknown what role the virome has during early-life immune development. Further, fungi, protozoans, and helminths, which are traditionally excluded from culture- and non-culture-based studies, are important and immunomodulatory members of the gut microbiome, albeit in smaller proportions than bacteria. Nonetheless, it was recently shown that fungi species are present at much higher diversity in the first months of life, compared to later months, and that this change in diversity inversely correlates with bacterial diversity [(14); Figure 2]. Future studies directed at exploring inter-kingdom gut microbial associations during early life and how these associations influence the host will provide a more global understanding of the microbial triggers influencing immune system development.

Eventually, the identification of the critical events and factors that influence microbiome resilience and function will enable the development of effective interventions aimed at maintaining and/or improving immune system development and disease prevention. Although an astounding amount of work has been carried out to understand the reliance of the immune system on the infant gut microbiome, much remains to be elucidated on the particular mechanisms responsible for this training. Improvements in our understanding will arise from continuing multidisciplinary joint efforts between immunologists, microbiologists, clinicians, bioinformaticians, and ecologists.

M-CA formulated the concept for this review, and IL-L wrote the first draft. Both authors co-wrote and revised the entire review article.

The authors declare that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.