In addition to protection provided by maternal factors, the neonatal immune system is quickly developing to help eliminate acute pathogenic threats. The first two waves of hematopoiesis from the yolk sac and fetal liver bring about a slew of innate effectors consisting of macrophages, mast cells, Natural Killer (NK) cells, innate B-1 cells, dendritic cells, innate lymphoid cells (ILCs), γδ T-cells, and mucosal-associated invariant T (MAIT) cells (39). Many of these cells undergo imprinting in early life that is dependent on the microbial environment.

Unconventional T-cell subsets which include MR1-restricted MAIT cells, CD1d-restricted NKT cells and CD1a/c/d-restricted γδ T-cells have been shown to be regulated by early-life microbial exposure and metabolites (40). By using the PLZF transcription factor to identify innate-like lymphocytes in mice, it was demonstrated that early-life antibiotic usage can decrease the frequency of PLZF-expressing cells in the thymus, a phenomenon that persists into adulthood (41). Antibiotics given in adult life did not impact thymic PLZF⁺ populations suggesting that the time frame for microbial influence is specific to early life (41). Likewise, a separate group showed that colonization of neonatal germ-free mice with riboflavin-synthesizing microbes induced MAIT cell development at barrier sites, but colonization of adult mice was not sufficient to generate these cells (42). The degree to which dysfunctional development of unconventional T-cells may influence disease is an area of ongoing research. A study in germ-free mice demonstrated accumulation of invariant NK T (iNKT) cells in colonic lamina propria and lung that resulted in increased morbidity for inducible models of IBD and allergic asthma (43). Conventional microbiota colonization of neonatal mice but not adult mice was sufficient to prevent iNKT accumulation and pathology (43) A different study found neonatal mice treated with broad-spectrum antibiotics developed an increased susceptibility for imiquimod-induced psoriasis mediated by γδ+ T-cells (44). Interestingly, mice treated with the same antibiotic regimen as adults demonstrated ameliorated psoriasis in comparison to the control group (44).

In addition to unconventional T-cells, unconventional B-cells also play a role in neonatal defense. B-1 cells are innate-like B-cells that develop in the neonatal period and express a highly cross-reactive BCR repertoire which binds both self and microbial antigens. In mice, exposure to bacterial antigens during a specific neonatal period is important for establishing clonal diversity in the GlcNAc-reactive B-1 repertoire (45). Whether this repertoire contributes to homeostatic mucosal IgA responses that can impact microbial colonization in adulthood is yet to be determined (46). In human neonates, polyreactive innate-like B-cells were shown to generate antibodies that are capable of binding commensal bacteria and may facilitate intestinal colonization (47).

Similar to B-1 cells, early-life CD8⁺ T-cells derived from fetal liver have been recognized as possessing distinct transcriptional profiles that favor low TCR diversity and increased rates of homeostatic proliferation (48, 49). This comes at the expense of memory formation but allows for an enhanced innate-like ability to respond broadly to inflammatory signaling. Historically, these cells were thought to be immature, but newer literature suggests these cells are intentionally poised to meet the needs of the neonate and contribute to rapid effector response in adulthood (49, 50). The microbiota also contributes to signaling that allows for protective immunity by CD8⁺ T-cells. In the postnatal period, the dominant conventional dendritic cells are of the cDC1 subtype (51). Upon exposure to microbiota at birth, monocytes and macrophages release myeloid-derived tumor necrosis factor (TNF) (52). This signal modifies fatty acid metabolism in neonatal pre-cDC1 and promotes maturation and increased IL-12p40 production that is important for promoting protective CD8⁺ T-cell responses (52). Maternal antibiotic treatment delays cDC1 differentiation and maintains cDC1s in a tolerogenic IL-10 producing state resulting in increased neonatal susceptibility to pathogens such as Listeria monocytogenes (52).

Altogether, early-life microbiota exposure is important for the maturation and function of cells that provide protective immunity to the developing neonate and disruption in microbial exposure may leave infants vulnerable to infection.

While strong effector responses are critical in protecting the vulnerable infant during development, a hyper-reactive immune response is equally dangerous and thus tolerogenic adaptive immunity quickly follows. Toward the end of gestational development, progenitors from the fetal liver and bone marrow seed the thymus and generate conventional CD4⁺ T-cells (39). In early life, the CD4⁺ T-cell response is skewed toward a Th2 and FOXP3-expressing Treg phenotype (53). Though the contribution of thymic-derived (tTregs) and peripherally-induced (pTregs) is debated due to lack of differentiating phenotypic surface markers, it is clear that both populations are important for suppressing effector T-cell responses to microbiota. Therefore, understanding the mechanisms by which these cells develop may provide important clues as to how tolerance is generated in the neonatal period.

In the intestine, RORγt⁺ pTregs have been shown to mediate tolerance toward commensal microbes and are selectively decreased in germ-free and antibiotic-treated mice (34, 54). Recently discovered, the APCs responsible for RORγt⁺ pTreg differentiation during the weaning reaction were found to display features of both medullary thymic epithelial cells and dendritic cells. These “thetis cells” expressing transcription factors RORγt and AIRE, were found to be enriched between 1-3 weeks of age and then quickly declined. Temporal ablation of MHCII on RORγt expressing APCs in early life but not adulthood resulted in a decline in pTregs (55). pTregs generated during this critical window imprint a durable microbiota-specific T-cell response which persists into adulthood, again suggesting a tightly regulated developmental window with long-lasting implications for the adult immune response (55). In addition to traditional intestinal APCs, a separate study indicated the importance of the intestinal epithelium in promoting pTreg induction while dampening commensal-specific Th17 responses (56). It was shown that microbiota-sensitive epithelial histone deacetylase (HDAC3) is capable of regulating MHCII expression on inter-epithelial cells (IECs). Loss of either epithelial HDAC3 or MHCII on IECs resulted in a significant reduction of commensal-specific Tregs and increase in commensal-specific Th17 cells (56).

Thymic-derived tTregs are also important for neonatal tolerance against microbes colonizing the skin and may also play a role in intestinal microbial tolerance (57, 58). By performing high-throughput TCR sequencing on mice with limited TCR repertoires, it was shown that tTregs constitute the majority of Tregs in intestinal organs (58). How intestinal microbial antigens may influence selection of T-cells in the thymus is still an outstanding question in the field, but recent work suggests that in early life, CX3CR1⁺ APCs can migrate from the intestine to the thymus to influence thymic microbiota-specific T-cell development (59). This area of research warrants further study because it is still unclear how signaling events downstream of interactions between developing T-cells and their cognate peptide-MHC ligands determine the threshold of TCR reactivity and T-cell responses toward commensal microbes later in life.

Anti-inflammatory signaling molecules are also important for maintaining tolerance in early life and can be influenced by microbial colonization. Transforming growth factor-β (TGF-β) is a pleiotropic cytokine which regulates IgA production and promotes the suppressor functions of both pTregs and tTregs (60, 61). TGF-β1 levels are reduced in the colon of germ-free mice as compared to Clostridia-colonized mice suggesting a role for microbiota in regulating its expression (62). Recent literature has identified the SCFA, butyrate, as the primary metabolite which acts to increase TGF-β production in intestinal epithelial cells via HDAC class 1 inhibition (63). Another example of microbial contribution to tolerance is the ability of the Bacteroides genus to enhance pTreg function and IL-10 expression by signaling through toll-like receptor 2 (TLR2) via polysaccharide A (PSA) (64).

While this section primarily focuses on Tregs during development, other cell types such as myeloid-derived suppressor cells (MDSCs) and B regulatory cells can also contribute to immune tolerance in the neonatal period and future studies may demonstrate how their development is impacted by early-life microbiota.

Given that this phase of development is centered around tolerance, vaccines administered at birth generally have reduced efficacy. Additionally, immunization at birth with diphtheria, pertussis and tetanus (DTaP) was associated with a significant reduction in antibody response to subsequent boosters compared with a routine immunization schedule suggesting that early neonatal vaccination might impair the response later in life (65). This being the case, the majority of vaccinations are administered after the neonatal period around 2 months of age, but with the substantial burden of respiratory syncytial virus (RSV)-associated hospitalizations, there is increasing demand for earlier and more effective intervention. Solutions being investigated include utilizing adjuvants that better engage the infant immune system and encouraging maternal vaccination to provide passive protection (66, 67). Another consideration for vaccination efficacy, is the tolerogenic response to environmental microbial exposures that may preclude vaccination efforts (68, 69). For example, the lowest efficacy rates of the Bacille Calmette-Guérin (BCG) vaccine against tuberculosis is seen in equatorial regions where exposure to environmental mycobacteria is common. A study investigating this phenomenon in mice found that they could circumvent this immunosuppressive response by changing the route of vaccine administration from oral to pulmonary (70).

Both protective and tolerogenic immune responses are important for healthy neonatal development but their imbalance may increase infection susceptibility or increase propensity to develop allergy and autoimmune disease. Alterations to microbiota exposure or composition in the neonatal period can have long-lasting consequences that impact immunity in adult life. Importantly, these disruptions cannot always be resolved once the critical window of opportunity closes. Multiple factors may result in deviation from the carefully constructed developmental pathway, so what options are there to restore early-life microbiota in the face of dysbiosis?