Microbiota-mediated mechanisms of mucosal immunity across the lifespan

Abstract

The microbiota plays a fundamental role in regulating homeostasis and inflammation across the barrier surfaces of the body. The gut is a unique bioreactor where the high concentration of microbes, microbial and dietary metabolites, microbial PAMPs, immune cells, stroma, and neurons all form a complex, highly interactive, and precisely regulated system. The mucosal immune system in the gut has profound local and systemic effects, influencing both health and disease. A critical period of immune imprinting occurs early in life, shaped by the neonatal microbiota and nutrition, to influence immune development and long-term disease susceptibility. Microbiota-derived metabolites play crucial roles in immune modulation, influencing epithelial integrity, oral tolerance, and inflammatory responses. This review explores the interactions between microbiota and the mucosal immune system from infancy to adulthood, highlighting the impact on health and disease. We also discuss therapeutic interventions, including microbiota-derived molecules, dietary metabolites, and emerging microbiome-based co-therapies.

Introduction

The microbiota serves as a key regulator of homeostasis and inflammation at body barrier surfaces. The gut is a complex bioreactor where microbes, microbial and dietary metabolites, immune cells, and neuronal networks interact dynamically. The gut mucosal immune system influences homeostasis and disease states in multiple organs across the body. The increasing incidence of inflammatory, autoimmune, metabolic, and neoplastic diseases all parallel the change in environmental, dietary, and lifestyle factors that affect the microbiome and host microbiome and host immunity¹.

The gastrointestinal barrier composed of epithelial cells and mucus separates microbes from direct contact with the host immune system. This surface acts as a firewall, allowing the assimilation of nutrients and microbial metabolites while preventing the spread of microbes throughout the body. Microbiota-produced metabolites, dietary components, and microbial cell constituents play a myriad of roles in immune regulation and health. The microbiota is crucial for development, immune imprinting, and shaping the immune system early in life. Bacteria, fungi, and viruses can also collectively fuel chronic inflammation and influence the pathophysiology of multiple diseases. Here, we will discuss key components of the mucosal immune system and its interactions with the microbiota early and later in life, that are crucial for maintaining the delicate balance at barrier surfaces in health and disease, along with recent scientific and therapeutic advancements in this field.

Main Text

Neonatal microbiota and immune regulation in early life

The interaction between the immune system and microbiota begins at birth and has a critical role in development. Immune imprinting that has a long-term impact on the immune response to pathogens, microbiota, dietary or self-antigens^2–4. After birth, the newborn’s gut is progressively populated by myeloid cells and B cells from the bone marrow and lymphocytes from the thymus ^5–7. These newly immigrated immune cells are functionally shaped by the unique gut bacteria and nutrients available in the neonatal gut, which drive the generation and expansion of effector T cells, including regulatory T cells (Tregs), IgA-producing plasma cells, innate lymphoid cells, as well as the formation of isolated lymphoid follicles (ILFs). Transcriptome profiling of human tissue T cells revealed that effector and memory T cells in infants are primarily localized to the gut and lung, suggesting that these mucosal surfaces provide critical initial sources of antigens, some likely from commensal bacteria ⁸. This highlights the importance of acquiring and developing a diverse and functionally adept gut microbiota during the early neonatal period to facilitate appropriate effector and memory T cells. In support of this, human neonatal antibiotic usage is associated with an increased risk of immune-related diseases in later life, including asthma, allergy, obesity, cancer, and inflammatory bowel disease^9–11

Microbiota influence on prenatal immune system development

While maternal health issues may, in rare cases, permit translocation of invasive gut bacteria to the fetus, live bacteria are highly unlikely to be present in the fetus during a normal pregnancy. Indeed, the concept of a placenta/fetal microbiota has been challenged by recent comprehensive studies indicating that prior sequencing-based reports of microbial presence in the human placenta likely resulted from contamination during DNA extraction and sequencing¹². However, microbial products from the mother can cross the placenta and may play a role in priming the fetal immune system. For example, in both human and mice, maternal microbiota-derived metabolites, including tryptophan-derivatives, are detected in the fetus intestine and suggested to play a role in facilitating fetal brain and intestinal development^13,14. In humans, memory CD4⁺ T cells have been identified in the fetal intestine and shown to respond to bacterial antigens likely originating from maternal microbiota^15,16, suggesting that maternal microbial antigens may play a role in the generation of long-term antigen-specific T cells. In mice, transient colonization of pregnant germ-free dams with Escherichia coli resulted in a greater number of intestinal IL-22 producing Group 3 innate lymphoid cells (ILC3s) and a transcriptionally more mature intestinal epithelium in the offspring¹⁷. Similarly, mild maternal enteric infection during pregnancy epigenetically imprinted the fetal intestine in an IL-6–dependent manner, enhancing resistance to enteric pathogens but increasing susceptibility to gut inflammation postnatally¹⁸.

Development of the neonatal gut microbiota

After birth, the newborn’s gut microenvironment vastly differs from that in adults, with slightly lower acidity, a relatively higher level of oxygen ¹⁹, and maternal milk as the main source of nutrients. The acidity, oxygen content, as well as nutrients change as the gut matures, this leads to a dynamic shift in the specific bacterial species that colonize and dominate in the neonatal gut over time ^20,21. Immediately after birth, via vaginal delivery, the slightly higher oxygen content in the newborn’s gut favors the colonization of facultative anaerobes, such as Enterobacteriaceae, Lactobacillaceae, and Streptococceae. Through aerobic respiration, these early colonizers play an important role in purging oxygen from the newborn’s gut, thus allowing subsequent colonization by strict anaerobes, including beneficial Bifidobacteriaceae, which also thrive on maternal milk oligosaccharides²⁰ (Box1). Later, with the introduction of solid foods with more complex carbohydrates and the loss of maternal milk antibodies, weaning induces a significant shift in the microbiota characterized by an overall increase in the abundance of bacteria that are capable of fermenting complex fibers, including Bacteroides and Clostridia^21,22. The earliest bacterial colonizers in the intestine, however, are different in newborns who were born via Cesarean section, who acquire microbes from the mother’s skin, including Staphylococcus species, or the hospital environment, leading to lower microbial diversity initially and reduced levels of beneficial Bifidobacteria and Bacteroides²³.

BOX 1. Microbial diversity and community stability increase during the post-weaning period, accompanied by shifts in specific bacterial and fungal species, and bacteriophages.

Innate immunity plays a pivotal role in the immune system maturation early in life. In infants, increased abundance of Bacteroides have been associated with a lower risk of autoimmune diseases, possibly due to the presence of structurally distinct LPS, which inhibits innate immune signaling and promotes endotoxin tolerance ²⁴. Microbial-associated molecular patterns (MAMPs) such as LPS and peptidoglycans engage pattern recognition receptors like TLRs and NOD-like receptors on innate immune cells driving macrophage maturation and function^25–29, while short-chain fatty acids from neonatal Bifidobacteria promote an anti-inflammatory M2-like phenotype via HDAC inhibition and IL-10 upregulation ³⁰, supporting immune tolerance in early life.

Finely orchestrated parallel maturation of the neonatal gut microenvironment and microbiota persists until about three years of age in humans when the balance between microbiota and the host is finally established. This process is disrupted in preterm infants who were born with underdeveloped intestines. The maturation of the gut microbiota in severely preterm infants (less than 25 weeks of gestation) tends to be severely delayed in part due to the relatively higher oxygen content, underdeveloped gut epithelium, and limited nutrients due to difficulties of breastfeeding². This typically leads to overabundance of opportunistic Enterobacteriaceae, including E. coli and Klebsiella pneumoniae, and delayed and/or reduced colonization of beneficial Bifidobacteriaceae and Bacteroidetes. In this case, high concentrations of proinflammatory lipopolysaccharide (LPS) from Gram-negative Enterobacteriaceae potentially induces massive inflammation in the premature gut, leading to life-threatening necrotizing enterocolitis (NEC)^31–33. In addition, over-bloom of E. coli and K. pneumoniae may also increase the risk of late-onset sepsis (LOS) in preterm infants³⁴.

These findings underscore the critical role of early microbial colonization in shaping gut health and immune development, with disruptions in this process posing significant risks to long-term health outcomes in preterm infants.

The weaning reaction

The neonatal gut microbiota prior to weaning is limited in its diversity, abundance, and colonization resistance to enteric bacterial pathogens (Box1). Gnotobiotic mice with pre-weaning gut microbiota showed poor induction of commensal bacteria-specific IgA antibodies and increased susceptibility to enteric pathogens^35,36. Maternal milk antibodies, including IgA and IgG, shape the development of the gut microbiota in part by keeping opportunistic Enterobacteriaceae in check prior to weaning and thereby limit proinflammatory IL-17-producing T cells that would be otherwise induced by Enterobacteriaceae ^37–39. Weaning is characterized by a reduction in maternal milk antibodies and introduction of solid foods, and massive influx of dietary antigens and an increasing abundance of a more diverse gut microbiota. This triggers a vigorous but transient immune response known as “weaning reaction”. In mice, the gut microbiota during weaning induces a transient surge of IFNγ and TNFα -producing T cells, which wane within one week; this is coupled with an expansion of RORγt⁺Tregs²² (Figure 1). Strong systemic effects by both bacterial ⁴⁰ and fungal ^41,42 microbiota have been described to occur during this window affecting metabolism and behavior. The regulation and function of IFNγ and TNFα -producing T cells induced during weaning remain unclear. In mice, IFNγ and TNFα synergistically regulate intestinal epithelial cell proliferation and apoptosis in part through modulation of β-catenin pathways ⁴³. Thus, the IFNγ and TNFα induced in the intestine during weaning might play a role in the development of the gut epithelium. RORγt⁺ Tregs promote tolerance to gut bacteria by dampening the development of proinflammatory Th17 helper cells in an antigen-specific manner ^44,45. The mechanisms governing the weaning reaction, however, are still not fully understood. RORγt⁺ antigen-presenting cells (APCs) are a unique cell type that was discovered to be necessary for immune tolerance to gut microbiota through MHCII and prevent spontaneous inflammation^46,47. Recent studies highlight an expanded family of RORγt⁺ APCs, including group 3 innate lymphoid cells (ILC3s), Janus cells, extra-thymic AIRE-expressing cells, Thetis cells, DC-like cells, and Prdm16⁺ APCs, which are essential to instruct the differentiation of microbiota-specific RORγt⁺ Tregs^{48–50 51,52 53 54}. These cells express MHC class II and are thought to facilitate Treg induction though interactions with commensal microbiota and dietary antigens, contributing to the prevention of spontaneous inflammation. Dietary metabolites, including tryptophan-derivatives and retinoic acid, have also been implicated in influencing the development of gut RORγt⁺ Tregs^22,55. The precise roles and heterogeneity of RORγt⁺ APC subsets in early-life immune imprinting continue to be explored, with ongoing discussions about their relative contributions to tolerance. The weaning reaction thus represents a pivotal phase in immune development, establishing tolerance to the rapidly diversifying gut microbiota while restraining inflammatory T cell responses to diverse bacteria and fungi.

Figure 1. Regulation of mucosal immunity circuits by gut microbiota and dietary components.

An interplay between diet and gut microbiota profoundly shape mucosal immunity. This occurs through fermentation of fiber or bile acids, which can directly lead to the recruitment or promotion of Tregs while suppressing Th17 cells. These pathways also impact innate lymphocytes, through the induction of IL-33, and subsequent engagement of a feed-forward circuit with IL-25 production by Tuft cells and IL-13 production by ILC2s. Other microbiota byproducts, such as tryptophan engages the aryl hydrocarbon receptor on immune and non-immune cell types, while succinate is directly sensed Tuft cells to engage in the ILC2 circuit. Finally, direct sensing of fungal or bacteria in the gut by myeloid or stromal cell responses engages circuits with innate lymphocytes. This includes production of IL-1b that engages in a circuit with ILC3s, to induce IL-2 and GM-CSF, that supports an optimal Treg response in the gut.

Oral immune tolerance in the neonatal gut

Increasing evidence suggests that colonization by a diverse gut microbiota during the postnatal period is critical for the establishment of a tolerogenic gut microenvironment necessary to promote immune tolerance to dietary antigens and thereby prevent food allergy and gut inflammation⁵⁶. Recent studies have shed light on the mechanisms of oral tolerance in the neonatal gut. For example, dietary antigens gain access to CD103⁺ conventional DCs (cDCs) in the lamina propria via goblet cell-associated passages (GAPs) or via microfold (M) cells in the villus epithelium. These CD103⁺ cDCs, specifically expressing Indoleamine 2,3-dioxygenase (IDO), migrate to draining mesenteric lymph nodes to induce antigen-specific Tregs that subsequently circulate systemically or to the gut lamina propria, exerting long-term protection against allergic reaction to the antigen^57,58. In addition, a recent mouse study showed that serotonin in the neonatal gut is derived mainly from unique bacteria in the neonatal gut and promotes the differentiation of Tregs in the neonatal gut by inhibition of mTOR activation to facilitate oral tolerance⁵⁹. Human milk oligosaccharides (HMOs) also contribute to the tolerogenic microenvironment in the neonatal gut. They are resistant to digestion by human digestive enzymes and favor the expansion of beneficial gut bacteria, including Bifidobacteria. For example, HMOs promote the expansion of Bifidobacterium infantis, an early colonizer of the neonatal gut, which dampens Th2 and Th17 cells by producing indole-3-lactic acid (ILA)⁶⁰. The tolerance to food antigens is initiated early in life and is dependent on the induction of peripheral regulatory T cells (pTregs) specific for dietary antigens. The induction of these food-specific pTregs has been linked to both classical dendritic cell (DC) subsets, cDC1 and cDC2^57,61. Using the LIPSTIC proximity labeling method, recent study described a sequence of events in dietary-antigen presentation in which RORγt⁺ APCs played an earlier role, likely via an early signal that primes naïve T cells toward a regulatory phenotype, followed by later engagement of cDCs⁶². Indeed, a series of recent studies revealed a non-redundant role of RORγt⁺ APCs in instructing the differentiation of dietary antigen-specific RORγt⁺ Tregs, as well as in restraining Th2 cell-mediated allergic inflammation^{51,54,63–65}. Further, in the context of helminth infections, these tolerogenic pathways are disrupted by reducing tolerogenic migratory cDC1s and RORγt⁺ APCs, while inducing cDC2s, which averted the presentation of dietary antigen. Therefore, the establishment of immune tolerance to dietary antigens involves diverse mechanisms, dependent on the gut microbiota, serotonin, human milk oligosaccharides, sIgA and specific populations of APCs, which promote food-specific regulatory T cells and long-term protection against food allergies and inflammation.

Altogether these studies underscore early life as a critical developmental window in which microbial colonization and immune system education intersect to shape long-term health trajectory.

The intestinal epithelial barrier: mechanism of protection, regeneration and cell death.

The intestinal epithelium is a key barrier responsible for vital processes, including renewal, response to injury, induction of tolerance, absorption of nutrients, and production of antimicrobial peptides and mucus, which regulate microbial access and host-microbial interactions.

Mucus production by goblet cells plays a crucial role in maintaining intestinal epithelial integrity and protection creating a dynamic barrier⁶⁶. MUC2 mucin form a mucus barrier in the small intestine and the colon, with an inner layer that is anchored to goblet cells to protect against bacterial infiltration. The outer colon mucus layer is the habitat for commensal microbiota where they get in contact with the host mucin glycans at the outer surface of the inner mucus layer⁶⁶. Disruptions in this protective barrier contribute to the pathophysiology of both Crohn’s disease (CD) and ulcerative colitis (UC), where bacteria penetrate the normally impenetrable inner mucus layer⁶⁷ (Figure 2).

Figure 2. Mechanisms of Cell Death.

Homeostatic apoptosis of IEC as part of natural cellular turnover engenders immune tolerance and anti-inflammation in the gut facilitated by CD103+ cDC1 sampling of apoptotic IEC, their migration to mLN and expansion of immunosuppressive FoxP3+ Tregs. Cell death of IECs is dysregulated in IBD due to increased expression of necroptosis mediators (RIPK3, MLK), pyroptosis effectors (GSDMD/GSDMDB/GSDMDE) or defects in cell death/autophagy regulators (XIAP, XBP1, ATG16L1). IBD patients exhibit patient-to-patient heterogeneity, showing either excessive apoptosis or necroptosis in the intestinal epithelium. IBD risk loci also impact repair pathways exacerbating pathology and curtailing mucosal healing (PTGER4, ERRFI1, HNF4A).

Mucus layer maturation is a gradual process, requiring weeks for full development post-colonization⁶⁸. A recent study has demonstrated that constitutive activation of autophagy via Beclin 1 enables the production of a thicker and less penetrable mucus layer by reducing endoplasmic reticulum (ER) stress. ER-stress-mediated control of mucus secretion is dependent on the microbiota and NOD2⁶⁹. Paneth cells contribute to mucosal defense by secreting antimicrobial peptides (AMPs) such as defensins and lysozyme ⁷⁰. AMPs are entrenched within the mucus layer and complement the protective function of the mucus by directly targeting and killing pathogens, preventing the breach of the epithelial barrier. Recent advances in machine learning have enabled large-scale identification of AMPs from microbial genomes, revealing novel candidates with potent antibacterial properties⁷¹. These findings not only expand our understanding of gut barrier function but also provide a valuable resource for antibiotic discovery. Finally, SIgA within the enteric mucus layer provide additional protection against enteric bacteria and fungi via high and low affinity binding to these microbes and/or their toxins^{72 73}. All of the above-described mechanisms of protection are synchronized and depend on the key process of epithelial cell renewal and death.

Epithelial death and renewal are in root processes crucial for the maintenance of the intestinal homeostasis. Central modes of programmed cell death, apoptosis, necroptosis, and pyroptosis have each been reported to occur in the intestinal epithelium. Apoptosis maintains the function of the intestinal epithelium under steady-state homeostatic conditions and contributes to immune tolerance and suppression of inflammation^74,75. Genetic deletion of apoptosis executioner caspases 3 and 7 in mice, however, has highlighted non-apoptotic extrusion of intestinal epithelial cells (IECs) to also contribute to normal IEC turnover and intestinal development⁷⁶ (Figure 2). Compared to apoptosis, both necroptosis and pyroptosis are inherently inflammatory. Necroptosis is orchestrated by the receptor-interacting protein kinase RIPK3, which phosphorylates mixed lineage kinase domain-like protein (MLKL) leading to its plasma membrane translocation and oligomerization to cause cell rupture⁷⁷. Pyroptosis is an effector response of canonical and noncanonical inflammasomes orchestrated by inflammatory caspase mediated cleavage of the cell death executor gasdermin D (GSDMD), a member of the gasdermin family of six paralogous genes⁷⁸, and downstream ninjurin 1 (NINJ1), which is ultimately responsible for plasma membrane rupture⁷⁹. The N-terminal fragment of cleaved GSDMD forms pores in the plasma membrane that mediate the release of inflammatory IL-1b from living cells^80,81. Both necroptosis and pyroptosis result in the release of damage associated molecular patterns (DAMPs) such as HMGB1 (high mobility group box 1), which trigger immune activation and inflammation^78,82.

Excessive cell death within the intestinal epithelium is characteristic of IBD where these processes are extensively studied. IEC death in IBD has been regarded as a consequence of the inflammation that furthers epithelial damage and perpetuates a cycle of cell death and immune activation, although recent understanding of the pathways of cell death have illustrated that cell death itself can drive inflammation. The nature of inflammation likely differs depending on the mode of cell death, although this remains to be carefully defined. Patient-to-patient heterogeneity has been reported in the prevailing form of cell death in both UC and Crohn’s CD such that either apoptosis or necroptosis dominates⁸³. Dysregulated cell death in the intestinal epithelium is linked to the severity of inflammation in IBD⁸⁴. Excessive IEC apoptosis reverses its homeostatic benefits leading to barrier defects, increased intestinal permeability and heightened susceptibility to IBD¹. Increased expression of RIPK3 and MLKL has been observed in intestinal tissues from patients with IBD. Dysregulation of necroptosis in IBD is thought to result from aberrant activation of RIPK1, RIPK3 and MLKL, and has been linked to the excessive inflammatory responses in IBD and may underlie the chronic inflammation, epithelial barrier breakdown and immune dysregulation associated with this disease¹¹. Although the contribution of pyroptosis by gasdermin proteins to chronic inflammatory diseases in humans such as IBD has been unclear^85,86, polymorphisms in GSDMB and GSDMA are strongly associated with susceptibility to IBD^77,87. GSDMD, GSDMB and GSDME expression are also increased in intestinal mucosal biopsies from IBD patients^80,88,89. Increased levels of N-terminal GSDME in inflamed colonic mucosa of CD patients has suggested that pyroptosis contributes to the mucosal inflammation in CD⁸⁹.

Several genetic factors influence susceptibility to cell death in the intestinal epithelium and predispose individuals to IBD. These genetic factors often disrupt the regulation of apoptosis or impair the balance between epithelial cell survival and death, contributing to epithelial barrier dysfunction and inflammation. Mutations in the nucleotide-binding oligomerization domain containing protein 2 (NOD2), which impair bacterial sensing and promote excessive apoptosis of intestinal epithelial cells are strongly associated with CD. Variants in ATG16L1 and IRGM, linked to both UC and CD, impair autophagy critical for clearing damaged organelles and controlling bacterial infections and can lead to uncontrolled apoptosis in response to cellular stress or microbial invasion⁹⁰. Mutations in XBP1, which is involved in the unfolded protein response (UPR) lead to endoplasmic reticulum (ER) stress and increased epithelial cell death, which in turn exacerbate and contribute to IBD pathology⁹¹. Mutations in X-linked inhibitor of apoptosis (XIAP) have been associated with very early onset IBD, which can often be more severe compared to typical forms of IBD and typically affects males⁹².

Wound healing and mucosal restitution are critical processes in maintaining and restoring the integrity of the intestinal epithelium following IEC cell death. Repair of the intestinal epithelium entails multiple processes relying on growth factors and restitutive signals that drive crypt stem cell proliferation and differentiation including WNT-b-catenin signaling, epidermal growth factor (EGF) and transforming growth factor-β (TGF-b) vital for promoting epithelial cell migration⁹³, restoration of extracellular matrix integrity by supporting fibroblast and myofibroblast activities⁹⁴, and resolution of inflammation through the actions of anti-inflammatory IL-10 and TFG-βb^95,96. Sampling of apoptotic cells by intestinal mononuclear phagocytes orchestrates distinct programs of immune suppression including the induction of negative regulators targeting multiple inflammatory innate immune pathways as well as the induction of FOXP3⁺ regulatory CD4 T cells². The secretome of apoptotic cells contains metabolites that induce specific programs of wound healing, cell proliferation and suppression of inflammation⁹⁷. Supernatants from pyroptotic cells contain signatures of genes involved in migration, cellular proliferation and wound healing, as well as oxylipins and metabolites linked to wound healing⁹⁸. IEC cell death can thus serve as a messenger signaling repair of the intestinal epithelium. Defects in key healing pathways have been identified in both CD and UC contribute to the chronic and relapsing nature of the condition. Risk loci associated with impaired epithelial repair include the E-type prostaglandin receptor encoding PTGER4 required for restitution, ERRGI1 a negative regulator of epidermal growth factor (EGF) receptor signaling, and HNF4A which orchestrates intestinal epithelial repair and IEC differentiation⁹⁹.

In summary, epithelial cell homeostasis, sIgA secretion and transportation across the epithelium, mucus and antimicrobial peptide production, are key for intestinal health. While the precise determinants for cell death heterogeneity in the intestine during diseases are not defined, extensive molecular characterization of the pathways mediating cell death, and the appreciation that cell death itself can elicit inflammation, has shifted focus to defining the potential triggers of cell death and gaining an understanding of the factors that prevent its resolution. The picture that emerges is that a combination of increased susceptibility to cell death and defective repair may thus underlie IBD pathology.

Immune regulation by bacteria-produced and dietary metabolites.

Gut commensal bacteria play a pivotal role in maintenance of the immune homeostasis via direct interaction with host structural and immune cells, or via the production of metabolites with immunomodulatory or other bioactive properties. The decrease in abundance of several bacterial groups such as Faecalibacterium prausnitzii, Christensenellaceae, Collinsella, Akkermansia,Clostridium spp., Roseburia, and Ruminococcus and a concomitant increase in Enterobacteriaceae, Fusobacterium, Ruminococcus, Streptococcus, Enterococcus, Campylobacter, Gammaproteobacteria, have been associated with multiple diseases ranging from IBD and colorectal cancer to metabolic and neurological disorders ^100–103. F. prausnitzii which is among the most abundant species in the human gut^104,105 is a key producer of the SCFA^{105 106}. Butyrate and other SCFAs such as acetic acid, propionate, valerate are synthesized via gut microbial fermentation of dietary fibers and amino acids¹⁰⁷, and play a key role in sustaining epithelial homeostasis, regulation innate and adaptive immune responses, and suppressing inflammation¹⁰⁷ (Figure 1). Propionate¹⁰⁸ and butyrate^109,110 influence intestinal and peripheral regulatory T (Treg) cell differentiation via the GPR43 fatty acid receptor, and limit inflammatory responses in the gut¹¹¹. Extrathymic pathway dependent on CNS1, an intronic enhancer required for extrathymic but not for the thymic differentiation of T regs¹⁰⁸ while antibiotics targeting butyrate-producing species suppresses signaling through the butyrate sensor peroxisome proliferator-activated receptor γ(PPAR γ), preventing the expansion of pathogenic Escherichia and Salmonella species¹¹². A reduction in SCFA-producing bacteria such as F. prausnitzii and R. hominis has been observed in IBD^113–115 that further leads to reduction in butyrate, propionate, and valerate/isovalerate during IBD dysbiosis¹¹⁴. Several SCFA have direct antibacterial and antifungal properties via intracellular acidification of bacterial and fungal cells^{116 117}.

Bile acids are another group of metabolites shown to exert profound effects on host immunity (Figure 1). Primary bile acids drive the enrichment of bacteria expressing bile acid metabolism genes, which plays a role in bile acid tolerance in newborns¹¹⁸. Primary bile acids such as cholic acid and chenodeoxycholic acid undergo bacterial modification to give rise to a diverse range of secondary bile acids, that influence metabolism and immune responses^119,120. Some secondary bile acids exert their effects on immunity via nuclear receptors such as the farnesoid X receptor (FXR), retinoid acid-related orphan receptor γ (RORγ), vitamin D receptor, Takeda G protein-coupled receptor 5 (TGR5) and nuclear receptor 4A1 (NR4A1)¹²¹ 3-oxolithocholic acid (3-oxoLCA), derived from lithocholic acid (LCA), inhibits intestinal T helper 17 (Th17) cell differentiation via binding to RORγ, while isoalloLCA enhances Treg cell differentiation by binding to NR4A1¹²². A consortia of 238 bacterial isolates (belonging to 12 genera) from the human gut produce 3-oxoLCA and isoLCA to negatively regulate Th17 responses through conversion of LCA into 3-oxoLCA and isoLCA¹²³. These findings indicate that such secondary bile acids can contribute to IBD by altering the Th17—IL-17 signaling axis¹²³.

Alterations in bile acid composition have also been shown to regulate gut RORγ⁺ Treg cell homeostasis via a vitamin D receptor-dependent mechanism¹²⁴. In patients with UC, reduced levels of secondary bile acids such as LCA have been described, while LCA supplementation reduced intestinal inflammation in a TGR5-dependent manner¹²⁵.

New bile-acid conjugations with amino acids (phenylalanocholic acid, tyrosocholic acid, and leucocholic acid) have also been identified through analysis of the metabolome in germ-free and specific pathogen-free mice¹²⁶. Several additional amino acid-conjugated bile acids have since been discovered using reverse metabolomics approaches¹²⁷. These metabolites are produced by bacteria belonging to the Bifidobacterium, Clostridium, and Enterococcus genera and are increased in patients with CD¹²⁷.

The gut microbiota metabolizes tryptophan to various indole metabolites, including indole propionic acid and indole lactic acid¹²⁸, which affect intestinal inflammation through aryl hydrocarbon receptor (AHR) signaling pathways¹²⁸. A new class of genotoxic metabolites derived from tryptophan was recently discovered, termed indolimines, which can induce DNA damage¹²⁹, These and other nutrient-derived genotoxic metabolites, such as glucosinolates may feed into this pathway^130,131. The presence of a microbiota-encoded decarboxylase gene (aspartate aminotransferase) facilitates the biosynthesis of this class of metabolites. Furthermore, colonization by indolimine-producing gut bacteria, such as Morganella morganii, increased intestinal permeability¹²⁹. Other bacterial species, including Clostridium perfringens and Clostridium ramosum, also produced small molecules that were able to induce DNA damage in cell-free assays and expression of the double-strand break marker γ-H2AX in epithelial cells¹²⁹. DNA damage induced by these metabolites was caused by a mechanism that was independent of colibactin-mediated DNA damage driven by selected E. coli strains¹³², indicating the presence of multiple bacteria-associated genotoxic metabolites. Conversely, a consortia of commensal bacterial strains isolated from healthy human stool was recently developed to counteract potentially harmful microbial species such as Klebsiella pneumoniae, by competitively depleting gluconate¹³³. In mice, this consortia reduced inflammation and restored intestinal colonization resistance without affecting non-Enterobacteriaceae species, highlighting a new mechanism of nutritional competition between beneficial commensals and intestinal pathobionts. Notably, the expansion of gluconate-utilizing Enterobacteriaceae is associated with intestinal inflammation in a cohort of IBD patients while gluconate-metabolizing bacteria correlated with gastrointestinal health¹³³.

CX3CR1⁺ mononuclear phagocytes (MNPs) detect bacterial and fungal cells and their metabolites in the gut. Under the steady state, CX3CR1⁺ MNPs regulate immune balance by restricting T helper cell expansion and supporting Treg cell development, whereas microbiota disruption leads to inflammatory T helper cell responses¹³⁴. CX3CR1⁺ MNPs secrete IL-1β in response to propanediol dehydratase (PduC)-mediated propionate produced by adherent-invasive E. coli to aggravate intestinal inflammation¹³⁵. Furthermore, CX3CR1⁺ MNPs play a critical role in initiating innate¹³⁶ and adaptive¹³⁷ immune responses to intestinal fungi via a Syk-dependent mechanism in the gut. Colonization of mice with bacterial strains from IBD patients aggravates colitis via the induction of Th17 cell and the suppression of protective Treg cell responses¹³⁸. Conversely, SFB-specific intestinal Th17 cells developed an anti-inflammatory phenotype characterized by IL-10 secretion and driven by the transcription factor c-MAF¹³⁹. SFB-induced Th17 cells further protected mice from obesity and metabolic syndrome, by regulating lipid absorption across intestinal epithelium¹⁴⁰. Dietary sugar led to a loss of these protective Th17 cells fostering the outgrowth of Faecalibaculum rodentium that displaced Th17-inducing SFB¹⁴⁰.

In addition to T cells, B cells play a crucial role in maintenance of the gut homeostasis by producing secretory IgA (sIgA), the most abundant immunoglobulin in the gastrointestinal tract (Figure 1). sIgA prevents direct contact between intestinal microbes and intestinal epithelial cells¹⁴¹. Polyreactive sIgA binds with low affinity to a broad range of gut bacteria¹⁴² or target fungi with a high affinity for fungal hyphae^73,143.

Intestinal ILCs are robustly primed to interface with the external environment prior to or soon after birth, and this is supported with substantive experimental evidence demonstrating key roles for these cell types in shaping the outcomes of host-microbe interactions. ILC2 circuits in the gut are dynamically influenced by diet, and may be further modified by resident microbiota and associated IL-33 responses to shape the magnitude of tissue-protective versus inflammatory type-2 allergic responses^144–147. Group 3 innate lymphoid cells (ILC3s), similarly engaged in dynamic cross talk with gut microbiota^148,149. Indeed, the study of the latter pathway revealed a non-redundant role for MHCII⁺ ILC3s, and perhaps other related RORγt⁺ antigen presenting cells, in the induction of microbiota-specific Tregs that co-express RORγt and are essential to drive immune tolerance in the gut^48–50. ILC3s are also critical to preventing the maladaptation of key cytokines networks in the gut.

Altogether these examples underscore the expanding role of various microbial and dietary metabolites in influencing the immunity and states of inflammation in the gut.

Fungi in mucosal immunity and inflammation.

Fungi are ubiquitous in the environment and reside on all body surfaces, including the gastrointestinal tract^{150–161 162}. In the past several years, significant progress has been made in understanding various aspects of antifungal immunity at the mucosal barriers^{162 163}. NETs produced by neutrophils during inflammation are sensed by MICL which acts as a negative regulator of inflammation affecting the outcome of joint inflammation and host response to Aspergillus fumigatus¹⁶⁴. Type I and III interferons, and GM-CSF mediate monocyte and neutrophil crosstalk during A. fumigatus infection in the lungs^165,166. GI colonization of mice with Candida albicans boosts granulopoiesis via IL-6 dependent mechanism, undermining the outcome of inflammation after SARS-CoV-2 infection and leaving a long-lasting mark on granulocyte myeloid progenitors of patients with severe COVID-19¹⁶⁷. Conversely, such innate activation of trained immunity by gut C. albicans colonization, protects against subsequent microbial infections¹⁶⁸.¹⁶⁹ Altogether, these examples highlight the complexity of antifungal immune mechanisms.

IL-17 and IL-22-dependent¹⁷⁰ or independent¹⁷¹ production of antimicrobial peptides limit C. albicans growth or hyphal formation. IL-22 activates regenerative signals on oral epithelial cells trough IL-22RA/IL-10RB to foster the proliferation and repair of IL-17R-expressing epithelial cells that respond to IL17 and mediate antifungal events ¹⁷². In the colon, mucosa-associated fungi induce IL-22 and IL-17 production by T helper cells that in protects against fungal dissemination and reinforces intestinal epithelial function to protect mice against intestinal injury⁴¹. A consortia of gut mucosa-associated fungal species can influence both intestinal barrier function and social behavior via IL-17R signaling on neurons⁴¹. In humans, circulating fungus-reactive memory Th17 cells recognize gastrointestinal C. albicans-derived antigens and cross-react with other fungi in pathologies such as CD or A. fumigatus-triggered allergic airway disease¹⁷³.

In addition to type 17 responses, enhanced type 1 immunity can also increase susceptibility to mucosal fungal infections. Aberrant T cell-driven type 1 mucosal inflammation in autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy (APECED) patients increase susceptibility to C. albicans invasion, while inhibition of interferon-γ or JAK-STAT signaling can prevent oral candidiasis in a mouse model of APECED¹⁷⁴.Gut C. albicans strains with ability to damage macrophages and gut epithelial cells, fuel pre-existing gut inflammation via IL-1R -dependent mechanisms induced by the toxin candidalysin¹. Complement component 5a (C5) ligation of C5aR1 promotes phagocyte effector and metabolic function to mediate fungal clearance during systemic candidiasis in mice, while impaired complement activation correlates with poor outcomes in candidemia patients¹⁷⁵. On the protective side, gut fungal symbionts shape the human IgG and IgA antibody repertoire through CARD9-dependent induction of antifungal antibodies that are protective against C. albicans and C. auris systemic infection in mice¹³⁷. The GI fungal commensal Kazachstania pintolopesii, which resides in the murine stomach, induces type 2 immunity via epithelial cell-produced IL-33 that is activated by the fungus¹⁴⁷ (Figure 1). K. weizmannii isolated from IL-23-deficient mice can outcompete C. albicans, protecting mice from systemic infection¹⁷⁶. Wild mice residing in suburban and urban areas carry K. pintolopesii, which provides protection against intestinal parasites and maintains its presence in the GI niche against other fungi¹⁴⁷. These studies position several intestinal fungi as key inducers of type 17 and type 2 immunity in the gastrointestinal tract. While these responses can be detrimental under certain pathological conditions, they can also be protective under homeostatic conditions, highlighting the dual nature of antifungal immunity at mucosal sites.

Viruses in mucosal immunity and inflammation.

Viruses affect mucosal immunity, inflammation or therapeutic outcomes^177–180 in multiple ways. For example, intestinal murine norovirus aggravates intestinal pathology in mice with a mutation in Atg16L1 leading to a Paneth cells defect, linking viral triggers with intestinal inflammation¹⁸¹.

Bacteriophage including dsDNA Caudovirales, ssDNA phage family Microviridae, and Enterovirus B, are present in the gut of patients with UC and CD¹⁸². In mice, intestinal bacteriophages can aggravate intestinal inflammation and colitis¹⁸³. Perturbations in the intestinal virome and an increase in enterovirus B species, exacerbate intestinal inflammation via nucleic acid sensing by RIG-I, MDA5, or cGAS¹⁷⁸, altogether demonstrating viral and phage-mediated triggers of intestinal immunity.

Parasites and protozoa in mucosal immunity and inflammation.

In response to intestinal helminths tuft cells engage in a feed-forward activation circuit with ILC2s, where subsequent IL-13 production by ILC2s drives Tuft cell hyperplasia, collectively orchestrate type-2 immunity to helminths in the intestines^184–186. This initial activation of Tuft cells can occur through sensing of the metabolite succinate via the succinate receptor, which is produced by intestinal helminths and gut microbiota that encompass both bacteria and protozoa^187–189. If chronically activated, this provokes an altered landscape of antimicrobial peptides, substantial tissue remodeling in the small bowel, and in some cases may protect from intestinal inflammation^188,190,191. These ILC2 circuits in the gut are also dynamically influenced by diet, and may be further modified by resident microbiota and associated IL-33 responses to shape the magnitude of tissue-protective versus inflammatory type-2 allergic responses^144–147. The GI fungal commensal Kazachstania pintolopesii protects mice against intestinal helminths through an IL-33-dependent mechanism¹⁴⁷, demonstrating how the immune response activated by one intestinal eukaryote can protect against another and revealing a level of eukaryotic interspecies interaction in the gut (Figure 1).

Microbiota-based therapeutic opportunities.

Insight into the molecular mechanisms underlying host-microbe interactions has paved the way for precision microbiome therapeutics (PMT)(Figure 3). Newly developed approaches that enable proteome-scale assessment of host exoproteome-microbiome interactions will help to expand the identification of targets for selectively blocking gut pathobionts contributing to inflammatory disease¹⁹². Unique metabolic pathways utilized by pathobionts including propanediol and ethanolamine utilization by Adherent-Invasive E. Coli (AIEC), which are expanded in Crohn’s disease, may serve as therapeutic targets for small molecules or xenobiotic modulation^{135,193 194}. Recent studies reporting the capability of targeted gene editing of E. coli and K. pneumonia (Kp) strains in the mouse gut in situ add to the arsenal of strategies for strain-specific targeting¹⁹⁵. Conversely, new approaches engineering native gut bacteria, which can achieve durable engraftment, enable functional transgenes to serve as therapy targeting inflammatory pathology¹⁹⁶. Gut pathobiont-specific bacteriophages also serve as a therapeutic strategy to target and reduce specific strains contributing to inflammatory disease. Pre-clinical studies using AIEC-specific bacteriophages confirming the selective targeting of AIEC in vivo and the subsequent reduction of colitis severity in mice support ongoing clinical studies evaluating the potential efficacy in CD¹⁹⁷(and NCT03808103). Similar strategies using Kp-specific phages confirm robust efficacy in reducing intestinal inflammation in pre-clinical models, and first-in-human studies showing efficient phage engraftment highlight the potential for rapid and patient-specific engineering of bacteriophage as PMT in IBD¹⁹⁸.

Figure 3. The intestinal microbiota is a critical regulator of inflammation and anti-tumor immunity.

The intestinal microbiota can enhance immune checkpoint inhibitor (ICI) therapy by translocating to secondary lymphoid organs to enhance T cell activation. Gut microbiota influences inflammatory bowel disease (IBD) severity and treatment response. The microbiota releases an array of metabolites (e.g., SCFA, IPA, inosine, muropeptides, STING agonists) into the circulation to influence inflammation outcomes or to improve T cell activation in lymph nodes and anti-tumor responses in the tumor microenvironment. Beneficial microbial metabolites that promote intestinal healing and impact inflammation outcomes, or promote gut microbiota-dependent enhancement of ICI therapy, can be influenced by dietary interventions (e.g., high-fiber diets) and nutritional supplements/prebiotics (e.g., castalagin).

The intestinal microbiota of patients who respond to ICI therapy can be transplanted via fecal microbiota transfer (FMT) into those previously non-responsive to promote responsiveness and tumor control. High-fiber diets and prebiotics (e.g., castalagin) support beneficial microbes, improving immune regulation. FMT in IBD influences positively dysbiosis, reduce inflammation and has been shown to drive steroid-free remission in UC. Microbiome-targeted interventions offer promising strategies to enhance both cancer immunotherapy and IBD management.

Immune checkpoint inhibitor (ICI) therapy restores the function of suppressed tumor-specific T cells and is a first-line therapy for many types of cancer¹⁹⁹ (Figure 3). However, not all patients respond, and not all responses are durable. The intestinal microbiota is a critical modulator of anti-tumor immunity and ICI responsiveness²⁰⁰. Landmark studies showed that antibiotics use correlated with impaired ICI therapy and that specific gut commensals were associated with improved ICI responsiveness in patients, which was causal in mouse models^{201–207 201,202,206–209 200}. Importantly, the gut microbiota directly promoted ICI therapy in patients in two pilot clinical trials^210,211. The microbiota of patients that had a complete response to ICI therapy was administered to advanced cancer patients that were refractory to ICI therapy. Remarkably, approximately 30% became responsive^210,211. Preclinical studies have shed light on the mechanisms involved (discussed later). Although ICI therapy has provided great benefit, it can also cause immune-related adverse events (irAE)²¹². IrAE occur commonly at barrier tissues such as the intestine and skin and can be caused by the microbiota in preclinical models ^213–215. Further, a small clinical trial (2 patients) showed that patients with refractory ICI therapy-associated colitis receiving an FMT from a healthy donor had complete resolution of clinical symptoms²¹⁶. This was associated with an enrichment in Bifidobacteria in patients, consistent with a report using mouse models of ICI therapy and colitis²¹⁷. These studies demonstrate the importance of the microbiota in modulating the efficacy of ICI therapy and associated irAE, and provide novel targets to improve outcomes for patients.

Microbial and immune mechanisms by which the microbiota promotes therapeutic outcomes.

While FMT is highly effective for treating recurrent Clostridium difficile infection, clinical outcomes in inflammatory diseases such as IBD have been variable²¹⁸ (Figure 3). In IBD, clinical remission has been linked with the presence of specific taxa—including Odoribacter splanchnicus, Fecalibacterium prausnitzii, Eubacterium hallii, and Roseburia inulivorans ^219–221—and recent studies show that lyophilized donor product can achieve clinical remission, supporting the sufficiency of specific taxa ²²². Strain-specific factors shape metabolic function and immune modulation, yet many of the key enzymes responsible for the metabolic targets remain poorly characterized. Emerging technologies capable of resolving individual genomes within complex microbial community ²²³ along with activity-based probes ^224,225 enable potential strategies for in situ identification of strain-specific enzymes. This functional mapping can uncover metabolic pathways to improve PMT and allow direct isolation of microbes based on enzymatic or metabolic activity ²²⁴.

FMT trials indicate that microbiota-mediated responsiveness to ICI therapy in patients correlated with increased DC activation, T cell tumor infiltration and function, and remodeling of the myeloid compartment^210,211. This is consistent with preclinical studies, which showed that the microbiota mediated these effects by translocating from the intestine to prime systemic immunity²²⁶, and via the production of metabolites by bacteria and fungi^{208,227–232}. Microbiota-derived metabolites can also increase suppressive immune ¹⁰⁸, which may play role in negatively regulating the efficacy of ICI therapy²³³. The microbiota can also express molecules (e.g., bacteriophage peptides) that mimic tumor antigens, thereby promoting cross-reactive anti-tumor T cell responses^234–236. In addition, unfavorable commensals can release LPS into the blood to cause chronic, aberrant inflammation that impairs anti-tumor immunity²⁰⁹. These studies provide a foundation to test therapeutic interventions that promote anti-tumor immunity in patients, such as administering commensal-derived metabolites in place of an FMT.

The role of the diet

Both dietary prebiotics and xenobiotics can modulate the therapeutic efficacy of PMT for inflammatory disease. Recent seminal studies demonstrate that fiber type and the host microbiome composition jointly shape distinct immunometabolic response^237–239. In subjects with metabolic syndrome, combining low-fermentable fiber with FMT improves insulin sensitivity ²⁴⁰, but the impact of fiber supplementation with FMT in IBD remains under investigation ^241,242. Pre-clinical mouse studies reveal that fiber regulates microbiome enzymatic function ²⁴³ and that downstream metabolites influence immune cell function ¹⁴⁴, helping to guide ongoing clinical trials. Beyond prebiotics, xenobiotics can also enhance the functional impact of microbial-based therapies. For example, sulfonamide-mediated inhibition of microbial folate synthesis promotes production of anti-inflammatory colipterins in E. coli ²⁴⁴, while sulfasalazine boosts butyrate production by F. prausnitzii ²⁴⁵. Clinical validation of xenobiotic-microbiome interventions in diverse inflammatory disease cohorts with varied diets is warranted.

Diet drastically reshape the gut microbiota, which could be used to alter a patient’s baseline microbiota prior to receiving ICI therapy to promote responsiveness^{144,246–250}. Diets high in fiber optimize anti-tumor immunity and ICI responses, which was associated with improved survival in cancer patients and was recapitulated in mouse models^230,251,252. Sufficient fiber intake enriches for the Ruminococcaceae family and Faecalibacterium genus and correlates with increased intra-tumoral IFNγ-producing CD8⁺ T cells^{201,205,209,251}. Caloric restriction enriches for acetate-producing Bifidobacteria that enhanced anti-tumor immunity in mice^253–255. Omega-3 and a Mediterranean diet correlated with increased ICI responses in cancer patients^252,256, and ketogenic and low-protein diets increases anti-tumor immunity and ICI responses in preclinical models, ^{249,257–259}, but the role of the microbiota in mediating these effects is unclear. Prebiotics can enhance anti-tumor immunity and ICI responses in mice, including the polyphenol castalagin and Ginseng extracts^260,261. Diet can also promote an unfavorable microbiota that accelerates tumor formation. For example, a ‘Western diet’ can enrich for commensals that promote colorectal cancer^262,263. However, a Western diet can counterintuitively promote ICI therapy responsiveness^264,265, highlighting the need for further mechanistic studies. Altogether, diet is an accessible approach to modulate the microbiota and anti-tumor immunity. Future research on individual responses to nutritional interventions that are simple to adhere to are required to rationally utilize diet to enrich for commensals that promote anti-tumor immunity^247,266.

Concluding remarks

The interactions between the host and its commensal microbiota are fundamental to maintaining and promoting host health. Unraveling these complex relationships will provide valuable insights into how commensal microbes shape immune function and inflammation, both in the intestine and at gut-distal sites. A deeper understanding of microbiota assembly in early life and its role in immune system imprinting could unlock lifelong health benefits. Exploring the functional characteristics of the microbiota and harnessing its vast repertoire of bioactive and immunomodulatory molecules may pave the way for novel therapeutic strategies. Investigating how diet and microbial metabolism influence immunity could lead to less invasive, preventive approaches that, alone or in combination with existing immunomodulatory drugs, would open new avenues to control inflammation and other processes underlying many chronic and neoplastic diseases.