Bifidobacterium mechanisms of immune modulation and tolerance

ABSTRACT Bifidobacterium is a widely distributed commensal bacterial genus that displays beneficial pro-homeostatic and anti-inflammatory immunomodulatory properties. Depletion or absence of Bifidobacterium in humans and model organisms is associated with autoimmune responses and impaired immune homeostasis. At the cellular level, Bifidobacterium upregulates suppressive regulatory T cells, maintains intestinal barrier function, modulates dendritic cell and macrophage activity, and dampens intestinal Th2 and Th17 programs. While there has been a large volume of literature characterizing the probiotic properties of various Bifidobacterial species, the likely multifactorial mechanisms underlying these effects remain elusive, in particular, its immune tolerogenic effect. However, recent work has shed light on Bifidobacterium surface structural polysaccharide and protein elements, as well as its metabolic products, as commensal mediators of immune homeostasis. This review aims to discuss several mechanisms Bifidobacterium utilizes for immune modulation as well as their indirect impact on the regulation of gut microbiome structure and function, from structural molecules to produced metabolites. These mechanisms are pertinent to an increasingly networked understanding of immune tolerance and homeostasis in health and disease.


PART I. Overview of Bifidobacterium immune modulatory abilities
Bifidobacterium is a gram-positive, anaerobic, saccharoclastic, non-motile commensal bacterial genus.It contains 10 phylogenetic clusters and has a broad host range within the mammalian gastrointestinal tract. 1,2In vitro culture of Bifidobacteria require highly specific conditions given their requirement for an anaerobic environment and metabolic precussors. 3,4Bifidobacterium exhibits genomic differences that partly reflect the gene acquisition events required to thrive in different host ecological niches.Bifidobacterium tropism and niche adaption underpin its diverse role in host immune regulation.
Bifidobacterium has been implicated as an immunomodulator or biomarker of human disease, serving as both a driver and protector.
Various strains are commonly used as live biotherapeutics 5 and demonstrate beneficial immunomodulatory and anti-inflammatory properties.These include upregulation of suppressive Foxp3+ regulatory T cells (Tregs), 6 improvement of intestinal barrier function, 7 and dampening of intestinal Th2 and Th17 programs. 8On the other hand, absence or reduction of Bifidobacterium species has been implicated in multiple autoimmune and autoinflammatory conditions in humans.For instance, decreased gut levels of several Bifidobacterium species in humans are associated with the microbiome fingerprint of treatmentnaïve Crohn's disease (CD). 9Decreased levels of gut Bifidobacterium infantis are correlated with Guillain-Barré Syndrome in humans. 10Mice treated with B. pseudolongum ATCC25526 and receiving allogeneic heterotopic heart transplants with long-term immunosuppression display improved long-term graft survival and decreased allograft inflammation. 11Contrary to its classical protective role, enrichment of Bifidobacterium is also associated with Parkinson's disease in a meta-analysis of patient gut microbiome data. 12Similarly, Bifidobacterium is enriched in the stool of patients with ulcerative colitis (UC) compared to healthy controls. 13These results suggest that Bifidobacteria possess both multifactorial immunomodulatory mechanisms and diverse strain-mediated immune effects.
Bifidobacteria also modulate immune responses at the level of the gut mucosa.In mice treated with Bifidobacterium adolescentis ATCC15703, the levels of pro-inflammatory cytokines TNFα, IL-6, IL-1β, IL-18, IL-22, and IL-9 in colon homogenates are lower than controls, 14 while antiinflammatory IL-10 and Th2-type cytokines IL-4 and IL-5 are higher.Tregs are also increased in the colons of colitic mice receiving B. adolescentis ATCC15703.Similarly, germ-free mice colonized with Bifidobacterium bifidum strain PRI1 have increased Tregs in the colonic lamina propria. 6his effect is facilitated by colon lamina propria dendritic cells (DCs), which have increased mRNA expression of IL-10, GM-CSF, TGFβ1, Indoleamine 2,3-dioxygenase, PTGS2, and PD-1, as well as the co-stimulatory molecules CD86 and CD40 after treatment with PRI1.In vitro treatment of DCs with PRI1 followed by co-culture with naïve CD4 T cells leads to enhanced Treg induction and IL-10 production. 6ifidobacterium immunomodulatory programs vary in phenotype and intensity at the species and strain levels, including pro-inflammatory effects. 15he immunomodulatory effect of Bifidobacterium was shown to be independent of its phylogeny. 16one marrow-derived DCs co-cultured with Bifidobacterium animalis subsp.lactis 5764 (Bl 5764) display high levels of maturation and costimulatory molecules CD40, CD86, and MHC II in comparison to non-stimulated cells.When pretreated DCs are co-cultured with naïve CD4 T cells, there is increased pro-inflammatory IL-17A and IL-17F production compared to control and groups treated with different bacterial species. 17Co-culture of the THP-1 human monocyte cell line with Bifidobacterium breve strains UCC2003 and JCM7017 results in MyD88-dependent NFκB and TNFα expression compared to untreated controls. 18Murine bone marrow derived macrophages express increased TNFα after co-culture with these B. breve strains, favoring an anti-inflammatory phenotype.While this coculture model provides a controlled environment to investigate potential interactions between Bifidobacterium and immune cells, the findings require careful interpretation and further validation using more sophisticated ex vivo or in vivo models.Strain-specific immunomodulation is likely rooted in niche adaption due to the different mammalian gut microenvironments from which each strain has been isolated.Thus, it is important to understand the properties of specific Bifidobacterium strains when planning for therapeutic application.

PART II. Bifidobacterium modulates immune responses by affecting gut microbiome
Microbial species interact through the metabolites they consume and secrete, a process known as cross-feeding.0][21][22][23][24][25][26] For example, Anaerostipes caccae L1-92 is an important butyrate producer that relies on metabolites produced by Bifidobacterium in order to establish itself in the infant gut. A. caccae L1-92 is unable to grow in monoculture, but co-culture with B. infantis ATCC15697 enables A. caccae L1-92 growth, which utilizes glucose and galactose along with the acetate generated by B. infantis ATCC15697 to produce butyrate. 20 In these co-cultures, Bifidobacterium is necessary for either the establishment of a butyrate producer or for enhancing butyrate production through the metabolism of human milk oligosaccharides (HMOs) into monosaccharides and the production of acetate. 19,21,228][29] Shortchain fatty acids (SCFAs), particularly butyrate, are important for colonic Treg homeostasis. 30,31ingh et al. demonstrated the role of butyrate and niacin in the suppression of colonic inflammation and carcinogenesis through the activation of Gpr109a.This stimulated DCs and macrophages to produce IL-10, leading to enhanced differentiation of Tregs. 32Furthermore, co-culture of F. prausnitzii A2-165 or ATCC 27,768 and B. catenulatum KCTC 3221 results in the reduction of pro-inflammatory cytokines produced by HT-29 human colorectal adenocarcinoma cells and RAW 264.7 murine macrophages in vitro as well as decreased IL-8 in the colons of colitic mouse models. 22These studies indicate the important role of Bifidobacterium metabolism in modulating immune homeostasis directly or via cross-feeding.Furthermore, Bifidobacterium does not possess polyamine biosynthetic machinery for putrescene, a known immunomodulator, but administration of B. animalis subsp.lactis LKM512 increases the concentration of luminal putrescene by sufficiently acidifying the intestine and allowing for activation of polyamine biosynthesis by endogenous gut microbiota. 33rom a cooperativity standpoint, Bifidobacteria can also negatively regulate the presence of proinflammatory metabolites such as trimethylamine N-oxide (TMAO), derived from gut microbiotaproduced choline, which is associated with the development of atherosclerosis. 34Supplementation with B. breve Bb4 as well as B. longum BL1 and BL7 decreases plasma TMAO levels in mice. 35These pathways exemplify the complex interactions between different Bifidobacterium species and the host intestinal environment, including available carbohydrate nutrient sources and other members of the colonic microbiota, and their subsequent downstream effects on host immune responses.
Cross-feeding relationships also exist among various species of Bifidobacterium (Table 1), which subsequently affect metabolic production and immune properties.Different Bifidobacterium  26 This mutualism highlights the intricate relationships between various bacterial species in the gut microbiome and demonstrates that these direct and indirect interactions are all critical for determining the overall environment for immune stimulation versus homeostasis.The diverse applications of Bifidobacterium as a live biotherapeutic are contingent upon an array of strain-specific traits as well as interactions within the host milieu.This encompasses the interplay between host and gut microbiota, metabolic functionalities, adherence to intestinal epithelial cells, resilience to gastric acids and bile, immunomodulatory capacity, and competitive antagonism with pathogenic bacteria.Consequently, the functional dynamics of individual Bifidobacterium strains emerge from a multifaceted interplay of these distinct attributes.

Solid organ transplant
Given its role as a marker and inducer of antiinflammatory and pro-tolerogenic immune effects, Bifidobacterium has been studied in solid organ transplant models.Mice receiving allogeneic heart transplants and Bifidobacterium pseudolongum ATCC25526 gavage along with daily tacrolimus immunosuppression display improved long-term allograft survival and decreased graft inflammation. 36Furthermore, treatment with B. pseudolongum ATCC25526 results in lymph node (LN) architectural changes, increasing the ratio between extracellular matrix glycoproteins laminin α4 and laminin α5, which is associated with tolerance. 37Treatment with Bifidobacteria also promotes intestinal homeostasis following transplantation.In a rat model of liver transplant, prolonged antibiotic use and semi-starvation for 4-5 weeks (associated with decreased ileocecal Bifidobacterium), and supplementation with Bifidobacterium and Lactobacillus-containing probiotic promotes partial restoration of intestinal microflora and improved intestinal barrier function. 38In a separate rat model of liver ischemia reperfusion injury, treatment with Bifidobacterium catenulatum ZYB0401 decreases serum TNFα and liver malondialdehyde and increases liver superoxide dismutase, which is associated with reduced liver injury. 39Rats administered B. longumcontaining probiotic cocktail followed by liver transplant without immunosuppression display increased intestinal Treg cells and TGFβ in the serum and liver, with concomitant decreases in CD4/CD8 T cell ratios and serum IL-2. 40xemplifying the dysbiotic state associated with immunosuppressant treatment, metagenomic analyses of human liver transplant recipients reveal deficits in beneficial Bifidobacteriaceae compared to healthy controls. 41,42Similarly, in renal transplant recipients, the gut microbiome displays a decreased abundance of multiple Bifidobacterium species 43 .However, the presence of the family Bifidobacteriaceae in the gut microbiomes of liver transplant recipients is associated with acute cellular rejection. 44As the family includes a large variety of species and strains that can have distinct immune modulatory properties, it is important to have species-and even strainlevel taxonomic resolution to assist in characterizing the immunomodulatory effects of specific microbiota species and strains (Table 2).

Gliadin-induced enteropathy
Bifidobacteria protect intestinal epithelial cells from damage in a gliadin-induced enteropathy (GIE) model of celiac disease, in which incomplete hydrolysis of dietary proteins leads to small intestinal inflammation, lymphocyte infiltration, villous atrophy, and crypt hyperplasia. 50Specifically, treatment of mice with B. longum CECT 7347 partially suppresses disease by inhibiting the production of inflammatory cytokines and CD4 T cell mediated immune responses.Similar anti-inflammatory effects are observed with B. longum ES1 and B. bifidum ES2 in vitro. 53While B. longum CECT 7347 restores intestinal structure without reversing cellular infiltration, 50 B. longum NCC2705 treatment prevents intraepithelial infiltration of lymphocytes in mice sensitized with gliadin. 52rther, in culture with epithelial cells, B. lactis counteracts the gliadin-induced permeability of intestinal epithelium, inhibited membrane ruffle formation, and protected tight junctions. 54While GIE leads to decreased NFκB and increased TNFα and IL-10 expression, B. longum CECT 7347 treatment restores baseline NFκB and IL-10 levels, but further increases TNFα. 50B. longum CECT 7347 treatment also modulates T cell differentiation by reducing overall CD4 and Treg populations while increasing CD8 T cells. 50In another study, B. longum CECT 7347 co-administration with gliadin to IFNγ-sensitized mice results in upregulated stress and intestinal absorption proteins along with downregulated cellular homeostasis proteins involved in cytoskeletal organization, protein transport, gene transcription, retinoic acid binding, and cell starvation. 51These observations reinforce the notion that the effects of Bifidobacterium treatment are pleiotropic and context dependent (Table 2).

Experimental autoimmune encephalomyelitis
Similar to EAMG, Bifidobacterium also ameliorates experimental autoimmune encephalomyelitis (EAE) in rats. 55Myelin basic protein (MBP)immunized rats, after injection with MBP-specific T cell blasts, have fewer of these cells localized to spinal cord tissue after treatment with B. animalis subsp.Lactis BB12 and LMG S-28195.Bifidobacterium in combination with Lactobacillus or as part of a larger cocktail (B.bifidum, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus reuteni, and Streptococcus thermophilus) prevents the progression of EAE in mice. 57,58AE and multiple sclerosis share many clinical and pathological features, with pathogenesis dependent on IL-17-producing T cells. 58Probiotic-treated EAE mice show reduced central nervous system inflammation with limited neuronal demyelination.Treatment also increases Tregs in peripheral LN (PLN) and spleen while inhibiting Th1 and Th17 polarization.IL-17, IL-6, and IFNγ are downregulated, while TGFβ, IL-4, and IL-10 are upregulated. 57,58In contrast, Bifidobacterium is enriched in patients with multiple sclerosis in both pediatric and adult human case-control series 59,60 (Table 2).

Food allergies
Bifidobacteria are more commonly found in the gut microbiomes of individuals who do not suffer from food allergies, suggesting the importance of Bifidobacteria in regulating allergic responses.In a mouse model of shrimp tropomyosin-induced allergy, treatment with either B. lactis or B. infantis 14.518 reduces allergic symptoms (including decreased serum IgE in both children with food allergies and mouse allergy models). 61,62ice treated with B. lactis exhibit increased Treg/ Th17 ratios 61 .Increased microbiome Dorea and decreased Ralstonia in treated animals correlates with elevated Treg/Th17 ratios, suggesting their involvement in the immunomodulatory response induced by Bifidobacterium administration.B. infantis 14.518 increases DC maturation and CD103+ tolerogenic DC accumulation in Peyer's patches and MLN.This leads to a similar increase in Tregs and suppression of Th2 responses. 62. infantis 14.518 partially restores gut microbiome richness in tropomyosin-sensitized animals. 62In mice with ovalbumin (Ova)-induced food allergy, gut dysbiosis, inflammation, and inflammatory cell infiltration are reduced or inhibited by concurrent treatment with B. breve M-16 V. 63 The probiotic modulates immune responses by inhibiting Th2 responses via ST2 blockade. 63A similar study on the effects of B. breve administration on Ovainduced rhinitis shows increased levels of splenic Treg and decreased Th2 responses, including serum IgE, IL-4, and IL-10. 65B. longum KACC 91,563 also synergizes with IgETRAP, a fusion protein of human high-affinity IgE receptor extracellular domain, hFcεRI, and an IgD/IgG4 hybrid Fc domain, to neutralize IgE and alleviate allergic responses in an Ova-induced food allergy model, including hypothermia, anaphylaxis score, serum IgE and MCPT-1, mast cell numbers, and goblet cell hyperplasia. 64This combined therapy does not alter the host microbiome, suggesting that B. longum KACC 91,563 does this without host colonization 64 (Table 2).

Exopolysaccharides
Exopolysaccharides (EPS) are carbohydrate polymers expressed on the cell surface or secreted by bacteria for both protection and interaction with the surrounding environment. 67,68Reflected by the large inter-and intra-species variability in gene clusters responsible for EPS biosynthesis, structure, and composition, these molecules play numerous roles in host-microbe interactions, including adhesion to the intestinal epithelium and protection from adverse environmental conditions. 69,701][72] Knocking out EPS expression in Bifidobacterium breve enhances the DC inflammatory phenotype by increasing the expression of co-stimulatory molecule genes Cd80 and Cd83. 18Cell surface β-glucan/galactan polysaccharides of B. bifidum PRI1 induce immunosuppressive Tregs via Toll-like receptor (TLR) 2 signaling on regulatory DCs. 6Even between strains of the same Bifidobacterium species, EPS structure and immune regulation differ.For example, B. breve UCC2003 has a thicker EPS layer and more anti-inflammatory phenotype than B. breve JCM7017 through modulation of macrophage IL-10 and TNFα and DC Tnfa, Il6, and Il23a expression. 18Murine B. pseudolongum UMB287 MBP-01 EPS increases intestinal Tregs compared to control, but EPS from porcine-derived B. pseudolongum ATCC25526 did not.EPS from both strains results in increased intestinal DC, MLN DC, and MLN macrophages. 73The cell surface components of B. longum strains NCC 2705, ATCC 15,707, and BIF53, but not BB536 or NCIMB 8809, stimulate the production of IL-10 and TNFα in isolated peripheral blood mononuclear cells. 74Given the contextdependence of bacterial EPS expression, its effects are likely only a portion of the Bifidobacterial immune modulatory mechanism (Table 3).

Pili
Pili are surface appendages that mediate adhesion to the host intestinal epithelium, bacterial cell aggregation, motility, electron transfer, biofilm formation, and immunomodulation. 77These structures display bacterial species-specific immunomodulatory properties. 69Bifidobacteria utilize sortase-dependent (SD) pili, consisting of covalently cross-linked pilin monomers anchored to the cell wall. 69,76These SD pili have roles in virulence, nutrient acquisition, mucin production, host adhesion, and immune signaling (Figure 1). 78Bifidobacteria also express type IVb tight adherence (Tad) pili, which mediate host surface adhesion. 76Expression of the B. bifidum PRL2010 SD pil3 PRL2010 gene cluster in the normally non-piliated Lactococcus lactis NZ9000 enhances adhesion to human intestinal enterocytes and evokes increased TNFα in a human macrophage-like cell line and mouse model compared to non-piliated control bacteria. 75Oral administration of piliated L. lactis-pil3PRL2010 results in decreased IL-10 compared with non-piliated controls in mice. 75Although B. bifidum PRL2010 pili induce higher TNFα expression, they act as weak inducers of other systemic proinflammatory cytokines such as IL-12, 69,75 suggesting that the immunomodulatory effects of SD pili may be limited to local mucosal immune responses. 75Given the presence of B. bifidum PRL2010 in the infant gut microbiome, the local pro-inflammatory effects of B. bifidum PRL2010 SD pili may prime the neonatal immune system 69,75 (Table 3).

Acetate
Metabolites from Bifidobacterium carbohydrate fermentation directly and indirectly influence host immune responses. 69Acetate is one of the most abundant metabolites produced by Bifidobacterium longum 5 1A reaching the systemic circulation. 79Acetate modulates host defenses and provide protection against various diseases.Mice pre-treated with live B. longum 5 1A or with acetate have elevated levels of IL-10 in lung tissue following Klebsiella pneumoniae infection compared to untreated infected mice, thus protecting the lungs from injury. 79The ability of Bifidobacterium species and strains to produce acetate varies considerably. 80This variability is influenced not only by the specific species or strain of Bifidobacterium, but also by the nutrient environment in which these bacteria reside.The specific genetic composition of each Bifidobacterium strain confers it with the capability to metabolize a different range of carbohydrate types, directly shaping its metabolic potential.For example, colonization of germfree mice with B. longum subsp.longum JCM 1217 or B. longum subsp.infantis 157F results in significantly higher concentrations of fecal acetate than colonization with B. longum subsp.infantis JCM 1222 T or B. adolescentis JCM 1275 T81 .Colonization with the former two strains conferred increased survival in mice inoculated with Shiga toxin-producing enteropathogenic E. coli O157. 81These B. longum strains increase acetate production via expression of ATP-binding cassette (ABC)-type carbohydrate transporters that increase sugar consumption for catabolism and acetate production, explaining how these bacteria can still produce acetate even in the fructose-limited distal colon. 81Protection against lethal enteropathogenic E. coli O157:H7 infection in the colon by Bifidobacterium longum subsp.longum JCM 1217 T is dependent on acetate production in the distal colon.This is accompanied by upregulation of host immune modulating genes (Apoe, C3, and Pla2g2a) and prevention of Shiga toxin translocation from the gut to the circulation. 81,82his also demonstrates how different strains of  84 This is demonstrated through serum amylase reduction, reduction of pancreatic lesions, and improved survival (Table 4).

Conjugated linoleic acid
Bifidobacterium-mediated conversion of linoleic acid (LA) into conjugated linoleic acid (CLA) is critical for the maintenance of intestinal homeostasis. 85LA induces redox stress and reduces growth of many different bacterial species, resulting in widespread metabolic reprogramming and dysbiosis. 86However, several Bifidobacterial strains are capable of converting free LA into CLA isomers. 85,86While the exact mechanism behind this bioconversion is unknown, production of CLA promotes gut homeostasis by protecting the microbiome from LA accumulation, which has been shown to confer protective effects in a number of disease models as well as to promote an antiinflammatory environment 85,86,95 (Table 4).

Inosine
The purine nucleoside inosine has best been characterized for its role as an anti-tumor immunomodulator. 93Inosine is produced by B. pseudolongum and enhances anti-tumor immunity via modulation of immune checkpoint blockade by anti-CTLA4 treatment.Inosine mediates CD4 Th1 differentiation via IFN-γ signaling through A 2A R on T cells.Immune checkpoint blockage (ICB) therapy is associated with elevated intestinal barrier permeability, which potentially facilitates translocation of inosine and other metabolites from the gut lumen into the systemic circulation.This augmented systemic translocation of metabolites may underpin the observed systemic effects of B. pseudolongum in the context of ICB therapy.Inosine also upregulates the IL-12 receptor on CD4 T cells, which is engaged by conventional DC-produced IL-12 89 (Table 4).

Tryptophan
Tryptophan metabolism is an important mediator of Bifidobacterial immune modulation.Bifidobacterium longum subsp.infantis ATCC 15,697, a "star colonizer" in breastmilk fed infant gut, produces aromatic lactic acids such as indole-3-lactic acid (ILA) from metabolizing tryptophan, which reduces inflammatory IL-8 production in the human H4 immature primary small intestinal epithelial cell line after IL-1β stimulation. 89,96Similarly, ILA pre-treatment significantly decreases LPS-induced IL-8 production in the Caco-2 intestinal epithelial cell line. 90ILA production is enriched in B. infantis ATCC 15,697 grown on HMO-supplemented media compared with lactosesupplemented media, suggesting that ILA production is driven by the metabolism of milk glycans. 90ILA pre-treatment inhibits LPS-induced NFκB activation in a dose-dependent manner in the murine RAWblue macrophage reporter cell line, engineered with secreted embryonic alkaline phosphatase. 90ILA signals through the aryl hydrocarbon receptor (AhR), which plays a role in regulating intestinal homeostasis and crosstalk with the cytoprotective Nrf2 pathway that reduces oxidative stress 90 (Table 4).Aromatic amino acid-derived aromatic lactic acids are ligands for AhR that influence gut homeostasis through enhanced mucosal barrier function, protection from pathogens, 97 and host metabolism. 98Bifidobacterial species convert aromatic amino acids (tryptophan, phenylalanine, and tyrosine) into their respective aromatic lactic acids (ILA, phenyllactic acid, and 4-hydroxyphenyllactic acid), which in turn activates AhR. 96Many Bifidobacterium-derived aromatic lactic acids are found in the infant gut from B. longum, B. bifidum, and B. breve. 96The ability of each species to produce aromatic lactic acids is tied to its ability to use HMOs as a carbohydrate source.In particular, ILA is enriched in the gut of breastfed infants, which modulates IL-22 production in CD4 T cells in vitro via AhR.B. longum CCFM1029 administration increases levels of the tryptophan metabolite indole-3-carbaldehyde (I3C) and improves atopic dermatitis symptoms in mice and humans by suppression of Th2 type immune responses via AhR activation. 91Indole-3-acetic acid (IAA) is another tryptophan metabolite and AhR ligand produced by Bifidobacterial species. 99n ankylosing spondylitis mice, IAA restores the ileal lamina propria Th17/Treg balance via AhR, resulting in increased levels of Foxp3 and downregulation of RORγt and STAT3 (Figure 2). 92However, there is a broad array of microbiota-derived molecules that act as AhR modulators apart from aromatic lactic acids.For example, the production of butyrate, which can be stimulated by Bifidobacterium as mentioned above, 19 acts as an HDAC inhibitor in human intestinal epithelial cell lines and colonic biopsies, increasing the recruitment of AhR to the target gene promoter in the presence of tryptophan-derived AhR agonists, 100 Furthermore, tryptophan metabolite indole-3-aldehyde (I3A), catabolized by Lactobacillus reuteri, found within the melanoma tumor microenvironment, facilitates immune checkpoint inhibition via AhR. 101

Vitamins
Bifidobacterium, along with lactic acid fermenting bacteria, have been reported to synthesize B-group and K-group vitamins de novo, providing an estimated 30% of the host's daily intake. 102Notably, several Bifidobacterium species are known to produce B vitamins, including B2 (riboflavin), B6, B9 (folate), and B12 (cobalamin).These vitamins are vital cofactors in numerous metabolic processes, including those integral to host immune function. 103The impact of these vitamins on the immune system is multifaceted: they aid in the development and function of lymphocytes, modulate cytokine production, and support the integrity of the mucosal barriers, forming the first line of defense against pathogens.Furthermore, Bifidobacterium's role in vitamin synthesis extends beyond B and K vitamins.A recent study showed commonly used human probiotic strain Bifidobacterium bifidum, along with other bacteria from Bacilli and Clostridia, converts dietary vitamin A to retinoic acid (RA) via aldehyde dehydrogenases. 104,105The upregulated RAdependent responses in intestinal epithelial cells can provide protection against pathogen colonization, and crucially, contribute to both immunological tolerance and the elicitation of adaptive immune responses. 106For example, RA generation from vitamin A occurs in intestinal epithelial cells and a subset of DCs, resulting in the conversion of naïve T cells into Tregs. 107The vitamin-producing capability of Bifidobacterium thus not only underscores its importance in maintaining gut health, but also highlights its broader influence in immune regulation.

PART VI. Bifidobacterium-influenced metabolites as immunomodulators
While Bifidobacterium does not possess all the metabolic machinery to produce all known immunomodulatory metabolites, it does contribute to an environment permissive toward production of these metabolites by other pro-homeostatic bacteria through mechanisms such as cross-feeding (Table 5).

Butyrate
Butyrate regulates the expression of antiinflammatory cytokine genes, such as TGFβ and IL-10, in antigen presenting cells (APCs) and intestinal epithelial cells (IECs) and stimulates Treg development. 108,109Butyrate has antioncogenic properties through induction of tumor cell apoptosis in colorectal cancer. 108imilarly, acetate induces genes involved in antiinflammatory responses 81 and acts on the colonic epithelium to enhance barrier function, thereby blocking the translocation of pathogens and toxins (Figure 2). 81While Bifidobacteria are unable to directly synthesize butyrate, Bifidobacterium and the acetate they produce may influence the activity and composition of other members of the gut microbiota that produce butyrate, thus stimulating a secondary butyrogenic effect. 19,69,115Co-culture of B. longum subsp.longum NCC2705, an arabinoxylan oligosaccharide (AXOS)-converting acetate producer, and Eubacterium rectale ATCC 33,656, an acetate-converting butyrate producer, in a growth medium with AXOS yields Bifidobacterium proliferation and butyrate production. 19This occurs due to butyryl-CoA: acetate CoA-transferase upregulation in E. rectale ATCC 33,656, which utilizes acetate as a co-substrate in the final step of butyrate biosynthesis (Table 5).

Secondary bile acids (BAs)
Secondary BAs also contribute to the regulation of immune homeostasis that are mostly known to be indirectly influenced by Bifidobacterium via modulating the gut environment and the microbial community structure 116 .Certain Bifidobacterium species can deconjugate bile salts, which can then influence the ability of other bacteria such as Bacteroides, Eubacterium, Ruminococcus, Clostridium, and Escherichia to further metabolize these bile components into secondary bile acids.The conversion of primary to secondary bile acids is a key process in the gut, which plays a significant role in maintaining gut homeostasis in addition to influencing metabolic and immune functions.The interplay between Bifidobacterium and these other bacterial species in the metabolism of bile components highlights the intricate and collaborative nature of the gut microbiome.Derived from cholesterol catabolism in the liver, primary BAs are secreted postprandially into the duodenum to aid the uptake of dietary fatty acids and fat-soluble vitamins. 87,117Most BAs return to the liver from the gut via the enterohepatic circulation.However, approximately 5% of BAs escape reabsorption in the ileum and are modified by resident enteric bacteria into secondary BAs via dehydroxylation, dehydrogenation, and deconjugation. 87,110,117econdary BAs can signal Tregs, Th17, or DCs in immune-mediated disorders. 87,118,119ifidobacterium have been identified in a highthroughput screen of human stool as a converter of 3-oxolithocholic acid (3-oxoLCA), a BA found in the human gut, to isoallolithocholic acid (isoalloLCA), a known immunomodulatory secondary BA. 87IsoalloLCA increases the rate of cellular oxygen consumption, inducing mitochondrial reactive oxygen species that increases the expression of FOXP3, thereby increasing Treg differentiation. 88espite these connections, few studies have directly investigated the production of secondary BAs by Bifidobacterial species or determined their effects on immune regulation (Table 5).
Anti-inflammatory secondary BA 3βhydroxydeoxycholic acid (isoDCA) decreases DC TNFα and IL-6 production. 110IsoDCA also promotes Foxp3 induction, increasing the number of peripheral Tregs (Figure 2).Although Bifidobacterium lack 7αdehydroxylation activity to produce isoDCA from cholic acid. 120Bifidobacterium encode bile salt hydrolases that deconjugate BAs into other secondary BAs that may have similar immunomodulatory roles. 111,112

Polyamines
Polyamines (i.e., putrescine, spermidine, and spermine) play a critical role in regulating immunity and inflammation. 121,122While Bifidobacterium does not possess polyamine biosynthetic machinery, its presence does enhance the production of polyamines from other bacterial sources.Administration of several different strains of Bifidobacterium can increase intralumenal putrescene via acidification, likely through acetate and lactate production, which augments putrescine production by E. coli and Enterococcus faecalis 33 .Spermine restrains innate immune responses by inhibiting M1 macrophage activation via suppression of ornithine decarboxylase and pro-inflammatory cytokine synthesis without perturbing antiinflammatory TGFβ and IL-10 (Figure 2). 113,114permidine also modulates systemic and mucosal adaptive immunity by modulating T cell differentiation. 122,123Clinically, N-acetyl putrescine and N-acetyl spermidine are enriched in allo-hematopoietic stem cell transplantation (HSCT) recipients free from graft versus host disease (GvHD) compared to those with GvHD 124 (Table 5).

PART VII. Future directions: therapeutics and technological pipeline
Initial investigations into the role of Bifidobacterium in human disease has been predominantly associative, yet recent studies have begun to delve into the immunomodulatory mechanisms and causal role Bifidobacterium plays in host immune responses.Recognizing a causal relationship not only lays the groundwork for the formulation of targeted therapeutic strategies but also illuminates the underpinnings of disease pathogenesis.This enhanced understanding paves the way for novel drug development, refined disease progression predictions, and innovative preventative strategies.
Though Bifidobacterium displays immunologic pleiotropy, varying across host species and specific strains, there is an increasing interest in its surface components and derived metabolites.These components present promising targets for host immune modulation.A better understanding of the metabolic perturbations due to Bifidobacterium and the influence of their metabolic products on pro-tolerant and pro-homeostatic immunity are critical, especially when considering clinical scenarios such as solid organ transplantation, autoimmunity, and anti-tumor responses.Furthermore, a deeper understanding of the role Bifidobacteria within the broader commensal gut microbiota communities is pivotal.Such knowledge will be instrumental in optimizing live biotherapeutics for the prophylaxis or treatment of disease conditions.

Live biotherapeutics
The absence of HMO-metabolizing Bifidobacterium in the infant gut correlates with Th2-and Th17-driven inflammation in the intestine, as well as both acute and chronic systemic immune disorders. 8,125Investigators have begun to test the impact of exogenous Bifidobacteria on improving overall immune health.Infants fed a diet supplemented with B. longum subsp.infantis EVC001, an optimized strain containing all HMO-utilization genes, display a higher abundance of Bifidobacterium within two months of administration. 125,126These infants have decreased intestinal inflammation, as evidenced by decreased fecal inflammatory cytokine and calprotectin levels 125 .Fecal water from EVC001treated infants also skews the polarization of naïve T cells cultured under Th0 conditions toward a Th1-like state, whereas fecal water from infants lacking B. infantis induces a Th2like state.When cultured under Th17 polarizing conditions and exposed to EVC001 fecal water, T cells show reduced levels of activation and proliferation markers compared to controls.This effect is recapitulated by B. infantis-derived metabolite ILA, which in addition to the effects mentioned above, upregulates CXCR3, granzyme B, and galectin-1 in cultured cells. 8Administration of EVC001 is also associated with an increase in intestinal IFNβ, a known inducer of Tregs. 8,127long with changes in cytokine and immune responses, EVC001 supplementation contributes to reducing the amount of virulence factors in the infant gut, restricting the establishment of pathogenic bacterial communities. 126This reinforces the importance of Bifidobacterium-derived metabolites as a mechanism of immunomodulation and as potential therapeutic avenues.
In adults with active UC, one month of treatment with B. longum and prebiotic (preferential inulin-oligofructose growth substrate) reduces endoscopic and histologic inflammation in the colon and reduces mucosal TNFα and IL1 mRNA levels. 128Ex vivo, heat-killed B. breve strain Yakult and B. bifidum strain Yakult both induce increased IL-10 levels in peripheral blood mononuclear cells from UC patients. 129Similarly, in CD patients, B. longum and prebiotic reduces disease activity indices, histologic scores, and mucosal TNFα levels. 130B. infantis 35624 demonstrates a clinical anti-inflammatory effect in UC, chronic fatigue syndrome, and psoriasis.After 6-8 weeks of administration, this probiotic strain reduces plasma CRP levels in all three conditions, reduces TNFα in chronic fatigue syndrome and psoriasis, and reduces IL-6 in UC and chronic fatigue syndrome. 131lthough there has been much interest and studies of the benefits of Bifidobacteria-based probiotics, colonization of strains and the direct mechanisms underlying these effects have been challenging to elucidate.Given the complex interactions that Bifidobacteria has with other members of the gut microbiome, off target effects of ongoing probiotic treatment are likely.However, small molecule mediators of the immune modulatory effects of Bifidobacterial strains have a demonstrated potential as therapeutics that have higher purity with more direct and consistent effects than probiotics.Furthermore, metabolites may be able to be administered in a more targeted, tissue-specific manner, compared to probiotics, which must be administered enterally.Future studies will necessarily need to focus on the tissuespecific effects of probiotics, but more specifically of individual metabolic alterations in response to probiotics.

Anti-tumor immunotherapy enhancement
In addition to its direct role in the treatment of autoimmune and inflammatory diseases, Bifidobacterium has also been implicated in modulating the effects of anti-cancer regimens and enhancing anti-tumor immunity.From a mechanistic standpoint, some of these effects may be mediated by Bifidobacterium-derived metabolites.B. pseudolongum promotes immune checkpoint blockade efficacy in mouse models of melanoma, bladder cancer, and colorectal cancer through its production of inosine, which acts on T cells via A 2A R. 94 In humans, stool samples from patients with improved clinical responses to antiprogrammed cell death protein 1 (αPD-1)-based immunotherapy for metastatic melanoma show Bifidobacterium longum enrichment. 132dministration of a Bifidobacterial cocktail containing B. breve and B. longum, both alone and in combination with anti-programmed cell death protein 1 ligand 1 (αPD-L1) to mice with B16.SIY melanoma results in increased gut Bifidobacterium and decreased tumor growth. 133he reduction in tumor growth is dependent on Bifidobacterial-stimulated DC maturation, which enhances CD8 T cell priming and accumulation in the tumor microenvironment. 133B. breve strain JCM92 also augments oxaliplatin's anti-tumor efficacy in MC38 colon carcinoma-bearing mice compared with oxaliplatin alone, with increased intratumor CD8 T cells as well as increased CD4/Treg and CD8/Treg ratios. 134This effect is also true with B. breve JCM92 PD-1 blockade, with increased intra-tumor CD8 T cells and CD8/Treg ratios compared to PD-1 blockade alone.At the transcriptional level, B. breve JCM92 results in higher IL-2, STAT5 signaling, and IFN-γ responses compared to mice treated with oxaliplatin or PD-1 blockade and control bacteria.Addition of B. infantis to monoclonal antibody and radiotherapy treatment of Lewis lung carcinoma in mice slows tumor growth and prolongs animal survival. 135In the same model, B. pseudolongum is significantly elevated in the gut of mice with delayed or absent tumorigenesis. 136n addition to augmenting anti-cancer therapies, Bifidobacteria has also been shown to have independent anti-tumor activity.B. breve lw01 administration suppresses head and neck squamous cell carcinoma growth in mice, mediated by the upregulation of CCL20, which is associated with increased migration of CD11c DCs to ileal villi and tumor microenvironment via upregulation of IL-12 137 .Furthermore, Bifidobacteria ameliorate checkpoint inhibitor-associated autoimmunity.For examples, mice receiving anti-CTLA-4 with superimposed DSS colitis also receiving a cocktail of Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium lactis, and Bifidobacterium breve show less colitis compared to no bacteria controls without impacting anti-tumor efficacy against B16F10 melanoma. 138Tregs are required for this protective effect as it is abrogated in Treg-depleted mice.Despite their broad tolerogenic immune function, Bifidobacteria are also capable of contributing alone or in concert with immunotherapies and chemotherapies to increase host anti-tumor activity.

Pipeline for discovery
Over the past several years, the use of untargeted metabolomics to detect alterations in systemic metabolism has greatly expanded.This technology has provided a hypothesis generating engine for labs studying the metabolite mediators generated or induced by members of the gut microbiota.Other groups have begun to employ spatial transcriptomics to compare local transcriptional changes in cells at the host microbiota interface. 139Application of a spatial omics approaches have proven critical to studying microbiota-gut interface given the significant artifacts that accompany cellular disaggregation protocols used to isolate mucosal cell populations for flow cytometry or singlecell RNA sequencing (scRNAseq).Depending on the protocol used to produce single-cell suspensions for assays, the composition of immune cells from similar starting samples will differ. 140owever, both tools only demonstrate association rather than causation.More recently, teams have begun to adopt a hybrid spatial metabolomics approach, which permits spatial resolution of metabolic changes in the host. 136Authors localized metabolic changes, particularly in glycolysis and amino acid catabolism, in tumor tissue in mice administered Lewis Cancer cells and subsequent Akkermansia muciniphila treatment.For example, lactic acid (highly expressed in tumor tissue) was downregulated in the tumors of mice treated with Akkermansia, with confirmed downregulation of lactate dehydrogenase-A enzyme via immunofluorescence.Again, as hypothesis generators, this omics pipeline identifies small molecules of interest both for mechanic studies in vitro and in vivo, but also for development of potential diagnostic biomarkers and therapeutic interventions.While these techniques have not been applied directly to the study of Bifidobacteria, these early studies provide a blueprint.

Disclosure statement
No potential conflict of interest was reported by the authors.

Table 1 .
Cross-feeding relationships between Bifidobacterium and other microbiota species.

Table 2 .
Bifidobacterium association with disease states and models.

Table 3 .
Bifidobacterium cell surface components and their immunomodulatory properties.