5.2 Th17 cells
Th17 cells are CD4+T cells that secrete cytokines, such as IL-17, IL-21, and IL-22 58. On the one hand, Th17 cells resist pathogens such as fungi, maintain the immune barrier integrity of digestive tract59. However, excessive inflammatory induction by Th17 cells leads to various disease 60, such as experimental autoimmune diseases and human inflammatory conditions 59.
After culturing with the LCA + E. lenta DSM2243 (3α HSDH+) supernatant, the differentiation of naive CD4+ T cells into Th17 cells was inhibited. After Citrobacter rodentium significantly induced Th1 and Th17 cells, 3-oxoLCA treatment resulted in decreased Th17 and IFN-γ +Th17 cell levels in the colon, while Th1 and Treg cells were unaffected61. LCA processing showed the opposite of the above results. 3-oxoLCA and isoLCA reduced the differentiation of Th17 cells by interacting with RORγt and inhibiting its transcriptional activity8,61(Fig. 1). Notably, isoalloLCA reduced the differentiation of Th17 cells without affecting RORγt expression and did d not impair cell viability8. The regulatory effects of 3-oxoLCA and isoalloLCA on Th17 and Treg cells did not affect intestinal commensal bacteria; they directly regulated Th17 and Treg cells in mice. However, these metabolites are not present in germ-free mice, implying that the production of these metabolites needs commensals8. More studies are needed to determine the identity of gut-residing bacteria and the corresponding enzymes that convert LCA into 3-oxoLCA and isoLCA 61. It has been shown that 3β-HSDH encoded by BF3538 in B. fragilis enables the production of isoLCA; 3α-HSDHs encoded by Elen_0690 and its homologues in E. lenta strains are required for 3-oxoLCA. The researchers used 3α-HSDH and 3β-HSDH positive and negative bacteria as tools to study the effects of the two LCA derivatives on TH17 cells. The study of the genes encoding various metabolism-related enzymes in the strain will help clarify the link between the intestinal flora and immune cells. It provides a reference for the development of engineered bacteria, which benefits scientific research and treatment of diseases.
Studies have shown that UDCA decreases Th17 cells and IL-17 via the pAMPK-SMILE pathway62(Fig. 1). This revealed the role of UDCA in regulating the balance between Th17 and Treg cells. BAs were cytotoxic to cells at high concentrations (63. CD4+ T effector cells (Teff cells) include Th1 cells that produce interferon-gamma (IFNγ) and Th17 cells, and studies have shown that the relationship between BAs and Teff cells, conjugated bile acids (CBA), drives oxidative stress in Teff cells and kills transformed epithelial cells. The xenobiotic transporter Mdr1, which is induced by CD103+ DCs, enforces T cell homeostasis in the presence of CBA64(Fig. 1). Abnormal concentrations of BAs affect the activity of T cells, and the interaction between DC and T cells can resist adverse effects, while changes in related genes are not conducive to the maintenance of normal homeostasis.
Gut flora need special enzymes and BAs conformations to regulate immunocytes
Diet directly controls the liver synthesis of BAs, whereas the gut flora and host mainly control the modification process of BAs in the gut. The appearance of SBAs in stool is the major difference between conventionalized and germ-free mice 36. The overall concentration of BAs in mice mono-colonized with gut microflora (E. coli and B. thetaiotaomicron) was higher, and SBAs were not detected. This indicates that the metabolism of PBAs requires special bacteria, and different gut microflora have their respective metabolic activities. Ridlon et al. also concluded that bile salt hydrolysis and hydroxy group dehydrogenation reactions are induced by extensive intestinal anaerobes65. However, BAs 7-dehydroxylation is restricted to a small part of intestinal anaerobes66.
Clostridium scindens has been widely used to convert PBAs into both LCA and DCA in previous studies. However, an established multi-strain also produces SBAs without this strain because Extibacter sp. GGCC_0201 provides a 7-dehydratase that converts CA and CDCA into DCA and LCA respectively 67. This suggests that specific enzymes are indispensable for bacterial metabolic activity. Human gut bacteria that convert LCA into 3-oxoLCA and related genes that encode 3α-/3β-HSDHs are negatively associated with Crohn’s Disease (CD) in humans61. The generation of isoDCA requires some key enzymes; researchers constructed several bacterial strains with key enzymes and produced a large amount of isoDCA, which also excluded bacterial strain backgrounds27. Building strains that metabolize specific molecules is a strong indication of the centrality of enzymes and proves the operability of developing therapeutic approaches.
IsoalloLCA enhanced FOXP3 expression, while other LCA isomers did not show the same effect, indicating that both the 3β-hydroxyl group and trans (5α-hydrogen) A–B ring configuration of isoalloLCA are required to regulate Treg cells 8. Paik et al. observed that isoLCA inhibited naive CD4+ T cells to differentiate into Th17 cells as efficiently as 3-oxoLCA, whereas isoDCA, which is abundant in 3β-OH, did not inhibit differentiation61. Campbell et al. reported that the spatial orientation of specific hydroxyl (-OH) groups is vital for isoDCA and ω-MCA to exert effects27. Therefore, microbial epimerization and specific spatial conformations bring about the unique properties of BAs to regulate immunity.
Roles of TGR5 and FXR in immune cells and intestinal epithelial cells
TGR5/GPBAR1 is a receptor located on the cell membranes of cells in the gallbladder, ileum, colon, and liver. FXR is a nuclear receptor involved in the regulation of BAs and lipid homeostasis. There are many studies on the influence of receptor activation on the immunocytes of the body.
7.1 Macrophage
7.1.1 TGR5
As important anti-tumor immune cells, macrophages have the ability to chemotaxis, phagocytosis, antigen presentation, and secretion of cytokines. The effect of TGR5 on macrophages has been extensively studied in organ (I/R) injury, IBD, and liver disease; however, it has been less studied in tumors.
BAR501 is an agonist of GPBAR1, and LPS-induced elevation of M1 markers (CD38, Fpr2, and Gpr18) was reversed after treatment with BAR501(Fig. 2). Moreover, BAR501 up-regulates the markers of M2 (Egr2 and c-myc)68. TGR5-deficient BMDMs showed higher levels of the M1 markers iNOS and IL-6 and lower levels of the M2 markers PPARγ and Arg-169. UDCA enhancement of TGR5 activation is beneficial for its anti-inflammatory effects and promotes M2 polarization. In vitro experiments showed that TGR5 inhibits macrophage migration by inhibiting Cat E69. Another study showed that TGR5 inhibits macrophage migration by inducing the differential expression of C/EBp-β via the mTOR complex70. The above studies demonstrated the effect of TGR5 activation on the macrophage phenotype and migration.
In non-small cell lung cancer(NSCLC), TGR5 promotes the formation of tumor-associated macrophages (TAMs) by activating the cAMP-STAT3/STAT6 signaling pathway. This inhibits CD8+ T cells and decreases the production of granzyme B, IFN-γ, and TNF-α, thus inhibiting anti-tumor immunity 71. The phagocytosis of tumor cells by TGR5 deficient macrophages is enhanced; therefore, we can reasonably speculate that TGR5 reduces the phagocytosis of macrophages. We can think that in intestinal tumors, the changes in microbiota metabolites, especially the BA molecules regulating TGR5, may affect the formation of TAMs, thus unfavorable to anti-tumor effects. In human patients with NSCLC tissue, TGR5 expression correlates with infiltration of TAMs, and their high expression is associated with poorer prognosis and shorter overall survival.71 Taurolithocholic acid (TLCA) inhibits the expression of LPS-induced IL-6, IL-12, TNF-α, and TNF-β72. The inhibitory effect of TGR5 on IFN-β expression by cAMPPKA is also reflected by the downregulation of the IFN-stimulated genes MxA and PKR in human macrophages72. IFN and T cells have intricate relationships and studies have shown that IFN-β increases the induction of Treg cells73. This suggested that the activation of TGR5 regulates the expression of interferon in macrophages. This information helps us study the coordination between macrophages and T cells to exert antitumor effects in the intestinal tumor immune microenvironment. In microglia, BAs, such as tauroursodeoxycholic acid (TUDCA) or taurolithocholic acid (TLCA), reduce PKM2 expression and regulate the glycolytic pathway74. This deepens our understanding of the energy regulation of BAs.
TCDCA acts on the TGR5 receptor and modulates inflammation through the cAMP-PKA-CREB signaling pathway. TCDCA reduces inflammatory cytokines, such as IL-1β, IL-6, IL-8, IL-12, and TNF-α by affecting NF-κB activity75. IBD patients have higher levels of TGR5 expression in intestinal mucosal lamina mononucleus cells (LPMCs), and TGR5 agonists and BAs (DCA and LCA) inhibit TNF-α production in macrophages through the TGR5- cAMP pathway. In this pathway, c-Fos phosphorylation is induced to regulate NF-κB p6576. TGR5 inhibited the upregulation of the TLR4-NF-κB pathway and activation of Caspase-8 in vivo77. In in vitro hypoxia/reoxygenation macrophage models, activation of TGR5 downregulates the expression of TNF-α and IL-6, and upregulates the expression of IL-1077. Another study showed that the activation of TGR5 alleviates inflammation through the Keap1- Nrf2 pathway. Specifically, it increased the expression of Nrf2 and Ho-1 in the nucleus, but decreased the expression of Keap19. Previous studies have shown that TGR5 inhibits NLRP3 inflammasome activation and caspase-1 cleavage78. A recent study found that BA supplementation activated the NLRP3 inflammasome in macrophages and promoted inflammation under noninflammatory conditions. However, BA inhibits NLRP3 inflammasomes and reduces inflammation in LPS-induced inflammatory macrophages79.
7.1.2 FXR
FXR regulates macrophage activation and oxidative stress and contributes to the anti-inflammatory phenotype and function of macrophages80. The relationship between the gut microbiome, BAs, FXR, and NLRP3 inflammasome remains uncertain. Gut microbiome metabolites act as natural FXR regulatory molecules and BAs are important components. In turn, FXR alters the BAs and gut microbiome composition81. BAs activate the NLRP3 inflammasome by promoting calcium influx, but FXR inhibits the NLRP3 inflammasome82.
Osteoclasts originate in the mononuclear macrophage system, which ich is a special type of terminally differentiated cell. FXR agonists inhibit the c-Jun N-terminal kinase (JNK) 1/2/nuclear factor of activated T-cells 1 (NFATc1) pathway JNK1/2/NFATc1 and alleviate subchondral osteoclast fusion83. Kupffer cells, which are liver macrophages, showed decreased TNF-α and increased IL-10 expression with the activation of FXR by GW406484. GW4064 increased FXR binding to the Abcb11 promoter and decreased the expression of genes related to recruitment and activation of macrophages in the liver. This is not conducive to the accumulation, activation, and infiltration of liver macrophages85,86. Loss of nuclear receptors FXR and SHP leads to YAP activation in mice with unbalanced bile acid homeostasis and spontaneous liver tumor87.
7.2 DC
The expression of TGR5 is down-regulated during the differentiation of monocytes into DC. BA-cultured DC produce lower levels of IL-12 and TNF-α under the stimulation of symbiotic bacterial antigens, which is mediated by the TGR5-cAMP pathway88. DCA regulated the function of DC through the TGR5-cAMP-PKA pathway and inhibited the activation of NF-kB (Fig. 3). DCA decreased the secretion of IL-1β, IL-6, IL-12, and TNF-α from LPS-induced bone marrow-derived DCs. It is noteworthy that the expression of the DC co-stimulatory molecules CD40, CD80, CD86, and MHC II was also inhibited. It is not conducive to T cell differentiation and Th1 and Th17 cell development, and the secretion of IL-17 and IFN-γ is reduced 89. The discovery that bile acids regulate T cell differentiation and function through DC suggests the potential research value of bile acid signaling molecules in the local immune microenvironment, which is conducive to promoting related research on inflammatory and tumor diseases, especially intestinal cancer.
7.3 NKT
Natural killer T (NKT) cells are a special T cell subgroup with both TCR and NK cell receptors on the cell surface. According to the constancy of the TCR, NKT cells are divided into types I and II. Type I NKT cells mainly play an antitumor role, while type II NKT cells can promote the development of tumors 90. Type I NKT cells include pro-inflammatory NKT1 subsets that produce IFN-γ, and regulatory NKT10 subsets that secrete IL-10. In a mouse model of hepatitis, ablation of GPBAR1 exacerbated liver damage and resulted in a phenotype of type I NKT cells biased towards NKT1. While activation of GPBAR1 rescues liver damage, NKT cells are polarized into NKT10. In addition, GPBAR1 excitation significantly expanded the IL-10-secreting type II NKT cell subsets91. LCA inhibits hematopoietic stem cell activation via the Smad pathway (reduces TGF-β) or MAPK-ERK pathway. LCA increases recruitment of NK cells and reduces activation of NKT cells. However, the effect of LCA weakened when antibiotics reduced the diversity and abundance of gut microbes92.
7.4 MDSC
Intravenous infusion of TDCA decreased serum proinflammatory cytokines, increased the number of granulocyte myeloid suppressor cells (MDSCLTS) in the spleen of septic mice, and inhibited T cell proliferation93. Activation of FXR enhances the immunosuppressive activity of MDSCs on T-cell proliferation by binding to and upregulating the PIR-B promoter. FXR activation drives the accumulation of MDSCs in the liver through upregulation of S100A8 mRNA expression94.
7.5 B cell
Immunoglobulin A (IgA) is present in mucosal tissue and resists the invasion of pathogens, and its absence leads to intestinal inflammation95. In PBA-fed rats, IgA levels in the ileum mucosa were increased96. UDCA belongs to SBAs, and in in vitro experiments, UDCA inhibits the production of IgM, IgG, and IgA in peripheral blood monocytes. UDCA also acts on B cells by suppressing Ig production in human B cells97. The effects of various BAs on local B cells and mucosal immunity in vivo warrant further investigation.
7.6 Intestinal epithelial cells
In an oxazolone-induced colitis model, the GPBAR1 agonist BAR501 alleviated the symptoms of enteritis, inhibited inflammatory markers such as IL-1β, IL-6, and IFN-γ, and increased the expression of TGF-β, IL-10, and Foxp368. Gut microbiota-BAs-TGR5 Axis is beneficial for restoring the integrity of the damaged colonic epithelium98. During organoid growth, TGR5 activation increases the activation of Yes-associated protein 1 (YAP1) and its upstream regulator SRC from intestinal stem cells (ISCs). TGR5 activates ISCs and promotes epithelial cell regeneration, causing them to renew and proliferate in response to injury99. Another study indicated that DCA retards wound healing in colon epithelial cells by activating the AKT pathway via GPCR5100.
Fibroblast growth factor 15/19 (FGF15/19) is a hormone released by ileal gut cells in response to stimulation by FXR (usually via absorbed BAs) that provides negative feedback for BAs synthesis in hepatocytes101. In intestinal epithelial cells, FXR activates the transcription of intestinal bile acid-binding protein (I-BABP) and FGF15, both of which regulate key aspects of the liver and intestinal homeostasis. SHP is a regulatory gene of FXR and the synthetic ligand of FXR can reduce organ damage and immune cell activation in vivo. Activation of FXR increased the expression of I-BABP, FXR, and SHP in the colon and decreased the expression of IL-1β, IL-6, TNF-α, iNOS, cyclooxygenase (COX)-1, and COX-2 in THP1 cells, reducing the severity of the disease102.
In mouse models of intestinal tumors and chronic colitis, loss of FXR promotes Wnt signaling in the intestinal mucosa through neutrophil and macrophage infiltration and TNF-α production, leading to early death and increased tumor progression. When activated, FXR induces apoptosis and clearance of genetically altered cells103. This suggests that strategies to reactivate FXR expression in colon tumors may be useful for the treatment of colon cancer. BA-activated FXR not only increases GLUT2 expression and controls glucose uptake through the FXR-S1PR2-ERk1/2 signaling cascade but also reduces cell energy production by inhibiting oxidative phosphorylation104. In the case of bowel cancer, how FXR regulates tumor growth by affecting energy is worthy of further investigation. DCA activates the STAT3 signaling pathway, interferes with the gastric microbiome and BA metabolism, and induces gastrointestinal metaplasia105. DCA reduces the expression of FXR, activates the Wnt signaling pathway, increases the levels of β-catenin and c-Myc, and promotes the proliferation and migration of colon cancer cells. The FXR agonist GW4064 reduces the proliferation of colon cancer cells by inhibiting the Wnt signaling pathway106. These studies suggest a relationship between DCA, FXR, and the Wnt signaling pathway. The authors highlighted the value of FXR agonists in intestinal cancers.
BAs, immune and disease
BAs promote the absorption of lipid nutrients in the intestine and act as signaling molecules. BAs also influence immune homeostasis and energy utilization. Disorder of intestinal flora and an imbalanced BAs pool lead to various diseases (Fig. 5).
Intestinal microbiota regulate enteric virus107,108 , norovirus infections are major causes of gastroenteritis 109 .  FXR is expressed differently in the gut, and the proximal bacterial biotransformed BAs initiate a type III interferon response and resist viral infection. Therefore, commensal bacteria inhibit viral infection of the proximal gut, while promoting viral infection of the distal gut110 . The cause of enteritis is complex. Studies have shown that dietary astragalus polysaccharide influences the gut microbiota, metabolites, and Th17/Treg balance in necrotic enteritis (NE) chickens111 . Primary sclerosing cholangitis (PSC)-IBD is also a result of a dysregulated Th17/Treg balance112. IBD is often accompanied by dysregulated bacterial flora, BAs, and pro-inflammatory factors113. Genes encoding various metabolism-related enzymes in the gut flora regulate anti-inflammatory BAs small molecules and their derivatives, and are negatively correlated with intestinal IBD27,61.
The intestinal flora and BAs are also closely associated with endocrine and metabolic diseases.   Glycodeoxycholic acid induces ILC3 to secrete IL-22, which improves the phenotype of Polycystic ovary syndrome114. Pigs hyocholic acid (HCA) acts on enteroendocrine cells, activates TGR5, and inhibits FXR to promote the production and secretion of glucagon-like peptide-1 (GLP-1), consequently improving glucose homeostasis and avoiding diabetes 115. In mice, feces from patients with coronary artery disease (CAD) promote intestinal inflammation by disturbing the lamina propria Th17/Treg cell balance and worsening gut barrier permeability 116. These studies offer a new perspective on the treatment of metabolic and endocrine system diseases.
Hydrophobic BAs damage intestinal cells and are risk factors for colorectal cancer (CRC)117. High-fat diet and APC mutation of WNT signaling change BAs profiles and promote Lgr5+ cancer stem cell cells (CSC) proliferation and DNA damage10. However, activation of intestinal FXR limits its growth and suppresses CRC progression. The role of FXR implicates it as a potential therapeutic target for CRC. Moreover, DCA and LCA derivatives that promote Treg cell differentiation and inhibit Th17 cell differentiation may also cause CRC. Studies on TGR5, FXR, and their activation in macrophages, DC, NKT cells, MDSC, B cells, and intestinal epithelial cells also provide references for the exploration of the etiology of CRC and for better cancer treatment.
Clinical applications