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