Bile acid‐activated receptors in innate and adaptive immunity: targeted drugs and biological agents

Bile acid‐activated receptors (BARs) such as a G‐protein bile acid receptor 1 and the farnesol X receptor are activated by bile acids (BAs) and have been implicated in the regulation of microbiota‐host immunity in the intestine. The mechanistic roles of these receptors in immune signaling suggest that they may also influence the development of metabolic disorders. In this perspective, we provide a summary of recent literature describing the main regulatory pathways and mechanisms of BARs and how they affect both innate and adaptive immune system, cell proliferation, and signaling in the context of inflammatory diseases. We also discuss new approaches for therapy and summarize clinical projects on BAs for the treatment of diseases. In parallel, some drugs that are classically used for other therapeutic purposes and BAR activity have recently been proposed as regulators of immune cells phenotype. Another strategy consists of using specific strains of gut bacteria to regulate BA production in the intestine.


Introduction
Bile acids (BAs), which are 24-carbon steroids, act as natural surfactants and are generated from cholesterol in the liver. They are then stored in the gall bladder and secreted into the duodenum [1]. BAs play a crucial role in lipid digestion, lipid absorption, bacterial defense, and cholesterol homeostasis [2,3]. The apical sodium dependent BA transporter (also known as IBAT), which is found in the distal ileum, reabsorbs around 95% of the biliary BAs produced in the intestine and circulates them back to the liver for release [4]. This process, known as enterohepatic cir-monocytes and macrophages, as well as in the human spleen, and may play an anti-inflammatory role in the immune system. At the same time, it showed clear beneficial effects in the regulation of BA metabolism, weight loss, and regulation of glucose homeostasis. Dysregulation of the interaction between BAs and immune cell, caused by both genetic and environmental factors, may promote response or predispose to diseases, such as cholestasis, in which increased levels of BAs in the liver lead to hepatic diseases [7]. Moreover, increasing evidence suggests the importance of BAs in maintaining innate immune response via receptors, such as autoimmune uveitis [8][9][10][11][12]. Li et al. revealed that restoration of the gut BAs pool could attenuate the severity of EAU by modulating the maturation of splenic dendritic cell (DCs). In addition, G-protein bile acid receptor 1 (GPBAR1) deficiency could reverse the inhibitory effect of lithocholic acid (LCA) and deoxycholic acid (DCA) on DCs [12,13]. Recent research also shows BA-activated receptor (BARs) regulate adaptive immunity. These observations identify the BA pathway as an attractive target for intervention in inflammatory diseases.

BA synthesis
The main mechanism for cholesterol catabolism is the BA production from cholesterol. At least 17 distinct enzymes catalyze the conversion of cholesterol into BA in the liver [14]. The BA synthesis is divided into two pathways, known as "classic/neutral" and "alternative/acidic." Cholesterol-7α-hydroxylase is the ratelimiting enzyme for the classic pathway (over than 75% of total BA pool). Cholesterol is converted to 7α-hydroxy-cholesterol by cholesterol-7α-hydroxylase. Then it can be metabolized to 7α-hydroxy-4-cholesten-3-one (C4) by a specific steroid dehydrogenase. The activity of the enzyme sterol 12α-hydroxylase (CYP8B1), which is essential for the production of cholic acid (CA), provides access to the dispersion of these two BAs. As a branching enzyme, the CYP8B1 can hydroxylate C4 and produce CA. In any other case, C4 would be metabolized to chenodeoxycholic acid (CDCA) with CYP8B1 [15]. The alternative pathway is a conversion of cholesterol to a component of 27-hydroxycholesterol by steroid 27-hydroxylase, followed by hydroxylation on ring B, metabolized by oxysterol 7α-hydroxylase, and side chain modification for the synthesis of CDCA. In addition, this pathway produces only CDCA, with less than 25% of total BA pool [16]. Primary BAs are bound to glycine or taurine at position C-24 via bile acyl synthetase and BA-CoA aa N-acyltransferase to produce tauro-CA, tauro-CDCA, glyco-CA, and glycolyl-CDCA, respectively, which are then secreted into the bile canaliculi. CA and CDCA contain glycine and taurine in the human liver in a ratio of 3:1. In mice, about 95% of primary BAs are tauro-conjugated. Bile salts are intermediate derivatives of primary BAs, which are released into the bile ducts and sent to the intestine for microbial modification (Fig. 1). Notably, the secondary BAs concentrations are in the hundreds of micromolar range in the healthy human gut [4]. Rats create CA and muricholic acids (MCAs), primarily beta-MCA, whereas humans primarily make CDCA and CA [17].

Gut microbiome, BAs, and adaptive immunity
The gut microbiome, which is the most densely populated natural ecosystem, consists of over 10 14 bacterial cells [18]. It is a community of commensal, symbiotic, and pathogenic microorganisms consisting of bacteria, archaea, fungi, and viruses that can inhabit the gastrointestinal tract and contribute to both human health and disease [19,20]. BAs play a protection role in the gut epithelium health and defense against pathogens such as Clostridium difficile [21]. In addition, a decreased amount of Ruminococcaceae and Lachnospiraceae have been identified as a potential cause of different autoimmune diseases, including inflammatory uveitis [22], inflammatory bowel disease (IBD) [23], enthesitis-related arthritis [24], pediatric MS [10], and type 1 diabetes [25]. Ruminococcaceae and Lachnospiraceae stimulate 7α-dehydroxylation, which converts the primary BA (CA and CDCA) into secondary BAs, such as DCA and LCA [26]. Interestingly, the intestinal microbiota also produces secondary BAs, the GPBAR1 Ls, and derivatives of oxo-BAs, the RORt Ls, from the breakdown of cholesterol. Recent studies have also found a reduced amount of fecal secondary BAs in germ-free mice compared to specific pathogen-free mice [27], providing further evidence of the importance of microbiota in BA metabolism.
The composition of human GM can be altered by various factors such as age, diet, lifestyle, disease, and antibiotics. These changes can lead to alternations in the BA pool which in turn affect host metabolism and homeostasis. BAs have both direct and indirect antimicrobial effects on gut microbes [26] and act as a major regulator of the GM.
BAs can directly increase cell membrane permeability and lead to cell damage that suppresses bacterial (such as Clostridium cluster XIVa) proliferation [28][29][30]. These can also damage macromolecules such as DNA, interfere with RNA secondary structures, and promote protein misfolding leading to cell damage [18,29,30]. Additionally, BAs can induce oxidative stress by chelating critical cellular ions [31]. DCA is one of the most effective antimicrobial BAs and has ten times higher bactericidal activity than CA, which can positively prohibit GM proliferation [32]. The indirect effect of BAs on farnesoid X receptor (FXR) and vitamin D receptor (VDR) could act as an antimicrobial [28, 33,34]. Inagaki et al. [34] revealed that the FXR agonist GW4064 prevented the overgrowth of aerobic and anaerobic bacteria in the ileum and cecum of mice. Moreover, FXR activation could induce antimicrobial peptide production and regulate the host immune response. Further studies have shown that BAs such as CDCA and ursodeoxycholic acid (UDCA) act through FXR and VDR to induce the expression of cathelicidin, an antimicrobial peptide stored in the lysosomes of immune cells [28, 35,36].
A body of evidence has shown that the influence of diet and GMs, as well as Bas, may play an essential role in modulating colonic regulatory T-cells (Tregs) in vivo. In particular, a study performed by Ramanan et al. demonstrated that a deficiency in BAs causes a reduction in Tregs, contributing to inflammatory colitis progression [37]. As transgene-derived BAs are involved in intestinal immunity and inflammation, Ramanan et al. first silenced the BA conversion gene in various GMs, before the modified and nonmodified microbes were placed into the specially bred mice with no microorganisms in their gut. The result demonstrated that the Treg-cell population was significantly lower in mice intestines lacking the GMs and the BA conversion gene [37]. The finding also suggests that both GMs and food-derived BAs are important factors in immune modulation, as germ-free mice with food intake also had low levels of Tregs. Importantly, research have recently shown that the microbiota balances type 2 responses through the local induction of Treg cells that express the transcription factor ROR-γt [38]. Additionally, another study has showed the nongenetic transfer of an important immunoregulatory trait by immunologic means, for which the entero-mammary axis provides the mechanistic underpinning of multigenerational matrilineal transmission [37].

BAs and bile acids-activated receptors (BARs)
BAs are natural Ls of various receptors and are subjected to biotransformation by the resident microbial community to their unconjugated forms, a process that is critical to metabolic homeostasis [28,31,39]. Unconjugated BAs can activate intracellular signaling by binding in BA receptors, including FXR, pregnane X receptor (PXR), constitutive androstane receptor, VDR, and Gprotein coupled receptor GPBAR1 [15,39,40]. In humans, CDCA (CA in mice) is considered the most potent FXR L, whereas secondary BAs DCA and LCA are Ls for GPBAR1, PXR, and VDR. In addition, remaining BAs, such as HCA, can bind to liver-X-receptor α and β [41]. These receptors are highly expressed by innate and adaptive immune cells, including DC, macrophages, innate lymphoid 3 cells (ILC3s), and T helper 17 (Th17) cells [42]. This suggests that different BA species may regulate intestinal immunity by influencing the intestinal T-cell response to microbial antigens. Hematopoietic and nonhematopoietic cells of the innate immune system are located at the interface of the host microbiome and can recognize microorganisms and their metabolic products to regulate the host's physiological responses and microecology [43]. Therefore, understanding the mechanisms that control dysbiosis, changes in the GM, and the composition of BA is essential to comprehend the emergence of metabolic disorders [44][45][46].

GPBAR1 (TGR5)
GPBAR1 is a cell membrane receptor for secondary Bas, and it binds with both DCA and LCA and their T and G derivatives [47]. GPBAR1 is regarded as a metabolic regulator in BA synthesis, glucose metabolism, energy homeostasis, and a potent immune regulator. Interestingly, some evidence confirmed that the total BA pool size in TGR −/− mice decreased compared with that of WT mice, although the reason is still unknown [48]. GPBAR1 is highly expressed by several innate immune cells, including macrophages, monocytes, DCs, NK cells, and NKT cells [48][49][50]. As reported, the expression of GPBAR1 is negatively related to modulating inflammation in liver diseases, nonalcoholic steato-hepatitis, type 2 diabetes, and atherosclerosis [3,5,41,51].
Monocytes are the "sentinel cells" of the innate immune system and can differentiate into populations of DCs and macrophages, which have implications for the homeostasis of cells surrounding macrophages [52]. Interestingly, Kawamata et al. selectively addressed the inhibitory role of BA: GPBAR1 signaling on CD14 + peripheral blood monocytes and observed that the expression of GPBAR1 was dramatically reduced during DC differentiation from monocytes [53]. Macrophages are major regulators of cytokine production in the gastrointestinal tract, and a body of evidence has suggested that GPBAR1 activation by BAs plays a key role in modulating MU phenotype. In the M1like pro-inflammatory phenotype, GPBAR1-dependent transactivation of EGFR is essential for triggering SRC activation and its downstream cascades (ERK1/2 and PKC) [54,55]. This transactivation occurs when EGFR and GPBAR1 are co-localized on caveolae, lipid rafts enriched with cholesterol and sphingolipid, which plays a key role in modulating intracellular signaling. In this regard, metalloproteinase-dependent cleavage of the EGFR L HB-EGF occurs following GPBAR1 activation [56]. GPBAR1dependent PKC activation further stimulates the NF-κB pathway and the autocrine activation of ERK1/2, causing the release of pro-inflammatory cytokines such as interleukin-1β (IL-1β), IL-6, and TNF-α. SRC-dependent activation of ERK1/2 in the M1-like phenotype further promotes the c-MYC upregulation and results in cell death [57].
Innate immunity is also triggered by pro-inflammatory M1like signaling, which comes from stimulating the conversion of naive T cells in Th17 cells (increased IL-1β, IL-6) and Th1 cells (increased IL-12, IFN-γ). Meanwhile, it suppresses immunosuppressive Treg cells (decreased IL-10) via the production of proinflammatory cytokines [58]. During BA-dependent activation of GPBAR1, the pro-inflammatory M1-like phenotype is inhibited. At the same time, STAT3 is activated through SRC-dependent signaling, which upregulates the anti-inflammatory effects. In this respect, the level of Th17 and Th1 cells, TNF-α, IFN-β, IL-6, and BAs are absorbed by enterocytes through apical sodium-dependent bile acid transporter (ASBT). After transporting through the portal blood, BAs can be transported back to the liver. Primary BAs are converted into secondary BAs by the gut microbiome through modifications such as dehydroxylation and oxidation. Gut microbial mediated secondary BAs regulate DCs to attenuate inflammation through G-protein bile acid receptor 1 (GPBAR1). DCs induce TH1 and T helper 17 (TH17) cells by producing pro-inflammatory cytokines such as IL-1β and IL-6. GPBAR1, a bile acid-activated receptor, is also highly expressed in NKTs and monocytes/macrophages cells.
Moreover, the activation of GPBAR1 by BAs triggers the cAMP pathway signaling independently of EGFR transactivation. The activation of PKA by cAMP exhibits increased cAMP response element binding protein (CREB) expression and activity (Fig. 2). cFos, a key target gene of the CREB pathway, can bind to the p65 subunit of activated NF-κB, inhibiting its translocation into the nucleus and eliciting an anti-inflammatory response mediated by GPBAR1 activation. Besides, BAs can also induce the antiinflammatory capability of human macrophages by increasing the IL-10/IL-12 ratio in response to PKA activation [59,60].

FXR
FXR activation has direct and indirect effects on monocytes and macrophages including interactions with the atypical nuclear receptor small heterodimer partner (SHP) [45,65,66]. SHP binding via protein-protein interactions is necessary for FXR activation and induction of its target genes [67]. Yang et al. showed that SHP occupation stabilizes an inhibitory complex binding on the promoter of the chemokine (C-C motif) L CCL 2, thereby inhibiting the recruitment of the NF-κB p65 subunit, leading to reduced CCL2 expression in macrophages and subsequently the inhibition of cell migration and invasion [68].
The primary BAs that activate FXR can trigger the activation of iNOS, TNF-α, and IL-1β to the promoters of metabolism through binding to the nuclear receptor corepressor 1 (NCor1) complex. The NCor1 complex binds to the promoters of these genes, repressing NF-κB at the basal level and maintaining a state of transcriptional inactivity [69]. The release of NCor1 from the promoters upregulates the transcription of these genes and causes an upregulation of Toll-like receptor-4 (TLR-4) [45]. However, treatment with obeticholic acid (OCA), also called INT-747, stabilizes the NCoR1 complex on the iNOS and IL-1β promoters, leading to transrepression [46]. Additionally, Massafra et al. also found that OCA treatment induced an anti-inflammatory immune state in an experimental model of IBD. The state included the retention of DCs in the spleen, which was associated with a reduction in colonic inflammation. Therefore, these findings support the hypothesis that pharmacological FXR activation is an attractive new drug target for the treatment of IBD [70].
Remarkably, definitive evidence for the potential use of OCA in acute hepatitis has recently been provided by studies in murine models. Treatment with OCA led to an inhibition of the infiltration of NKT cells. It was shown that the gut microbiome uses BAs as messengers to control the mechanism of chemokine-dependent accumulation of hepatic NKT cells and hepatic antitumor immunity [71]. Besides, Willart et al. demonstrated that UDCA promotes DC-driven Th1 development by acting on the FXR [72]. Further findings also suggest that the activation of the bile salt nuclear receptor FXR is inhibited by pro-inflammatory cytokines through the activation of NF-κB signaling in the gut [73].
Recent investigation has also shown the effects of FXR in AIM2, NLRP1, NLRP3, and NLRC4 inflammasomes. They are a class of cytoplasmic multiprotein complexes that can sense endogenous and exogenous pathogen-or damage-associated molecular patterns (PAMP and DAMP, respectively). The typical inflammasome consists of nt-binding structural domains, leucine-rich repeat containing proteins (NLRs), and is absent in melanoma 2-like receptors (AIM2), which are in complex with the adapter protein ASC and caspase-1 [74]. Caspase-1 is a protease that triggers the cleavage of the cytokine precursors IL-1β and IL-18, which help the host to defend against infection [75]. NLRP3 is a known inflammasome that has been recognized in several diseases. Studies have shown that FXR functions as a negative modulator of NLRP3 assembly through an interaction with NLRP3 and caspase 1. Furthermore, SHP has been shown to suppress the formation of NLRP3 [76]. Notably, increased BA concentrations, which are often seen in patients or models of obstructive cholestasis, mediate the activation of the inflammasome [77]. A summary of BARs expression on immune cells and their mode of action is shown in Table 1.

The retinoid-related orphan receptors (RORs)
RORs are members of the nuclear receptor family of intracellular transcription factors. Three forms of ROR (ROR-α, -β, and -γ) are available and mapped to human chromosomes 15q22.2, 9q21.13, and 1q21.3, respectively [78]. Two different isoforms of ROR-γ are produced, ROR-γ1 and ROR-γt (or γ2), encoded by the RORC gene. RORs show a regulatory role in the circadian expression of clock genes and downstream targets in adipose tissues and the liver [79].
Interestingly, a major breakthrough led by immunologists Hang and Song has demonstrated the role of microbial-derived BA metabolites in modulating gut ROR-γ + Treg-cell homeostasis [27,80]. It has been shown that ROR-γt can bind to some oxygen derivatives of BAs, such as 3-oxo-LCA, as an inverse agonist. Indeed, ROR-γt is an essential transcription factor for thymic Tcell development, secondary lymphoid tissue organogenesis, and peripheral immune cell [81,82]. ROR-γt is expressed by Th17 cells, ILC3s, and γδ T cells [83,84]. In CD4 + T cells, ROR-γt is important for the differentiation and production of Th17 cells and for IL-17 production by ILC3 [85] (Table 1). Although RORs are thought to be orphan nuclear receptors, various oxygen steroids Table 1. A summary of bile acid-activated receptors (BARs) expression and mode of action in immunity is shown in Table 1, along with the expression and role of G-protein bile acid receptor 1 (GPBAR1), farnesoid-X-receptor (FXR), and ROR-γt in immune cells.

PXR
The pregnane X receptor (PXR) is a member of the nuclear receptor superfamily and is expressed in the liver and intestine as well as in other tissues and cells. The PXR is activated by a variety of endogens, dietary compounds, and drugs [87]. This nuclear receptor is a major transcriptional regulator of CYP3A isozymes and also regulates a large number of enzymes and transporters involved in the pharmacokinetics of endogenous and exogenous drugs [88]. PXR affects energy balance by regulating glucolipid metabolism. Activation of the PXR also protects the liver from toxic BA. A novel role for the PXR in intrahepatic homeostasis, IBD, hepatic steatosis, and fibrous formation has been demonstrated. PXR directly regulates the expression of multidrug resistance protein 1, and other important proteins involved in drug metabolism [89].

Drugs and agents: profiling of bars
Considering all research highlighting the anti-inflammatory activity of BA over the last recent years, the interest of the pharmaceutical industry has led to the development of several specific agonists for BARs. Table 2 summarizes clinical projects on BAs for the treatment of diseases. In parallel, some drugs that are classically used for other therapeutic purposes (e.g., rifaximin) and BAR activity have recently been proposed as regulators of immune cells phenotype. Another strategy consists of using specific strains of gut bacteria to regulate BA production in the intestine. Additionally, dietary supplements can be adapted to the target treatments to optimize BA production and BA metabolites.

Synthetic agonists of BARs
Given that several studies have pinpointed a potential beneficial effect of BA for the treatment of inflammatory diseases, recent research has focused on developing synthetic agonists of BARs to improve their bioavailability and modulate immunological response to improve the therapeutic outcome of inflammatory diseases, such as primary biliary cholangitis and IBDs.

FXR
Among the synthetic FXR agonist, OCA, INT-767, and GW4064 have shown the best pharmacological activity and therapeutic outcomes in preclinical and translational studies. The representative FXR agonist is OCA that has been the studied compounds in this category in recent years. It was first developed in 2002 and is semisynthetically produced based on CDCA, with an affinity for FXR that is more than two orders of magnitude higher than that of CDCA. It was approved by the FDA in 2016 for clinical use in the treatment of adult PBC patients who have had poor results with UDCA alone or who are intolerant to UDCA alone. For instance, oral administration of OCA promoted a good therapeutic response in experimental models of colitis [90,91]. Such an effect was accompanied by decreased leukocyte infiltration and reduction of pro-inflammatory cytokines in the colon, such as IL-1β, IL-6, and MCP-1 [90]. In vitro analysis demonstrated that OCA prevented TNF-α secretion by peripheral blood mononuclear cells (PBMCs), monocytes, DCs [90], and mucosal-associated invariant T cells [92]. Further, OCA inhibited the secretion of IFN-γ, IL-17, TNF-α, and IL-1β by lamina propria-derived mononuclear cells [90,91]. Similarly, INT-767 improved disease progression in experimental models of nonalcoholic liver diseases [93,94], such effects have been associated with the inhibition of NF-κB activation, reduced TNF-α production [93], and inhibition of MU recruitment to the liver [94]. Beside, in vitro INT-767 treatment inhibited the production of pro-inflammatory cytokines (TNF-α, IL-1β, and MCP-1) and increased IL-10 by BM-derived macrophages [94]. The synthetic FXR agonist GW064 has shown promising results in preclinical studies. For instance, oral administration of GW4064 (twice/daily) increased fasting plasma corticosteroid levels in C57BL/6 mice [95]. Treatment with GW4064 prevented exacerbated MU infiltration in the liver of a murine model of endotoxin-induced hepatic inflammation in nonalcoholic fatty liver disease [96]. Furthermore, the authors showed that LPSinduced mRNA expression of TNF-α, MCP-1, IL-1β, and IL-6 in high-fat diet fed mice was attenuated by GW4064 treatment [96]. In addition, in a murine model of diet-induced obesity and hepatic steatosis, GW4064 treatment significantly suppressed the diet-induced elevation of MU markers in the liver, such as F4/80, CD68, CD11c, and CD11b [97]. The protective effect of GW4064 on liver diseases was further confirmed by demonstrating that GW4064 attenuated LPS-induced expression of TNF-α and increased the expression of IL-10 in murine Kupffer cells, the resident macrophages of the liver [98]. Remarkably, the FXR agonist OCA showed a beneficial effect in mice subjected to experimental autoimmune encephalomyelitis (EAE), an in vivo model of MS [99]. In part, this effect was mediated by the inhibition of lymphocyte activation, in particular a decrease in CD4 + T cells and CD19 + B cells associated with downregulation of VLA4 (α4β1 integrin), which is necessary for lymphocyte extravasation across the blood-brain barrier [99].

GPBAR1
Several preclinical studies have shown the beneficial effects of synthetic GPBAR1 agonists in modulating immune responses and improving therapeutic outcomes. In particular, INT-777 has been extensively used due its anti-inflammatory activity in immune cells. For instance, pretreatment of BM-derived macrophages with INT-777 in vitro prevented the LPS-induced expression of TNF-α, IL-6, CXCL-10, and MCP-1, while increasing the expression of the anti-inflammatory cytokine IL-10 [99,100]. Pretreatment of mouse peritoneal exudate macrophages (PEMs) with INT-777 in vitro inhibited vesicular stomatitis virus infection in a concentration dependent manner. Besides, INT-777 treatment-induced IFN-β expression in peritoneal exudate macrophages exposed to vesicular stomatitis virus and herpes simplex virus type 1 (HSV-1) via AKT-mediated IRF3 activation [100,101]. Treatment of primary macrophages with INT-777 reduced the ability of MU recruitment toward the chemokine CCL2. Further, INT-777 treatment attenuated the LPS-dependent genic upregulation of CCL2, CC3, and CCL4 in BM-derived macrophages which was mediated by AKT-dependent activation of mTOR complex 1 [102]. Besides, its protective effect on in vitro studies INT-777 also has protective effects on experimental disease models. In a mouse model of acute pancreatitis induced by caerulein, INT-777 reduced serum levels of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) and inhibited the activation of the NLRP3 inflammasome pathway [103]. By using a murine model of Parkinson's disease, intranasal administration of INT-777 ameliorated motor deficits and cognitive impairment. Further, the authors have found that INT-777 prevented Parkinson's disease-induced microglial activation, which was associated with the downregulation of TNF-α and chemokines CCL3 and CCL6 in the brain, preventing leukocyte infiltration into the brain [104]. In a rat model of subarachnoid hemorrhage, intranasal administration of INT-777 prevented microglial activation and neuroinflammation, as evidenced by a reduction in Iba-1 and CD68 positive microglia. Additionally, the authors observed that INT-777 treatment inhibited NLRP3 activation and expression of IL-6, IL-1β, and TNF-α, resulting in reduced neutrophil infiltration in the brain [105]. Lastly, in rats subjected to middle cerebral artery occlusion (MCAO), an experimental model of stroke, intranasal administration of INT-777 reduced infarct volume and improved neurological scores. Further analysis of brain homogenates showed that INT-777 treatment prevented middle cerebral artery occlusion-induced NLRP3 activation and production of TNF-α, IL-1β, and IL-18 [106].
Festa et al. designed and synthesized 6b-Ethyl-3a,7bdihydroxy-5b-cholan-24-ol (BAR501), an alcohol produced on a methoxycholine scaffold. This represents the first example of a UDCA derivative substituted at C-6 with a β-directed ethyl group and is a rather efficient L for GPBAR1 [107]. Biagioli et al. showed that BAR501 protects against colitis induced by TNBS. Ablation of the GPBAR1 gene enhanced the recruitment of classically activated macrophages in the colonic layer and exacerbated the severity of inflammation [108]. Exposure to BAR501 shifted intestinal macrophages from a classically activated (CD11b + , CCR7 + , F4/802) to an alternative activated (CD11b + , CCR72, F4/80 + ) phenotype and reduced the expression of inflammatory genes.

PXR
Although the synthetic PXR agonists have not been as extensively described as FXR and GPBAR1 agonists, recent studies showed the promising effects of such drugs. Rifaximin, a poorly absorbed oral antimicrobial agent, has been recently used in the treatment of IBDs due to its agonistic activity on PXR in the gut. Its antiinflammatory activity was confirmed by in vitro studies showing that pretreatment with rifaximin prevented LPS-induced production of TNF-α, IL-8, PGE2, and RANTES in supernatants of human colon epithelial cells. Such effect was associated with the suppression of LPS-induced NF-κB DNA binding activity, which possibly was mediated through the increased association between PXR and NF-κB p65 [109]. In a murine model of DSS-induced colitis, treatment with the synthetic PXR agonist PCN attenuated body weight loss, diarrhea, rectal bleeding, and reduction of colon length [110]. Further mRNA analysis of colonic tissue has shown that PCN treatment downregulated DSS-induced genic expression of several inflammatory markers, such as IL-1β, TNF-α, IL-6, iNOS, and MCP-1. In vitro studies using colon cells (HCT116 cells) suggested that such effects were possibly mediated by the inhibition of NF-κB activation [110]. The relevance of PXR agonists on the regulation of immune response was confirmed by a study showing that synthetic PXR agonists PCN and RU-486 decreased the expression of the cell surface activation marker CD25 in murine T lymphocytes, whereas rifampicin and RU-486 reduced the expression of CD25 in Jurkat T cells [111]. Besides, RU486 treatment decreased T lymphocyte-derived IFN in a PXR-dependent manner, suggesting that PXR L compromised T lymphocyte function by suppressing CD25 expression and IFN-γ. Additionally, PCN treatment reduced the level of phosphorylated p65 NF-κB and MEK1/2 in activated lymphocytes, which are important signals for lymphocyte proliferation [111].

VDR
The anti-inflammatory activity of VDR has led to the development of specific and stable agonists. For instance, EB1089 promotes the conversion of Th2 cells into Tregs, which possess immune suppressive functions. In vitro treatment of CD4 + , CD25 + , and CD127 + cells from ulcerative colitis with EB1089 resulted in their conversion into FoxP3 + Tregs, which can suppress effector T cell proliferation [112]. Synthetic VDR Ls LY2108491 and LY2109866 were effective in preventing the genic upregulation of IL-2 and IL-4 induced by TPA and PHA, whereas they increased anti-inflammatory IL-10 in PBMCs. Additionally, VDR agonists decreased the Th1 cytokine response in vivo, as demonstrated by reduced IFN-γ and IL-2 production by splenocytes from mice treated with LY2109866 [113].

Strategies modulating BA metabolism
Recently, a body of evidence has proposed that the treatment for some diseases such as cholestatic liver disease, fatty liver disease, diabetes mellitus, gallstones, obesity, and metabolic syndrome may benefit from modulating the FXR, PXR, constitutive androstane receptor, and VDR targets [114]. They make excellent therapeutic targets because they participate in the transport and metabolism of BAs, which are connected to BA signaling and BA pool size and composition. For instance, phases II and III clinical trials are currently evaluating the therapeutic effect of UDCA treatment and the synthetic FXR agonist PX-102 [115]. The FDA has approved the BA derivative OCA for the treatment of Primary Biliary Cholangitis (PBC) in adults who have an unsatisfactory response to UDCA or as a monotherapy in individuals who are unable to tolerate UDCA [116].
Further studies have described the 24-norursodeoxycholic acid, a UDCA derivative produced by removing a methylene group, which is more hydrophilic and less toxic than UDCA, and it can be passively absorbed by cholangiocytes [117]. As a consequence of 24-norursodeoxycholic acid-induced secretion of bicarbonate (HCO 3 − ) from biliary cells, an alkaline "umbrella" is formed on the apical surface of cholangiocytes, which inhibits the entry of apolar hydrophobic BAs into cholangiocytes and hepatocytes [117].
Noticeably, emerging studies have demonstrated the potential therapeutic advantages of GM in managing hyperammonemia. For instance, the administration of genetically modified Lactobacillus plantarum, which consumes ammonia, has showed benefits compared to WT bacteria [118]. Mice treated with the ammonia-hyperconsuming L. plantarum strain had reduced ammonia levels in the blood and feces as well as increased survival rates [118]. Furthermore, microencapsulated L. plantarum 80 (pCBH1) was found to effectively degrade and eliminate BAs (GDCA and TDCA) in vitro, supporting this theory [119]. Such techniques could be more advantageous than modulating the entire gut microbiome as genetically modified strains are more productive and can be designed to target specific objectives.
The diverse compositions of BA in mice and humans present various physical challenges and intervention effects. For example, mice produce tauro-MCA, a potent FXR antagonist that can preserve intestinal integrity and act as an intestinal FXR antagonist due to it is resistance to bacterial bile salt hydrolase [120]. As a result, oral administration of the tauro-MCA derivative glycine-MCA ameliorated insulin resistance, hepatic steatosis, and obesity linked to lower blood ceramide levels [120]. However, mousebased data on BA control in glycemic reactions may not necessarily translate to human, and such findings should be carefully analyzed before clinical trials.
Lastly, SCFAs are recognized as the primary byproduct of saccharide microbe fermentation in the gut [121]. SCFAs have the potential to reduce the severity of immune-mediated and endotoxin-induced uveitis [122]. Recent investigations have also shown that people with bowel disease had high amounts of fecal acetate and low levels of fecal butyrate [123]. Additionally, the feces of people with bowel diseases showed decreased levels of bacteria that produce butyrate [11].

Conclusions and perspectives
BAs serve as signaling molecules that facilitate communication between the intestinal immune system and the GM. Leuko-cytes play a crucial role in the formation of immune cells and express FXR and GPBAR1, which are essential for intestinal MU immunological tolerance based on gene KO studies. However, the role of BAs and BARs in regulating intestinal immunity is still being explored. BARs, including FXR, GPBAR1, VDR, and liver-X-receptors, are highly expressed in innate immune cells (monocytes, macrophages, DCs, NKs, and NKTs), but to a lesser extent in adaptive immune cells (T and B cells). It is unclear whether this phenomenon can result in the regulation of functional activity in pathological conditions. Activation of BARs in macrophages, DCs, and NKTs causes regulatory functions that are inhibitory in nature. Moreover, GPBAR1 and FXR contribute to fine-tuning the tolerogenic state of the liver and intestine innate immunity in response to bacterial and endogenous antigens [41,124].
Recent research has demonstrated that BA metabolism contribute to modulating T-cell innate immunity by balancing proinflammatory T-helper cells and anti-inflammatory cells (Treg cells) in both the intestine and periphery [125][126][127][128][129]. In addition, various BA oxo-derivatives regulate the adaptive immune system by influencing ROR-γt activity. These BA oxo-derivatives function as ROR-γt inverse agonists, specifically inhibiting Th17 cell-specific inflammation [130].
Dual and selective GPBAR1/FXR agonists have been shown to effectively reverse inflammation in mice with vascular inflammation and autoimmune encephalitis. BAR Ls can be regarded as potential drugs in the treatment of clinical disorders caused by metabolic and immune dysfunctions [131][132][133].
For FXR, it provides counterregulatory signals in macrophages, DC, and NK cells that are partly exerted by SHP. However, it is still unknown whether these effects manifest in patients administered with an FXR agonist. Activation of GPBAR1 induces the transcription of IL-10 in macrophages and prevents the accumulation of activated macrophages [108,134,135]. In addition to macrophages, GPBAR1 is expressed in DCs to regulate innate immunity, and its activation attenuated DCs polarization toward an IL-12 and TNF-α-biased phenotype [41,59,136].
FXR and GPBAR1 have been regarded as effective drugs. However, the severe side effects such as those observed with FXR agonists for NASH treatment cannot be ignored [137]. GPBAR1 has recently attracted researchers' interest due to its beneficial effects on inflammation. Recent findings suggest that selective GPBAR1 or dual FXR/GPBAR1 Ls may be developed, rather than GPBAR1 acting solely as a mediator of itching in humans. This approach could potentially overcome some of the side effects observed in clinical trials. Further understanding of BAs and their receptors, including FXR, GPBAR1, and ROR-γt, can help us comprehend the communication between the innate and adaptive immune system and the intestinal microbiota [41].