Role of HCA2 in Regulating Intestinal Homeostasis and Suppressing Colon Carcinogenesis

Hydroxycarboxylic acid receptor 2 (HCA2) is vital for sensing intermediates of metabolism, including β-hydroxybutyrate and butyrate. It also regulates profound anti-inflammatory effects in various tissues, indicating that HCA2 may serve as an essential therapeutic target for mediating inflammation-associated diseases. Butyrate and niacin, endogenous and exogenous ligands of HCA2, have been reported to play an essential role in maintaining intestinal homeostasis. HCA2, predominantly expressed in diverse immune cells, is also present in intestinal epithelial cells (IECs), where it regulates the intricate communication network between diet, microbiota, and immune cells. This review summarizes the physiological role of HCA2 in intestinal homeostasis and its pathological role in intestinal inflammation and cancer.


INTRODUCTION
The intestinal tract is an organ system with specialized architecture that functions to digest food, and extract and absorb energy and nutrients. It also secretes over 20 different hormones and harbors more than 640 different species of bacteria (1). Physiological and pathophysiological events that trigger the breakdown of intestinal homeostasis negatively impact intestinal health, and may result in intestinal disorders including inflammatory bowel disease (IBD) and colitis-associated cancer. IBD is a chronic and life-threating disease characterized by prolonged inflammation of the digestive tract (2,3). IBD encompasses two conditions, Crohn's disease and ulcerative colitis. Crohn's disease can affect any part and layer of the gastrointestinal tract, while ulcerative colitis is usually limited to the innermost layer of the colon and rectum (4). Both Crohn's disease and ulcerative colitis are characterized by episodes of fatigue, abdominal cramping, rectal bleeding, diarrhea, weight loss, and the influx of immune cells that produce cytokines, proteolytic enzymes, and free radicals (5,6). Patients with IBD are at increased risk of developing colitis-associated cancer which is difficult to treat and has high mortality (>50%) (7,8). In 2015, an estimated 1.3% of US adults reported living with IBD, with cases increasing worldwide (9,10). The global spread of IBD is associated with the host genetic background, intestinal microbiota, diets, environments and immunological dysregulation (4,11,12).
The intestinal tract represents the largest compartment of the immune system in the body (13), with intestinal health implicated in controlling disease development not only within itself but also throughout the body. To maintain intestinal homeostasis, a multi-pronged approach including the immune system, microbial ecosystem and diet is necessary. A versatile receptor, hydroxycarboxylic acid receptor 2 (HCA 2 ), is capable of both nutrient sensing and immunomodulation, lending to its popularity as a potential target for the promotion of intestine health.
In 1993, HCA 2 was identified as an orphan receptor (GPR109A) (14,15), and later described in mice as a "protein upregulated in macrophages by interferon-gamma (IFN-γ)" (PUMA-G) (16). In 2003, several studies reported that HCA 2 is a receptor for niacin and functions to mediate its antilipolytic effects in adipocytes (17)(18)(19). Benyó et al. and Hanson et al. subsequently demonstrated that binding of niacin to HCA 2 on Langerhans cells and keratinocytes is also responsible for the niacin-induced cutaneous flushing reaction, involving release of prostaglandin D 2 (PGD 2 ) and prostaglandin E 2 (PGE 2 ) (20,21). In 2005, the ketone body β-hydroxybutyrate (β-OHB) was identified as an endogenous ligand of HCA 2 (22). This resulted in the deorphanization of the receptor, which was subsequently renamed hydroxycarboxylic acid receptor 2 (HCA 2 ) (23). Most recently, butyrate, a short-chain fatty acid (SCFA) bacterial product in the colon lumen generated at high concentrations (10-20 mM) from dietary fiber fermentation, was recognized as an endogenous ligand of HCA 2 (24). Butyrate activation of HCA 2 plays an important role in the maintenance of intestinal homeostasis (24). New synthetic ligands of HCA 2 have been developed, such as acipimox, GSK256073 and derivatives of pyrazole-3-carboxylic acid or cyclopentapyrazole (25)(26)(27).
HCA 2 is widely expressed in various tissues and cell types, including adipose tissue, spleen, lung, lymph node and intestine. HCA 2 is predominantly expressed not only in both white and brown adipocytes, but also in diverse immune cells, including dendritic cells (DCs), monocytes, macrophages, neutrophils and epidermal Langerhans cells, but not lymphocytes (16,18,21,28,29). Interestingly, several cytokines show the ability to regulate the expression of HCA 2 in immune cells. HCA 2 expression is upregulated in macrophages and monocytes after IFN-γ treatment (16), and the expression of HCA 2 in macrophages is significantly increased by proinflammatory stimulants lipopolysaccharide (LPS), interleukin (IL)-6 and IL-1β (30). Colony-stimulating factor 2 (CSF2) increases HCA 2 expression level in neutrophils (29). HCA 2 is also present in intestinal epithelial cells (IECs), retinal pigment epithelium, hepatocytes, keratinocytes and microglia (21,(31)(32)(33)(34). Notably, both mRNA and protein levels of HCA 2 in IECs are drastically reduced in germ-free mice compared to conventional mice, due to the absence of gut bacteria. These changes are reversed when the intestinal tract of germ-free mice is re-colonized (35).

INTESTINAL HOMEOSTASIS AND HCA 2 : IMMUNE CELLS, INTESTINAL EPITHELIUM, MICROBIOME, AND METABOLITES
The intestinal tract, comprised of small intestine, large intestine/colon, and rectum, is the central location for nutrient and water absorption. It harbors more than 10 13 microorganisms, contains over 20 different hormones, and serves as the single largest immune compartment in the body (13,59). Consequently, building and maintaining a homeostatic intestinal tract is a highly complex and broad concept that encompasses a multi-disciplinary approach including the immune system, host cells, gut microbiota and nutrients. Further complexity arises from the mutual interactions between the intestinal tract and other organ systems.
The intestinal mucosa, a crucial site of innate and adaptive immune regulation, is comprised of IECs, lamina propria and muscularis mucosa (Figure 2). IECs are specialized epithelia comprised of many different cell types: epithelial stem cells which continuously self-renew by dividing and generate all differentiated intestinal cell types, enterocytes which absorb water and nutrients, goblet cells which secrete mucins to form a mucus layer boundary between the gut microbiota and host tissue, Paneth cells which secrete anti-microbial peptides, enteroendocrine cells which secrete hormones and cytokines capable of systemic or local effects, and microfold cells (M cells) which connect to the intestinal lymphoid follicles (60)(61)(62)(63). The intestinal epithelium is bound together by tight junction proteins, which regulate the paracellular permeability and are essential for the integrity of the epithelial barrier. Tight junction proteins prevent harmful substances such as LPS, foreign antigens, toxins and microorganisms from entering into the blood stream (64). In adipocytes, activation of HCA 2 triggers a Gαi-mediated inhibition of adenylate cyclase activity, which leads to lower intracellular cAMP levels, reduced protein kinase A (PKA) activity, and further reduces the activity of hormone sensitive lipase (HSL), an important lipolytic enzyme. This inhibition of lipolysis results in a decreased release of free fatty acids into the circulation. (B) In hepatocytes, activation of HCA 2 mediates the protein kinase C (PKC)-extracellular signal-regulated kinase (ERK) signaling pathway, leading to phosphorylation of AMP-activated protein kinase (AMPK) and inhibition of acetyl CoA carboxylase (ACC). This results in an inhibition of hepatic de novo lipogenesis and a remarkable decrease of lipid accumulation in liver. (C) In colonocytes, ligand binding to HCA 2 suppresses NF-κB activation and activates NLRP3 inflammasome, which recruits caspase-1 and promotes the maturation of IL-18 for secretion. (D) In retinal pigment epithelium, HCA 2 exerts either an anti-inflammatory response through the Gαi/cAMP/NF-κB pathway, or apoptosis through the Gβγ/Ca 2+ /NOX4/ROS/JNK pathway. (E) Within Langerhans cells, HCA 2 -mediated Gα i activation primarily results in the Gβγ-complex released from activated Gα i , thereby increasing intracellular calcium concentration by mobilizing Ca 2+ release from the endoplasmic reticulum and ultimately driving the formation of PGD 2 and PGE 2 , which are released to the dermal layer and cause cutaneous flushing reaction. (F) In macrophages, activation of HCA 2 involves inhibition of NF-κB, thereby exerting an anti-inflammatory effec. HCA 2 activation in macrophages also represses F-actin and blocks Gβγ signaling to inhibit chemokine-induced migration of macrophages. HCA 2 also shows anti-inflammatory effects in Parkinson's disease models by inhibiting the phosphorylation of the NF-κB p65 signaling pathway in microglia. HCA 2 suppresses LPS-induced NF-κB activation in human monocytes, resulting in decreased transcription of IL-6, TNFα, and MCP-1. In DCs, HCA 2 activation decreases IL-6 level and increases IL-10 level, also upregulates expression of RALDH1, which synthesizes RA from retinol. RA and IL-10 promote Treg cell proliferation. In neutrophils, niacin-mediated HCA 2 activation inhibits PKA activity and subsequently increase BAD levels, which drives apoptosis of neutrophils.
IECs are well-equipped to recognize luminal pathogens by expressing different pattern recognition receptors, including NOD-like receptors (NLRs) in the cytosol and Toll-like receptors (TLRs) on the apical membrane and in endosomes, with the capacity to sample gram-positive and gram-negative infectious bacteria (65,66). Additionally, various immune cells, including intraepithelial γδ-T cells and specialized mucosal macrophages, reside intercalated in the IEC layer, and function to sample pathogens from the lumen (67). IECs also express multimeric protein complexes known as inflammasomes that are important for intestinal immune homeostasis, inflammation, and tumorigenesis. Ligand stimulation of HCA 2 expression is associated with increased NLRP3 inflammasome activation, which processes the proIL-18 into IL-18, an anti-inflammatory cytokine which is critical in regulating mucosal immunity and epithelial integrity (46) (Figure 2). Recent studies demonstrate that mice deficient in IL-18 have increased pathogenesis of colitis and colon cancer, and dysregulation of IL-1β expression exacerbates IBD (48,68).
Immune cells are found in intestinal epithelium (intraepithelial lymphocytes) as well as in organized lymphoid tissues/organs, such as the Peyer's Patches (PPs) and mesenteric lymph nodes (MLNs). Substantial amounts of scattered innate and adaptive effector immune cells are also widely distributed in the lamina propria, which is a loosely packed connective tissue layer underlying the IEC layer (69-71) (Figure 2). Collectively, the lamina propria and IECs form FIGURE 2 | HCA 2 regulates gut immune homeostasis. Butyrate produced by gut microbiota fermentation activates HCA 2 expressed on IECs, macrophages, and dendritic cells. In IECs, HCA 2 stimulation is associated with increased inflammasome activation, which processes pro-IL-18 into mature IL-18, which is critical in regulating mucosal immunity and epithelial integrity. IL-18 and ligand induced anti-inflammatory IL-10 (from intestinal macrophages and dendritic cells) promote naïve T cells differentiation and proliferation into immunomodulator Treg cells, which protect intestine from inflammation and colitis associated cancer. IL-18 and IL-10 also decrease the proinflammatory Th1 and Th17 cell number. In addition, HCA 2 decreases proinflammatory cytokine IL-6 and TNF-α expression in intestinal macrophages and inhibits dendritic cell-induced IL-23 production to suppress ILC3-associated colonic inflammation. a unique immunological compartment which contains the largest population of immune cells in the body, as well as supply the nerve, blood and lymph drainage for the entire mucosa (71). The lamina propria contains lymphocytes and numerous innate immune system-related cell populations, including eosinophils, macrophages, DCs, immunoglobulin (Ig) A secreting plasma cells, mast cells and innate lymphoid cells (ILCs) (71-73) (Figure 2). ILCs are a family of three innate effector cells (ILC1, ILC2, and ILC3) that are critical modulators of mucosal immunity (74). Particularly, ILC3 is implicated in innate intestinal inflammation though production of IFN-γ, IL-17, and IL-22 under induction by IL-1β and IL-23 (75). Depletion of ILC3 abrogates innate colitis, suggesting ILC3 is responsible for the intestinal pathogenesis (75). Bhatt et al. showed that HCA 2 signaling limits IL-23 production by DCs, which further suppresses ILC3-mediated colonic inflammation (76) (Figure 2). Activation of HCA 2 expressed on immune cells in colon lamina propria also modulates the frequency and number of Treg cells and IL-10 producing T cells (34) (Figure 2).
The gut microbiota is considered a commensal metabolic organ with critical roles in energy salvaging and nutrient absorption. It also functions in systemic immunity regulation and protection of the colonized host by eliminating pathogenic bacteria (77). Tan et al. compared fecal microbiota composition between WT and HCA 2 −/− mice fed a high-fiber diet, and determined that loss of HCA 2 alters microbiota composition dramatically (57). Specifically, HCA 2 −/− mice show an increase of Verrucomicrobiae, Alphaproteobacteria, and Bacilli, and a decrease of Bacteroidia (57). Germ-free animals show extensive impaired maturation of isolated lymphoid follicles, PPs and MLNs, and are also defective in antibody production and cytokine secretion compared to conventional animals (78). The status of germ-free animals converts after colonization with normal gut microbiota, suggesting a dynamic relationship between the commensal organism and host immune system. Gut microbiota also plays an irreplaceable role in the regulation of host intestinal gene expression with around 700 genes altered remarkably in mice under germ-free conditions. Among them, the expression of Hca 2 is reduced significantly in the ileum and colon under germ-free conditions, which is restored to normal levels after introduction of gut bacteria (35). When the balance of gut microbiota ecosystems is disturbed (dysbiosis), tight junction barrier is compromised. Antigens, toxins and microorganisms can pass through the epithelium and trigger the immune response. Intestinal dysbiosis is commonly associated with a series of intestinal and extraintestinal pathological disorders, including obesity, diabetes mellitus, multisystem organ failure, allergy, asthma, colitisassociated cancer and IBD (77,79). Specifically, IBD patients shift their gut microbiota composition to an enrichment of Desulfovibrio, Enterobacteriaceae, Ruminococcus gnavus, and depletion of Akkermansia Faecalibacterium prausnitzii, and Lachnospiraceae (80).
Multiple evidence suggests that the composition of the intestinal microbiota can be altered by diet within hours to days, leading to aberrant immune responses (81)(82)(83). Extensive studies have demonstrated that the structure and function of the gut microbiota rapidly shifts and intestinal atrophy and low-grade inflammation occur under Western diets conditions within 1 day (84)(85)(86)(87)(88)(89). Nevertheless, this influence is largely eliminated by manipulating the dietary fiber content in Western diets, allowing for protection against microbiota depletion, amelioration of the inflammation and restoration of colon length (84,88). These beneficial aspects of fiber are largely attributed to bacterial fermentation products (SCFAs), including acetate, propionate and butyrate. SCFAs are sensed by specific immunomodulating receptors, including HCA 2 , GPR41, and GPR43, which are involved in intestinal immunoactivity, intestinal motility regulation and cytokine secretion (90).
Among the SCFAs, butyrate/HCA 2 -mediated signaling has received the most attention for its effects on intestinal homeostasis and may provide an important molecular link between gut bacteria and the host (91)(92)(93)(94). Numerous studies have confirmed that antibiotic treatment causes gut microbiota dysbiosis by perturbing intestinal immune regulation, evidenced by a reduction in Treg cell numbers within the colon (95,96). Niacin and HCA 2 agonist supplementation efficiently rescues Treg cell depletion in antibiotic-treated WT mice, but this effect is nullified in HCA 2 −/− mice (34). HCA 2 −/− mice also show an inflammatory intestinal phenotype and enhanced susceptibility to azoxymethane (AOM) + dextran sulfate sodium (DSS)induced colitis-associated colon cancer (34). Clinically, patients with ulcerative colitis and colitis-associated cancer suffer a remarkable depletion in the total amount of butyrate-producing bacteria in colon (97,98), while irrigating the colon with butyrate significantly suppresses intestinal inflammation during ulcerative colitis (99). Hence, HCA 2 is a critical link in the network of diet, microbiota, immune cells, and host cells which are necessary for the maintenance of intestinal homeostasis.

ROLE OF HCA 2 IN INTESTINAL INFLAMMATION
The role of HCA 2 in regulating intestinal immunological response and inflammation is multifaceted. Singh et al. found that the colons of mice lacking HCA 2 present a unique status of CD4 + T cells, also known as T helper cells (Th cells) (34). These cells play an important role in immune regulation, where they mediate the activation of other immune cells though the release of various cytokines. Among the CD4 + T cells, Tregs express the transcription factor Forkhead box protein P3 (Foxp3), which is capable of potently suppressing immune responses. In colonic lamina propria of HCA 2 −/− mice, the amount of Foxp3 + /Treg cells among CD4 + T cells and antiinflammatory IL-10 producing CD4 + T cells is significantly less than WT mice, while the frequency and number of CD4 + T cells producing the inflammatory cytokine IL-17 are increased (34). In contrast, a similar fraction of those cells distribute in splenic T cells from both WT and HCA 2 −/− mice, suggesting that only the colon CD4 + T cells are specifically influenced by a lack of HCA 2 −/− (34). Singh et al. reasoned that this proinflammatory phenotype of the HCA 2 −/− mice colon is dependent on colonic DCs and macrophages, since they both express HCA 2 and are critical inducers of naive T cell differentiation (100,101). They addressed this by testing the ability of colonic DCs and macrophages from both WT and HCA 2 −/− mice to induce differentiation of naive CD4 + T cells. As expected, HCA 2 −/− colonic DCs and macrophages are defective in expression of retinaldehyde dehydrogenase 1 (RALDH1) and immunosuppressive cytokine IL-10, and express more proinflammatory cytokine IL-6 compared to WT DCs and macrophages. This change in expression leads naive CD4 + T cells to differentiate into proinflammatory Th17 cells, but not Treg cells and IL-10-producing CD4 + T cells (34). Likewise, HCA 2 −/− is necessary to maintain normal anti-inflammatory IL-18 levels, as both mRNA and protein expression of IL-18 are significantly decreased in IECs of HCA 2 −/− mice (34). Consistent with this evidence, Singh et al. also demonstrated that niacin treatment restored colonic Treg cell numbers in antibiotic-treated WT mice, and butyrate and niacin induced IL-10 and RALDH1 expression and promoted naïve T cells differentiation into Treg cells in macrophages and DCs in an HCA 2 -dependent manner (34) (Figure 2). In addition, butyrate and niacin increased expression of IL-18 in colonic epithelium of WT mice but not HCA 2 −/− mice (34). Bhatt et al. recently described another anti-inflammatory effect of HCA 2 in restraining microbiota-induced IL-23 production to suppress ILC3-associated colonic inflammation (76). To diminish the influence of the adaptive immune system, they bred HCA 2 −/− mice with recombination activating gene 1 (RAG1) deficient mice [no mature B and T lymphocytes (102) (76). Further detailed studies on the role of HCA 2 in DSSinduced colitis treatment demonstrate that HCA 2 −/− mice are highly susceptible to colitis development, with all experimental animals succumbing to death 10 days after DSS administration (3). In contrast, WT counterparts all survive through the entirety of the DSS treatment (34). Sodium butyrate markedly reduces inflammation and improves IECs barrier integrity by activating HCA 2 signaling and suppressing the AKT-NF-κB p65 signaling pathway in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis, a model that resembles Crohn's disease (51,52). In a similar study, a sodium butyratecontaining diet attenuates diarrhea symptoms and facilitates tight junction protein expression in the colon of piglets by acting on Akt signaling pathway in an HCA 2 -dependent manner (103). Another source of butyrate, tributyrin, is a chemically stable structured lipid that could be administered orally (104). Tributyrin supplementation prevents mice from chronic and acute ethanol-induced gut injury by improving gut barrier function (occludin, ZO-1) and increasing the expression of HCA 2 in both ileum and proximal colon (105). In accordance with this, niacin administration attenuates iodoacetamideinduced colitis by a reduction in colon weight and colonic myeloperoxidase activity (a hallmark of colonic inflammation), and restores normal levels of colonic IL-10, tumor necrosis factor alpha (TNF-α), angiostatin and endostatin in a rat model (106). This beneficial effect of niacin is largely abolished by mepenzolate bromide, a HCA 2 receptor blocker, indicating niacin/ HCA 2 signaling ameliorates iodoacetamide-induced colitis (106). In addition to it oral pharmacologic activity, niacin is also a microbial-derived metabolite, produced by specific gut microbiota, including Lactobacillus acidophilus, Bacteroides fragilis, Prevotella copri, Fusobacterium varium, Clostridium difficile, Bifidobacterium infantis, and Ruminococcus lactaris (76,107,108). Niacin deficiency is associated with intestinal inflammation and diarrhea (76).
Overall, these reports provide compelling evidence that HCA 2 signaling modulates immune cells to inhibit production of several inflammatory cytokines, pathways, and enzymes, leading to the suppression of experimental models of colitis.

ROLE OF HCA 2 IN COLON CANCER
HCA 2 not only plays a critical role in the suppression of intestinal inflammation, but also has a significant effect on colonic cancer development and progression. Expression of HCA 2 is silenced in colon cancer cell lines, and in both mice and humans with colon cancer (24). The tumor-associated silencing of HCA 2 involves DNA methyltransferase 1 (DNMT1)mediated DNA methylation (24) (Figure 3A). Reexpression of HCA 2 in cancer cell lines induces apoptosis by inhibiting Bcell lymphoma (Bcl)-2, B-cell lymphoma-extra-large (Bcl-xL), cyclin D1 and NF-κB activity and upregulating the death receptor pathway in a ligand-dependent manner ( Figure 3B). Butyrate is also an inhibitor of histone deacetylases, but this HCA 2 -mediated effort in colon cancer cells does not involve repressing histone deacetylation (24). Strikingly, Bardhan et al. discovered that IFN-γ reverses DNA methylation-mediated HCA 2 silencing without altering the methylation status of the HCA 2 promoter in colon carcinoma cells (109). Signal transducer and activator of transcription 1 (STAT1) is rapidly activated by IFN-γ and binds to the p300 promoter to activate p300 transcription. p300 is a histone acetyltransferase and a master transcriptional mediator in mammalian cells, resulting in a permissive chromatin conformation at the HCA 2 promoter to allow STAT1 to activate HCA 2 transcription despite DNA methylation (109) (Figure 3B).
Consistently, HCA 2 −/− mice are more susceptible to the development of colon cancer (34,46,106,110). In mouse models of inflammation-associated colon cancer caused by AOM and DSS, colons of HCA 2 −/− mice shrink with highly increased myeloperoxidase activity, upregulated expression of colon cancer-promoting genes such as cyclin-D1, cyclin-B1, and cyclin-dependent kinase 1, decreased tight junction proteins expression and decreased expression of genes that inhibit colitis and colon carcinogenesis, such as transforming growth factor beta (Tgfb)1, Tgfb2, Solute Carrier Family 5 Member 8 (Slc5a8), MutS Homolog (Msh)2, and Msh3 (34) (Figure 3C). In addition, HCA 2 −/− mice exhibit a severe impairment of IL-10 and IL-18 production when compared to WT counterparts (34,111) (Figure 3C). Histologically, crypt and epithelium structure damage, mucosa ulcerations and large amount of immune cell infiltration is observed in colons of AOM+DSS treated promoter. STAT1 is rapidly activated by IFN-γ and binds to the p300 promoter to activate p300 transcription, resulting in a permissive chromatin conformation at the HCA 2 promoter to allow HCA 2 transcription in colon carcinoma cells. Reexpression of HCA 2 in cancer cell lines induces apoptosis by inhibiting of Bcl-2, Bcl-xL, cyclin D1, and NF-κB activity and upregulating the death receptor pathway in a ligand-dependent manner. (C) In mouse models of inflammation-associated colon cancer caused by AOM and DSS, the intestinal epithelium of HCA 2 −/− mice display upregulated expression of colon cancer-promoting genes and decreased genes that inhibit colitis and colon carcinogenesis. HCA 2 −/− mice also exhibit a severe impairment of IL-10 and IL-18 production, which leads to a decrease in Treg cell number.
HCA 2 −/− mice group, indicating epithelial barrier breakdown (34). Systemically, levels of both colonic and serum cytokines that promote colonic inflammation and carcinogenesis such as amyloid A, chemokine (C-X-C motif) ligand (CXCL) 1, C-C motif chemokine ligand (CCL) 2, IL-1β, IL-6, and IL-17 are all elevated. At the end of the AOM+DSS treatment regime, HCA 2 −/− mice demonstrate anemia and increased number of large polyps on colon (34). Remarkably, niacin administration suppresses colon tumor development in antibiotic-treated microbiota-depleted WT mice (34). However, it also promotes colitis-associated cancer in HCA 2 −/− mice, which is associated with an expansion of bacteria in Prevotellaceae family and TM7 phylum (34), suggesting microbiota/niacin protective effect is HCA 2 -dependent. In the same report, Singh et al. manipulated another mouse model of intestinal carcinogenesis, Apc Min/+ , in which multiple intestinal neoplasia (Min) is a mutant allele of the murine adenomatous polyposis coli (Apc) locus (110). Apc Min/+ mice show significantly enlarged colonic polyp numbers, which were rescued by niacin treatment. However, niacin was not able to decrease the development of colonic polyps in HCA 2 −/− Apc Min/+ (34). Taken together, these data demonstrate that HCA 2 mediates cancer development and progression by promoting intestine mucosal immunity and decreasing cancer-promoting genes.

ANTI-INFLAMMATORY EFFECTS OF HCA 2 IN OTHER DISEASES
HCA 2 signaling plays an essential role in preventing and reducing inflammation in the intestine. In addition, HCA 2 has also been associated with anti-inflammatory effects in numerous disease states. In particular, various studies report that activation of HCA 2 reduces inflammation in atherosclerosis (36), diabetes mellitus (25), diabetic retinopathy (31), neurodegenerative diseases (37,38), sepsis (39), mammary cancer (40) and pancreatitis (41). Activation of HCA 2 on immune cells in the vasculature by niacin reduces the progression of atherosclerosis and suppresses macrophage recruitment to atherosclerotic plaques (36). Chronic activation of HCA 2 by niacin increases serum adiponectin in obese men with metabolic syndrome, suggesting a role in diabetes mellitus and obesity (112,113). Additionally, in pancreatic islets of diabetic db/db mice as well as in type 2 diabetic (T2D) patients, HCA 2 expression is decreased (114). Administration of GSK256073, a HCA 2 agonist, notably reduced serum glucose and non-esterified fatty acids without inducing the niacin-associated side effect of cutaneous flushing in diabetic patients (25). In retinal pigmented epithelial cells, niacinmediated activation of HCA 2 suppresses TNF-α-induced NF-κB activation and IL-6 and monocyte chemoattractant protein-1 (MCP-1) secretion (31). HCA 2 ligands have been also reported to attenuate inflammation in neurodegenerative diseases such as Parkinson's disease (115), Huntington's disease (38), Alzheimer's disease (116), multiple sclerosis (37), ischemic stroke (117) and traumatic brain injury (118), although, the mechanisms behind many of these beneficial effects have yet to be fully elucidated. In sepsis, niacin attenuated kidney and lung inflammation by decreasing NF-κB activation and subsequently decreasing inflammatory cytokines (39,119,120). As was the case in colon cancer, HCA 2 functions as a tumor suppressor in mammary cancer via inhibition of genes involved in cell survival and anti-apoptotic pathways in human breast cancer cell lines (40). In pancreatitis, β-OHB supplementation inhibits macrophage NF-κB activation in an HCA 2 -dependent manner, and limits sterile inflammation (41). Moreover, HCA 2 plays an antiviral role in reducing the Zika virus replication. HCA 2 expression is significantly induced by Zika virus infection, while depletion of HCA 2 resulted in significant increase of Zika virus RNA levels and viral yields, indicating that HCA 2 can serve as a restriction factor for Zika virus and providing a potential target for anti-Zika virus therapeutic (121).

CONCLUSION
There is mounting evidence summarized in this review that HCA 2 plays an important role in modulating inflammation and carcinogenesis in the intestine. Ligands for the HCA 2 receptor mediate a wide variety of inflammation-suppressing signaling events. NF-κB, NLRP3 and prostaglandins PGD 2 and PGE 2 have all been implicated as downstream targets of the HCA 2 receptor, suggesting activation of one pathway may have beneficial or undesirable effects that are tissuedependent.
Therefore, tissue-specific, pharmacologic ligands which trigger bias signaling cascades, and therefore minimize less desirable downstream effects, are required. In addition, these pathways interweave the process of inflammatory and metabolic disorders through HCA 2 . Thus, this interplay of gut microbiota, HCA 2 signaling and immune responses is a double-edged sword of inducing inflamed intestinal diseases or colon cancer and promoting intestinal homeostasis.

AUTHOR CONTRIBUTIONS
ZL was primarily responsible for researching and writing the manuscript (including the generation of figures). KM was responsible for writing specific sections and reviewing the manuscript. RJ proposed the topic of the review and supervised the writing and review of the manuscript. All authors contributed to the article and approved the submitted version.