Human intestinal bitter taste receptors regulate innate immune responses and metabolic regulators in obesity.

Bitter taste receptors (taste 2 receptors, TAS2Rs) serve as warning sensors in the lingual system against the ingestion of potentially poisonous food. Here, we investigated the functional role of TAS2Rs in the human gut and focused on their potential to trigger an additional host defense pathway in the intestine. Human jejunal crypts, especially those from individuals with obesity, responded to bitter agonists by inducing the release of antimicrobial peptides (α-defensin 5 and regenerating islet-derived protein 3 α [REG3A]) but also regulated the expression of other innate immune factors (mucins, chemokines) that affected E. coli growth. We found that the effect of aloin on E. coli growth and on the release of the mucus glycoprotein CLCA1, identified via proteomics, was affected by TAS2R43 deletion polymorphisms and thus confirmed a role for TAS2R43. RNA-Seq revealed that denatonium benzoate induced an NRF2-mediated nutrient stress response and an unfolded protein response that increased the expression of the mitokine GDF15 but also ADM2 and LDLR, genes that are involved in anorectic signaling and lipid homeostasis. In conclusion, TAS2Rs in the intestine constitute a promising target for treating diseases that involve disturbances in the innate immune system and body weight control. TAS2R polymorphisms may be valuable genetic markers to predict therapeutic responses.

Free Feeding of CpG-Oligodeoxynucleotide Particles Prophylactically Attenuates Allergic Airway Inflammation and Hyperresponsiveness in Mice.

CpG-oligodeoxynucleotides (CpG-ODNs) constitute an attractive alternative for asthma treatment. However, very little evidence is available from studies on the oral administration of CpG-ODNs in animals. Previously, we developed acid-resistant particles (named ODNcap) as an oral delivery device for ODNs. Here, we showed that free feeding of an ODNcap-containing feed prophylactically attenuates allergic airway inflammation, hyperresponsiveness, and goblet cell hyperplasia in an ovalbumin-induced asthma model. Using transcriptomics-driven approaches, we demonstrated that injury of pulmonary vein cardiomyocytes accompanies allergen inhalation challenge, but is inhibited by ODNcap feeding. We also showed the participation of an airway antimicrobial peptide (Reg3γ) and fecal microbiota in the ODNcap-mediated effects. Collectively, our findings suggest that daily oral ingestion of ODNcap may provide preventive effects on allergic bronchopulmonary insults via regulation of mechanisms involved in the gut-lung connection.

Secretome screening reveals immunomodulating functions of IFNα-7, PAP and GDF-7 on regulatory T-cells.

Regulatory T cells (Tregs) are the key cells regulating peripheral autoreactive T lymphocytes. Tregs exert their function by suppressing effector T cells. Tregs have been shown to play essential roles in the control of a variety of physiological and pathological immune responses. However, Tregs are unstable and can lose the expression of FOXP3 and suppressive functions as a consequence of outer stimuli. Available literature suggests that secreted proteins regulate Treg functional states, such as differentiation, proliferation and suppressive function. Identification of secreted proteins that affect Treg cell function are highly interesting for both therapeutic and diagnostic purposes in either hyperactive or immunosuppressed populations. Here, we report a phenotypic screening of a human secretome library in human Treg cells utilising a high throughput flow cytometry technology. Screening a library of 575 secreted proteins allowed us to identify proteins stabilising or destabilising the Treg phenotype as suggested by changes in expression of Treg marker proteins FOXP3 and/or CTLA4. Four proteins including GDF-7, IL-10, PAP and IFNα-7 were identified as positive regulators that increased FOXP3 and/or CTLA4 expression. PAP is a phosphatase. A catalytic-dead version of the protein did not induce an increase in FOXP3 expression. Ten interferon proteins were identified as negative regulators that reduced the expression of both CTLA4 and FOXP3, without affecting cell viability. A transcriptomics analysis supported the differential effect on Tregs of IFNα-7 versus other IFNα proteins, indicating differences in JAK/STAT signaling. A conformational model experiment confirmed a tenfold reduction in IFNAR-mediated ISG transcription for IFNα-7 compared to IFNα-10. This further strengthened the theory of a shift in downstream messaging upon external stimulation. As a summary, we have identified four positive regulators of FOXP3 and/or CTLA4 expression. Further exploration of these Treg modulators and their method of action has the potential to aid the discovery of novel therapies for both autoimmune and infectious diseases as well as for cancer.

IL-22-dependent dysbiosis and mononuclear phagocyte depletion contribute to steroid-resistant gut graft-versus-host disease in mice.

Efforts to improve the prognosis of steroid-resistant gut acute graft-versus-host-disease (SR-Gut-aGVHD) have suffered from poor understanding of its pathogenesis. Here we show that the pathogenesis of SR-Gut-aGVHD is associated with reduction of IFN-γ+ Th/Tc1 cells and preferential expansion of IL-17-IL-22+ Th/Tc22 cells. The IL-22 from Th/Tc22 cells causes dysbiosis in a Reg3γ-dependent manner. Transplantation of IFN-γ-deficient donor CD8+ T cells in the absence of CD4+ T cells produces a phenocopy of SR-Gut-aGVHD. IFN-γ deficiency in donor CD8+ T cells also leads to a PD-1-dependent depletion of intestinal protective CX3CR1hi mononuclear phagocytes (MNP), which also augments expansion of Tc22 cells. Supporting the dual regulation, simultaneous dysbiosis induction and depletion of CX3CR1hi MNP results in full-blown Gut-aGVHD. Our results thus provide insights into SR-Gut-aGVHD pathogenesis and suggest the potential efficacy of IL-22 antagonists and IFN-γ agonists in SR-Gut-aGVHD therapy.

Microbiota-derived short chain fatty acid promotion of Amphiregulin expression by dendritic cells is regulated by GPR43 and Blimp-1.

Dendritic cells (DC) are the most important antigen-presenting cells, which guide T cell activation and function, and dysregulated DC function might be one of the crucial causes of inflammatory bowel disease (IBD). It has been well-known that microbiota and their metabolites play an essential role in regulating the biology and function of DC, thus contributing to the pathogenesis of IBD. However, the underlying mechanisms remain largely unknown. Amphiregulin (AREG), a molecule of the epidermal growth factor (EGF) family, is primarily described as an epithelial cell-derived cytokine and recognized as a critical regulator of cell proliferation and tissue repair. Here, we found that DC expression of AREG depended on butyrate (a microbiota-derived short chained fatty acid), which required the interaction between butyrate and G-protein-coupled receptor 43 (GPR43). Furthermore, we found that butyrate-GPR43 interaction failed to induce AREG expression in DC deficient in B lymphocyte induced maturation protein 1 (Blimp-1). Notably, DC-derived AREG was indispensable for the protection against experimental colitis in mice. Additionally, AREG expression was significantly decreased in DC from IBD patients. Our data provide novel evidences to interpret how AREG expression is regulated in DC, and shed new light on the mechanisms whereby microbiota regulate DC function.

Anti-tumor effect of single-chain antibody to Reg3a in colorectal cancer.

BACKGROUND: Regenerating protein 3a (Reg3a) is a trophic factor that functions as a stimulus in cell proliferation and neogenesis. Previous studies showed that Reg3a is ectopically upregulated in a majority of colorectal cancers (CRC) and detectable in the serum. METHODS: Single-chain variable fragment targeting Reg3a (scFv-Reg3a) was screened from a phage library. The bioactivity of recombinant Reg3a (rReg3a) and scFv-Reg3a were tested in LoVo and RKO cell lines using MTT, flow cytometry, wound healing and transwell analyses. Whether scFv-Reg3a inhibits tumor growth and enhances 5-fluorouracil (5-FU)-caused cell death were further examined in LoVo cell-transplanted nude BALB/c mice. RESULTS: A scFv-Reg3a from clone C2 was obtained and its binding affinity (KD) to rReg3a was determined to be 4.44 × 10-10. In cultured LoVo and RKO cells, rReg3a promoted but scFv-Reg3a inhibited cell proliferation, survival, migration and invasion. In LoVo cell-xenografted nude mice, administration of rReg3a accelerated tumor growth while scFv-Reg3a suppressed cell proliferation and reinforced 5-FU-induced cell death. CONCLUSION: The newly developed scFv-Reg3a is an anti-cancer agent which is potent to suppress CRC cell proliferation and survival. The use of scFv-Reg3a could enhance the effectiveness of 5-FU-based chemotherapy in the cancerous treatment.

MicroRNA-10a-3p mediates Th17/Treg cell balance and improves renal injury by inhibiting REG3A in lupus nephritis.

BACKGROUND: The therapeutic approaches guided toward microRNAs (miRNAs) have been extensively explored in lupus nephritis (LN), but the precise position of miR-10a-3p posted in disease is not translated thoroughly. Therein, this work pivoting on miR-10a-3p was launched with the involvement of regenerating islet-derived 3 α (REG3A). METHODS: Peripheral blood samples from LN patients and healthy controls (n = 132) were collected. miR-10a-3p and REG3A expression in peripheral blood mononuclear cells were tested. Mice were injected with miR-10a-3p agomir, miR-10a-3p antagomir and/or REG3A low expression vector for presentation of their roles in renal function, T helper cell 17 (Th17)/regulatory cell (Treg) balance, renal pathological damage, JAK2/STAT3 pathway activation and renal injury in LN. The relation between miR-10a-3p and REG3A was tested. RESULTS: MiR-10a-3p was down-regulated while REG3A was up-regulated in LN. Restoring miR-10a-3p or silencing REG3A decreased Th17/Treg ratio in CD4+ T cells, inhibited JAK2/STAT3 pathway activation, ameliorated renal function, improved renal pathological damage and alleviated renal injury in LN. REG3A depletion negated the effects of down-regulated miR-10a-3p on LN. MiR-10a-3p targeted REG3A. CONCLUSION: The work elucidates that miR-10a-3p restoration decreases Th17/Treg ratio and attenuates renal injury in LN via inhibiting REG3A and the activation of JAK2/STAT3 pathway, which renews the therapeutic reference for LN management.

Role of regenerating islet-derived proteins in inflammatory bowel disease.

Inflammatory bowel disease (IBD) is an inflammatory disorder of the gastrointestinal tract that affects millions of patients worldwide. It has a complex and multifactorial etiology leading to excessive exposure of intestinal epithelium to microbial antigens, inappropriate activation of the immune system and ultimately to the damage of intestinal tissues. Although numerous efforts have been made to improve the disease management, IBD remains persistently recurring and beyond cure. This is due largely to the gaps in our understanding of the pathogenesis of IBD that hamper the development of timely diagnoses and effective treatment. However, some recent discoveries, including the beneficial effects of interleukin-22 (IL-22) on the inflamed intestine, have shed light on a self-protective mechanism in IBD. Regenerating islet-derived (REG/Reg) proteins are small secretory proteins which function as IL-22's downstream effectors. Mounting studies have demonstrated that IBD patients have significantly increased REG expressions in the injured intestine, but with undefined mechanisms and roles. The reported functions of REG/Reg proteins in intestinal homeostasis, such as those of antibacterial, anti-inflammatory and tissue repair, lead us to discuss their potential mechanisms and clinical relevance in IBD in order to advance IBD research and management.

Gut Microbiota-Derived Propionate Regulates the Expression of Reg3 Mucosal Lectins and Ameliorates Experimental Colitis in Mice.

BACKGROUND AND AIMS: Regenerating islet-derived protein type 3 [Reg3] lectins are antimicrobial peptides at mucosal surfaces of the gut, whose expression is regulated by pathogenic gut microbes via interleukin-22- or Toll-like receptor signalling. In addition to antimicrobial effects, tissue protection is hypothesized, but has been poorly investigated in the gut. METHODS: We applied antibiotic-induced microbiota perturbations, gnotobiotic approaches and a dextran-sodium sulfate [DSS] colitis model to assess microbial Reg3 regulation in the intestines and its role in colitis. We also used an intestinal organoid model to investigate this axis in vitro. RESULTS: First, we studied whether gut commensals are involved in Reg3 expression in mice, and found that antibiotic-mediated reduction of Clostridia downregulated intestinal Reg3B. A loss in Clostridia was accompanied by a significant reduction of short-chain fatty acids [SCFAs], and knock-out [KO] mice for SCFA receptors GPR43 and GPR109 expressed less intestinal Reg3B/-G. Propionate was found to induce Reg3 in intestinal organoids and in gnotobiotic mice colonized with a defined, SCFA-producing microbiota. Investigating the role of Reg3B as a protective factor in colitis, we found that Reg3B-KO mice display increased inflammation and less crypt proliferation in the DSS colitis model. Propionate decreased colitis and increased proliferation. Treatment of organoids exposed to DSS with Reg3B or propionate reversed the chemical injury with a loss of expression of the stem-cell marker Lgr5 and Olfm4. CONCLUSIONS: Our results suggest that Clostridia can regulate Reg3-associated epithelial homeostasis through propionate signalling. We also provide evidence that the Reg3-propionate axis may be an important mediator of gut epithelial regeneration in colitis.

Melatonin controls microbiota in colitis by goblet cell differentiation and antimicrobial peptide production through Toll-like receptor 4 signalling.

Microbial dysbiosis has long been postulated to be associated with the pathogenesis of inflammatory bowel disease (IBD). Although evidence supporting the anti-colitic effects of melatonin have been accumulating, it is not clear how melatonin affects the microbiota. Herein, we investigated the effects of melatonin on the microbiome in colitis and identified involvement of Toll-like receptor (TLR) 4 signalling in the effects. Melatonin improved dextran sulfate sodium (DSS)-induced colitis and reverted microbial dysbiosis in wild-type (WT) mice but not in TLR4 knockout (KO) mice. Induction of goblet cells was observed with melatonin administration, which was accompanied by suppression of Il1b and Il17a and induction of melatonin receptor and Reg3ß, an antimicrobial peptide (AMP) against Gram-negative bacteria. In vitro, melatonin treatment of HT-29 intestinal epithelial cells promotes mucin and wound healing and inhibits growth of Escherichia coli. Herein, we showed that melatonin significantly increases goblet cells, Reg3ß, and the ratio of Firmicutes to Bacteriodetes by suppressing Gram-negative bacteria through TLR4 signalling. Our study suggests that sensing of bacteria through TLR4 and regulation of bacteria through altered goblet cells and AMPs is involved in the anti-colitic effects of melatonin. Melatonin may have use in therapeutics for IBD.

Single-chain Antibody Against Reg4 Suppresses Gastric Cancer Cell Growth and Enhances 5-FU-induced Cell Death in vitro.

BACKGROUND: Regenerating islet-derived gene family member 4 (Reg4), a well-investigated growth factor in the regenerative pancreas, has recently been reported to be highly associated with a majority of gastrointestinal cancers. Pathological hyper-expression or artificial over-expression of Reg4 causes acceleration of tumor growth, migration, and resistance to chemotherapeutic 5-Fluorouracil (5-FU). Until now, no method has been successfully established for eliminating the effects of Reg4 protein. METHODS: This study reports the production of an engineered immunoglobin, a single-chain variable fragment (scFv-Reg4), to specifically bind Reg4 and block the bioactivity. The complementary-determining regions (CDRs) against Reg4 were assigned using MOE and ZDOCK servers. The binding affinity (KD) was determined by bio-layer interferometry (BLI). MKN45 and AGS cell proliferation was determined by Thiazolyl blue tetrazolium bromide (MTT) method and the cell apoptosis was detected by flow cytometry assay. RESULTS: The KD of scFv-Reg4 to Reg4 was determined to be 1.91×10-8. In MKN45 and AGS cell lines, scFv- Reg4 depressed Reg4-stimulated cell proliferation and the inhibitory rates were 27.7±1.5% and 17.3±2.6%, respectively. Furthermore, scFv significantly enhanced 5-FU-induced cell death, from 23.0±1.0% to 28.4±1.2% in MKN45 and 28.2±0.7% to 36.6±0.6% in AGS cells. Treatment with scFv alone could lyse cancer cells to a certain extent, but no significance has been observed. CONCLUSION: The single-chain antibody (scFv-Reg4) significantly inhibited gastric cancer cell proliferation and synergistically enhanced the lethal effect of 5-FU. Thus, traditional chemo-/radio- therapeutics supplemented with scFv-Reg4 may provide advances in the strategy for gastrointestinal cancer treatment.

Bacteria engineered to produce IL-22 in intestine induce expression of REG3G to reduce ethanol-induced liver disease in mice.

OBJECTIVE: Antimicrobial C-type lectin regenerating islet-derived 3 gamma (REG3G) is suppressed in the small intestine during chronic ethanol feeding. Our aim was to determine the mechanism that underlies REG3G suppression during experimental alcoholic liver disease. DESIGN: Interleukin 22 (IL-22) regulates expression of REG3G. Therefore, we investigated the role of IL-22 in mice subjected to chronic-binge ethanol feeding (NIAAA model). RESULTS: In a mouse model of alcoholic liver disease, we found that type 3 innate lymphoid cells produce lower levels of IL-22. Reduced IL-22 production was the result of ethanol-induced dysbiosis and lower intestinal levels of indole-3-acetic acid (IAA), a microbiota-derived ligand of the aryl hydrocarbon receptor (AHR), which regulates expression of IL-22. Importantly, faecal levels of IAA were also found to be lower in patients with alcoholic hepatitis compared with healthy controls. Supplementation to restore intestinal levels of IAA protected mice from ethanol-induced steatohepatitis by inducing intestinal expression of IL-22 and REG3G, which prevented translocation of bacteria to liver. We engineered Lactobacillus reuteri to produce IL-22 (L. reuteri/IL-22) and fed them to mice along with the ethanol diet; these mice had reduced liver damage, inflammation and bacterial translocation to the liver compared with mice fed an isogenic control strain and upregulated expression of REG3G in intestine. However, L. reuteri/IL-22 did not reduce ethanol-induced liver disease in Reg3g-/- mice. CONCLUSION: Ethanol-associated dysbiosis reduces levels of IAA and activation of the AHR to decrease expression of IL-22 in the intestine, leading to reduced expression of REG3G; this results in bacterial translocation to the liver and steatohepatitis. Bacteria engineered to produce IL-22 induce expression of REG3G to reduce ethanol-induced steatohepatitis.

Acceleration of pancreatic tumorigenesis under immunosuppressive microenvironment induced by Reg3g overexpression.

Reg3g is a potential risk for pancreatic ductal adenocarcinoma (PDAC). We previously demonstrated that Reg3g promoted pancreatic carcinogenesis via a STAT3 signaling pathway in a murine model of chronic pancreatitis. Whether the immune response is involved in tumorigenesis induced by Reg3g remains unknown. In this study, Reg3g-regulated tumor immunity was evaluated in tumor-implanted murine models, immune cells, and tumor microenvironment. In mice that had been orthotopically or ectopically implanted with Panc02 cells, Reg3g overexpression increased EGFR and Ki67, diminished MHC-I and caspase-3 expression, and accelerated growth of tumors. By interacting with PD-1/PD-L1, Reg3g also promoted differentiation of Tregs and recruitment of MDSC, retarded maturation of DCs and inactivation of CD8+ T cells, and suppressed cross-priming of CD8+ T-cell responses by DCs in tumor-bearing mice. Knockdown of Reg3g delayed tumor development in normal mice, but not in CD8+ T-cell-deficient mice. In vitro, Reg3g upregulated EGFR in DCs, activated heme oxygenase-1 (Hmox1) involved JAK2/STAT3 signaling, raised levels of Th2 cytokines in and suppressed maturation of DCs, and enhanced tumor cell proliferation. These results reveal a novel role of Reg3g as an immunosuppressive promoter that weakens tumor-specific antigenicity and suppresses antitumor effects of CD8+ T cells in a murine model of pancreatic cancer. Reg3g produces these effects by activating the JAK2/STAT3 signaling pathway in DCs, triggering the generation of an immunosuppressive tumor microenvironment.

Hyperglycaemia inhibits REG3A expression to exacerbate TLR3-mediated skin inflammation in diabetes.

Dysregulated inflammatory responses are known to impair wound healing in diabetes, but the underlying mechanisms are poorly understood. Here we show that the antimicrobial protein REG3A controls TLR3-mediated inflammation after skin injury. This control is mediated by REG3A-induced SHP-1 protein, and acts selectively on TLR3-activated JNK2. In diabetic mouse skin, hyperglycaemia inhibits the expression of IL-17-induced IL-33 via glucose glycation. The decrease in cutaneous IL-33 reduces REG3A expression in epidermal keratinocytes. The reduction in REG3A is associated with lower levels of SHP-1, which normally inhibits TLR3-induced JNK2 phosphorylation, thereby increasing inflammation in skin wounds. To our knowledge, these findings show for the first time that REG3A can modulate specific cutaneous inflammatory responses and that the decrease in cutaneous REG3A exacerbates inflammation in diabetic skin wounds.

Epithelial IL-23R Signaling Licenses Protective IL-22 Responses in Intestinal Inflammation.

A plethora of functional and genetic studies have suggested a key role for the IL-23 pathway in chronic intestinal inflammation. Currently, pathogenic actions of IL-23 have been ascribed to specific effects on immune cells. Herein, we unveil a protective role of IL-23R signaling. Mice deficient in IL-23R expression in intestinal epithelial cells (Il23R(ΔIEC)) have reduced Reg3b expression, show a disturbed colonic microflora with an expansion of flagellated bacteria, and succumb to DSS colitis. Surprisingly, Il23R(ΔIEC) mice show impaired mucosal IL-22 induction in response to IL-23. αThy-1 treatment significantly deteriorates colitis in Il23R(ΔIEC) animals, which can be rescued by IL-22 application. Importantly, exogenous Reg3b administration rescues DSS-treated Il23R(ΔIEC) mice by recruiting neutrophils as IL-22-producing cells, thereby restoring mucosal IL-22 levels. The study identifies a critical barrier-protective immune pathway that originates from, and is orchestrated by, IL-23R signaling in intestinal epithelial cells.