Consumption of Fructooligosaccharides Influences the Action of Fecal MicroRNAs in Altering the Structure of Cultured Gut Microbiota in Mice.

Fecal microRNAs (miRNAs) derived from intestinal epithelial cells have been suggested to influence gut microbiota homeostasis. We recently showed that supplementing murine fecal small RNAs, most likely miRNAs, alters the structure of cultured fecal microbiota in a sequence-dependent manner. The present study investigated the effect of consuming fructooligosaccharides (FOS) on the action of fecal small RNAs in altering the structure of cultured fecal microbiota. Female C57BL/6J mice were allowed free access to AIN-93G diet, and tap water supplemented with or without 4% (w/v) FOS for 2 wk. As assessed by 16S rRNA gene sequence analysis in cecal contents, the gut microbiota structure differed between mice supplemented with and without FOS. Fecal bacteria isolated from the cecal contents of mice without FOS supplementation were cultured for 24 h under anaerobic conditions. The structure of cultured microbiota differed between the cultures supplemented with small RNAs isolated from the cecal contents of mice supplemented with and without FOS. Microarray analysis showed that the miRNA profile in the cecal contents differed between mice supplemented with and without FOS. We propose that FOS consumption influences the action of intestinal epithelial cell-derived miRNAs in altering the structure of cultured gut microbiota, and such FOS action is associated with changes in the profile of miRNAs. It may be possible that intestinal epithelial cell-derived miRNAs contribute, at least in part, to diet-induced alteration of gut microbiota.

Murine fecal microRNAs alter the composition of cultured gut microbiota.

Fecal microRNAs (miRNAs) derived from intestinal epithelial cells have been suggested to influence gut microbiota homeostasis. The present study examined whether fecal miRNAs alter the structure of cultured gut microbiota. Fecal bacteria isolated from murine cecal contents were cultured for 24 h under anaerobic conditions. Supplementation with fecal small RNAs isolated from cecal contents altered the structure of cultured fecal microbiota as assessed by 16S rRNA gene sequence analysis. In particular, fecal small RNAs increased Enterococcus spp. Fractionation of fecal small RNAs by ultrafiltration showed that small RNAs smaller than 10 kDa significantly increased enterococci compared to those larger than 10 kDa, as assessed by quantitative PCR, suggesting that the increase in enterococci by fecal small RNAs can mainly be attributed to miRNAs. Negative control miRNA that has low homology to miRNA sequences of human, mouse, and rat, failed to increase enterococci. Therefore, the findings from the present study employing cultured fecal bacteria suggest that fecal small RNAs, most likely host-derived miRNAs, alter gut microbiota structure by expanding enterococci in a sequence-dependent manner.

Role of microRNAs in the crosstalk between the gut microbiota and intestinal immune system.

MicroRNAs (miRNAs) are small non-coding RNA species involved in diverse physiological processes, including immunity. Accumulating evidence suggests that miRNA-induced gene silencing plays a significant role in the regulation of the intestinal immune system by the gut commensal microbiota. This review aims to provide an overview of the intestinal miRNA-mediated crosstalk between the gut microbiota and the host intestinal immune system. First, we describe the role of miRNAs in regulating the intestinal immune system. Then we describe the effect of the gut microbiota on intestinal miRNA expression. Subsequently, we describe the role of miRNAs in the modulation of the intestinal immune system by the gut microbiota. Finally, we describe the effect of host miRNAs on the gut microbiota. Although the entire picture of this complex crosstalk remains unclear, efforts to unravel it will contribute significantly to developing new strategies for preventing and treating intestinal immune disorders such as inflammatory bowel disease.

Administration of Bifidobacterium pseudolongum suppresses the increase of colonic serotonin and alleviates symptoms in dextran sodium sulfate-induced colitis in mice.

Previous studies suggested that altered gut serotonin (5-HT) signaling is implicated in the pathophysiology of inflammatory bowel disease (IBD). Indeed, 5-HT administration reportedly exacerbated the severity of murine dextran sodium sulfate (DSS)-induced colitis that mimics human IBD. Our recent study suggested that Bifidobacterium pseudolongum, one of the most predominant bifidobacterial species in various mammals, reduces the colonic 5-HT content in mice. The present study thus tested whether the administration of B. pseudolongum prevents DSS-induced colitis in mice. Colitis was induced by administering 3% DSS in drinking water in female BALB/c mice, and B. pseudolongum (109 CFU/day) or 5-aminosalicylic acid (5-ASA, 200 mg/kg body weight) was intragastrically administered once daily throughout the experimental period. B. pseudolongum administration reduced body weight loss, diarrhea, fecal bleeding, colon shortening, spleen enlargement, and colon tissue damage and increased colonic mRNA levels of cytokine genes (Il1b, Il6, Il10, and Tnf) almost to an extent similar to 5-ASA administration in DSS-treated mice. B. pseudolongum administration also reduced the increase of colonic 5-HT content, whereas it did not alter the colonic mRNA levels of genes that encode the 5-HT synthesizing enzyme, 5-HT reuptake transporter, 5-HT metabolizing enzyme, and tight junction-associated proteins. We propose that B. pseudolongum is as beneficial against murine DSS-induced colitis as the widely used anti-inflammatory agent 5-ASA. However, further studies are needed to clarify the causal relationship between the reduced colonic 5-HT content and reduced severity of DSS-induced colitis caused by B. pseudolongum administration.

Consumption of 1-Kestose Upregulates MicroRNA-200 and -192/215 Families in Lamina Propria Leukocytes of the Murine Large Intestine.

By comparing germ-free mice and specific pathogen-free mice, we recently demonstrated that the presence of gut commensals upregulates microRNA-200 family members in lamina propria leukocytes (LPL) of the murine large intestine. The present study tested whether the consumption of 1-kestose (KES), an indigestible oligosaccharide that alters gut microbiota composition, influences the microRNA expression in the LPL. Supplementation of KES (4%) in drinking water for 2 wk increased the levels of miR-182-5p, -205-5p, -290a-5p, miR-200 family members (miR-141-3p, -200a-3p, -200b-3p, -200c-3p, and -429-3p) as well as miR-192/215 family members (miR-192-5p, -194-5p, and -215-5p) as determined by microarray analysis in large intestinal LPL of C57BL/6 mice. Quantitative reverse transcription-PCR further confirmed the increase in miR-192-5p, -194-5p, -200a-3p, -200b-3p, -200c-3p, -205-5p, and 215-5p. KES consumption significantly increased Bifidobacterium pseudolongum in the cecal contents. In a separate experiment, intragastric administration of B. pseudolongum (109 CFU/d) for 7 d increased the levels of miR-182-5p, -194-5p, and -200a-3p and tended to increase the levels of miR-200b-3p, -215-5p, and -429-3p. These results suggest that dietary KES influences miRNA expression in the large intestinal LPL, which may be associated with the increased population of B. pseudolongum.

Local free fatty acids trigger the expression of lipopolysaccharide-binding protein in murine white adipose tissue.

Although lipopolysaccharide (LPS)-binding protein (LBP) is an acute-phase protein mainly produced by hepatocytes, it has also been proposed to be a pro-inflammatory adipokine. Obesity and the consumption of a high-fat diet (HFD) are reportedly associated with elevated levels of LPS in plasma and free fatty acids (FFAs) in white adipose tissue (WAT). We examined whether circulating LPS or local FFAs are responsible for the HFD-induced increase of LBP in WAT. Male C57BL/6J mice were fed either a normal-fat diet (NFD) or an HFD. The mRNA levels in the liver and mesenteric WAT (mWAT), total FFA content in mWAT, and LBP and LPS concentrations in plasma were determined. The Lbp mRNA level in mWAT was higher in mice fed the HFD than in those fed the NFD for 3, 7, or 28 days or 14 weeks, whereas the hepatic Lbp mRNA level did not differ between the groups. The Lbp mRNA level in mWAT was also increased by the HFD in germ-free mice, which do not have gut microbiota, the source of LPS. The plasma LPS level did not show a significant correlation with the mWAT Lbp mRNA level. The total FFA content in mWAT was higher in mice fed the HFD than in those fed the NFD and positively correlated with the Lbp mRNA level. Supplementation with palmitic acid increased the Lbp mRNA level in 3T3-L1 adipocytes. We propose that local FFAs, but not circulating LPS, are the trigger for increased Lbp expression in mWAT of mice fed the HFD.

Consumption of indigestible saccharides and administration of Bifidobacterium pseudolongum reduce mucosal serotonin in murine colonic mucosa.

SCFA increase serotonin (5-hydroxytryptamine, 5-HT) synthesis and content in the colon in vitro and ex vivo, but little is known in vivo. We tested whether dietary indigestible saccharides, utilised as a substrate to produce SCFA by gut microbiota, would increase colonic 5-HT content in mice. Male C57BL/6J mice were fed a purified diet and water supplemented with 4 % (w/v) 1-kestose (KES) for 2 weeks. Colonic 5-HT content and enterochromaffin (EC) cell numbers were lower in mice supplemented with KES than those without supplementation, while monoamine oxidase A activity and mRNA levels of tryptophan hydroxylase 1 (Tph1), chromogranin A (Chga), Slc6a4 and monoamine oxidase A (Maoa) genes in the colonic mucosa, serum 5-HT concentration and total 5-HT content in the colonic contents did not differ between groups. Caecal acetate concentration and Bifidobacterium pseudolongum population were higher in KES-supplemented mice. Similar trends were observed in mice supplemented with other indigestible saccharides, that is, fructo-oligosaccharides, inulin and raffinose. Intragastric administration of live B. pseudolongum (108 colony-forming units/d) for 2 weeks reduced colonic 5-HT content and EC cell numbers. These results suggest that changes in synthesis, reuptake, catabolism and overflow of 5-HT in the colonic mucosa are not involved in the reduction of colonic 5-HT content by dietary indigestible saccharides in mice. We propose that gut microbes including B. pseudolongum could contribute to the reduction of 5-HT content in the colonic mucosa via diminishing EC cells.

Gut commensals suppress interleukin-2 production through microRNA-200/BCL11B and microRNA-200/ETS-1 axes in lamina propria leukocytes of murine large intestine.

The role of microRNAs (miRNAs) in how microbiota influence the host intestinal immune system is not fully understood. We compared the expression profiles of miRNAs and mRNAs in lamina propria leukocytes (LPL) in the large intestines of germ-free (GF) and specific pathogen-free (SPF) mice. Microarray analysis revealed different expression profiles of miRNAs and mRNAs between GF and SPF mice. Quantitative real time-PCR (qRT-PCR) showed that the level of miR-200 family members was significantly higher in SPF mice than in GF mice. In silico prediction followed by qRT-PCR suggested that Bcl11b, Ets1, Gbp7, Stat5b, and Zeb1 genes were downregulated by the miR-200 family. Western blotting revealed that the expression of BCL11B and ETS-1, but not ZEB1, in large intestinal LPL was significantly lower in SPF mice than in GF mice. Interleukin (IL)-2 production in cultured LPL upon stimulation with phorbol 12-myristate 13-acetate and ionomycin for 24 h was significantly lower in SPF mice than in GF mice. Conventionalization of GF mice substantially recapitulated SPF mice in terms of the expression of miR-200 family members and their target genes and IL-2 production in large intestinal LPL. Considering that BCL11B and ETS-1 reportedly function as transcription factors to activate the Il2 gene, we propose that the presence of gut commensals suppresses IL-2 production in large intestinal LPL, at least in part through post-transcriptional downregulation of Bcl11b and Ets1 genes by miR-200 family members.

Expression of serotonin receptor HTR4 in glucagon-like peptide-1-positive enteroendocrine cells of the murine intestine.

Serotonin (5-hydroxytryptamine [5-HT]) synthesized and released in enterochromaffin (EC) cells participates in various functions in the gastrointestinal tract by acting on a diverse range of 5-HT receptors (HTRs) expressed on smooth muscle, enteric neurons, and epithelial cells. We previously observed that genes encoding HTR2A, HTR2B, and HTR4 are expressed in murine intestinal organoids, suggesting the expression of these HTRs in intestinal epithelial cells. The present study investigated the localization of these HTRs in the murine intestine by immunofluorescence staining. HTR2A, HTR2B, and HTR4 localized in individual solitary cells in the epithelium, while HTR2C was observed in the lamina propria. In the epithelium, HTR2A, HTR2B, and HTR4 colocalized with 5-HT, and HTR4 colocalized with glucagon-like peptide 1 (GLP-1) and peptide YY (PYY). Murine intestinal organoids show a colocalization pattern that is similar to in vivo HTR2A and HTR4 with 5-HT, GLP-1, and PYY. Intraperitoneal and intragastric administration of tegaserod, an HTR4 agonist, failed to alter plasma GLP-1 levels in fasted mice. However, intragastric but not intraperitoneal administration of tegaserod reduced dietary lipid-induced increases of plasma GLP-1 levels. This action of tegaserod was inhibited by co-administration of RS39604, an HTR4 antagonist. These results suggest that murine ileal GLP-1/PYY-producing enteroendocrine (EE) cells express HTR4, while 5-HT-producing EC cells express HTR2A, HTR2B, and HTR4. In addition, the observations regarding in vivo GLP-1 secretion suggest that HTR4 signaling in ileal EE cells suppresses dietary lipid-induced GLP-1 secretion. We thus propose that EC and EE cells may interact with each other through paracrine signaling mechanisms.

Repurposing existing drugs: identification of irreversible IMPDH inhibitors by high-throughput screening.

Inosine 5'-monophosphate dehydrogenase (IMPDH) is an essential enzyme for the production of guanine nucleotides. Disruption of IMPDH activity has been explored as a therapeutic strategy for numerous purposes, such as for anticancer, immunosuppression, antiviral, and antimicrobial therapy. In the present study, we established a luciferase-based high-throughput screening system to identify IMPDH inhibitors from our chemical library of known bioactive small molecules. The screening of 1400 compounds resulted in the discovery of three irreversible inhibitors: disulfiram, bronopol, and ebselen. Each compound has a distinct chemical moiety that differs from other reported IMPDH inhibitors. Further evaluation revealed that these compounds are potent inhibitors of IMPDHs with kon values of 0.7 × 104 to 9.3 × 104 M-1·s-1. Both disulfiram and bronopol exerted similar degree of inhibition to protozoan and mammalian IMPDHs. Ebselen showed an intriguing difference in mode of inhibition for different IMPDHs, with reversible and irreversible inhibition to each Cryptosporidium parvum IMPDH and human IMPDH type II, respectively. In the preliminary efficacy experiment against cryptosporidiosis in severe combined immunodeficiency (SCID) mouse, a decrease in the number of oocyst shed was observed upon the oral administration of disulfiram and bronopol, providing an early clinical proof-of-concept for further utilization of these compounds as IMPDH inhibitors.

Murine intestinal organoids resemble intestinal epithelium in their microRNA profiles.

Intestinal organoids were established as an ex vivo model of the intestinal epithelium. We investigated whether organoids resemble the intestinal epithelium in their microRNA (miRNA) profiles. Total RNA samples were obtained from crypt and villus fractions in murine intestine and from cultured organoids. Microarray analysis showed that organoids largely resembled intestinal epithelial cells in their miRNA profiles. In silico prediction followed by qRT-PCR suggested that six genes are regulated by corresponding miRNAs along the crypt-villus axis, suggesting miRNA regulation of epithelial cell renewal in the intestine. However, such expression patterns of miRNAs and their target mRNAs were not reproduced during organoids maturation. This might be due to lack of luminal factors and endocrine, nervous, and immune systems in organoids and different cell populations between in vivo epithelium and organoids. Nevertheless, we propose that intestinal organoids provide a useful in vitro model to investigate miRNA expression in intestinal epithelial cells.

Structural and thermodynamic analyses reveal critical features of glycopeptide recognition by the human PILRα immune cell receptor.

Before entering host cells, herpes simplex virus-1 uses its envelope glycoprotein B to bind paired immunoglobulin-like type 2 receptor α (PILRα) on immune cells. PILRα belongs to the Siglec (sialic acid (SA)-binding immunoglobulin-like lectin)-like family, members of which bind SA. PILRα is the only Siglec member to recognize not only the sialylated O-linked sugar T antigen (sTn) but also its attached peptide region. We previously determined the crystal structure of PILRα complexed with the sTn-linked glycopeptide of glycoprotein B, revealing the simultaneous recognition of sTn and peptide by the receptor. However, the contribution of each glycopeptide component to PILRα binding was largely unclear. Here, we chemically synthesized glycopeptide derivatives and determined the thermodynamic parameters of their interaction with PILRα. We show that glycopeptides with different sugar units linking SA and peptides (i.e. "GlcNAc-type" and "deoxy-GlcNAc-type" glycopeptides) have lower affinity and more enthalpy-driven binding than the wild type (i.e. GalNAc-type glycopeptide). The crystal structures of PILRα complexed with these glycopeptides highlighted the importance of stereochemical positioning of the O4 atom of the sugar moiety. These results provide insights both for understanding the unique O-glycosylated peptide recognition by the PILRα and for the rational design of herpes simplex virus-1 entry inhibitors.

Production of Single-Chain Fv Antibodies Specific for GA-Pyridine, an Advanced Glycation End-Product (AGE), with Reduced Inter-Domain Motion.

Due to their lower production cost compared with monoclonal antibodies, single-chain variable fragments (scFvs) have potential for use in several applications, such as for diagnosis and treatment of a range of diseases, and as sensor elements. However, the usefulness of scFvs is limited by inhomogeneity through the formation of dimers, trimers, and larger oligomers. The scFv protein is assumed to be in equilibrium between the closed and open states formed by assembly or disassembly of VH and VL domains. Therefore, the production of an scFv with equilibrium biased to the closed state would be critical to overcome the problem in inhomogeneity of scFv for industrial or therapeutic applications. In this study, we obtained scFv clones stable against GA-pyridine, an advanced glycation end-product (AGE), by using a combination of a phage display system and random mutagenesis. Executing the bio-panning at 37 °C markedly improved the stability of scFvs. We further evaluated the radius of gyration by small-angle X-ray scattering (SAXS), obtained compact clones, and also visualized open.