Unabsorbed Fecal Fat Content Correlates with a Reduction of Immunoglobulin a Coating of Gut Bacteria in High-Lard Diet-Fed Mice.

SCOPE: Immunoglobulin A (IgA) selectively coats gut bacteria and contributes to regulatory functions in gastrointestinal inflammation and glucose metabolism. Excess intake of lard leads to decrease in the IgA coating of gut bacteria, although the underlying mechanisms remain unknown. This study validates how unabsorbed fat derived from a high-lard diet in the gut affects the IgA coating of bacteria, as assessed in mouse models using three types of dietary fat (lard, medium-, and long-chain triglycerides [MLCTs], and medium-chain triglycerides [MCTs]) exhibiting different digestibilities. METHODS AND RESULTS: C57BL/6J mice are maintained on diets containing lard, MLCTs, or MCTs at 7% or 30% w/w for 10 weeks (n = 6 per group). The fecal fatty acid concentration is measured to quantify unabsorbed fat content. The ratio of IgA-coated bacteria to total bacteria (IgA coating ratio) in the feces is measured by flow cytometry. Compared to lard-fed mice, MLCT- and MCT-fed mice exhibit lower fecal concentrations of palmitic acid, stearic acid, and oleic acid and higher IgA coating ratios at both 7% and 30% dietary fat, and these parameters exhibit significant negative correlations. CONCLUSION: Unabsorbed fat content in the gut may result in attenuated IgA coating of bacteria in high-lard diet-fed mice.

Development of an improved colonization system for human-derived Bifidobacterium longum subsp. longum in conventional mice through the feeding of raffinose or 1-kestose.

How bifidobacteria colonize and survive in the intestine is not fully understood. The administration of bifidobacteria to conventional mice can be used to evaluate their ability to colonize the intestine in the presence of endogenous gut microbiota. However, human-derived bifidobacteria do not readily colonize the intestines of conventional mice, and although colonization by Bifidobacterium breve UCC2003 has been achieved, the viability of such populations requires improvement. Therefore, we aimed to establish a colonization system with human-derived bifidobacteria of high viability in conventional mice using Bifidobacterium longum subsp. longum 105-A. Lactose, raffinose, and 1-kestose were identified as the preferred carbohydrate sources for the growth of this strain in culture. The administration of B. longum 105-A to conventional BALB/c mice fed these carbohydrates showed that diets containing 6% (w/w) raffinose or 1-kestose facilitated colonization with >108 colony-forming units/g feces for 2 weeks. The population of this strain was more stable in the raffinose-fed group than in the 1-kestose-fed group. The ingestion of these prebiotics had a greater impact on the composition of the microbiota than the administration of B. longum 105-A. The ingestion of these prebiotics also increased the fecal concentrations of organic acids, which was indicative of greater intestinal fermentation. Collectively, we established a colonization system for B. longum 105-A with high viability in conventional mice by feeding the mice raffinose or 1-kestose. This system should be useful for elucidation of the mechanisms of colonization and survival of bifidobacteria in the intestines in the presence of the endogenous 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.

White adipose tissue undergoes browning during preweaning period in association with microbiota formation in mice.

Beige adipocytes are transiently induced during early postnatal period in mice. Previous studies have suggested that, unlike in adults, the induction is independent of the sympathetic nerve activity; however, the mechanism is yet unknown. Here, we showed that beige adipocytes are induced during the preweaning period in association with the formation of microbiota in mice. Alteration of gut microbiota composition in preweaning mice by maternal treatment with antibiotics or high-fat diet feeding substantially suppressed WAT browning. The suppression was also found in pups transplanted cecal microbiota from pups of high-fat diet-fed dams. These treatments reduced the hepatic expression of genes involved in bile acid synthesis and the serum bile acids level. The abundance of Porphyromonadaceae and Ruminococcaceae in microbiota showed a positive and negative correlation with the induction of beige adipocytes, respectively. This finding may provide comprehensive understanding of the association between gut microbiota and adipose tissue development in the neonatal period.

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.

Cyclic Nigerosylnigerose Attenuates High-Fat Diet-Induced Fat Deposition, Colonic Inflammation, and Abnormal Glucose Metabolism and Modifies Gut Immunoglobulin a Reactivity to Commensal Bacteria.

SCOPE: High-fat diet (HFD) intake induces gut dysbiosis, inflammation in the peripheral tissues, and a reduction in immunoglobulin A (IgA) coating of gut bacteria, which is related to HFD-induced insulin resistance (IR). This study evaluates the effect of cyclic nigerosylnigerose (CNN), a dietary fiber that prevents gut inflammation and promotes IgA coating of gut bacteria, on the above-mentioned HFD-induced disorders. METHODS AND RESULTS: Balb/c mice are fed an HFD and administered CNN for 20 weeks. CNN administration reduces mesenteric adipose tissue weight, colonic tumor necrosis factor α (TNFα) mRNA expression, and serum endotoxin levels and ameliorates HFD-induced abnormal glucose metabolism. Additionally, CNN administration promotes gut bacteria-specific IgA secretion and alters IgA reactivity to gut bacteria. The alterations of IgA reactivity to specific bacteria such as Erysipelatoclostridium, Escherichia, Faecalibaculum, Lachnospiraceae genera, and Stenotrophomonas are correlated with mesenteric adipose tissue weight, colonic TNFα mRNA expression, serum endotoxin levels, and a homeostasis model assessment for IR. CONCLUSION: CNN-induced alterations in IgA reactivity to gut bacteria may be related to the suppression of HFD-induced fat deposition, colonic inflammation, endotoxemia, and IR. These observations indicate that dietary fiber that modulates IgA reactivity to gut bacteria may be useful in preventing HFD-induced disorders.

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.

Aicda deficiency exacerbates high-fat diet-induced hyperinsulinemia but not gut dysbiosis in mice.

Immunoglobulin A (IgA) is a major antibody in the gut. We previously observed that a high-fat diet (HFD) reduces IgA reactivity to gut microbiota, but the physiological implications have yet to be elucidated. We hypothesized that a reduction of IgA reactivity to gut microbiota induced by a HFD may contribute to development of gut dysbiosis and inflammation that accompanies HFD feeding. To test our hypothesis, we used Aicda deficient mice, which have a deficiency in IgA production. Aicda deficient mice and wild-type mice were fed normal-fat diet or HFD for 12 weeks. We found that HFD feeding but not Aicda deficiency altered the fecal microbiota composition. Meanwhile, Aicda deficiency significantly increased gene expression of inflammatory cytokines in the ileum, but not in the colon despite no significant difference between diets. These results suggest that a reduction of IgA reactivity to gut microbiota induced by HFD partly contributes to development of inflammation in the ileum, but not to gut dysbiosis. We also found that the fasting blood insulin level was significantly increased by Aicda deficiency only under HFD feeding. Furthermore, the gene expression of monocyte chemoattractant protein1, a major chemokine responsible for the onset of hyperinsulinemia, in the liver was significantly increased by HFD feeding and tended to be increased by Aicda deficiency. These findings suggest that a reduction of IgA reactivity to gut microbiota induced by HFD contributes to hyperinsulinemia partly via increasing monocyte chemoattractant protein-1 expression in the liver.

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.

Intestinal microbiota transplantation reveals the role of microbiota in dietary regulation of RegIIIß and RegIIIγ expression in mouse intestine.

RegIIIß and RegIIIγ are antimicrobial peptides expressed in intestinal epithelial cells. Expression of these peptides is reportedly decreased by high-fat diet (HFD) and increased by indigestible oligosaccharides in mice. Clearly, these dietary regimens change the structure of intestinal microbiota. We employed an intestinal microbiota transplantation (IMT) to test whether diet-induced changes in the expression of these peptides are mediated by gut microbiota. C57BL/6J mice were fed either a normal-fat diet (NFD), a HFD, or a NFD supplemented with or without 1-kestose (KES), an indigestible oligosaccharide. Ileal RegIIIß and RegIIIγ mRNA levels were lower in mice receiving IMT from HFD-fed mice than in those receiving NFD-fed mice and higher in mice receiving IMT from KES-supplemented mice than in those receiving the mice without KES supplementation. Western blot analysis showed that serum RegIIIß levels changed in parallel with the ileal mRNA levels. We propose that HFD- and KES-induced changes in the ileal RegIIIß and RegIIIγ expression and in the circulating RegIIIß levels are mediated, at least in part, by intestinal microbiota.

Application of Recombinase-Based In Vivo Expression Technology to Bifidobacterium longum subsp. longum for Identification of Genes Induced in the Gastrointestinal Tract of Mice.

Bifidobacteria are one of the major components in human gut microbiota and well-known as beneficial microbes. However, clarification of commensal mechanisms of bifidobacteria in the intestines is still ongoing, especially in the presence of the gut microbiota. Here, we applied recombinase-based in vivo expression technology (R-IVET) using the bacteriophage P1 Cre/loxP system to Bifidobacterium longum subsp. longum 105-A (B. longum 105-A) to identify genes that are specifically expressed in the gastrointestinal tract of conventionally raised mice. Oral administration of the genomic DNA library of B. longum 105-A to conventionally raised mice resulted in the identification of 73 in vivo-induced genes. Four out of seven tested genes were verified in vivo-specific induction at least in the cecum by quantitative reverse transcription PCR. Although there is still room for improvement of the system, our findings can contribute to expanding our understanding of the commensal behavior of B. longum in the gut ecosystem.

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.

Extra-adrenal glucocorticoids contribute to the postprandial increase of circulating leptin in mice.

Leptin, an adipokine secreted by white adipocytes, is known for its function in regulating food intake and energy expenditure, but the mechanisms regulating its circulating levels is not fully understood. Our previous findings suggest that as yet unidentified humoral factors released from enterocytes are involved. The present study tested glucocorticoids (GCs) as candidate factors. Supplementation of corticosterone and cortisol promoted leptin production in murine adipocytes from the 3T3-L1 cell strain and human adipocytes from the Simpson Golabi-Behmel syndrome (SGBS) cell strain, respectively. These changes were observed in the absence but not presence of the GC-receptor antagonist mifepristone. The cortisol concentration in conditioned medium (CM) of human enterocyte-like Caco-2 cells was increased by phorbol-12-myristate 13-acetate and decreased by metyrapone. When SGBS adipocytes were cultured in these CMs, leptin production was positively associated with cortisol concentrations. During a 2-h refeeding after fasting, plasma leptin levels continued to increase in sham-operated mice, transiently increased at 60 min in adrenalectomized mice, and were unchanged in mifepristone-administered mice. These results suggest that extra-adrenal GCs contribute to the GC-receptor signaling-dependent increase of postprandial circulating leptin, whereas further studies will be required to determine whether enterocytes participate in the GCs-mediated increase of postprandial circulating leptin.

Fructo-oligosaccharide-Induced Transient Increases in Cecal Immunoglobulin A Concentrations in Rats Are Associated with Mucosal Inflammation in Response to Increased Gut Permeability.

Background: The mechanism underlying transient increases in immunoglobulin (Ig) A concentrations in the cecal contents of rats fed fructo-oligosaccharide (FOS) is unclear.Objective: This study was designed to test whether increased IgA concentrations represent one aspect of the inflammatory response to increased permeability induced by FOS in the cecum.Methods: Seven-week-old male Wistar rats were fed a fiber-free semipurified diet (FFP) with or without supplemental FOS (60 g/kg diet) for 9 or 58 d [experiment (expt.) 1], 7 d (expt. 2), or 7 or 56 d (expt. 3). In addition to measuring IgA concentrations in cecal content, we assessed gut permeability, inflammatory responses (expt. 1), the number of IgA plasma cells in the cecal lamina propria, polymeric Ig receptor (pIgR) expression in the cecal mucosa (expt. 2), and the condition of the cecal mucus layer (expt. 3).Results: The cecal IgA concentration in the FOS-fed rats was 15-fold higher than that of the rats fed FFP for 9 d (P < 0.05). Gut permeability estimated by urinary chromium-EDTA excretion, bacterial translocation to mesenteric lymph nodes, myeloperoxidase activity, and expression of inflammatory cytokine genes in the cecal mucosa was greater in the FOS-fed rats than in the rats fed FFP for 9 d. These effects were not observed in the rats fed FOS for 58 d (expt. 1). Accompanying the higher cecal IgA concentration, pIgR protein and the number of IgA plasma cells in the cecal mucosa were higher in the FOS-fed rats than in the rats fed FFP for 7 d (expt. 2). Destruction of the mucus layer on the epithelial surface, as evidenced by Alcian blue staining in the cecal sections, was evident in the rats fed FOS for 7 d, but the mucus layer appeared normal in the rats fed FOS for 56 d (expt. 3).Conclusions: These findings suggest that transient increases in cecal IgA concentrations induced by FOS in rats are associated with mucosal inflammation in response to increased gut permeability; these are presumably evoked by disruption of the cecal mucus barrier. The observed responses could contribute to the maturation of the gut immune system.

Exosomes isolated from sera of mice fed Lactobacillus strains affect inflammatory cytokine production in macrophages in vitro.

Orally administered Lactobacillus strains, including L. plantarum No.14 and L. rhamnosus GG, reportedly reduce inflammatory cytokine production in mice. The present study tested our idea that circulating exosomes mediate the action of Lactobacillus strains. The lipopolysaccharide-induced production of TNF-α and IL-6 in vitro was attenuated in peritoneal exudate cells (PECs) isolated from C57BL/6N mice that had been fed L. plantarum No.14. When PECs were cultured for 24 h with exosomes isolated from the serum of mice fed L. plantarum No.14 or L. rhamnosus GG, accumulation of both TNF-α and of the corresponding mRNA was lowered. Growth in the presence of these exosomes also decreased the production of TNF-α and IL-6 by the murine macrophage cell line RAW264.7. In contrast, supplementation with exosome-depleted serum of mice fed L. plantarum No.14 or L. rhamnosus GG failed to affect the production of TNF-α and IL-6 by RAW264.7 cells. When PECs and RAW264.7 cells were cultured for 24 h with PKH67-labeled exosomes isolated from murine serum, fluorescent signal was observed inside the cells, suggesting that these cells incorporate serum exosomes. We propose that the anti-inflammatory activity of orally administered L. plantarum No.14 and L. rhamnosus GG is mediated, at least in part, by circulating exosomes.

Organoids as an ex vivo model for studying the serotonin system in the murine small intestine and colon epithelium.

Intestinal organoids were recently established as an ex vivo model of the intestinal epithelium. The present study investigated the serotonin (5-hydroxytryptamine, 5-HT) system using organoids. Organoids from murine small intestinal and colonic crypts were successfully cultured. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that small intestinal and colonic organoids express mRNAs encoding tryptophan hydroxylase-1 (TPH1) (the rate-limiting enzyme of 5-HT synthesis), serotonin reuptake transporter (SERT), 5-HT receptor (HTR)2A, HTR2B, and HTR4. SERT mRNA levels were significantly higher in the small intestine than in the colon in both the mucosal tissues and organoids, as estimated by quantitative real-time RT-PCR. Although the 5-HT concentration and levels of chromogranin A (CgA) (an enteroendocrine cell marker), TPH1, and HTR4 mRNAs were significantly higher in the colonic mucosa than the small intestinal mucosa, they were the same in small intestinal and colonic organoids. There were no significant differences in HTR2A and HTR2B mRNA levels between the small intestine and colon in either the mucosal tissues or organoids. Immunofluorescence staining showed that the number of CgA-positive cells in the colonic organoids appeared to increase upon culturing with acetate. Acetate supplementation significantly increased CgA, TPH1, and HTR4 mRNA levels in the colonic organoids. We propose that organoids are useful for investigating the 5-HT system in the intestinal epithelium, even though colonic organoids may require gut microbiota-derived factors such as short-chain fatty acids.