Intestinal commensal microbiota and cytokines regulate Fut2+ Paneth cells for gut defense.

Paneth cells are intestinal epithelial cells that release antimicrobial peptides, such as α-defensin as part of host defense. Together with mesenchymal cells, Paneth cells provide niche factors for epithelial stem cell homeostasis. Here, we report two subtypes of murine Paneth cells, differentiated by their production and utilization of fucosyltransferase 2 (Fut2), which regulates α(1,2)fucosylation to create cohabitation niches for commensal bacteria and prevent invasion of the intestine by pathogenic bacteria. The majority of Fut2- Paneth cells were localized in the duodenum, whereas the majority of Fut2+ Paneth cells were in the ileum. Fut2+ Paneth cells showed higher granularity and structural complexity than did Fut2- Paneth cells, suggesting that Fut2+ Paneth cells are involved in host defense. Signaling by the commensal bacteria, together with interleukin 22 (IL-22), induced the development of Fut2+ Paneth cells. IL-22 was found to affect the α-defensin secretion system via modulation of Fut2 expression, and IL-17a was found to increase the production of α-defensin in the intestinal tract. Thus, these intestinal cytokines regulate the development and function of Fut2+ Paneth cells as part of gut defense.

Reactive granulopoiesis depends on T-cell production of IL-17A and neutropenia-associated alteration of gut microbiota.

Granulopoiesis in the bone marrow adjusts cellular output as demand for neutrophils changes. Reactive granulopoiesis is induced by profound neutropenia, but its mechanism remains to be clarified. We herein explored its mechanisms using mouse models of syngeneic hematopoietic stem cell transplantation (SCT) and 5-fluorouracil-induced neutropenia. After SCT, T cell production of IL-17A was up-regulated. Neutrophil recovery was significantly delayed in IL-17A-deficient or T cell-deficient RAG1-/- mice, and adoptive transfer of wild-type (WT) T cells facilitated neutrophil engraftment. Gut decontamination with oral antibiotics suppressed T cell production of IL-17A and impaired neutrophil recovery. Transplantation of fecal microbiota collected from neutropenic, not naive, mice promoted neutrophil recovery in these mice, suggesting that neutropenia-associated microbiota had a potential to stimulate reactive granulopoiesis. Our study uncovered a cross talk between gut microbiota and neutropenia after SCT and chemotherapy.

Association between oral frailty and Prevotella percentage in the oral microbiota of community-dwelling older adults who participated in the CHEER Iwamizawa project, Japan.

BACKGROUND: Prevotella bacteria are associated with inherent diseases of the oral cavity, such as periodontal disease, and systemic diseases. Oral frailty (OF) has been associated with nursing necessity and death. However, the relationship between OF and oral microbiota has not been fully clarified. OBJECTIVE: This cross-sectional study investigated the association between OF and Prevotella percentage in the oral microbiota of community-dwelling older adults. METHODS: Oral bacteria species from saliva were identified in 208 community-dwelling older individuals aged ≥60 years in Japan. The proportion of Prevotella in the oral microbiota was classified into three tertile groups, and its relationship with each test item for OF (number of remaining teeth, masticatory performance, oral diadochokinesis, tongue pressure, difficulties eating tough foods, difficulties swallowing tea or soup, number of applicable OF judgement items, and existence of OF) was examined using ordinal logistic regression analysis. RESULTS: The Prevotella proportions were classified into lower, middle and upper groups, comprising 70, 69 and 69 participants, respectively. The three groups showed a significant relationship between the number of remaining teeth (odds ratio [OR]: 0.946, 95% confidence interval [CI]: 0.915-0.977), masticatory performance (OR: 0.897, 95% CI: 0.844-0.953), number of applicable OF judgement items (OR: 1.477, 95% CI: 1.14-1.915), and existence of OF (OR: 4.194, 95% CI: 1.519-11.576). CONCLUSION: The proportion of Prevotella in oral microbiota was high in individuals with OF. Among the older adults, the type of oral microbiota and systemic diseases may be related to the examination and management of oral function decline.

Efficient recombinant production of mouse-derived cryptdin family peptides by a novel facilitation strategy for inclusion body formation.

BACKGROUND: A number of antimicrobial peptides (AMPs) hold promise as new drugs owing to their potent bactericidal activity and because they are often refractory to the development of drug resistance. Cryptdins (Crps) are a family of antimicrobial peptides found in the small intestine of mice, comprising six isoforms containing three sets of disulfide bonds. Although Crp4 is actively being investigated, there have been few studies to date on the other Crp isoforms. A prerequisite for detailed characterization of the other Crp isoforms is establishment of efficient sample preparation methods. RESULTS: To avoid degradation during recombinant expression of Crps in E. coli, co-expression of Crps with the aggregation-prone protein human α-lactalbumin (HLA) was used to promote the formation of stable inclusion bodies. Using this method, the production of Crp4 and Crp6 by the BL21 strain was effective, but the expression of other Crp isoforms was not as efficient. The results of a cell-free system study suggested that Crps were degraded, even though a substantial amounts of Crps were synthesized. Therefore, using the Origami™ B strain, we were able to significantly increase the expression efficiency of Crps by promoting the formation of erroneous intermolecular disulfide bonds between HLA and Crps, thereby promoting protein aggregation and inclusion body formation, which prevented degradation. The various Crp isoforms were successfully refolded in vitro and purified using reversed-phase HPLC. In addition, the yield was further improved by deformylation of formyl-Crps. We measured the antibacterial activity of Crps against both Gram-positive and Gram-negative bacteria. Each Crp isoform exhibited a completely different trend in antimicrobial activity, although conformational analysis by circular dichroism did not reveal any significant steric differences. CONCLUSION: In this study, we established a novel and efficient method for the production of the cryptdin family of cysteine-containing antimicrobial peptides. Additionally, we found that there were notable differences in the antibacterial activities of the various Crp family members. The expression system established in this study is expected to provide new insights regarding the mechanisms underlying the different antibacterial activities of the Crp family of peptides.

Efficient and simple genetic engineering of enteroids using mouse isolated crypts for investigating intestinal functions.

Intestinal epithelial cells separate subepithelial tissues from luminal environment formed with food, incoming pathogens, and resident intestinal microbiota, etc., and elicit various intestinal function. Enteroid, a three-dimensional culture system of small intestinal epithelial cells, has been widely used for analyzing the intestinal function, further a transgenic enteroid was developed to investigate the molecular mechanisms. However, conventional transgenic enteroid production method, which transfer gene into single stem cells, has limitations including low efficiency and time-consuming. Here we show that by gene transfer into small intestinal isolated crypts maintaining stem cell niche, a transgenic enteroid was obtained quickly and efficiently. Isolated crypts were transfected by lentiviral vector without separating into single cells, and transgenic enteroid composed of all lineages of intestinal epithelial cells was generated at day 7 with yield of 56%, maintaining the intestinal function in drug transport and innate immunity. Our efficient and simple transgenic enteroid generation method enables high-throughput investigation of intestinal epithelial cells and contributes to understanding intestinal function.

Potent bactericidal activity of reduced cryptdin-4 derived from its hydrophobicity and mediated by bacterial membrane disruption.

Defensin is a cysteine-rich antimicrobial peptide with three disulphide bonds under normal oxidative conditions. Cryptdin-4 (Crp4) is a defensin secreted by Paneth cells in the small intestine of mice, and only reduced Crp4 (Crp4red) shows activity against enteric commensal bacteria, although both oxidised Crp4 (Crp4ox) and Crp4red can kill non-commensal bacteria. To investigate the molecular factors that affect the potent antimicrobial activity of Crp4red, the bactericidal activities of Crp4ox and Crp4red, Crp4 with all Cys residues substituted with Ser peptide (6C/S-Crp4), and Crp4 with all thiol groups modified by N-ethylmaleimide (NEM-Crp4) were assessed. All peptides showed bactericidal activity against non-commensal bacteria, whereas Crp4red and NEM-Crp4 showed bactericidal activity against commensal bacteria. These potent peptides exhibited high hydrophobicity, which was strongly correlated with membrane insertion. Intriguingly, Crp4ox formed electrostatic interactions with the membrane surface of bacteria, even without exerting bactericidal activity. Moreover, the bactericidal activity of both oxidised and reduced forms of Crp4 was abolished by inhibition of electrostatic interactions; this finding suggests that Crp4red targets bacterial membranes. Finally, a liposome leakage assay against lipids extracted from commensal bacteria demonstrated a correlation with bactericidal activity. These results suggest that the potent bactericidal activity of Crp4red is derived from its hydrophobicity, and the bactericidal mechanism involves disruption of the bacterial membrane. Findings from this study provide a better understanding of the bactericidal mechanism of both Crp4ox and Crp4red.

Oral frailty and carriage of oral Candida in community-dwelling older adults (Check-up to discover Health with Energy for senior Residents in Iwamizawa; CHEER Iwamizawa).

OBJECTIVE: To examine the association between oral frailty and oral Candida carriage as a general indicator of deteriorating oral function in older adults. BACKGROUND: Older adults exhibit an elevated risk of oral candidiasis caused by Candida. Although many studies have identified factors associated with oral Candida carriage, none have evaluated its relationship with oral function. MATERIALS AND METHODS: This study included 210 community-dwelling older adults aged ≥60 years who participated in wellness checks. Fungal flora expression in saliva samples was evaluated to identify oral C. albicans and C. glabrata. Participants were categorised by detection of neither strain (group 1), either one of the strains (group 2), or both strains (group 3). The relationship between oral Candida carriage and oral frailty was evaluated by multinomial logistic regression analysis. RESULTS: The participants included 58 men and 152 women with a mean age of 74.2 ± 6.1 years. A total of 88 (41.9%), 94 (44.8%) and 28 (13.3%) participants were assigned to groups 1, 2 and 3 respectively. In the multinomial logistic regression analysis, significant associations were observed between group 1 and group 2 for "Have you choked on your tea or soup recently?" and the number of applicable oral frailty items. Between group 1 and group 3, significant associations were observed for the number of remaining teeth, masticatory performance and the number of applicable oral frailty items. CONCLUSION: We obtained basic data useful for intervention studies aimed at verifying whether oral function management prevents deterioration of the oral bacterial flora.

Simultaneous real-time analysis of Paneth cell and intestinal stem cell response to interferon-γ by a novel stem cell niche tracking method.

Paneth cells and Lgr5+ intestinal stem cells (Lgr5+ ISCs) constitute the stem cell niche and maintain small intestinal epithelial integrity by recognizing various niche factors derived from subepithelial cells and external antigens. Although it has been known that interferon-γ (IFN-γ), a Th1 cytokine, is associated with intestinal epithelial disruption during inflammation as a niche factor, dynamics of Paneth cells and Lgr5+ ISCs in response to IFN-γ remain to be understood. Here we show that CAG-tdTomato;Lgr5-EGFP (CT-LE) mice generated in this study enable to identify Paneth cells and Lgr5+ ISCs separately by fluorescence signals. Lgr5+ ISCs underwent cell death a little earlier than Paneth cells in response to IFN-γ by simultaneous tracking using CT-LE mice. In addition, the timing of cell death in most Paneth cells overlapped with Lgr5+ ISCs, suggesting that Paneth cell depletion is induced directly by IFN-γ. Taken together, we established a novel simultaneous stem cell niche tracking method and clarified the involvement of both Paneth cells and Lgr5+ ISCs in stem cell niche damage induced by IFN-γ, further contribute to understanding the mechanism for maintaining intestinal homeostasis by stem cell niche.

Disease progression-associated alterations in fecal metabolites in SAMP1/YitFc mice, a Crohn's disease model.

INTRODUCTION: Crohn's disease (CD) is a chronic, relapsing inflammatory bowel disease affecting the gastrointestinal tract. Although its precise etiology has not been fully elucidated, an imbalance of the intestinal microbiota has been known to play a role in CD. Fecal metabolites derived from microbiota may be related to the onset and progression of CD OBJECTIVES: This study aimed to clarify the transition of gut microbiota and fecal metabolites associated with disease progression using SAMP1/YitFc mice, a model of spontaneous CD METHODS: The ileum tissues isolated from SAMP1/YitFc mice at different ages were stained with hematoxylin-eosin for histologic characterization with CD progression. Feces from control, Institute of Cancer Research (ICR; n = 6), and SAMP1/YitFc (n = 8) mice at different ages were subjected to microbial analysis and 1H nuclear magnetic resonance (NMR) analysis to investigate fluctuations in gut microbiota and fecal metabolites with CD progression RESULTS: Relative abundance of the Lachnospiraceae, Ruminococcaceae, Bacteroidaceae, and Bacteroidales S24-7 at family-level gut microbiota and fecal metabolites, such as short-chain fatty acids, lactate, glucose, xylose, and choline, dramatically fluctuated with histologic progression of intestinal inflammation in SAMP1/YitFc mice. Unlike the other metabolites, fecal taurine concentration in SAMP1/YitFc mice was higher than ICR mice regardless of age CONCLUSION: The fecal metabolites showing characteristic fluctuations may help to understand the inflammatory mechanism associated with CD, and might be utilized as potential biomarkers in predicting CD pathology.

Entamoeba histolytica Alters Ileal Paneth Cell Functions in Intact and Muc2 Mucin Deficiency.

Enteric α-defensins, termed cryptdins (Crps) in mice, and lysozymes secreted by Paneth cells contribute to innate host defense in the ileum. Antimicrobial factors, including lysozymes and ß-defensins, are often embedded in luminal glycosylated colonic Muc2 mucin secreted by goblet cells that form the protective mucus layer critical for gut homeostasis and pathogen invasion. In this study, we investigated ileal innate immunity against Entamoeba histolytica, the causative agent of intestinal amebiasis, by inoculating parasites in closed ileal loops in Muc2+/+ and Muc2-/- littermates and quantifying Paneth cell localization (lysozyme expression) and function (Crp secretion). Relative to Muc2+/+ littermates, Muc2-/- littermates showed a disorganized mislocalization of Paneth cells that was diffusely distributed, with elevated lysozyme secretion in the crypts and on villi in response to E. histolytica Inhibition of E. histolytica Gal/GalNAc lectin (Gal-lectin) binding with exogenous galactose and Entamoeba histolytica cysteine proteinase 5 (EhCP5)-negative E. histolytica had no effect on parasite-induced erratic Paneth cell lysozyme synthesis. Although the basal ileal expression of Crp genes was unaffected in Muc2-/- mice in response to E. histolytica, there was a robust release of proinflammatory cytokines and Crp peptide secretions in luminal exudates that was also present in the colon. Interestingly, E. histolytica-secreted cysteine proteinases cleaved the proregion of Crp4 but not the active form. These findings define Muc2 mucin as an essential component of ileal barrier function that regulates the localization and function of Paneth cells critical for host defense against microbes.

Ploidy-dependent change in cyclin D2 expression and sensitization to cdk4/6 inhibition in human somatic haploid cells.

Near-haploidy is observed in certain cancer types, but ploidy-dependent alterations in gene regulation in the haploid state remain elusive. Here, by comparative transcriptome analysis between human isogenic haploid and diploid cell lines, we found lowering of cyclin D2 level in haploids. Acute genome duplication in haploids restored cyclin D2 expression to diploid level, indicating that the regulation of cyclin D2 expression is directly linked to ploidy. Downstream pathways of cyclin D2, such as Rb phosphorylation and p27 sequestration remained intact in haploids, suggesting that they adapt to lowered cyclin D level. Interestingly, however, haploid cells were more susceptible to cdk4/6 inhibition compared to diploids. Our finding indicates feasibility of selective growth suppression of haploid cells based on ploidy-linked gene regulation.

Efficient production of a correctly folded mouse α-defensin, cryptdin-4, by refolding during inclusion body solubilization.

Mammalian α-defensins contribute to innate immunity by exerting antimicrobial activity against various pathogens. To perform structural and functional analysis of α-defensins, large amounts of α-defensins are essential. Although many expression systems for the production of recombinant α-defensins have been developed, attempts to obtain large amounts of α-defensins have been only moderately successful. Therefore, in this study, we applied a previously developed aggregation-prone protein coexpression method for the production of mouse α-defensin cryptdin-4 (Crp4) in order to enhance the formation of inclusion bodies in Escherichia coli expression system. By using this method, we succeeded in obtaining a large amount of Crp4 in the form of inclusion bodies. Moreover, we attempted to refold Crp4 directly during the inclusion-body solubilization step under oxidative conditions. Surprisingly, even without any purification, Crp4 was efficiently refolded during the solubilization step of inclusion bodies, and the yield was better than that of the conventional refolding method. NMR spectra of purified Crp4 suggested that it was folded into its correct tertiary structure. Therefore, the method described in this study not only enhances the expression of α-defensin as inclusion bodies, but also eliminates the cumbersome and time-consuming refolding step.

Bacterial cell wall components regulate adipokine secretion from visceral adipocytes.

Recent studies suggest a relationship between intestinal microbiota and metabolic syndromes; however, the underlying mechanism remains unclear. To clarify this issue, we assessed the effects of bacterial cell wall components on adiponectin, leptin and resistin secretion from rat visceral adipocytes in vitro. We also measured the relative population of Firmicutes and Bacteroidetes in fecal microbiota and the amount of fecal mucin as an intestinal barrier function, when mice were fed a high-fat diet. In the present study, we demonstrated that bacterial cell wall components affect the secretion of adipokines, depending on the presence of antigens from gram-positive or gram-negative bacteria. Lipopolysaccharide markedly inhibited adiponectin, leptin, and resistin secretion, whereas peptidoglycan increased adiponectin secretion and decreased resistin secretion in vitro. In vivo experiments showed that the high-fat diet increased the population of Firmicutes and decreased that of Bacteroidetes. In contrast, the high-fat diet downregulated the stool output and fecal mucin content. These results demonstrate that bacterial cell wall components affect the onset of metabolic syndromes by mediating the secretion of adipokines from visceral adipose tissue. Furthermore, we believe that metabolic endotoxemia is not due to the increasing dominance of gram-negative bacteria, Bacteroidetes, but due to the depression of intestinal barrier function.

Graft-versus-host disease disrupts intestinal microbial ecology by inhibiting Paneth cell production of α-defensins.

Allogeneic hematopoietic stem cell transplantation (SCT) is a curative therapy for various hematologic disorders. Graft-versus-host disease (GVHD) and infections are the major complications of SCT, and their close relationship has been suggested. In this study, we evaluated a link between 2 complications in mouse models. The intestinal microbial communities are actively regulated by Paneth cells through their secretion of antimicrobial peptides, α-defensins. We discovered that Paneth cells are targeted by GVHD, resulting in marked reduction in the expression of α-defensins, which selectively kill noncommensals, while preserving commensals. Molecular profiling of intestinal microbial communities showed loss of physiologic diversity among the microflora and the overwhelming expansion of otherwise rare bacteria Escherichia coli, which caused septicemia. These changes occurred only in mice with GVHD, independently on conditioning-induced intestinal injury, and there was a significant correlation between alteration in the intestinal microbiota and GVHD severity. Oral administration of polymyxin B inhibited outgrowth of E coli and ameliorated GVHD. These results reveal the novel mechanism responsible for shift in the gut flora from commensals toward the widespread prevalence of pathogens and the previously unrecognized association between GVHD and infection after allogeneic SCT.

Effects of exposure to the neonicotinoid pesticide clothianidin on α-defensin secretion and gut microbiota in mice.

The mechanism by which the neonicotinoid pesticide clothianidin (CLO) disrupts the intestinal microbiota of experimental animals is unknown. We focused on α-defensins, which are regulators of the intestinal microbiota. Subchronic exposure to CLO induced dysbiosis and reduced short-chain fatty acid-producing bacteria in the intestinal microbiota of mice. Levels of cryptdin-1 (Crp1, a major α-defensin in mice) in feces and cecal contents were lower in the CLO-exposed groups than in control. In Crp1 immunostaining, Paneth cells in the jejunum and ileum of the no-observed-adverse-effect-level CLO-exposed group showed a stronger positive signal than control, likely due to the suppression of Crp1 release. Our results showed that CLO exposure suppresses α-defensin secretion from Paneth cells as part of the mechanism underlying CLO-induced dysbiosis.

Reciprocal expression of enteric antimicrobial proteins in intestinal graft-versus-host disease.

We recently demonstrated that expression of α-defensins, the major antimicrobial peptides produced by Paneth cells, was severely suppressed in mice with graft-versus-host disease (GVHD). In this study, we found that antibacterial lectin, regenerating islet-derived IIIγ (RegIIIγ) was upregulated in villous enterocytes, thus demonstrating the reciprocal control of enteric antimicrobial proteins in GVHD. Upregulation of RegIIIγ was mediated by a mechanism independent upon radiation-induced intestinal tract damage. MyD88-mediated signaling in intestinal epithelium was required for RegIIIγ upregulation in GVHD and antibiotic therapy downregulated RegIIIγ expression. These results suggest that MyD88-mediated sensing of the intestinal microbes disregulated in GVHD induces RegIIIγ upregulation in GVHD and argue a role for RegIIIγ in the pathogenesis of GVHD.

A monoclonal antibody-based sandwich enzyme-linked immunosorbent assay for detection of secreted α-defensin.

Paneth cells at the base of small intestinal crypts secrete α-defensins, which contribute to innate immunity and shape composition of enteric microbiota. Efforts to establish a relationship between secreted α-defensins and disease have been hampered by a lack of sensitive assays to quantify luminal α-defensins. Here we report on a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for the mouse Paneth cell α-defensin cryptdin-4 (Crp4) in varied sources, including luminal contents rinsed from stomach to distal colon and fecal pellets. One pair of monoclonal antibodies (mAbs), selected from 10 rat hybridomas secreting Crp4-specific mAbs, was optimized for Crp4 detection and specificity in the sandwich ELISA. In CD1 mice, luminal Crp4 levels increased gradually from 6.8 ± 5.2 ng/ml in proximal small intestine to 54.3 ± 10.3 ng/ml in distal small intestine, and the peptide was detected in colonic lumen and feces. Secreted Crp4 was reduced significantly in feces of IL10 null mice, a model of inflammatory bowel disease (IBD) when compared with wild-type controls. This Crp4 sandwich ELISA enables accurate determinations of luminal α-defensins as biomarkers of Paneth cell function and enteric integrity in diverse disease states such as IBD, infectious disease, graft versus host disease, and obesity in association with dysbiosis of the intestinal microbiota.

Associations between Maternal Diet, Human Milk Macronutrients, and Breast-Fed Infant Growth during the First Month of Life in the SMILE Iwamizawa in Japan.

Maternal diet may affect human milk macronutrients, but it remains to be elucidated whether this is also influential in infant growth. This study aimed to examine (1) how maternal diet influences human milk macronutrients, and (2) to what extent the variation in milk macronutrients affects infant growth during the first month of life. In 71 Japanese lactating women, maternal dietary information was collected from the brief-type self-administered diet history questionnaire, and anthropometry of mother-infant dyads was collected from medical records. Macronutrients in milk were analyzed by a Human Milk Analyzer. Maternal retinol intake was associated with the carbohydrate content in human milk at 1-month postpartum (standardized ß coefficient: 0.287; p = 0.038). Moreover, the energy content in human milk was associated with an increase in the weight standard deviation score based on the WHO growth standard at 1 month of age (standardized ß coefficient: 0.399; p = 0.046). Nevertheless, the milk macronutrient was not associated with the risk of infant growth abnormalities. In conclusion, a part of the maternal diet impacts macronutrient contents in human milk, but milk macronutrients have a limited effect on infant growth only within the normal growth curve during the first month of life.

Shorter sleep time relates to lower human defensin 5 secretion and compositional disturbance of the intestinal microbiota accompanied by decreased short-chain fatty acid production.

Sleep is essential for our health. Short sleep is known to increase disease risks via imbalance of intestinal microbiota, dysbiosis. However, mechanisms by which short sleep induces dysbiosis remain unknown. Small intestinal Paneth cell regulates the intestinal microbiota by secreting antimicrobial peptides including α-defensin, human defensin 5 (HD5). Disruption of circadian rhythm mediating sleep-wake cycle induces Paneth cell failure. We aim to clarify effects of short sleep on HD5 secretion and the intestinal microbiota. Fecal samples and self-reported sleep time were obtained from 35 healthy middle-aged Japanese (41 to 60-year-old). Shorter sleep time was associated with lower fecal HD5 concentration (r = 0.354, p = 0.037), lower centered log ratio (CLR)-transformed abundance of short-chain fatty acid (SCFA) producers in the intestinal microbiota such as [Ruminococcus] gnavus group (r = 0.504, p = 0.002) and Butyricicoccus (r = 0.484, p = 0.003), and lower fecal SCFA concentration. Furthermore, fecal HD5 positively correlated with the abundance of these genera and SCFA concentration. These findings suggest that short sleep relates to disturbance of the intestinal microbiota via decreased HD5 secretion.