Effects of subchronic and mild social defeat stress on the intestinal microbiota and fecal bile acid composition in mice.

The gut microbiota plays a crucial role in both the pathogenesis and alleviation of host depression by modulating the brain-gut axis. We have developed a murine model of human depression called the subchronic and mild social defeat stress (sCSDS) model, which impacts not only behavior but also the host gut microbiota and gut metabolites, including bile acids. In this study, we utilized liquid chromatography/mass spectrometry (LC/MS) to explore the effects of sCSDS on the mouse fecal bile acid profile. sCSDS mice exhibited significantly elevated levels of deoxycholic acid (DCA) and lithocholic acid (LCA) in fecal extracts, leading to a notable increase in total bile acids and 7α-dehydroxylated secondary bile acids. Consequently, a noteworthy negative correlation was identified between the abundances of DCA and LCA and the social interaction score, an indicator of susceptibility in stressed mice. Furthermore, analysis of the colonic microbiome unveiled a negative correlation between the abundance of CDCA and Turicibacter. Additionally, DCA and LCA exhibited positive correlations with Oscillospiraceae and Lachnospiraceae but negative correlations with the Eubacterium coprostanoligenes group. These findings suggest that sCSDS impacts the bidirectional interaction between the gut microbiota and bile acids and is associated with reduced social interaction, a behavioral indicator of susceptibility in stressed mice.

Indole-3-Pyruvic Acid, an Aryl Hydrocarbon Receptor Activator, Suppresses Experimental Colitis in Mice.

Aryl hydrocarbon receptor (AHR) agonists are promising immunomodulators that potentially maintain immune tolerance. In this study, we examined the ability of indole-3-pyruvic acid (IPA), a major precursor of microbiota-derived AHR agonists and a proagonist of AHR, to activate AHR. The anti-inflammatory effects of IPA were also evaluated in a mouse model of colitis in comparison with other aromatic pyruvic acids (phenylpyruvic acid and 4-hydroxyphenylpyruvic acid). Among them, IPA showed the strongest ability to activate AHR in vitro and in vivo, and only IPA improved chronic inflammation in an experimental colitis model. IPA attenuated the expression of genes encoding Th1 cytokines and enhanced Il-10 gene expression in the colon. Oral administration of IPA decreased the frequency of IFN-γ+ IL-10- CD4+ T cells and increased that of IFN-γ- IL-10+ CD4+ T cells in the colon lamina propria in a T cell-mediated colitis model. IPA directly promoted the differentiation of type 1 regulatory T cells in vitro. Furthermore, IPA administration attenuated the ability of dendritic cells (DCs) in the mesenteric lymph nodes (MLN) to induce IFN-γ-producing T cells, increased the frequency of CD103+ CD11b- DCs, and decreased the frequency of CD103- CD11b+ DCs in the MLN. Adoptive transfer of MLN CD103+ CD11b- DCs significantly improved the severity of colon inflammation. Treatment with an AHR antagonist inhibited IPA-induced differentiation of type 1 regulatory T cells and the IPA-induced increase in CD103+ CD11b- DCs and attenuated the anti-inflammatory effect of IPA. These findings suggest that IPA potently prevents chronic inflammation in the colon by activating AHR.

Reduced fucosylation in the distal intestinal epithelium of mice subjected to chronic social defeat stress.

Psychological stress can cause dysfunction of the gastrointestinal tract by regulating its interaction with central nervous system (brain-gut axis). Chronic social defeat stress (CSDS) is widely used to produce a rodent model of stress-induced human mood disorders and depression. We previously showed that CSDS significantly affects the intestinal ecosystem including cecal and fecal microbiota, intestinal gene expression profiles and cecal metabolite profiles. Here, we investigated whether the glycosylation pattern in the intestinal epithelium was affected in C57BL/6 mice exposed to CSDS (hereinafter referred to as CSDS mice). A lectin microarray analysis revealed that CSDS significantly reduced the reactivity of fucose-specific lectins (rAOL, TJA-II, rAAL, rGC2, AOL, AAL, rPAIIL and rRSIIL) with distal intestinal mucosa, but not with mucosa from proximal intestine and colon. Flow cytometric analysis confirmed the reduced TJA-II reactivity with intestinal epithelial cells in CSDS mice. In addition, distal intestine expression levels of the genes encoding fucosyltransferase 1 and 2 (Fut1 and Fut2) were downregulated in CSDS mice. These findings suggest that CSDS alters the fucosylation pattern in the distal intestinal epithelium, which could be used as a sensitive marker for CSDS exposure.

The distinct effects of orally administered Lactobacillus rhamnosus GG and Lactococcus lactis subsp. lactis C59 on gene expression in the murine small intestine.

The molecular mechanisms of strain-specific probiotic effects and the impact of the oral administration of probiotic strains on the host's gene expression are not yet well understood. The aim of this study was to investigate the strain-specific effects of probiotic strain intake on gene expression in the murine small intestine. Two distinct strains of lactic acid bacteria, Lactobacillus rhamnosus GG (GG) and Lactococcus lactis subsp. lactis C59 (C59), were orally administered to BALB/c mice, daily for 2 weeks. The total RNA was isolated from the upper (including the duodenum) and lower (the terminal ileum) small intestine, and gene expression was assessed by microarray analysis. The data revealed (1) oral administration of C59 and GG markedly down-regulated the expression of genes encoding fibrinogen subunits and plasminogen in the upper small intestine; (2) administration of more than 1 × 107 CFU/day of GG changed the gene expression of the host ileum. (3) strain- and dose-related effects on various GO biological processes; and (4) enrichment for B cell-related Gene Ontology terms among up-regulated genes in the terminal ileum of mice administered the 1 × 109 CFU/day of GG. The distinct effects of GG and C59 on gene expression in the intact small intestine provide clues to understand how the health beneficial effects of specific strains of probiotic bacteria are mediated by interactions with intestinal cells.

Promoting effect of lactoferrin on barrier function and epithelial differentiation of human keratinocytes.

The purpose of this study was to elucidate the effects of bovine lactoferrin on keratinocyte differentiation and barrier function. Addition of bovine lactoferrin to differentiating HaCaT human keratinocytes led to increased transepithelial electrical resistance (TER), a marker of epithelial barrier function. This elevation was followed by upregulation of two differentiation markers, involucrin and filaggrin. The expression level of sterol regulatory element-binding protein-1 was also enhanced by bovine lactoferrin. The lactoferrin-induced upregulation of involucrin and filaggrin expression were confirmed in normal human epidermal keratinocytes (NHEK). Treatment with SB203580, a p38 mitogen-activated protein kinase (MAPK) α inhibitor, impaired the upregulation of involucrin and filaggrin expression in response to lactoferrin. The elevation of p38 MAPK phosphorylation was further enhanced by lactoferrin in the initial stage of differentiation of HaCaT keratinocytes. The findings suggest that bovine lactoferrin promotes epithelial differentiation by a p38-MAPK-dependent mechanism.

Role of CXC chemokine receptor type 4 as a lactoferrin receptor.

Lactoferrin exerts its biological activities by interacting with receptors on target cells, including LDL receptor-related protein-1 (LRP-1/CD91), intelectin-1 (omentin-1), and Toll-like receptor 4 (TLR4). However, the effects mediated by these receptors are not sufficient to fully explain the many functions of lactoferrin. C-X-C-motif cytokine receptor 4 (CXCR4) is a ubiquitously expressed G-protein coupled receptor for stromal cell-derived factor-1 (SDF-1/CXCL12). Lactoferrin was found to be as capable as SDF-1 in blocking infection by an HIV variant that uses CXCR4 as a co-receptor (X4-tropic HIV), suggesting that lactoferrin interacts with CXCR4. We addressed whether CXCR4 acts as a lactoferrin receptor using HaCaT human keratinocytes and Caco-2 human intestinal cells. We found that bovine lactoferrin interacted with CXCR4-containing lipoparticles, and that this interaction was not antagonized by SDF-1. In addition, activation of Akt in response to lactoferrin was abrogated by AMD3100, a small molecule inhibitor of CXCR4, or by a CXCR4-neutralizing antibody, suggesting that CXCR4 functions as a lactoferrin receptor able to mediate activation of the PI3K-Akt signaling pathway. Lactoferrin stimulation mimicked many aspects of SDF-1-induced CXCR4 activity, including receptor dimerization, tyrosine phosphorylation, and ubiquitination. Cycloheximide chase assays indicated that turnover of CXCR4 was accelerated in response to lactoferrin. These results indicate that CXCR4 is a potent lactoferrin receptor that mediates lactoferrin-induced activation of Akt signaling.

Omics Studies of the Murine Intestinal Ecosystem Exposed to Subchronic and Mild Social Defeat Stress.

The microbiota-gut-brain axis plays an important role in the development of stress-induced mental disorders. We previously established the subchronic and mild social defeat stress (sCSDS) model, a murine experimental model of depression, and investigated the metabolomic profiles of plasma and liver. Here we used omics approaches to identify stress-induced changes in the gastrointestinal tract. Mice exposed to sCSDS for 10 days showed the following changes: (1) elevation of cholic acid and reduction of 5-aminovaleric acid among cecal metabolites; (2) downregulation of genes involved in the immune response in the terminal ileum; (3) a shift in the diversity of the microbiota in cecal contents and feces; and (4) fluctuations in the concentrations of cecal metabolites produced by gut microbiota reflected in plasma and hepatic metabolites. Operational taxonomic units within the family Lachnospiraceae showed an inverse correlation with certain metabolites. The social interaction score correlated with cecal metabolites, IgA, and cecal and fecal microbiota, suggesting that sCSDS suppressed the ileal immune response, altering the balance of microbiota, which together with host cells and host enzymes resulted in a pattern of accumulated metabolites in the intestinal ecosystem distinct from that of control mice.

Protective effect of indole-3-pyruvate against ultraviolet b-induced damage to cultured HaCaT keratinocytes and the skin of hairless mice.

Previous investigations demonstrated that pyruvate protects human keratinocytes against cell damage stemming from exposure to ultraviolet B (UVB) radiation. This study endeavoured to elucidate the protective capacity of aromatic pyruvates (e.g., phenylpyruvate (PPyr), 4-hydroxyphenylpyruvate (HPPyr), and indole-3-pyruvate (IPyr)) against UVB-induced injury to skin cells, both in vitro and in vivo. Cultured human HaCaT keratinocytes were irradiated with UVB light (60 mJ/cm2) and maintained with or without test compounds (1-25 mM).In addition, the dorsal skin of hairless mice (HR-1) was treated with test compounds (10 µmol) and exposed to UVB light (1 J/cm2) twice [corrected]. The ability of the test compounds to ameliorate UVB-induced cytotoxicity and inflammation was then assessed. Aromatic pyruvates reduced cytotoxicity in UVB-irradiated HaCaT keratinocytes, and also diminished the expression of interleukin 1ß (IL-1ß) and interleukin 6 (IL-6). IPyr was more efficacious than either PPyr or HPPyr. Furthermore, only IPyr inhibited cyclooxygenase-2 (Cox-2) expression at both the mRNA and the protein level in UVB-treated keratinocytes. Topical application of IPyr to the dorsal skin of hairless mice reduced the severity of UVB-induced skin lesions, the augmentation of dermal thickness, and transepithelial water loss. Overproduction of IL-1ß and IL-6 in response to UVB radiation was also suppressed in vivo by the topical administration of IPyr. These data strongly suggest that IPyr might find utility as a UVB-blocking reagent in therapeutic strategies to lessen UVB-induced inflammatory skin damage.

Prevention of UVB-induced production of the inflammatory mediator in human keratinocytes by lactic acid derivatives generated from aromatic amino acids.

The anti-inflammatory effects of lactic acid derivatives were investigated on ultraviolet B (UVB)-irradiated HaCaT human keratinocytes. A pretreatment with indole-3-lactic acid (ILA) and 4-hydroxyphenyllactic acid (HPLA) inhibited the UVB-induced production of interlekin-6 (IL-6). The inhibitory effect of L-HPLA was equivalent to that of a corresponding racemic mixture (DL-HPLA), suggesting that optical isomerism did not affect the anti-inflammatory activity of HPLA.

Protective effect of pyruvate against UVB-induced damage in HaCaT human keratinocytes.

The protective effect of pyruvate against ultraviolet B (UVB)-induced damage was investigated in human immortalized keratinocytes (HaCaT cells). Although pyruvate did not inhibit UVB-induced stimulation of intracellular reactive oxygen species (ROS) levels, it did improve the survival rate of UVB-irradiated HaCaT cells. Furthermore, pyruvate suppressed the UVB-induced mRNA expression of inflammatory mediators such as interleukin (IL)-1α, IL-1ß, IL-6 and cyclooxygenase-2 (Cox-2). This decrease was associated with the reduced secretion of IL-1α, IL-1ß, IL-6 and prostaglandin E2 (PGE2) into culture media. In addition, pyruvate reversed the phosphorylation of p38 mitogen-activated protein kinase (MAPK), induced by UVB-irradiation, in HaCaT cells but increased p38 MAPK phosphorylation in sham-irradiated cells. UVB-induced production of IL-6 was inhibited by SB203580, a p38 MAPK inhibitor. These results suggested that pyruvate inhibits UVB-mediated inflammatory response by inhibiting the p38 MAPK activation.

Roles of lactoferrin on skin wound healing.

Skin wound healing is a complex biological process that requires the regulation of different cell types, including immune cells, keratinocytes, fibroblasts, and endothelial cells. It consists of 5 stages: hemostasis, inflammation, granulation tissue formation, re-epithelialization, and wound remodeling. While inflammation is essential for successful wound healing, prolonged or excess inflammation can result in nonhealing chronic wounds. Lactoferrin, an iron-binding glycoprotein secreted from glandular epithelial cells into body fluids, promotes skin wound healing by enhancing the initial inflammatory phase. Lactoferrin also exhibits anti-inflammatory activity that neutralizes overabundant immune response. Accumulating evidence suggests that lactoferrin directly promotes both the formation of granulation tissue and re-epithelialization. Lactoferrin stimulates the proliferation and migration of fibroblasts and keratinocytes and enhances the synthesis of extracellular matrix components, such as collagen and hyaluronan. In an in vitro model of wound contraction, lactoferrin promoted fibroblast-mediated collagen gel contraction. These observations indicate that lactoferrin supports multiple biological processes involved in wound healing.

Lactoferrin promotes hyaluronan synthesis in human dermal fibroblasts.

Bovine lactoferrin (BLF) enhanced production of hyaluronan in normal human dermal fibroblasts. The elevation of hyaluronan was accompanied by elevation of HAS2 (hyaluronan synthase 2) mRNA transcription and HAS2 protein expression. The promoting effect of BLF was not observed for HAS1. In addition, COL1A1 transcription and collagen synthesis were enhanced by BLF. These observations suggest that BLF promotes wound healing by increasing hyaluronan and type-I collagen synthesis.

Inhibitory effect of lactoferrin on hypertrophic differentiation of ATDC5 mouse chondroprogenitor cells.

The skeleton is formed by two different mechanisms. In intramembranous ossification, osteoblasts form bone directly, whereas in endochondral ossification, chondrocytes develop a cartilage template, prior to osteoblast-mediated skeletogenesis. Lactoferrin is an iron-binding glycoprotein belonging to the transferrin family. It is known to promote the growth and differentiation of osteoblasts. In this study, we investigated the effects of bovine lactoferrin on the chondrogenic differentiation of ATDC5 chondroprogenitor cells. This mouse embryonic carcinoma-derived clonal cell line provides an in vitro model of chondrogenesis. Lactoferrin treatment of differentiating ATDC5 cells promoted cell proliferation in the initial stage of the differentiation process. However, lactoferrin treatment resulted in inhibition of hypertrophic differentiation, characterized by suppression of alkaline phosphatase activity, aggrecan synthesis and N-cadherin expression. This inhibitory effect was accompanied by sustained Sox9 expression, as well as increased Smad2/3 expression and phosphorylation, suggesting that lactoferrin regulates chondrogenic differentiation by up-regulating the Smad2/3-Sox9 signaling pathway.

LRP1 regulates architecture of the vascular wall by controlling PDGFRbeta-dependent phosphatidylinositol 3-kinase activation.

BACKGROUND: Low density lipoprotein receptor-related protein 1 (LRP1) protects against atherosclerosis by regulating the activation of platelet-derived growth factor receptor beta (PDGFRbeta) in vascular smooth muscle cells (SMCs). Activated PDGFRbeta undergoes tyrosine phosphorylation and subsequently interacts with various signaling molecules, including phosphatidylinositol 3-kinase (PI3K), which binds to the phosphorylated tyrosine 739/750 residues in mice, and thus regulates actin polymerization and cell movement. METHODS AND PRINCIPAL FINDINGS: In this study, we found disorganized actin in the form of membrane ruffling and enhanced cell migration in LRP1-deficient (LRP1-/-) SMCs. Marfan syndrome-like phenotypes such as tortuous aortas, disrupted elastic layers and abnormally activated transforming growth factor beta (TGFbeta) signaling are present in smooth muscle-specific LRP1 knockout (smLRP1-/-) mice. To investigate the role of LRP1-regulated PI3K activation by PDGFRbeta in atherogenesis, we generated a strain of smLRP1-/- mice in which tyrosine 739/750 of the PDGFRbeta had been mutated to phenylalanines (PDGFRbeta F2/F2). Spontaneous atherosclerosis was significantly reduced in the absence of hypercholesterolemia in these mice compared to smLRP1-/- animals that express wild type PDGFR. Normal actin organization was restored and spontaneous SMC migration as well as PDGF-BB-induced chemotaxis was dramatically reduced, despite continued overactivation of TGFbeta signaling, as indicated by high levels of nuclear phospho-Smad2. CONCLUSIONS AND SIGNIFICANCE: Our data suggest that LRP1 regulates actin organization and cell migration by controlling PDGFRbeta-dependent activation of PI3K. TGFbeta activation alone is not sufficient for the expression of the Marfan-like vascular phenotype. Thus, regulation of PI3 Kinase by PDGFRbeta is essential for maintaining vascular integrity, and for the prevention of atherosclerosis as well as Marfan syndrome.

Effect of lactoferrin-embedded collagen membrane on osteogenic differentiation of human osteoblast-like cells.

Lactoferrin (LF) has the ability to promote the proliferation and differentiation of osteoblasts, suggesting its potential utility as an osteogenic growth factor in bone tissue engineering. However, this type of application requires improved drug delivery system (DDS) technology at the target site. In this study, we report enhanced calcium deposition and alkaline phosphatase (ALP) activity using the type I collagen membrane during osteogenic differentiation of MG63 human osteoblast-like cells, indicating that type I collagen not only acts as a site for calcification but also promotes the expression of differentiated phenotypes. We also used this membrane as a drug delivery carrier for bovine LF. Approximately 27% of LF embedded on the type I collagen membrane was released within the first hour in cell-free condition. This initial burst release of LF was followed by a slower release from the collagen membrane. Bovine LF embedded in the type I collagen membrane promoted its calcification during osteogenic differentiation of MG63 cells without the loss of LF bioactivity. Taken together, ALP activity and osteocalcin production were enhanced in the MG63 cells plated on the LF-embedded collagen membrane, suggesting that LF incorporated in the collagen membrane promoted bone-like tissue formation by MG63 cells. These observations suggest that the type I collagen membrane is useful as a drug delivery carrier for LF in bone tissue engineering.

Effect of bovine lactoferrin on extracellular matrix calcification by human osteoblast-like cells.

Osteoblast-mediated calcium deposition to the extracellular matrix (ECM) is a critical step in bone tissue generation. Bovine lactoferrin enhanced the calcium deposition by MG63 human osteoblast-like cells cultured on collagen-coated plates. Lactoferrin also promoted the alkaline phosphatase activity and osteocalcin production during the calcification process, whereas it had little effect on the growth of the cells on the collagen-coated plates.

Collagen vitrigel membrane useful for paracrine assays in vitro and drug delivery systems in vivo.

We previously succeeded in converting a soft and turbid disk of type-I collagen gel into a strong and transparent vitrigel membrane utilizing a concept for the vitrification of heat-denatured proteins and have demonstrated its protein-permeability and advantage as a scaffold for reconstructing crosstalk models between two different cell types. In this study, we observed the nano-structure of the type-I collagen vitrigel membrane and verified its utility for paracrine assays in vitro and drug delivery systems in vivo. Scanning electron microscopic observation revealed that the vitrigel membrane was a dense network architecture of typical type-I collagen fibrils. In the crosstalk model between PC-12 pheochromocytoma cells and L929 fibroblasts, nerve growth factor (NGF) secreted from L929 cells passed through the collagen vitrigel membrane and induced the neurite outgrowth of PC-12 cells by its paracrine effect. Also, the collagen vitrigel membrane containing vascular endothelial growth factor (VEGF) showed sustained-release of VEGF in vitro and its subcutaneous transplantation into a rat resulted in remarkable angiogenesis. These data suggest that the collagen vitrigel membrane is useful for paracrine assays in vitro and drug delivery systems in vivo.

A protein-permeable scaffold of a collagen vitrigel membrane useful for reconstructing crosstalk models between two different cell types.

Soft and turbid collagen gel disks were previously converted into strong and transparent gel membranes utilizing a concept for the vitrification of heat-denatured of proteins. This novel stable and transparent gel has been termed 'vitrigel'. By encompassing the collagen vitrigel membrane in a nylon frame, it can be easily handled with tweezers, and functions as an excellent scaffold for three-dimensional cell culture models, as cells can be cultured on both sides. Here, we investigated the molecular permeability of the collagen vitrigel membrane in a time course-dependent manner using glucose and serum proteins. Glucose penetrated through the collagen vitrigel membrane to the opposite side, and concentrations on each side were found to be equilibrated within 24 h. Serum proteins up to a molecular weight >100 kDa also gradually passed through the collagen vitrigel membrane. In addition, human microvascular endothelial cells (HMVECs) were cultured on one surface of the collagen vitrigel membrane with a nylon frame, and human dermal fibroblasts (HDFs) or HT-29 (a human colon carcinoma cell line) cells were cocultured on the opposite surface. Histomorphological observations revealed the formation of three-dimensional crosstalk models composed of HMVECs and HDFs or HMVECs and HT-29 cells. Resulting data suggest that the protein-permeable scaffold composed of the collagen vitrigel membrane is useful for the reconstruction and/or modeling of 'crosstalk' between two different cells types. Hereafter, such crosstalk models in vitro could be applied to research not only of paracrine factors, but also to epithelial- or endothelial-mesenchymal transitions.

LRP1 functions as an atheroprotective integrator of TGFbeta and PDFG signals in the vascular wall: implications for Marfan syndrome.

BACKGROUND: The multifunctional receptor LRP1 controls expression, activity and trafficking of the PDGF receptor-beta in vascular smooth muscle cells (VSMC). LRP1 is also a receptor for TGFbeta1 and is required for TGFbeta mediated inhibition of cell proliferation. METHODS AND PRINCIPAL FINDINGS: We show that loss of LRP1 in VSMC (smLRP(-)) in vivo results in a Marfan-like syndrome with nuclear accumulation of phosphorylated Smad2/3, disruption of elastic layers, tortuous aorta, and increased expression of the TGFbeta target genes thrombospondin-1 (TSP1) and PDGFRbeta in the vascular wall. Treatment of smLRP1(-) animals with the PPARgamma agonist rosiglitazone abolished nuclear pSmad accumulation, reversed the Marfan-like phenotype, and markedly reduced smooth muscle proliferation, fibrosis and atherosclerosis independent of plasma cholesterol levels. CONCLUSIONS AND SIGNIFICANCE: Our findings are consistent with an activation of TGFbeta signals in the LRP1-deficient vascular wall. LRP1 may function as an integrator of proliferative and anti-proliferative signals that control physiological mechanisms common to the pathogenesis of Marfan syndrome and atherosclerosis, and this is essential for maintaining vascular wall integrity.