Reiji Aoki

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.

Oral administration of live Lactococcus lactis C59 suppresses IgE antibody production in ovalbumin-sensitized mice via the regulation of interleukin-4 production

To explore the potential probiotic effects of diary starter strains to suppress an IgE allergic response, 10 strains of live dairy lactic acid bacteria were screened for their ability to stimulate the T-helper (Th) type 1 response that counteracts the Th2 response. Four strains with distinct patterns of interleukin(IL)-12p70 and interferon-γ production by murine splenocytes were then orally administered to Balb/c mice, and serum IgE antibody production was examined after ovalbumin sensitization. Oral administration of live Lactococcus lactis strain C59 significantly reduced the total IgE antibody levels, whereas oral administration of the other three strains had no effect on the total IgE levels in ovalbumin-sensitized mice. This inhibitory effect on IgE antibody production was lost when heat-killed C59 was used for oral administration. Ex vivo experiments showed that IL-4 production upon stimulation with the anti-CD3 antibody was significantly reduced in splenocytes of mice with an oral administration of live strain C59 compared with the control. These results indicate that the inhibition of IgE antibody production in mice treated with live strain C59 was due to the suppression of IL-4 production.

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 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-1β, IL-6 and cyclooxygenase-2 (Cox-2). This decrease was associated with the reduced secretion of IL-1α, IL-1β, 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.

Oral intake of heat-killed cells of Lactococcus lactis strain H61 promotes skin health in women.

We conducted a double-blind, placebo-controlled trial to evaluate the effect of heat-killed cells of Lactococcus lactis strain H61 on various skin properties of Japanese women. Volunteers (age 31–62 years) were randomly assigned to receive test food with or without 60 mg of heat-killed strain H61 (fifteen women in each group; H61 and control groups, respectively) daily for 8 weeks. Results were analysed for three age categories (30s, 40s and 50–60s). Compared with that at week 0, skin hydration at the inner forearm at weeks 4 and 8 decreased in all volunteers (except those in their 50–60s) because of the environmental change from autumn to winter. The oldest H61 group maintained skin hydration at the inner forearm throughout the study. Skin elasticity and melanin content in the cheek decreased and sebum content increased throughout the test period due to seasonal environmental change, regardless of age or H61 treatment. Self-evaluation scores for apparent hair follicles and dryness of the throat at week 8 were higher in the overall H61 group than in the combined placebo group. The 30s H61 group noted marked improvements in self-surveyed skin elasticity at week 8 compared with at week 0 and with the placebo group at week 8. The results of the present study indicate that oral intake of heat-killed cells of L. lactis strain H61 can improve some skin properties and body characteristics in women. This strain would probably be useful in increasing the quality of life in an ageing population.

Lactobacillus rhamnosus GG increases Toll-like receptor 3 gene expression in murine small intestine ex vivo and in vivo

Administration of Lactobacillus rhamnosus GG (LGG) has been reported to be therapeutically effective against acute secretory diarrhoea resulting from the structural and functional intestinal mucosal lesions induced by rotavirus infection; however, the underlying mechanisms remain to be completely elucidated. Because Toll-like receptor 3 (TLR3) plays a key role in the innate immune responses following the recognition of rotavirus, the present study examined whether LGG influences TLR3 gene expression in murine small intestine ex vivo and in vivo. We employed cultured intestinal organoids derived from small intestinal crypts as an ex vivo tissue model. LGG supplementation increased TLR3 mRNA levels in the intestinal organoids, as estimated by quantitative real-time polymerase chain reaction. Likewise, single and 7-day consecutive daily administrations of LGG increased TLR3 mRNA levels in the small intestine of C57BL/6N mice. The mRNA levels of other TLRs were not substantially altered both ex vivo and in vivo. In addition, LGG supplementation increased the mRNA levels of an antiviral type 1 interferon, interferon-α (IFN-α), and a neutrophil chemokine, CXCL1, upon stimulation with a synthetic TLR3 ligand, poly(I:C) in the intestinal organoids. LGG administration did not alter IFN-α and CXCL1 mRNA levels in the small intestine in vivo. Supplementation of other bacterial strains, Bifidobacterium bifidum and Lactobacillus paracasei, failed to increase TLR3 and poly(I:C)-stimulated CXCL1 mRNA levels ex vivo. We propose that upregulation of TLR3 gene expression may play a pivotal role in the therapeutic efficacy of LGG against rotavirus-associated diarrhoea. In addition, we demonstrated that intestinal organoids may be a promising ex vivo tissue model for investigating host-pathogen interactions and the antiviral action of probiotics in the intestinal epithelium.

First Complete Genome Sequence of the Skin-Improving Lactobacillus curvatus Strain FBA2, Isolated from Fermented Vegetables, Determined by PacBio Single-Molecule Real-Time Technology

ABSTRACT The first complete genome sequence of Lactobacillus curvatus was determined by PacBio RS II. The single circular chromosome (1,848,756 bp, G+C content of 42.1%) of L. curvatus FBA2, isolated from fermented vegetables, contained low G+C regions (26.9% minimum) and 43 sets of >1,000-bp identical sequence pairs. No plasmids were detected.

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 (100 µmol) and exposed to UVB light (1 J/cm2) for two times. 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 1β (IL-1β) 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-1β 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.

Intraspecies discrimination of Lactobacillus paraplantarum by PCR

Lactobacillus paraplantarum is a species phenotypically close to Lactobacillus plantarum. Several PCR methods were evaluated to discriminate L. paraplantarum strains and among them, a PCR using an enterobacterial repetitive intergenic consensus (ERIC) sequence differentiated L. paraplantarum from other Lactobacillus species. In addition, a combination of ERIC and random amplified polymorphic DNA (RAPD) analysis distinguished among seven strains of L. paraplantarum tested. ERIC-PCR profiles showed several strain-specific DNA fragments in L. paraplantarum, among them, a 2.2-kb ERIC marker, termed LpF1, found to be specific to strain FBA1, which improved the skin integrity in an animal model. The LpF1 encodes three proteins similar to Lactobacillus fermentum AroA, TyrA, and AroK, which are involved in the shikimate pathway. A primer pair specific to FBA1 based on the internal sequence of LpF1 amplified a 950-bp FBA1-specific fragment LpF2. Southern blot analysis of Dra I-digested genomic DNA of L. paraplantarum strains using LpF2 as a probe showed that LpF2 is distinctive of strain FBA1 among 16 L. paraplantarum strains. Because both ERIC- and RAPD-PCR are fast and technically simple methods, they are useful for the rapid discrimination of L. paraplantarum strains and for the development of new strain-specific DNA markers for identifying industrially important strains.

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.