Oral Administration of Lipopolysaccharide Enhances Insulin Signaling-Related Factors in the KK/Ay Mouse Model of Type 2 Diabetes Mellitus.

Lipopolysaccharide (LPS), an endotoxin, induces systemic inflammation by injection and is thought to be a causative agent of chronic inflammatory diseases, including type 2 diabetes mellitus (T2DM). However, our previous studies found that oral LPS administration does not exacerbate T2DM conditions in KK/Ay mice, which is the opposite of the response from LPS injection. Therefore, this study aims to confirm that oral LPS administration does not aggravate T2DM and to investigate the possible mechanisms. In this study, KK/Ay mice with T2DM were orally administered LPS (1 mg/kg BW/day) for 8 weeks, and blood glucose parameters before and after oral administration were compared. Abnormal glucose tolerance, insulin resistance progression, and progression of T2DM symptoms were suppressed by oral LPS administration. Furthermore, the expressions of factors involved in insulin signaling, such as insulin receptor, insulin receptor substrate 1, thymoma viral proto-oncogene, and glucose transporter type 4, were upregulated in the adipose tissues of KK/Ay mice, where this effect was observed. For the first time, oral LPS administration induces the expression of adiponectin in adipose tissues, which is involved in the increased expression of these molecules. Briefly, oral LPS administration may prevent T2DM by inducing an increase in the expressions of insulin signaling-related factors based on adiponectin production in adipose tissues.

Lipopolysaccharide Derived from Pantoea agglomerans Directly Promotes the Migration of Human Keratinocytes.

BACKGROUND/AIM: Because the skin is exposed to the external environment, it is important that wound healing processes proceed and terminate rapidly to minimize the risk of infection. A previous case report described the promotion of wound healing by transdermal administration of lipopolysaccharide derived from Pantoea agglomerans (LPSp). However, whether the wound healing-promoting effect of LPSp was due to direct activity on skin cells or indirect effects involving macrophages remained unclear. Therefore, this study investigated the wound healing-promoting effect of LPSp, particularly the promotion of keratinocyte migration. MATERIALS AND METHODS: The migration of HaCaT human keratinocytes over time with and without LPSp was assayed using a cell migration assay kit. Migration was also analyzed using HaCaT cells treated with LPSp and an antibody against Toll-like receptor (TLR) 4, a receptor for LPS. RESULTS: Addition of LPSp significantly enhanced cell migration compared to no LPSp addition. Migration was inhibited by the addition of anti-TLR4 antibody. CONCLUSION: LPSp acts directly on epidermal cells to promote migration and may be one mechanism by which LPSp promotes wound healing.

Restoration of Spatial Learning Through Oral Administration of Lipopolysaccharides in Diabetes-related Cognitive Dysfunction.

BACKGROUND/AIM: In a previous report, our group showed that oral administration of lipopolysaccharides (LPS) from Pantoea agglomerans can prevent the progression of streptozotocin (STZ)-induced diabetes-related cognitive dysfunction (DRCD) in mice without causing significant side-effects. However, the treatment effects of oral administration of LPS to DRCD remain unknown. MATERIALS AND METHODS: We modified our previous animal experimental model to investigate whether oral administration of LPS can recover cognitive function after DRCD onset. RESULTS: The Morris water maze (MWM) revealed a significant decrease in learning and memory abilities at 13 days after intracerebroventricular administration of STZ, thereby providing evidence of the occurrence of DRCD in the animal model. Oral administration of LPS (1 mg/kg per day) started after cognitive impairment was observed. After 28 days of treatment, mice receiving LPS via the oral route showed significant recovery of spatial learning ability, a symptom of early dementia, while only a trend toward recovery was seen for spatial memory compared to the untreated group. CONCLUSION: These results, limited to MWM, suggest that oral administration of LPS is a promising therapeutic strategy for restoring decreased spatial learning ability.

Baseline data collections of lipopolysaccharide content in 414 herbal extracts and its role in innate immune activation.

Some herbal extracts contain relatively high amounts of lipopolysaccharide (LPS). Because orally administered LPS activates innate immunity without inducing inflammation, it plays a role as an active ingredient in herbal extracts. However, the LPS content in herbal extracts remains extensively unevaluated. This study aimed to create a database of LPS content in herbal extracts; therefore, the LPS content of 414 herbal extracts was measured and the macrophage activation potential was evaluated. The LPS content of these hot water extracts was determined using the kinetic-turbidimetric method. The LPS concentration ranged from a few ng/g to hundreds of µg/g (Standard Escherichia coli LPS equivalent). Twelve samples had a high-LPS-content of > 100 µg/g, including seven samples from roots and three samples from leaves of the herbal extracts. These samples showed high phagocytosis and NO production capacity, and further investigation using polymyxin B, an LPS inhibitor, significantly inhibited macrophage activation. This study suggests that some herbal extracts contain sufficient LPS concentration to activate innate immunity. Therefore, a new approach to evaluate the efficacy of herbal extracts based on their LPS content was proposed. A database listing the LPS content of different herbal extracts is essential for this approach.

Orally administered LPS enhances head kidney macrophage activation with down-regulation of IL-6 in common carp (Cyprinus carpio).

Immunostimulants represent a promising aquaculture tool for enhancing disease and stress resistance in cultured fish. Moreover, the term and dose for acting immunostimulants is an important thing for fish farmer. This study investigated the immune parameters of common carp after oral administration of LPS (5, 10, 20 µg/kg/days) for 30 and 60 days, which is considered to be the proper time period for acting in aquaculture. Phagocytic and bactericidal activities of head kidney macrophages and serum lysozyme activities were significantly enhanced in LPS-fed carp. Orally administered LPS augmented the expression of interleukin (IL)-1ß and TNF-α mRNAs but reduced the expression of IL-6 mRNA in head kidney. Although LPS was detected in the serum and liver after a high-dose (>15 mg/kg) oral administration, it was not detected by administered LPS-specific ELISA after a low-dose (<20 µg/kg) administration. It is speculated that orally administered LPS enhances the eliminating functions of head kidney macrophages with down-regulation of IL-6.

Genome sequence of Pantoea agglomerans strain IG1.

Pantoea agglomerans is a gram-negative bacterium that grows symbiotically with various plants. Here we report the 4.8-Mb genome sequence of P. agglomerans strain IG1. The lipopolysaccharides derived from P. agglomerans IG1 have been shown to be effective in the prevention of various diseases, such as bacterial or viral infection, lifestyle-related diseases. This genome sequence represents a substantial step toward the elucidation of pathways for production of lipopolysaccharides.

Anti-inflammatory and Insulin Signaling Phenotype Induced by Repeated Lipopolysaccharide Stimulation in 3T3-L1 Adipocytes.

BACKGROUND/AIM: Lipopolysaccharide (LPS) is thought to be a causative agent of type 2 diabetes, because it has been shown that a single LPS stimulation in vitro induces chronic inflammation and reduces insulin signaling in adipocytes. However, oral LPS administration prevents type 2 diabetes, and this effect does not correspond to a single LPS adipocyte stimulation. In this study, the response of adipocytes to single and repeated stimulation with LPS was examined. MATERIALS AND METHODS: 3T3-L1 cells were differentiated into adipocytes and stimulated with LPS once or thrice every 24 h. The expression levels of inflammatory and anti-inflammatory factors and insulin sensitivity-related factors were measured. RESULTS: Single stimulation with LPS increased the mRNA and protein expression of inflammatory factors (interleukin-6 and monocyte chemotactic protein 1), but this increase was inhibited by repeated stimulation. In contrast, the mRNA expression levels of anti-inflammatory factors (proliferator-activated receptor γ and peroxisome proliferator-activated receptor gamma coactivator1 α) were increased by repeated LPS stimulation. Additionally, the mRNA expression levels of insulin sensitivity-related factors (glucose transporter type 4, insulin receptor, insulin receptor substrate 1 and thymoma viral proto-oncogene 2) in adipocytes were increased upon repeated LPS stimulation. CONCLUSION: Repetitive LPS stimulation, unlike single stimulation of adipocytes, upregulates anti-inflammatory and insulin signaling-related factors.

Low-dose lipopolysaccharide as an immune regulator for homeostasis maintenance in the central nervous system through transformation to neuroprotective microglia.

Microglia, which are tissue-resident macrophages in the brain, play a central role in the brain innate immunity and contribute to the maintenance of brain homeostasis. Lipopolysaccharide is a component of the outer membrane of gram-negative bacteria, and activates immune cells including microglia via Toll-like receptor 4 signaling. Lipopolysaccharide is generally known as an endotoxin, as administration of high-dose lipopolysaccharide induces potent systemic inflammation. Also, it has long been recognized that lipopolysaccharide exacerbates neuroinflammation. In contrast, our study revealed that oral administration of lipopolysaccharide ameliorates Alzheimer's disease pathology and suggested that neuroprotective microglia are involved in this phenomenon. Additionally, other recent studies have accumulated evidence demonstrating that controlled immune training with low-dose lipopolysaccharide prevents neuronal damage by transforming the microglia into a neuroprotective phenotype. Therefore, lipopolysaccharide may not a mere inflammatory inducer, but an immunomodulator that can lead to neuroprotective effects in the brain. In this review, we summarized current studies regarding neuroprotective microglia transformed by immune training with lipopolysaccharide. We state that microglia transformed by lipopolysaccharide preconditioning cannot simply be characterized by their general suppression of proinflammatory mediators and general promotion of anti-inflammatory mediators, but instead must be described by their complex profile comprising various molecules related to inflammatory regulation, phagocytosis, neuroprotection, anti-apoptosis, and antioxidation. In addition, microglial transformation seems to depend on the dose of lipopolysaccharide used during immune training. Immune training of neuroprotective microglia using low-dose lipopolysaccharide, especially through oral lipopolysaccharide administration, may represent an innovative prevention or treatment for neurological diseases; however more vigorous studies are still required to properly modulate these treatments.

Prevention of Diabetes-Associated Cognitive Dysfunction Through Oral Administration of Lipopolysaccharide Derived From Pantoea agglomerans.

Diabetes-related cognitive dysfunction (DRCD) is a serious complication induced by diabetes. However, there are currently no specific remedies for DRCD. Here, we show that streptozotocin-induced DRCD can be prevented without causing side effects through oral administration of lipopolysaccharide (LPS) derived from Pantoea agglomerans. Oral administration of LPS (OAL) prevented the cerebral cortex atrophy and tau phosphorylation induced by DRCD. Moreover, we observed that neuroprotective transformation of microglia (brain tissue-resident macrophages) is important for preventing DRCD through OAL. These findings are contrary to the general recognition of LPS as an inflammatory agent when injected systemically. Furthermore, our results strongly suggest that OAL promotes membrane-bound colony stimulating factor 1 (CSF1) expression on peripheral leukocytes, which activates the CSF1 receptor on microglia, leading to their transformation to the neuroprotective phenotype. Taken together, the present study indicates that controlling innate immune modulation through the simple and safe strategy of OAL can be an innovative prophylaxis for intractable neurological diseases such as DRCD. In a sense, for modern people living in an LPS-depleted environment, OAL is like a time machine that returns microglia to the good old LPS-abundant era.

Prevention of streptozotocin­induced Neuro­2a cell death by C8­B4 microglia transformed with repetitive low­dose lipopolysaccharide.

Diabetes­associated neuronal dysfunction (DAND) is one of the serious complications of diabetes, but there is currently no remedy for it. Streptozotocin [2­deoxy­2­(3­methy1­3­nitrosoureido) D­glucopyranose; STZ] is one of the most well­established diabetes inducers and has been used in vivo and in vitro DAND models. The aim of the present study was to demonstrate that C8­B4 microglia transformed by the stimulus of repetitive low­dose lipopolysaccharide (LPSx3­microglia) prevent STZ­induced Neuro­2a neuronal cell death in vitro. The ELISA results showed that neurotrophin­4/5 (NT­4/5) secretion was promoted in LPSx3­microglia and the cell viability assay with trypan blue staining revealed that the culture supernatant of LPSx3­microglia prevented STZ­induced neuronal cell death. In addition, reverse transcription­quantitative PCR showed that neurons treated with the culture supernatant of LPSx3­microglia promoted the gene expression of B­cell lymphoma­extra large and glucose­dependent insulinotropic polypeptide receptor. Furthermore, the inhibition of tyrosine kinase receptor B, a receptor of NT­4/5, suppressed the neuroprotective effect of LPSx3­microglia. Taken together, the present study demonstrated that LPSx3­microglia prevent STZ­induced neuronal death and that NT­4/5 may be involved in the neuroprotective mechanism of LPSx3­microglia.

Oral Administration of Lipopolysaccharide Prevents Cognitive Impairment in Streptozotocin-induced Diabetic Mice in a Blood Glucose-independent Manner.

BACKGROUND/AIM: Diabetes is a risk factor for dementia. However, no radical preventive method for diabetes-associated dementia has yet been developed. Our previous study revealed that oral administration of lipopolysaccharide (LPS) prevents high-fat diet-induced cognitive impairment. Therefore, we investigated here whether oral administration of LPS (OAL) could also prevent diabetes-associated dementia. MATERIALS AND METHODS: Diabetic mice were produced by intraperitoneal administration of streptozotocin (STZ), and then mice were orally administered LPS. Cognitive ability was evaluated using the Morris water maze, and gene expression was analyzed in isolated microglia. RESULTS: OAL prevented STZ-induced diabetic cognitive impairment, but did not affect blood glucose levels. Moreover, OAL promoted the expression of neuroprotective genes in microglia, such as heat shock protein family 40 (HSP40) and chemokine CCL7. CONCLUSION: OAL prevents diabetes-associated dementia, potentially via promotion of HSP40 and CCL7 expression in microglia.

Immune recovery effects of immunopotentiator from Pantoea agglomerans 1 (IP-PA1) on low antibody productions in response to Salmonella enteritidis vaccine and sheep red blood cells in dexamethasone-treated stressed chicken models.

Considering the usefulness of the immunopotentiator from Pantoea agglomerans 1 (IP-PA1), which is a purified lipopolysaccharide (LPS) derived from symbiotic gram-negative bacteria of food crops, in controlling immunosuppression in poultry husbandry, in this study, we examined its immune-recovery effects in dexamethasone-treated stressed chicken models. Three-week-old chickens daily administered 10 microg/kg of dexamethasone for 35 days to induce stress showed more whole body weight loss; relative thymic, bursal, and splenic weight losses; and decrease in the number of peripheral blood lymphocytes, as compared with the control chickens on day 35; the IP-PA1-pretreated, dexamethasone-treated chickens showed reduced weight losses. Five- to eight-week-old chickens administered 5 mg/kg of dexamethasone showed excessive apoptosis of thymic and bursal lymphocytes 24 hr after a single dexamethasone treatment; apoptosis was inhibited in the IP-PA1-pretreated, dexamethasone-treated chickens. Chickens daily administered 10 microg/kg of dexamethasone for 35 days and injected with commercial Salmonella Enteritidis (SE) vaccine or sheep red blood cells (SRBC) on days 7 and 21 showed about 8- or 2-fold lower antibody production in response to SE or SRBC, respectively, as compared with the control chickens on day 35; the antibody production in response to SE or SRBC was increased in the IP-PA1-pretreated, dexamethasone-treated chickens. These results indicate that IP-PA1 exerts inhibitory effects on dexamethasone-induced immunosuppression and that it may be useful in controlling immunosuppression in poultry husbandry.

Attempt to Construct an In Vitro Model of Enhancement of Macrophage Phagocytosis Via Continuous Administration of LPS.

BACKGROUND: Continuous oral administration of lipopolysaccharide (LPS) enhances the phagocytic ability of macrophages, which is useful for preventing various diseases. Here, we attempted to create an in vitro model of continuous administration of LPS. MATERIALS AND METHODS: RAW264.7 cells were stimulated with LPS three times every 24 h (repeated stimulation), and phagocytic ability and inflammatory cytokine [interleukin-6 (IL6) and tumor necrosis factor-α (TNFα)] production were measured. RESULTS: The phagocytic ability was increased by a single stimulation with LPS and was maintained by repeated stimulation. IL6 production increased with a single stimulation with LPS; however, IL6 production by repeated stimulation with LPS was comparable to that of non-stimulation with LPS. On the other hand, the amount of TNFα was significantly increased by single and repeated stimulation with LPS. CONCLUSION: Repeated stimulation with LPS in RAW264.7 cells triggered a phenotype that was similar to that of macrophages after continuous oral administration of LPS. This suggests that this study model may reproduce the enhancement of macrophage phagocytosis, an effect afforded by continuous oral administration of LPS.

A Novel Anti-inflammatory Phenotype Transformed by Repetitive Low-dose Lipopolysaccharide in Primary Peritoneal Tissue-resident Macrophages.

BACKGROUND/AIM: Our previous studies suggested that oral administration of lipopolysaccharide (LPS) regulates the progression of various diseases via transformation of tissue-resident macrophages (MΦ). Recently, we characterized microglia transformed by repetitive low-dose LPS treatment (REPELL-microglia) in vitro, and this response was similar to that observed in response to oral administration of LPS in vivo. Here, we examined the characteristics of peritoneal tissue-resident MΦ (pMΦ) transformed by repetitive low-dose LPS treatment (REPELL-pMΦ). MATERIALS AND METHODS: Primary pMΦ were treated with low-dose LPS (1 ng/ml) three times; subsequently, phagocytic activity and gene expression were evaluated. RESULTS: REPELL-pMΦ exhibited high phagocytic activity and elevated expression of Arg1, Gipr, Gdnf, and Fpr2. The gene expression profiles observed in REPELL-pMΦ were distinct from those of REPELL-microglia. CONCLUSION: REPELL-pMΦ have the potential to promote clearance of xenobiotics and to suppress inflammation. The present study also demonstrates the diversity of tissue-resident MΦ transformation that reflect their tissue origin.

Mastication Affects Transcriptomes of Mouse Microglia.

BACKGROUND/AIM: We investigated whether mastication affects microglia, whose activity is thought to be associated with cognition and brain tumor progression. MATERIALS AND METHODS: We kept mice by feeding either a hard or soft diet for 2, 4 or 8 months. After each period, we removed the whole brains and isolated microglia. The total RNA extracted from each brain's microglia was subjected to DNA microarray analysis. RESULTS: Many genes were found to be significantly differentially expressed between hard- and soft-diet-fed mice in each group of the same feeding period. The expression of several genes involved in the regulation of actin cytoskeleton was down-regulated in the soft-diet-fed mice. CONCLUSION: Mastication may affect microglia's roles in cognition as well as their neuroimmune activity through their activity of patrolling the brain.

A unique hybrid characteristic having both pro- and anti-inflammatory phenotype transformed by repetitive low-dose lipopolysaccharide in C8-B4 microglia.

Although lipopolysaccharide (LPS) is regarded as an inducer of inflammation, previous studies have suggested that repetitive low-dose LPS has neuroprotective effects via immunomodulation of microglia, resident macrophages of brain. However, microglia transformed by the stimulus of repetitive low-dose LPS (REPELL-microglia) are not well characterized, whereas microglia transformed by repetitive high-dose LPS are well studied as an endotoxin tolerance model in which the induction of pro-inflammatory molecules is suppressed. In this study, to characterize REPELL-microglia, the gene expression and phagocytic activity of REPELL-microglia were analyzed with the murine C8-B4 microglia cell line. The REPELL-microglia were characterized by a high expression of pro-inflammatory molecules (Nos2, Ccl1, IL-12B, and CD86), anti-inflammatory molecules (IL-10, Arg1, Il13ra2, and Mrc1), and neuroprotective molecules (Ntf5, Ccl7, and Gipr). In addition, the phagocytic activity of REPELL-microglia was promoted as high as that of microglia transformed by single low-dose LPS. These results suggest the potential of REPELL-microglia for inflammatory regulation, neuroprotection, and phagocytic clearance. Moreover, this study revealed that gene expression of REPELL-microglia was distinct from that of microglia transformed by repetitive high-dose LPS treatment, suggesting the diversity of microglia transformation by different doses of LPS.

Two types of tumor necrosis factor-alpha in bluefin tuna (Thunnus orientalis) genes: Molecular cloning and expression profile in response to several immunological stimulants.

Tumor necrosis factor-alpha (TNF-alpha) is a key inflammatory mediator and has also the potential as a prominent biomarker of innate immunity. In this study, we identified and characterized TNF-alpha from bluefin tuna, which is an important cultured species. Two types of TNF-alpha were also cloned incidentally (TNF1 and TNF2). The open reading frame of TNF1 and TNF2 cDNA encoded 247 and 245 amino acids, respectively. The amino acid sequence identity among sea perch, red sea bream, and tiger puffer was 73, 70, 59% for TNF1 and 49, 51, 45% for TNF2, respectively. The identity between TNF1 and TNF2 amino acid sequences of the bluefin tuna was only 43%. The positions of cysteine residues, transmembrane sequence, and protease cleavage site in bluefin tuna TNFs were similar with other reported fish and mammalian TNF-alpha. In a phylogenetic analysis, TNF1 is grouped with other reported Perciformes TNF-alpha. On the other hand, TNF2 is grouped with ayu TNF and is quite distant from the fish TNF-alpha group and lymphotoxin-beta group. While TNF1 mRNA showed no significant difference in all tissues, TNF2 mRNA was expressed significantly higher in the blood than in the gill, intestine, head kidney, spleen, heart, and ovary. In peripheral blood leucocytes (PBL), expressions of TNF2 mRNA were significantly increased by stimulation with lipopolysaccharide, phytohemagglutinin, concanavalin A, pokeweed mitogen, phorbol myristate acetate in vitro, but those of TNF1 were not. Recombinant mature TNF1 and TNF2 proteins significantly enhanced phagocytic activity of PBL. Our results suggest that bluefin tuna possess two types of TNF-alpha homologue, and TNF2 is a potential biomarker for innate immunity.

ROS and innate immunity.

Oxygen is converted into reactive oxygen (RO) by radiation, light, the electron transport system in mitochondria, or by other enzymes and is regulated by the action of antioxidative enzymes which convert RO into an inactive state. Reactive oxygen species (ROS) have a biocidal effect on invading bacteria and they can also injure the cells of the host. For this reason, RO is considered as a general cause of aging and contributes to lifestyle-related diseases and cancer. However, for any organism that uses oxygen as an energy source, RO is inevitably produced and has important biological significance. Apart from the direct activity of RO, recent studies have shown that it functions as a second messenger of signal transduction. In this review, the recent findings related to ROS/nitric oxide (NO) and especially of its relationship to innate immunity are summarized.

High-dose vitamin C (ascorbic acid) therapy in the treatment of patients with advanced cancer.

Vitamin C (ascorbic acid, ascorbate) has a controversial history in cancer treatment. Emerging evidence indicates that ascorbate in cancer treatment deserves re-examination. As research results concerning ascorbate pharmacokinetics and its mechanisms of action against tumor cells have been published, and as evidence from case studies has continued to mount that ascorbate therapy could be effective if the right protocols were used, interest among physicians and scientists has increased. In this review, high-dose vitamin C therapy in cancer treatment is re-evaluated.

Utility and safety of LPS-based fermented flour extract as a macrophage activator.

The immune system is part of the homeostasis system. Our research is focused on ways to maintain health, with an emphasis on the role of macrophages. We have hypothesized that tissue macrophages form a systemic network which we believe contributes to the homeostasis system, and have named it the 'macrophage network.' This network creates a dynamic equilibrium situation where macrophages control homeostasis. Our research is based on this macrophage network theory as we believe that the innate immune system provides the foundation for the homeostasis system. As part of our research, we have demonstrated that macrophage activation can provide protection and therapeutic effects for various diseases. Therefore, we have also focused on lipopolysaccharide (LPS). We proved that the LPS of Pantoea agglomerans (which we have named IP-PA1) was useful in preventing various health disorders and in restoring health when administered via the oral or transdermal route. We also developed a 'fermented flour extract', which consists largely of IP-PA1. For LPS to become a valuable commodity, it is very important to provide assurance about safety (when administered orally or transdermally) to build confidence. For this reason, we tested fermented flour extract (in which the major component was IP-PA1) to confirm that it was safe. The results of these safety trials confirmed that oral and transdermal administration of fermented flour extract was very safe. Thus, we believe that fermented flour extract is a new substance that will have applications in health food, cosmetics, animal feeds, fisheries feeds and drugs industries.