Toll-like receptor (TLR) recognizes viral and bacterial specific components to activate immune responses. TLR plays an essential role for a pathogen sensor. TLR can be divided two groups by their localization. One is cell surface TLR which localizes in cell membrane to recognize mainly bacterial cell wall components. The other is nucleic acid-sensing TLRs, which localize in endosome or lysosome in immune cells to recognize viral and bacterial nucleic acids. We have investigated about TLR trafficking to regulate TLR7 activation. First, I want to introduce TLR4 trafficking and activation mechanism as a cell surface receptor which recognizes gram-negative bacterial cell wall component LPS. Second, I want to introduce TLR7 trafficking and activation mechanism as an endo-lysosomal TLR to recognize bacterial and viral RNAs.

 Classical passive immunization has provided the benefit to prevent against infectious diseases for over century. Although lots of antibody-drugs have been developed for cancer and autoimmune disease, those against infectious disease are rarely available. Because it has complex reasons including the current availability of antimicrobial drugs, small markets, high costs, and microbial antigenic variation.

 We firstly demonstrated long-prophylaxis against influenza virus (A/Puerto Rico/8/34, IAV) using plasmids encoding neutralizing IgG monoclonal antibodies. Antibody gene-based injection could induce stable and high expression level of the neutralizing antibodies, which was possible that single inoculation protected the mice against a lethal dose of IAV infection. We proposed that this method could dissolve problems of high cost to the purification, limited supply for pandemic, and the risk of using viral vectors.

 We also succeeded to treatment against IAV infection using antibody gene-based injection by hydrodynamics (HD), which was involving rapid inoculation of a large volume of plasmid-DNA solution into mice via the tail vein. HD could rapidly induce the potent level of neutralizing antibodies in the serum within 24 hours. We demonstrated that a single HD completely protected the mice even after a lethal dose of IAV infection. Finally, we also generated other isotypes of antibody-gene to exchange from IgG to IgA, IgM, IgD, and IgE to retain the variable region. The neutralizing IgA was most effective at reducing upper respiratory tract IAV infection. Thus, our passive immunotherapy could provide a new prophylaxis/therapeutic strategy of targeting IAV infection.

 Various extrinsic and endogenous mediators are produced and released not only in the local injured and infected foci, but in the systemic circulation after surgical stress and sepsis. Recent progress of lipid research has figured out that lipid mediators have pleiotropic immunological properties and are associated with various disease pathophysiology. Therefore, lipid mediators can play a major role of regulating host responses after surgical stress and sepsis. In this review, we focused on important lipid mediators, including specialized proresolving lipid mediators and lysophospholipids, and discuss the role for surgical host responses from perspectives of clinical application.

 Bacteroides fragilis is a member of normal intestinal flora and is known as an opportunistic infectious bacterium causing intraperitoneal abscess or septicemia. It has been shown that the bacterium activates the intestinal innate immune system via Toll-like receptor 2 (TLR2), and the relation with intestinal disease has been drawing attention.

 The cell surface of this bacterium is composed of capsular polysaccharide (CPS), lipopolysaccharide (LPS) and lipoprotein (LP). Capsular polysaccharide A (PSA), a type of CPS, is a zwitterionic polysaccharide and is known to induce regulatory T cells in the intestinal tract and inhibit enteritis. In recent years, it has been reported that PSA activates TLR2 which is an innate immune receptor. However, the structure of PSA is largely different from the general recognition structure of TLR2, and the possibility of contamination could be considered. In this study, we separated the PSA fraction from B. fragilis and examined the substances involved in TLR2 activation.

 B. fragilis cells were subjected to hot water-phenol extraction and followed by hydrophobic chromatography separation to obtain a PSA fraction having the ability to activate TLR2. The fraction was further subjected to SDS-PAGE separation based on their molecular weight. TLR2-stimulating activity was scarcely observed in the high molecular mass area where PSA was present, and high activities were observed in the low molecular mass area. The active substances were found to be proteins because they were digested with proteolytic enzymes. These results indicate that contaminating proteins are substances responsible for TLR2 activation in the PSA fraction derived from B. fragilis.

 Low Endotoxin Recovery (LER) is a phenomenon of endotoxin activity decrease in a matrix containing a chelating agent and a detergent, and is a controversial topic in the biopharmaceutical field. The mechanism of LER is not fully elucidated. When endotoxin in LER solutions was diluted with water, the activity was decreased. The activity was maintained for a long time at 4°C, and was recovered by magnesium dilution and direct addition to the Limulus amebocyte lysate (LAL). The size of endotoxin in LER solution was not changed after the activity was decreased. Considering these results, a new LER mechanism was proposed. A chelating agent removes divalent cations from the surface of endotoxin aggregates, and endotoxin molecules on the surface of the aggregates are replaced with detergent molecules. The reduction of the surface area of endotoxin aggregates causes decrease of the endotoxin activity to the LAL.

 Ulcerative colitis (UC) is an idiopathic and chronic inflammatory disorder of the colonic mucosa. Damage to epithelial barrier function due to ulceration and colitis leads to increased permeability of the intestine. This barrier loss causes increased uptake of luminal antigens such as endotoxin (ET). We evaluated ET levels detected by Endotoxin activity assay (EAA) in patients with UC. We measured EAA 8 cases (refractory UC 6 and cancer/dysplasia UC 2) of UC required surgical treatments. Various clinicopathological parameters were reviwed. The severity of UC was evaluated by Seo’s index. Pre-operative EAA was 0.14 in patients with cancer/dysplasia UC and 0.4 in patients with refractory UC. EAA levels increased according to severity of UC, mild 0.095, moderate 0.39, severe 0.42 without statistical significance. There was a significantly positive relationship between EAA and Seo’s index (Spearman’s rank correlation coefficient 0.8333, p=0.0102). EAA appeared to be useful biomarker to evaluate patients’severity with UC. Large scale study need to clarify the significance of EAA in patients with UC.

 Peroxiredoxin 1 (PRDX1) is an antioxidant that detoxifies hydrogen peroxide and peroxynitrite. Compared with wild-type (WT) mice, Prdx1-deficient (Prdx1^−/−) mice showed increased susceptibility to Mycobacterium tuberculosis (Mtb). Infection with Mtb led to significantly shorter mean postinfection (p. i.) survival in Prdx1^−/− (80.8±10.0 days) than in WT mice (143.4±17.5 days). The numbers of viable Mtb in the lungs at 9 weeks p. i. were 10-fold higher in Prdx1^−/− than in WT lungs. Histological examinations showed auramine-rhodamine-stained Mtb was present predominantly in the alveolar sacs of Prdx1^−/− lungs, but few lesions were observed in WT lungs. IFN-γ-activated Prdx1^−/− bone marrow-derived macrophages (BMDMs) did not kill Mtb effectively. Nitric oxide (NO) production levels were lower, and arginase activity and arginase 1 (Arg1) expression levels were higher in IFN-γ-activated Prdx1^−/− than WT BMDMs after Mtb infection. An arginase inhibitor, N^ω-hydroxy-nor-arginine, restored antimicrobial activity and NO production in IFN-γ-activated Prdx1^−/− BMDMs after Mtb infection. At 9 wk p. i., Arg1 mRNA levels were significantly higher in the lungs of Prdx1^−/− than WT mice. These results suggest that PRDX1 contributes to host defenses against Mtb. PRDX1 positively regulates NO production by suppressing Arg1 expression in macrophages infected with Mtb.

 The lipoteichoic acid (LTA) is a specific polymer on Gram-positive bacterial cell surfaces, and is not found in Gram-negative bacterial cells. LTA is an amphipathic anionic polymer typically comprised of poly-glycerophosphate (GroP) linked to glycolipid of cell membrane. In general, hydroxyl groups of GroP residues in polymer region are often substituted by D-alanine and/or carbohydrates. Glycolipid anchor typically comprises diglycosylglycerolipid containing two acyl groups. On the other hand, structural variation on species-or strain-level has been observed in degree of polymerization, substitution ratio of the GroP repeating unit, and numbers of carbohydrate and acyl groups in glycolipid moiety. However, structural information has not sufficiently accumulated to discuss unity and diversity of LTA structures.

 We determined chemical structure of LTA derived from seven strains of Lactobacillus gasseri, an intestinal lactic acid bacterium. All strains shared common structure. Polymer chain comprised poly-GroP with partial substitution with D-alanine. Glycolipid anchor contained novel tetrasaccharide structure and two or three acyl groups. The three acyl group-containing glycolipid anchor is characteristic LTA structure in lactic acid bacteria, including Lactobacillus genus, Lactococcus genus and Leuconostoc genus. The tetrasaccharide structure has not been reported in other Gram-positive bacterial LTA. In conclusion, the tetrasaccharide-containing glycolipid anchor is unique and species-specific LTA structure in L. gasseri.

 Toll-like receptors (TLRs) trigger innate immune responses by recognizing microbe-associated molecular patterns such as lipopolysaccharides and nucleic acids. However, overactivation or dysregulation of TLR system may lead to exaggerated inflammation and host tissue injury. TLR signaling can be controlled by other intracellular signaling, including Rho GTPases and RhoGEFs signaling. Knowledge of the molecular mechanisms underlying cross-talk of TLR and other signaling pathways can, therefore, be used for tailored therapeutic approaches to regulate local and temporal host immunity. In this review, we will focus on RhoGEFs which affect TLR signal transduction, and further we will discuss about the mechanisms of the cross-talk between TLRs and RhoGEFs. Future interests in this research field include establishing mechanistic understanding of TLRs and RhoGEFs cross-talk. This may furthermore open up novel therapeutic options for inflammatory or autoimmune diseases.

 Toll-like receptors (TLRs) are a family of type Ⅰ membrane proteins, classified to pattern recognition receptors (PRR). TLRs are expressed in cell surface or endosomal compartment of immune cells. TLRs are activated by recognition of pathogen specific components called pathogen-associated molecular patterns (PAMPs). Signal of TLRs induces the innate immune response and adaptive immune response. Therefore, TLRs play important roles in host defense system against viral or bacterial infection. In human, 10 kinds of TLR (TLR1 to TLR10) have been found. Each TLR recognizes different PAMP ; lipoprotein for TLR1/6 and TLR2/6 ; nucleic acid components for TLR3, -7, -8, and -9 ; lipopolysaccharide (LPS) for TLR4. TLRs are also activated by the specific endogenous danger signal (called damage-associated molecular pattern, DAMPs) produced by damaged tissue, which is thought to cause various chronic inflammatory diseases. On the other hand, low molecular weight TLR ligands have been found from natural and synthetic compounds, those can be candidates for development of chemical probes and drugs. In this review, focusing on TLR4, we describe recent progress in exploring TLR4 ligands with agonistic or antagonistic activity and our recent study on the structure-activity relationship of pyrimido [5,4-b] indole derivatives.

 Complement system, one of the innate immunities, is a strong immune system, which destroys invasive microbes by making a hole in the cell membranes. On the other hands, several proteins inhibit complement system to protect the host cells from the attack. C4b-binding protein (C4BP), one of the inhibitory proteins, constitutively exists at high density called 200 μg/mL in serum. C4BP binds to C4b or C3b and helps the degradation, followed by prevention against the activation of complement system.

 Recently, we found that C4BP associates with Toll-like Receptor (TLR) 2, one of the pathogen recognition receptors (PRRs), and regulates the activation response through TLR2. Furthermore, we found that C4BP also binds to TLR4, which was other PRR, and regulates the activation response through TLR4. A competition assay by using two kinds of anti-TLR4 mAb revealed that C4BP prevents an interaction between TLR4/MD-2 and its ligand. These findings indicate that C4BP binds to TLRs on the cell surface and inhibits the interaction between TLRs and the ligands, followed by inhibition of TLR activation.

 Here we explain the function of C4BP to affect complement and TLR, which is called the two major immune systems. We also give an outline about a unique other function of C4BP and influence on disease onset.

 Lipid A is known to exhibit various immunostimulating activities, and acyloxyacyl structure formed by 3-hydroxy and non-polar fatty acids is important for the activity. Although the relationship between the activity and the acyloxyacyl structure has been precisely investigated using chemically synthesized lipid A, modifications of lipid A by genetic methods have not been much attempted. In the present study, the double-knockout mutant of Escherichia coli with disrupted lauroyl-and palmitoyltransferase genes (KGu0377) was constructed, and transformed with palmitoyltransferase gene from Salmonella (pagP) or from Campylobacter jejuni to obtain strains KGu0431 and KGu0441, respectively. The mutant produced the lipid A without acyloxyacyl structure, showed slower growth at 37°C, and was very sensitive to polymyxin B. The transformant KGu0431 could grow as rapid as the wild-type strain and showed the partially restored resistance to polymyxin B. Another transformant KGu0441 also showed the similar recovery of polymyxin B resistance, but its growth was still slower than the wild-type strain. Mass spectrometric analysis revealed that KGu0431 produced considerable amount of penta-acylated lipid A with palmitic acid. On the other hand, KGu0441 produced a small amount of hexa-acylated lipid A with palmitic and myristic acids. The IL-6 inducing activity of KGu0377 LPS was much weaker than that of the wild-type strain, as expected, and the activity of LPS from the transformants was at the same level as that of KGu0377 or only slightly restored. The present study showed the possibility to produce lipid A with novel structures using various acyltransferases from other bacteria.

 LL-37 is the only known member of the cathelicidin family of antimicrobial peptides in humans. In addition to its broad spectrum of antimicrobial activities, LL-37 can modulate various inflammatory reactions. We previously revealed that LL-37 improves the survival of a murine cecal ligation and puncture (CLP) sepsis model. In the present study, we elucidated the mechanism for the protective action of LL-37 using the CLP model, focusing on the effect of LL-37 on the release of neutrophil extracellular traps (NETs). The results indicated that the intravenous administration of LL-37 suppressed the increase of damage-associated molecular patterns, DAMPs (such as histone-DNA complex and HMGB1) as well as IL-1β, TNF-α and soluble triggering receptor expressed on myeloid cells (TREM-1) in plasma and peritoneal fluids. Interestingly, LL-37 significantly suppressed the decrease of mononuclear cell number in blood, and the increase of polymorphonuclear cell (neutrophil) number in the peritoneal cavity during sepsis. Moreover, LL-37 reduced the bacterial burdens in blood and peritoneal fluids. Notably, LL-37 increased the level of NETs (myeloperoxidase-DNA complex) in plasma and peritoneal fluids. In addition, we confirmed that LL-37 induces the release of NETs from neutrophils, and NETs possess the bactericidal activity. Together, these observations suggest that LL-37 improves the survival of CLP septic mice by possibly suppressing the inflammatory responses as evidenced by the inhibition of the increase of cytokines, soluble TREM-1 and DAMPs (host cell death) and the alteration of inflammatory cell numbers, and the bacterial growth via the release of NETs with the bactericidal activity.

 HSR incites pulmonary inflammation that leads to acute respiratory distress syndrome (ARDS). However, there have been no definitive pharmacological therapies against ARDS. CO is a toxic gas due to the generation of carboxyhemoglobin (COHb). However, trace amount of CO is endogenously produced by the enzymatic reaction of heme oxygenase-1 (HO-1) that is induced by oxidative stress to confer protection against various inflammatory disorders. Recent studies have indicated that low dose of CO exerts potent cytoprotective effects on inflammatory organ damage in animal models by its anti-inflammatory property. We also demonstrated that CO inhalation at 250 ppm ameliorated HSR-induced pulmonary injury in rats. However, this dose of CO increased blood COHb level to approximately 20% that may be toxic to humans. Very recently, to overcome the disadvantage, CORMs have been developed by coordinating CO with a transition metal carbonyl complexes. Among various types of CORMs, CORM-3, a water-soluble CORM, spontaneously liberated and deliver CO to various tissues under physiological condition through intravenous administration. We found that CORM-3 treatment mitigated HSR-induced lung injury without any increase in blood COHb levels through its anti-inflammatory property. We propose that CO/CORMs are possible pharmacological agent to treat ARDS.

 In the inflammatory disease chronic periodontitis, the periodontium is exposed to a high density and diversity of periodontal bacteria. Neutrophils are the first line of defense in the periodontium. Circulating neutrophils adhere to endothelial cells by adhesion molecules, such as β2-integrin, following their migration across endothelial cells into the inflamed periodontium. Endothelial cell-derived developmental endothelial locus-1 (Del-1) acts as a negative regulator of neutrophil extravasation that inhibits β2-integrin-dependent adhesion to vascular endothelial cells. Neutrophil extracellular traps (NETs) are extracellular web-like DNA structures that contain bactericidal substances such as histones, human neutrophil elastase, and cathelicidin antimicrobial peptides. NETs are produced by many bacterial species, but it remains unknown whether the development of periodontal disease is due to periodontal bacteria-induced NET production. Fusobacterium nucleatum is a Gram-negative anaerobic bacterium associated with periodontal disease. We examined the effects of F. nucleatum-induced NET stimulation on the induction of Del-1 in human endothelial cells. A transendothelial migration assay was performed to evaluate the number of transmigrated neutrophils. Stimulation of human umbilical vein endothelial cells (HUVECs) with F. nucleatum-infected NET fragments enhanced the transendothelial migration of neutrophils. Furthermore, the Del-1 production induced by HUVECs was attenuated in NET fragments-primed cells. Thus, F. nucleatum-induced NETs may enhance neutrophil extravasation in a manner dependent on the attenuation of Del-1 production in endothelial cells. This study suggests that NETs are associated with the pathogenesis of periodontal disease.