Maintenance of homeostasis by TLR4 ligands.

Immunotherapy is renowned for its capacity to elicit anti-infective and anti-cancer effects by harnessing immune responses to microbial components and bolstering innate healing mechanisms through a cascade of immunological reactions. Specifically, mammalian Toll-like receptors (TLRs) have been identified as key receptors responsible for detecting microbial components. The discovery of these mammalian Toll-like receptors has clarified antigen recognition by the innate immune system. It has furnished a molecular foundation for comprehending the interplay between innate immunity and its anti-tumor or anti-infective capabilities. Moreover, accumulating evidence highlights the crucial role of TLRs in maintaining tissue homeostasis. It has also become evident that TLR-expressing macrophages play a central role in immunity by participating in the clearance of foreign substances, tissue repair, and the establishment of new tissue. This macrophage network, centered on macrophages, significantly contributes to innate healing. This review will primarily delve into innate immunity, specifically focusing on substances targeting TLR4.

Development of a Water Soluble Self-assembling Analogue of Vizantin.

Vizantin, 6,6'-bis-O-(3-nonyldodecanoyl)-α,α'-trehalose, has been developed as a safe immunostimulator on the basis of a structure-activity relationship study with trehalose 6,6'-dicorynomycolate. Our recent study indicated that vizantin acts as an effective Toll-like receptor-4 (TLR4) partial agonist to reduce the lethality of an immune shock caused by lipopolysaccharide (LPS). However, because vizantin has low solubility in water, the aqueous solution used in in vivo assay systems settles out in tens of minutes. Here, vizantin was chemically modified in an attempt to facilitate the preparation of an aqueous solution of the drug. This paper describes the concise synthesis of a water-soluble vizantin analogue in which all the hydroxyl groups of the sugar unit were replaced by sulfates. The vizantin derivative displayed micelle-forming ability in water and potent TLR-4 partial agonist activity.

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.

Effects of Erythromycin on Osteoclasts and Bone Resorption via DEL-1 Induction in Mice.

Macrolides are used to treat various infectious diseases, including periodontitis. Furthermore, macrolides are known to have immunomodulatory effects; however, the underlying mechanism of their action remains unclear. DEL-1 has emerged as an important factor in homeostatic immunity and osteoclastogenesis. Specifically, DEL-1 is downregulated in periodontitis tissues. Therefore, in the present study, we investigated whether the osteoclastogenesis inhibitory effects of erythromycin (ERM) are mediated through upregulation of DEL-1 expression. We used a ligature-induced periodontitis model in C57BL/6Ncrl wild-type or DEL-1-deficient mice and in vitro cell-based mechanistic studies to investigate how ERM inhibits alveolar bone resorption. As a result of measuring alveolar bone resorption and gene expression in the tooth ligation model, ERM treatment reduced bone loss by increasing DEL-1 expression and decreasing the expression of osteoclast-related factors in wild-type mice. In DEL-1-deficient mice, ERM failed to suppress bone loss and gene expression of osteoclast-related factors. In addition, ERM treatment downregulated osteoclast differentiation and calcium resorption in in vitro experiments with mouse bone marrow-derived macrophages. In conclusion, ERM promotes the induction of DEL-1 in periodontal tissue, which may regulate osteoclastogenesis and decrease inflammatory bone resorption. These findings suggest that ERM may exert immunomodulatory effects in a DEL-1-dependent manner.

Broad spectrum in vitro microbicidal activity of benzoyl peroxide against microorganisms related to cutaneous diseases.

The in vitro microbicidal activity of benzoyl peroxide against Cutibacterium acnes, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Malassezia furfur, Malassezia restricta, and Malassezia globosa was investigated. These strains were incubated for 1 h in the presence of 0.25, 0.5, 1, or 2 mmol/L benzoyl peroxide in phosphate buffered saline supplemented with 0.1% glycerol and 2% Tween 80. After exposure to benzoyl peroxide, counts of viable Gram-positive bacteria and fungi were markedly decreased, whereas counts of Gram-negative bacteria were unchanged. Transmission electron microscopy images showed a decrease in electron density and the destruction of C. acnes and M. restricta cell walls after exposure to 2 mmol/L benzoyl peroxide. In conclusion, this study showed that benzoyl peroxide has a potent and rapid microbicidal activity against Gram-positive bacteria and fungi that are associated with various cutaneous diseases. This suggests that the direct destruction of bacterial cell walls by benzoyl peroxide is an essential mechanism of its rapid and potent microbicidal activity against microorganisms.

Sulfated vizantin causes detachment of biofilms composed mainly of the genus Streptococcus without affecting bacterial growth and viability.

BACKGROUND: Sulfated vizantin, a recently developed immunostimulant, has also been found to exert antibiofilm properties. It acts not as a bactericide, but as a detachment-promoting agent by reducing the biofilm structural stability. This study aimed to investigate the mechanism underlying this activity and its species specificity using two distinct ex vivo oral biofilm models derived from human saliva. RESULTS: The biofilm, composed mainly of the genus Streptococcus and containing 50 µM of sulfated vizantin, detached significantly from its basal surface with rotation at 500 rpm for only 15 s, even when 0.2% sucrose was supplied. Expression analyses for genes associated with biofilm formation and bacterial adhesion following identification of the Streptococcus species, revealed that a variety of Streptococcus species in a cariogenic biofilm showed downregulation of genes encoding glucosyltransferases involved in the biosynthesis of water-soluble glucan. The expression of some genes encoding surface proteins was also downregulated. Of the two quorum sensing systems involved in the genus Streptococcus, the expression of luxS in three species, Streptococcus oralis, Streptococcus gordonii, and Streptococcus mutans, was significantly downregulated in the presence of 50 µM sulfated vizantin. Biofilm detachment may be facilitated by the reduced structural stability due to these modulations. As a non-specific reaction, 50 µM sulfated vizantin decreased cell surface hydrophobicity by binding to the cell surface, resulting in reduced bacterial adherence. CONCLUSION: Sulfated vizantin may be a candidate for a new antibiofilm strategy targeting the biofilm matrix while preserving the resident microflora.

Erythromycin inhibits neutrophilic inflammation and mucosal disease by upregulating DEL-1.

Macrolide antibiotics exert antiinflammatory effects; however, little is known regarding their immunomodulatory mechanisms. In this study, using 2 distinct mouse models of mucosal inflammatory disease (LPS-induced acute lung injury and ligature-induced periodontitis), we demonstrated that the antiinflammatory action of erythromycin (ERM) is mediated through upregulation of the secreted homeostatic protein developmental endothelial locus-1 (DEL-1). Consistent with the anti-neutrophil recruitment action of endothelial cell-derived DEL-1, ERM inhibited neutrophil infiltration in the lungs and the periodontium in a DEL-1-dependent manner. Whereas ERM (but not other antibiotics, such as josamycin and penicillin) protected against lethal pulmonary inflammation and inflammatory periodontal bone loss, these protective effects of ERM were abolished in Del1-deficient mice. By interacting with the growth hormone secretagogue receptor and activating JAK2 in human lung microvascular endothelial cells, ERM induced DEL-1 transcription that was mediated by MAPK p38 and was CCAAT/enhancer binding protein-ß dependent. Moreover, ERM reversed IL-17-induced inhibition of DEL-1 transcription, in a manner that was dependent not only on JAK2 but also on PI3K/AKT signaling. Because DEL-1 levels are severely reduced in inflammatory conditions and with aging, the ability of ERM to upregulate DEL-1 may lead to a novel approach for the treatment of inflammatory and aging-related diseases.

Lipopolysaccharide-Deficient Acinetobacter baumannii Due to Colistin Resistance Is Killed by Neutrophil-Produced Lysozyme.

Acinetobacter baumannii causes nosocomial infections due to its multidrug resistance and high environmental adaptability. Colistin is a polypeptide antibacterial agent that targets lipopolysaccharide (LPS) and is currently used to control serious multidrug-resistant Gram-negative bacterial infections, including those caused by A. baumannii. However, A. baumannii may acquire colistin resistance by losing their LPS. In mouse models, LPS-deficient A. baumannii have attenuated virulence. Nevertheless, the mechanism through which the pathogen is cleared by host immune cells is unknown. Here, we established colistin-resistant A. baumannii strains and analyzed possible mechanisms through which they are cleared by neutrophils. Colistin-resistant, LPS-deficient strains harbor mutations or insertion sequence (IS) in lpx genes, and introduction of intact lpx genes restored LPS deficiency. Analysis of interactions between these strains and neutrophils revealed that compared with wild type, LPS-deficient A. baumannii only weakly stimulated neutrophils, with consequent reduced levels of reactive oxygen species (ROS) and inflammatory cytokine production. Nonetheless, neutrophils preferentially killed LPS-deficient A. baumannii compared to wild-type strains. Moreover, LPS-deficient A. baumannii strains presented with increased sensitivities to antibacterial lysozyme and lactoferrin. We revealed that neutrophil-secreted lysozyme was the antimicrobial factor during clearance of LPS-deficient A. baumannii strains. These findings may inform the development of targeted therapeutics aimed to treat multidrug-resistant infections in immunocompromised patients who are unable to mount an appropriate cell-mediated immune response.

Sulfated vizantin inhibits biofilm maturation by Streptococcus mutans.

Streptococcus mutans is the main pathogen of dental caries and adheres to the tooth surface via soluble and insoluble glucans produced by the bacterial glucosyltransferase enzyme. Thus, the S. mutans glucosyltransferase is an important virulence factor for this cariogenic bacterium. Sulfated vizantin effectively inhibits biofilm formation by S. mutans without affecting its growth. In this study, less S. mutans biofilm formation occurred on hydroxyapatite discs coated with sulfated vizantin than on noncoated discs. Sulfated vizantin showed no cytotoxicity against the human gingival cell line Ca9-22. Sulfated vizantin dose-dependently inhibited the extracellular release of cell-free glucosyltransferase from S. mutans and enhanced the accumulation of cell-associated glucosyltransferase, compared with that observed with untreated bacteria. Sulfated vizantin disrupted the localization balance between cell-associated glucosyltransferase and cell-free glucosyltransferase, resulting in inhibited biofilm maturation. These results indicate that sulfated vizantin can potentially serve as a novel agent for preventing dental caries.

Role of Sphingomyelinase in the Pathogenesis of Bacillus cereus Infection.

Bacillus cereus is well known as a causative agent of food poisoning but it also causes bacteremia and endophthalmitis in nosocomial infections. However, as an environmental bacterium that lives in soil, it is often treated as simple contamination by hospitals. In recent years, highly pathogenic B. cereus strains that are similar to Bacillus anthracis have been detected in hospitals. The B. cereus sphingomyelinase contributes to its pathogenicity, as do sphingomyelinases produced by Staphylococcus aureus, Pseudomonas aeruginosa, Helicobacter pylori, and B. anthracis. Highly pathogenic B. cereus produces a large amount of sphingomyelinase. In this review, we describe the regulation of sphingomyelinase expression through the PlcR-PapR system, the pathogenicity of bacterial sphingomyelinases, and their potential as therapeutic drug targets.

Synthesis and Antimicrobial Evaluation of Side-Chain Derivatives based on Eurotiumide A.

Side-chain derivatives of eurotiumide A, a dihydroisochroman-type natural product, have been synthesized and their antimicrobial activities described. Sixteen derivatives were synthesized from a key intermediate of the total synthesis of eurotiumide A, and their antimicrobial activities against two Gram-positive bacteria, methicillin-susceptible and methicillin-resistant Staphylococcus aureus (MSSA and MRSA), and a Gram-negative bacterium, Porphyromonas gingivalis, were evaluated. The results showed that derivatives having an iodine atom on their aromatic ring instead of the prenyl moiety displayed better antimicrobial activity than eurotiumide A against MSSA and P. gingivalis. Moreover, we discovered that a derivative with an isopentyl side chain, which is a hydrogenated product of eurotiumide A, is the strongest antimicrobial agent against all three strains, including MRSA.

Sulfated vizantin suppresses mucin layer penetration dependent on the flagella motility of Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa is an opportunistic pathogen that causes severe infections, such as pneumonia and bacteremia. Several studies demonstrated that flagellar motility is an important virulence factor for P. aeruginosa infection. In this study, we determined whether sulfated vizantin affects P. aeruginosa flagellar motility in the absence of direct antimicrobial activity. We found that 100 µM sulfated vizantin suppressed P. aeruginosa PAO1 from penetrating through an artificial mucin layer by affecting flagellar motility, although it did not influence growth nor bacterial protease activity. To further clarify the mechanism in which sulfated vizantin suppresses the flagellar motility of P. aeruginosa PAO1, we examined the effects of sulfated vizantin on the composition of the flagellar filament and mRNA expression of several flagella-related genes, finding that sulfated vizantin did not influence the composition of the flagellar complex (fliC, motA, and motB) in P. aeruginosa PAO1, but significantly decreased mRNA expression of the chemotaxis-related genes cheR1, cheW, and cheZ. These results indicated that sulfated vizantin is an effective inhibitor of flagellar motility in P. aeruginosa.

Mechanism of Macrolide-Induced Inhibition of Pneumolysin Release Involves Impairment of Autolysin Release in Macrolide-Resistant Streptococcus pneumoniae.

Streptococcus pneumoniae is a leading cause of community-acquired pneumonia. Over the past 2 decades, macrolide resistance among S. pneumoniae organisms has been increasing steadily and has escalated at an alarming rate worldwide. However, the use of macrolides in the treatment of community-acquired pneumonia has been reported to be effective regardless of the antibiotic susceptibility of the causative pneumococci. Although previous studies suggested that sub-MICs of macrolides inhibit the production of the pneumococcal pore-forming toxin pneumolysin by macrolide-resistant S. pneumoniae (MRSP), the underlying mechanisms of the inhibitory effect have not been fully elucidated. Here, we show that the release of pneumococcal autolysin, which promotes cell lysis and the release of pneumolysin, was inhibited by treatment with azithromycin and erythromycin, whereas replenishing with recombinant autolysin restored the release of pneumolysin from MRSP. Additionally, macrolides significantly downregulated ply transcription followed by a slight decrease of the intracellular pneumolysin level. These findings suggest the mechanisms involved in the inhibition of pneumolysin in MRSP, which may provide an additional explanation for the benefits of macrolides on the outcome of treatment for pneumococcal diseases.

Streptococcus pyogenes CAMP factor promotes calcium ion uptake in RAW264.7 cells.

Streptococcus pyogenes is a bacterium that causes systemic diseases such as pharyngitis and toxic shock syndrome. S. pyogenes produces molecules that inhibit the function of the human immune system, thus allowing growth and spread of the pathogen in tissues. It is known that S. pyogenes CAMP factor induces vacuolation in macrophages; however, the mechanism remains unclear. In the current study, the mechanism by which CAMP factor induces vacuolation in macrophages was investigated. CAMP factor was found to induce calcium ion uptake in murine macrophage RAW264.7 cells. In addition, EDTA inhibited calcium ion uptake and vacuolation in the cells. The L-type voltage-dependent calcium ion channel blockers nifedipine and verapamil reduced vacuolation. Furthermore, the phosphoinositide 3-kinase inhibitors LY294002 and wortmannin also inhibited the vacuolation induced by CAMP factor. Fluorescent microscopy revealed that clathrin localized to the vacuoles. These results suggest that the vacuolation is related to calcium ion uptake by RAW264.7 cells via L-type voltage-dependent calcium ion channels. Therefore, it was concluded that the vacuoles induced by S. pyogenes CAMP factor in macrophages are clathrin-dependent endosomes induced by activation of the phosphoinositide 3-kinase signaling pathway through calcium ion uptake.

Neutrophil Elastase Subverts the Immune Response by Cleaving Toll-Like Receptors and Cytokines in Pneumococcal Pneumonia.

Excessive activation of neutrophils results in the release of neutrophil elastase (NE), which leads to lung injury in severe pneumonia. Previously, we demonstrated a novel immune subversion mechanism involving microbial exploitation of this NE ability, which eventually promotes disruption of the pulmonary epithelial barrier. In the present study, we investigated the effect of NE on host innate immune response. THP-1-derived macrophages were stimulated with heat-killed Streptococcus pneumoniae or lipopolysaccharide in the presence or absence of NE followed by analysis of toll-like receptor (TLR) and cytokine expression. Additionally, the biological significance of NE was confirmed in an in vivo mouse intratracheal infection model. NE downregulated the gene transcription of multiple cytokines in THP-1-derived macrophages through the cleavage of TLRs and myeloid differentiation factor 2. Additionally, NE cleaved inflammatory cytokines and chemokines. In a mouse model of intratracheal pneumococcal challenge, administration of an NE inhibitor significantly increased proinflammatory cytokine levels in bronchoalveolar lavage fluid, enhanced bacterial clearance, and improved survival rates. Our work indicates that NE subverts the innate immune response and that inhibition of this enzyme may constitute a novel therapeutic option for the treatment of pneumococcal pneumonia.

Sulfated vizantin induces formation of macrophage extracellular traps.

Vizantin is an insoluble adjuvant that activates macrophages and lymphocytes. Recently, 2,2',3,3',4,4'-hexasulfated-vizantin (sulfated vizantin), which enables solubilization of vizantin, was developed by the present team. Sulfated vizantin was found to enhance bactericidal activity against multi-drug resistant Pseudomonas aeruginosa in RAW264.7 cells. In addition, spread of P. aeruginosa was inhibited in RAW264.7 cells treated with sulfated vizantin. When only sulfated vizantin and P. aeruginosa were incubated, sulfated vizantin did not affect growth of P. aeruginosa. Formation of DNA-based extracellular traps (ETs), a novel defense mechanism in several types of innate immune cells, helps to eliminate pathogens. In the present study, ET-forming macrophages constituted the majority of immune cells. Sulfated vizantin induced ET formation in RAW264.7 cells, whereas a Ca-chelating reagent, EDTA, and T-type calcium channel blocker, tetrandrine, inhibited ET formation and attenuated inhibition of spread of P. aeruginosa in sulfated vizantin-treated cells. Thus, sulfated vizantin induces ET formation in phagocytic cells in a Ca-dependent manner, thus preventing spread of P. aeruginosa. Hence, sulfated vizantin may be useful in the management of infectious diseases.

Pneumococcal DNA-binding proteins released through autolysis induce the production of proinflammatory cytokines via toll-like receptor 4.

Streptococcus pneumoniae is a leading cause of bacterial pneumonia. Our previous study suggested that S. pneumoniae autolysis-dependently releases intracellular pneumolysin, which subsequently leads to lung injury. In this study, we hypothesized that pneumococcal autolysis induces the leakage of additional intracellular molecules that could increase the pathogenicity of S. pneumoniae. Liquid chromatography tandem-mass spectrometry analysis identified that chaperone protein DnaK, elongation factor Tu (EF-Tu), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were released with pneumococcal DNA by autolysis. We demonstrated that recombinant (r) DnaK, rEF-Tu, and rGAPDH induced significantly higher levels of interleukin-6 and tumor necrosis factor production in peritoneal macrophages and THP-1-derived macrophage-like cells via toll-like receptor 4. Furthermore, the DNA-binding activity of these proteins was confirmed by surface plasmon resonance assay. We demonstrated that pneumococcal DnaK, EF-Tu, and GAPDH induced the production of proinflammatory cytokines in macrophages, and might cause host tissue damage and affect the development of pneumococcal diseases.

Streptococcus pyogenes CAMP factor promotes bacterial adhesion and invasion in pharyngeal epithelial cells without serum via PI3K/Akt signaling pathway.

Streptococcus pyogenes is a bacterium that causes systemic diseases, such as pharyngitis and toxic shock syndrome, via oral- or nasal-cavity infection. S. pyogenes produces various molecules known to function with serum components that lead to bacterial adhesion and invasion in human tissues. In this study, we identified a novel S. pyogenes adhesin/invasin. Our results revealed that CAMP factor promoted streptococcal adhesion and invasion in pharyngeal epithelial Detroit562 cells without serum. Recombinant CAMP factor initially localized on the membranes of cells and then became internalized in the cytosol following S. pyogenes infection. Additionally, CAMP factor phosphorylated phosphoinositide 3-kinase and serine-threonine kinase in the cells. ELISA results demonstrate that CAMP factor affected the amount of phosphorylated phosphoinositide 3-kinase and serine-threonine kinase in Detroit562 cells. Furthermore, CAMP factor did not reverse the effect of phosphoinositide 3-kinase knockdown by small interfering RNA in reducing the level of adhesion and invasion of S. pyogenes isogenic cfa-deficient mutant. These results suggested that S. pyogenes CAMP factor activated the phosphoinositide 3-kinase/serine-threonine kinase signaling pathway, promoting S. pyogenes invasion of Detroit562 cells without serum. Our findings suggested that CAMP factor played an important role on adhesion and invasion in pharyngeal epithelial cells.

Cellular Entry of Clostridium perfringens Iota-Toxin and Clostridium botulinum C2 Toxin.

Clostridium perfringens iota-toxin and Clostridium botulinum C2 toxin are composed of two non-linked proteins, one being the enzymatic component and the other being the binding/translocation component. These latter components recognize specific receptors and oligomerize in plasma membrane lipid-rafts, mediating the uptake of the enzymatic component into the cytosol. Enzymatic components induce actin cytoskeleton disorganization through the ADP-ribosylation of actin and are responsible for cell rounding and death. This review focuses upon the recent advances in cellular internalization of clostridial binary toxins.

Streptococcus pyogenes Phospholipase A2 Induces the Expression of Adhesion Molecules on Human Umbilical Vein Endothelial Cells and Aorta of Mice.

The Streptococcus pyogenes phospholipase A2 (SlaA) gene is highly conserved in the M3 serotype of group A S. pyogenes, which often involves hypervirulent clones. However, the role of SlaA in S. pyogenes pathogenesis is unclear. Herein, we report that SlaA induces the expression of intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) via the arachidonic acid signaling cascade. Notably, recombinant SlaA induced ICAM1 and VCAM1 expression in human umbilical vein endothelial cells (HUVECs), resulting in enhanced adhesion of human monocytic leukemia (THP-1) cells. However, C134A, a variant enzyme with no enzymatic activity, did not induce such events. In addition, culture supernatants from S. pyogenes SSI-1 enhanced the adhesion of THP-1 cells to HUVECs, but culture supernatants from the ΔslaA isogenic mutant strain had limited effects. Aspirin, a cyclooxygenase 2 inhibitor, prevented the adhesion of THP-1 cells to HUVECs and did not induce ICAM1 and VCAM1 expression in HUVECs treated with SlaA. However, zileuton, a 5-lipoxygenase inhibitor, did not exhibit such effects. Furthermore, pre-administration of aspirin in mice intravenously injected with SlaA attenuated the transcriptional abundance of ICAM1 and VCAM1 in the aorta. These results suggested that SlaA from S. pyogenes stimulates the expression of adhesion molecules in vascular endothelial cells. Thus, SlaA contributes to the inflammation of vascular endothelial cells upon S. pyogenes infection.