Rab32 and Rab38 maintain bone homeostasis by regulating intracellular traffic in osteoclasts.

Osteoclasts play a crucial role in bone homeostasis by forming resorption pits on bone surfaces, resulting in bone resorption. The osteoclast expression of Rab38 protein is highly induced during differentiation from macrophages. Here we generated mice with double knockout (DKO) of Rab38 and its paralogue, Rab32, to investigate the roles of these proteins in osteoclasts. Bone marrow-derived macrophages from Rab32/38 DKO mice differentiated normally into osteoclasts in vitro. However, DKO osteoclasts showed reduced bone resorption activity. These osteoclasts also demonstrated defective secretion of tartrate-resistant acid phosphatase and cathepsin K into culture medium. Furthermore, the plasma membrane localization of a3, an osteoclast-specific a subunit of V-ATPase, was abrogated in DKO mice, substantiating the reduced resorption activity. In vivo, Rab32- and Rab38-positive cells were attached to the bone surface. Eight-week-old DKO mice showed significantly thickened trabecular bones in micro-CT and histomorphometry analysis, as well as reduced serum levels of cross-linked C-telopeptide of type I collagen, indicating diminished bone resorption in vivo. In DKO male mice over 10 weeks of age, hyperostosis appeared at the talofibular syndesmosis, the distal junction of the tibia and fibula. Furthermore, middle-aged mice (10 to 12 months of age) exhibited kyphosis, which is not usually observed in wild-type male mice until around 24 months of age. These results indicate that Rab32 and Rab38 contribute to osteoclast function by supporting intracellular traffic, thereby maintaining normal bone homeostasis.Key words: Rab32, Rab38, osteoclast, lysosome-related organelle, secretory lysosome.

Opposing roles of RUBCN isoforms in autophagy and memory B cell generation.

RUBCN (also known as Rubicon) was originally identified as a negative regulator of autophagy, a process by which cells degrade and recycle damaged components or organelles and that requires the activity of the class III PI3K VPS34 and the mTORC1 protein complex. Here, we characterized the role of a shorter isoform, RUBCN100, as an autophagy-promoting factor in B cells. RUBCN100 was translated from alternative translation initiation sites and lacked the RUN domain of the longer, previously characterized RUBCN130 isoform. Specific deficiency of RUBCN130 in B cells enhanced autophagy, which promoted memory B cell generation. In contrast to RUBCN130, which is localized in late endosomes and lysosomes and suppresses the enzymatic activity of VPS34, an effect thought to mediated by its RUN domain, RUBCN100 was preferentially located in early endosomes and enhanced VPS34 activity, presumably because of the absence of the RUN domain. Furthermore, RUBCN100, but not RUBCN130, enhanced autophagy and suppressed mTORC1 activation. Our findings reveal that the opposing roles of two RUBCN isoforms are critical for autophagy regulation and memory B cell differentiation.

Characterization of Rab32- and Rab38-positive lysosome-related organelles in osteoclasts and macrophages.

Both the biogenesis and functions of osteoclasts and macrophages involves dynamic membrane traffic. We screened transcript levels for Rab family small GTPases related to osteoclasts and identified Rab38. Rab38 expression is upregulated during osteoclast differentiation and maturation. In osteoclasts, both Rab38 and its paralog, Rab32, colocalize to lysosome-related organelles (LROs). In macrophages, Rab32 is also found in LROs. LROs are part of the endocytic pathway but are distinct from lysosomes. After receptor activator of NF-κB ligand stimulation, LROs contain cathepsin K and tartrate-resistant acid phosphatase inside and help both proteins to accumulate around bone resorption pits. After osteoclast maturation, these enzymes are hardly found within LROs. In macrophages derived from Rab32 and Rab38 double knockout mice, both acidification and V-ATPase a3 localization were severely compromised. Both the double knockout macrophage and bafilomycin-treated wildtype macrophage show an increase in Lamp1-positive organelles, implying that biogenesis of lysosomes and LROs are related. These results indicate that Rab32 and Rab38 both play a crucial role in LRO biogenesis in macrophages and in osteoclasts.

VEGF-Mediated Augmentation of Autophagic and Lysosomal Activity in Endothelial Cells Defends against Intracellular Streptococcus pyogenes.

Group A Streptococcus (GAS), a deleterious human-pathogenic bacterium, causes life-threatening diseases such as sepsis and necrotic fasciitis. We recently reported that GAS survives and replicates within blood vessel endothelial cells because these cells are intrinsically defective in xenophagy. Because blood vessel endothelial cells are relatively germfree environments, specific stimulation may be required to sufficiently induce xenophagy. Here, we explored how vascular endothelial growth factor (VEGF) promoted xenophagy and lysosomal activity in endothelial cells. These effects were achieved by amplifying the activation of TFEB, a transcriptional factor crucial for lysosome/autophagy biogenesis, via cAMP-mediated calcium release. In a mouse model of local infection with GAS, the VEGF level was significantly elevated at the infection site. Interestingly, low serum VEGF levels were found in a mouse model of invasive bacteremia and in patients with severe GAS-induced sepsis. Moreover, the administration of VEGF improved the survival of GAS-infected mice. We propose a novel theory regarding GAS infection in endothelial cells, wherein VEGF concentrations in the systemic circulation play a critical role. IMPORTANCE Sepsis caused by Streptococcus pyogenes is a life-threatening condition. Blood vessel endothelial cells should serve as a barrier to infection, although we recently reported that endothelial cells allow intracellular GAS proliferation due to defective xenophagy. In this study, we revealed that administration of VEGF augmented both xenophagy and lysosomal activity in these cells, leading to the efficient killing of intracellular GAS. By comparison, the opposite relationship was observed in vivo, as low serum VEGF concentrations were accompanied by high-severity sepsis in both a mouse model and in human patients. Administration of VEGF reduced mortality in the GAS sepsis model. Based on these findings, we hypothesize that during acute infection, strong VEGF stimulation boosts the intracellular defense system of the endothelium to provide a stronger blood vessel barrier, thereby helping to prevent bacterial dissemination.

Quercetin in Tartary Buckwheat Induces Autophagy against Protein Aggregations.

Tartary buckwheat is used as an ingredient in flour and tea, as well as in traditional Chinese medicine for its antioxidant effects. Here, we found that an ethanol extract of tartary buckwheat (TBE) potently induced autophagy flux in HeLa cells by suppressing mTORC1 activity, as revealed by dephosphorylation of the mTORC1 substrates Ulk1, S6K, and 4EBP, as well as by the nuclear translocation of transcriptional factor EB. In addition to non-selective bulk autophagy, TBE also induced aggrephagy, which is defined as autophagy against aggregated proteins. Quercetin is a flavonol found at high levels in TBE. We showed that quercetin induced both non-selective bulk autophagy and aggrephagy. These effects were also observed in Huh-7 cells derived from hepatocytes. Thus, aggrephagy induction by TBE and quercetin may relieve alcoholic hepatitis, which is closely linked to the accumulation of protein aggregations called Mallory-Denk bodies.

Starvation-induced autophagy via calcium-dependent TFEB dephosphorylation is suppressed by Shigyakusan.

Kampo, a system of traditional Japanese therapy utilizing mixtures of herbal medicine, is widely accepted in the Japanese medical system. Kampo originated from traditional Chinese medicine, and was gradually adopted into a Japanese style. Although its effects on a variety of diseases are appreciated, the underlying mechanisms remain mostly unclear. Using a quantitative tf-LC3 system, we conducted a high-throughput screen of 128 kinds of Kampo to evaluate the effects on autophagy. The results revealed a suppressive effect of Shigyakusan/TJ-35 on autophagic activity. TJ-35 specifically suppressed dephosphorylation of ULK1 and TFEB, among several TORC1 substrates, in response to nutrient deprivation. TFEB was dephosphorylated by calcineurin in a Ca2+ dependent manner. Cytosolic Ca2+ concentration was increased in response to nutrient starvation, and TJ-35 suppressed this increase. Thus, TJ-35 prevents the starvation-induced Ca2+ increase, thereby suppressing induction of autophagy.

Nicotinamide Increases Intracellular NAD+ Content to Enhance Autophagy-Mediated Group A Streptococcal Clearance in Endothelial Cells.

Group A streptococcus (GAS) is a versatile pathogen that causes a wide spectrum of diseases in humans. Invading host cells is a known strategy for GAS to avoid antibiotic killing and immune recognition. However, the underlying mechanisms of GAS resistance to intracellular killing need to be explored. Endothelial HMEC-1 cells were infected with GAS, methicillin-resistant Staphylococcus aureus (MRSA) and Salmonella Typhimurium under nicotinamide (NAM)-supplemented conditions. The intracellular NAD+ level and cell viability were respectively measured by NAD+ quantification kit and protease-based cytotoxicity assay. Moreover, the intracellular bacteria were analyzed by colony-forming assay, transmission electron microscopy, and confocal microscopy. We found that supplementation with exogenous nicotinamide during infection significantly inhibited the growth of intracellular GAS in endothelial cells. Moreover, the NAD+ content and NAD+/NADH ratio of GAS-infected endothelial cells were dramatically increased, whereas the cell cytotoxicity was decreased by exogenous nicotinamide treatment. After knockdown of the autophagy-related ATG9A, the intracellular bacterial load was increased in nicotinamide-treated endothelial cells. The results of Western blot and transmission electron microscopy also revealed that cells treated with nicotinamide can increase autophagy-associated LC3 conversion and double-membrane formation during GAS infection. Confocal microscopy images further showed that more GAS-containing vacuoles were colocalized with lysosome under nicotinamide-supplemented conditions than without nicotinamide treatment. In contrast to GAS, supplementation with exogenous nicotinamide did not effectively inhibit the growth of MRSA or S. Typhimurium in endothelial cells. These results indicate that intracellular NAD+ homeostasis is crucial for controlling intracellular GAS infection in endothelial cells. In addition, nicotinamide may be a potential new therapeutic agent to overcome persistent infections of GAS.

Group A Streptococcus Induces LAPosomes via SLO/ß1 Integrin/NOX2/ROS Pathway in Endothelial Cells That Are Ineffective in Bacterial Killing and Suppress Xenophagy.

Group A streptococcus (GAS) is an important human pathogen which can cause fatal diseases after invasion into the bloodstream. Although antibiotics and immune surveillance are the main defenses against GAS infection, GAS utilizes internalization into cells as a major immune evasion strategy. Our previous findings revealed that light chain 3 (LC3)-associated single membrane GAS-containing vacuoles in endothelial cells are compromised for bacterial clearance due to insufficient acidification after fusion with lysosomes. However, the characteristics and the activation mechanisms of these LC3-positive compartments are still largely unknown. In the present study, we demonstrated that the LC3-positive GAS is surrounded by single membrane and colocalizes with NADPH oxidase 2 (NOX2) complex but without ULK1, which are characteristics of LC3-associated phagocytosis (LAP). Inhibition of NOX2 or reactive oxygen species (ROS) significantly reduces GAS multiplication and enhances autolysosome acidification in endothelial cells through converting LAP to conventional xenophagy, which is revealed by enhancement of ULK1 recruitment, attenuation of p70s6k phosphorylation, and formation of the isolation membrane. We also clarify that the inactivation of mTORC1, which is the initiation signal of autophagy, is inhibited by NOX2- and ROS-activated phosphatidylinositol 3-kinase (PI3K)/AKT and MEK/extracellular signal-regulated kinase (ERK) pathways. In addition, streptolysin O (SLO) of GAS is identified as a crucial inducer of ROS for ß1 integrin-mediated LAP induction. After downregulation of ß1 integrin, GAS multiplication is reduced, accompanied with LAP inhibition and xenophagy induction. These results demonstrate that GAS infection preferentially induces ineffective LAP to evade xenophagic killing in endothelial cells through the SLO/ß1 integrin/NOX2/ROS pathway.IMPORTANCE Our previous reports showed that the LC3-associated GAS-containing single membrane vacuoles are inefficient for bacterial clearance in endothelial cells, which may result in bacteremia. However, the characteristics and the induction mechanisms of these LC3-positive vacuoles are still largely unknown. Here we provide the first evidence that these LC3-positive GAS-containing single membrane compartments appear to be LAPosomes, which are induced by NOX2 and ROS. Through NOX2- and ROS-mediated signaling, GAS preferentially induces LAP and inhibits bacteriostatic xenophagy in endothelial cells. We also provide the first demonstration that ß1 integrin acts as the receptor for LAP induction through GAS-produced SLO stimulation in endothelial cells. Our findings reveal the underlying mechanisms of LAP induction and autophagy evasion for GAS multiplication in endothelial cells.

Galectin-3 Inhibits Galectin-8/Parkin-Mediated Ubiquitination of Group A Streptococcus.

Group A streptococcus (GAS) is an important human pathogen that causes a wide variety of cutaneous and systemic infections. Although originally thought to be an extracellular bacterium, numerous studies have demonstrated that GAS can trigger internalization into nonimmune cells to escape from immune surveillance or antibiotic-mediated killing. Epithelial cells possess a defense mechanism involving autophagy-mediated targeting and killing of GAS within lysosome-fused autophagosomes. In endothelial cells, in contrast, we previously showed that autophagy is not sufficient for GAS killing. In the present study, we showed higher galectin-3 (Gal-3) expression and lower Gal-8 expression in endothelial cells than in epithelial cells. The recruitment of Gal-3 to GAS is higher and the recruitment of Gal-8 to GAS is lower in endothelial cells than in epithelial cells. We further showed that Gal-3 promotes GAS replication and diminishes the recruitment of Gal-8 and ubiquitin, the latter of which is a critical protein for autophagy sequestration. After knockdown of Gal-3 in endothelial cells, the colocalization of Gal-8, parkin, and ubiquitin-decorated GAS is significantly increased, as is the interaction of Gal-8 and parkin, an E3 ligase. Furthermore, inhibition of Gal-8 in epithelial cells attenuates recruitment of parkin; both Gal-8 and parkin contribute to ubiquitin recruitment and GAS elimination. Animal studies confirmed that Gal-3-knockout mice develop less-severe skin damage and that GAS replication can be detected only in the air pouch and not in organs and endothelial cells. These results demonstrate that Gal-3 inhibits ubiquitin recruitment by blocking Gal-8 and parkin recruitment, resulting in GAS replication in endothelial cells.IMPORTANCE In epithelial cells, GAS can be efficiently killed within the lysosome-fused autophaosome compartment. However, we previously showed that, in spite of LC-3 recruitment, the autophagic machinery is not sufficient for GAS killing in endothelial cells. In this report, we provide the first evidence that Gal-3, highly expressed in endothelial cells, blocks the tagging of ubiquitin to GAS by inhibiting recruitment of Gal-8 and parkin, leading to an enhancement of GAS replication. We also provide the first demonstration that Gal-8 can interact with parkin, the critical E3 ligase, for resistance to intracellular bacteria by facilitating the decoration of bacteria with ubiquitin chains. Our findings reveal that differential levels of Gal-3 and Gal-8 expression and recruitment to GAS between epithelial cells and endothelial cells may contribute to the different outcomes of GAS elimination or survival and growth of GAS in these two types of cells.

Endothelial cells are intrinsically defective in xenophagy of Streptococcus pyogenes.

Group A Streptococcus (GAS) is deleterious pathogenic bacteria whose interaction with blood vessels leads to life-threatening bacteremia. Although xenophagy, a special form of autophagy, eliminates invading GAS in epithelial cells, we found that GAS could survive and multiply in endothelial cells. Endothelial cells were competent in starvation-induced autophagy, but failed to form double-membrane structures surrounding GAS, an essential step in xenophagy. This deficiency stemmed from reduced recruitment of ubiquitin and several core autophagy proteins in endothelial cells, as demonstrated by the fact that it could be rescued by exogenous coating of GAS with ubiquitin. The defect was associated with reduced NO-mediated ubiquitin signaling. Therefore, we propose that the lack of efficient clearance of GAS in endothelial cells is caused by their intrinsic inability to target GAS with ubiquitin to promote autophagosome biogenesis for xenophagy.

Insufficient Acidification of Autophagosomes Facilitates Group A Streptococcus Survival and Growth in Endothelial Cells.

UNLABELLED: Group A streptococcus (GAS) is an important human pathogen, and its invasion via blood vessels is critically important in serious events such as bacteremia or multiorgan failure. Although GAS was identified as an extracellular bacterium, the internalization of GAS into nonphagocytic cells may provide a strategy to escape from immune surveillance and antibiotic killing. However, GAS has also been reported to induce autophagy and is efficiently killed within lysosome-fused autophagosomes in epithelial cells. In this study, we show that GAS can replicate in endothelial cells and that streptolysin O is required for GAS growth. Bacterial replication can be suppressed by altering GAS gene expression in an acidic medium before internalization into endothelial cells. The inhibitory effect on GAS replication can be reversed by treatment with bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase. Compared with epithelial cells in which acidification causes autophagy-mediated clearance of GAS, there was a defect in acidification of GAS-containing vesicles in endothelial cells. Consequently, endothelial cells fail to maintain low pH in GAS-containing autophagosomes, thereby permitting GAS replication inside LAMP-1- and LC3-positive vesicles. Furthermore, treatment of epithelial cells with bafilomycin A1 resulted in defective GAS clearance by autophagy, with subsequent bacterial growth intracellularly. Therefore, low pH is a key factor for autophagy-mediated suppression of GAS growth inside epithelial cells, while defective acidification of GAS-containing vesicles results in bacterial growth in endothelial cells. IMPORTANCE: Previous reports showed that GAS can induce autophagy and is efficiently killed within lysosome-fused autophagosomes in epithelial cells. In endothelial cells, in contrast, induction of autophagy is not sufficient for GAS killing. In this study, we provide the first evidence that low pH is required to prevent intracellular growth of GAS in epithelial cells and that this mechanism is defective in endothelial cells. Treatment of GAS with low pH altered GAS growth rate and gene expression of virulence factors and resulted in enhanced susceptibility of GAS to intracellular lysosomal killing. Our findings reveal the existence of different mechanisms of host defense against GAS invasion between epithelial and endothelial cells.

Kallistatin modulates immune cells and confers anti-inflammatory response to protect mice from group A streptococcal infection.

Group A streptococcus (GAS) infection may cause severe life-threatening diseases, including necrotizing fasciitis and streptococcal toxic shock syndrome. Despite the availability of effective antimicrobial agents, there has been a worldwide increase in the incidence of invasive GAS infection. Kallistatin (KS), originally found to be a tissue kallikrein-binding protein, has recently been shown to possess anti-inflammatory properties. However, its efficacy in microbial infection has not been explored. In this study, we transiently expressed the human KS gene by hydrodynamic injection and investigated its anti-inflammatory and protective effects in mice via air pouch inoculation of GAS. The results showed that KS significantly increased the survival rate of GAS-infected mice. KS treatment reduced local skin damage and bacterial counts compared with those in mice infected with GAS and treated with a control plasmid or saline. While there was a decrease in immune cell infiltration of the local infection site, cell viability and antimicrobial factors such as reactive oxygen species actually increased after KS treatment. The efficiency of intracellular bacterial killing in neutrophils was directly enhanced by KS administration. Several inflammatory cytokines, including tumor necrosis factor alpha, interleukin 1ß, and interleukin 6, in local infection sites were reduced by KS. In addition, KS treatment reduced vessel leakage, bacteremia, and liver damage after local infection. Therefore, our study demonstrates that KS provides protection in GAS-infected mice by enhancing bacterial clearance, as well as reducing inflammatory responses and organ damage.

Regulatory role of GSK-3 ß on NF- κ B, nitric oxide, and TNF- α in group A streptococcal infection.

Group A streptococcus (GAS) imposes a great burden on humans. Efforts to minimize the associated morbidity and mortality represent a critical issue. Glycogen synthase kinase-3 ß (GSK-3 ß) is known to regulate inflammatory response in infectious diseases. However, the regulation of GSK-3 ß in GAS infection is still unknown. The present study investigates the interaction between GSK-3 ß , NF- κ B, and possible related inflammatory mediators in vitro and in a mouse model. The results revealed that GAS could activate NF- κ B, followed by an increased expression of inducible nitric oxide synthase (iNOS) and NO production in a murine macrophage cell line. Activation of GSK-3 ß occurred after GAS infection, and inhibition of GSK-3 ß reduced iNOS expression and NO production. Furthermore, GSK-3 ß inhibitors reduced NF- κ B activation and subsequent TNF- α production, which indicates that GSK-3 ß acts upstream of NF- κ B in GAS-infected macrophages. Similar to the in vitro findings, administration of GSK-3 ß inhibitor in an air pouch GAS infection mouse model significantly reduced the level of serum TNF- α and improved the survival rate. The inhibition of GSK-3 ß to moderate the inflammatory effect might be an alternative therapeutic strategy against GAS infection.

Plasma kallistatin levels in patients with severe community-acquired pneumonia.

INTRODUCTION: Community-acquired pneumonia (CAP) requiring intensive care unit (ICU) treatment commonly causes acute respiratory failure with high mortality. Kallistatin, an endogenous tissue kallikrein inhibitor, has been reported to be protective in various human diseases. The aim of this study was to assess the correlations of kallistatin with other biomarkers and to determine whether kallistatin levels have a prognostic value in severe CAP. METHODS: Plasma samples and clinical data were prospectively collected from 54 patients with severe CAP requiring ICU admission. Seventeen healthy control subjects were included for comparison. Plasma kallistatin, kallikrein, and other biomarkers of inflammation (tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, IL-8, C-reactive protein (CRP)), and anti-coagulation (protein C, anti-thrombin III) were measured on days 1 and 4 of ICU admission. Comparison between survivors (n = 41) and nonsurvivors (n = 13) was performed. RESULTS: Plasma kallistatin was significantly consumed in severe CAP patients compared with healthy individuals. Lower day 1 kallistatin levels showed a strong trend toward increased mortality (P = 0.018) and higher day 1 CURB-65 scores (P = 0.004). Plasma kallistatin levels on day 1 of ICU admission were significantly decreased in patients who developed septic shock (P = 0.017) and who had acute respiratory distress syndrome (P = 0.044). In addition, kallistatin levels were positively correlated with anti-thrombin III and protein C and inversely correlated with IL-1ß, IL-6, and CRP levels. In a multivariate logistic regression analysis, higher day 1 CURB-65 scores were independent predictors of mortality (odds ratio = 29.9; P = 0.009). Also, higher day 1 kallistatin levels were independently associated with a decreased risk of death (odds ratio, 0.1) with a nearly significant statistical difference (P = 0.056). Furthermore, we found that a cutoff level of 6.5 µg/ml of day 1 kallistatin determined by receiver operating characteristic curves could be used to distinguish between patients who survived in 60 days and those who did not. CONCLUSIONS: These results suggest that kallistatin may serve as a novel marker for severe CAP prognosis and may be involved in the pathogenesis of CAP through antiinflammatory and anticoagulation effects.

Synergistic effects of streptolysin S and streptococcal pyrogenic exotoxin B on the mouse model of group A streptococcal infection.

Streptococcus pyogenes is a group A streptococcus (GAS) and an important human pathogen that causes a variety of diseases. Streptococcal pyrogenic exotoxin B (SPE B) and streptolysin S (SLS) are important virulence factors involved in GAS infection, but it is not clear which one is more virulent. Using an air pouch infection model, the wild-type strain NZ131, its isogenic mutants, and complementary mutants were used to examine the effects of SPE B and SLS on GAS infection. The results of the skin lesion and mouse mortality assays showed that although SPE B and SLS had a synergistic effect on GAS infection, SPE B played a more important role in local tissue damage while SLS had a more prominent effect on mouse mortality. Surveys of the exudates from the air pouch revealed that the expression of inflammatory cytokines was significantly inhibited in the sagB/speB-double-mutant JM4-infected mice. Furthermore, in vivo and in vitro studies showed that the isogenic mutant strains were more susceptible to the immune cell killing than the wild-type strain and that the sagB/speB-double-mutant JM4 was the most sensitive among these strains. Moreover, infection with the sagB/speB-double-mutant JM4 strain caused the least amount of macrophage apoptosis compared to infection with the wild-type NZ131 and the other complementary strains, which express only SPE B or SLS or both. Taken together, these results indicate that both SPE B and SLS contributed to GAS evasion from immune cell killing, local tissue damage, and mouse mortality.

Size-dependent attenuation of TLR9 signaling by gold nanoparticles in macrophages.

Gold nanoparticles (GNPs), which are generally thought to be bio-inert and non-cytotoxic, have become one of the most ideal nanomaterials for medical applications. Once engulfed by phagocytes, the immunological effects of GNPs are still of concern and require detailed investigation. Therefore, this study explored the immunological significance of GNPs on TLR-mediated innate immunity in murine macrophages. GNP causes specific inhibition of TLR9 (CpG oligodeoxynucleotides; CpG-ODNs) signal in macrophages. The impaired CpG-ODN-induced TNF-α production is GNP concentration- and size-dependent in murine Raw264.7 cells: a GNP of 4 nm in size is more potent than a GNP of 11, 19, 35, or 45 nm in size. Consistent with cytokine inhibition, the CpG-ODN-induced phosphorylation of NF-κB and JNK as well as NF-κB activation are suppressed by GNPs. GNPs accumulate in lysosomes after phagocytosis and also increase TLR9-associated lysosomal cathepsin expression and activities, but this is irrelevant to TLR9 inhibition by GNPs in our studies. In addition, GNPs affected TLR9 translocation in response to CpG-ODNs and to phagosomes. Further exploring how GNPs inhibited TLR9 function, we found that GNPs could bind to high-mobility group box-1 (which is involved in the regulation of TLR9 signaling) inside the lysosomes. The current studies demonstrate that size-dependent inhibition of TLR9 function by GNP may be attributed to its binding to high-mobility group box-1.

Dextromethorphan efficiently increases bactericidal activity, attenuates inflammatory responses, and prevents group a streptococcal sepsis.

Group A streptococcus (GAS) is an important human pathogen that causes a wide spectrum of diseases, ranging from mild throat and skin infections to severe invasive diseases such as necrotizing fasciitis and streptococcal toxic shock syndrome. Dextromethorphan (DM), a dextrorotatory morphinan and a widely used antitussive drug, has recently been reported to possess anti-inflammatory properties. In this study, we investigated the potential protective effect of DM in GAS infection using an air pouch infection mouse model. Our results showed that DM treatment increased the survival rate of GAS-infected mice. Bacterial numbers in the air pouch were lower in mice treated with DM than in those infected with GAS alone. The bacterial elimination efficacy was associated with increased cell viability and bactericidal activity of air-pouch-infiltrating cells. Moreover, DM treatment prevented bacterial dissemination in the blood and reduced serum levels of the proinflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and IL-1ß and the chemokines monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 2 (MIP-2), and RANTES. In addition, GAS-induced mouse liver injury was reduced by DM treatment. Taken together, DM can increase bacterial killing and reduce inflammatory responses to prevent sepsis in GAS infection. The consideration of DM as an adjunct treatment in combination with antibiotics against bacterial infection warrants further study.