Shikonin ameliorates lipoteichoic acid­induced acute lung injury via promotion of neutrophil apoptosis.

Shikonin is the major active component in Lithospermum erythrorhizon and has pharmacological effects including reducing inflammation, aiding resistance to bacteria and promoting wound healing. However, the effect of shikonin on lipoteichoic acid (LTA)­induced acute lung injury (ALI) remains to be elucidated. ALI is a serious illness resulting from significant pulmonary inflammation caused by various diseases, such as sepsis, acid aspiration and trauma. The present study found that shikonin significantly attenuated LTA­induced ALI. Following shikonin treatment, the accumulation of pulmonary neutrophils and expression of TNFα, IL­1ß and IL­6 were decreased in mice with LTA­induced ALI. Furthermore, Shikonin promoted neutrophil apoptosis by increasing the activation of caspase­3 and reducing the expression of the antiapoptotic myeloid cell leukemia­1 (Mcl­1) protein. However, shikonin treatment did not influence the expression of B­cell lymphoma­2. The findings of the present study demonstrated that shikonin protected against LTA­induced ALI by promoting caspase-3 and Mcl­1­related neutrophil apoptosis, suggesting that shikonin is a potential agent that can be used in the treatment of sepsis­mediated lung injury.

Characterising lipoteichoic acid as an in vitro model of acute neuroinflammation.

Toll-like receptor 2 (TLR2) is a primary sensor for pathogens, including those derived from gram-positive bacteria. It can also mediate the effects of endogenous inflammatory signals such as ß-amyloid peptide (Aß), thus promoting the microglial activation and subsequent neuronal dysfunction, characteristic of chronic neuroinflammatory conditions. More recently, a role for TLR2 has been proposed in the pathogenesis of disorders associated with acute inflammation, including anxiety and depression. The current study aims to characterise the acute effects of the TLR2 agonist lipoteichoic acid (LTA) on microglial activation and neuronal integrity, and to evaluate the influence of LTA exposure on sensitivity to the inflammation and neuronal dysfunction associated with Aß. Using BV2 and N2a cells as an in vitro model, we highlight that acute exposure to LTA robustly promotes inflammatory cytokine and nitric oxide (NO) production in microglia but also in neurons, similar to that reported under longer-term and chronic inflammatory conditions. Moreover, we find that exposure to LTA can enhance sensitivity to subthreshold Aß, promoting an 'M1'-like phenotype in microglia and provoking dysregulation of neuronal activity in acute hippocampal slices. Anti-inflammatory agents, including mimetics of brain-derived neurotrophic factor (BDNF), have proven effective at alleviating chronic neuroinflammatory complications. We further examined the effects of 7,8,3-trihydroxyflavone (7,8,3-THF), a small-molecule TrkB agonist, on LTA-induced microglial activation. We report that 7,8,3-THF can significantly ameliorate interleukin (IL)-6 and NO production in LTA-stimulated BV2 cells. Taken together, our findings offer support for exploration of TLR2 as a potential target for therapeutic intervention into acute neuroinflammatory conditions. Moreover we propose that exposure to gram-positive bacterial pathogens may promote sensitivity to the inflammatory changes characteristic of the aged brain.

Epinecidin-1: An orange-spotted grouper antimicrobial peptide that modulates Staphylococcus aureus lipoteichoic acid-induced inflammation in macrophage cells.

Orange-spotted grouper (Epinephelus coioides) is among the most economically important of all fish species farmed in Asia. This species expresses an antimicrobial peptide called epinecidin-1 (EPI), which is considered to be a host defense factor due to its strong bacterial killing activity. Antimicrobial peptides usually possess both bacterial killing and immunomodulatory activity, however, the modulatory activity of EPI on Gram-positive bacterial lipoteichoic acids (LTA)-induced inflammation has not been previously reported. In this study, we found that EPI effectively suppressed LTA-induced production of proinflammatory factors in macrophages. Mechanistically, EPI attenuated LTA-induced inflammation by inhibiting Toll-like receptor (TLR) 2 internalization and subsequent downstream signaling (reactive oxygen species, Akt, p38 and Nuclear factor κB). However, protein abundance of TLR2 was not altered by EPI or LTA. Taken together, our findings reveal for the first time that EPI possesses inhibitory activity toward LTA-induced inflammation in macrophages.

Inhibition of inflammatory response in human keratinocytes by magnetic nanoparticles functionalized with PBP10 peptide derived from the PIP2-binding site of human plasma gelsolin.

BACKGROUND: Human plasma gelsolin (pGSN) is a multifunctional actin-binding protein involved in a variety of biological processes, including neutralization of pro-inflammatory molecules such as lipopolysaccharide (LPS) and lipoteichoic acid (LTA) and modulation of host inflammatory response. It was found that PBP10, a synthetic rhodamine B-conjugated peptide, based on the phosphoinositide-binding site of pGSN, exerts bactericidal activity against Gram-positive and Gram-negative bacteria, interacts specifically with LPS and LTA, and limits microbial-induced inflammatory effects. The therapeutic efficiency of PBP10 when immobilized on the surface of iron oxide-based magnetic nanoparticles was not evaluated, to date. RESULTS: Using the human keratinocyte cell line HaCaT stimulated by bacterially-derived LPS and LTA as an in vitro model of bacterial infection, we examined the anti-inflammatory effects of nanosystems consisting of iron oxide-based magnetic nanoparticles with aminosilane (MNP@NH2) or gold shells (MNP@Au) functionalized by a set of peptides, derived from the phosphatidylinositol 4,5-bisphosphate (PIP2)-binding site of the human plasma protein gelsolin, which also binds LPS and LTA. Our results indicate that these nanosystems can kill both Gram-positive and Gram-negative bacteria and limit the production of inflammatory mediators, including nitric oxide (NO), reactive oxygen species (ROS), and interleukin-8 (IL-8) in the response to heat-killed microbes or extracted bacterial cell wall components. The nanoparticles possess the potential to improve therapeutic efficacy and are characterized by lower toxicity and improved hemocompatibility when compared to free peptides. Atomic force microscopy (AFM) showed that these PBP10-based nanosystems prevented changes in nanomechanical properties of cells that were otherwise stimulated by LPS. CONCLUSIONS: Neutralization of endotoxemia-mediated cellular effects by gelsolin-derived peptides and PBP10-containing nanosystems might be considered as potent therapeutic agents in the improved therapy of bacterial infections and microbial-induced inflammation.

Cytotoxicity of intracanal dressings on apical papilla cells differ upon activation with E. faecalis LTA

Abstract Objective The aim of this study was to investigate the cytotoxic effects of modified triple antibiotic paste and an experimental composition using calcium hydroxide on lipoteichoic acid (LTA)-primed apical papilla cells (APC). Material and Methods Human APC were tested for in vitro cytotoxicity of modified Triple Antibiotic Paste (mTAP - Ciprofloxacin, Metronidazole and Cefaclor at 1:1:1) and of a paste of Ciprofloxacin, Metronidazole and Calcium hydroxide (CMC - 1:1:2) and modified CMC (mCMC - 2:2:1) by using MTT assay. The substances were reconstituted in DMEM at 1,000 µg/mL and » serially diluted before being kept in contact with cells for 1, 3, 5 and 7 days. Further, cells were primed with 1 µg/mL of Enterococcus faecalis LTA for 7 days prior to the viability test with 1,000 µg/mL of each substance. Statistical analysis was performed using one-way analysis of variance (ANOVA) and two-way ANOVA respectively followed by Tukey's post-test. Significance levels were set at p<0.05. Results In the first assay, the higher cytotoxic rates were reached by mTAP for all experimental periods. CMC was found toxic for APC at 5 and 7 days, whereas mCMC did not affect the cell viability. Only CMC and mCMC were able to induce some cellular proliferation. In the second assay, when considering the condition with medium only, LTA-primed cells significantly proliferated in comparison to LTA-untreated ones. At this context, mTAP and CMC showed similar cytotoxicity than the observed for LTA-untreated cells, while mCMC was shown cytotoxic at 7 days only for LTA-primed APC. Comparing the medications, mTAP was more cytotoxic than CMC and mCMC. Conclusion mTAP showed higher cytotoxicity than CMC and mCMC and the effect of topic antimicrobials might differ when tested against apical papilla cells under physiological or activated conditions.

Low-density lipoprotein (LDL)-dependent uptake of Gram-positive lipoteichoic acid and Gram-negative lipopolysaccharide occurs through LDL receptor.

Lipoteichoic acid (LTA) and lipopolysaccharide (LPS) are bacterial lipids that stimulate pro-inflammatory cytokine production, thereby exacerbating sepsis pathophysiology. Proprotein convertase subtilisin/kexin type 9 (PCSK9) negatively regulates uptake of cholesterol by downregulating hepatic lipoprotein receptors, including low-density lipoprotein (LDL) receptor (LDLR) and possibly LDLR-related protein-1 (LRP1). PCSK9 also negatively regulates Gram-negative LPS uptake by hepatocytes, however this mechanism is not completely characterized and mechanisms of Gram-positive LTA uptake are unknown. Therefore, our objective was to elucidate the mechanisms through which PCSK9 regulates uptake of LTA and LPS by investigating the roles of lipoproteins and lipoprotein receptors. Here we show that plasma PCSK9 concentrations increase transiently over time in septic and non-septic critically ill patients, with highly similar profiles over 14 days. Using flow cytometry, we demonstrate that PCSK9 negatively regulates LDLR-mediated uptake of LTA and LPS by HepG2 hepatocytes through an LDL-dependent mechanism, whereas LRP1 and high-density lipoprotein do not contribute to this uptake pathway. Bacterial lipid uptake by hepatocytes was not associated with cytokine production or hepatocellular injury. In conclusion, our study characterizes an LDL-dependent and LDLR-mediated bacterial lipid uptake pathway regulated by PCSK9, and provides evidence in support of PCSK9 inhibition as a potential therapeutic strategy for sepsis.

(3R)-5,6,7-trihydroxy-3-isopropyl-3-methylisochroman-1-one reduces lipoteichoic acid-induced damage in rat cardiomyoblast cells.

OBJECTIVE: Infective endocarditis is usually caused by Streptococcus sanguinis and characterized by inflammatory responses in the endocardium. This study aimed to investigate if the new compound (3R)-5,6,7-trihydroxy-3-isopropyl-3-methylisochroman-1-one (TIM) isolated from Alpinia katsumadai Hayata could provide protection against lipoteichoic acid (LTA)-induced cell damage in embryonic rat heart cells (H9c2). METHODS: LTA-induced cell damage was established in H9c2, and the protective effects of TIM against the cell damage were examined at different concentrations (0.1-2.5 µM). The inflammatory response and oxidative stress in H9c2 cells were also measured. RESULTS: Treatment with TIM (0.1-2.5 µM) significantly decreased LTA-induced toxicity in H9c2 cells, which was indicated by increase in cell viability, elevation in the mitochondrial membrane potential, decrease in the release of cytochrome-c and DNA damage, inhibition of caspase-3/9 activities, and change in apoptosis-related protein expression in LTA-treated H9c2 cells. TIM treatment also significantly attenuated the redox imbalance in H9c2 cells by decreasing malondialdehyde and intracellular reactive oxygen species levels as well as by enhancing superoxide dismutase activities and glutathione levels by increasing nuclear factor (erythroid-derived 2)-like 2 protein expression. Moreover, TIM treatment decreased interleukin 1 ß, interleukin 12, and tumor necrosis factor α levels by inhibiting nuclear factor kappa B protein expression. CONCLUSION: Our data indicated that TIM protected H9c2 cells against LTA-induced toxicity, at least partially through inhibiting the inflammatory response and oxidative stress, providing scientific rational to develop TIM to treat infective endocarditis.

Polydatin reduces Staphylococcus aureus lipoteichoic acid-induced injury by attenuating reactive oxygen species generation and TLR2-NFκB signalling.

Staphylococcus aureus (S. aureus) causes severe inflammation in various infectious diseases, leading to high mortality. The clinical application of antibiotics has gained a significant curative effect. However, it has led to the emergence of various resistant bacteria. Therefore, in this study, we investigated the protective effect of polydatin (PD), a traditional Chinese medicine extract, on S. aureus lipoteichoic acid (LTA)-induced injury in vitro and in vivo. First, a significant improvement in the pathological conditions of PD in vivo was observed, suggesting that PD had a certain protective effect on LTA-induced injury in a mouse model. To further explore the underlying mechanisms of this protective effect of PD, LTA-induced murine macrophages were used in this study. The results have shown that PD could reduce the NF-κB p65, and IκBα phosphorylation levels increased by LTA, resulting in a decrease in the transcription of pro-inflammatory factors, such as TNF-α, IL-1ß and IL-6. However, LTA can not only activate NF-κB through the recognition of TLR2 but also increase the level of intracellular reactive oxygen species (ROS), thereby activating NF-κB signalling. We also detected high levels of ROS that activate caspases 9 and 3 to induce apoptosis. In addition, using a specific NF-κB inhibitor that could attenuate apoptosis, namely NF-κB p65, acted as a pro-apoptotic transcription factor in LTA-induced murine macrophages. However, PD could inhibit the generation of ROS and NF-κB p65 activation, suggesting that PD suppressed LTA-induced injury by attenuating ROS generation and TLR2-NFκB signalling.

Short communication: Differential loss of bovine mammary epithelial barrier integrity in response to lipopolysaccharide and lipoteichoic acid.

In the mammary gland, the blood-milk barrier prevents an uncontrolled intermixture of blood and milk constituents and hence maintains the osmotic gradient to draw water into the mammary secretion. During mastitis, the permeability of the blood-milk barrier is increased, which is reflected by the transfer of blood constituents into milk and vice versa. In this study, we aimed to investigate changes in the barrier function of mammary epithelial cells in vitro as induced by cell wall components of different pathogens. Primary bovine mammary epithelial cells from 3 different cows were grown separately on Transwell (Corning Inc., Corning, NY) inserts. The formation of tight junctions between adjacent epithelial cells was shown by transmission electron microscopy and by immunofluorescence staining of the tight junction protein zona occludens-1. The integrity of the epithelial barrier was assayed by means of transepithelial electrical resistance, as well as by diffusion of the fluorophore Lucifer yellow across the cell layer. The release of lactate dehydrogenase (LDH) was used as an indicator for cytotoxic effects. In response to a 24-h challenge with bacterial endotoxin, barrier integrity was reduced after 3 or 7h, respectively, in response to 0.5mg/mL lipopolysaccharide (LPS) from Escherichia coli or 20mg/mL lipoteichoic acid (LTA) from Staphylococcus aureus. No paracellular leakage was observed in response to 0.2mg/mL LPS or 2mg/mL LTA. Although LPS and LTA affected barrier permeability, most likely by opening the tight junctions, only LPS caused cell damage, reflected by increased LDH concentrations in cell culture medium. These results prove a pathogen-specific loss of blood-milk barrier integrity during mastitis, which is characterized by tight junction opening by both LPS and LTA and by additional epithelial cell destruction through LPS.

Barrier protection via Toll-like receptor 2 signaling in porcine intestinal epithelial cells damaged by deoxynivalnol.

Intestinal barrier is the first line of defense inside the body and comprises intercellular tight junction (TJ) proteins that regulate paracellular permeability. Deoxynivalenol (DON), a fungal metabolite often found in the contaminated food of domestic animals, is known to impair intestinal barrier function and may be involved in intestinal inflammation. Unlike in humans and mice, the importance of Toll-like receptor (TLR) 2 expressed in porcine intestinal epithelial cells is largely unclear. Therefore, the aim of the present study was to investigate whether TLR2 stimulation enhances intestinal barrier function and protects against DON exposure. We found that the cells treated with TLR2 ligands decreased the epithelial barrier permeability and enhanced TJ protein expression in intestinal porcine epithelial cells (IPEC-J2). In addition, pretreatment with TLR2 ligand, including Pam3CSK4 (PCSK) and lipoteichoic acid from Bacillus subtilis, prevented DON-induced barrier dysfunction by increasing the expression of TJ proteins via the PI3K-Akt-dependent pathway. It is likely that the DON-disrupted intestinal barrier caused biological changes of immune cells in the lamina propria. Thus, we conducted co-culture of differentiated IPEC-J2 cells in the upper well together with peripheral blood mononuclear cells in the bottom well and found that apical TLR2 stimulation of IPEC-J2 cells could alleviate the reduction in cell survival and proliferation of immune cells. Conclusively, TLR2 signaling on intestinal epithelial cells may enhance intestinal barrier function and prevent DON-induced barrier dysfunction of epithelial cells.

Inhibition of UDP/P2Y6 purinergic signaling prevents phagocytosis of viable neurons by activated microglia in vitro and in vivo.

Microglia activated through Toll-like receptor (TLR)-2 or -4 can cause neuronal death by phagocytosing otherwise-viable neurons-a form of cell death called "phagoptosis." UDP release from neurons has been shown to provoke microglial phagocytosis of neurons via microglial P2Y6 receptors, but whether inhibition of this process affects neuronal survival is unknown. We tested here whether inhibition of P2Y6 signaling could prevent neuronal death in inflammatory conditions, and whether UDP signaling can induce phagoptosis of stressed but viable neurons. We find that delayed neuronal loss and death in mixed neuronal/glial cultures induced by the TLR ligands lipopolysaccharide (LPS) or lipoteichoic acid was prevented by: apyrase (to degrade nucleotides), Reactive Blue 2 (to inhibit purinergic signaling), or MRS2578 (to specifically block P2Y6 receptors). In each case, inflammatory activation of microglia was not affected, and the rescued neurons remained viable for at least 7 days. Blocking P2Y6 receptors with MRS2578 also prevented phagoptosis of neurons induced by 250 nM amyloid beta 1-42, 5 µM peroxynitrite, or 50 µM 3-morpholinosydnonimine (which releases reactive oxygen and nitrogen species). Furthermore, the P2Y6 receptor agonist UDP by itself was sufficient to stimulate microglial phagocytosis and to induce rapid neuronal loss that was prevented by eliminating microglia or inhibiting phagocytosis. In vivo, injection of LPS into rat striatum induced microglial activation and delayed neuronal loss and blocking P2Y6 receptors with MRS2578 prevented this neuronal loss. Thus, blocking UDP/P2Y6 signaling is sufficient to prevent neuronal loss and death induced by a wide range of stimuli that activate microglial phagocytosis of neurons.

Pathogen associated molecular pattern-induced TNF, IL-1ß, IL-6 and CXCL-8 production from feline whole blood culture.

Whole blood culture (C(wb)) is a method to evaluate leukocyte response to stimuli. We used C(wb) to evaluate the inflammatory response to pathogen associated molecular patterns (PAMPs) in cats. Blood was collected from diluted with RPMI and stimulated with various concentrations of lipopolysaccharide (LPS), lipoteichoic acid (LTA), peptidoglycan (PG) or control (PBS). Multiple concentrations of LPS, LTA and PG significantly stimulated tumor necrosis factor (TNF), interleukin (IL)-1ß and CXC chemokine ligand (CXCL)-8 in feline C(wb). All PAMPs failed to stimulate IL-6 production and PG failed to stimulate CXCL-8 production. Lipopolysaccharide was a more potent inducer of IL-1ß and CXCL-8 than LTA or PG and LTA is a more potent inducer of CXCL-8 than PG. Based on these data, PAMPs from gram positive and negative bacteria induce TNF, IL-1ß and CXCL-8 production in feline whole blood. Cats appear to be relatively more sensitive to gram negative compared to gram positive bacteria.

Ikaros expression in tongue sole macrophages: a marker for lipopolysaccharide- and lipoteichoic acid-induced inflammatory responses.

Ikaros, an important transcription factor plays a role in the development of hemato-lymphoid system, yet its functional importance in fish macrophages remains unknown. In this study, an Ikaros cDNA was cloned from the half-smooth tongue sole Cynoglossus semilaevis. The cDNA contained an open reading frame of 1,290 nucleotides that encoded a 430 amino acid protein. The deduced protein is structurally similar to dul from other species, for example human, axolotl, and possesses 3-zinc finger and 2-zinc finger domains at its N- and C-termini, respectively. Phylogenetic analysis revealed C. semilaevis Ikaros to be grouped with all the fish Ikaros, but branching from other Ikaros family members. Both semi-quantitative PCR and quantitative real-time PCR indicated Ikaros to be predominantly expressed in the immune-relevant tissues such as kidney, thymus, spleen and liver. In the macrophages cultured from C. semilaevis head kidney and challenged with lipopolysaccharide and lipoteichoic acid not only induced expression of the proinflammatory cytokines tumor necrosis factor-alpha and interleukin 1-beta but also caused up-regulation of Ikaros in a dose- and time-dependent fashions. All these data suggest that Ikaros might be a useful marker for inflammatory responses in C. semilaevis.

Muramyl dipeptide synergizes with Staphylococcus aureus lipoteichoic acid to recruit neutrophils in the mammary gland and to stimulate mammary epithelial cells.

Staphylococcus aureus, a major pathogen for the mammary gland of dairy ruminants, elicits the recruitment of neutrophils into milk during mastitis, but the mechanisms are incompletely understood. We investigated the response of the bovine mammary gland to muramyl dipeptide (MDP), an elementary constituent of the bacterial peptidoglycan, alone or in combination with lipoteichoic acid (LTA), another staphylococcal microbial-associated molecular pattern (MAMP). MDP induced a prompt and marked influx of neutrophils in milk, and its combination with LTA elicited a more intense and prolonged influx than the responses to either stimulus alone. The concentrations of several chemoattractants for neutrophils (CXCL1, CXCL2, CXCL3, CXCL8, and C5a) increased in milk after challenge, and the highest increases followed challenge with the combination of MDP and LTA. MDP and LTA were also synergistic in inducing in vitro chemokine production by bovine mammary epithelial cells (bMEpC). Nucleotide-binding oligomerization domain 2 (NOD2), a major sensor of MDP, was expressed (mRNA) in bovine mammary tissue and by bMEpC in culture. The production of interleukin-8 (IL-8) following the stimulation of bMEpC by LTA and MDP was dependent on the activation of NF-κB. LTA-induced IL-8 production did not depend on platelet-activating factor receptor (PAFR), as the PAFR antagonist WEB2086 was without effect. In contrast, bMEpC and mammary tissue are known to express Toll-like receptor 2 (TLR2) and to respond to TLR2 agonists. Although the levels of expression of the inflammatory cytokines tumor necrosis factor alpha (TNF-α) and IL-1ß were increased by LTA and MDP at the mRNA level, no protein could be detected in the bMEpC culture supernatant. The level of induction of IL-6 was low at both the mRNA and protein levels. These results indicate that MDP and LTA exert synergistic effects to induce neutrophilic inflammation in the mammary gland. These results also show that bMEpC could contribute to the inflammatory response by recognizing LTA and MDP and secreting chemokines but not proinflammatory cytokines. Overall, this study indicates that the TLR2 and NOD2 pathways could cooperate to trigger an innate immune response to S. aureus mastitis.

Cobalt protoporphyrin inhibition of lipopolysaccharide or lipoteichoic acid-induced nitric oxide production via blocking c-Jun N-terminal kinase activation and nitric oxide enzyme activity.

In the present study, low doses (0.5, 1, and 2 microM) of cobalt protoporphyrin (CoPP), but not ferric protoporphyrin (FePP) or tin protoporphyrin (SnPP), significantly inhibited lipopolysaccharide (LPS) or lipoteichoic acid (LTA)-induced inducible nitric oxide (iNOS) and nitric oxide (NO) production with an increase in heme oxygenase 1 (HO-1) protein in RAW264.7 macrophages under serum-free conditions. IC(50) values of CoPP inhibition of NO and iNOS protein individually induced by LPS and LTA were around 0.25 and 1.7 microM, respectively. This suggests that CoPP is more sensitive at inhibiting NO production than iNOS protein in response to separate LPS and LTA stimulation. NO inhibition and HO-1 induction by CoPP were blocked by the separate addition of fetal bovine serum (FBS) and bovine serum albumin (BSA). Decreasing iNOS/NO production and increasing HO-1 protein by CoPP were observed with CoPP pretreatment, CoPP co-treatment, and CoPP post-treatment with LPS and LTA stimulation. LPS- and LTA-induced NOS/NO productions were significantly suppressed by the JNK inhibitor, SP600125, but not by the ERK inhibitor, PD98059, through a reduction in JNK protein phosphorylation. Transfection of a dominant negative JNK plasmid inhibited LPS- and LTA-induced iNOS/NO production and JNK protein phosphorylation, suggesting that JNK activation is involved in LPS- and LTA-induced iNOS/NO production. Additionally, CoPP inhibition of LPS- and LTA-induced JNK, but not ERK, protein phosphorylation was identified in RAW264.7 cells. Furthermore, CoPP significantly reduced NO production in a cell-mediated, but not cell-free, iNOS enzyme activity assay accompanied by HO-1 induction. However, attenuation of HO-1 protein stimulated by CoPP via transfection of HO-1 siRNA did not affect NO's inhibition of CoPP against LPS stimulation. CoPP effectively suppressing LPS- and LTA-induced iNOS/NO production through blocking JNK activation and iNOS enzyme activity via a HO-1 independent manner is first demonstrated herein.

Changes in neurotransmitter levels and proinflammatory cytokine mRNA expressions in the mice olfactory bulb following nanoparticle exposure.

Recently, there have been increasing reports that nano-sized component of particulate matter can reach the brain and may be associated with neurodegenerative diseases. Previously, our laboratory has studied the effect of intranasal instillation of nano-sized carbon black (CB) (14 nm and 95 nm) on brain cytokine and chemokine mRNA expressions and found that 14-nm CB increased IL-1 beta, TNF-alpha, CCL2 and CCL3 mRNA expressions in the olfactory bulb, not in the hippocampus of mice. To investigate the effect of a single administration of nanoparticles on neurotransmitters and proinflammatory cytokines in a mouse olfactory bulb, we performed in vivo microdialysis and real-time PCR methods. Ten-week-old male BALB/c mice were implanted with guide cannula in the right olfactory bulb and, 1 week later, were instilled vehicle or CB (14 nm, 250 microg) intranasally. Six hours after the nanoparticle instillation, the mice were intraperitoneally injected with normal saline or 50 mug of bacteria cell wall component lipoteichoic acid (LTA), which may potentiate CB-induced neurologic effect. Extracellular glutamate and glycine levels were significantly increased in the olfactory bulb of CB-instilled mice when compared with vehicle-instilled control mice. Moreover, we found that LTA further increased glutamate and glycine levels. However, no alteration of taurine and GABA levels was observed in the olfactory bulb of the same mice. We also detected immunological changes in the olfactory bulb 11 h after vehicle or CB instillation and found that IL-1 beta mRNA expression was significantly increased in CB- and LTA-treated mice when compared with control group. However, TNF-alpha mRNA expression was increased significantly in CB- and saline-treated mice when compared with control group. These findings suggest that nanoparticle CB may modulate the extracellular amino acid neurotransmitter levels and proinflammatory cytokine IL-1 beta mRNA expressions synergistically with LTA in the mice olfactory bulb.

Intrapulmonary delivery of ethyl pyruvate attenuates lipopolysaccharide- and lipoteichoic acid-induced lung inflammation in vivo.

Ethyl pyruvate (EP) is a stable pyruvate derivative that has been shown to exert anti-inflammatory effects in various models of systemic inflammation including endotoxemia. We here sought to determine the local effects of EP, after intrapulmonary delivery, in models of lung inflammation induced by instillation via the airways of either lipopolysaccharide (LPS, a constituent of the gram-negative bacterial cell wall) or lipoteichoic acid (LTA, a component of the gram-positive bacterial cell wall). For this, we first established that EP dose dependently reduced the responsiveness of mouse MH-S alveolar macrophages and mouse MLE-15 and MLE-12 respiratory epithelial cells to stimulation with LPS or LTA in vitro. We then showed that intranasal administration of EP dose dependently inhibited tumor necrosis factor alpha release in bronchoalveolar lavage fluid of mice challenged with either LPS or LTA via the airways. Moreover, EP reduced the recruitment of neutrophils into the bronchoalveolar space after either LPS or LTA administration. These data suggest that intrapulmonary delivery of EP diminishes lung inflammation induced by LPS or LTA, at least in part by targeting alveolar macrophages and respiratory epithelial cells.

Effect of ultrafine carbon black particles on lipoteichoic acid-induced early pulmonary inflammation in BALB/c mice.

We studied the interaction effects of a single intratracheal instillation of ultrafine carbon black (CB) particles and staphylococcal lipoteichoic acid (LTA) on early pulmonary inflammation in male BALB/c mice. We examined the cellular profile, cytokine and chemokine levels in the bronchoalveolar lavage (BAL) fluid, and expression of chemokine and toll-like receptor (TLR) mRNAs in lungs. LTA produced a dose-related increase in early pulmonary inflammation, which was characterized by (1) influx of polymorphonuclear neutrophils (PMNs) and (2) induction of interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, macrophage inflammatory protein (MIP)-1alpha/CCL3, but no effect on monocyte chemoattractant protein (MCP)-1/CCL2 at 24 h after instillation. Levels of some proinflammatory indicators and TLR2-mRNA expression were significantly increased by 14 nm or 95 nm CB (125 microg) and low-dose LTA (10 microg) treatment compared to CB or LTA alone at 4 h after instillation. Notably, PMN levels and production of IL-6 and CCL2 in the 14 nm CB + LTA were significantly higher than that of 95 nm CB + LTA at 4 h after instillation. However, at 24 h after instillation, only PMN levels were significantly higher in the 14 nm CB + LTA than 95 nm CB + LTA but not the cytokines and chemokines. These data show additive as well as synergistic interaction effects of 14 nm or 95 nm ultrafine CB particles and LTA. We suggest that early pulmonary inflammatory responses in male BALB/c mice may be induced in a size-specific manner of the CB particles used in our study.

Effects of diesel exhaust particles on human alveolar macrophage ability to secrete inflammatory mediators in response to lipopolysaccharide.

Ambient particulate matter (PM) has been shown to be associated with mortality and morbidity. Diesel exhaust particles (DEP) contribute to ambient PM. Alveolar macrophages (AM) are important targets for PM effects in the lung. The effects of DEP exposure on human AM response to lipopolysachharide (LPS; from gram-negative bacteria) challenge in vitro were determined by monitoring the production of interleukin 8 (IL-8), tumor necrosis factor-alpha (TNF-alpha) and prostaglandin E(2) (PGE(2)). The roles of organic compounds and carbonaceous core of DEP in response to LPS were evaluated by comparing the DEPs effect to that of carbon black (CB), a carbonaceous particle with few adsorbed organic compounds. AMs were exposed in vitro to Standard Reference Material (SRM) DEP 2975, SRM DEP 1650, SRM 1975 (a dichloromethane extract of SRM DEP 2975) and CB particles for 24 h. DEPs induced a decreased secretion of IL-8, TNF-alpha and PGE(2) in response to a subsequent LPS stimulation. DEPs also show suppressive effect on the release of inflammatory mediators when stimulated with lipoteichoic acid, a product of gram positive bacteria. In summary, in vitro exposure of human AM to DEPs significantly suppress AM responsiveness to gram-negative and positive bacterial products, which may be a contributing factor to the impairment of pulmonary defense.

Impaired adrenal stress response in Toll-like receptor 2-deficient mice.

Septicemia is one of the major health concerns worldwide, and rapid activation of adrenal steroid release is a key event in the organism's first line of defense during this form of severe illness. The family of Toll-like receptors (TLRs) is critical in the early immune response upon bacterial infection, and TLR polymorphisms are frequent in humans. Here, we demonstrate that TLR-2 deficiency in mice is associated with reduced plasma corticosterone levels and marked cellular alterations in adrenocortical tissue. TLR-2-deficient mice have an impaired adrenal corticosterone release after inflammatory stress induced by bacterial cell wall compounds. This defect appears to be mediated by a decrease in systemic and intraadrenal cytokine expression, including IL-1, tumor necrosis factor alpha, and IL-6. Our data demonstrate a link between the innate immune system and the endocrine stress response. The critical role of TLR-2 in adrenal glucocorticoid regulation needs to be considered in patients with inflammatory disease.