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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 LTA‑induced ALI. Following shikonin treatment, the accumulation of pulmonary neutrophils and expression of TNFα, IL‑1β and IL‑6 were decreased in mice with LTA‑induced ALI. Furthermore, Shikonin promoted neutrophil apoptosis by increasing the activation of caspase‑3 and reducing the expression of the antiapoptotic myeloid cell leukemia‑1 (Mcl‑1) protein. However, shikonin treatment did not influence the expression of B‑cell lymphoma‑2. The findings of the present study demonstrated that shikonin protected against LTA‑induced ALI by promoting caspase-3 and Mcl‑1‑related neutrophil apoptosis, suggesting that shikonin is a potential agent that can be used in the treatment of sepsis‑mediated lung injury.

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 β-amyloid peptide (Aβ), 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 Aβ. 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 Aβ, 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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective: Excessive inflammatory responses in the endocardium are related to progression of infectious endocarditis. This study aimed to investigate whether (Z)-7,4'-dimethoxy-6-hydroxy-aurone-4-O-β-glucopyranoside (DHAG), a compound isolated from the endophytic fungus Penicillium citrinum of Bruguiera gymnorrhiza , could attenuate cell damage caused by lipoteichoic acid (LTA) in embryonic rat heart cells (H9c2)., Methods: LTA-induced cell damage occurred in H9c2 cells and the protective effects of DHAG at different concentrations (1-10  µM) were assessed. Indicators of oxidative stress and inflammatory responses in H9c2 cells were measured., Results: DHAG (1-10  µM) significantly attenuated LTA-induced damage in H9c2 cells, as evidenced by increased cell viability and mitochondrial membrane potential, decreased cytochrome c release and DNA fragmentation, inhibition of caspase-3 and -9 activity, and altered expression of apoptosis-related proteins. DHAG also decreased oxidative stress by increasing protein expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Furthermore, DHAG inhibited inflammatory responses by decreasing protein expression of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs)., Conclusion: DHAG exerted protective effects against LTA-induced cell damage, at least partially by decreasing oxidative stress and inhibiting inflammatory responses. Our results provide a scientific rational for developing DHAG as a therapy against infectious endocarditis.

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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Individual susceptibility differences to fungal infection following invasive and/or immunosuppressive medical interventions are an important clinical issue. In order to explore immune response-related factors that may be linked to fungal infection susceptibility, we have compared the response of inbred C57BL/6J and outbred CD1 mouse strains to different experimental models of fungal sepsis. The challenge of animals with the zymosan-induced generalised inflammation model revealed poorer survival rates in C57BL/6J, consistent with lower Th1 cytokine interferon (IFN)-γ serum levels, compared with CD1 mice. Likewise, ex vivo exposure of C57BL/6J splenocytes to zymosan but also bacterial lipopolisaccharide or lipoteichoic acid, resulted in lower IFN-γ secretion compared with CD1 mice. C57BL/6J susceptibility could be reverted by rescue infusion of relative low IFN-γ doses (0.2 μg/kg) either alone or in combination with the ß-glucan-binding CD5 protein (0.7 mg/kg) leading to improved post zymosan-induced generalised inflammation survival. Similarly, low survival rates to systemic Candida albicans infection (2.86 × 10 4  CFU/gr) were ameliorated by low-dose IFN-γ infusion in C57BL/6J but not CD1 mice. Our results highlight the importance of strain choice in experimental fungal infection models and provide a susceptibility rationale for more specific antifungal immunotherapy designs., (© 2018 John Wiley & Sons Ltd.)

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 ¼ 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.

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@NH 2 ) 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.

Objective: Exposure to oxidative stress will lead to the progression of retinal degenerative diseases, and unfortunately the exact mechanisms have not been fully understood. In this study, the protective effects of (3R)-5,6,7-trihydroxy-3-isopropyl-3-methylisochroman-1-one (TIM) against the lipoteichoic acid (LTA)-induced cell damage in mouse photoreceptor-derived 661W cells were investigated., Methods: 661W cells were pre-treated with TIM at different concentrations (0.1-2.5 μM) before exposure to LTA. The oxidative stress and inflammatory response were detected in 661W cells., Results: Pre-treatment of 661W cells with TIM (0.1-2.5 μM) for 4 h significantly decreased the LTA-induced toxicity. Meanwhile, pre-treatment with TIM could attenuate the imbalance state of redox in 661W cells by decreasing the levels of intracellular ROS and MDA, as well as enhancing the SOD activity and the level of GSH, through increasing the protein expression of Nrf2. Moreover, TIM pre-treatment decreased pro-inflammatory factors IL-1β, IL-12 and TNFα, through inhibiting the nuclear factor kappa B. Pre-treatment with TIM also suppressed Egr1, Fosl1, and Lox12 gene expression., Conclusion: These results suggested that TIM may exert its protective effects against LTA-induced toxicity in 661W cells, through counteracting the oxidative stress and inhibiting inflammatory response. Our findings provided the scientific rational to develop TIM in the treatment of oxidative stress-induced photoreceptor cell damage.

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.

Drug-resistant microorganism infections cause serious disease and can lead to mortality and morbidity. In particular, Staphylococcus aureus induces pyrogenic and toxigenic infections, and drug-resistance occurs rapidly. Multidrug-resistant S. aureus, such as methicillin-resistant S. aureus and methicillin-sensitive S. aureus, can also cause immunodeficiency and immune deficiency syndrome from lipoteichoic acid. However, antimicrobial peptides, such as KW4, have strong antimicrobial activity, low cytotoxicity, and high neutralization activity against endotoxin substances from Gram-negative bacteria. The objective of this study was to use a synthetic KW4 antimicrobial peptide to evaluate the inhibition of drug-resistance development, antimicrobial activity, and neutralizing activity in S. aureus Gram-positive bacteria. The KW4 peptide showed strong antimicrobial activity against drug-resistant S. aureus strains and significantly increased the anti-neutralizing activity of lipoteichoic acid in S. aureus 1630 drug-resistant bacteria. In addition, S. aureus ATCC 29213 did not develop resistance to KW4 as with other antibiotic drugs. These results suggest that the KW4 peptide is an effective antibiotic and anti-neutralizing agent against multidrug-resistant S. aureus strains.

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.

Enteric viruses encounter diverse environments as they migrate through the gastrointestinal tract to infect their hosts. The interaction of eukaryotic viruses with members of the host microbiota can greatly impact various aspects of virus biology, including the efficiency with which viruses can infect their hosts. Mammalian orthoreovirus, a human enteric virus that infects most humans during childhood, is negatively affected by antibiotic treatment prior to infection. However, it is not known how components of the host microbiota affect reovirus infectivity. In this study, we show that reovirus virions directly interact with Gram positive and Gram negative bacteria. Reovirus interaction with bacterial cells conveys enhanced virion thermostability that translates into enhanced attachment and infection of cells following an environmental insult. Enhanced virion thermostability was also conveyed by bacterial envelope components lipopolysaccharide (LPS) and peptidoglycan (PG). Lipoteichoic acid and N-acetylglucosamine-containing polysaccharides enhanced virion stability in a serotype-dependent manner. LPS and PG also enhanced the thermostability of an intermediate reovirus particle (ISVP) that is associated with primary infection in the gut. Although LPS and PG alter reovirus thermostability, these bacterial envelope components did not affect reovirus utilization of its proteinaceous cellular receptor junctional adhesion molecule-A or cell entry kinetics. LPS and PG also did not affect the overall number of reovirus capsid proteins σ1 and σ3, suggesting their effect on virion thermostability is not mediated through altering the overall number of major capsid proteins on the virus. Incubation of reovirus with LPS and PG did not significantly affect the neutralizing efficiency of reovirus-specific antibodies. These data suggest that bacteria enhance reovirus infection of the intestinal tract by enhancing the thermal stability of the reovirus particle at a variety of temperatures through interactions between the viral particle and bacterial envelope components.

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-1β 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., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Purpose: To apply quantitative Toll-like receptors (TLR) cell assays to compare lipopolysaccharides (LPSs) and lipoteichoic acids (LTAs) from different oral bacterial strains for potential pathogenicity in vitro., Methods: The potency of LPS and LTA from different bacteria on activation of TLR reporter genes in HEK-tlr cell lines was examined. P. gingivalis LPS mix, P. gingivalis 1690 LPS, P. gingivalis 1435/50 LPS, E. coli LPS (E. coli K12), B. subtilis LTA, S. aureus LTA, E. hirae LTA and S. pyogenes LTA were examined in both TLR2 and TLR4 HEK cell line reporter assays. Solutions of LPS and LTA from selected bacteria were applied in a dose response fashion to the TLR reporter cells under standard culture conditions for mammalian cells. Reporter gene secreted-embryonic-alkaline-phosphatase (SEAP) was measured, and half maximal effective concentration (EC50) was determined for each sample. Concentration dependent TLR activation was compared to similar responses to LPS and LTA for commercial BODIPY-TR-Cadaverine and LAL biochemical (non cell based) assays., Results: All LPS from P. gingivalis activated both TLR2 and TLR4 responses. E. coli LPS is a strong activator for TLR4 but not for TLR2 responses. In contrast, both B. subtilis and S. aureus LTA provoked responses only in TLR2, but not in the TLR4 assay. Interestingly, E. hirae LTA and S. pyogenes LTA did not stimulate strong TLR2 responses. Instead, both E. hirae LTA and S. pyogenes LTA mounted a reasonable response in TLR4 reporter gene assay. Both LPS and LTA showed deactivation of fluorescence in BODIPY-TR-Cadaverine while only LPS was active in LAL. As with biochemical assays, an EC50 could be determined for LPS and LTA from various bacterial strains. The EC50 is defined as a concentration of LPS or LTA that provokes a response halfway between the baseline and maximum responses. Lower EC50 means higher potency in promoting TLR responses, and in principle indicates greater toxicity to the host., Clinical Significance: InvivoGen TLR2 and TLR4 assays distinguish specific types of microbial products, such as LPS and LTA from different bacteria. Application of EC50 determinations creates a means for quantitative and comparisons of LPS and LTA virulence in a cellular-based assay and combinations of TLR reporter cell assays along with biochemical evaluation of LPS#47;LTA in BODIPY-TR-Cadaverine and LPS in LAL assays provides a means to quantitate virulence of plaque samples with respect to both LPS and LTA. These learnings have long-term implications for patient care in that understanding the virulence of patients' plaque provides important information to assess risk of oral diseases., Competing Interests: All authors are employees of Procter $ Gamble Co.

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., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

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.

The emergence of multi-drug resistant (MDR) microbes leads to urgent demands for novel antibiotics exploration. We demonstrated a cDNA from amphioxus Branchiostoma japonicum, designated Bjamp1, encoded a protein with features typical of antimicrobial peptides (AMPs), which is not homologous to any AMPs currently discovered. It was found that Bjamp1 was expressed in distinct tissues, and its expression was remarkably up-regulated following challenge with LPS and LTA. Moreover, the synthesized putative mature AMP, mBjAMP1, underwent a coil-to-helix transition in the presence of TFE or SDS, agreeing well with the expectation that BjAMP1 was a potential AMP. Functional assays showed that mBjAMP1 inhibited the growth of all the bacteria tested, and induced membrane/cytoplasmic damage. ELISA indicated that mBjAMP1 was a pattern recognition molecule capable of identifying LPS and LTA. Importantly, mBjAMP1 disrupted the bacterial membranes by a membranolytic mechanism. Additionally, mBjAMP1 was non-cytotoxic to mammalian cells. Collectively, these data indicate that mBjAMP1 is a new AMP with a high bacterial membrane selectivity, rendering it a promising template for the design of novel peptide antibiotics against MDR microbes. It also shows for the first time that use of signal conserved sequence of AMPs is effective identifying potential AMPs across different animal classes.

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., (© 2014 The Authors. Glia Published by Wiley Periodicals, Inc.)

Pyrogens are components of microorganisms, like bacteria, viruses or fungi, which can induce a complex inflammatory response in the human body. Pyrogen contamination on medical devices prior operation is still critical and associated with severe complications for the patients. The aim of our study was to develop a reliable test, which allows detection of pyrogen contamination on the surface of medical devices. After in vitro pyrogen contamination of different medical devices and incubation in a rotation model, the human whole blood monocyte activation test (MAT), which is based on an IL-1β-specific ELISA, was employed. Our results show that when combining a modified MAT protocol and a dynamic incubation system, even smallest amounts of pyrogens can be directly detected on the surface of medical devices. Therefore, screening of medical devices prior clinical application using our novel assay, has the potential to significantly reduce complications associated with pyrogen-contaminated medical devices.

Background: Gram-positive and gram-negative bacteria are common causative agents of respiratory tract infection. Lipopolysaccharide (LPS) is a component of the gram-negative cell wall and a strong inducer of inflammation. The main proinflammatory component of the gram-positive bacterial cell wall is lipoteichoic acid (LTA). The protein kinase p38 mitogen activated protein kinase (MAPK) plays an important role in the inflammatory process induced by these two bacterial structures., Aim: We here sought to establish the impact of local p38 MAPK inhibition on lung inflammatory responses induced by LPS and LTA. We investigated the effects of direct intrapulmonary delivery of a p38 MAPK inhibitor on local LPS and LTA induced airway inflammation in mice., Results: In vitro, BIRB 796 reduced LPS induced p38 MAPK phosphorylation in alveolar macrophage and respiratory epithelial cell lines and diminished cytokine/chemokine release. In vivo, BIRB 796 circumvented p38 MAPK phosphorylation in both LPS and LTA induced inflammation. Cellular influx was not affected. Lung TNFα, IL-6, MIP-2 and LIX production was reduced in LPS induced inflammation but not in lung inflammation by LTA. BIRB 796 reduced total protein and IgM in bronchoalveolar lavage fluid after LTA instillation, while enhancing TATc and d-dimers in LPS- and LTA induced inflammation., Conclusion: These results taken together with earlier studies on systemic administration of p38 MAPK inhibitors in rodents and humans suggest that direct intrapulmonary delivery of a p38 MAPK inhibitor is less effective in inhibiting inflammation and is associated with unexpected procoagulant effects in the bronchoalveolar space., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Objectives: In gram-positive sepsis, lipoteichoic acid (LTA) can induce alterations of haemostasis, potentially leading to disseminated intravascular coagulation., Patients and Methods: Here, we demonstrate the effects of LTA on haemostasis in an in vitro model of gram-positive sepsis based on rotation thromboelastrography (ROTEM)., Results: In this model, LTA leads to time- and dose-dependent shortening of the clotting time (CT), whereas other ROTEM parameters are unaffected. Following heat shock simulation, the LTA effect was blunted with equal CTs in the presence and in the absence of LTA. In addition, the shortening of CT by LTA was inhibited by addition of the protein synthesis inhibitor., Conclusion: Our work demonstrates that the ROTEM system is capable of detecting the LTA effect on haemostasis and provides a sensitive in vitro tool for research into the links between gram-positive sepsis and coagulation.

Gastric aspiration increases the risks for developing secondary bacterial pneumonia. Cytokine elaboration through pathogen recognition receptors (PRRs) is an important mechanism in initiating innate immune host response. Effects of low pH stress, a critical component of aspiration pathogenesis, on the PRR pathways were examined, specifically toll-like receptor-2 (TLR2) and TLR4, using isolated rat alveolar macrophages (aMØs). We assessed the ability of aMØs after brief exposure to acidified saline to elaborate proinflammatory cytokines in response to lipopolysaccharide (LPS) and lipoteichoic acid (LTA) stimulation, known ligands of TLR4 and TLR2, respectively. Low pH stress reduced LPS- and LTA-mediated cytokine release (CINC-1, MIP-2, TNF-α, MCP-1, and IFN-β). LPS and LTA increased intracellular Ca²⁺ concentrations while Ca²⁺ chelation by BAPTA decreased LPS- and LTA-mediated cytokine responses. BAPTA blocked the effects of low pH stress on most of LPS-stimulated cytokines but not of LTA-stimulated responses. In vivo mouse model demonstrates suppressed E. coli and S. pneumoniae clearance following acid aspiration. In conclusion, low pH stress inhibits antibacterial cytokine response of aMØs due to impaired TLR2 (MyD88 pathway) and TLR4 signaling (MyD88 and TRIF pathways). The role of Ca²⁺ in low pH stress-induced signaling is complex but appears to be distinct between LPS- and LTA-mediated responses.

Pyrogens are heterogeneous group of fever-inducing substances derived from Gram-positive and Gram-negative bacteria, fungi, and viruses. They incite immune response by producing endogenous pyrogens such as prostaglandins and other proinflammatory cytokines like IL-1β, IL-6, and TNF-α. The present study was to analyze the influence of cryopreservation in IL-1β release, a marker for inflammatory response from human lymphocytes, in response to exogenous pyrogenic stimulants. Lymphocytes isolated from pooled blood of multiple healthy individuals were cryopreserved in DMSO and glycerol for periods of 7, 14, 30, and 60 days and were challenged with LPS and LTA in vitro. The inflammatory cytokine, IL-1β release, was measured by ELISA method. It was observed that the release of IL-1β increases instantaneously after the initiation of incubation and reaches a maximum at 3 to 5 hours and then gradually decreases and gets stabilized for both pyrogens. Moreover it was also observed that the effect of cryoprotectants, DMSO (10%) and glycerol (10%), showed almost similar results for short-term storage, but DMSO-preserved lymphocytes yielded a better viability for long-term storage. Thus, the isolated cryopreserved lymphocytes system can be a promising approach for the total replacement/alteration to animal experimentation for pyrogenicity evaluation.

Tunneled central venous catheters (TCVCs) are used for dialysis access in 82% of new hemodialysis patients and are rapidly colonized with Gram-positive organism (e.g. Staphylococcus aureus) biofilm, a source of recurrent infections and chronic inflammation. Lipoteichoic acid (LTA), a cell wall ribitol polymer from Gram-positive organisms, mediates inflammation through the Toll-like receptor 2 (TLR2). The effect of LTA on lung endothelial permeability is not known. We tested the hypothesis that LTA from Staphylococcus aureus induces alterations in the permeability of pulmonary microvessel endothelial monolayers (PMEM) that result from activation of TLR2 and are mediated by reactive oxygen/nitrogen species (RONS). The permeability of PMEM was assessed by the clearance rate of Evans blue-labeled albumin, the activation of the TLR2 pathway was assessed by Western blot, and the generation of RONS was measured by the fluorescence of oxidized dihydroethidium and a dichlorofluorescein derivative. Treatment with LTA or the TLR2 agonist Pam((3))CSK((4)) induced significant increases in albumin permeability, IκBα phosphorylation, IRAK1 degradation, RONS generation, and endothelial nitric oxide synthase (eNOS) activation (as measured by the p-eNOS(ser1177):p-eNOS(thr495) ratio). The effects on permeability and RONS were effectively prevented by co-administration of the superoxide scavenger Tiron, the peroxynitrite scavenger Urate, or the eNOS inhibitor L-NAME and these effects as well as eNOS activation were reduced or prevented by pretreatment with an IRAK1/4 inhibitor. The results indicate that the activation of TLR2 and the generation of ROS/RNS mediates LTA-induced barrier dysfunction in PMEM.

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.

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)-1β 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-1β 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-1β and CXCL-8 production in feline whole blood. Cats appear to be relatively more sensitive to gram negative compared to gram positive bacteria., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

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-1β 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.

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.

The response of the bovine mammary gland to lipoteichoic acid (LTA), which is a major pathogen-associated molecular pattern of Gram-positive bacteria, was investigated by infusing purified Staphylococcus aureus LTA in the lumen of the gland. LTA was able to induce clinical mastitis at the dose of 100 microg/quarter, and a subclinical inflammatory response at 10 microg/quarter. The induced inflammation was characterized by a prompt and massive influx of neutrophils in milk. LTA proved to induce strongly the secretion of the chemokines CXCL1, CXCL2, CXCL3 and CXCL8, which target mainly neutrophils. The complement-derived chemoattractant C5a was generated in milk only with the highest dose of LTA (100 microg). The pro-inflammatory cytokine IL-1beta was induced in milk, but there was very little if any TNF-alpha and no IFN-gamma. The re-assessment of CXCL8 concentrations in milk whey of quarters previously challenged with S. aureus, by using an ELISA designed for bovine CXCL8, showed that this chemokine was induced in milk, contradicting previous reports. Overall, S. aureus LTA elicited mammary inflammatory responses that shared several attributes with S. aureus mastitis. Purified LTA looks promising as a convenient tool to investigate the inflammatory and immune responses of the mammary gland to S. aureus.

Beta2-adrenergic receptors are expressed on different cell types in the lung, including respiratory epithelial cells, smooth muscle cells, and macrophages. The aim of the current study was to determine the role of beta-adrenergic receptors in the regulation of lung inflammation induced by instillation via the airways of lipopolysaccharide (LPS) (a constituent of the gram-negative bacterial cell wall) or lipoteichoic acid (LTA) (a component of the gram-positive bacterial cell wall). Mice inhaled the beta-adrenergic antagonist propranolol or saline 30 minutes before and 3 hours after intranasal LPS or LTA administration. LPS and LTA induced a profound inflammatory response in the lungs as reflected by an influx of neutrophils and the release of proinflammatory cytokines and chemokines into bronchoalveolar lavage fluid (BALF). Propranolol inhalation resulted in enhanced LPS-induced lung inflammation, which was reflected by a stronger secretion of TNF-alpha, IL-6, and monocyte chemoattractant protein-1 into BALF and by enhanced coagulation activation (thrombin-antithrombin complexes). In LTA-induced lung inflammation, propranolol did not influence cytokine release but potentiated activation of coagulation. Propranolol did not alter neutrophil recruitment in either model. This study suggests that beta-adrenergic receptors, which are widely expressed in the lungs, serve as negative regulators of pulmonary cytokine release and coagulation induced by LPS and less so during LTA-induced pulmonary inflammation.

Lipoteichoic acid (LTA), as a primary immunostimulus, triggers the systematic inflammatory responses. Our hypothesis is that ApoA-I can neutralize LTA toxicity, like its effect on LPS. BALB/c mice were challenged with LTA, followed by human ApoA-I administration. We found that ApoA-I could attenuate LTA-induced acute lung injury and inflammation and significantly inhibit LTA-induced IL-1beta and TNF-alpha accumulation in the serum (P<0.01 and P<0.05, respectively), as well as in bronchoalveolar lavage (BAL) fluid (P<0.01 and P<0.05, respectively). Moreover, ApoA-I could significantly reduce the L-929 cell mortality caused by LTA-activated macrophages in a dose-dependent fashion. Furthermore, ApoA-I treatment could diminish LTA-mediated NFkappaB nuclear translocation in macrophages. An in vitro binding assay indicated that ApoA-I can bind LTA. These results clearly indicated that ApoA-I can effectively protect against LTA-induced sepsis and acute lung damage. The mechanism might be related to the binding and neutralization of LTA.

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.

There are no predictive factors for peritoneal dialysis-associated peritonitis; however, its resolution correlates with a cell-mediated Th1 immune response. We tested the hypothesis that induction of receptor-interacting protein 2 (RIP2), an assumed kinase linked with Th1 responses, is a useful marker in this clinical setting. Basal RIP2 expression was measured in human immune cells and during dialysis-associated peritonitis. RIP2 increased with bacterial toxin cell activation and the temporal profile for this differed depending on immune cell involvement in the innate or adaptive phases of the response. Importantly, RIP2 expression increased in peritoneal immune cells during dialysis-associated peritonitis and this upregulation correlated with clinical outcome. An early induction in peritoneal CD14(+) cells correlated with rapid resolution, whereas minimal induction correlated with protracted infection and with catheter loss in 36% of patients. These latter patients had higher levels of MCP-1 consistent with a delayed transition from innate to adaptive immunity. Our study shows that upregulation of RIP2 is a useful marker to monitor dialysis-associated peritonitis and in predicting the clinical outcome of these infections.

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.

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.

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.

Inflammatory processes occur in the central nervous system (CNS) through mechanisms that differ from other inflammation, and with distinct cellular effects. Neuronal injury in bacterial meningitis is not a monocausal event, but is mediated by several factors. One is possible direct toxicity of bacterial compounds. Lipoteichoic acid (LTA) is a cell wall component unique to Gram-positive bacteria. In a previous report, LTA could interact with CD14 to induce NF-kappaB activation, which is involved in transcriptional regulation of adhesion molecules, enzymes and cytokines. Although there are many aspects to neuroinflammation, the pathways involving the cyclooxygenase (COX)-2 and subsequent generation of prostaglandin clearly play a role. LTA has been shown to stimulate inflammatory responses in a number of in vivo and in vitro experimental models. However, little was known about the molecular mechanisms of LTA implicated in inflammatory responses in neurons. In this study, we characterized the mechanisms underlying signaling transduction in rat cortical neuronal cells challenged by LTA. Here, we first showed that in rat cortical neuronal cells, LTA might activate protein tyrosine kinase (PTK), phosphatidylcholine-specific phospholipase C (PC-PLC), and phosphatidylinositol-specific phospholipase C (PI-PLC) to induce protein kinase Cepsilon activation, which in turn induces extracellular signal-regulated kinase (ERK) activation, finally inducing PGE(2) release and COX-2 synthesis.

Activated hepatic stellate cells (HSCs) secrete extracellular matrix components during hepatic fibrosis, but recent studies have shown that HSCs can also release a variety of proinflammatory cytokines. Moreover, bacterial endotoxemia is not only associated with systemic complications in the late stages of liver failure but is also a direct cause of liver damage, activating resident inflammatory cells. In this study, we investigated whether HSCs can respond directly to bacterial cell wall products acquiring a new phenotype. RT-PCR and immunocytochemistry assays were used to show that murine HSCs expressed specific mRNA transcripts and proteins for LPS and lipoteichoic acid (LTA) receptor systems and peptidoglycan recognition proteins. Exposing HSCs to bacterial endotoxins led to phosphorylation of mitogen-activated protein kinase ERK1 and the development of a proinflammatory phenotype. After exposure to LPS, LTA, or N-acetyl muramyl peptide, transforming growth factor-beta1, IL-6, and monocyte chemoattractant protein-1 (MCP-1) mRNA specific transcripts and proteins increased significantly in HSCs, as assayed by quantitative real-time RT-PCR and ELISA. These LPS-mediated effects in HSCs were receptor dependent, because LPS-induced ERK1 phosphorylation, IL-6, and MCP-1 mRNA and protein level upregulation were significantly less pronounced in HSCs isolated from C3H/HeJ mice lacking Toll-like receptor 4. In conclusion, our results show that murine HSCs express functional receptors for bacterial endotoxins, and HSCs exposed to bacterial products develop a strong proinflammatory phenotype. We speculate that high levels of bacterial endotoxins in the portal vein may directly induce a proinflammatory phenotype in HSCs that contributes to liver damage.

Various bacterial components (e.g., endotoxin, teichoic and lipoteichoic acids, peptidoglycans, DNA) induce or enhance inflammation by stimulating the innate immune system and/or are directly toxic in eukariotic cells (e.g., hemolysins). When antibiotics which inhibit bacterial protein synthesis kill bacteria, smaller quantities of proinflammatory or toxic compounds are released in vitro and in vivo than during killing of bacteria by beta-lactams and other cell-wall active drugs. In general, high antibiotic concentrations liberate lower quantities of bacterial proinflammatory or toxic compounds than concentrations close to the minimum inhibitory concentration. In animal models of Escherichia coli Pseudomonas aeruginosa and Staphylococcus aureus peritonitis/sepsis and of Streptococcus pneumoniae meningitis, a lower release of proinflammatory bacterial compounds was associated with a reduced mortality or neuronal injury. Pre-treatment with a bacterial protein synthesis inhibitor reduced the strong release of bacterial products usually observed during treatment with a beta-lactam antibiotic. Data available strongly encourage clinical trials comparing antibiotic regimens with different release of proinflammatory/toxic bacterial products. The benefit of the approach to reduce the liberation of bacterial products should be greatest in patients with a high bacterial load.

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.

In the present study, the signal pathways involved in NO formation and iNOS expression in RAW 264.7 macrophages stimulated by LTA were investigated. We also compared the relative inhibitory activities and mechanisms of PMC, a novel potent antioxidant of alpha-tocopherol derivatives, with those of YC-1, an sGC activator, on the induction of iNOS expression by LTA in cultured macrophages in vitro and LTA-induced hypotension in vivo. LTA induced concentration (0.1-50 microg/mL)- and time (4-24 hr)-dependent increases in nitrite (an indicator of NO biosynthesis) in macrophages. Both PMC (50 microM) and YC-1 (10 microM) inhibited NO production, iNOS protein, mRNA expression, and IkappaBalpha degradation upon stimulation by LTA (20 microg/mL) in macrophages. On the other hand, PMC (50 microM) almost completely suppressed JNK/SAPK activation, whereas YC-1 (10 microM) only partially inhibited its activation in LTA-stimulated macrophages. Moreover, PMC (10 mg/kg, i.v.) and YC-1 (5 mg/kg, i.v.) significantly inhibited the fall in MAP stimulated by LTA (10 mg/kg, i.v.) in rats. In conclusion, we demonstrate that YC-1 shows more-potent activity than PMC at abrogating the expression of iNOS in macrophages in vitro and reversing delayed hypotension in rats with endotoxic shock stimulated by LTA. The inhibitory mechanisms of PMC may be due to its antioxidative properties, with a resulting influence on JNK/SAPK and NF-kappaB activations. YC-1 may be mediated by increasing cyclic GMP, followed by, at least partly, inhibition of JNK/SAPK and NF-kappaB activations, thereby leading to inhibition of iNOS expression.

Streptococcus pneumoniae is a leading cause of gram-positive sepsis, and lipoteichoic acid (LTA) may be important in causing gram-positive bacterial septic shock. Even though pneumococcal LTA is structurally distinct from the LTA of other gram-positive bacteria, the immunological properties of pneumococcal LTA have not been well characterized. We have investigated the ability of LTAs to stimulate human monocytes by using highly pure and structurally intact preparations of pneumococcal LTA and its two structural variants. The variants were pneumococcal LTA with only one acyl chain (LTA-1) and completely deacylated LTA (LTA-0). The target cells used in the study were peripheral blood mononuclear cells (PBMCs) and two model cell lines (CHO/CD14/TLR2 and CHO/CD14/TLR4) that express human CD25 protein in response to Toll-like receptor 2 (TLR2) and TLR4 stimulation, respectively. Intact pneumococcal LTA and LTA-1 stimulated PBMC and CHO/CD14/TLR2 cells in a dose-dependent manner but did not stimulate CHO/CD14/TLR4 cells. Pneumococcal LTA was about 100-fold less potent than Staphylococcus aureus LTA in stimulating the CHO/CD14/TLR2 cells and PBMCs. LTA-0 (or pneumococcal teichoic acid) stimulated neither CHO/CD14/TLR2 nor CHO/CD14/TLR4 cells even at high concentrations. Excess teichoic acid, LTA-0, antibodies to phosphocholine, or antibodies to TLR4 did not inhibit the LTA-induced TLR2 stimulation. However, antibodies to CD14, TLR1, or TLR2 suppressed tumor necrosis factor alpha (TNF-alpha) production by PBMCs in response to LTA or LTA-1. These results suggest that pneumococcal LTA with one or both acyl chains stimulates PBMCs primarily via TLR2 with the help of CD14 and TLR1.

Lipoteichoic acid (LTA) derived from Streptococcus pneumoniae, purified employing a chloroform/methanol protocol, and from Staphylococcus aureus, prepared by the recently described butanol extraction procedure, was investigated regarding its interaction with lipopolysaccharide (LPS)-binding protein (LBP), CD14, Toll-like receptors (TLRs)-2 and -4, and MD-2. LTA from both organisms induced cytokine synthesis in human mononuclear phagocytes. Activation was LBP- and CD14-dependent, and formation of complexes of LTA with LBP and soluble CD14 as well as catalytic transfer of LTA to CD14 by LBP was verified by PhastGel(TM) native gel electrophoresis. Human embryonic kidney (HEK) 293/CD14 cells and Chinese hamster ovary (CHO) cells were responsive to LTA only after transfection with TLR-2. Additional transfection with MD-2 did not affect stimulation of these cells by LTA. Our data suggest that innate immune recognition of LTA via LBP, CD14, and TLR-2 represents an important mechanism in the pathogenesis of systemic complications in the course of infectious diseases brought about by the clinically most important Gram-positive pathogens. However, the involvement of TLR-4 and MD-2 in this process was ruled out.

Chylomicrons have been shown to protect against endotoxin-induced lethality. LPS-binding protein (LBP) is involved in the inactivation of bacterial toxin by lipoproteins. The current study examined the interaction among LBP, chylomicrons, and bacterial toxin. LBP was demonstrated to associate with chylomicrons and enhance the amount of LPS binding to chylomicrons in a dose-dependent fashion. In addition, LBP accelerated LPS binding to chylomicrons. This LBP-induced interaction of LPS with chylomicrons prevented endotoxin toxicity, as demonstrated by reduced cytokine secretion by PBMC. When postprandial circulating concentrations of chylomicrons were compared with circulating levels of low density lipoprotein, very low density lipoprotein, and high density lipoprotein, chylomicrons exceeded the other lipoproteins in LPS-inactivating capacity. Furthermore, highly purified lipoteichoic acid, an immunostimulatory component of Gram-positive bacteria, was detoxified by incubation with LBP and chylomicrons. In conclusion, our results indicate that LBP associates with chylomicrons and enables chylomicrons to rapidly bind bacterial toxin, thereby preventing cell activation. Besides a role in the detoxification of bacterial toxin present in the circulation, we believe that LBP-chylomicron complexes may be part of a local defense mechanism of the intestine against translocated bacterial toxin.

Bacterial lipopolysaccharides (LPS) activate cells of innate immunity, such as macrophages, by stimulating signaling through toll-like receptor 4 (TLR4). We and others have hypothesized that LPS derived from different bacterial species may function through TLR4-independent mechanisms. To test this hypothesis, we have generated using a nonviral transformation procedure a bone marrow-derived macrophage cell line called 10ScNCr/23 from mouse strain C57BL/10ScNCr. This mouse strain has a deletion of the TLR4 locus, causing the mouse strain to be nonresponsive to stimulation by LPS from Escherichia coli while responding normally to other bacterial substrates, such as lipoteichoic acid (LTA) from Staphylococcus aureus, which signal TLR4 independently. Stimulation with LTA induces five- and sixfold increases in 10ScNCr/23 cell line tumor necrosis factor alpha and macrophage inflammatory protein-2 (MIP-2) secretion, but no increases in either cytokine were found when cells were stimulated with E. coli LPS. Bacteroides fragilis-derived LPS, however, can effectively stimulate MIP-2 expression in the absence of functional TLR4 in the 10ScNCr/23 cell line. This gives rise to the notion that LPS from some bacterial species will utilize alternative receptors to stimulate the innate immune response.

The natural antibiotic molecules, beta-defensins 1 and 2 (HBD1/2) and secretory leukocyte protease inhibitor (SLPI), have an important role in mucosal defence and are present in the uterus. This study details their regulation in primary endometrial epithelial cells and in two endometrial cell lines (MFE/HES). Cells were treated with proinflammatory molecules and mimics of infection [lipopolysaccharide (LPS) and lipoteichoic acid (LTA)]. mRNA for HBD1, HBD2 and SLPI was detected in primary endometrial epithelial cells using real-time quantitative PCR. HBD1 mRNA was present at very low levels preventing conclusive study of its regulation. However, HBD2 mRNA expression was increased by interferon-gamma, interleukin (IL)-1beta alone and IL-1beta+tumour necrosis factor (TNF)-alpha. SLPI mRNA was not affected by proinflammatory mediators, although protein levels fell in the presence of IL-1beta+TNFalpha. LPS had little effect on antimicrobial expression. However, there was a trend towards increased expression with LTA treatment for 4-8 h. Antimicrobial expression in endometrial cell lines was similar to that in primary cells, although SLPI was increased by IL-1beta+TNFalpha treatment. These results suggest that in endometrium some natural antibiotics (e.g. SLPI) may be constitutively expressed providing a basal level of protection, while others (e.g. HBD2) are inducible allowing maximal antimicrobial activity during infection. Natural antimicrobials will have an important role in endometrium in protecting against infection.

Lipoteichoic acid (LTA) is a surface-associated adhesion amphiphile from Gram-positive bacteria and regulator of autolytic wall enzymes (muramidases). It is released from the bacterial cells mainly after bacteriolysis induced by lysozyme, cationic peptides from leucocytes, or beta-lactam antibiotics. It binds to target cells either non-specifically, to membrane phospholipids, or specifically, to CD14 and to Toll-like receptors. LTA bound to targets can interact with circulating antibodies and activate the complement cascade to induce a passive immune kill phenomenon. It also triggers the release from neutrophils and macrophages of reactive oxygen and nitrogen species, acid hydrolases, highly cationic proteinases, bactericidal cationic peptides, growth factors, and cytotoxic cytokines, which may act in synergy to amplify cell damage. Thus, LTA shares with endotoxin (lipopolysaccharide) many of its pathogenetic properties. In animal studies, LTA has induced arthritis, nephritis, uveitis, encephalomyelitis, meningeal inflammation, and periodontal lesions, and also triggered cascades resulting in septic shock and multiorgan failure. Binding of LTA to targets can be inhibited by antibodies, phospholipids, and specific antibodies to CD14 and Toll, and in vitro its release can be inhibited by non-bacteriolytic antibiotics and by polysulphates such as heparin, which probably interfere with the activation of autolysis. From all this evidence, LTA can be considered a virulence factor that has an important role in infections and in postinfectious sequelae caused by Gram-positive bacteria. The future development of effective antibacteriolitic drugs and multidrug strategies to attenuate LTA-induced secretion of proinflammatory agonists is of great importance to combat septic shock and multiorgan failure caused by Gram-positive bacteria.

We have recently shown that highly purified lipoteichoic acid (LTA) represents a major immunostimulatory principle of Staphylococcus aureus. In order to test whether this translates to other bacterial species, we extracted and purified LTA from 12 laboratory-grown species. All LTA induced the release of TNF-alpha, IL-1beta, IL-6 and IL-10 in human whole blood. Soluble CD14 (sCD14) inhibited monokine induction by LTA but failed to confer LTA responsiveness for IL-6 and IL-8 release of human umbilical vein endothelial cells (HUVEC). In a competitive LPS-binding protein (LBP) binding assay, the IC(50) of the tested LTA preparations was up to 3,230-fold higher than for LPS. LBP enhanced TNF-alpha release of human peripheral blood mononuclear cells (PBMC) upon LPS but not LTA stimulation. These data demonstrate a differential role for the serum proteins LBP and sCD14 in the recognition of LPS and LTA. Different efficacies of various anti-CD14 antibodies against LPS vs. LTA-induced cytokine release suggest that the recognition sites of CD14 for LPS and LTA are distinct with a partial overlap. While the maximal achievable monokine release in response to LTA was comparable to LPS, all LTA induced significantly less IL-12 and IFN-gamma. IL-12 substitution increased LTA-inducible IFN-gamma release up to 180-fold, suggesting a critical role of poor LTA-inducible IL-12 for IFN-gamma formation. Pretreatment with IFN-gamma rendered galactosamine-sensitized mice sensitive to challenge with LTA. In conclusion, LTA compared to LPS, are weak inducers of IL-12 and subsequent IFN-gamma formation which might explain their lower toxicity in vivo.