Lipopolysaccharide Activation Research Articles

LPS inactivation by a host lipase allows lung epithelial cell sensitization for allergic asthma.

Allergic asthma is a chronic inflammatory disease primarily mediated by Th2 immune mechanisms. Numerous studies have suggested that early life exposure to lipopolysaccharide (LPS) is negatively associated with allergic asthma. One proposed mechanism invokes desensitization of lung epithelial cells by LPS. We report here that acyloxyacyl hydrolase (AOAH), a host lipase that degrades and inactivates LPS, renders mice more susceptible to house dust mite (HDM)-induced allergic asthma. Lung epithelial cells from Aoah-/- mice are refractory to HDM stimulation, decreasing dendritic cell activation and Th2 responses. Antibiotic treatment that diminished commensal LPS-producing bacteria normalized Aoah-/- responses to HDM, while giving LPS intrarectally ameliorated asthma. Aoah-/- mouse feces, plasma, and lungs contained more bioactive LPS than did those of Aoah+/+ mice. By inactivating commensal LPS, AOAH thus prevents desensitization of lung epithelial cells. An enzyme that prevents severe lung inflammation/injury in Gram-negative bacterial pneumonia has the seemingly paradoxical effect of predisposing to a Th2-mediated airway disease.

Effect of CD14/TLR4 antagonist on GnRH/LH secretion in ewe during central inflammation induced by intracerebroventricular administration of LPS

BackgroundImmune stress induced by lipopolysaccharide (LPS) influences the gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) secretion. Presence of LPS interacting Toll-like receptor (TLR) 4 in the hypothalamus may enable the direct action of LPS on the GnRH/LH secretion. So, the aim of the study was to investigate the influence of intracerebroventricular (icv) injection of TLR4 antagonist on GnRH/LH secretion in anestrous ewes during LPS-induced central inflammation. Animals were divided into three groups icv-treated with: Ringer-Locke solution, LPS and TLR4 antagonist followed by LPS.ResultsIt was demonstrated that TLR4 antagonist reduced LPS-dependent suppression of GnRH gene expression in the preoptic area and in the medial basal hypothalamus, and suppression of receptor for GnRH gene expression in the anterior pituitary gland. It was also shown that TLR4 antagonist reduced suppression of LH release caused by icv injection of LPS. Central administration of LPS stimulated TLR4 gene expression in the medial basal hypothalamus.ConclusionsIt was indicated that blockade of TLR4 prevents the inhibitory effect of centrally acting LPS on the GnRH/LH secretion. This suggests that some negative effects of bacterial infection on the hypothalamic-pituitary-gonadal axis activity at the hypothalamic level may be caused by central action of LPS acting through TLR4.

A synthetic diosgenin primary amine derivative attenuates LPS-stimulated inflammation via inhibition of NF-κB and JNK MAPK signaling in microglial BV2 cells

Diosgenin, a precursor of steroid hormones in plants, is known to exhibit diverse pharmacological activities including anti-inflammatory properties. In this study, (3β, 25R)‑spirost‑5‑en‑3‑oxyl (2‑((2((2‑aminoethyl)amino)ethyl)amino)ethyl) carbamate (DGP), a new synthetic diosgenin derivative incorporating primary amine was used to investigate its anti-inflammatory effects and underlying mechanisms of action in lipopolysaccharide (LPS)-stimulated microglial BV2 cells. Pretreatment with DGP resulted in significant inhibition of nitric oxide (NO) synthesis, and down-regulation of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in LPS-stimulated microglial BV2 cells. In addition, DGP decreased the production of reactive oxygen species (ROS) and pro-inflammatory cytokines such as interleukin (IL)-6, IL-1β, and tumor necrosis factor alpha (TNF-α). The inhibitory effects of DGP on these inflammatory mediators in LPS-stimulated microglial BV2 cells were regulated by NF-κB signaling through blocking p65 nuclear translocation and NF-κB p65/DNA binding activity. DGP also blocked the phosphorylation of c-Jun amino-terminal kinase (JNK), but not p38 kinase or extracellular signal-regulated kinases (ERK). The NF-κB inhibitor JSH-23 and JNK-specific inhibitor SP600125 significantly decreased NO production and IL-6 release in LPS-stimulated BV2 cells, respectively. The overall results demonstrate that DGP has anti-inflammatory effects on LPS-stimulated BV2 cells via inhibition of NF-κB and JNK activation, suggesting that DGP is a potential prophylactic agent in various neurodegenerative disorders.

Serum amyloid A primes microglia for ATP-dependent interleukin-1\u03b2 release

BackgroundAcute-phase response is a systemic reaction to environmental/inflammatory insults and involves production of acute-phase proteins, including serum amyloid A (SAA). Interleukin-1β (IL-1β), a master regulator of neuroinflammation produced by activated inflammatory cells of the myeloid lineage, in particular microglia, plays a key role in the pathogenesis of acute and chronic diseases of the peripheral nervous system and CNS. IL-1β release is promoted by ATP acting at the purinergic P2X7 receptor (P2X7R) in cells primed with toll-like receptor (TLR) ligands.MethodsPurified (> 99%) microglia cultured from neonatal rat cortex and cerebellum were first primed with the putative TLR4/TLR2 agonist SAA (recombinant human Apo-SAA) or the established TLR4 agonist lipopolysaccharide (LPS) followed by addition of ATP. Expression of genes for the NLRP3 inflammasome, IL-1β, tumor necrosis factor-α (TNF-α), and SAA1 was measured by quantitative real-time polymerase chain reaction (q-PCR). Intracellular and extracellular amounts of IL-1β were determined by ELISA.ResultsApo-SAA stimulated, in a time-dependent manner, the expression of NLRP3, IL-1β, and TNF-α in cortical microglia, and produced a concentration-dependent increase in the intracellular content of IL-1β in these cells. A 2-h ‘priming’ of the microglia with Apo-SAA followed by addition of ATP for 1 h, resulting in a robust release of IL-1β into the culture medium, with a concomitant reduction in its intracellular content. The selective P2X7R antagonist A740003 blocked ATP-dependent release of IL-1β. Microglia prepared from rat cerebellum displayed similar behaviors. As with LPS, Apo-SAA upregulated SAA1 and TLR2 mRNA, and downregulated that of TLR4. LPS was less efficacious than Apo-SAA, perhaps reflecting an action of the latter at TLR4 and TLR2. The TLR4 antagonist CLI-095 fully blocked the action of LPS, but only partially that of Apo-SAA. Although the TLR2 antagonist CU-CPT22 was inactive against Apo-SAA, it also failed to block the TLR2 agonist Pam3CSK4.ConclusionsMicroglia are central to the inflammatory process and a major source of IL-1β when activated. P2X7R-triggered IL-1β maturation and export is thus likely to represent an important contributor to this cytokine pool. Given that SAA is detected in Alzheimer disease and multiple sclerosis brain, together with IL-1β-immunopositive microglia, these findings propose a link between P2X7R, SAA, and IL-1β in CNS pathophysiology.

Lactic acid suppresses LPS-induced cytokine production in mast cells by limiting glycolysis and ATP availability

Abstract Despite a mortality rate of 30–50%, there are no targeted molecular treatments for sepsis. Our understanding of the secondary immunosuppressive phase is recent, and it is unclear whether many mediators are helpful or detrimental to patient survival. Elevated blood lactate and slow lactate clearance are associated with mortality, however, any regulatory roles are currently unknown. Because mast cells can be activated systemically and contribute to sepsis, this project aims to elucidate lactic acid effects on mast cell activation and signaling. Lactic acid significantly suppressed LPS-induced cytokine and chemokine production as well as NFκB transcription in bone marrow derived and peritoneal mast cells. These effects were dependent upon pH and MCT-1 transporter activity. Further, lactic acid significantly suppressed cytokine and chemokine induction following LPS-induced septic shock in vivo. We also examined the role of lactic acid as a feedback regulator of glucose metabolism. Lactic acid treatment reduced glucose uptake, lactate export, and hexokinase 2 expression following LPS activation. The glycolytic inhibitors 2-deoxyglucose, sodium oxamate, or dichloracetate mimicked lactic acid effects, suppressing LPS-induced cytokine production and NFκB transcription. Furthermore, adding ATP to the culture media at the time of LPS activation reversed lactic acid effects. These results indicate that suppressing glycolysis and ATP production are necessary and sufficient for lactic acid effects. Future studies should target enhancing glycolysis and ATP production to improve immune function and counteract lactic acid effects in sepsis.

Novel Pycard gene polymorphism impairs Nlpr3 inflammasome-induced IL-1β production in mice selected for low inflammatory response.

Abstract A SNP based linkage study in mouse lines phenotypically selected for maximal (AIRmax) or minimal (AIRmin) acute inflammatory response (AIR), mapped a major locus, Inflammatory response modulator 1 (Irm1) at 128Mb (3.5Mb interval) in chr 7 controlling AIR, measured by leukocyte and IL-6 levels in exudates and IL-1β production by circulating leukocytes after Nlpr3 inflammasome activation. Sequencing of this region in mice with extreme high or low IL-1β levels revealed 14 SNPs between the two groups, narrowing the locus interval to 420Kb. Candidate genes at Irm1 include Pycard with an undescribed exon 3 (C/T) mutation leading to E19K substitution at the pyrin domain, and Itgam, Rgs10 and BC017158 with intronic SNPs. In Pycard, the C allele was fixed in high responder AIRmax mice whereas the C and T alleles frequencies were 39% and 61%, respectively in AIRmin. We then investigated the effect of this novel Pycard SNP in inflammation phenotypes. Methods AIRmin mice bearing the 3 genotypes at Pycard: CC, CT, TT were produced by genotype-assisted mating. Inflammatory response was measured in AIRmax and in the 3 AIRmin sublines by the number of infiltrating cells and IL-6 concentration in the 24h exudate induced by sc Biogel P-100 bead injection and ex vivo IL-1β production by circulating leukocytes after E coli LPS (1 ug) and ATP (5mM) activation. Results IL-1β levels were similar in AIRmaxCC (4.5-±0.4 ng/ml) and AIRminCC (3.4±2.4 ng/ml) whereas AIRminCT produced 0.3±0.5 and AIRminTT <0.05 ng/ml IL-1β. Leukocyte influx and IL-6 levels in inflammatory exudates were not affected. Conclusion The E19K substitution in Pycard causes a negative effect in inflammasome activation for IL-1β production, without interfering in other inflammation phenotypes.

Diverse pro-inflammatory endotoxin recognition systems of mammalian innate immunity.

In humans and other mammals, recognition of endotoxins—abundant surface lipopolysaccharides (LPS) of Gram-negative bacteria—provides a potent stimulus for induction of inflammation and mobilization of host defenses. The structurally unique lipid A region of LPS is the principal determinant of this pro-inflammatory activity. This region of LPS is normally buried within the bacterial outer membrane and aggregates of purified LPS, making even more remarkable its picomolar potency and the ability of discrete variations in lipid A structure to markedly alter the pro-inflammatory activity of LPS. Two recognition systems—MD-2/TLR4 and “LPS-sensing” cytosolic caspases—together confer LPS responsiveness at the host cell surface, within endosomes, and at sites physically accessible to the cytosol. Understanding how the lipid A of LPS is delivered and recognized at these diverse sites is crucial to understanding how the magnitude and character of the inflammatory responses are regulated.

Quantitative assessment of surface functionality effects on microglial uptake and retention of PAMAM dendrimers

Dendrimers are a promising class of polymeric nanoparticles for delivery of therapeutics and diagnostics. Polyamidoamine (PAMAM) dendrimers have shown significant efficacy in many animal models, with performance dependent on surface functionalities. Understanding the effects of end groups on biological interactions is critical for rational design of dendrimer-mediated therapies. In this study, we quantify the cellular trafficking kinetics (endocytosis and exocytosis) of generation 4 neutral (D4-OH), cationic (D4-NH2), anionic (D3.5-COOH), and generation 6 neutral (D6-OH) PAMAM dendrimers to investigate the nanoscale effects of surface functionality and size on cellular interactions. Resting and LPS-activated microglia were studied due to their central roles in dendrimer therapies for central nervous system disorders. D4-OH exhibits greater cellular uptake and lower retention than the larger D6-OH. D4-OH and D3.5-COOH exhibit similar trafficking kinetics, while D4-NH2 exhibits significant membrane interactions, resulting in faster cell association but lower internalization. Cationic charge may also enhance vesicular escape for greater cellular retention and preferential partitioning to nuclei. LPS activation further improves uptake of dendrimers, with smaller and cationic dendrimers experiencing the greatest increases in uptake compared to resting microglia. These studies have implications for the dependence of trafficking pathway on dendrimer properties and inform the design of dendrimer constructs tailored to specific therapeutic needs. Cationic dendrimers are ideal for delivering genetic materials to nuclei, but toxicity may be a limiting factor. Smaller, neutral dendrimers are best suited for delivering high levels of therapeutics in acute neuroinflammation, while larger or cationic dendrimers provide robust retention for sustained release of therapeutics in longer-term diseases.

Magnesium deficiency heightens lipopolysaccharide-induced inflammation and enhances monocyte adhesion in human umbilical vein endothelial cells.

Given a possible anti-inflammatory role of magnesium in endothelial cells, the aim of this study was to investigate the effects of magnesium on human umbilical vein endothelial cell (HUVEC) viability, gene expression, and the pro-inflammatory response caused by a bacterial endotoxin (LPS). HUVECs were cultured at three different concentrations of magnesium sulphate (0.1mM; control-1mM; 5mM) for 72 hours. Exposing the cells to LPS reduced cell viability in culture with low magnesium, but high magnesium protected the HUVECs from LPS-induced cell death. LPS-treated HUVECs cultured in low magnesium showed up-regulation of mRNA expression for pro-inflammatory factors and the expression of cytokine proteins, including IL-2, IL-3, IL-8, IL-15 and MCP-1. This was associated with greater adhesion of monocytes to the cells. In contrast, high magnesium decreased the expression of inflammatory factors and cytokines. The study found that LPS activation of the expression of many pro-inflammatory factors is exacerbated in the presence of low magnesium concentration whilst a high magnesium concentration partly inhibited the inflammatory response to LPS.

Expression and regulation of alarmin cytokine IL-1α in human retinal pigment epithelial cells

Human retinal pigment epithelial (hRPE) cells play important immune-regulatory roles in a variety of retinal pathologic processes, including the production of inflammatory cytokines that are essential mediators of the innate immune response within the ocular microenvironment. The pro-inflammatory “alarmin” cytokine IL-1α has been implicated in both infectious and non-infectious retinal diseases, but its regulation in the retina is poorly understood. The purpose of this study was to elucidate the expression and regulation of IL-1α within hRPE cells. To do this, IL-1α mRNA and protein in hRPE cells was assessed by RT-PCR, qPCR, ELISA, Western blot, and immunofluorescence following treatment with a variety of stimuli and inhibitors. ER stress, LPS, IL-1β, and TLR2 activation all significantly increased intracellular IL-1α protein. Increasing intracellular calcium synergized both LPS- and Pam3CSK4-induced IL-1α protein production. Accordingly, blocking calcium signaling and calpain activity strongly suppressed IL-1α protein expression. Significant but more moderate inhibition occurred following blockage of TLR4, caspase-4, or caspase-1. Neutralizing antibodies to IL-1β and TLR2 partially eliminated LPS- and TLR2 ligand Pam3CSK4-stimulated IL-1α protein production. IFN-β induced caspase-4 expression and activation, and also potentiated LPS-induced IL-1α expression, but IFN-β alone had no effect on IL-1α protein production. Interestingly, all inhibitors targeting the PI3K/Akt pathway, with the exception of Ly294002, strongly increased IL-1α protein expression. This study improves understanding of the complex mechanisms regulating IL-1α protein expression in hRPE cells by demonstrating that TLR4 and TLR2 stimulation and exposure to IL-1β, ER stress and intracellular calcium all induce hRPE cells to produce intracellular IL-1α, which is negatively regulated by the PI3K/Akt pathway. Additionally, the non-canonical inflammasome pathway was shown to be involved in LPS-induced hRPE IL-1α expression through caspase-4 signaling.

Oxysterol levels and metabolism in the course of neuroinflammation: insights from in vitro and in vivo models

BackgroundOxysterols are cholesterol derivatives that have been suggested to play a role in inflammatory diseases such as obesity, atherosclerosis, or neuroinflammatory diseases. However, the effect of neuroinflammation on oxysterol levels has only been partially studied so far.MethodsWe used an HPLC-MS method to quantify over ten oxysterols both in in vitro and in vivo models of neuroinflammation. In the same models, we used RT-qPCR to analyze the expression of the enzymes responsible for oxysterol metabolism. Using the BV2 microglial cell line, we explored the effect of lipopolysaccharide (LPS)-induced (M1-type) and IL-4-induced (M2-type) cell activation on oxysterol levels. We also used LPS-activated co-cultures of mouse primary microglia and astrocytes. In vivo, we induced a neuroinflammation by administering LPS to mice. Finally, we used a mouse model of multiple sclerosis, namely the experimental autoimmune encephalomyelitis (EAE) model, that is characterized by demyelination and neuroinflammation.ResultsIn vitro, we found that LPS activation induces profound alterations in oxysterol levels. Interestingly, we could discriminate between control and LPS-activated cells based on the changes in oxysterol levels both in BV2 cells and in the primary co-culture of glial cells. In vivo, the changes in oxysterol levels were less marked than in vitro. However, we found in both models increased levels of the GPR183 agonist 7α,25-dihydroxycholesterol. Furthermore, we studied in vitro the effect of 14 oxysterols on the mRNA expression of inflammatory markers in LPS-activated co-culture of microglia and astrocytes. We found that several oxysterols decreased the LPS-induced expression of pro-inflammatory markers.ConclusionsThese data demonstrate that inflammation profoundly affects oxysterol levels and that oxysterols can modulate glial cell activation. This further supports the interest of a large screening of oxysterol levels when studying the interplay between neuroinflammation and bioactive lipids.

Investigating the CYP2E1 Potential Role in the Mechanisms Behind INH/LPS-Induced Hepatotoxicity.

Tuberculosis (TB) is one of the oldest infectious diseases that affected humankind and remains one of the world’s deadliest communicable diseases that could be considered as global emergency, but the discovery and development of isoniazid (INH) in the 1950s paved the way to an effective single and/or combined first-line anti-TB therapy. However, administration of INH induces severe hepatic toxicity in some patients. Previously, we establish a rat model of INH hepatotoxicity utilizing the inflammatory stress theory, in which bacterial lipopolysaccharide (LPS) potentially enhanced INH toxicity. These enhancing activities ranged between augmenting the inflammatory stress, oxidative stress, alteration of bile acid homeostasis, and CYP2E1 over-expression. Although pre-treatment with dexamethasone (DEX) helped overcome both inflammatory and oxidative stress which ended-up in alleviation of LPS augmenting effects, but still minor toxicities were being detected, alongside with CYP2E1 over expression. This finding positively indicated the corner-stone role played by CYP2E1 in the pathogenesis of INH/LPS-induced liver damage. Therefore, we examined whether INH/LPS co-treatment with CYP2E1 inhibitor diallyl sulfide (DAS) and DEX can protect against the INH/LPS-induced hepatotoxicity. Our results showed that pre-administration of both DAS and DEX caused significant reduction in serum TBA, TBil, and gamma-glutamyl transferase levels. Furthermore, the histopathological analysis showed that DAS and DEX could effectively reverse the liver lesions seen following INH/LPS treatment and protect against hepatic steatosis as indicated by absence of lipid accumulation. Pre-treatment with DAS alone could not completely block the CYP2E1 protein expression following INH/LPS treatment, as appeared in the immunoblotting and immunohistochemistry results. This is probably due to the fact that the combined enhancement activities of both INH and LPS on CYP2E1 protein expression levels might resist the blocking probabilities of DAS. In the meantime, addition of DEX to the DAS/INH/LPS combination caused a significant reduction in CYP2E1 protein expression as revealed by the immunoblotting and fading coloration in immunohistochemistry results. Thus, addition of DEX and DAS together caused strong protection against INH/LPS-induced hepatic damage. These findings reveal the potential therapeutic value of combining DAS and DEX with INH in TB management for reducing the potential risk and incidences of hepatotoxicity.

Hypoacylated LPS from Foodborne Pathogen Campylobacter jejuni Induces Moderate TLR4-Mediated Inflammatory Response in Murine Macrophages.

Toll-like receptor 4 (TLR4) initiates immune response against Gram-negative bacteria upon specific recognition of lipid A moiety of lipopolysaccharide (LPS), the major component of their cell wall. Some natural differences between LPS variants in their ability to interact with TLR4 may lead to either insufficient activation that may not prevent bacterial growth, or excessive activation which may lead to septic shock. In this study we evaluated the biological activity of LPS isolated from pathogenic strain of Campylobacter jejuni, the most widespread bacterial cause of foodborne diarrhea in humans. With the help of hydrophobic chromatography and MALDI-TOF mass spectrometry we showed that LPS from a C. jejuni strain O2A consists of both hexaacyl and tetraacyl forms. Since such hypoacylation can result in a reduced immune response in humans, we assessed the activity of LPS from C. jejuni in mouse macrophages by measuring its capacity to activate TLR4-mediated proinflammatory cytokine and chemokine production, as well as NFκB-dependent reporter gene transcription. Our data support the hypothesis that LPS acylation correlates with its bioactivity.

Analysis of NO-suppressing activity of Strawberry Wine supplemented with ball-milled achenes.

Inflammation is generally thought to be involved in the development of several chronical diseases, therefore, phytochemicals to modulate immune responses has attracted great interests. The objective of the present study was to evaluate the potential anti-inflammatory effects of wine supplemented using ball-milled achene on modulating NO production and inducible nitric oxide synthase (iNOS) expression. Ball-milled achenes were added in strawberry must prior to fermentation, and the wine samples were then concentrated and extracted with water and/or ethanol prior to analysis. Bioactivities of wine extracts were evaluated using the cell viability assay, cell cycle measurements, NO production and iNOS expression in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Treatments of achenes supplemented strawberry wine extract up to 100μg/mL inhibited the proliferation of LPS-stimulated RAW264.7 cell via affecting the progression of cell cycle. Moreover, no detectable cytotoxicity in RAW264.7 cells was observed. The supplemented wine extract suppressed the action of LPS and led to a decreased NO production in stimulated cells. The inhibitory effect of the wine extract on NO production was determined to be a 25-40% decrease in the level of 25-100μg/mL, in contrast to a 10% decrease for conventional wine samples. Additionally, an alcoholic wine extract (100μg/mL) led to a 40.31% decrease in iNOS expression in LPS-stimulated cells, which was more effective than the same dose of tocopherol. The results show that strawberry wine supplemented with ball-milled achenes causes a substantial inhibition of NO production, and this biofunction is exerted via the down-regulation of iNOS expression.

Interleukin-8 favors pro-inflammatory activity of human monocytes/macrophages

Interleukin-8 (IL-8, CXCL8) belongs to major chemokines to stimulate migration of neutrophils and monocytes/macrophages (Mc/Mphs) into the inflammation sites. We studied the direct effects of IL-8 on the functionality of human Mc/Mphs in vitro. CD14-positive cells were isolated from human peripheral blood mononuclear cells (PBMCs) by positive magnetic separation and were further cultured with or without lipopolysaccharide (LPS, 1.0 μg/ml) for 24 h. We showed that upon LPS activation of Mc/Mphs, IL-8 reduced markedly both the percentages and median fluorescence intensity (MFI) of CD16 (FcγRIII)-positive cells among CD14high cells, as well as in cells that reduced the expression of СD14 during their culturing. IL-8 was also found to be capable of reducing the expression of СD124 (IL-4 receptor subunit alpha, IL-4RA), with concomitant enhancement of the expression of both CD119 (interferon-gamma receptor 1) and CD197 (CCR7) in Mph cells. In addition, IL-8 up-regulated production of IL-6 and IL-1β [but not tumor necrosis factor-α (TNF-α) and IL-10] by activated Mc/Mphs. Our results suggest the ability for IL-8 to directly favor pro-inflammatory M1-type Mph activity.

The impact of probiotics and n-3 long-chain polyunsaturated fatty acids on intestinal permeability in pregnancy: a randomised clinical trial.

A disruption in intestinal barrier integrity may predispose individuals to metabolic aberrations, particularly during the vulnerable period of pregnancy. We investigated whether intestinal permeability, as measured by serum zonulin concentration, changes over the duration of pregnancy and whether this change is reflected in lipopolysaccharide (LPS) activity. Second, we tested in a randomised double-blind placebo controlled clinical trial the impact of consuming dietary probiotics and/or long chain polyunsaturated fatty acid (LC-PUFA) supplements in lowering serum zonulin concentration and LPS activity. The probiotic supplement was a combination of two bacteria, Bifidobacterium animalis ssp. lactis 420 and Lactobacillus rhamnosus HN001. This study included 200 overweight pregnant women participating in an on-going study; participants were randomised to consume either (1) probiotics, (2) LC-PUFA, (3) probiotics and LC-PUFA, or (4) placebo for each supplement. Blood samples were obtained at early, the baseline, and late pregnancy (mean 14 and 35 weeks of gestation, respectively). Serum zonulin concentration increased from early (mean (standard deviation): 62.7 (12.9) ng/ml) to late pregnancy by 5.3 (95%CI 3.7-6.9) ng/ml, and LPS activity increased from (0.16 (0.04) EU/ml) by 0.04 (95%CI 0.03-0.05) EU/ml. No differences among the intervention groups were detected in the change from early to late pregnancy in serum zonulin concentration (P=0.8) or LPS activity (P=0.2). The change in serum zonulin concentration during the pregnancy was associated with the weeks of follow up (r=0.25, P<0.001). Serum LPS activity was correlated with higher maternal weight gain (r=0.19, P=0.008). As a conclusion, intestinal permeability increased with the progression of pregnancy in overweight and obese women and was reflected in LPS activity. No efficacy of supplementation with probiotics and/or LC-PUFA was demonstrated in pregnancy-induced changes in serum zonulin concentration or LPS activity.

Inhibitory effect of Lactococcus lactis on the bioactivity of periodontopathogens.

Lactococcus lactis is a probiotic bacterium that produces various bacteriocins. Periodontopathogens induce inflammation and halitosis through the actions of lipopolysaccharide (LPS) and trypsin-like enzymes. The purpose of this study was to investigate the inhibitory effects of L. lactis on the bioactivity of periodontopathogens. To investigate the antimicrobial peptide of L. lactis, the spent culture medium (SCM) of L. lactis was treated with or without proteinase K after collection by centrifugation, and the antibacterial activity of SCM against periodontopathogens was assessed. To evaluate the neutralizing effect of L. lactis on halitosis, SCM of periodontopathogens was mixed with an L. lactis suspension, and the levels of volatile sulfur compounds (VSCs) were measured by gas chromatography. LPS from the periodontopathogens was extracted by an LPS extraction kit with little modification, and THP-1 cells as a monocytic cell line were treated with the extracted LPS in the presence or absence of UV-killed L. lactis. The production of inflammatory cytokines was analyzed by ELISA. The SCM of L. lactis exhibited antimicrobial activity against the periodontopathogens, whereas the proteinase K-treated SCM showed little antimicrobial activity. In addition, the L. lactis suspension had a neutralizing effect on the VSCs produced by periodontopathogens, and UV-killed L. lactis inhibited the production of IL-6 and TNF-α induced by the LPS. These results suggest that L. lactis may be a useful probiotic to prevent and treat periodontitis and halitosis.

Reticulon-1 and Reduced Migration toward Chemoattractants by Macrophages Differentiated from the Bone Marrow of Ultraviolet-Irradiated and Ultraviolet-Chimeric Mice.

The ability of macrophages to respond to chemoattractants and inflammatory signals is important for their migration to sites of inflammation and immune activity and for host responses to infection. Macrophages differentiated from the bone marrow (BM) of UV-irradiated mice, even after activation with LPS, migrated inefficiently toward CSF-1 and CCL2. When BM cells were harvested from UV-irradiated mice and transplanted into naive mice, the recipient mice (UV-chimeric) had reduced accumulation of elicited monocytes/macrophages in the peritoneal cavity in response to inflammatory thioglycollate or alum. Macrophages differentiating from the BM of UV-chimeric mice also had an inherent reduced ability to migrate toward chemoattractants in vitro, even after LPS activation. Microarray analysis identified reduced reticulon-1 mRNA expressed in macrophages differentiated from the BM of UV-chimeric mice. By using an anti-reticulon-1 Ab, a role for reticulon-1 in macrophage migration toward both CSF-1 and CCL2 was confirmed. Reticulon-1 subcellular localization to the periphery after exposure to CSF-1 for 2.5 min was shown by immunofluorescence microscopy. The proposal that reduced reticulon-1 is responsible for the poor inherent ability of macrophages to respond to chemokine gradients was supported by Western blotting. In summary, skin exposure to erythemal UV radiation can modulate macrophage progenitors in the BM such that their differentiated progeny respond inefficiently to signals to accumulate at sites of inflammation and immunity.

Modulating inflammation in a cutaneous chronic wound model by IL-10 released from collagen-silica nanocomposites via gene delivery.

Cutaneous chronic wounds remain a major clinical challenge which requires the development of novel wound dressings. Previously, we showed that collagen-silica nanocomposites consisting of polyethyleneimine (PEI)-DNA complexes associated with silica nanoparticles (SiNP), collagen hydrogel and 3T3 fibroblasts, can work as a local "cell factory". Indeed, the "in-gel" transfection leads to a sustained production and release of biomolecules. Herein, we further explored the possibility for nanocomposites to deliver interleukin-10 (IL-10), a potent anti-inflammatory cytokine, which favors tissue repair. Its anti-inflammatory effect was evaluated in an in vitro inflammation model carried out by LPS (lipopolysaccharide) activation of macrophages embedded in collagen gel. The IL-10 synthesis from nanocomposites was detected over one week in the range of 200-400 pg mL-1 and reached a maximum at day 5 without any observed cytotoxic effects. PEI10-SiNP outperformed free PEI10 and PEI25-SiNP, implying that the introduction of SiNP improved the transfection efficiency of low Mw of PEI. In addition, the structure and mechanical properties of collagen-silica nanocomposites were stable over one week. Subsequently, the ability of nanocomposites to modulate inflammation was tested in a 3D model of inflammation. The decrease of TNF-α and IL-1β gene expression by 20-80% indicated successful inhibition of inflammation by IL-10 released from nanocomposites. Taken together, the nanocomposites are capable of producing effective doses of IL-10 which inhibit the synthesis of pro-inflammatory cytokines and favor the expression of wound healing cytokines. Therefore, the as-constructed 3D gene delivery system represents a promising strategy for the controlled release of therapeutic biomolecules favoring cutaneous wound healing.

Cangju Qinggan Jiangzhi Decoction Reduces the Development of NonAlcoholic Steatohepatitis and Activation of Kupffer Cells

Background/Aims: Nonalcoholic steatohepatitis (NASH) is defined as lipid accumulation with hepatic injury, inflammation and early to moderate fibrosis. Kupffer cells play a crucial role in promoting hepatic inflammation, which further facilitates the development of NASH. Here we investigated the effects of Cangju Qinggan Jiangzhi decoction (CQJD) on high fat diet (HFD) and methionine-choline deficient (MCD) induced mouse NASH pathogenesis. Methods: Mouse NASH models were developed by HFD and MCD diet. The treated mice were divided into three groups: the control group (n = 10), the low-dose CQJD treatment group (n = 10) and the high-dose CQJD treatment group (n = 10). The hepatic injury, inflammation, and apoptotic molecules were evaluated by H&E staining, immunohistochemistry and real-time PCR. Kupffer cells were isolated from control mice and CQJD-treated mice after stimulation by lipopolysaccharide (LPS) and/or palmitic acid. The level of the inflammatory cytokines TNFα, IL1β, and CCL2 was measured by ELISA. Results: The HFD-fed mice displayed significant metabolic, inflammatory, and oxidative stress-related alterations due to hepatic lipid accumulation. CQJD treatment largely normalized the hepatic injury, lowered the ALT/AST level, and reduced the severity of liver inflammation, as revealed by the decreased inflammatory cytokines levels. In vitro, CQJD blocked the activation of LPS- or palmitic acid-primed Kupffer cells in a dose-dependent manner. In the MCD diet-induced NASH mice, similar therapeutic effects of CQJD were also observed. Conclusion: CQJD ameliorates mouse nonalcoholic steatohepatitis. The reduction in liver injury and inflammation induced by CQJD is associated with reduced activation of Kupffer cells. Our results suggest that CQJD is a promising therapeutic strategy in clinical steatohepatitis.