Lipopolysaccharide Activation Research Articles

Abstract 463: Linoleate Epoxides, Epomes, Are Potent Anti-inflammatory Fatty Acid Epoxides and Nearly Equivalent to Arachidonate-derived Epetres

Background: Epoxides of arachidonic acid (EpETrEs, also commonly EETs) are potent anti-inflammatory lipid mediators and are associated with hypotensive and protective vascular actions. Marine omega-3 versions (epoxides of eicosapentaenoic and docosahexaenoic acids) have similar action. Surprisingly, none of these epoxides are as abundant in tissues or in plasma as epoxides of linoleic acid (EpOMEs) are. EpOMEs are up to one-hundred times more abundant than EpETrEs. Hence, EpOMEs could exert physiologic effects even with potency two-orders of magnitude higher, provided they have comparable efficacy. Few studies consider EpOMEs when assessing outcomes. Objective: To compare the potency and efficacy of EpOMEs to EpETrEs in a model of inflammation relevant to vascular disease, the RAW 264.7 macrophage. Approach and results: The suppression of inflammatory cytokines (IL-6 and TNFa) in RAW 264.7 macrophages responding to LPS treatment (500 nM, 24 hrs.) was measured in the presence of 1 hour pretreatment with 9(10)-EpOME and 11(12)-EpETrE over 4-orders of magnitude (0.03 nM to 100 nM). The resulting dose-response curves were used to estimate potency (IC 50 ), and efficacy (suppression as percent of maximal LPS activation). Least-square-mean and 95% CIs are reported. The IC 50 for suppression of cytokines by EpOME and EpETrE were not different, regardless of cytokine, yielding an estimate of 4.2 [2.6, 6.8] nM and indicating that EpOMEs are equipotent to EpETrEs in cytokine suppression. While EpOMEs had the same effectiveness in suppressing IL-6 to 59% [54, 64] of max, they were not as effective in suppressing TNFa, to 83% [80, 86] of max versus 78% [75, 81] for EpETrEs (p<0.01). Conclusions: Given their great abundance in tissue and plasma, linoleate epoxides would need to be more than 100 times less potent than arachidonate epoxides to be physiologically irrelevant. Here, we find they are equipotent for suppression of two pro-inflammatory cytokines, with moderately lower efficacy for one cytokine, TNFa. We find little reason to ignore linoleate-derived EpOMEs and conclude they are a physiologically relevant pool of epoxides whose levels should be measured along with EpETrE and other epoxides when estimating overall epoxide effects.

Bovine and soybean milk bioactive compounds: Effects on inflammatory response of human intestinal Caco-2 cells

In this study the effects of commercial bovine and soybean milks and their bioactive compounds, namely genistein, daidzein and equol, on the inflammatory responses induced by lipopolysaccharide (LPS) treatment of human intestinal Caco-2 cells were examined, in terms of nitric oxide (NO) release and inducible nitric oxide synthetase (iNOS) expression.Both milks and their bioactive compounds significantly inhibited, dose-dependently, the expression of iNOS mRNA and protein, resulting in a decreased NO production. The NF-κB activation in LPS-stimulated intestinal cells was also examined. In all cases we observed that cell pre-treatment before LPS activation inhibited the IkB phosphorylation. Accordingly, quantification of bioactive compounds by solid phase microextraction coupled with liquid chromatography has shown that they were absorbed, metabolized and released by Caco-2 cells in culture media.In conclusion, we demonstrated that milks and compounds tested are able to reduce LPS-induced inflammatory responses from intestinal cells, interfering with NF-kB dependent molecular mechanisms.

In vitro therapeutic index application in high throughput screening of the anti‐inflammatory effects of natural products evaluation in different cell lines

One thousand and four hundred crude organic extracts from natural plants, botanical chemicals were evaluated for anti‐inflammatory activity LPS treated cell lines: BV‐2 microligal cells, RAW 264.7 macrophages and C6 glioma cells. LPS is commonly used to invoke in vitro inflammation. However, studies using immortalized macrophages, microglia or astrocytoma (malignant phenotype) could result in an interfering variable when evaluating natural products which are both anti‐inflammatory and anti‐cancer (pro‐apoptotic) at similar/close concentrations. In this study, a high throuput HTP study was conducted on inflammatory parameters using an in vitro therapeutic index (iTI) = ratio of toxicity (LC50) / anti‐inflammatory potency (IC50) compared to reference controls (e.g. hydrocortisone, dexamethasone, iNOS inhibitor N6‐(1‐iminoethyl)‐L‐lysine (L‐NIL) and other OTC NSAIDs). Preliminary profiling studies showed (1) LPS activation of BV2 cells resulted > 10‐fold rise in IL‐6, MIP‐2, MIP‐1g, RANTES and nitric oxide (NO); (2) LPS activated RAW 264.7 cells generated > 10‐fold rise in sTNFR2, MCP‐1, IL‐6, GCSF, RANTES and NO (3) LPS/IFN‐g activated C6 glioma initiated >10‐fold rise in MCP‐1, CINC1, CINC2a, CINC3 and NO. Using NO2‐ production as a guideline molecule for HTP screening, the data show that of all compounds tested : the highest anti‐inflammatory differential LC50/IC50s (Ti ) with efficacy < 250μg/mL occurred in the presence of Ashwaganda, Elecampane, Feverfew, Tansy, Turmeric, Bayleaf, Rosemary, Green Tea, Yerba Santa and Centipeda Herb. Likewise, naturally derived substances with the highest anti‐inflammatory differential LC50/IC50s (Ti ) were established for cardamonin and to a lesser extent: butein, apigenin, EGCG, curcumin and quercetin. These substances had capacity to reduce NO, downregulate iNOS protein expression, significantly attenuate IL‐6 in activated RAW/BV2 cells and reduce levels of cytokine‐induced neutrophil chemoattractant CINC‐1/3, in C6 cells. These particular natural substances seem to hold therapeutic value above many others tested, with possible applications for future prevention of neuro inflammatory conditions / treatment of general inflammatory chronic disorders and possibly drug development for strategies to prevent multi‐organ damages associated with endotoxic shock.Support or Funding InformationNIMHD Grants G12MD007582 and Grant Number 1P20 MD006738

Influence of Two‐Hit Lipopolysaccharide (LPS) Administration on Acute Hypoxic Ventilatory Response (HVR) in Urethane‐Anesthetized Spontaneously Breathing Adult Male C57BL/6 Mice

In chronic lung/airway disease, there is often a combination of systemic inflammation with an airway/lung‐mediated exacerbation. Under these conditions, patients may experience bouts of acute hypoxia; however, to date, there is limited information regarding the influence of this inflammation paradigm, including its accompanying neuroinflammation, on the acute hypoxic ventilatory response (HVR). To begin to address this issue, we used a ‘two‐hit’ lipopolysaccharide (LPS) administration protocol in which systemic LPS (ip, 3 mg/kg) was administered ~24 hr prior to an IT LPS (0.5 mg/kg) injection in spontaneously breathing adult male C57BL/6 mice, and at 2–6 hr post ‘two‐hit’ LPS administration, we recorded EMG activities from diaphragm (EMGDia) and genioglossus (EMGGG) muscles under baseline conditions (40% O2 in a balance of N2), during an acute bout of hypoxia (HVR, 10% O2 for 90 s), and during recovery from hypoxia. A similar recording protocol was used in control experiments in which mice received two‐hit ip‐IT saline injections. We found that ip‐IT LPS administration not only altered basal inspiratory motor output, but markedly altered the acute HVR. Specifically, ‘two‐hit’ LPS administration blunted the hypoxia‐induced amplitude response, such that amplitude increased by ≤10% (versus 30–50% increase in control mice), and produced an exaggerated frequency response, such that frequency increased by ≥30% (versus~15–20% increase in control mice). In addition, in LPS treated mice in which a dysrhythmic breathing pattern was observed, hypoxia either worsened the dysrhythmia or improved it to yield a more normal (but higher frequency) rhythm. Finally, ‘two‐hit’ LPS administration slightly altered the EMG burst pattern and attenuated the reduction in inspiratory burst complexity (as reflected by a decrease in the approximate entropy (ApEn) value) when compared to changes observed in control mice. During recovery from hypoxia, the inspiratory motor activity in both LPS treated and control mice returned toward baseline levels. These findings indicate that ‘two‐hit’ administration of LPS significantly alters the acute HVR, and further support the use of a ‘two‐hit’ administration approach for studying the role of lung/airway inflammation and its accompanying neuroinflammation in central respiratory control, including the chemical drive to breath.

Monophosphoryl lipid A-induced pro-inflammatory cytokine expression does not require CD14 in primary human dendritic cells.

To elucidate if TLR4-mediated MyD88 and TRIF signalling by the clinically applicable Lipopolysaccharide (LPS)-derivative monophosphoryl lipid A (MPLA) in primary human dendritic cells requires LPS cofactors LPS-binding protein (LBP) and CD14. Cytokine production by monocyte-derived DCs stimulated with MPLA or LPS was determined using ELISA. To investigate involvement of CD14 for action of LPS or MPLA, CD14 was inhibited using blocking antibodies or down-modulated using specific siRNA. To assess involvement of LBP monocyte-derived DCs were stimulated in serum-free culture medium in absence or presence of purified LBP. LBP and CD14 are not required for and do not enhance the capacity of MPLA to induce MyD88- and TRIF-dependent pro-inflammatory IL-6 and TNF-α. Interestingly, although CD14 is required for TRIF-dependent downstream events in mice, we show that in human CD14 is redundant for MPLA-induced TRIF-dependent chemokine production. These findings provide novel insight in the modes of action of MPLA in human and show that, compared to LPS, MyD88 and TRIF signalling in dendritic cells by MPLA is not mediated nor amplified by TLR4 cofactors. This gives insight why MPLA induces immune activation without provoking toxicity in human and clarifies why MPLA can be used as activating compound for clinically applicable immuno-activatory cellular products grown in serum-free regimens.

Low-dose polymyxin: an option for therapy of Gram-negative sepsis.

Endotoxins are the major components of the outer membrane of most Gram-negative bacteria and are one of the main targets in inflammatory diseases. The presence of endotoxins in blood can provoke septic shock in case of pronounced immune response. Here we show in vitro inactivation of endotoxins by polymyxin B (PMB). The inflammatory activity of the LPS–PMB complex in blood was examined in vitro in freshly drawn blood samples. Plasma protein binding of PMB was determined by ultracentrifugation using membranes with different molecular cut-offs, and PMB clearance during dialysis was calculated after in vitro experiments using the AV1000S filter. The formed LPS–PMB complex has lower inflammatory activity in blood, which results in highly reduced cytokine secretion. According to in vitro measurements, the appropriate plasma level of PMB for LPS inactivation is between 100 and 200 ng/ml. Furthermore, the combination of cytokine removal by adsorbent treatment with LPS inactivation by PMB dosage leads to strong suppression of inflammatory effects in blood in an in vitro model. Inactivation of endotoxins by low-dose intravenous PMB infusion or infusion into the extracorporeal circuit during blood purification can be applied to overcome the urgent need for endotoxin elimination not only in treatment of sepsis, but also in liver failure.

The binding capability of plasma phospholipid transfer protein, but not HDL pool size, is critical to repress LPS induced inflammation.

Phospholipid transfer protein (PLTP) participates in high density lipoprotein (HDL) metabolism. Increased plasma PLTP activity was observed in lipopolysaccharide (LPS) triggered acute inflammatory diseases. This study aimed to determine the exact role of PLTP in LPS induced inflammation. HDL pool size was shrunk both in PLTP deficient mice (PLTP−/−) and PLTP transgenic mice (PLTP-Tg). PLTP displayed a strong protective effect on lethal endotoxemia in mice survival study. Furthermore, after LPS stimulation, the expression of pro-inflammatory cytokines were increased in bone marrow derived macrophage (BMDM) from PLTP−/−, while decreased in BMDM from PLTP-Tg compared with BMDM from wild-type mice (WT). Moreover, LPS induced nuclear factor kappa-B (NFκB) activation was enhanced in PLTP−/− BMDM or PLTP knockdown RAW264.7. Conversely, PLTP overexpression countered the NFκB activation in LPS challenged BMDM. Additionally, the activation of toll like receptor 4 (TLR4) induced by LPS showed no alteration in PLTP−/− BMDM. Finally, PLTP could bind to LPS, attenuate the pro-inflammatory effects of LPS, and improve the cell viability in vitro. To sum up, these findings elucidated that PLTP repressed LPS induced inflammation due to extracellular LPS binding capability, and the protective effects were not related to HDL pool size in mice.

Toll-like Receptor 4 Ligands Down-regulate Fcγ Receptor IIb (FcγRIIb) via MARCH3 Protein-mediated Ubiquitination

Monocytes and macrophages are critical for the effectiveness of monoclonal antibody therapy. Responses to antibody-coated tumor cells are largely mediated by Fcγ receptors (FcγRs), which become activated upon binding to immune complexes. FcγRIIb is an inhibitory FcγR that negatively regulates these responses, and it is expressed on monocytes and macrophages. Therefore, deletion or down-regulation of this receptor may substantially enhance therapeutic outcomes. Here we screened a panel of Toll-like receptor (TLR) agonists and found that those selective for TLR4 and TLR8 could significantly down-regulate the expression of FcγRIIb. Upon further examination, we found that treatment of monocytes with TLR4 agonists could lead to the ubiquitination of FcγRIIb protein. A search of our earlier microarray database of monocytes activated with the TLR7/8 agonist R-848 (in which FcγRIIb was down-regulated) revealed an up-regulation of membrane-associated ring finger (C3HC4) 3 (MARCH3), an E3 ubiquitin ligase. Therefore, we tested whether LPS treatment could up-regulate MARCH3 in monocytes and whether this E3 ligase was involved with LPS-mediated FcγRIIb down-regulation. The results showed that LPS activation of TLR4 significantly increased MARCH3 expression and that siRNA against MARCH3 prevented the decrease in FcγRIIb following LPS treatment. These data suggest that activation of TLR4 on monocytes can induce a rapid down-regulation of FcγRIIb protein and that this involves ubiquitination.

Supramolecular structure of enterobacterial wild-type lipopolysaccharides (LPS), fractions thereof, and their neutralization by Pep19-2.5.

Lipopolysaccharides (LPS) belong to the strongest immune-modulating compounds known in nature, and are often described as pathogen-associated molecular patterns (PAMPs). In particular, at higher concentrations they are responsible for sepsis and the septic shock syndrome associated with high lethality. Since most data are indicative that LPS aggregates are the bioactive units, their supramolecular structures are considered to be of outmost relevance for deciphering the molecular mechanisms of its bioactivity. So far, however, most of the data available addressing this issue, were published only for the lipid part (lipid A) and the core-oligosaccharide containing rough LPS, representing the bioactive unit. By contrast, it is well known that most of the LPS specimen identified in natural habitats contain the smooth-form (S-form) LPS, which carry additionally a high-molecular polysaccharide (O-chain). To fill this lacuna and going into a more natural system, here various wild-type (smooth form) LPS including also some LPS fractions were investigated by small-angle X-ray scattering with synchrotron radiation to analyze their aggregate structure. Furthermore, the influence of a recently designed synthetic anti-LPS peptide (SALP) Pep19-2.5 on the aggregate structure, on the binding thermodynamics, and on the cytokine-inducing activity of LPS were characterized, showing defined aggregate changes, high affinity binding and inhibition of cytokine secretion. The data obtained are suitable to refine our view on the preferences of LPS for non-lamellar structures, representing the highest bioactive forms which can be significantly influenced by the binding with neutralizing peptides such as Pep19-2.5.

Lipopolysaccharide Compromises Human Sperm Function by Reducing Intracellular cAMP.

A worldwide decline in the quality of human semen is currently occurring. In mammals, sperm are produced from diploid stem-cell spermatogonia by spermatogenesis in testes and become mature in epididymis. Nevertheless, these biological processes can be affected by Gram-negative bacterial infection mediated by lipopolysaccharide (LPS), the major endotoxin of Gram-negative bacteria. It is well known that LPS can disturb spermatogenesis and affect sperm maturation and quality in vivo. However, the effect of LPS on the ejaculated mature sperm in vitro remains unclear. Thus, this study aimed to assess the in vitro toxicity of LPS on human sperm function and to elucidate the underlying mechanism. Human sperm were incubated with LPS (0.1-100 μg/ml) for 1-12 h in vitro and, subsequently, sperm viability, motility and capacitation, and the acrosome reaction were examined. LPS dose-dependently inhibited total and progressive motility and the ability to move through a viscous medium of the sperm but did not affect sperm viability, capacitation, and the acrosome reaction. To explore the underlying mechanism of LPS's actions, we examined the effects of LPS on the intracellular concentrations of cyclic adenosine monophosphate (cAMP) and calcium ([Ca(2+)]i) and protein-tyrosine phosphorylation of human sperm, which are key regulators of human sperm function. LPS decreased intracellular cAMP dose-dependently but had no effect on [Ca(2+)]i and protein-tyrosine phosphorylation of human sperm. These findings suggest that LPS inhibits human sperm motility by decreasing intracellular cAMP.

Effect of LPS on the Viability and Proliferation of Human Oral and Esophageal Cancer Cell Lines

The esophagus and mouth tumors are very frequent malignancies worldwide. Lipopolysaccharides (LPS) are capable of regulating gene expression of pro-inflammatory cytokines by binding to toll-like receptor 4 (TLR4). Recent studies show that LPS can increase the migration ability of human esophageal cancer cell line HKESC-2 by increasing its adhesion properties. However, the effect of LPS has not been tested on viability of human esophageal and oral cancer cells. This study aimed to determine the action of LPS on the cell proliferation and viability in OE19 (adenocarcinoma) and OE21 (squamous carcinoma) cell lines, representative of human esophageal cancer, and HN30 cell line, representative of human oral carcinoma. LPS was used as treatment to OE19 and OE21 cells, and PgLPS (Porphyromonasgingivalis lipopolysaccharide) to HN30 cells. Viability was assessed by MTT assay and proliferation by cell counting. TLR4 expression was evaluated by real-time PCR. LPS at higher concentrations decreased significantly cell viability in both cell lines, adenocarcinoma (OE19) and squamous esophageal carcinoma (OE21) at different times of treatment. In addition, both cell lines, OE19 and OE21, expressed TLR4 receptor. Taken together, our data demonstrated that LPS at high concentrations might contribute to tumor death, in agreement with previously data.

Hemolysis Controls Pro-Inflammatory Monocyte Priming Via Induction of and Interaction with Circadian Clock Protein Rev-Erbα in Sickle Cell Disease

Background: Patients with SCD tolerate a systemic pro-inflammatory vascular milieu created by chronic ischemia/reperfusion injury and profound erythrocyte hemolysis. In addition to this chronic low level inflammation, exposure to relatively innocuous, sub-clinical inflammatory stimuli appears to ignite an exaggerated, potentially fatal inflammatory response in patients. The etiology of this inflammatory hyper-reactivity is not well understood. There is ample evidence that, in steady state, a cadre of inflammatory cells, especially monocytes, exhibit a primed phenotype. Such priming, or propensity to activate, likely contributes to baseline inflammation, and is requisite for the inflated inflammatory response. Monocytes are quite unique amongst the leukocytes in that their inflammatory potential, including Il-6 release, is governed by the mammalian circadian clock. A role for the rhythmic oscillation of clock proteins as a controller of inflammation in SCD has never been demonstrated. However, a binding partner for heme, the nuclear receptor rev-erbα, is implicated as a regulator of clock controlled genes.Objective: To test the hypothesis that hemolysis, via heme-induced perturbation of the clock protein Rev-erbα, forms the basis for an enhanced inflammatory response in the monocyte.Methods: Intraperitoneal low dose lipopolysaccharide (LPS) was used to elicit an inflammatory response in the Townes mouse model of SCD. Plasma from the mice was acquired 6 hours after LPS injection. Analysis of 25 cytokines was accomplished using luminex methods. Monocytes were modeled in vitro using THP-1 cells. Simultaneous analysis of 84 induced inflammatory genes was conducted via qRT-PCR using the Qiagen RT Profiler PCR array. Inflammatory cytokine levels in cell supernatants were determined via ELISA.Results: We challenged the mice with low dose LPS (<10ng). Interrogation of the inflammatory cytokines in these mice revealed no change in any cytokine tested in the AA mice, but 20 out of 25 inflammatory cytokines were upregulated in mice with the SS genotype. The monocyte-based cytokines were clearly target of LPS activation in the SS mice. TNF-α and Il-1β were both upregulated 20 fold and 80 fold respectively in the SS mice. KC levels (the murine equivalent of Il-8) levels were increased 80 fold in the SS mice treated with LPS. Il-6 levels, however, were the most pronounced with a 40,000 fold increase over PBS injected SS mice. We then evaluated the role of hemolysis on monocyte inflammatory potential in vitro. Sustained monocyte exposure to physiological levels of heme in SCD alone could induce a low level of inflammatory gene expression and Il-6 release. However, sustained exposure to heme dramatically increased Il-6 release from the monocyte in response to LPS. Expression of the Il-6 gene was also increased, but the peak gene expression was time delayed compared to LPS treatment alone. In fact, we noted this phase shifting of inflammatory gene expression in the heme primed cells. LPS induced the release of significantly more TNF-α and Il-1β into the culture media in the presence of heme - consistent with the notion of heme setting a hyperactive threshold in response to LPS. We also noted that heme induced expression of the clock gene rev-erbα, and that antagonizing the activity of rev-erbα ablated the enhanced inflammatory response induced by LPS in the heme primed cells.Conclusion: These data provide evidence that hemolysis may play an important role in the hyper-inflammatory monocyte response via heme- induced dysregulation of the circadian clock. These novel observations provide entirely new avenues of anti-inflammatory therapy in SCD. DisclosuresNo relevant conflicts of interest to declare.

Natural IgM Switches the Function of Lipopolysaccharide-Activated Murine Bone Marrow-Derived Dendritic Cells to a Regulatory Dendritic Cell That Suppresses Innate Inflammation.

We have previously shown that polyclonal natural IgM protects mice from renal ischemia/reperfusion injury (IRI) by inhibiting the reperfusion inflammatory response. We hypothesized that a potential mechanism involved IgM modulation of dendritic cells (DC), as we observed high IgM binding to splenic DC. To test this hypothesis, we pretreated bone marrow-derived DC (BMDC) with polyclonal murine or human IgM prior to LPS activation and demonstrated that 0.5 × 10(6) IgM/LPS-pretreated BMDC, when injected into wild-type C57BL/6 mice 24 h before renal ischemia, protect mice from developing renal IRI. We show that this switching of LPS-activated BMDC to a regulatory phenotype requires modulation of BMDC function that is mediated by IgM binding to nonapoptotic BMDC receptors. Regulatory BMDC require IL-10 and programmed death 1 as well as downregulation of CD40 and p65 NF-κB phosphorylation to protect in renal IRI. Blocking the programmed death ligand 1 binding site just before i.v. injection of IgM/LPS-pretreated BMDC or using IL-10 knockout BMDC fails to induce protection. Similarly, IgM/LPS-pretreated BMDC are rendered nonprotective by increasing CD40 expression and phosphorylation of p65 NF-κB. How IgM/LPS regulatory BMDC suppress in vivo ischemia-induced innate inflammation remains to be determined. However, we show that suppression is dependent on other in vivo regulatory mechanisms in the host, that is, CD25(+) T cells, B cells, IL-10, and circulating IgM. There was no increase in Foxp3(+) regulatory T cells in the spleen either before or after renal IRI. Collectively, these findings show that natural IgM anti-leukocyte Abs can switch BMDC to a regulatory phenotype despite the presence of LPS that ordinarily induces BMDC maturation.

Ferritin-Mediated Iron Sequestration Stabilizes Hypoxia-Inducible Factor-1α upon LPS Activation in the Presence of Ample Oxygen.

Both hypoxic and inflammatory conditions activate transcription factors such as hypoxia-inducible factor (HIF)-1α and nuclear factor (NF)-κB, which play a crucial role in adaptive responses to these challenges. In dendritic cells (DC), lipopolysaccharide (LPS)-induced HIF1α accumulation requires NF-κB signaling and promotes inflammatory DC function. The mechanisms that drive LPS-induced HIF1α accumulation under normoxia are unclear. Here, we demonstrate that LPS inhibits prolyl hydroxylase domain enzyme (PHD) activity and thereby blocks HIF1α degradation. Of note, LPS-induced PHD inhibition was neither due to cosubstrate depletion (oxygen or α-ketoglutarate) nor due to increased levels of reactive oxygen species, fumarate, and succinate. Instead, LPS inhibited PHD activity through NF-κB-mediated induction of the iron storage protein ferritin and subsequent decrease of intracellular available iron, a critical cofactor of PHD. Thus, hypoxia and LPS both induce HIF1α accumulation via PHD inhibition but deploy distinct molecular mechanisms (lack of cosubstrate oxygen versus deprivation of co-factor iron).

Structural Relationship of the Lipid A Acyl Groups to Activation of Murine Toll-Like Receptor 4 by Lipopolysaccharides from Pathogenic Strains of Burkholderia mallei, Acinetobacter baumannii, and Pseudomonas aeruginosa.

Toll-like receptor 4 (TLR4) is required for activation of innate immunity upon recognition of lipopolysaccharide (LPS) of Gram-negative bacteria. The ability of TLR4 to respond to a particular LPS species is important since insufficient activation may not prevent bacterial growth while excessive immune reaction may lead to immunopathology associated with sepsis. Here, we investigated the biological activity of LPS from Burkholderia mallei that causes glanders, and from the two well-known opportunistic pathogens Acinetobacter baumannii and Pseudomonas aeruginosa (causative agents of nosocomial infections). For each bacterial strain, R-form LPS preparations were purified by hydrophobic chromatography and the chemical structure of lipid A, an LPS structural component, was elucidated by HR-MALDI-TOF mass spectrometry. The biological activity of LPS samples was evaluated by their ability to induce production of proinflammatory cytokines, such as IL-6 and TNF, by bone marrow-derived macrophages. Our results demonstrate direct correlation between the biological activity of LPS from these pathogenic bacteria and the extent of their lipid A acylation.

Murine germinal center B cells require functional Fms-like tyrosine kinase 3 signaling for IgG1 class-switch recombination

Switched antibody classes are important for efficient immune responses. Aberrant antibody production to otherwise harmless antigens may result in autoimmunity. The protein kinase fms-like tyrosine kinase 3 receptor (Flt3) has an important role during early B-cell development, but the role of Flt3 in peripheral B cells has not been assessed before. Herein we describe a previously unappreciated role for Flt3 in IgG1 class-switch recombination (CSR) and production. We show that Flt3 is reexpressed on B-cell lymphoma 6(+) germinal center B cells in vivo and following LPS activation of peripheral B cells in vitro. Absence of Flt3 signaling in Flt3 ligand-deficient mice results in impaired IgG1 CSR and accumulation of IgM-secreting plasma cells. On activated B cells, Flt3 is coexpressed and functions in synergy with the common-gamma chain receptor family. B cells from Flt3 ligand-deficient mice have impaired IL-4R signaling, with reduced phosphorylation of signal transducer and activator of transcription (Stat) 6, and demonstrate a failure to initiate CSR to IgG1 with low expression of γ1 germ-line transcripts, resulting in impaired IgG1 production. Thus, functional synergy between Flt3 and IL-4R signaling is critical for Stat-mediated regulation of sterile γ1 germ-line transcripts and CSR to IgG1.

Impact of Cadmium Exposure on the Association between Lipopolysaccharide and Metabolic Syndrome.

Cadmium (Cd) is an environmental contaminant that has a direct impact on the gut microbiome. Perturbations in the gut microbiome have been linked to metabolic disorders associated with inflammation generated by lipopolysaccharide (LPS). We investigated the impact of Cd on the association between LPS and metabolic syndrome. The study population consisted of 200 apparently healthy subjects (30–64 years of age; 96 men, 104 women). Serum LPS and blood Cd concentrations were measured by ELISA and graphite furnace-atomic absorption spectrophotometry (GF-AAS), respectively. The highest LPS quartile was associated with a greater prevalence of metabolic syndrome in men. There was a significant association between LPS activity and metabolic syndrome in men with blood Cd concentrations higher than the 50th percentile (OR = 3.05, 95% CI = 1.39–6.70); however, this relationship was not significant in men with blood Cd concentrations lower than the 50th percentile. The results of this study provide evidence for a strong association between high LPS activity and the prevalence of metabolic syndrome in men with relatively high blood Cd concentrations. Therefore, exposure to Cd may potentiate the association between LPS and metabolic syndrome in men.

Hyaluronan ameliorates LPS-induced acute lung injury in mice via Toll-like receptor (TLR) 4-dependent signaling pathways

Toll-like receptor-4 (TLR4) signaling has been implicated in innate immunity and acute inflammation following acute lung injury (ALI). As such, modulating inflammatory response through TLR4 represents an attractive therapeutic approach to treat ALI. Increasing evidence demonstrates that hyaluronan (HA) can modulate TLR4 activation and has shown early promise as a therapeutic agent in ALI. However, the mechanism associated with HA has not been fully elucidated. In the current study, we sought to determine the effects of HA on lipopolysaccharide (LPS)-induced inflammatory response and gain insights into the mechanism of action in mice with intratracheal instillation of LPS. Our results demonstrate that in contrast to mice challenged with LPS, pretreatment with HA significantly inhibited inflammatory cell recruitment, attenuated lung injury and suppressed the level of cytokine/chemokine in bronchial alveolar lavage fluid (BALF). Investigation of the mechanism responsible for inhibition of LPS activation showed HA treatment significantly inhibited the nuclear translocation of NF-κB p65 and protein expression of myeloid differentiation primary response protein (MyD88) and TIR-domain-containing adapter-inducing interferon-β (TRIF) and p38 MAPK, JNK and ERK activation in lung tissue. Furthermore, we compared the protection effect of HA in TLR4-deficient mice with those of genetically matched wild type (WT) mice in an acute model of lung injury. However, in TLR4-deficient mice, HA pretreatment before LPS instillation fail to affect the LPS response. Therefore, our findings suggest that HA pretreatment attenuated LPS-induced ALI and the anti-inflammatory function of HA was partial dependent on TLR4, which shed new light on potential elements that regulate the lung injury response.

Metformin Inhibits the Production of Reactive Oxygen Species from NADH:Ubiquinone Oxidoreductase to Limit Induction of Interleukin-1β (IL-1β) and Boosts Interleukin-10 (IL-10) in Lipopolysaccharide (LPS)-activated Macrophages

Metformin, a frontline treatment for type II diabetes mellitus, decreases production of the pro-form of the inflammatory cytokine IL-1β in response to LPS in macrophages. We found that it specifically inhibited pro-IL-1β production, having no effect on TNF-α. Furthermore, metformin boosted induction of the anti-inflammatory cytokine IL-10 in response to LPS. We ruled out a role for AMP-activated protein kinase (AMPK) in the effect of metformin because activation of AMPK with A769662 did not mimic metformin here. Furthermore, metformin was still inhibitory in AMKPα1- or AMPKβ1-deficient cells. The activity of NADH:ubiquinone oxidoreductase (complex I) was inhibited by metformin. Another complex I inhibitor, rotenone, mimicked the effect of metformin on pro-IL-1β and IL-10. LPS induced reactive oxygen species production, an effect inhibited by metformin or rotenone pretreatment. MitoQ, a mitochondrially targeted antioxidant, decreased LPS-induced IL-1β without affecting TNF-α. These results, therefore, implicate complex I in LPS action in macrophages.

Endoplasmic reticulum stress collaborates with lipopolysaccharide to promote the inflammatory response in macrophages

To investigate the protective mechanism of endoplasmic reticulum stress (ERS) inhibition against inflammation-induced acute liver injury using a mouse model. Marrow-derived stem cells were isolated from mouse femur and used to derive macrophages for analysis in experimental inflammation conditions, established by exposure to LPS and consequent activation of TLR4. Tunicamycin, an ERS chemical inducer, was applied to interfere the inflammation model condition.Affect on the inflammation-related factor MAPK was detected by western blot, and affects on gene expression of inflammatory factors were measured by real-time quantitative PCR. Affect on TNFa was also measured by ELISA. Expression of TNFa, IL-6 and IL-1b was induced upon exposure to LPS, with the peak levels being reached at 4 hours of exposure (TNFa, 0.82+/-0.24; IL-1 b, 2.20+/-0.69; IL-6, 0.330+/-0.150). Tunicamycin significantly enhanced the LPS-induced up-regulation of TNFa, IL-6 and IL-1b expression (TNFa, 1.44+/-0.38, t=2.8, P<0.05; IL-1b, 16.063.40, t =7.93, P<0.05; IL-6, 31.1610.60, t=5.08, P<0.05). The tuniamycin treatment also enhanced the LPS-induced up-regulation of the protein expression of phospo-p38, phospho-JNK and phoshpo-ERK. ERS collaborates with LPS to promote the TLR4-mediated inflammatory response of macrophages, and this collaboration may be a pathogenic mechanism underlying progressive development of acute liver injury.