Dose Of LPS Research Articles

The effects of aspirin and ticagrelor on Toll-like receptor (TLR)-mediated platelet activation: results of a randomized, cross-over trial.

Platelet activation underlies the pathology of an acute myocardial infarction (AMI), and dual antiplatelet therapy (DAPT) is administered post-AMI to limit this activation. Platelets express Toll-like receptors (TLRs) 1, 2, and 4 and become potently activated in response to TLR2/1 and TLR4 stimulation. However, it is unknown whether antiplatelet agents can protect against platelet activation via these TLR pathways. This study aimed to determine the extent to which TLR-mediated platelet activation can be inhibited by currently used antiplatelet agents. Ten healthy subjects were enrolled into a single-blinded randomized cross-over trial. Subjects received either aspirin monotherapy or DAPT (aspirin in combination with ticagrelor) for 1week, were washed out, and crossed over to the other drug regimen. Platelet activation was assessed in response to Pam3CSK4 (a TLR2/1 agonist) and lipopolysaccharide (LPS; a TLR4 agonist) at baseline and after each antiplatelet drug regimen. Platelet-surface expression of CD62p and PAC1 by flow cytometry was measured as markers of platelet activation. At baseline, expression of CD62p and PAC1 increased significantly in response to high-dose LPS and in a dose-dependent manner in response to Pam3CSK4. Aspirin monotherapy did not inhibit platelet activation in response to any TLR agonist tested. DAPT with aspirin and ticagrelor only modestly inhibited expression of both activation markers in response to high doses of Pam3CSK4 and LPS. However, incubation with these TLR agonists led to substantial platelet activation despite treatment with these anti-platelet agents. Platelet-TLR2/1 and platelet-TLR4 represent intact on-treatment platelet activation pathways, which may contribute to on-going platelet activation post-AMI.

NEW ASPECTS OF SEPSIS AND SEPTIC SHOCK PATHOGENESIS IN CHILDREN. THE COMPLEMENT SYSTEM AS TARGET FOR AN EFFECTIVE THERAPY

Nowadays sepsis is grave complication of infection end the cause of death reanimation. In this survey, we would like to emphasize the importance of the control over the activation over the compliment system. It has been proved of animal model a complement one of the key role in the development of hyperactive immunity response, later resulting in violation of immunity homeostasis. Mice which had C3–/–, C4–/– deficit, aft receiving a LPS dose intraperitoneallis showed a better survival to compare with the control group of animals. There exist clinical data which confirm active participation of the compliment in the chain of the septic process. The research showed the patient affected by sepsis, had protein C3 and C4 concentration correlating which mortality at the time of diagnosis. The is chemoattractants, protein C3a and C5a, turn tube the result of complements pathway activation. The chemoattractants, provoke the extraction a big number of cytokines. Vessels permeability increase and DIC-syndrome activation wis it, multiple organ dysfunction develops. Ishemiareperfusion launch triggers aseptic inflammation, which appears decentralization and DIC-syndrome. C1-INH controls the work of classical way complement and Hemostasis System. Researchers the deficit in C1-INH animals and patients affected bay sepsis, which is proved in laboratory and clinics. The remedy C1-INH (Berinert, CSL Behring) appeared over 25 years ago and was used and therapy hereditary angioedema. For the lasted years we accumulated a considerable quantity of fasts of C1-INH use which after pathologies: heart attack, Ischemia-reperfusion injury, trauma provoked by cardiopulmonary bypass. The use of C1-INH on animal models septic in clinical research their efficacy and safety.

Exacerbated liver injury of antithyroid drugs in endotoxin-treated mice

Drug-induced liver injury is a major concern in clinical studies as well as in post-marketing surveillance. Previous evidence suggested that drug exposure during periods of inflammation could increase an individual’s susceptibility to drug hepatoxicity. The antithyroid drugs, methimazole (MMI) and propylthiouracil (PTU) can cause adverse reactions in patients, with liver as a usual target. We tested the hypothesis that MMI and PTU could be rendered hepatotoxic in animals undergoing a modest inflammation. Mice were treated with a nonhepatotoxic dose of LPS (100 µg/kg, i.p) or its vehicle. Nonhepatotoxic doses of MMI (10, 25 and 50 mg/kg, oral) and PTU (10, 25 and 50 mg/kg, oral) were administered two hours after LPS treatment. It was found that liver injury was evident only in animals received both drug and LPS, as estimated by increases in serum alanine aminotransferase (ALT), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and TNF-α. An increase in liver myeloperoxidase (MPO) enzyme activity and tissue lipid peroxidation (LPO) in addition of liver glutathione (GSH) depletion were also detected in LPS and antithyroid drugs cotreated animals. Furthermore, histopathological changes including, endotheliitis, fatty changes, severe inflammatory cells infiltration (hepatitis) and sinusoidal congestion were detected in liver tissue. Methyl palmitate (2 g/kg, i.v, 44 hours before LPS), as a macrophage suppressor, significantly alleviated antithyroids hepatotoxicity in LPS-treated animals. The results indicate a synergistic liver injury from antithyroid drugs and bacterial lipopolysaccharide coexposure.

Low doses of LPS exacerbate the inflammatory response and trigger death on TLR3-primed human monocytes

TLR sensing of pathogens triggers monocyte activation to initiate the host innate immune response to infection. Monocytes can dynamically adapt to different TLR agonists inducing different patterns of inflammatory response, and the sequence of exposure to TLRs can dramatically modulate cell activation. Understanding the interactions between TLR signalling that lead to synergy, priming and tolerance to TLR agonists may help explain how prior infections and inflammatory conditioning can regulate the innate immune response to subsequent infections. Our goal was to investigate the role of MyD88-independent/dependent TLR priming on modulating the monocyte response to LPS exposure. We stimulated human blood monocytes with agonists for TLR4 (LPS), TLR3 (poly(I:C)) and TLR7/8 (R848) and subsequently challenged them to low doses of endotoxin. The different TLR agonists promoted distinct inflammatory signatures in monocytes. Upon subsequent LPS challenge, LPS- and R848-primed monocytes did not enhance the previous response, whereas poly(I:C)-primed monocytes exhibited a significant inflammatory response concomitant with a sharp reduction on cell viability. Our results show that TLR3-primed monocytes are prompted to cell death by apoptosis in the presence of low endotoxin levels, concurrent with the production of high levels of TNFα and IL6. Of note, blocking of TNFR I/II in those monocytes did reduce TNFα production but did not abrogate cell death. Instead, direct signalling through TLR4 was responsible of such effect. Collectively, our study provides new insights on the effects of cross-priming and synergism between TLR3 and TLR4, identifying the selective induction of apoptosis as a strategy for TLR-mediated host innate response.

Ibrutinib has a novel immunomodulatory effect by enhancing DC maturation both in vivo and in a model of inflammation

Abstract Myeloid derived suppressor cells (MDSCs) can inhibit T cell activation by altering the characteristics and activation of dendritic cells (DCs). We had previously reported that Ibrutinib enhances the expression of co-stimulatory molecules on DCs and promotes DC-mediated T cell activation. In the present study we further determined the mechanisms of DC immunomodulation by Ibrutinib. We evaluated the effect of Ibrutinib on (i) DC maturation and migration from the injection site to draining lymph nodes, (ii) T cell proliferation in vivo, and (iii) monocyte maturation to DCs using a mouse inflammation model. DCs were generated from mouse bone marrow-derived cells in the presence of vehicle or 10μM lbrutinib. CFES-labelled DCs were injected subcutaneously into the footpads of naive mice, and their migration to the regional lymph nodes and maturation profile was analyzed by flow cytometry. To evaluate T cell priming, CFSE-labelled OVA-specific T cells isolated from OT-II mice were injected into the tail vein of syngeneic mice that were prior injected with mature DCs loaded with OVA peptide. After four days, T cell proliferation was evaluated by CFSE dilution. In the inflammation model, C57BL6 mice were treated with vehicle or 6mg/kg/day Ibrutinib orally for a week. A sub-lethal dose of LPS was injected in the dorsal skin and the frequency of inflammatory monocytes and mature DCs was determined. We found that Ibrutinib significantly improves DC maturation and migration, inducing T cell activation and proliferation in vivo. In addition, Ibrutinib enhances monocyte maturation to DCs in the skin. The results suggest a novel effect of Ibrutinib, which may have therapeutic use for cancers characterized by a high MDSC infiltration.

Fasciola hepatica Glutathione S-tranferase suppresses inflammatory cytokines and chemokines in vivo against lethal doses of lipopolysaccharide on C57Bl/6 mice.

Abstract The world-wide helminth parasite, Fasciola hepatica, is an excellent immunomodulator. It survives in the host by preventing immune responses against itself by using excretory-secretory products (ESPs). One of the ESPs well-known for its anti-oxidant properties is Glutathione S-transferase (GST). Although it has been studied as a vaccine, the function GST plays in immunomodulation has scarcely been studied. Previously, we reported how the use of GST prior to TLR ligands significantly decreased TLR activation in vitro and boosted the survival rate of mice injected intraperitoneally with GST before a lethal dose of LPS by 80%. In this study, we investigate whether these C57BL/6 mice that survived after a lethal dose of LPS experienced a reduction in inflammatory cytokines and/or chemokines. For this, C57BL/6 6–8 week-old mice were allotted into two groups of 12 animals each. The control group (n=12) was injected intraperitoneally with 0.1M endotoxin free PBS and allowed to rest 1 hr, while the experimental group (n=12) was injected intraperitoneally with 15 ug of GST and allowed to rest 1 hour. Afterwards, both groups were injected with a lethal dose (1 mg) of E.coli LPS (serotype 0111:B4; Sigma) and monitored during a 48 hour period. Sera from mice in the experimental group were compared to the control group by using a Cytokine 20-Plex Mouse Panel kit (Invitrogen) for a wide array of cytokines and chemokines. Our results demonstrate that injecting 15ug of GST intraperitoneally 1 hour before a lethal dose of LPS gives the surviving 80% of C57BL/6 mice significantly reduced amounts of pro-inflammatory cytokines and chemokines, such as IL-1β (p = 0.0004), IL-12p70 (p = 0.0001) and MIP-1α (p = 0.0024), after 48 hrs.

Xianyu decoction attenuates the inflammatory response of human lung bronchial epithelial cell

BackgroundXianyu decoction (XD), a Chinese experience recipe, shows inhibitory effects on lung cancer. However, the potential functions of XD on pneumonia were unknown. This study aimed to investigate the effect of XD on inflammatory response of childhood pneumonia. MethodsHuman lung bronchial epithelial cell line BEAS-2B was cultured in different doses of LPS with or without XD treatment. The expression of miR-15a and IKBKB were altered by transfection assay. RT-PCR and western blot were used to evaluate the effects of XD and miR-15a mimic/inhibitor on the expression levels of miR-15a, IKBKB, p65 and IκBα. ELISA was used to determine the levels of CRP, IL-6 and IL-8. ResultsHigh expression of miR-15a was observed in serum and cell model of pneumonia. miR-15a promoted the expression of inflammatory cytokines IL-6, IL-8, CRP and IKBKB in vitro. XD treatment downregulated the level of miR-15a in pneumonia children. In addition, XD reduced the expression of inflammatory cytokines and the phosphorylation levels of p65 and IκBα by inhibition of miR-15a and IKBKB expression in LPS-stimulated BEAS-2B cells. ConclusionXD downregulated the level of miR-15a in serum of pneumonia children. Additionally, XD inhibited inflammatory response in LPS-stimulated BEAS-2B cells possibly by blocking IKBKB/NF-κB signal pathway which was regulated by miR-15a.

Intrinsic anti‐inflammatory properties of serum in deep‐diving seals

Diving seals are repeatedly exposed to hypoxia‐reoxygenation and other pro‐inflammatory stimuli such as shear stresses in alveoli from lung collapse and re‐expansion. Moreover, they have a known hyperlipidemic status, which is associated with vascular inflammatory stress in humans and mice. To investigate potential protective strategies in free‐living populations of deep‐diving seals, we examined baseline levels of cytokine biomarkers, as well as the inflammatory response of IL6 gene expression in seal blood cells exposed to an endotoxin (LPS, lipopolysaccharide, 1–1000ng/mL). Despite aspects of their basic physiology that predisposes other mammals to inflammatory stress, cytokine production in healthy Weddell seals (adults and pups) and Northern elephant seals (adults only) following LPS incubation was blunted (10–100X increase in plasma IL6), compared to human samples assayed under the same conditions (>1000X increase). These plasma samples, further assayed using a cytokine panel validated for pinnipeds, revealed that many cytokines were below detectable levels in Weddell seals, with only IL6, IL10 and TNFα exhibiting a dose‐response to LPS. Under isolated, in vitro conditions, Weddell seal monocytes maintained the expected, robust response to stimulation with LPS endotoxin. However, when this response was compared under typical cell culture conditions (using media supplemented with FBS) versus media supplemented with an equivalent amount of Weddell seal serum (WSS), IL6 expression from stimulated monocytes exposed to WSS declined significantly (LPS dose*serum‐type 2way repeated‐measures ANOVA across all doses: F1,6= 8.5, p=0.027 for FBS vs WSS), as did cytokines detected in the cell culture media. To further elucidate the potential anti‐inflammatory properties of seal serum, we repeated the experiment comparing FBS to de‐complemented WSS in RAW cells, a mouse macrophage line. These results validated our findings from seal monocytes, demonstrating that mouse cytokine expression (IL6, TNFα, and Mcp1), was consistently reduced in cells cultured with WSS compared to FBS. Finally, we confirmed that these results held in RAW cells exposed to other TLR ligands, including Pam‐3‐Cys, Poly I:C and CpG DNA (TNFα expression in WSS<FBS, p=0.0001 in each experiment). These findings suggest that seal serum innately possesses anti‐inflammatory properties, which may protect these deep divers from naturally occurring inflammatory challenges such as dive‐induced hypoxia‐reoxygenation and lung collapse.Support or Funding InformationFunded by the National Science Foundation (Office of Polar Programs).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Recombinant Fasciola hepatica Fatty Acid Binding Protein Downregulates Inflammatory Cytokines/Chemokines in a Mouse Model of Septic Shock and Shows a Wide Spectrum of Action

Sepsis is the leading cause of death from infections. It involves, both pathogen and host factors and it is characterized by an exacerbation of inflammatory cytokines and chemokines and organ dysfunction and failure. Recently, we reported that a recombinant Fasciola hepatica fatty acid binding protein, termed Fh15, is capable of suppressing the LPS induced expression of inflammatory cytokines such as IL‐1β and TNFα in murine bone marrow derived macrophages in vitro. We also demonstrated that such anti‐inflammatory effect is achieved by targeting specifically the CD14 co‐receptor, which results in a complete suppression of TLR4 activation and makes macrophages irresponsive to subsequent LPS‐stimuli. In the current study, we evaluated the capacity of Fh15 to suppress the TLR4‐activation in vitro, as well as its capacity to therapeutically suppress the cytokine storm in a mouse model of septic shock. We also tested the ability of Fh15 to suppress the activation of multiple TLRs in response to Gram‐positive and Gram‐negative whole bacteria extracts in vitro. For in vivo studies, we used female BALB/c mice that received a lethal intraperitoneal (i.p.) injection of LPS‐E. coli and 1h later received a single i.p. injection with 50μg Fh15. Animals were sacrificed 12h after the LPS challenge. For in vitro experiments, we used THP1‐CD14 cells, which were cultured with Fh15 prior to stimulation with optimized concentrations of 1×108 cells of Klebsiella pneumoniae and Enterococcus faecalis. Results demonstrated that, although Fh15 was unable to stimulate the secretion of any inflammatory cytokine, it significantly suppressed the levels of serum IFNγ (p=0.0425), TNFα (p=0.0008), IL‐1β (p=0.0021) in all animals exposed to lethal doses of LPS. Fh15 also significantly suppressed the levels in serum of two macrophage inflammatory proteins MIP‐1α (p=0.0027) and MIP‐1β (p<0.0001), two chemotactic chemokines that play relevant roles in the inflammatory process. Moreover, we also demonstrated that Fh15 inhibits the stimulation of various TLRs in response to whole bacteria extracts, suggesting that Fh15 could have a broad spectrum of action. These results support the possibility of using Fh15 as an excellent alternative for an anti‐inflammatory drug in pre‐clinical studies in the near future.Support or Funding InformationThis study was supported by MBRS‐RISE R25GM061838‐13, and NIH grants G12MD007600 and 2P40OD012217.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sphingosine Kinase 2 Expression in CD11b+ Macrophages Negatively Regulates cGASSTING Activity and Resolves Lung Injury

Lung macrophages (MΦ) are important in triggering the immune response and thereby resolve acute lung injury (ALI) by sensing pathogens through pattern recognizing receptors including TLRs. However, the role of macrophages in resolving inflammation and lung injury is less well understood. Herein, we used transgenic mice expressing human diphtheria toxin receptor (DTR) under the control of Cd11b‐promoter (DTR) mice to assess their specific role in regulating lung inflammatory injury. Administration of diphtheria toxin (DT) in DTR mice selectively depletes CD11b cells by causing their apoptosis. We first assessed alteration in lung macrophages (gated using CD11b+, CD11c+, Gr1−, CD64+) using LyzM‐GFP mice (green myeloid cells) following lung injury. Flow cytometry analysis showed a significant decline in CD11C+/CD11b+ MΦ at the peak of lung injury at 4h while these cells increased in proportion during resolution at 24h and 48h respectively. Next, we administered DT after LPS‐induced lung injury which only depleted CD11b+ MΦ by 50% in DTR mice at 24 h without inducing neutropenia. Loss of Cd11b+ MΦ did not alter the magnitude of LPS‐induced injury in DTR mice but markedly impaired resolution of injury. Survival curve of cecal ligation puncture and high dose of LPS in DTR mice also indicated that CD11b+ MΦ are critical during resolution of lung injury from sepsis. The recognition of endogenous DNA released during cell death is a major mechanism to activate innate immune responses. Cyclic GMP‐AMP (cGAMP) synthase (cGAS) is a critical sensor of cytosolic dsDNA and stimulator of interferon gene (STING) in the endoplasmic reticulum which leads to induction of IFNb and downstream inflammatory signaling. Intriguingly, we found that loss of CD11b MΦ augmented IFN‐β production by LPS in addition to canonical NF‐kB mediated inflammatory signaling in DTR mice. Further, we also observed that LPS administration in DTR mice induced TBK1 phosphorylation indicating STING activation. Sphingosine‐1‐phosphate (S1P) generated by the action of sphingosine kinases (SPHK1 and SPHK2) plays a critical role in dampening inflammation and thereby lung injury. Therefore, we addressed if altered SPHK1 or SPHK2 expression in CD11b MΦ was responsible for maladaptive inflammatory response in DTR mice. Transplantation of SPHK2 or SPHK1 bone marrow into lethally irradiated wild type (WT) mice indicated that SPHK2 expression in hematopoietic cells play a critical role in resolving lung injury. Thus, we adoptively transferred WT or SPHK2−/−CD11b+ bone marrow cells in DTR mice following LPS and DT challenge. We observed that WT‐CD11b cells resolved inflammatory injury in DTR mice while CD11b cells lacking SPHK2 could not. Moreover, SPHK2 null bone marrow derived cells showed increased IFN‐β production by LPS in association with increase in TBK1‐phosphorylation. Thus, these data demonstrated that following lung injury SPHK2 is activated in CD11b MΦ to suppress STING activity and thereby dampens IFN‐β generation leading to resolution of ALI.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Development and Characterization of an Endotoxemia Model in Zebra Fish.

Endotoxemia is a condition in which endotoxins enter the blood stream and cause systemic and sometimes lethal inflammation. Zebra fish provides a genetically tractable model organism for studying innate immunity, with additional advantages in live imaging and drug discovery. However, a bona fide endotoxemia model has not been established in zebra fish. Here, we have developed an acute endotoxemia model in zebra fish by injecting a single dose of LPS directly into the circulation. Hallmarks of human acute endotoxemia, including systemic inflammation, extensive tissue damage, circulation blockade, immune cell mobilization, and emergency hematopoiesis, were recapitulated in this model. Knocking out the adaptor protein Myd88 inhibited systemic inflammation and improved zebra fish survival. In addition, similar alternations of pathways with human acute endotoxemia were detected using global proteomic profiling and MetaCore™ pathway enrichment analysis. Furthermore, treating zebra fish with a protein tyrosine phosphatase nonreceptor type 11 (Shp2) inhibitor decreased systemic inflammation, immune mobilization, tissue damage, and improved survival in the endotoxemia model. Together, we have established and characterized the phenotypic and gene expression changes of a zebra fish endotoxemia model, which is amenable to genetic and pharmacological discoveries that can ultimately lead to a better mechanistic understanding of the dynamics and interplay of the innate immune system.

Systemic inflammation inhibits serotonin receptor 2-induced phrenic motor facilitation upstream from BDNF/TrkB signaling.

Although systemic inflammation induced by even a low dose of lipopolysaccharide (LPS, 100 μg/kg) impairs respiratory motor plasticity, little is known concerning cellular mechanisms giving rise to this inhibition. Phrenic motor facilitation (pMF) is a form of respiratory motor plasticity elicited by pharmacological agents applied to the cervical spinal cord, or by acute intermittent hypoxia (AIH; 3, 5-min hypoxic episodes); when elicited by AIH, pMF is known as phrenic long-term facilitation (pLTF). AIH consisting of moderate hypoxic episodes (mAIH, arterial Po2 = 35-55 mmHg) elicits pLTF via the Q pathway to pMF, a mechanism that requires spinal serotonin (5HT2) receptor activation and new brain-derived neurotrophic factor (BDNF) protein synthesis. Although mild systemic inflammation attenuates mAIH-induced pLTF via spinal p38 MAP kinase activation, little is known concerning how p38 MAP kinase activity inhibits the Q pathway. Here, we confirmed that 24 h after a low LPS dose (100 μg/kg ip), mAIH-induced pLTF is greatly attenuated. Similarly, pMF elicited by intrathecal cervical injections of 5HT2A (DOI; 100 μM; 3 × 6 μl) or 5HT2B receptor agonists (BW723C86; 100 μM; 3 × 6 μl) is blocked 24 h post-LPS. When pMF was elicited by intrathecal BDNF (100 ng, 12 μl), pMF was actually enhanced 24 h post-LPS. Thus 5HT2A/2B receptor-induced pMF is impaired downstream from 5HT2 receptor activation, but upstream from BDNF/TrkB signaling. Mechanisms whereby LPS augments BDNF-induced pMF are not yet known. NEW & NOTEWORTHY These experiments give novel insights concerning mechanisms whereby systemic inflammation undermines serotonin-dependent, spinal respiratory motor plasticity, yet enhances brain-derived neurotrophic factor (BDNF)/TrkB signaling in phrenic motor neurons. These insights may guide development of new strategies to elicit functional recovery of breathing capacity in patients with respiratory impairment by reducing (or bypassing) the impact of systemic inflammation characteristic of clinical disorders that compromise breathing.

Fragmented hyaluronan has no alarmin function assessed in arthritis synovial fibroblast and chondrocyte cultures.

Hyaluronan (HA) is a large polymer and an important component of the extracellular matrix. During homeostasis, high molecular mass HA is the predominant form, but upon inflammation, degradation products of HA accumulate. These HA fragments (HA-fs) have been reported to possess pro-inflammatory activities and thus act as alarmins, notifying immune cells of danger via TLR4 and CD44. HA is found in large quantities in synovial joint fluid. In order to reveal a potential role of HA-fs in arthritis pathogenesis, the in vitro effects of HA of various molecular masses (from 1680 kDa to oligosaccharide HA) on synovial fibroblasts and chondrocytes from rheumatoid arthritis patients, and on peripheral blood mononuclear cells from healthy donors, were investigated. TLR4 and CD44 surface expression was confirmed by immunocytochemistry, and cell activation was determined based on cytokine and chemokine production. While the cell types investigated expressed TLR4 and CD44, no increased release of IL-1ß, IL-6, IL-8, IL-10, IL-12 or TNF-α was detected after HA stimulation. Similarly, HA did not enhance activation after priming cells with low doses of LPS or by forming complexes with LPS. Hence, this study does not support the common view of HA-fs being pro-inflammatory mediators and it is not likely that HA-fs generated during arthritis contribute to disease pathogenesis.

Age-related cognitive impairment is associated with long-term neuroinflammation and oxidative stress in a mouse model of episodic systemic inflammation

BackgroundMicroglia function is essential to maintain the brain homeostasis. Evidence shows that aged microglia are primed and show exaggerated response to acute inflammatory challenge. Systemic inflammation signals to the brain inducing changes that impact cognitive function. However, the mechanisms involved in age-related cognitive decline associated to episodic systemic inflammation are not completely understood. The aim of this study was to identify neuropathological features associated to age-related cognitive decline in a mouse model of episodic systemic inflammation.MethodsYoung and aged Swiss mice were injected with low doses of LPS once a week for 6 weeks to induce episodic systemic inflammation. Sickness behavior, inflammatory markers, and neuroinflammation were assessed in different phases of systemic inflammation in young and aged mice. Behavior was evaluated long term after episodic systemic inflammation by open field, forced swimming, object recognition, and water maze tests.ResultsEpisodic systemic inflammation induced systemic inflammation and sickness behavior mainly in aged mice. Systemic inflammation induced depressive-like behavior in both young and aged mice. Memory and learning were significantly affected in aged mice that presented lower exploratory activity and deficits in episodic and spatial memories, compared to aged controls and to young after episodic systemic inflammation. Systemic inflammation induced acute microglia activation in young mice that returned to base levels long term after episodic systemic inflammation. Aged mice presented dystrophic microglia in the hippocampus and entorhinal cortex at basal level and did not change morphology in the acute response to SI. Regardless of their dystrophic microglia, aged mice produced higher levels of pro-inflammatory (IL-1β and IL-6) as well as pro-resolution (IL-10 and IL-4) cytokines in the brain. Also, higher levels of Nox2 expression, oxidized proteins and lower antioxidant defenses were found in the aged brains compared to the young after episodic systemic inflammation.ConclusionsOur data show that aged mice have increased susceptibility to episodic systemic inflammation. Aged mice that showed cognitive impairments also presented higher oxidative stress and abnormal production of cytokines in their brains. These results indicate that a neuroinflammation and oxidative stress are pathophysiological mechanisms of age-related cognitive impairments.

Establishment of different experimental asthma models in mice.

Asthma is a complex disease. The heterogeneity of airway inflammation during asthma indicates there are different mechanisms involved. In order to further study the mechanisms of asthma, different mouse models were established to mimic corresponding subtypes of asthma in clinic. Eosinophilic asthma was established by intraperitoneal injections of ovalbumin (OVA) on day 0 and day 7, followed by inhalation of aerosolized OVA on days 14–17. Neutrophilic asthma was established by transtracheal administration of a high dose of lipopolysaccharide (LPS; 10 µg) on days 15 and 17 in combination with OVA sensitization and challenge as described previously. Mix-granulocytic asthma was established by transtracheal administration of a low dose of LPS (1 µg) on day 15, in combination with OVA sensitization and challenge as described previously. Compared with healthy controls, increased numbers of eosinophils, elevated levels of T helper (Th)2 cytokines in bronchoalveolar lavage fluid (BALF), and moderated inflammation of lung tissues was observed in eosinophilic asthma. Increased numbers of neutrophils, elevated levels of Th1 and Th17 cytokines in BALF and severe inflammation of lung tissues was observed in neutrophilic asthma. Increased numbers of both eosinophils and neutrophils, elevated levels of Th1, Th2 and Th17 cytokines in BALF and severe inflammation of lung tissues was observed in mix-granulocytic asthma. Airway hyperresponsiveness, increased bronchial mucus secretion, and elevated serum levels of immunoglobin (Ig)E and OVA-IgE were detected in all three asthma models. Dexamethasone reduced the pathogenic symptoms of the mice in eosinophilic asthma, however had no effect on neutrophilic asthma or mix-granulocytic asthma. Each model of asthma established in the present study represents corresponding subtypes of asthma in clinic.

The expression of microRNA-155 and 127 in the neonatal SD rats with acute lung injury induced by lipopolysaccharide

Aim: This study was to observe the changes of the gene expression of microRNA-155 and 127 in the neonatal rats with Acute Lung Injury (ALI). Methods: Eighty neonatal SD rats were randomly divided into experimental groups (intraperitoneal injection with LPS, n=60) and control (intraperitoneal injection with NS, n=20). Neonatal rats in experimental groups (LPS 1 mg, 2 mg and 3 mg group, n=20) were received corresponding dose of LPS (1, 2 and 3 mg/kg, diluted with saline to 0.2 ml), while neonatal rats in control group were injected 0.2 ml saline respectively. Then we killed rats at 1 h, 6 h, 12 h and 24 h respectively in each group. The lung general pathological changes were observed. The expression of microRNA-155 and 127 were detected by semi-quantitative reverse transcription-polymerase chain reaction. Tumor Necrosis Factor-α (TNF-α) and Interleukelin-6 (IL-6) in Bronchoalveolar Lavage Fluid (BALF) were detected by enzyme-linked immunosorbent assay. The expression of TNF-α and IL-6 protein in lung tissue were detected by western blotting. Results: The expression of microRNA-155 was up-regulated in the tissues and BALF in a dose and timedependent manner and microRNA-127 down-regulated. The difference were statistically significant when compared with the control group (all P<0.05). The TNF-α and IL-6 level of BALF rats with ALI in LPS groups were increased compared with the control group in a dose-dependent manner (all P<0.05). A similar trend had been seen in TNF-α and IL-6 protein level of lung tissues, the difference was statistically significant. Conclusion: The expression of microRNA-155 and 127 are associated with severity of the ALI in a dose and time-dependent manner, and they might be considered as potential biomarkers for early diagnosis of ALI.

MRP14 is dispensable for LPS-induced shock in BALB/c mice

Myeloid-related protein (MRP) 14 and MRP8 are abundantly expressed by myeloid cells and are involved in various inflammatory disorders. Although accumulating evidence revealed the roles of MRP14 and MRP8 in inflammatory responses by using MRP14-knockout (KO) mice, the KO mice were only available in the C57BL/6 background. We established BALB/c-background MRP14-KO mice to examine if its biological functions are conserved in mice with a different genetic background. MRP14-KO BALB/c mice showed different phenotypes from the reported MRP14-KO C57BL/6 mice in terms of bone marrow cell response to LPS and peripheral leukocyte population. When an acute lethal dose of LPS was injected, the survival rate was not different between MRP14-KO and WT mice, which was also different from results previously reported on C57BL/6 mice. These results suggest that immunological functions of MRP14, and possibly also the associated molecule MRP8, are different between BALB/c and C57BL/6 mice, at least in the response to LPS.

Lipopolysaccharide Potentiates Insulin-Driven Hypoglycemic Shock.

Critically ill patients typically present with hyperglycemia. Treatment with conventional insulin therapy (targeting 144-180 mg/dl) improves patient survival; however, intensive insulin therapy (IIT) targeting normal blood glucose levels (81-108 mg/dl) increases the incidence of moderate and severe hypoglycemia, and increases mortality. Septic patients are especially prone to IIT-induced hypoglycemia, but the mechanism remains unknown. Here, we show that codelivery of insulin with otherwise sublethal doses of LPS induced hypoglycemic shock in mice within 1-2 h. LPS impaired clearance of insulin, which amplified insulin receptor signaling. These effects were mediated by caspase-11, TLR4, and complement, each of which trigger eicosanoid production that potentiates insulin signaling. Finally, in an animal model of sepsis, we observed that Salmonella typhimurium-infected mice exhibited simultaneous impaired insulin clearance coexisting with insulin resistance. Our results raise the possibility that septic patients have impaired insulin clearance, which could increase their susceptibility to hypoglycemia during IIT, contraindicating its use.

Abstract 20934: Chronic Polarization of Inflammatory Monocytes by Super-Low Grade Endotoxin Aggravates the Pathogenesis of Atherosclerosis

The subclinical levels of Gram negative bacterial endotoxin, LPS, circulating in the blood are prevalent in the individuals with adverse health conditions or lifestyles. LPS is a potent agent in the dynamic programming of monocytes, and altered innate immune homeostasis mediated by non-resolving inflammatory monocytes has long been suspected in the pathogenesis of atherosclerosis. However, the mechanisms responsible for the sustainment of inflammatory monocytes during atherosclerosis are poorly understood. We hypothesized that the selective modulation of molecular switches in monocytes by super-low dose LPS may facilitate the establishment of non-resolving inflammation during atherosclerosis. We challenged high-fat-diet (HFD) fed ApoE deficient mice with chronic LPS administration, and observed that LPS effectively induced expansion of Ly6C high pro-inflammatory monocytes with elevated levels of CCR5, MCP-1, and reduced SR-B1. Compare to PBS administration, chronic injection of super-low dose LPS significantly elevated the lipid deposition within the atherosclerotic plaques in HFD fed ApoE deficient mice, while induced remarkable reduction in collagen content of the plaques. To test whether the LPS reprogrammed monocytes, instead of systemic effects of LPS, may contribute the exacerbation of atherosclerosis, we performed adoptive transfer experiment. Bone-marrow monocytes harvested from ApoE deficient mice were primed in vitro with super-low dose LPS for 5 days, and then intravenously injected into HFD fed ApoE once weekly for 1 month. We observed significant increases of the plaque sizes as well as lipid deposition in atherosclerotic lesions, indicating that programmed monocytes directly aggravated atherosclerosis progression. At the molecular level, we observed that monocytes programmed by super-low dose LPS maintained higher levels of transcription factor IRF5 and reduced levels of a critical inflammatory repressor, Blimp-1. Intriguingly, the inflammatory monocyte priming by super-low dose LPS was dependent upon TRAM/TRIF but not MyD88. In conclusion, our data suggest super-low grade LPS may program sustained low-grade inflammatory monocytes that are conducive for the chronic pathogenesis of atherosclerosis.

Endogenous pleiotrophin and midkine regulate LPS-induced glial responses.

Pleiotrophin (PTN) and Midkine (MK) are two growth factors that modulate neuroinflammation. PTN overexpression in the brain prevents LPS-induced astrocytosis in mice but potentiates microglial activation. The modest astrocytic response caused by a low dose of LPS (0.5mg/kg) is blocked in the striatum of MK-/- mice whereas microglial response is unaffected. We have now tested the effects of an intermediate dose of LPS (7.5mg/kg) in glial response in PTN-/- and MK-/- mice. We found that LPS-induced astrocytosis is prevented in prefrontal cortex and striatum of both PTN-/- and MK-/- mice. Some of the morphological changes of microglia induced by LPS tended to increase in both genotypes, particularly in PTN-/- mice. Since we previously showed that PTN potentiates LPS-induced activation of BV2 microglial cells, we tested the activation of FYN kinase, a substrate of the PTN receptor RPTPβ/ζ, and the subsequent ERK1/2 phosphorylation on LPS and PTN-treated BV2 cells. LPS effects on BV2 cells were not affected by the addition of PTN, suggesting that PTN does not recruit the FYN-MAP kinase signaling pathway in order to modulate LPS effects on microglial cells. Taking together, evidences demonstrate that regulation of astroglial responses to LPS administration are highly dependent on the levels of expression of PTN and MK. Further studies are needed to clarify the possible roles of endogenous expression of PTN and MK in LPS-induced microglial responses.