LPS-induced Lung Inflammation Research Articles

Inhibition of miRNA-34a Promotes M2 Macrophage Polarization and Improves LPS-Induced Lung Injury by Targeting Klf4.

Acute respiratory distress syndrome (ARDS) is an outcome of an accelerated immune response that starts initially as a defensive measure, however, due to non-canonical signaling, it later proves to be fatal not only to the affected tissue but to the whole organ system. microRNAs are known for playing a decisive role in regulating the expression of genes involved in diverse functions such as lung development, repair, and inflammation. In-silico analyses of clinical data and microRNA databases predicted a probable interaction between miRNA-34a (miR-34a), mitogen-activated protein kinase 1 (ERK), and kruppel like factor 4 (Klf4). Parallel to in silico results, here, we show that intra-tracheal instillation of lipopolysaccharides (LPS) to mice enhanced miR-34a expression in lung macrophages. Inhibition of miR-34a significantly improved lung histology, whereas over-expression of miR-34a worsened the lung injury phenotype. miR-34a over-expression in macrophages were also demonstrated to favour pro-inflammatory M1 phenotype and inhibition of M2 polarization. In a quest to confirm this likely interaction, expression profiles of Klf4 as the putative target were analyzed in different macrophage polarizing conditions. Klf4 expression was found to be prominent in the miR-34a inhibitor-treated group but down-regulated in the miR-34a mimic treated group. Immuno-histopathological analyses of lung tissue from the mice treated with miR-34a inhibitor also showed reduced inflammatory M1 markers as well as enhanced cell proliferation. The present study indicates that miR-34a intensified LPS-induced lung injury and inflammation by regulating Klf4 and macrophage polarization, which may serve as a potential therapeutic target for acute lung injury/ARDS.

Treatment of acute lung inflammation by pulmonary delivery of anti-TNF-α siRNA with PAMAM dendrimers in a murine model

To improve the efficacy of nucleic acid-based therapeutics, e.g., small interfering RNA (siRNA), transfection agents are needed for efficient delivery into cells. Several classes of dendrimers have been found useful as transfection agents for the delivery of siRNA because their surface can readily be functionalized, and the size of the dendriplexes they form with siRNA is within the range of conventional nanomedicine. In this study, commercially available generation 3 poly(amidoamine) (PAMAM) dendrimer was investigated for pulmonary delivery of siRNA directed against tumor necrosis factor (TNF) α for the treatment of acute lung inflammation. Delivery efficiency was assessed in vitro in the RAW264.7 macrophage cell line activated with lipopolysaccharide (LPS), and efficacy was evaluated in vivo in a murine model of LPS-induced lung inflammation upon pre-treatment with TNF-α siRNA. The PAMAM dendrimer-siRNA complexes (dendriplexes) displayed strong siRNA condensation and high cellular uptake in macrophages compared with non-complexed siRNA. Q-PCR analyses showed that the dendriplexes mediated efficient and specific TNF-α silencing in vitro, as compared to non-complexed siRNA and dendriplexes with negative control siRNA. Also in vivo, the PAMAM dendriplexes induced efficacious TNF-α siRNA inhibition, as compared to non-complexed siRNA, upon pulmonary administration to mice with LPS-induced lung inflammation. Hence, these data suggest that PAMAM dendrimers are promising for the local delivery of TNF-α siRNA in the treatment of lung inflammation via pulmonary administration.

Correction to: A role for mast cells and mast cell tryptase in driving neutrophil recruitment in LPS-induced lung inflammation via protease-activated receptor 2 in mice

The original article can be found online.

A role for mast cells and mast cell tryptase in driving neutrophil recruitment in LPS-induced lung inflammation via protease-activated receptor 2 in mice.

This study aims to investigate the role of protease-activated receptor (PAR) 2 and mast cell (MC) tryptase in LPS-induced lung inflammation and neutrophil recruitment in the lungs of C57BL/6 mice. C57BL/6 mice were pretreated with the PAR2 antagonist ENMD-1068, compound 48/80 or aprotinin prior to intranasal instillation of MC tryptase or LPS. Blood leukocytes, C-X-C motif chemokine ligand (CXCL) 1 production leukocytes recovered from bronchoalveolar lavage fluid (BALF), and histopathological analysis of the lung were evaluated 4h later. Furthermore, we performed experiments to determine intracellular calcium signaling in RAW 264.7 cells stimulated with LPS in the presence or absence of a protease inhibitor cocktail or ENMD-1068 and evaluated PAR2 expression in the lungs of LPS-treated mice. Pharmacological blockade of PAR2 or inhibition of proteases reduced neutrophils recovered in BALF and LPS-induced calcium signaling. PAR2 blockade impaired LPS-induced lung inflammation, PAR2 expression in the lung and CXCL1 release in BALF, and increased circulating blood neutrophils. Intranasal instillation of MC tryptase increased the number of neutrophils recovered in BALF, and MC depletion with compound 48/80 impaired LPS-induced neutrophil migration. Our study provides, for the first time, evidence of a pivotal role for MCs and MC tryptase in neutrophil migration, lung inflammation and macrophage activation triggered by LPS, by a mechanism dependent on PAR2 activation.

Lung and general health effects of Toll-like receptor-4 (TLR4)-interacting SPA4 peptide

BackgroundA surfactant protein-A-derived peptide, which we call SPA4 peptide (amino acids: GDFRYSDGTPVNYTNWYRGE), alleviates lung infection and inflammation. This study investigated the effects of intratracheally administered SPA4 peptide on systemic, lung, and health parameters in an outbred mouse strain, and in an intratracheal lipopolysaccharide (LPS) challenge model.MethodsThe outbred CD-1 mice were intratracheally administered with incremental doses of SPA4 peptide (0.625–10 μg/g body weight) once every 24 h, for 3 days. Mice left untreated and those treated with vehicle were included as controls. Mice were euthanized after 24 h of last administration of SPA4 peptide. In order to assess the biological activity of SPA4 peptide, C57BL6 mice were intratracheally challenged with 5 μg LPS/g body weight and treated with 50 μg SPA4 peptide via intratracheal route 1 h post LPS-challenge. Mice were euthanized after 4 h of LPS challenge. Signs of sickness and body weights were regularly monitored. At the time of necropsy, blood and major organs were harvested. Blood gas and electrolytes, serum biochemical profiles and SPA4 peptide-specific immunoglobulin G (IgG) antibody levels, and common lung injury markers (levels of total protein, albumin, and lactate, lactate dehydrogenase activity, and lung wet/dry weight ratios) were determined. Lung, liver, spleen, kidney, heart, and intestine were examined histologically. Differences in measured parameters were analyzed among study groups by analysis of variance test.ResultsThe results demonstrated no signs of sickness or changes in body weight over 3 days of treatment with various doses of SPA4 peptide. It did not induce any major toxicity or IgG antibody response to SPA4 peptide. The SPA4 peptide treatment also did not affect blood gas, electrolytes, or serum biochemistry. There was no evidence of injury to the tissues and organs. However, the SPA4 peptide suppressed the LPS-induced lung inflammation.ConclusionsThese findings provide an initial toxicity profile of SPA4 peptide. Intratracheal administration of escalating doses of SPA4 peptide does not induce any significant toxicity at tissue and organ levels. However, treatment with a dose of 50 μg SPA4 peptide, comparable to 2.5 μg/g body weight, alleviates LPS-induced lung inflammation.

Ficolin A derived from local macrophages and neutrophils protects against lipopolysaccharide-induced acute lung injury by activating complement.

Ficolins are important and widely distributed pattern recognition molecules that can induce lectin complement pathway activation and initiate the innate immune response. Although ficolins can bind lipopolysaccharide (LPS) in vitro, the sources, dynamic changes and roles of local ficolins in LPS-induced pulmonary inflammation and injury remain poorly understood. In this study, we established a ficolin knockout mouse model by clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology, and used flow cytometry and hematoxylin and eosin staining to study the expressions and roles of local ficolins in LPS-induced pulmonary inflammation and injury. Our results show that besides ficolin B (FcnB), ficolin A (FcnA) is also expressed in leukocytes from the bone marrow, peripheral blood, lung and spleen. Further analyses showed that macrophages and neutrophils are the main sources of FcnA and FcnB, and T and B cells also express a small amount of FcnB. The intranasal administration of LPS induced local pulmonary inflammation with the increased recruitment of macrophages and neutrophils. LPS stimulation induced increased expression of FcnA and FcnB in neutrophils at the acute stage and in macrophages at the late stage. The severity of the lung injury and local inflammation of Fcna-/- mice was increased by the induction of extracellular complement activation. The recovery of LPS-induced local lung inflammation and injury was delayed in Fcnb-/- mice. Hence, these findings suggested that the local macrophage- and neutrophil-derived FcnA protects against LPS-induced acute lung injury by mediating extracellular complement activation.

Potential Moracin M Prodrugs Strongly Attenuate Airway Inflammation In Vivo

This study aims to develop new potential therapeutic moracin M prodrugs acting on lung inflammatory disorders. Potential moracin M prodrugs (KW01-KW07) were chemically synthesized to obtain potent orally active derivatives, and their pharmacological activities against lung inflammation were, for the first time, examined in vivo using lipopolysaccharide (LPS)-induced acute lung injury model. In addition, the metabolism of KW02 was also investigated using microsomal stability test and pharmacokinetic study in rats. When orally administered, some of these compounds (30 mg/kg) showed higher inhibitory action against LPS-induced lung inflammation in mice compared to moracin M. Of them, 2-(3,5-bis((dimethylcarbamoyl)oxy)phenyl)benzofuran-6-yl acetate (KW02) showed potent and dose-dependent inhibitory effect on the same animal model of lung inflammation at 1, 3, and 10 mg/kg. This compound at 10 mg/kg also significantly reduced IL-1β concentration in the bronchoalveolar lavage fluid of the inflamed-lungs. KW02 was rapidly metabolized to 5-(6-hydroxybenzofuran-2-yl)-1,3-phenylene bis(dimethylcarbamate) (KW06) and moracin M when it was incubated with rat serum and liver microsome as expected. When KW02 was administered to rats via intravenous or oral route, KW06 was detected in the serum as a metabolite. Thus, it is concluded that KW02 has potent inhibitory action against LPS-induced lung inflammation. It could behave as a potential prodrug of moracin M to effectively treat lung inflammatory disorders.

Acupoint Catgut Embedding Improves the Lipopolysaccharide-Induced Acute Respiratory Distress Syndrome in Rats.

Background This study investigated the potential therapeutic effects of acupoint catgut embedding (ACE) at ST36 and BL13 on lipopolysaccharide- (LPS-) induced acute respiratory distress syndrome (ARDS) in rats. Materials and Methods Male Sprague-Dawley rats were randomized into the normal saline (NS group with a sham procedure), lipopolysaccharide (LPS group with a sham procedure), and LPS plus ACE (LPS+ACE with ACE at bilateral BL13 and ST36 acupoints one day before LPS injection) groups. After intratracheal instillation of normal saline or LPS (0.5 mg/kg), all rats were subjected to mechanical ventilation for 4 h. Their blood gas was analyzed before and after lung injury, and their lung pressure-volumes were measured longitudinally. The levels of TNF-α, IL-6, IL-10, and phosphatidylcholine (PC) and total proteins (TP) in bronchial alveolar lavage fluid (BALF) were assessed. Their wet to dry lung weight ratios, histology, myeloperoxidase (MPO), superoxide dismutase (SOD) activity, and malondialdehyde (MDA) levels were measured. Their lung aquaporin 1 (AQP1) and Occludin protein levels were analyzed. Results LPS administration significantly decreased the ratios of PaO2/FiO2 and pressure-volumes and induced lung inflammation and injury by increased concentrations of TNF-α, IL-6, IL-10, and TP in BALF and MPO and MDA in the lung but decreased PC in BALF and SOD activity in the lungs. LPS also reduced AQP1 and Occludin protein levels in the lung of rats. In contrast, ACE significantly mitigated the LPS-induced lung injury, inflammation, and oxidative stress and preserved the AQP1 and Occludin contents in the lung of rats. Conclusions ACE significantly improved respiratory function by mitigating inflammation and oxidative stress and preserving AQP1 and Occludin expression in the lung in a rat model of LPS-induced ARDS.

The pterocarpanquinone LQB 118 inhibits inflammation triggered by zymosan in vivo and in vitro

LQB 118, a hydride molecule, has been described as an antineoplastic and antiparasitic drug. Recently, LQB118 was also shown to display anti-inflammatory properties using an LPS-induced lung inflammation model. However, LQB 118 effects on the inflammatory response induced by zymosan has not been demonstrated. In this study, swiss mice were LQB 118 intraperitoneally (i.p.) treated and zymosan was used to induce peritoneal inflammation. Peritoneal fluid was collected and used for cell counting and proinflammatory cytokines quantification (IL-1β, IL-6, and TNF-α) by immunoenzymatic assay (ELISA). For in vitro studies, peritoneal macrophages zymosan-stimulated were used. Results demonstrated that LQB 118 treatment reduced polymorphonuclear cell migration and TNF-α, IL-1β, and IL-6 levels in the peritoneal cavity. In macrophages, LQB 118 treatment display no cytotoxic effect and is also able to reduce cytokines levels. To investigate LQB 118 putative mechanism of action, TLR2, CD69, and P-p38 MAPK expression were evaluated. LQB 118 treatment reduced CD69 expression and p38 phosphorylation induced by zymosan. Furthermore, LQB 118 was able to negatively modulate TLR2 expression in the presence of inflammatory stimulus. Thus, our study provide new evidences for the mechanisms related to the anti-inflammatory effect of LQB 118 in vivo and in vitro.

Does p-coumaric acid improve cardiac injury following LPS-induced lung inflammation through miRNA-146a activity?

In cardiovascular diseases, inflammatory response plays an important role and affects heart function. As a flavonoid compound, p-coumaric acid (pCA), commonly exists in many fruits and vegetables and has a therapeutic effect on inflammatory diseases due to its anti-inflammatory properties. The purpose of the present study was to investigate pCA anti-inflammatory effect and the miRNAs (miRs) signaling pathway involved in cardiac inflammation following lipopolysaccharide-induced acute lung injury (ALI). Thirty-two Sprague-Dawley male rats were divided into 4 groups: control (received saline for 10 days, i.p.), LPS (received saline for 10 days+5 mg/kg LPS on day 8, intratracheally), pCA (received pCA 100 mg/kg for 10 days, ip), and LPS+pCA (received LPS+pCA). The level of IL-1β, IL-18 in heart tissue and IL-1β in bronchoalveolar lavage fluid (BALF) was determined by ELISA kits. Also the level of lactate dehydrogenase (LDH) in heart tissue and myeloperoxidase (MPO) in lung tissue were measured, and pCA effect on miR- 146a in heart tissue was analyzed. Data showed that 100 mg/kg of pCA significantly suppressed LDH activity (p<0.05), IL-18 (p<0.05) and IL-1β (p<0.01) level in heart tissue. Also, in BAL, IL-1β and MPO levels were significantly reduced (p<0.001). Finally, pCA modulated activation of miR-146a (p<0.05) in LPS -induced cardiac injury. These findings indicated that LPS causes cardiac dysfunction and pre-treatment with pCA, as an anti-inflammatory agent, improved cardiac inflammation through modulation of miR-146a, and reducing cytokines and LDH activity.

MicroRNA-486-5p Promotes Acute Lung Injury via Inducing Inflammation and Apoptosis by Targeting OTUD7B.

The aim of this article is to study the effect of miR-486-5p in acute lung injury (ALI). MiR-486-5p expression in peripheral blood was determined in ALI patients and healthy volunteers by qRT-PCR. ALI mouse model were reproduced by LPS treatment, and miR-486-5p NC and miRNA-486 inhibitors were injected through trachea. ALI patients' peripheral blood and LPS-induced acute lung injury in mice had significantly higher miR-486-5p levels than control subjects. Inhibition of miR-486-5p by injection with antagomiR-486-5p markedly reduced LPS-induced lung inflammation. Moreover, knockdown of miR-486-5p can reduce protects A549 cell against LPS-induced injury and its corresponding inflammatory response. In addition, Mechanistic analysis indicated that miR-486-5p on the occurrence of ALI is related to the inhibition of OTUD7B activity, which induces the downregulation of inflammatory in ALI. Our results identified miR-486-5p independently associated with ALI. miR-486-5p can mediate the formation of ALI by promoting inflammation.

Effect of Intratracheal Instillation of ZnO Nanoparticles on Acute Lung Inflammation Induced by Lipopolysaccharides in Mice.

Although studies have shown toxic effects of zinc oxide (ZnO) particles following inhalation, additional effects on injured lungs, which are characterized by dysfunction of the alveolar-capillary barriers, remain uncharacterized. To explore these additional effects, nano-sized ZnO (nZnO) and bulk-sized ZnO were applied to lipopolysaccharide (LPS)-challenged mouse lungs, which were used as a disease model of acute lung inflammation. An elevated Zn2+ concentration was detected in lung tissue after LPS plus nZnO exposure. Exposure to nZnO in LPS-challenged mice resulted in higher total cell number, proportion of neutrophils, and total protein level in bronchoalveolar lavage fluid. Intratracheal instillation of nZnO intensively aggravated LPS-induced lung inflammation that was accompanied by enhanced expression of interleukin-1β, interleukin-6, monocyte chemotactic protein-1α, and granulocyte-macrophage colony stimulating factor. Catalase, glutathione, and total superoxide dismutase levels were significantly decreased, and the malondialdehyde level was obviously increased in the LPS plus nZnO group. 8-Hydroxyguanosine, a marker for DNA damage, was highly concentrated in the lungs from the LPS plus nZnO group. Furthermore, nZnO increased lung apoptosis in an acute lung inflammation model. Taken together, this evidence indicates that nZnO aggravates lung inflammation related to LPS. This enhancement effect may be mediated via oxidative stress, which can lead to DNA damage and apoptosis. This work is important because of the ever-increasing exposure of people to ZnO nanoparticles in industry. The identification of the toxic effects of nZnO and possible mechanisms revealed in this study provide valuable information for future studies.

WNK4–SPAK modulates lipopolysaccharide-induced macrophage activation

Dysregulation of alveolar macrophage activation has been recognized as the major mechanism in the pathogenesis of acute lung injury. The aim of the present study was to investigate the role of NKCC1 regulating mechanism in modulating macrophage activation. Knockout (SPAK−/− and WNK4−/−) and knockin (WNK4D561A/+) mice were used in this study. LPS induced expression of p-NKCC1 and activation of NFκB in the primary culture of alveolar macrophages. WNK4 or SPAK knockout suppressed p-NKCC1 expression and inflammation cascade activation, whereas WNK4 knockin enhanced these responses. Intrapulmonary administration of LPS induced in vivo expression and phosphorylation of NKCC1 in alveolar inflammation cells and caused a shift in the cell population from macrophage to neutrophil predominance. WNK4 or SPAK knockout attenuated the LPS-induced alveolar cell-population shifting, macrophage NKCC1 phosphorylation, and acute lung injury, whereas WNK4 knockin augmented the inflammatory response. In summary, our results demonstrated the presence of NKCC1 in alveolar macrophage, which is inducible by lipopolysaccharide. Our results also showed showed that the WNK4–SPAK–NKCC1 cascade plays an important role in modulating macrophage activation to regulate LPS-induced lung inflammation and lung injury.

CHF6001 Inhibits NF-κB Activation and Neutrophilic Recruitment in LPS-Induced Lung Inflammation in Mice.

Inhibitors of phosphodiesterase 4 (PDE4) are potent anti-inflammatory agents, inhibiting the production of inflammatory mediators through the elevation of intracellular cAMP concentrations. We studied the activity of a novel PDE4 inhibitor, CHF6001, both in vitro in human cells and in vivo, using bioluminescence imaging (BLI) in mice lung inflammation. Mice transiently transfected with the luciferase gene under the control of an NF-κB responsive element (NF-κB-luc) have been used to assess the in vivo anti-inflammatory activity of CHF6001 in lipopolysaccharide (LPS)-induced lung inflammation. BLI as well as inflammatory cells and the concentrations of pro-inflammatory cytokines were monitored in bronchoalveolar lavage fluids (BALF) while testing in vitro its ability to affect the production of leukotriene B4 (LTB4), measured by LC/MS/MS, by LPS/LPS/N-formyl--methionyl--leucyl-phenylalanine (fMLP)-activated human blood. CHF6001 inhibited the production of LTB4 in LPS/fMLP-activated human blood at sub-nanomolar concentrations. LPS-induced an increase of BLI signal in NF-κB-luc mice, and CHF6001 administered by dry powder inhalation decreased in parallel luciferase signal, cell airway infiltration, and pro-inflammatory cytokine concentrations in BALF. The results obtained provide in vitro and in vivo evidence of the anti-inflammatory activity of the potent PDE4 inhibitor CHF6001, showing that with a topical administration that closely mimics inhalation in humans, it efficiently disrupts the NF-κB activation associated with LPS challenge, an effect that may be relevant for the prevention of exacerbation episodes in chronic obstructive pulmonary disease subjects.

Montelukast, Leukotriene Inhibitor, Reduces LPS-Induced Acute Lung Inflammation and Human Neutrophil Activation

ObjectivesSome pro-inflammatory lipids derived from 1 lipooxygenase enzyme are potent neutrophil chemoattractant, a cell centrally involved in acute respiratory distress syndrome (ARDS); a syndrome lacking effective treatment. Considering the beneficial effects of the leukotriene receptor inhibitor, montelukast, on other lung diseases, whether montelukast attenuates inflammation in a mouse model of ARDS, and whether it reduces LPS stimulated activation of human neutrophils was investigated. MethodsThirty-five C57Bl/6 mice were distributed into control (PBS)+24h, LPS+24h (10μg/mouse), control+48h, LPS+48h, and LPS 48h+Montelukast (10mg/kg). In addition, human neutrophils were incubated with LPS (1μg/mL) and treated with montelukast (10μM). ResultsOral-tracheal administration of montelukast significantly attenuated total cells (P<.05), macrophages (P<.05), neutrophils (P<.01), lymphocytes (P<.001) and total protein levels in BAL (P<.05), as well as IL-6 (P<.05), CXCL1/KC (P<.05), IL-17 (P<.05) and TNF-α (P<.05). Furthermore, montelukast reduced neutrophils (P<.001), lymphocytes (P<.01) and macrophages (P<.01) in the lung parenchyma. In addition, montelukast restored BAL VEGF levels (P<.05). LTB4 receptor expression (P<.001) as well as NF-κB (P<.001), a downstream target of LPS, were also reduced in lung parenchymal leukocytes. Furthermore, montelukast reduced IL-8 (P<.001) production by LPS-treated human neutrophils. ConclusionIn conclusion, montelukast efficiently attenuated both LPS-induced lung inflammation in a mouse model of ARDS and in LPS challenged human neutrophils.

Ficolin A Derived from Local Macrophages and Neutrophils Protects Against LPS-Induced Acute Lung Injury by Activating Complement

Background: Ficolins are important and widely distributed secreted pattern recognition molecules that can induce lectin complement pathway activation and initiate the innate immune response. Although ficolins can bind lipopolysaccharide (LPS) in vitro, the sources, dynamic changes and roles of ficolins in LPS-induced local pulmonary inflammation and injury remain poorly understood. Methods: C57BL/6 and Ficolins-KO mice were intranasally administrated 10 μg LPS to induce acute lung inflammation and injury. Flow cytometry and immunofluorescence were performed to reveal the expression of ficolins. Lung injury was analyzed by hematoxylin & eosin staining. Complement activation was confirmed by immunohistochemistry and sandwich ELISA. Findings: In this study, we found that ficolin A and ficolin B are expressed in leukocytes from the bone marrow, peripheral blood, lung and spleen. Further analyses showed that macrophages and neutrophils are the main sources of ficolin A and ficolin B, and T and B cells also express a small amount of ficolin B. The intranasal administration of LPS induced local pulmonary inflammation with the increased recruitment of macrophages and neutrophils. LPS stimulation induced increased expression of ficolin A and ficolin B in neutrophils at the acute stage, and in macrophages at the late stage. The severity of the lung injury and local inflammation of Fcna−/− mice was increased via the induction of extracellular complement activation. The recovery of LPS-induced local lung inflammation and injury was delayed in Fcnb−/− mice. Interpretation: The local macrophage- and neutrophil-derived ficolin A protects against LPS-induced acute lung injury by mediating extracellular complement activation. These findings suggested that ficolins might be novel therapeutic targets for gram-negative bacteria infection induced pneumonia. Funding Statement: This work was supported by National Science Grant for Distinguished Young Scholars (81425001/H0104), National Natural Science Foundation of China, China (81373114), Beijing Municipal Natural Science Foundation, China (7182013), CAMS Innovation Fund for Medical Sciences, China (CIFMS 2018-I2M-1-003, CIFMS 2016-2M-1-014, 2016-12M-006), the Chinese National Major S & T Project (2017ZX10304402-001). Declaration of Interests: All authors declare no conflicts of interest associated with this manuscript. Ethics Approval Statement: All experiments were conducted with the approval of the Institutional Animal Care and Use Committee (IACUC) of Capital Medical University.

Abstract # 3234 The role of neutrophil granulocytes in immune-to-brain communication during LPS-induced systemic versus lung inflammation

Chronic prenatal stress contributes to poor birth outcomes for women and infants. Importantly, poor birth outcomes are most common among minority and low income women. To investigate underlying mechanisms, we tested the hypothesis that chronic stress related to minority or low income status is associated with glucocorticoid resistance as indicated by disruption in the cytokine-glucocorticoid feedback circuit. Home visits were conducted during which 3rd trimester pregnant women completed stress and depression surveys and provided blood for pro- and anti-inflammatory cytokines. Saliva was collected 5 times the preceding day for diurnal cortisol levels. For statistical analyses, women were grouped 3 ways, by race, income, and the presence or absence of either of those risk factors; this last group was labeled high or low general risk. Immune regulation was evaluated by evidence of a functioning negative feedback relationship between cytokines and cortisol. Of 96 participants, 18 were minority, 22 of low income, and 29 either minority or low income (high general risk). Pearson partial correlation identified a significant negative relationship between cortisol area under the curve (AUC) and pro- to anti-inflammatory cytokine ratios in the low general risk women (i.e., Caucasian, higher income) including IFNγ/IL10 (r = −0.73, p < 0.0001), IL6/IL10 (r = −0.38, p = 0.01), IL1β/IL10 (r = −0.44, p = 0.004) and TNFα/IL10 (r = −0.41; p = 0.005); no such correlations existed in the high general risk women (i.e., minority, low income) for (IFNγ/IL10: r = −0.25, p = 0.43; IL6/IL10: r = 0.12, p = 0.70; IL1 β/IL10: r = 0.05, p = 0.87; TNFα/IL10: r = 0.10; p = 0.75), suggestive of glucocorticoid resistance. Cortisol levels throughout the day also were higher in minority and high general risk groups (p < 0.05). Without cytokine glucocorticoid feedback, a pregnant woman's ability to regulate inflammation is limited, potentially contributing to adverse maternal and infant outcomes.

1-Palmitoyl-2-Linoleoyl-3-Acetyl-rac-Glycerol (PLAG) Rapidly Resolves LPS-Induced Acute Lung Injury Through the Effective Control of Neutrophil Recruitment.

Acute lung injury (ALI) is an acute respiratory failure that is associated with excessive neutrophil recruitment and high mortality. To assess the efficacy of 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) as a therapeutic agent for ALI, this compound was administered orally to mice challenged with an intranasal dose of lipopolysaccharide (LPS). Using this model, we found that PLAG promotes resolution of ALI through effective control of LPS-induced neutrophil infiltration, endothelial permeability, and inflammatory chemokine production. In addition, the Toll like Receptor 4 (TLR4) endocytosis/exocytosis cycle was significantly accelerated in Raw 264.7 cells co-treated with PLAG/LPS, as compared to cells treated only with LPS. During this cycle, a PLAG-induced exotoxin clearance pathway was observed to occur through the prompt assembly of nicotinamide adenine dinucleotide phosphate (NADPH) units and production of reactive oxygen species (ROS), which ultimately lead to earlier LPS clearance. We further detected reduced expression, as well as faster return to homeostatic levels, of macrophage inflammatory protein (MIP)-2, in PLAG/LPS- vs. LPS-treated cells. MIP-2 is a main inducer of neutrophil migration that is mainly controlled by interferon regulatory factor 3 (IRF3) activation and is involved in the TLR4 endosomal-signaling pathway. PLAG induced TLR4-mediated TRIF-related adaptor molecules/Toll-interleukin receptor (TIR) domain-containing adaptor protein including interferon (IFN)-β/IRF3 endosomal signaling, leading to rapid association of TRAM/TRIF and TLR4 and earlier IRF3 phosphorylation in PLAG/LPS-treated vs. LPS-treated cells. PLAG specificity was further verified with PLAG analogs and metabolites known to control excessive neutrophil infiltration, suggesting that this acetylated diacylglycerol has a unique biological role in neutrophil motility. Thus, our data indicate that PLAG may represent a potential therapeutic agent for resolution of LPS-induced lung inflammation through effective MIP-2 modulation.

C-Abl-Mediated Tyrosine Phosphorylation of PARP1 Is Crucial for Expression of Proinflammatory Genes.

Poly(ADP-ribosyl)ation is a rapid and transient posttranslational protein modification mostly catalyzed by poly(ADP-ribose) polymerase-1 (PARP1). Fundamental roles of activated PARP1 in DNA damage repair and cellular response pathways are well established; however, the precise mechanisms by which PARP1 is activated independent of DNA damage, and thereby playing a role in expression of inflammatory genes, remain poorly understood. In this study, we show that, in response to LPS or TNF-α exposure, the nonreceptor tyrosine kinase c-Abl undergoes nuclear translocation and interacts with and phosphorylates PARP1 at the conserved Y829 site. Tyrosine-phosphorylated PARP1 is required for protein poly(ADP-ribosyl)ation of RelA/p65 and NF-κB-dependent expression of proinflammatory genes in murine RAW 264.7 macrophages, human monocytic THP1 cells, or mouse lungs. Furthermore, LPS-induced airway lung inflammation was reduced by inhibition of c-Abl activity. The present study elucidated a novel signaling pathway to activate PARP1 and regulate gene expression, suggesting that blocking the interaction of c-Abl with PARP1 or pharmaceutical inhibition of c-Abl may improve the outcomes of PARP1 activation-mediated inflammatory diseases.

Punica granatum and Citrillus colocynthis Aqueous Extracts Protect Mice from LPS-Induced Lung Inflammation and Inhibit Metalloproteinases-2 and -9

Punica granatum and Citrillus colocynthis Aqueous Extracts Protect Mice from LPS-Induced Lung Inflammation and Inhibit Metalloproteinases-2 and -9