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1.  The novel role of IL-7 ligation to IL-7R in myeloid cells of rheumatoid arthritis and collagen induced arthritis 
Although role of IL-7 and IL-7R has been implicated in the pathogenesis of Rheumatoid arthritis (RA), the majority of the studies have focused on the impact of IL-7/IL-7R in T cell development and function. Our novel data, however, document that RA patients with greater disease activity have higher levels of IL-7, IL-7R and TNF-α in RA monocytes suggesting a feedback regulation between IL-7/IL-7R and TNF-α cascades in myeloid cells which is linked to chronic disease progression. Investigations into the involved mechanism showed that IL-7 is a novel and potent chemoattractant which attracts IL-7R+ monocytes through activation of the PI3K/AKT1 and ERK pathways at similar concentrations of IL-7 detected in RA synovial fluid. To determine whether ligation of IL-7 to IL-7R is a potential target for RA treatment and to identify their mechanism of action, collagen induced arthritis (CIA) was therapeutically treated with anti-IL-7 antibody or IgG control. Anti-IL-7 antibody treatment significantly reduces CIA monocyte recruitment and osteoclast differentiation as well as potent joint monocyte chemoattractants and bone erosion markers suggesting that both direct and indirect pathways may contribute to the observed effect. We also demonstrate that reduction in joint MIP-2 levels is responsible for suppressed vascularization detected in anti-IL-7 antibody treated mice compared to the control group. In conclusion we show for the first time that expression of IL-7/IL-7R in myeloid cells is strongly correlated with RA disease activity and that ligation of IL-7 to IL-7R contributes to monocyte homing, differentiation of osteoclasts and vascularization in the CIA effector phase.
doi:10.4049/jimmunol.1201675
PMCID: PMC3686279  PMID: 23606539
monocyte migration; collagen induced arthritis; disease correlation; IL-7; IL-7R and TNF-α
2.  Nrf2 is essential for the expression of lipocalin–prostaglandin D synthase induced by prostaglandin D2 
Free radical biology & medicine  2013;65:1134-1142.
Nrf2 is a transcription factor that protects against inflammatory diseases, but the underlying mechanism of this effect remains unclear. Here, we report that Nrf2 uses lipocalin–prostaglandin D synthase (L-PGDS) as a mechanism for suppressing inflammation. Exogenously added prostaglandin D2 (PGD2) induced L-PGDS expression in bone-marrow-derived macrophages (BMDMs), suggesting a positive feedback loop between L-PGDS expression and PGD2. Unlike lipopolysaccharide (LPS)-induced L-PGDS expression, PGD2-mediated expression was independent of MAPK, PU.1, or TLR4. Sequence analysis located a putative Nrf2 binding site in the murine L-PGDS promoter, to which Nrf2 bound when treated with PGD2. Chemical activation, or overexpression, of Nrf2 was sufficient to induce L-PGDS expression in macrophages, BMDMs, or lungs of Nrf2-knockout (KO) mice, but treatment with PGD2 failed to do so, suggesting a pivotal role for Nrf2 in the expression of L-PGDS. Consistent with this, expression of Nrf2 in the lungs of Nrf2-KO mice was sufficient to induce the expression of L-PGDS and to reduce neutrophilic lung inflammation elicited by LPS. Furthermore, expression of L-PGDS in mouse lungs decreased neutrophilic infiltration, ameliorating lung inflammation in mice. Together, our results show that Nrf2, activated by PGD2, induced L-PGDS expression, resulting in decreased inflammation. We suggest that the positive feedback induction of L-PGDS by PGD2 is part of the mechanism by which Nrf2 regulates inflammation.
doi:10.1016/j.freeradbiomed.2013.08.192
PMCID: PMC3972891  PMID: 24029383
Nrf2; Lung inflammation; Lipocalin-prostaglandin D synthase; Prostaglandin D2; Gene expression; Free radicals
3.  Method Development and Validation for Ultra-High Pressure Liquid Chromatography/Tandem Mass Spectrometry Determination of Multiple Prostanoids in Biological Samples 
Journal of AOAC International  2013;96(1):67-76.
Following oxygenation of arachidonic acid by cyclooxygenase to form prostaglandin H2 (PGH2), a variety of prostanoids can be generated with diverse physiologic effects on pain, inflammation, allergy, cardiovascular system, cancer, etc. To facilitate the quantitative analysis of prostanoids in human serum of cell culture, an ultra-high pressure LC (UHPLC)/MS/MS method was developed and validated for the measurement of six eicosanoids belonging to the cyclooxygenase pathway: PGE2, PGD2, 8-iso-PGF2α, PGF2α, 6-keto-PGF1α, and thromboxane B2 (TXB2). Selectivity, matrix effects, calibration model, precision, and accuracy (intraday and interday), lower limit of quantitation (LLOQ), recovery, stability, and sample dilution were evaluated. Fast UHPLC separation was carried out in only 0.5 min with isocratic elution, and each prostanoid was measured using negative electrospray ionization MS with collision-induced dissociation and selected reaction monitoring. UHPLC/MS/MS provided high throughput with peak widths of approximately 3 s and an LLOQ of 0.020 ng/mL for PGE2, 0.027 ng/mL for PGD2, 0.152 ng/mL for 8-iso-PGF2α, 0.179 ng/mL for PGF2α and 6-keto-PGF1α, and 0.013 ng/mL for TXB2.
PMCID: PMC3723117  PMID: 23513960
4.  Therapeutic Effect of the Tuber of Alisma orientale on Lipopolysaccharide-Induced Acute Lung Injury 
Although Alisma orientale, an ethnic herb, has been prescribed for treating various diseases in Asian traditional medicine, experimental evidence to support its therapeutic effects is lacking. Here, we sought to determine whether A. orientale has a therapeutic effect on acute lung injury (ALI). Ethanol extract of the tuber of A. orientale (EEAO) was prepared and fingerprinted by HPLC for its constituents. Mice received an intraperitoneal (i.p.) injection of lipopolysaccharide (LPS) for the induction of ALI. At 2 h after LPS treatment, mice received an intratracheal (i.t.) spraying of various amounts of EEAO to the lung. Bioluminescence imaging of transgenic NF-κB/luciferase reporter mice shows that i.t. EEAO posttreatment suppressed lung inflammation. In similar experiments with C57BL/6 mice, EEAO posttreatment significantly improved lung inflammation, as assessed by H&E staining of lung sections, counting of neutrophils in bronchoalveolar lavage fluid, and semiquantitative RT-PCR analyses of proinflammatory cytokines and Nrf2-dependent genes in the inflamed lungs. Furthermore, EEAO posttreatment enhanced the survival of mice that received a lethal dose of LPS. Together, our results provide evidence that A. orientale has a therapeutic effect on ALI induced by sepsis.
doi:10.1155/2013/863892
PMCID: PMC3745860  PMID: 23983806
5.  Protective Role of Reactive Oxygen Species in Endotoxin-Induced Lung Inflammation through Modulation of IL-10 Expression 
Reactive Oxygen Species (ROS) generated by NADPH oxidase are generally known to be pro-inflammatory, and it seems to be counterintuitive that ROS play a critical role in regulating the resolution of the inflammatory response. However, we observed that deficiency of the p47phox component of NADPH oxidase in macrophages was associated with a paradoxical accentuation of inflammation in a whole animal model of non-infectious sepsis induced by endotoxin. We have confirmed this observation by interrogating four separate in vivo models that employ complementary methodology including the use of p47phox−/− mice, p47phox−/− bone marrow chimera mice, adoptive transfer of macrophages from p47phox−/− mice, and an isolated perfused lung edema model that all point to a relationship between excessive acute inflammation and p47phox deficiency in macrophages. Mechanistic data indicate that ROS deficiency in both cells and mice results in decreased production of IL-10 in response to treatment with LPS, at least in part, through attenuation of the Akt-GSK3-β signal pathway and that it can be reversed by the administration of recombinant IL-10. Our data support the innovative concept that generation of ROS is essential for counter-regulation of acute lung inflammation.
doi:10.4049/jimmunol.1101323
PMCID: PMC3358534  PMID: 22547702
NADPH oxidase; p47phox; NF-kB; macrophage; endotoxin; sepsis
6.  Dangkwisoo-san, an herbal medicinal formula, ameliorates acute lung inflammation via activation of Nrf2 and suppression of NF-κB 
Journal of ethnopharmacology  2012;140(1):107-116.
Ethnopharmacological relevance
Dangkwisoo-san (DS), an herbal medicinal formula, has long been used in Korea for the treatment of inflammatory complications caused by physical trauma. Although the therapeutic effect of DS is likely associated with anti-inflammatory activity, the precise underlying mechanisms are largely unknown. Here we sought to elucidate the possible mechanisms of anti-inflammatory activity of DS.
Materials and Methods
The water extract of DS was orally fed to C57BL/6 mice for 14 days prior to LPS intranasal instillation for lung inflammation. The effects of DS on lung inflammation were determined by differential cell counting, lung histology, and semi-quantitative RT-PCR of lung sections. The effects of DS on the activities of Nrf2 and NF-κB were assessed by western blotting, semi-quantitative RT-PCR, and luciferase reporter assays in RAW 264.7, an NF-κB reporter cell line, and HEK 293 transfected with an NF-κB reporter construct.
Results
Mice that were treated with a water extract of DS showed significant attenuation of lung inflammation induced by intranasal lipopolysaccharide (LPS) compared to control mice treated with vehicle. In vitro experiments show that DS activated Nrf2, an anti-oxidant transcription factor that protects from various inflammatory diseases, and inducedNrf2-regulated genes including GCLC, NQO-1 and HO-1. In addition, DS suppressed NF-κB activity and reduced the production of pro-inflammatory cytokines. Transfection experiment indicates that inhibition of NF-κB likely occurred upstream of IKK complex. Furthermore, DS enhanced the expression of HO-1 and suppressed that of IL-1β and TNF-α in inflamed mouse lungs.
Conclusions
These results suggest that the therapeutic effects of DS are related with suppression of inflammation, which is, at least in part, mediated by activation of anti-inflammatory factor Nrf2 and inhibition of pro-inflammatory factor NF-κB.
doi:10.1016/j.jep.2011.12.043
PMCID: PMC3616192  PMID: 22230470
acute lung inflammation; anti-inflammation; Nrf2; NF-κB; Dangkwisoo-san; lipopolysaccharide
7.  ent-kaur-16-en-19-oic acid, isolated from the roots of Aralia continentalis, induces activation of Nrf2 
Journal of ethnopharmacology  2011;137(3):1442-1449.
Aim of the study
Excessive inflammation can lead to tissue damage and dysfunction of vital organs. Hence, regulating inflammatory response is a viable therapeutic approach. In Asian countries, various inflammatory diseases have often effectively been treated with herbal remedies including the root extract of Aralia continentalis Kitagawa (Araliaceae). Here, we investigated the effect of kaurenoic acid (ent-kaur-16-en-19-oic acid: KA), a diterpenoid that is extracted from Aralia continentalis Kitagawa root, on inflammation.
Results
Western blot and RT-PCR analyses show that KA induced the nuclear localization of Nrf2 as low as 1 nM in concentration and that KA treatment induced the expression of Nrf2 dependent genes such as GCLC and HO-1. On the other hand, KA did not affect the degradation of cytoplasmic IκB-α, the nuclear localization of RelA (p65), and NF-κB transcriptional activity in RAW 264.7 cells treated with endotoxin. Consistent with these data, KA treatment failed to suppress gene expression of representative pro-inflammatory mediators including COX-2, nitric oxide, IL-1β, TNF-α, and IL-12, indicating that KA did not have an important impact on NF-κB activation.
Conclusion
Together, these results show that KA was an effective activator of Nrf2, and suggest that the beneficial effects of A. continentalis Kitagawa root extract are, at least in part, mediated by activating Nrf2.
doi:10.1016/j.jep.2011.08.024
PMCID: PMC3616193  PMID: 21884778
Inflammation; Kaurenoic acid; Herbal treatment; anti-inflammation; Nrf2
8.  Local delivery of Granulocyte/Macrophage Colony Stimulating Factor protects mice from lethal pneumococcal pneumonia1 
The growth factor granulocyte/macrophage-colony stimulating factor (GM-CSF) has an important role in pulmonary surfactant metabolism and the regulation of antibacterial activities of lung sentinel cells. However, the potential of intra-alveolar GM-CSF to augment lung protective immunity against inhaled bacterial pathogens has not been defined in preclinical infection models. We hypothesized that transient overexpression of GM-CSF in the lungs of mice by adenoviral gene transfer (Ad-GM-CSF) would protect mice from subsequent lethal pneumococcal pneumonia. Our data show that intra-alveolar delivery of Ad-GM-CSF led to sustained increased pSTAT5 expression and PU.1 protein expression in alveolar macrophages during a 28 day observation period. Pulmonary Ad-GM-CSF delivery two or four weeks prior to infection of mice with S. pneumoniae significantly reduced mortality rates relative to control vector treated mice. This increased survival was accompanied by increased iNOS expression, antibacterial activity and a significant reduction in caspase 3 dependent apoptosis and secondary necrosis of lung sentinel cells. Importantly, therapeutic treatment of mice with recombinant GM-CSF improved lung protective immunity and accelerated bacterial clearance after pneumococcal challenge. We conclude that prophylactic delivery of GM-CSF triggers long-lasting immunostimulatory effects in the lung in vivo and rescues mice from lethal pneumococcal pneumonia by improving antibacterial immunity. These data support use of novel antibiotic-independent immunostimulatory therapies to protect patients against bacterial pneumonias.
doi:10.4049/jimmunol.1101413
PMCID: PMC3595102  PMID: 22003204
GM-CSF; S. pneumoniae; PU.1; pneumonia; therapy; infection
9.  Role for the guanine nucleotide exchange factor P-Rex1 in platelet secretion and aggregation 
Objective
Recent studies have shown a role for Rac1 in regulating platelet functions, but how Rac1 is activated in platelets remains unclear. PIP3-dependent Rac exchanger 1 (P-Rex1) was originally identified in neutrophils that regulates phagocyte functions. We sought to examine whether P-Rex1 plays a role in platelet activation.
Methods and Results
Western blotting showed P-Rex1 expression in mouse and human platelets. Mice lacking P-Rex1 exhibited prolonged bleeding time and increased re-bleeding. When challenged with low doses of the G protein-coupled receptor (GPCR) agonists U46619 and thrombin, P-Rex1-/- platelets displayed significantly reduced secretion and aggregation compared to WT platelets. Increasing the concentration of these agonists could overcome the defect. Platelet aggregation induced by collagen, a non-GPCR agonist, was also compromised in the absence of P-Rex1. Along with these phenotypic changes were impaired Rac1 activation, reduced ATP secretion, decreased phosphorylation of Akt, JNK and p38 MAPK in P-Rex1-/- platelets upon agonist stimulation.
Conclusion
These results demonstrate for the first time the presence of P-Rex1 in platelets and its role in platelet secretion as well as aggregation induced by low-dose agonists for GPCR and by collagen.
doi:10.1161/ATVBAHA.111.243675
PMCID: PMC3288658  PMID: 22207728
platelets; P-Rex1; Rac1; secretion; aggregation
10.  Anthrax Lethal Factor Activates K+ Channels To Induce IL-1β Secretion in Macrophages 
Anthrax lethal toxin (LeTx) is a virulence factor of Bacilillus anthracis that is a bivalent toxin, containing lethal factor (LF) and protective Ag proteins, which causes cytotoxicity and altered macrophage function. LeTx exposure results in early K+ efflux from macrophages associated with caspase-1 activation and increased IL-1β release. The mechanism of this toxin-induced K+ efflux is unknown. The goals of the current study were to determine whether LeTx-induced K+ efflux from macrophages is mediated by toxin effects on specific K+ channels and whether altered K+-channel activity is involved in LeTx-induced IL-1β release. Exposure of macrophages to LeTx induced a significant increase in the activities of two types of K+ channels that have been identified in mouse macrophages: Ba2+-sensitive inwardly rectifying K+ (Kir) channels and 4-aminopyridine–sensitive outwardly rectifying voltage-gated K+ (Kv) channels. LeTx enhancement of both Kir and Kv required the proteolytic activity of LF, because exposure of macrophages to a mutant LF-protein (LFE687C) combined with protective Ag protein had no effect on the currents. Furthermore, blocking Kir and Kv channels significantly decreased LeTx-induced release of IL-1β. In addition, retroviral transduction of macrophages with wild-type Kir enhanced LeTx-induced release of IL-1β, whereas transduction of dominant-negative Kir blocked LeTx-induced release of IL-1β. Activation of caspase-1 was not required for LeTx-induced activation of either of the K+ channels. These data indicate that a major mechanism through which LeTx stimulates macrophages to release IL-1β involves an LF-protease effect that enhances Kir and Kv channel function during toxin stimulation.
doi:10.4049/jimmunol.1001078
PMCID: PMC3583536  PMID: 21421849
11.  Dendritic Cell Depletion and Repopulation in the Lung after Irradiation and Bone Marrow Transplantation in Mice 
Dendritic cells (DCs) are essential for innate and adaptive immunity, but are purported to exhibit variable radiosensitivity in response to irradiation in various bone marrow transplantation (BMT) protocols. To address this controversy, we analyzed the magnitude of depletion and repopulation of both lung CD11bpos DC and CD103pos DC subsets in response to irradiation and BMT in a murine model. In our study, CD45.2pos donor bone marrow cells were transplanted into irradiated CD45.1pos recipient mice to examine the depletion of recipient DC subsets and the repopulation of donor DC subsets. We observed an apoptosis-mediated and necrosis-mediated depletion (> 90%) of the recipient CD103pos DC subset, and only a 50–60% depletion of recipient CD11bpos DCs from lung parenchymal tissue on Days 3 and 5, whereas recipient alveolar and lung macrophages were much less radiosensitive, showing an approximately 50% depletion by Days 14–21 after treatment. A repopulation of lung tissue with donor DC subsets had occurred by Days 10 and 28 for CD11bpos DCs and CD103pos DCs, whereas alveolar and lung macrophages were repopulated by 6 and 10 weeks after treatment. Furthermore, the infection of mice with Streptococcus pneumoniae further accelerated the turnover of lung DCs and lung macrophage subsets. Our data illustrate the vulnerability of lung CD103pos DCs and CD11bpos DCs to irradiation, and indicate that an accelerated turnover of lung DC subsets occurs, relative to pulmonary and lung macrophages. Our findings may have important implications in the development of adjuvant immune-stimulatory protocols that could reduce the risk of opportunistic infections in patients undergoing BMT.
doi:10.1165/rcmb.2010-0279OC
PMCID: PMC3361352  PMID: 21177980
dendritic cell; macrophage; pneumonia; Streptococcus pneumoniae; CD103
12.  Inhibitory κB Kinase 2 Activates Airway Epithelial Cells to Stimulate Bone Marrow Macrophages 
It has not been resolved whether macrophages or airway epithelial cells primarily respond to infectious and inflammatory stimuli and initiate a cell-to-cell inflammatory interaction within the airways. We hypothesized that the airway epithelial cells are primary responders that activate macrophages in response to environmental stimuli. To investigate the unilateral contribution of airway epithelial cells in the activation of macrophages, we developed an in vitro system in which the primary mouse tracheal epithelial cells (MTEC) and primary bone marrow–derived macrophages (BMDM) were incubated together for a brief period of time in a Transwell culture plate. MTEC were transfected with adenoviral vectors that express a constitutively active form of IKK2 (Ad-cIKK2), Ad-β-Gal, or PBS for 48 h before incubating with the macrophages. Macrophage activation was determined by measuring surface expression of CD11b, activation of NF-κB, phagocytic activity and production of reactive oxygen species, and cyclooxygenase (COX)-2 gene expression and production of prostaglandins. Macrophage adherence to epithelial layer was confirmed by CD68 immunostaining and scanning electron microscopy. MTEC cells transfected with Ad-cIKK2 produced increased amounts of IL-6, mouse GRO-α, TNF-α, and prostaglandin (PG)E2. Exposure of BMDM to MTEC, transfected with Ad-cIKK2, led to an increase in the CD11b expression and increased adherence of macrophages to the epithelial cell layer. NF-κB activation, COX-2 gene expression, and PGD2 synthesis were also increased in BMDM that were incubated with MTEC transfected with Ad-cIKK2. These data suggest that airway epithelial cells potentially play a primary role in generating inflammatory signals that result in activation of macrophages.
doi:10.1165/rcmb.2006-0245OC
PMCID: PMC1899334  PMID: 17204585
airway epithelium; macrophages; lung; inflammation
13.  Protective Effect of the Fruit Hull of Gleditsia sinensis on LPS-Induced Acute Lung Injury Is Associated with Nrf2 Activation 
The fruit hull of Gleditsia sinensis (FGS) has been prescribed as a traditional eastern Asian medicinal remedy for the treatment of various respiratory diseases, but the efficacy and underlying mechanisms remain poorly characterized. Here, we explored a potential usage of FGS for the treatment of acute lung injury (ALI), a highly fatal inflammatory lung disease that urgently needs effective therapeutics, and investigated a mechanism for the anti-inflammatory activity of FGS. Pretreatment of C57BL/6 mice with FGS significantly attenuated LPS-induced neutrophilic lung inflammation compared to sham-treated, inflamed mice. Reporter assays, semiquantitative RT-PCR, and Western blot analyses show that while not affecting NF-κB, FGS activated Nrf2 and expressed Nrf2-regulated genes including GCLC, NQO-1, and HO-1 in RAW 264.7 cells. Furthermore, pretreatment of mice with FGS enhanced the expression of GCLC and HO-1 but suppressed that of proinflammatory cytokines in including TNF-α and IL-1β in the inflamed lungs. These results suggest that FGS effectively suppresses neutrophilic lung inflammation, which can be associated with, at least in part, FGS-activating anti-inflammatory factor Nrf2. Our results suggest that FGS can be developed as a therapeutic option for the treatment of ALI.
doi:10.1155/2012/974713
PMCID: PMC3312273  PMID: 22474532
14.  Krüppel Like Factor 4 Promoter Undergoes Active Demethylation during Monocyte/Macrophage Differentiation 
PLoS ONE  2014;9(4):e93362.
The role of different lineage specific transcription factors in directing hematopoietic cell fate towards myeloid lineage is well established but the status of epigenetic modifications has not been defined during this important developmental process. We used non proliferating, PU.1 inducible myeloid progenitor cells and differentiating bone marrow derived macrophages to study the PU.1 dependent KLF4 transcriptional regulation and its promoter demethylation during monocyte/macrophage differentiation. Expression of KLF4 was regulated by active demethylation of its promoter and PU.1 specifically bound to KLF4 promoter oligo harboring the PU.1 consensus sequence. Methylation specific quantitative PCR and Bisulfite sequencing indicated demethylation of CpG residues most proximal to the transcription start site of KLF4 promoter. Cloned KLF4 promoter in pGL3 Luciferase and CpG free pcpgf-bas vectors showed accentuated reporter activity when co-transfected with the PU.1 expression vector. In vitro methylation of both KLF4 promoter oligo and cloned KLF4 promoter vectors showed attenuated in vitro DNA binding activity and Luciferase/mouse Alkaline phosphotase reporter activity indicating the negative influence of KLF4 promoter methylation on PU.1 binding. The Cytosine deaminase, Activation Induced Cytidine Deaminase (AICDA) was found to be critical for KLF4 promoter demethylation. More importantly, knock down of AICDA resulted in blockade of KLF4 promoter demethylation, decreased F4/80 expression and other phenotypic characters of macrophage differentiation. Our data proves that AICDA mediated active demethylation of the KLF4 promoter is necessary for transcriptional regulation of KLF4 by PU.1 during monocyte/macrophage differentiation.
doi:10.1371/journal.pone.0093362
PMCID: PMC3973678  PMID: 24695324
16.  Importance of CXC Chemokine Receptor 2 in Alveolar Neutrophil and Exudate Macrophage Recruitment in Response to Pneumococcal Lung Infection▿  
Infection and Immunity  2010;78(6):2620-2630.
Sustained neutrophilic infiltration is known to contribute to organ damage, such as acute lung injury. CXC chemokine receptor 2 (CXCR2) is the major receptor regulating inflammatory neutrophil recruitment in acute and chronic inflamed tissues. Whether or not the abundant neutrophil recruitment observed in severe pneumonia is essential for protective immunity against Streptococcus pneumoniae infections is incompletely defined. Here we show that CXCR2 deficiency severely perturbs the recruitment of both neutrophils and exudate macrophages associated with a massive bacterial outgrowth in distal airspaces after infection with S. pneumoniae, resulting in 100% mortality in knockout (KO) mice within 3 days. Moreover, irradiated wild-type mice reconstituted with increasing amounts of CXCR2 KO bone marrow (10, 25, 50, and 75% KO) have correspondingly decreased numbers of both neutrophils and exudate macrophages, which is associated with a stepwise increase in bacterial burden and a reciprocal stepwise decrease in survival in S. pneumoniae-induced pulmonary infection. Finally, application of the CXCR2 antagonist SB-225002 resulted in decreased alveolar neutrophil and exudate macrophage recruitment in mice along with increased lung bacterial loads after infection with S. pneumoniae. Together, these data show that CXC chemokine receptor 2 serves a previously unrecognized nonredundant role in the regulation of both neutrophil and exudate macrophage recruitment to the lung in response to S. pneumoniae infection. In addition, we demonstrate that a threshold level of 10 to 25% of reduced neutrophil recruitment is sufficient to cause increased mortality in mice infected with S. pneumoniae.
doi:10.1128/IAI.01169-09
PMCID: PMC2876546  PMID: 20368349
17.  Genetic determination of the role of PU.1 in macrophage gene expression 
PU.1, an Ets family transcription factor, mediates macrophage effector function in inflammation by regulating gene expression. But, the extent and nature of PU.1 function in gene expression has not been genetically determined because ablation of PU.1 gene abolishes macrophage development. Here, we epigenetically suppressed PU.1 by stably expressing PU.1 specific siRNA in macrophages, and determined the effect of PU.1 deficiency on expressions of key inflammatory genes: Toll-like receptor 4 (TLR4), cyclooxygenease-2 (COX-2), and macrophage inflammatory protein-1α (MIP-1α). PU.1-silenced cell lines expressed lower TLR4 mRNA and COX-2 protein, but higher MIP-1α protein, than controls. Over-expression of PU.1 suppressed LPS-induced MIP-1α production. PU.1 occupied proximal and distal cognate sites in the endogenous MIP-1α promoter, but dissociated only from the distal sites in response to lipopolysaccharide, suggesting a novel negative regulatory mechanism by PU.1. Together, our results defined PU.1 function in differentially regulating expressions of TLR4, COX-2 and MIP-1α.
doi:10.1016/j.bbrc.2008.04.189
PMCID: PMC2494535  PMID: 18485892
PU.1; epigenetic suppression; TLR4; COX-2; MIP-1α; gene regulation; macrophages; inflammation
18.  An improved LC-MS-MS method for the quantification of prostaglandins E2 and D2 production in biological fluids 
Analytical biochemistry  2007;372(1):41-51.
We report an improved LC-MS-MS assay that accurately measures prostaglandins D2 (PGD2) and E2 (PGE2) in cell culture supernatants and other biological fluids. The limit of detection for each prostaglandin was 20 pg/mL (0.20 pg; 0.55 fmol on-column), and the inter-day and intra-day coefficients of variation were less than 5%. Both d4-PGE2 and d4-PGD2 were used as surrogate standards to control for differential loss and degradation of the analytes. Stability studies indicated that sample preparation time should be less than 8 h to measure PGD2 accurately, whereas preparation time did not affect PGE2 measurement due to its greater stability in biological samples. As an application of the method, PGD2 and PGE2 were measured in culture supernatants from A549 cells and RAW 264.7 cells. The human lung alveolar cell line A549 was found to produce PGE2 but no PGD2 while the murine macrophage cell line RAW 264.7 produced PGD2 and only trace amounts of PGE2. This direct comparison showed that COX-2 gene expression can lead to differential production of PGD2 and PGE2 by epithelial cells and macrophages. Since PGE2 is anti-asthmatic and PGD2 is pro-asthmatic, we speculate that the balance of production of these eicosanoids by epithelial cells and macropahges in the lung contributes to the pathogenesis of COPD, bronchiectasis, asthma, and lung cancer.
doi:10.1016/j.ab.2007.08.041
PMCID: PMC2276632  PMID: 17976507
Prostaglandins; PGD2; PGE2; cell culture; A549 cells; RAW264.7 cells; LC-MS-MS
19.  Ambient particulate matter accelerates coagulation via an IL-6–dependent pathway 
The Journal of Clinical Investigation  2007;117(10):2952-2961.
The mechanisms by which exposure to particulate matter increases the risk of cardiovascular events are not known. Recent human and animal data suggest that particulate matter may induce alterations in hemostatic factors. In this study we determined the mechanisms by which particulate matter might accelerate thrombosis. We found that mice treated with a dose of well characterized particulate matter of less than 10 μM in diameter exhibited a shortened bleeding time, decreased prothrombin and partial thromboplastin times (decreased plasma clotting times), increased levels of fibrinogen, and increased activity of factor II, VIII, and X. This prothrombotic tendency was associated with increased generation of intravascular thrombin, an acceleration of arterial thrombosis, and an increase in bronchoalveolar fluid concentration of the prothrombotic cytokine IL-6. Knockout mice lacking IL-6 were protected against particulate matter–induced intravascular thrombin formation and the acceleration of arterial thrombosis. Depletion of macrophages by the intratracheal administration of liposomal clodronate attenuated particulate matter–induced IL-6 production and the resultant prothrombotic tendency. Our findings suggest that exposure to particulate matter triggers IL-6 production by alveolar macrophages, resulting in reduced clotting times, intravascular thrombin formation, and accelerated arterial thrombosis. These results provide a potential mechanism linking ambient particulate matter exposure and thrombotic events.
doi:10.1172/JCI30639
PMCID: PMC1978421  PMID: 17885684
20.  Pathogen–Host Interactions in Pseudomonas aeruginosa Pneumonia 
Pseudomonas aeruginosa is an important pathogen causing a wide range of acute and chronic infections. P. aeruginosa rarely causes infection in the normal host, but is an efficient opportunistic pathogen causing serious infections in patients who are mechanically ventilated, individuals who are immunocompromised, and patients with malignancies or HIV infection. Among these risk groups, the most vulnerable hosts are neutropenic and patients who are mechanically ventilated. In addition, P. aeruginosa is the most prevalent chronic infection contributing to the pathogenesis of cystic fibrosis. Because of the ubiquitous nature of P. aeruginosa and its ability to develop resistance to antibiotics, it continues to be problematic from a treatment perspective. The pathogenicity of P. aeruginosa is largely caused by multiple bacterial virulence factors and genetic flexibility enabling it to survive in varied environments. Lung injury associated with P. aeruginosa infection results from both the direct destructive effects of the organism on the lung parenchyma and exuberant host immune responses. This article focuses on the major bacterial virulence factors and important aspects of the host immunity that are involved in the pathogenesis of serious P. aeruginosa infection. In addition to antibiotic therapy, strategies directed toward enhancing host defense and/or limiting excessive inflammation could be important to improve outcome in P. aeruginosa lung infections.
doi:10.1164/rccm.200408-1044SO
PMCID: PMC2718459  PMID: 15695491
cystic fibrosis; cytokines; epithelium; host defense; nosocomial
21.  Endotoxin induced peritonitis elicits monocyte immigration into the lung: implications on alveolar space inflammatory responsiveness 
Respiratory Research  2006;7(1):30.
Background
Acute peritonitis developing in response to gram-negative bacterial infection is known to act as a trigger for the development of acute lung injury which is often complicated by the development of nosocomial pneumonia. We hypothesized that endotoxin-induced peritonitis provokes recruitment of monocytes into the lungs, which amplifies lung inflammatory responses to a second hit intra-alveolar challenge with endotoxin.
Methods
Serum and lavage cytokines as well as bronchoalveolar lavage fluid cells were analyzed at different time points after intraperitoneal or intratracheal application of LPS.
Results
We observed that mice challenged with intraperitoneal endotoxin developed rapidly increasing serum and bronchoalveolar lavage fluid (BALF) cytokine and chemokine levels (TNFα, MIP-2, CCL2) and a nearly two-fold expansion of the alveolar macrophage population by 96 h, but this was not associated with the development of neutrophilic alveolitis. In contrast, expansion of the alveolar macrophage pool was not observed in CCR2-deficient mice and in wild-type mice systemically pretreated with the anti-CD18 antibody GAME-46. An intentional two-fold expansion of alveolar macrophage numbers by intratracheal CCL2 following intraperitoneal endotoxin did not exacerbate the development of acute lung inflammation in response to intratracheal endotoxin compared to mice challenged only with intratracheal endotoxin.
Conclusion
These data, taken together, show that intraperitoneal endotoxin triggers a CCR2-dependent de novo recruitment of monocytes into the lungs of mice but this does not result in an accentuation of neutrophilic lung inflammation. This finding represents a previously unrecognized novel inflammatory component of lung inflammation that results from endotoxin-induced peritonitis.
doi:10.1186/1465-9921-7-30
PMCID: PMC1388208  PMID: 16503998
22.  Hepatitis C Virus Core Protein Suppresses NF-κB Activation and Cyclooxygenase-2 Expression by Direct Interaction with IκB Kinase β 
Journal of Virology  2005;79(12):7648-7657.
In addition to hepatocytes, hepatitis C virus (HCV) infects immune cells, including macrophages. However, little is known concerning the impact of HCV infection on cellular functions of these immune effector cells. Lipopolysaccharide (LPS) activates IκB kinase (IKK) signalsome and NF-κB, which leads to the expression of cyclooxygenase-2 (COX-2), which catalyzes production of prostaglandins, potent effectors on inflammation and possibly hepatitis. Here, we examined whether expression of HCV core interferes with IKK signalsome activity and COX-2 expression in activated macrophages. In reporter assays, HCV core inhibited NF-κB activation in RAW 264.7 and MH-S murine macrophage cell lines treated with bacterial LPS. HCV core inhibited IKK signalsome and IKKβ kinase activities induced by tumor necrosis factor alpha in HeLa cells and coexpressed IKKγ in 293 cells, respectively. HCV core was coprecipitated with IΚΚβ and prevented nuclear translocation of IKKβ. NF-κB activation by either LPS or overexpression of IKKβ was sufficient to induce robust expression of COX-2, which was markedly suppressed by ectopic expression of HCV core. Together, these data indicate that HCV core suppresses IKK signalsome activity, which blunts COX-2 expression in macrophages. Additional studies are necessary to determine whether interrupted COX-2 expression by HCV core contributes to HCV pathogenesis.
doi:10.1128/JVI.79.12.7648-7657.2005
PMCID: PMC1143634  PMID: 15919917
23.  Impaired Pulmonary NF-κB Activation in Response to Lipopolysaccharide in NADPH Oxidase-Deficient Mice 
Infection and Immunity  2001;69(10):5991-5996.
Reactive oxygen species (ROS) are thought to be involved in intracellular signaling, including activation of the transcription factor NF-κB. We investigated the role of NADPH oxidase in the NF-κB activation pathway by utilizing knockout mice (p47phox−/−) lacking the p47phox component of NADPH oxidase. Wild-type (WT) controls and p47phox−/− mice were treated with intraperitoneal (i.p.) Escherichia coli lipopolysaccharide (LPS) (5 or 20 μg/g of body weight). LPS-induced NF-κB binding activity and accumulation of RelA in nuclear protein extracts of lung tissue were markedly increased in WT compared to p47phox−/− mice 90 min after treatment with 20 but not 5 μg of i.p. LPS per g. In another model of lung inflammation, RelA nuclear translocation was reduced in p47phox−/− mice compared to WT mice following treatment with aerosolized LPS. In contrast to NF-κB activation in p47phox−/− mice, LPS-induced production of macrophage inflammatory protein 2 in the lungs and neutrophilic lung inflammation were not diminished in these mice compared to WT mice. We conclude that LPS-induced NF-κB activation is deficient in the lungs of p47phox−/− mice compared to WT mice, but this abnormality does not result in overt alteration in the acute inflammatory response.
doi:10.1128/IAI.69.10.5991-5996.2001
PMCID: PMC98726  PMID: 11553535
24.  Lipopolysaccharide-Induced Expression of Microsomal Prostaglandin E Synthase-1 Mediates Late-Phase PGE2 Production in Bone Marrow Derived Macrophages 
PLoS ONE  2012;7(11):e50244.
Cyclooxygenase (COX)-2 expression and release of prostaglandins (PGs) by macrophages are consistent features of lipopolysaccharide (LPS)-induced macrophage inflammation. The two major PGs, PGE2 and PGD2, are synthesized by the prostanoid isomerases, PGE synthases (PGES) and PGD synthases (PGDS), respectively. Since the expression profile and the individual role of these prostanoid isomerases-mediated inflammation in macrophages has not been defined, we examined the LPS-stimulated PGs production pattern and the expression profile of their synthases in the primary cultured mouse bone marrow derived macrophages (BMDM). Our data show that LPS induced both PGE2 and PGD2 production, which was evident by ∼8 hrs and remained at a similar ratio (∼1∶1) in the early phase (≤12 hrs) of LPS treatment. However, PGE2 production continued increase further in the late phase (16–24 hrs); whereas the production of PGD2 remained at a stable level from 12 to 24 hrs post-treatment. In response to LPS-treatment, the expression of both COX-2 and inducible nitric oxide synthase (iNOS) was detected within 2 to 4 hrs; whereas the increased expression of microsomal PGES (mPGES)-1 and a myeloid cell transcription factor PU.1 did not appear until later phase (≥12 hrs). In contrast, the expression of COX-1, hematopoietic-PGDS (H-PGDS), cytosolic-PGES (c-PGES), or mPGES-2 in BMDM was not affected by LPS treatment. Selective inhibition of mPGES-1 with either siRNA or isoform-selective inhibitor CAY10526, but not mPGES-2, c-PGES or PU.1, attenuated LPS-induced burst of PGE2 production indicating that mPGES-1 mediates LPS-induced PGE2 production in BMDM. Interestingly, selective inhibition of mPGES-1 was also associated with a decrease in LPS-induced iNOS expression. In summary, our data show that mPGES-1, but not mPGES-2 or c-PGES isomerase, mediates LPS-induced late-phase burst of PGE2 generation, and regulates LPS-induced iNOS expression in BMDM.
doi:10.1371/journal.pone.0050244
PMCID: PMC3511480  PMID: 23226252
25.  NADPH Oxidase Limits Innate Immune Responses in the Lungs in Mice 
PLoS ONE  2010;5(3):e9631.
Background
Chronic granulomatous disease (CGD), an inherited disorder of the NADPH oxidase in which phagocytes are defective in generating superoxide anion and downstream reactive oxidant intermediates (ROIs), is characterized by recurrent bacterial and fungal infections and by excessive inflammation (e.g., inflammatory bowel disease). The mechanisms by which NADPH oxidase regulates inflammation are not well understood.
Methodology/Principal Findings
We found that NADPH oxidase restrains inflammation by modulating redox-sensitive innate immune pathways. When challenged with either intratracheal zymosan or LPS, NADPH oxidase-deficient p47phox−/− mice and gp91phox-deficient mice developed exaggerated and progressive lung inflammation, augmented NF-κB activation, and elevated downstream pro-inflammatory cytokines (TNF-α, IL-17, and G-CSF) compared to wildtype mice. Replacement of functional NADPH oxidase in bone marrow-derived cells restored the normal lung inflammatory response. Studies in vivo and in isolated macrophages demonstrated that in the absence of functional NADPH oxidase, zymosan failed to activate Nrf2, a key redox-sensitive anti-inflammatory regulator. The triterpenoid, CDDO-Im, activated Nrf2 independently of NADPH oxidase and reduced zymosan-induced lung inflammation in CGD mice. Consistent with these findings, zymosan-treated peripheral blood mononuclear cells from X-linked CGD patients showed impaired Nrf2 activity and increased NF-κB activation.
Conclusions/Significance
These studies support a model in which NADPH oxidase-dependent, redox-mediated signaling is critical for termination of lung inflammation and suggest new potential therapeutic targets for CGD.
doi:10.1371/journal.pone.0009631
PMCID: PMC2838778  PMID: 20300512

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