Myungsoo Joo

NADPH Oxidase Limits Innate Immune Responses in the Lungs in Mice

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.

Targeted Immunomodulation of the NF-κB Pathway in Airway Epithelium Impacts Host Defense against Pseudomonas aeruginosa

We investigated the impact of inflammatory signaling in airway epithelial cells on host defense against Pseudomonas aeruginosa, a major cause of nosocomial pneumonia. In mice, airway instillation of P. aeruginosa resulted in NF-κB activation in the lungs that was primarily localized to the bronchial epithelium at 4 h, but was present in a variety of cell types by 24 h. We modulated NF-κB activity in airway epithelium by intratracheal delivery of adenoviral vectors expressing RelA (AdRelA) or a dominant inhibitor of NF-κB before P. aeruginosa infection. Bacterial clearance was enhanced by up-regulation of NF-κB activity following AdRelA administration and was impaired by treatment with a dominant inhibitor of NF-κB. The TNF-α concentration in lung lavage was increased by AdRelA treatment and beneficial effects of NF-κB up-regulation were abrogated in TNF-α-deficient mice. In contrast, NF-κB inhibition reduced MIP-2 expression and neutrophil influx following P. aeruginosa infection. Therefore, inflammatory signaling through the NF-κB pathway in airway epithelial cells critically regulates the innate immune response to P. aeruginosa.

Hepatitis C Virus Core Protein Suppresses NF-κB Activation and Cyclooxygenase-2 Expression by Direct Interaction with IκB Kinase β

ABSTRACT 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.

Bacterial clearance of Pseudomonas aeruginosa is enhanced by the inhibition of COX-2

Prostanoids generated by COX‐2 are involved in the regulation of inflammation but their exact role in the innate immune response has not been defined. We investigated whether COX‐2 is involved in host defense against Pseudomonas aeruginosa pneumonia. In vitro studies, in a macrophage cell line, showed that cytotoxic strain of P aeruginosa (PA103) induced significant COX‐2 protein expression and enzymatic function. In vivo data showed that infection with PA103 increased COX‐2 protein production in whole lung tissue compared to mice that were infected with mutant bacteria that lack ExoU (ΔU) or ExoU and ExoT (ΔUT). COX‐2–/– mice had accentuated clearance of cytotoxic P. aeruginosa from the lungs. We further tested the effects of COX‐2 products such as prostaglandin E2 on the function of phagocytic cells. Our studies indicate that prostaglandin E2 may be involved through interacting with the EP2 receptors in modulating the host response because treatment of macrophages with prostaglandin E2 suppressed production of reactive oxygen species. Furthermore there was enhanced bacterial clearance in EP2 receptor–/– mice compared to the wild‐type controls. Thus it is possible that inhibition of COX‐2 or EP2 receptors could be an effective adjunctive treatment for severe or resistant P. aeruginosa pneumonia.

A Central Role for Tumor-derived Monocyte Chemoattractant Protein-1 in Malignant Pleural Effusion

BACKGROUND Tumor cells in malignant pleural effusions (MPEs) are an important source of monocyte chemoattractant protein (MCP)-1. However, the role of tumor-derived MCP-1 in the pathogenesis and progression of MPE has not been determined. METHODS B16 mouse skin melanoma cells, which are deficient in MCP-1 expression, and mouse Lewis lung cancer (LLC) cells, which express high levels of MCP-1, were engineered to stably express MCP-1 and short hairpin RNAs (shRNAs) targeting the MCP-1 transcript, respectively. Cells were injected into the pleural cavities of syngeneic immunocompetent mice, and MPE volume and pleural tumors were quantified at necropsy (day 14). MCP-1 and other mediators were determined by cytometric bead array and enzyme-linked immunosorbent assay, and mononuclear and endothelial cells were identified by immunolabeling of F4/80 and factor VIII-related antigen respectively. Mouse survival was assessed using Kaplan-Meier analysis. Vascular permeability in mice with MPE was assessed using albumin-binding Evans blue. Statistical tests were two-sided. RESULTS LLC cells expressing shRNA against MCP-1 elaborated less than 5% of the MCP-1 level in cells expressing nonspecific shRNA (control cells), and intrapleural delivery of these cells resulted in less MPE (mean MPE volume = 86 and 585 muL, respectively; difference = 499 muL; 95% confidence interval [CI] = 331 to 669 muL; P < .001), reduced MCP-1 levels in the pleural fluid, and lower mortality than when control cells were delivered. Overexpression of MCP-1 in intrapleurally injected B16 melanoma cells led to increased MPE and reduced survival. In mice with MPE, MCP-1 was a potent inducer of vascular permeability, mononuclear recruitment, and, in pleural tumors, of angiogenesis. CONCLUSION MCP-1 produced by tumor cells is an important determinant of their capacity to induce the formation of MPE and may be a useful target for the treatment of malignant pleural disease.

Ethanol extract of Alismatis Rhizoma reduces acute lung inflammation by suppressing NF-κB and activating Nrf2

ETHNOPHARMACOLOGICAL RELEVANCE The tuber of Alisma orientale Juzepzuk, a medicinal herb that has been used for the treatment of various disorders in Korea, has an anti-inflammatory effect. Here, we investigated a possible underlying mechanism and a protective effect on acute lung injury (ALI). MATERIALS AND METHODS Alisma orientale tuber was extracted in 80% ethanol and dried. The powder of the ethanol extract of Alisma orientale tuber (EEAO) was dissolved in PBS. The effect of EEAO on NF-κB and Nrf2 activities was analyzed with RAW 264.7 cells. The effect of EEAO on lung inflammation was determined by histologic and molecular biological analyses of the lung tissue of C57BL/6 mice that were gavaged once a day with 0.3 or 1.2 g/kg of EEAO for 14 days, prior to an intranasal administration of LPS (0.01 g/kg) for inducing ALI. RESULTS EEAO pre-treatment of RAW 264.7 cells suppressed NF-κB activity and the expression of its dependent genes including COX-2, IL-1β and iNOS. Similar treatment enhanced Nrf2 activity and the expression of Nrf2-regulated genes including NQO-1, HO-1 and GCLC. LPS instillation induced acute neutrophilic lung inflammation, which was significantly suppressed by pre-treatment with EEAO. Analysis of the lungs revealed that EEAO pre-treatment induced the expression of Nrf2-regulated genes, with concomitant down-regulation of inflammatory gene expression. CONCLUSIONS EEAO attenuated lung inflammation in LPS-induced ALI mice, which was associated with differential regulation of NF-κB and Nrf2 activities. We suggest that EEAO can be developed as a potential therapeutics for the treatment of ALI.

PGD Synthase and PGD2 in Immune Resposne

PGD2 is formed from arachidonic acid by successive enzyme reactions: oxygenation of arachidonic acid to PGH2, a common precursor of various prostanoids, catalyzed by cyclooxygenase, and isomerization of PGH2 to PGD2 by PGD synthases (PGDSs). PGD2 can be either pro- or anti-inflammatory depending on disease process and etiology. The anti-inflammatory and immunomodulatory attributes of PGDS/PGD2 provide opportunities for development of novel therapeutic approaches for resistant infections and refractory inflammatory diseases. This paper highlights the role of PGD synthases and PGD2 in immune inflammatory response.

Induction and Function of Lipocalin Prostaglandin D Synthase in Host Immunity

Although mainly expressed in neuronal cells, lipocalin-type PGD synthase (L-PGDS) is detected in the macrophages infiltrated to atherosclerotic plaques. However, the regulation and significance of L-PGDS expression in macrophages are unknown. Here, we found that treatment of macrophages with bacterial endotoxin (LPS) or Pseudomonas induced L-PGDS expression. Epigenetic suppression of L-PGDS expression in macrophages blunted a majority of PGD2 produced after LPS treatment. Chromatin immunoprecipitation assays show that L-PGDS induction was regulated positively by AP-1, but negatively by p53. L-PGDS expression was detected in whole lung and alveolar macrophages treated with LPS or Pseudomonas. L-PGDS overexpressing transgenic mice improved clearance of Pseudomonas from the lung compared with nontransgenic mice. Similarly, intratracheal instillation of PGD2 enhanced removal of Pseudomonas from the lung in mice. In contrast, L-PGDS knockout mice were impaired in their ability to remove Pseudomonas from the lung. Together, our results identify induction of L-PGDS expression by inflammatory stimuli or bacterial infection, the regulatory mechanism of L-PGDS induction, and the protective role of L-PGDS expression in host immune response. Our study suggests a potential therapeutic usage of L-PGDS or PGD2 against Pseudomonas pneumonia.

Rehmannia glutinosa suppresses inflammatory responses elicited by advanced glycation end products.

Fresh rhizome of Rehmannia glutinosa Libosch. (Saeng-jihwang in Korean: SJH) has been prescribed for the treatment of diabetes-associated complications. The purpose of the present study is to investigate the underlying mechanisms of the efficacy of SJH in diabetes-related complications. Decoction was obtained after boiling SJH in water and subsequent lyophilization. The cellular toxicity of SJH was determined by MTT assay. The antioxidant activity of SJH was measured by DPPH and DCFH-DA assays. The effects of SJH on inflammatory responses elicited by AGEs were assessed by western blotting and semi-quantitative RT-PCR analyses. The water extract of SJH had a high free radical scavenging activity in vitro and decreased the level of intracellular ROS in THP-1 cells treated with AGEs. SJH suppressed the expression of pro-inflammatory genes, including TNF-α, MCP-1, IP-10, COX-2, and iNOS; the activation of NF-κB; and the expression of RAGE, a receptor for AGEs, where the expressions of which were induced by AGEs. These results suggest the possibility that SJH can be an alternative therapeutics for diabetes-associated diseases.

Peptidylarginine deiminase 2 suppresses inhibitory kappa B kinase activity in lipopolysaccharide-stimulated RAW 264.7 macrophages

Peptidylarginine deiminases (PADs) are enzymes that convert arginine to citrulline in proteins. In this study, we examined PAD-mediated citrullination and its effect on pro-inflammatory activity in the macrophage cell line RAW 264.7. Citrullination of 45–65-kDa proteins was induced when cells were treated with lipopolysaccharide (LPS; 1 μg/ml). Protein citrullination was suppressed by the intracellular calcium chelator BAPTA/AM (30 μm). LPS treatment up-regulated COX-2 levels in cells. Interestingly, overexpressing PAD2 reduced LPS-mediated COX-2 up-regulation by 50%. PAD2 overexpression also reduced NF-κB activity, determined by NF-κB-driven luciferase activity. The effect of PAD2 on NF-κB activity was further examined by using HEK 293 cells transfected with NF-κB luciferase, IκB β/γ kinase (IKKβ/γ) subunits, and PAD2. IKKβ increased NF-κB activity, but this increase was markedly suppressed when PAD2 was present in cells. IKKβ-mediated NF-κB activation was further enhanced by IKKγ in the presence of calcium ionophore A23187. However, this stimulatory effect of IKKβ/γ was abolished by PAD2. Coimmunoprecipitation of cell lysates showed that IKKγ and PAD2 can coimmunoprecipitate in the presence of the Ca2+ ionophore. IKKγ coimmunoprecipitated truncation mutants, PAD2(1–385) and PAD2(355–672). The substitution of Gln-358 (a putative ligand for Ca2+ binding) with an Ala abolished coimmunoprecipitation. Conversely, PAD2 coimmunoprecipitated truncation mutants IKKγ(1–196) and IKKγ(197–419). In other experiments, treating RAW 264.7 cells with LPS induced citrullination in the immunoprecipitates of IKKγ. In vitro citrullination assay showed that incubation of purified PAD2 and IKKγ proteins in the presence of Ca2+ citrullinated IKKγ. These results demonstrate that PAD2 interacts with IKKγ and suppresses NF-κB activity.