LPS-Treated Macrophage Cytokines Repress Surfactant Protein–B in Lung Epithelial Cells (original) (raw)
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Journal of Experimental Medicine, 2013
Pulmonary alveolar proteinosis (PAP) results from a dysfunction of alveolar macrophages (AMs), chiefly due to disruptions in the signaling of granulocyte macrophage colony–stimulating factor (GM-CSF). We found that mice deficient for the B lymphoid transcription repressor BTB and CNC homology 2 (Bach2) developed PAP-like accumulation of surfactant proteins in the lungs. Bach2 was expressed in AMs, and Bach2-deficient AMs showed alterations in lipid handling in comparison with wild-type (WT) cells. Although Bach2-deficient AMs showed a normal expression of the genes involved in the GM-CSF signaling, they showed an altered expression of the genes involved in chemotaxis, lipid metabolism, and alternative M2 macrophage activation with increased expression of Ym1 and arginase-1, and the M2 regulator Irf4. Peritoneal Bach2-deficient macrophages showed increased Ym1 expression when stimulated with interleukin-4. More eosinophils were present in the lung and peritoneal cavity of Bach2-defic...
The American Journal of Pathology, 2013
Although inflammation plays a central role in the pathogenesis of acute lung injury, the molecular mechanisms underlying inflammatory responses in acute lung injury are poorly understood, and therapeutic options remain limited. CCAAT/enhancer-binding proteins, C/EBPb and C/EBPd, are expressed in the lung and have been implicated in the regulation of inflammatory mediators. However, their functions in lung pathobiological characteristics are not well characterized. Herein, we show that C/EBPb and C/EBPd are activated in mouse lung after intrapulmonary deposition of lipopolysaccharide (LPS). Mice carrying a targeted deletion of the C/EBPd gene displayed significant attenuation of the lung permeability index (lung vascular leak of albumin), lung neutrophil accumulation (myeloperoxidase activity), and neutrophils in bronchial alveolar lavage fluids compared with wild-type mice. These phenotypes were consistent with morphological evaluation of lung, which showed reduced inflammatory cell influx and minimal intra-alveolar hemorrhage. Moreover, mutant mice expressed considerably less tumor necrosis factor-a, IL-6, and macrophage inflammatory protein-2 in bronchial alveolar lavage fluids in LPS-injured lung compared with wild-type mice. In contrast, C/EBPb deficiency had no effect on LPS-induced lung injury. By using small-interfering RNAemediated knockdown for C/EBPd, we demonstrate, for the first time to our knowledge, that C/EBPd plays a critical role for the tumor necrosis factor-a, IL-6, and macrophage inflammatory protein-2 production in LPS-stimulated alveolar macrophages. These findings demonstrate that C/EBPd, but not C/EBPb, plays an important role in LPSinduced lung inflammatory responses and injury. (Am J Pathol 2013, 182: 420e430; http:// dx.
Transcriptional profiling of the LPS induced NF-?B response in macrophages
Bmc Immunol, 2007
Background: Exposure of macrophages to bacterial products such as lipopolysaccharide (LPS) results in activation of the NF-κB transcription factor, which orchestrates a gene expression programme that underpins the macrophage-dependent immune response. These changes include the induction or repression of a wide range of genes that regulate inflammation, cell proliferation, migration and cell survival. This process is tightly regulated and loss of control is associated with conditions such as septic shock, inflammatory diseases and cancer. To study this response, it is important to have in vitro model systems that reflect the behaviour of cells in vivo. In addition, it is necessary to understand the natural differences that can occur between individuals. In this report, we have investigated and compared the LPS response in macrophage derived cell lines and peripheral blood mononuclear cell (PBMC) derived macrophages. Results: Gene expression profiles were determined following LPS treatment of THP-1 cells for 1 and 4 hours. LPS significantly induced or repressed 72 out of 465 genes selected as being known or putative NF-κB target genes, which exhibited 4 temporal patterns of expression. Results for 34 of these genes, including several genes not previously identified as LPS target genes, were validated using real time PCR. A high correlation between microarray and real time PCR data was found. Significantly, the LPS induced expression profile of THP-1 cells, as determined using real time PCR, was found to be very similar to that of human PBMC derived macrophages. Interestingly, some differences were observed in the LPS response between the two donor PBMC macrophage populations. Surprisingly, we found that the LPS response in U937 cells was dramatically different to both THP-1 and PBMC derived macrophages. Conclusion: This study revealed a dynamic and diverse transcriptional response to LPS in macrophages, involving both the induction and repression of gene expression in a time dependent manner. Moreover, we demonstrated that the LPS induced transcriptional response in the THP-1 cell line is very similar to primary PBMC derived macrophages. Therefore, THP-1 cells represent a good model system for studying the mechanisms of LPS and NF-κB dependent gene expression.
The Journal of Immunology, 2003
Human -defensin (hBD)-2, a cationic antimicrobial peptide primarily induced in epithelial cells in response to inflammatory stimuli, plays an important role in host defense. To elucidate the expression mechanism of hBD-2 in the lung, we investigated the modulation of hBD-2 transcription in pulmonary epithelial cells by mononuclear phagocytes stimulated with LPS. Coculture of A549 pulmonary epithelial cells with Mono-Mac-6 monocytic cells in the presence of Escherichia coli LPS markedly up-regulated hBD-2 promoter activity, whereas A549 alone did not respond to LPS to activate the hBD-2 promoter. Furthermore, IL-1 and TNF-␣ in the culture supernatants from LPS-stimulated monocytic cells activated the hBD-2 promoter in A549 cells. Of note, IL-1 was more potent than TNF-␣ in this effect. In addition, a mutation of the NF-B site at ؊200 (pB1 site) completely abolished this IL-1-and TNF-␣-induced hBD-2 promoter activation, whereas NF-B inhibitors (MG-132 and helenalin) strongly suppressed it. Moreover, electrophoretic mobility shift assay suggested that NF-B, consisting of p65-p50 heterodimer, could bind to the pB1 site in cytokine-stimulated A549 cells. Interestingly, flow cytometric analysis revealed that A549 cells expressed CD14 but lacked Toll-like receptor 4, which may account for the hyporesponsiveness of A549 cells to LPS. Taken together, these results suggest that hBD-2 expression in pulmonary epithelial cells is modulated by NF-B via the actions of IL-1 and TNF-␣ produced by LPS-stimulated mononuclear phagocytes.
The Journal of Immunology, 2003
To reveal the causal role of airway epithelial NF-B activation in evoking airway inflammation, a transgenic mouse was created expressing a mutant version of the inhibitory protein I-B␣. This I-B␣ superrepressor (I-B␣ SR) acts to repress NF-B activation exclusively in airway epithelial cells, under the transcriptional control of the rat CC10 promoter (CC10-I-B␣ SR). Compared with transgene-negative littermates, intranasal instillation of LPS did not induce nuclear translocation of NF-B in airway epithelium of CC10-I-B␣ SR transgenic mice. Consequently, the influx of neutrophils into the airways and secretion of the NF-B-regulated neutrophilic chemokine, macrophage-inflammatory protein-2, and the inflammatory cytokine, TNF-␣, were markedly reduced in CC10-I-B␣ SR mice relative to the transgene-negative mice exposed to LPS. Despite an inability to activate NF-B in airway epithelium, resident alveolar macrophages from transgene-positive mice were capable of activating NF-B in a manner indistinguishable from transgene-negative mice. These findings demonstrate that airway epithelial cells play a prominent role in orchestrating the airway inflammatory response to LPS and suggest that NF-B signaling in these cells is important for modulating innate immune responses to microbial products.
GM-CSF Regulates a PU.1-Dependent Transcriptional Program Determining the Pulmonary Response to LPS
American Journal of Respiratory Cell and Molecular Biology, 2007
Alveolar macrophages (AMs) normally respond to lipopolysaccharide (LPS) by activating Toll-like receptor (TLR)-4 signaling, a mechanism critical to lung host defense against gram-negative bacteria such as Pseudomonas aeruginosa. Because granulocyte macrophage colony-stimulating factor (GM-CSF)-deficient (GM ؊/؊) mice are hyporesponsive to LPS, we evaluated the role of GM-CSF in TLR-4 signaling in AMs. Pulmonary TNF-␣ levels and neutrophil recruitment 4 h after intratracheal administration of Pseudomonas LPS were reduced in GM ؊/؊ compared with wild-type (GM ϩ/ϩ) mice. Secretion of TNF-␣ by AMs exposed to LPS ex vivo was also reduced in GM Ϫ/Ϫ mice and restored in mice expressing GM-CSF specifically in the lungs (SPC-GM ϩ/ϩ /GM Ϫ/Ϫ mice). LPS-dependent NF-B promoter activity, TNF-␣ secretion, and neutrophil chemokine release were reduced in AM cell lines derived from GM Ϫ/Ϫ mice (mAM) compared with GM ϩ/ϩ (MH-S). Retroviral expression of PU.1 in mAM cells, which normally lack PU.1, rescued all of these AM defects. To determine whether GM-CSF, via PU.1, regulated expression of TLR-4 pathway components, mRNA and protein levels for key components were evaluated in MH-S cells (GM ϩ/ϩ , PU.1 Positive), mAM cells (GM Ϫ/Ϫ , PU.1 Negative), and mAMPU.1؉ cells (GM Ϫ/Ϫ , PU.1 Positive). Cluster of differentiation antigen-14, radioprotective 105, IL-1 receptor-associated kinase (IRAK)-M mRNA, and protein were dependent upon GM-CSF and restored by expression of PU.1. In contrast, expression of other TLR-4 pathway components (myeloid differentiation-2, TLR-4, IRAK-1, IRAK-2, Toll/IL-1 receptor domain containing adapter protein/MyD88 adaptor-like, myeloid differentiation primary-response protein 88, IRAK-4, TNF receptorassociated factor-6, NF-B, inhibitor of NF-B kinase) were not GM-CSF or PU.1-dependent. These results show that GM-CSF, via PU.1, enables AM responses to P. aeruginosa LPS by regulating expression of a specific subset of components of the TLR-4 signaling pathway.
American Journal of Physiology-Lung Cellular and Molecular Physiology, 2000
Surfactant protein B (SP-B) is essential for the maintenance of biophysical properties and physiological function of pulmonary surfactant. Tumor necrosis factor-α (TNF-α), an important mediator of lung inflammation, inhibits surfactant phospholipid and surfactant protein synthesis in the lung. In the present study, we investigated the TNF-α inhibition of rabbit SP-B promoter activity in a human lung adenocarcinoma cell line (NCI-H441). Deletion experiments indicated that the TNF-α response elements are located within −236 bp of SP-B 5′-flanking DNA. The TNF-α response region contained binding sites for nuclear factor-κB (NF-κB), Sp1/Sp3, thyroid transcription factor (TTF)-1, and hepatocyte nuclear factor (HNF)-3 transcription factors. Inhibitors of NF-κB activation such as dexamethasone and N-tosyl-l-phenylalanine chloromethyl ketone and mutation of the NF-κB element did not reverse TNF-α inhibition of SP-B promoter, indicating that TNF-α inhibition of SP-B promoter activity occurs ...
Reversibility of lung inflammation caused by SP-B deficiency
AJP: Lung Cellular and Molecular Physiology, 2005
Whereas decreased concentrations of surfactant protein (SP)-B are associated with lung injury and respiratory distress, potential causal relationships between SP-B deficiency and lung inflammation remain unclear. A transgenic mouse in which human SP-B expression was placed under conditional control of doxycycline via the CCSP promoter was utilized to determine the role of SP-B in the initiation of pulmonary inflammation. Adult mice, made SP-B deficient by removal of doxycycline, developed severe respiratory failure within 4 days. Deficiency of SP-B was associated with increased minimal surface tension of the surfactant and perturbed lung mechanics. Four days of SP-B deficiency did not alter SP-C content or surfactant phospholipid content or composition. SP-B deficiency was associated with lung inflammation and increased soluble L-selectin, STAT-3, and phosphorylated STAT-3 in alveolar macrophages and alveolar epithelial cells. Alveolar IL-6, IL-1β, and macrophage inflammatory protei...
Respiratory Research, 2010
Background: Lung cancer often develops in association with chronic pulmonary inflammatory diseases with an influx of neutrophils. More detailed information on inflammatory pathways and the role of neutrophils herein is a prerequisite for understanding the mechanism of inflammation associated cancer. Methods: In the present study, we used microarrays in order to obtain a global view of the transcriptional responses of the lung to LPS in mice, which mimics an acute lung inflammation. To investigate the influence of neutrophils in this process, we depleted mice from circulating neutrophils by treatment with anti-PMN antibodies prior to LPS exposure. Results: A total of 514 genes was greater than 1.5-fold differentially expressed in the LPS induced lung inflammation model. 394 of the 514 were up regulated genes mostly involved in cell cycle and immune/ inflammation related processes, such as cytokine/chemokine activity and signalling. Down regulated genes represented nonimmune processes, such as development, metabolism and transport. Notably, the number of genes and pathways that were differentially expressed, was reduced when animals were depleted from circulating neutrophils, confirming the central role of neutrophils in the inflammatory response. Furthermore, there was a significant correlation between the differentially expressed gene list and the promutagenic DNA lesion M 1 dG, suggesting that it is the extent of the immune response which drives genetic instability in the inflamed lung. Several genes that were specifically regulated by the presence of activated neutrophils could be identified and these were mostly involved in interferon signalling, oxidative stress response and cell cycle progression. The latter possibly refers to a higher rate of cell turnover in the inflamed lung with neutrophils, suggesting that the neutrophil influx is associated with a higher risk for the accumulation and fixation of mutations. Conclusion: Gene expression profiling identified specific genes and pathways that are related to neutrophilic inflammation and could be associated to cancer development and indicate an active role of neutrophils in mediating the LPS induced inflammatory response in the mouse lung.