Martha M. Monick

The p38 Mitogen-activated Protein Kinase Is Required for NF-κB-dependent Gene Expression THE ROLE OF TATA-BINDING PROTEIN (TBP)

Endotoxin-induced cytokine gene transcription in monocytes and macrophages is regulated in part by NF-κB. We have previously shown that the p38 mitogen-activated protein (MAP) kinase is necessary for endotoxin-induced cytokine gene transcription. Due to the fact that most cytokine promoter sequences have active NF-κB sites, we hypothesized that the p38 MAP kinase was necessary for NF-κB-dependent gene expression. We found that NF-κB-dependent gene expression was reduced to near control levels with either SB 203580 or a dominant-negative p38 MAP kinase expression vector. Inhibition of the p38 MAP kinase did not alter NF-κB activation at any level, but it significantly reduced the DNA binding of TATA-binding protein (TBP) to the TATA box. The dominant-negative p38 MAP kinase expression vector interfered with the direct interaction of native TFIID (TBP) with a co-transfected p65 fusion protein. Likewise, this dominant-negative plasmid also interfered with the direct interaction of a co-transfected TBP fusion protein with the native p65 subunit. The p38 kinase also phosphorylated TFIID (TBP) in vitro, and SB 203580 inhibited phosphorylation of TFIID (TBP) in vivo. Thus, the p38 MAP kinase regulates NF-κB-dependent gene transcription, in part, by modulating activation of TFIID (TBP).

Respiratory Epithelial Cells Convert Inactive Vitamin D to Its Active Form: Potential Effects on Host Defense

The role of vitamin D in innate immunity is increasingly recognized. Recent work has identified a number of tissues that express the enzyme 1α-hydroxylase and are able to activate vitamin D. This locally produced vitamin D is believed to have important immunomodulatory effects. In this paper, we show that primary lung epithelial cells express high baseline levels of activating 1α-hydroxylase and low levels of inactivating 24-hydroxylase. The result of this enzyme expression is that airway epithelial cells constitutively convert inactive 25-dihydroxyvitamin D3 to the active 1,25-dihydroxyvitamin D3. Active vitamin D that is generated by lung epithelium leads to increased expression of vitamin D-regulated genes with important innate immune functions. These include the cathelicidin antimicrobial peptide gene and the TLR coreceptor CD14. dsRNA increases the expression of 1α-hydroxylase, augments the production of active vitamin D, and synergizes with vitamin D to increase expression of cathelicidin. In contrast to induction of the antimicrobial peptide, vitamin D attenuates dsRNA-induced expression of the NF-κB-driven gene IL-8. We conclude that primary epithelial cells generate active vitamin D, which then influences the expression of vitamin D-driven genes that play a major role in host defense. Furthermore, the presence of vitamin D alters induction of antimicrobial peptides and inflammatory cytokines in response to viruses. These observations suggest a novel mechanism by which local conversion of inactive to active vitamin D alters immune function in the lung.

Respiratory Syncytial Virus Up-regulates TLR4 and Sensitizes Airway Epithelial Cells to Endotoxin

Airway epithelial cells are unresponsive to endotoxin (lipopolysaccharide (LPS)) exposure under normal conditions. This study demonstrates that respiratory syncytial virus (RSV) infection results in increased sensitivity to this environmental exposure. Infection with RSV results in increased expression of Toll-like receptor (TLR) 4 mRNA, protein, and increased TLR4 membrane localization. This permits significantly enhanced LPS binding to the epithelial monolayer that is blocked by disruption of the Golgi. The increased TLR4 results in an LPS-induced inflammatory response as demonstrated by increased mitogen-activated protein (MAP) kinase activity, IL-8 production, and tumor necrosis factor α production. RSV infection also allowed for tumor necrosis factor α production subsequent to TLR4 cross-linking with an immobilized antibody. These data suggest that RSV infection sensitizes airway epithelium to a subsequent environmental exposure (LPS) by altered expression and membrane localization of TLR4. The increased interaction between airway epithelial cells and LPS has the potential to profoundly alter airway inflammation.

Respiratory Syncytial Virus Induces TLR3 Protein and Protein Kinase R, Leading to Increased Double-Stranded RNA Responsiveness in Airway Epithelial Cells

Respiratory syncytial virus (RSV) preferentially infects airway epithelial cells, causing bronchiolitis, upper respiratory infections, asthma exacerbations, chronic obstructive pulmonary disease exacerbations, and pneumonia in immunocompromised hosts. A replication intermediate of RSV is dsRNA. This is an important ligand for both the innate immune receptor, TLR3, and protein kinase R (PKR). One known effect of RSV infection is the increased responsiveness of airway epithelial cells to subsequent bacterial ligands (i.e., LPS). In this study, we examined a possible role for RSV infection in increasing amounts and responsiveness of another TLR, TLR3. These studies demonstrate that RSV infection of A549 and human tracheobronchial epithelial cells increases the amounts of TLR3 and PKR in a time-dependent manner. This leads to increased NF-κB activity and production of the inflammatory cytokine IL-8 following a later exposure to dsRNA. Importantly, TLR3 was not detected on the cell surface at baseline but was detected on the cell surface after RSV infection. The data demonstrate that RSV, via an effect on TLR3 and PKR, sensitizes airway epithelial cells to subsequent dsRNA exposure. These findings are consistent with the hypothesis that RSV infection sensitizes the airway epithelium to subsequent viral and bacterial exposures by up-regulating TLRs and increasing their membrane localization.

Vitamin D Decreases Respiratory Syncytial Virus Induction of NF-κB–Linked Chemokines and Cytokines in Airway Epithelium While Maintaining the Antiviral State

Epidemiological studies suggest that low vitamin D levels may increase the risk or severity of respiratory viral infections. In this study, we examined the effect of vitamin D on respiratory syncytial virus (RSV)-infected human airway epithelial cells. Airway epithelium converts 25-hydroxyvitamin D3 (storage form) to 1,25-dihydroxyvitamin D3 (active form). Active vitamin D, generated locally in tissues, is important for the nonskeletal actions of vitamin D, including its effects on immune responses. We found that vitamin D induces IκBα, an NF-κB inhibitor, in airway epithelium and decreases RSV induction of NF-κB–driven genes such as IFN-β and CXCL10. We also found that exposing airway epithelial cells to vitamin D reduced induction of IFN-stimulated proteins with important antiviral activity (e.g., myxovirus resistance A and IFN-stimulated protein of 15 kDa). In contrast to RSV-induced gene expression, vitamin D had no effect on IFN signaling, and isolated IFN induced gene expression. Inhibiting NF-κB with an adenovirus vector that expressed a nondegradable form of IκBα mimicked the effects of vitamin D. When the vitamin D receptor was silenced with small interfering RNA, the vitamin D effects were abolished. Most importantly we found that, despite inducing IκBα and dampening chemokines and IFN-β, there was no increase in viral mRNA or protein or in viral replication. We conclude that vitamin D decreases the inflammatory response to viral infections in airway epithelium without jeopardizing viral clearance. This suggests that adequate vitamin D levels would contribute to reduced inflammation and less severe disease in RSV-infected individuals.

Lipopolysaccharide Activates Akt in Human Alveolar Macrophages Resulting in Nuclear Accumulation and Transcriptional Activity of β-Catenin

Exposure of human alveolar macrophages to bacterial LPS results in activation of a number of signal transduction pathways. An early event after the alveolar macrophage comes in contact with LPS is activation of the phosphatidylinositol 3 kinase (PI 3-kinase). This study evaluates the downstream effects of that activation. We observed that LPS exposure results in phosphorylation of Akt (serine 473). We found this using both phosphorylation-specific Abs and also by in vivo phosphorylation with 32P-loaded cells. AKT activation resulted in the phosphorylation-dependent inactivation of glycogen synthase kinase (GSK-3) (serine 21/9). We found that both of these events were linked to PI 3-kinase because the PI 3-kinase inhibitors, wortmannin and LY294002, inhibited LPS-induced phosphorylation of both AKT and GSK-3. Inactivation of GSK-3 has been shown to reduce the ubiquitination of β-catenin, resulting in nuclear accumulation and transcriptional activity of β-catenin. Consistent with this, we found that LPS caused an increase in the amounts of PI 3-kinase-dependent nuclear β-catenin in human alveolar macrophages and expression of genes that require nuclear β-catenin for their activation. This is the first demonstration that LPS exposure activates AKT, inactivates GSK-3, and causes accumulation and transcriptional activity of β-catenin in the nucleus of any cell, including alveolar macrophages.

Coordinated Changes in AHRR Methylation in Lymphoblasts and Pulmonary Macrophages from Smokers

Smoking is associated with a wide variety of adverse health outcomes including cancer, chronic obstructive pulmonary disease, diabetes, depression, and heart disease. Unfortunately, the molecular mechanisms through which these effects are conveyed are not clearly understood. To examine the potential role of epigenetic factors in these processes, we examined the relationship of smoking to genome wide methylation and gene expression using biomaterial from two independent samples, lymphoblast DNA and RNA (n = 119) and lung alveolar macrophage DNA (n = 19). We found that in both samples current smoking status was associated with significant changes in DNA methylation, in particular at the aryl hydrocarbon receptor repressor (AHRR), a known tumor suppressor. Both baseline DNA methylation and smoker associated DNA methylation signatures at AHRR were highly correlated (r = 0.94 and 0.45, respectively). DNA methylation at the most differentially methylated AHRR CpG residue in both samples, cg0557592, was significantly associated with AHRR gene expression. Pathway analysis of lymphoblast data (genes with most significant methylation changes) demonstrated enrichment in protein kinase C pathways and in TGF beta signaling pathways. For alveolar macrophages, pathway analysis demonstrated alterations in inflammation‐related processes. We conclude that smoking is associated with functionally significant genome wide changes in DNA methylation in both lymphoblasts and pulmonary macrophages and that further integrated investigations of these epigenetic effects of smoking on carcinogenesis and other related co‐morbidities are indicated.

Identification of an Autophagy Defect in Smokers’ Alveolar Macrophages

Alveolar macrophages are essential for clearing bacteria from the alveolar surface and preventing microbe-induced infections. It is well documented that smokers have an increased incidence of infections, in particular lung infections. Alveolar macrophages accumulate in smokers’ lungs, but they have a functional immune deficit. In this study, we identify an autophagy defect in smokers’ alveolar macrophages. Smokers’ alveolar macrophages accumulate both autophagosomes and p62, a marker of autophagic flux. The decrease in the process of autophagy leads to impaired protein aggregate clearance, dysfunctional mitochondria, and defective delivery of bacteria to lysosomes. This study identifies the autophagy pathway as a potential target for interventions designed to decrease infection rates in smokers and possibly in individuals with high environmental particulate exposure.

Protein Kinase C ζ Plays a Central Role in Activation of the p42/44 Mitogen-Activated Protein Kinase by Endotoxin in Alveolar Macrophages

Human alveolar macrophages respond to endotoxin (LPS) by activation of a number of mitogen-activated protein kinase pathways, including the p42/44 (extracellular signal-related kinase (ERK)) kinase pathway. In this study, we evaluated the role of the atypical protein kinase C (PKC) isoform, PKC ζ, in LPS-induced activation of the ERK kinase pathway. Kinase activity assays showed that LPS activates PKC ζ, mitogen-activated protein/ERK kinase (MEK, the upstream activator of ERK), and ERK. LPS did not activate Raf-1, the classic activator of MEK. Pseudosubstrate-specific peptides with attached myristic acid are cell permeable and can be used to block the activity of specific PKC isoforms in vivo. We found that a peptide specific for PKC ζ partially blocked activation of both MEK and ERK by LPS. We also found that this peptide blocked in vivo phosphorylation of MEK after LPS treatment. In addition, we found that LPS caused PKC ζ to bind to MEK in vivo. These observations suggest that MEK is an LPS-directed target of PKC ζ. PKC ζ has been shown in other systems to be phosphorylated by phosphatidylinositol (PI) 3-kinase-dependent kinase. We found that LPS activates PI 3-kinase and causes the formation of a PKC ζ/PI 3-kinase-dependent kinase complex. These data implicate the PI 3-kinase pathway as an integral part of the LPS-induced PKC ζ activation. Taken as a whole, these studies suggest that LPS activates ERK kinase, in part, through activation of an atypical PKC isoform, PKC ζ.

The effect of smoking on DNA methylation of peripheral blood mononuclear cells from African American women

BackgroundRegular smoking is associated with a wide variety of syndromes with prominent inflammatory components such as cancer, obesity and type 2 diabetes. Heavy regular smoking is also associated with changes in the DNA methylation of peripheral mononuclear cells. However, in younger smokers, inflammatory epigenetic findings are largely absent which suggests the inflammatory response(s) to smoking may be dose dependent. To help understand whether peripheral mononuclear cells have a role in mediating these responses in older smokers with higher cumulative smoke exposure, we examined genome-wide DNA methylation in a group of well characterized adult African American subjects informative for smoking, as well as serum C-reactive protein (CRP) and interleukin-6 receptor (IL6R) levels. In addition, complementary bioinformatic analyses were conducted to delineate possible pathways affected by long-term smoking.ResultsGenome-wide DNA methylation analysis with respect to smoking status yielded 910 significant loci after Benjamini-Hochberg correction. In particular, two loci from the AHRR gene (cg05575921 and cg23576855) and one locus from the GPR15 gene (cg19859270) were identified as highly significantly differentially methylated between smokers and non-smokers. The bioinformatic analyses showed that long-term chronic smoking is associated with altered promoter DNA methylation of genes coding for proteins mapping to critical sub-networks moderating inflammation, immune function, and coagulation.ConclusionsWe conclude that chronic regular smoking is associated with changes in peripheral mononuclear cell methylation signature which perturb inflammatory and immune function pathways and may contribute to increased vulnerability for complex illnesses with inflammatory components.