Dyslipidemia induces opposing effects on intrapulmonary and extrapulmonary host defense through divergent TLR response phenotypes.

Dyslipidemia influences innate immune responses in the bloodstream, but whether and how pulmonary innate immunity is sensitive to circulating lipoproteins is largely unknown. To define whether dyslipidemia impacts responses to bacteria in the airspace and, if so, whether differently from its effects in other tissues, airspace, bloodstream, and i.p. responses to LPS and Klebsiella pneumoniae were investigated using murine models of dyslipidemia. Dyslipidemia reduced neutrophil (PMN) recruitment to the airspace in response to LPS and K. pneumoniae by impairing both chemokine induction in the airspace and PMN chemotaxis, thereby compromising pulmonary bacterial clearance. Paradoxically, bacteria were cleared more effectively from the bloodstream during dyslipidemia. This enhanced systemic response was due, at least in part, to basal circulating neutrophilia and basal TLR4/MyD88-dependent serum cytokine induction and enhanced serum cytokine responses to systemically administered TLR ligands. Dyslipidemia did not globally impair PMN transvascular trafficking to, and host defense within all loci, because neutrophilia, cytokine induction, and bacterial clearance were enhanced within the infected peritoneum. Peritoneal macrophages from dyslipidemic animals were primed for more robust TLR responses, reflecting increased lipid rafts and increased TLR4 expression, whereas macrophages from the airspace, in which cholesterol was maintained constant during dyslipidemia, had normal responses and rafts. Dyslipidemia thus imparts opposing effects upon intra- and extrapulmonary host defense by inducing tissue-divergent TLR response phenotypes and dysregulating airspace/blood compartmental levels of PMNs and cytokines. We propose that the airspace is a "privileged" site, thereby uniquely sensitive to dyslipidemia.

Glucocorticoids regulate tristetraprolin synthesis and posttranscriptionally regulate tumor necrosis factor alpha inflammatory signaling.

Glucocorticoids are used to treat various inflammatory disorders, but the mechanisms underlying these actions are incompletely understood. The zinc finger protein tristetraprolin (TTP) destabilizes several proinflammatory cytokine mRNAs by binding to AU-rich elements within their 3' untranslated regions, targeting them for degradation. Here we report that glucocorticoids induce the synthesis of TTP mRNA and protein in A549 lung epithelial cells and in rat tissues. Dexamethasone treatment leads to a sustained induction of TTP mRNA expression that is abrogated by RU486. Glucocorticoid induction of TTP mRNA is also blocked by actinomycin D but not by cycloheximide, suggesting a transcriptional mechanism which has been confirmed by transcription run-on experiments. The most widely characterized TTP-regulated gene is the AU-rich tumor necrosis factor alpha (TNF-alpha) gene. Dexamethasone represses TNF-alpha mRNA in A549 cells and decreases luciferase expression of a TNF-alpha 3' untranslated region reporter plasmid in an orientation-dependent manner. Small interfering RNAs to TTP significantly prevent this effect, and a cell line stably expressing a short-hairpin RNA to TTP conclusively establishes that TTP is critical for dexamethasone inhibition of TNF-alpha mRNA expression. These studies provide the molecular evidence for glucocorticoid regulation of human TTP and reflect a novel inductive anti-inflammatory signaling pathway for glucocorticoids that acts via posttranscriptional mechanisms.

Glucocorticoids and tumor necrosis factor alpha cooperatively regulate toll-like receptor 2 gene expression.

Tumor necrosis factor alpha (TNF-alpha) and glucocorticoids are widely recognized as mutually antagonistic regulators of adaptive immunity and inflammation. Surprisingly, we show here that they cooperatively regulate components of innate immunity. The Toll-like receptor 2 (TLR2) gene encodes a transmembrane receptor critical for triggering innate immunity. Although TLR2 mRNA and protein are induced by inflammatory molecules such as TNF-alpha, we show that TLR2 is also induced by the anti-inflammatory glucocorticoids in cells where they also regulate MKP-1 mRNA and protein levels. TNF-alpha and glucocorticoids cooperate to regulate the TLR2 promoter, through the involvement of a 3' NF-kappaB site, a STAT-binding element, and a 3' glucocorticoid response element (GRE). Molecular studies show that the IkappaBalpha superrepressor or a STAT dominant negative element prevented TNF-alpha and dexamethasone stimulation of TLR2 promoter. Similarly, an AF-1 deletion mutant of glucocorticoid receptor or ablation of a putative GRE notably reduced the cooperative regulation of TLR2. Using chromatin immunoprecipitation assays, we demonstrate that all three transcription factors interact with both endogenous and transfected TLR2 promoters after stimulation by TNF-alpha and dexamethasone. Together, these studies define novel signaling mechanism for these three transcription factors, with a profound impact on discrimination of innate and adaptive immune responses.

Mechanisms of glucocorticoid receptor signaling during inflammation.

Glucocorticoids are among the most widely prescribed anti-inflammatory drugs. They act by binding to the glucocorticoid receptor (GR) that, upon activation, translocates to the nucleus and either stimulates or inhibits gene expression. GR inhibition of many proinflammatory response genes occurs through induction of the synthesis of anti-inflammatory proteins as well as through repression of proinflammatory transcription factors, such as nuclear factor-kappaB (NF-kappaB) or activator protein-1 (AP-1). In this review, we discuss the molecular mechanisms underlying GR inhibition of inflammatory responses, with an emphasis on repression of NF-kappaB and AP-1 and their respective signaling pathways.

Myeloid differentiation primary response protein 88 couples reverse cholesterol transport to inflammation.

Crosstalk exists in mammalian cells between cholesterol trafficking and innate immune signaling. Apolipoprotein A-I (apoA-I), a serum apolipoprotein that induces antiatherogenic efflux of macrophage cholesterol, is widely described as anti-inflammatory because it neutralizes bacterial lipopolysaccharide. Conversely, lipopolysaccharide-induced inflammation is proatherogenic. However, whether innate immunity plays an endogenous, physiological role in host cholesterol homeostasis in the absence of infection is undetermined. We report that apoA-I signals in the macrophage through Toll-like receptor (TLR)2, TLR4, and CD14, utilizing myeloid differentiation primary response protein 88 (MyD88)-dependent and -independent pathways, to activate nuclear factor-kappaB and induce cytokines. MyD88 plays a critical role in reverse cholesterol transport in vitro and in vivo, in part through promoting ATP-binding cassette A1 transporter upregulation. Taken together, this work identifies apoA-I as an endogenous stimulus of innate immunity that couples cholesterol trafficking to inflammation through MyD88 and identifies innate immunity as a physiologic signal in cholesterol homeostasis.

Effects of liver X receptor agonist treatment on pulmonary inflammation and host defense.

Liver X receptor (LXR) alpha and beta are members of the nuclear receptor superfamily of ligand-activated transcription factors. Best known for triggering "reverse cholesterol transport" gene programs upon their activation by endogenous oxysterols, LXRs have recently also been implicated in regulation of innate immunity. In this study, we define a role for LXRs in regulation of pulmonary inflammation and host defense and identify the lung and neutrophil as novel in vivo targets for pharmacologic LXR activation. LXR is expressed in murine alveolar macrophages, alveolar epithelial type II cells, and neutrophils. Treatment of mice with TO-901317, a synthetic LXR agonist, reduces influx of neutrophils to the lung triggered by inhaled LPS, intratracheal KC chemokine, and intratracheal Klebsiella pneumoniae and impairs pulmonary host defense against this bacterium. Pharmacologic LXR activation selectively modulates airspace cytokine expression induced by both LPS and K. pneumoniae. Moreover, we report for the first time that LXR activation impairs neutrophil motility and identify inhibition of chemokine-induced RhoA activation as a putative underlying mechanism. Taken together, these data define a novel role for LXR in lung pathophysiology and neutrophil biology and identify pharmacologic activation of LXR as a potential tool for modulation of innate immunity in the lung.

Basal and reovirus-induced beta interferon (IFN-beta) and IFN-beta-stimulated gene expression are cell type specific in the cardiac protective response.

Viral myocarditis is an important human disease, with a wide variety of viruses implicated. Cardiac myocytes are not replenished yet are critical for host survival and thus may have a unique response to infection. Previously, we determined that the extent of reovirus induction of beta interferon (IFN-beta) and IFN-beta-mediated protection in primary cardiac myocyte cultures was inversely correlated with the extent of reovirus-induced cardiac damage in a mouse model. Surprisingly, and in contrast, the IFN-beta response did not determine reovirus replication in skeletal muscle cells. Here we compared the IFN-beta response in cardiac myocytes to that in primary cardiac fibroblast cultures, a readily replenished cardiac cell type. We compared basal and reovirus-induced expression of IFN-beta, IRF-7 (an interferon-stimulated gene [ISG] that further induces IFN-beta), and another ISG (561) in the two cell types by using real-time reverse transcription-PCR. Basal IFN-beta, IRF-7, and 561 expression was higher in cardiac myocytes than in cardiac fibroblasts. Reovirus T3D induced greater expression of IFN-beta in cardiac myocytes than in cardiac fibroblasts but equivalent expression of IRF-7 and 561 in the two cell types (though fold induction for IRF-7 and 561 was higher in fibroblasts than in myocytes because of the differences in basal expression). Interestingly, while reovirus replicated to equivalent titers in cardiac myocytes and cardiac fibroblasts, removal of IFN-beta resulted in 10-fold-greater reovirus replication in the fibroblasts than in the myocytes. Together the data suggest that the IFN-beta response controls reovirus replication equivalently in the two cell types. In the absence of reovirus-induced IFN-beta, however, reovirus replicates to higher titers in cardiac fibroblasts than in cardiac myocytes, suggesting that the higher basal IFN-beta and ISG expression in myocytes may play an important protective role.

Interferon regulatory factor-1, interferon-beta, and reovirus-induced myocarditis.

Viral myocarditis is an important human disease, and reovirus-induced myocarditis in mice provides an excellent model to study direct viral damage to the heart. Previously, we showed that reovirus induction of and sensitivity to interferon-beta (IFN-beta) is an important determinant of viral pathogenicity in the heart and that the transcription factor interferon regulatory factor-3 (IRF-3) is required for reovirus induction of IFN-beta in primary cardiac myocyte cultures. Given several lines of evidence suggesting a possible distinctive environment for IRFs in the heart, we have now focused on IRF-1. Previous studies demonstrated that viruses, double-stranded-RNA (dsRNA), and IFN-alpha/beta can each induce IRF-1 and that IRF-1 plays a role in dsRNA, but perhaps not viral, induction of IFN-alpha/beta. Importantly, none of these studies used a virus with a dsRNA genome (such as reovirus), none of them used a highly differentiated nonlymphoid cell type, and none of them addressed whether viral induction of IRF-1 is direct or is mediated through viral induction of IFN-beta. Indeed, as recently as this year it has been assumed that viral induction of IRF-1 is direct. Here, we found that reovirus induced IRF-1 in primary cardiac myocyte cultures, but that IRF-1 was not required for reovirus induction of IFN-beta. Surprisingly, we found that reovirus failed to induce IRF-1 in the absence of the IFN-alpha/beta response. This provides the first evidence that viruses may not induce IRF-1 directly. Finally, nonmyocarditic reovirus strains induced more cardiac lesions in mice deficient for IRF-1 than they did in wildtype mice, directly demonstrating a protective role for IRF-1. Together, the results indicate that while IRF-1 is downstream of the IFN-beta response, it plays an important protective role against viral myocarditis.