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Tumor necrosis factor alpha (TNF-␣) 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-␣, 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-␣ and glucocorticoids cooperate to regulate the TLR2 promoter, through the involvement of a 3 NF-B site, a STAT-binding element, and a 3 glucocorticoid response element (GRE). Molecular studies show that the IB␣ superrepressor or a STAT dominant negative element prevented TNF-␣ 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-␣ 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.

Liver X receptor (LXR) α and β 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.

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-␣) gene. Dexamethasone represses TNF-␣ mRNA in A549 cells and decreases luciferase expression of a TNF-␣ 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-␣ 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.

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Summary Crosstalk exists in mammalian cells between cholesterol trafficking and innate immunity signaling. Apolipoprotein A-I (apoA-I), a serum apolipoprotein that induces anti-atherogenic efflux of macrophage cholesterol, is widely described as anti-inflammatory because it neutralizes bacterial lipopolysaccharide. Conversely, lipopolysaccharide-induced inflammation is pro-atherogenic. 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-κB 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.

Dyslipidemia influences innate immune responses in the bloodstream, but whether and how pulmonary innate immunity is sensitive to circulating lipoproteins is largely unknown. In order 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 intraperitoneal responses to lipopolysaccharide and Klebsiella pneumoniae were investigated using murine models of dyslipidemia. Dyslipidemia reduced neutrophil recruitment to the airspace in response to lipopolysaccharide and K. pneumoniae by impairing both chemokine induction in the airspace and neutrophil 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 Toll like Receptor 4 (TLR4)/Myeloid Differentiation Primary Response Protein 88-dependent serum cytokine induction, and enhanced serum cytokine responses to systemically administered TLR ligands. Dyslipidemia did not globally impair neutrophil transvascular trafficking to, and host defense within all loci, as 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 neutrophils and cytokines. We propose that the airspace is a ‘privileged’ site, thereby uniquely sensitive to dyslipidemia.

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