Cross-talk between sterol regulatory pathways and inflammatory pathways has been demonstrated to significantly impact the development of both atherosclerosis and infectious disease. The oxysterol ...25-hydroxycholesterol (25HC) plays multiple roles in lipid biosynthesis and immunity. We recently used a systems biology approach to identify 25HC as an innate immune mediator that had a predicted role in atherosclerosis and we demonstrated a role for 25HC in foam cell formation. Here, we show that this mediator also has several complex roles in the antiviral response. The host response to viruses involves gene regulatory circuits with multiple feedback loops and we show here that 25HC acts as an amplifier of inflammatory signaling in macrophages. We determined that 25HC amplifies inflammatory signaling, at least in part, by mediating the recruitment of the AP-1 components FBJ osteosarcoma oncogene (FOS) and jun proto-oncogene (JUN) to the promoters of a subset of Toll-like receptor-responsive genes. Consistent with previous reports, we found that 25HC inhibits in vitro infection of airway epithelial cells by influenza. Surprisingly, we found that deletion of Ch25h , the gene encoding the enzyme responsible for 25HC production, is protective in a mouse model of influenza infection as a result of decreased inflammatory-induced pathology. Thus, our study demonstrates, for the first time to our knowledge, that in addition to its direct antiviral role, 25HC also regulates transcriptional responses and acts as an amplifier of inflammation via AP-1 and that the resulting alteration in inflammatory response leads to increased tissue damage in mice following infection with influenza.
One quarter of strokes are of unknown cause, and subclinical atrial fibrillation may be a common etiologic factor. Pacemakers can detect subclinical episodes of rapid atrial rate, which correlate ...with electrocardiographically documented atrial fibrillation. We evaluated whether subclinical episodes of rapid atrial rate detected by implanted devices were associated with an increased risk of ischemic stroke in patients who did not have other evidence of atrial fibrillation.
We enrolled 2580 patients, 65 years of age or older, with hypertension and no history of atrial fibrillation, in whom a pacemaker or defibrillator had recently been implanted. We monitored the patients for 3 months to detect subclinical atrial tachyarrhythmias (episodes of atrial rate >190 beats per minute for more than 6 minutes) and followed them for a mean of 2.5 years for the primary outcome of ischemic stroke or systemic embolism. Patients with pacemakers were randomly assigned to receive or not to receive continuous atrial overdrive pacing.
By 3 months, subclinical atrial tachyarrhythmias detected by implanted devices had occurred in 261 patients (10.1%). Subclinical atrial tachyarrhythmias were associated with an increased risk of clinical atrial fibrillation (hazard ratio, 5.56; 95% confidence interval CI, 3.78 to 8.17; P<0.001) and of ischemic stroke or systemic embolism (hazard ratio, 2.49; 95% CI, 1.28 to 4.85; P=0.007). Of 51 patients who had a primary outcome event, 11 had had subclinical atrial tachyarrhythmias detected by 3 months, and none had had clinical atrial fibrillation by 3 months. The population attributable risk of stroke or systemic embolism associated with subclinical atrial tachyarrhythmias was 13%. Subclinical atrial tachyarrhythmias remained predictive of the primary outcome after adjustment for predictors of stroke (hazard ratio, 2.50; 95% CI, 1.28 to 4.89; P=0.008). Continuous atrial overdrive pacing did not prevent atrial fibrillation.
Subclinical atrial tachyarrhythmias, without clinical atrial fibrillation, occurred frequently in patients with pacemakers and were associated with a significantly increased risk of ischemic stroke or systemic embolism. (Funded by St. Jude Medical; ASSERT ClinicalTrials.gov number, NCT00256152.).
Metabolic reprogramming powers and polarizes macrophage functions, but the nature and regulation of this response during infection with pathogens remain controversial. In this study, we characterize ...the metabolic and transcriptional responses of murine macrophages to Mycobacterium tuberculosis (Mtb) in order to disentangle the underlying mechanisms. We find that type I interferon (IFN) signaling correlates with the decreased glycolysis and mitochondrial damage that is induced by live, but not killed, Mtb. Macrophages lacking the type I IFN receptor (IFNAR) maintain glycolytic flux and mitochondrial function during Mtb infection in vitro and in vivo. IFNβ itself restrains the glycolytic shift of inflammatory macrophages and initiates mitochondrial stress. We confirm that type I IFN acts upstream of mitochondrial damage using macrophages lacking the protein STING. We suggest that a type I IFN-mitochondrial feedback loop controls macrophage responses to mycobacteria and that this could contribute to pathogenesis across a range of diseases.
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•Live, but not killed, Mtb induces type I IFN-mediated metabolic changes in macrophages•IFNβ restrains glycolysis and drives mitochondrial stress in activated macrophages•Type I IFN signaling in vivo limits pulmonary macrophage metabolic responses to Mtb•STING signaling is upstream of mitochondrial damage during mycobacterial infection
The mechanisms controlling macrophage metabolism during bacterial infections remain poorly characterized. Olson et al. show that type I interferon, rather than direct bacterial virulence factors, restrains macrophage metabolism during Mycobacterium tuberculosis infection. IFNβ itself prevents the shift to aerobic glycolysis in inflammatory macrophages and drives mitochondrial dysfunction and stress.
Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of lipid-loaded macrophages in the arterial wall. We demonstrate that macrophage lipid body formation can be ...induced by modified lipoproteins or by inflammatory Toll-like receptor agonists. We used an unbiased approach to study the overlap in these pathways to identify regulators that control foam cell formation and atherogenesis. An analysis method integrating epigenomic and transcriptomic datasets with a transcription factor (TF) binding site prediction algorithm suggested that the TF ATF3 may regulate macrophage foam cell formation. Indeed, we found that deletion of this TF results in increased lipid body accumulation, and that ATF3 directly regulates transcription of the gene encoding cholesterol 25-hydroxylase. We further showed that production of 25-hydroxycholesterol (25-HC) promotes macrophage foam cell formation. Finally, deletion of ATF3 in Apoe(-/-) mice led to in vivo increases in foam cell formation, aortic 25-HC levels, and disease progression. These results define a previously unknown role for ATF3 in controlling macrophage lipid metabolism and demonstrate that ATF3 is a key intersection point for lipid metabolic and inflammatory pathways in these cells.
Progress in tuberculosis vaccine development is hampered by an incomplete understanding of the immune mechanisms that protect against infection with Mycobacterium tuberculosis (Mtb), the causative ...agent of tuberculosis. Although the M72/ASOE1 trial yielded encouraging results (54% efficacy in subjects with prior exposure to Mtb), a highly effective vaccine against adult tuberculosis remains elusive. We show that in a mouse model, establishment of a contained and persistent yet non-pathogenic infection with Mtb ("contained Mtb infection", CMTB) rapidly and durably reduces tuberculosis disease burden after re-exposure through aerosol challenge. Protection is associated with elevated activation of alveolar macrophages, the first cells that respond to inhaled Mtb, and accelerated recruitment of Mtb-specific T cells to the lung parenchyma. Systems approaches, as well as ex vivo functional assays and in vivo infection experiments, demonstrate that CMTB reconfigures tissue resident alveolar macrophages via low grade interferon-gamma exposure. These studies demonstrate that under certain circumstances, the continuous interaction of the immune system with Mtb is beneficial to the host by maintaining elevated innate immune responses.
Macrophages are versatile immune cells that can detect a variety of pathogen-associated molecular patterns through their Toll-like receptors (TLRs). In response to microbial challenge, the ...TLR-stimulated macrophage undergoes an activation program controlled by a dynamically inducible transcriptional regulatory network. Mapping a complex mammalian transcriptional network poses significant challenges and requires the integration of multiple experimental data types. In this work, we inferred a transcriptional network underlying TLR-stimulated murine macrophage activation. Microarray-based expression profiling and transcription factor binding site motif scanning were used to infer a network of associations between transcription factor genes and clusters of co-expressed target genes. The time-lagged correlation was used to analyze temporal expression data in order to identify potential causal influences in the network. A novel statistical test was developed to assess the significance of the time-lagged correlation. Several associations in the resulting inferred network were validated using targeted ChIP-on-chip experiments. The network incorporates known regulators and gives insight into the transcriptional control of macrophage activation. Our analysis identified a novel regulator (TGIF1) that may have a role in macrophage activation.
We report the first systems biology investigation of regulators controlling arterial plaque macrophage transcriptional changes in response to lipid lowering in vivo in two distinct mouse models of ...atherosclerosis regression. Transcriptome measurements from plaque macrophages from the Reversa mouse were integrated with measurements from an aortic transplant-based mouse model of plaque regression. Functional relevance of the genes detected as differentially expressed in plaque macrophages in response to lipid lowering in vivo was assessed through analysis of gene functional annotations, overlap with in vitro foam cell studies, and overlap of associated eQTLs with human atherosclerosis/CAD risk SNPs. To identify transcription factors that control plaque macrophage responses to lipid lowering in vivo, we used an integrative strategy--leveraging macrophage epigenomic measurements--to detect enrichment of transcription factor binding sites upstream of genes that are differentially expressed in plaque macrophages during regression. The integrated analysis uncovered eight transcription factor binding site elements that were statistically overrepresented within the 5' regulatory regions of genes that were upregulated in plaque macrophages in the Reversa model under maximal regression conditions and within the 5' regulatory regions of genes that were upregulated in the aortic transplant model during regression. Of these, the TCF/LEF binding site was present in promoters of upregulated genes related to cell motility, suggesting that the canonical Wnt signaling pathway may be activated in plaque macrophages during regression. We validated this network-based prediction by demonstrating that β-catenin expression is higher in regressing (vs. control group) plaques in both regression models, and we further demonstrated that stimulation of canonical Wnt signaling increases macrophage migration in vitro. These results suggest involvement of canonical Wnt signaling in macrophage emigration from the plaque during lipid lowering-induced regression, and they illustrate the discovery potential of an epigenome-guided, systems approach to understanding atherosclerosis regression.
LXR–cofactor complexes activate the gene expression program responsible for cholesterol efflux in macrophages. Inflammation antagonizes this program, resulting in foam cell formation and ...atherosclerosis; however, the molecular mechanisms underlying this antagonism remain to be fully elucidated. We use promoter enrichment‐quantitative mass spectrometry (PE‐QMS) to characterize the composition of gene regulatory complexes assembled at the promoter of the lipid transporter Abca1 following downregulation of its expression. We identify a subset of proteins that show LXR ligand‐ and binding‐dependent association with the Abca1 promoter and demonstrate they differentially control Abca1 expression. We determine that NCOA5 is linked to inflammatory Toll‐like receptor (TLR) signaling and establish that NCOA5 functions as an LXR corepressor to attenuate Abca1 expression. Importantly, TLR3–LXR signal crosstalk promotes recruitment of NCOA5 to the Abca1 promoter together with loss of RNA polymerase II and reduced cholesterol efflux. Together, these data significantly expand our knowledge of regulatory inputs impinging on the Abca1 promoter and indicate a central role for NCOA5 in mediating crosstalk between pro‐inflammatory and anti‐inflammatory pathways that results in repression of macrophage cholesterol efflux.
Synopsis
NCOA5 is a key target of the crosstalk between pro‐inflammatory TLR3 signaling and the anti‐inflammatory LXR pathway. Here, NCOA5 functions as an LXR corepressor to attenuate expression of the lipid transporter Abca1, thereby providing a mechanism whereby inflammatory signaling leads to repression of macrophage cholesterol efflux.
Promoter enrichment‐quantitative mass spectrometry (PE‐QMS) maps the dynamic assembly of protein complexes at the Abca1 promoter.
PE‐QMS identifies the LXR ligand‐stimulated and LXR binding‐induced occupancy of NCOA5 at the Abca1 promoter.
In response to TLR3‐LXR signal crosstalk, NCOA5 is recruited to the Abca1 promoter, where it functions as an LXR corepressor.
NCOA5 inhibits Abca1 gene expression by disrupting RNA Polymerase II recruitment and function.
Inflammatory signaling impinges on LXR–NCOA5 dependent mechanisms controlling lipid transporter expression and macrophage cholesterol efflux.
Patients infected with influenza are at high risk of secondary bacterial infection, which is a major proximate cause of morbidity and mortality. We have shown that in mice, prior infection with ...influenza results in increased inflammation and mortality upon Staphylococcus aureus infection, recapitulating the human disease. Lipidomic profiling of the lungs of superinfected mice revealed an increase in CYP450 metabolites during lethal superinfection. These lipids are endogenous ligands for the nuclear receptor PPARα, and we demonstrate that Ppara−/− mice are less susceptible to superinfection than wild-type mice. PPARα is an inhibitor of NFκB activation, and transcriptional profiling of cells isolated by bronchoalveolar lavage confirmed that influenza infection inhibits NFκB, thereby dampening proinflammatory and prosurvival signals. Furthermore, network analysis indicated an increase in necrotic cell death in the lungs of superinfected mice compared to mice infected with S. aureus alone. Consistent with this, we observed reduced NFκB-mediated inflammation and cell survival signaling in cells isolated from the lungs of superinfected mice. The kinase RIPK3 is required to induce necrotic cell death and is strongly induced in cells isolated from the lungs of superinfected mice compared to mice infected with S. aureus alone. Genetic and pharmacological perturbations demonstrated that PPARα mediates RIPK3-dependent necroptosis and that this pathway plays a central role in mortality following superinfection. Thus, we have identified a molecular circuit in which infection with influenza induces CYP450 metabolites that activate PPARα, leading to increased necrotic cell death in the lung which correlates with the excess mortality observed in superinfection.
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