29 research outputs found
Investigating the molecular mechanism of the anti-inflammatory effects of HDL in macrophages
Inflammation is critical for clearing microbial infections, however, excessive or pro-longed inflammation is an underlying component of many disease states, including atherosclerosis. Macrophages recognise sterile or microbial ligands via receptors such as Toll Like Receptors (TLRs). Activation of macrophages by TLR ligands leads to inflammation, mediated by cytokines and chemokines, which amplify the immune response. Elevated levels of High Density Lipoprotein (HDL) are inversely correlated with cardiovascular disease (CVD) risk and atherosclerosis progression. The protective effect of HDL in CVD is mainly attributed to its ability to remove excess cholesterol from peripheral tissues. In addition, HDL has anti-inflammatory properties, and fittingly, is also protective against inflammatory disorders such as sepsis. However, the molecular mechanisms by which HDL mediates these anti-inflammatory effects remain poorly characterised. Considering the key role that macrophages, TLRs and pro-inflammatory cytokines play in inflammation, the effect of HDL on TLR-mediated macrophage activation was investigated. Human and mouse normo-cholesterolemic macrophages were analysed, so that the anti-inflammatory effects of HDL could be applied to a range of inflammatory diseases beyond atherosclerosis. HDL inhibited TLR-induced pro-inflammatory cytokine expression, at the level of transcription. The transcriptional modulator ATF3 was identified as being responsible for HDL's anti-inflammatory effects on macrophage activation. HDL induced ATF3 expression both in vitro and in vivo, increased ATF3 binding across the genome, as analysed by ChIP Sequencing, and was no longer able to inhibit TLR-induced pro-inflammatory cytokine secretion in ATF3-deficient macrophages. The mechanism by which HDL induces ATF3 is still unclear, however some evidence presented here suggests that this is independent of cholesterol efflux and may require lysosomal catabolism of HDL's phospholipids. This work identifies a new biology for HDL as an anti-inflammatory modulator of macrophage activation via the transcription factor ATF3
A Method for the Acute and Rapid Degradation of Endogenous Proteins.
Methods for the targeted disruption of protein function have revolutionized science and greatly expedited the systematic characterization of genes. Two main approaches are currently used to disrupt protein function: DNA knockout and RNA interference, which act at the genome and mRNA level, respectively. A method that directly alters endogenous protein levels is currently not available. Here, we present Trim-Away, a technique to degrade endogenous proteins acutely in mammalian cells without prior modification of the genome or mRNA. Trim-Away harnesses the cellular protein degradation machinery to remove unmodified native proteins within minutes of application. This rapidity minimizes the risk that phenotypes are compensated and that secondary, non-specific defects accumulate over time. Because Trim-Away utilizes antibodies, it can be applied to a wide range of target proteins using off-the-shelf reagents. Trim-Away allows the study of protein function in diverse cell types, including non-dividing primary cells where genome- and RNA-targeting methods are limited
Optimization of transcription factor binding map accuracy utilizing knockout-mouse models
Genome-wide assessment of protein-DNA interaction by chromatin immunoprecipitation followed by massive parallel sequencing (ChIP-seq) is a key technology for studying transcription factor (TF) localization and regulation of gene expression. Signal-to-noise-ratio and signal specificity in ChIP-seq studies depend on many variables, including antibody affinity and specificity. Thus far, efforts to improve antibody reagents for ChIP-seq experiments have focused mainly on generating higher quality antibodies. Here we introduce KOIN (knockout implemented normalization) as a novel strategy to increase signal specificity and reduce noise by using TF knockout mice as a critical control for ChIP-seq data experiments. Additionally, KOIN can identify \u27hyper ChIPable regions\u27 as another source of false-positive signals. As the use of the KOIN algorithm reduces false-positive results and thereby prevents misinterpretation of ChIP-seq data, it should be considered as the gold standard for future ChIP-seq analyses, particularly when developing ChIP-assays with novel antibody reagents
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High density lipoprotein mediates anti-inflammatory transcriptional reprogramming of macrophages via the transcriptional repressor ATF3
High Density Lipoprotein (HDL) mediates reverse cholesterol transport and it is known to be protective against atherosclerosis. In addition, HDL has potent anti-inflammatory properties that may be critical for protection against other inflammatory diseases. The molecular mechanisms of how HDL can modulate inflammation, particularly in immune cells such as macrophages, remain poorly understood. Here we identify the transcriptional repressor ATF3, as an HDL-inducible target gene in macrophages that down-regulates the expression of Toll-like receptor (TLR)-induced pro-inflammatory cytokines. The protective effects of HDL against TLR-induced inflammation were fully dependent on ATF3 in vitro and in vivo. Our findings may explain the broad anti-inflammatory and metabolic actions of HDL and provide the basis for predicting the success of novel HDL-based therapies
Ancestral SARS-CoV-2, but not Omicron, replicates less efficiently in primary pediatric nasal epithelial cells
Children typically experience more mild symptoms of Coronavirus Disease 2019 (COVID-19) when compared to adults. There is a strong body of evidence that children are also less susceptible to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection with the ancestral viral isolate. However, the emergence of SARS-CoV-2 variants of concern (VOCs) has been associated with an increased number of pediatric infections. Whether this is the result of widespread adult vaccination or fundamental changes in the biology of SARS-CoV-2 remain to be determined. Here, we use primary nasal epithelial cells (NECs) from children and adults, differentiated at an air-liquid interface to show that the ancestral SARS-CoV-2 replicates to significantly lower titers in the NECs of children compared to those of adults. This was associated with a heightened antiviral response to SARS-CoV-2 in the NECs of children. Importantly, the Delta variant also replicated to significantly lower titers in the NECs of children. This trend was markedly less pronounced in the case of Omicron. It is also striking to note that, at least in terms of viral RNA, Omicron replicated better in pediatric NECs compared to both Delta and the ancestral virus. Taken together, these data show that the nasal epithelium of children supports lower infection and replication of ancestral SARS-CoV-2, although this may be changing as the virus evolves
Ancestral SARS-CoV-2, but not Omicron, replicates less efficiently in primary pediatric nasal epithelial cells
Children typically experience more mild symptoms of Coronavirus Disease 2019 (COVID-19) when compared to adults. There is a strong body of evidence that children are also less susceptible to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection with the ancestral viral isolate. However, the emergence of SARS-CoV-2 variants of concern (VOCs) has been associated with an increased number of pediatric infections. Whether this is the result of widespread adult vaccination or fundamental changes in the biology of SARS-CoV-2 remain to be determined. Here, we use primary nasal epithelial cells (NECs) from children and adults, differentiated at an air-liquid interface to show that the ancestral SARS-CoV-2 replicates to significantly lower titers in the NECs of children compared to those of adults. This was associated with a heightened antiviral response to SARS-CoV-2 in the NECs of children. Importantly, the Delta variant also replicated to significantly lower titers in the NECs of children. This trend was markedly less pronounced in the case of Omicron. It is also striking to note that, at least in terms of viral RNA, Omicron replicated better in pediatric NECs compared to both Delta and the ancestral virus. Taken together, these data show that the nasal epithelium of children supports lower infection and replication of ancestral SARS-CoV-2, although this may be changing as the virus evolves.Peer reviewe
Resource Transcriptome-Based Network Analysis Reveals a Spectrum Model of Human Macrophage Activation
Transcriptome-Based Network Analysis Reveals a Spectrum Model of Human Macrophage Activation
SummaryMacrophage activation is associated with profound transcriptional reprogramming. Although much progress has been made in the understanding of macrophage activation, polarization, and function, the transcriptional programs regulating these processes remain poorly characterized. We stimulated human macrophages with diverse activation signals, acquiring a data set of 299 macrophage transcriptomes. Analysis of this data set revealed a spectrum of macrophage activation states extending the current M1 versus M2-polarization model. Network analyses identified central transcriptional regulators associated with all macrophage activation complemented by regulators related to stimulus-specific programs. Applying these transcriptional programs to human alveolar macrophages from smokers and patients with chronic obstructive pulmonary disease (COPD) revealed an unexpected loss of inflammatory signatures in COPD patients. Finally, by integrating murine data from the ImmGen project we propose a refined, activation-independent core signature for human and murine macrophages. This resource serves as a framework for future research into regulation of macrophage activation in health and disease
Innate immunity and neurodegeneration
The innate immune system plays diverse roles in health and disease. It represents the first line of defense against infection and is involved in tissue repair, wound healing, and clearance of apoptotic cells and cellular debris. Excessive or nonresolving innate immune activation can lead to systemic or local inflammatory complications and cause or contribute to the development of inflammatory diseases. In the brain, microglia represent the key innate immune cells, which are involved in brain development, brain maturation, and homeostasis. Impaired microglial function, either through aberrant activation or decreased functionality, can occur during aging and during neurodegeneration, and the resulting inflammation is thought to contribute to neurodegenerative diseases. This review highlights recent advances in our understanding of the influence of innate immunity on neurodegenerative disorders such as Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, and Huntington's disease
Interferons and inflammasomes: cooperation and counterregulation in disease
Interferons and the IL-1 family of cytokines have important roles in host defense against invading viruses and bacteria. Inflammasomes, multimeric cytosolic sensors of infection, are required for IL-1β and IL-18 processing and release. Interferons, IL-1β, and IL-18 are also implicated in autoimmune disease and chronic inflammation. Although independent but complementary pathways induce these cytokine subsets during infection, in some circumstances the cross-talk between these key inflammatory mediators is a particular requirement for effective host defense. In this review we will summarize recent discoveries concerning the potentiation of inflammasome responses by type I interferons, particularly in patients with gram-negative bacterial infections, and reflect on the molecular mechanisms of IFN-β's immunosuppressive effects through modulation of inflammasome and IL-1β signaling in patients with tuberculosis\ua0and multiple sclerosis