Cholesterol crystal formation is a unifying pathogenic mechanism in the development of diabetic retinopathy.

AIMS/HYPOTHESIS: Hyper-reflective crystalline deposits found in retinal lesions have been suggested to predict the progression of diabetic retinopathy, but the nature of these structures remains unknown. METHODS: Scanning electron microscopy and immunohistochemistry were used to identify cholesterol crystals (CCs) in human donor, pig and mouse tissue. The effects of CCs were analysed in bovine retinal endothelial cells in vitro and in db/db mice in vivo using quantitative RT-PCR, bulk RNA sequencing, and cell death and permeability assays. Cholesterol homeostasis was determined using 2H2O and 2H7-cholesterol. RESULTS: We identified hyper-reflective crystalline deposits in human diabetic retina as CCs. Similarly, CCs were found in the retina of a diabetic mouse model and a high-cholesterol diet-fed pig model. Cell culture studies demonstrated that treatment of retinal cells with CCs can recapitulate all major pathogenic mechanisms leading to diabetic retinopathy, including inflammation, cell death and breakdown of the blood-retinal barrier. Fibrates, statins and α-cyclodextrin effectively dissolved CCs present in in vitro models of diabetic retinopathy, and prevented CC-induced endothelial pathology. Treatment of a diabetic mouse model with α-cyclodextrin reduced cholesterol levels and CC formation in the retina, and prevented diabetic retinopathy. CONCLUSIONS/INTERPRETATION: We established that cholesterol accumulation and CC formation are a unifying pathogenic mechanism in the development of diabetic retinopathy.

Retention of the NLRP3 Inflammasome-Primed Neutrophils in the Bone Marrow Is Essential for Myocardial Infarction-Induced Granulopoiesis.

BACKGROUND: Acute myocardial infarction (MI) results in overzealous production and infiltration of neutrophils to the ischemic heart. This is mediated in part by granulopoiesis induced by the S100A8/A9-NLRP3-IL-1ß signaling axis in injury-exposed neutrophils. Despite the transcriptional upregulation of the NLRP3 (Nod Like Receptor Family Pyrin Domain-Containing 3) inflammasome and associated signaling components in neutrophils, the serum levels of IL-1ß (interleukin-1ß), the effector molecule in granulopoiesis, were not affected by MI, suggesting that IL-1ß is not released systemically. We hypothesize that IL-1ß is released locally within the bone marrow (BM) by inflammasome-primed and reverse-migrating neutrophils. METHODS: Using a combination of time-dependent parabiosis and flow cytometry techniques, we first characterized the migration patterns of different blood cell types across the parabiotic barrier. We next induced MI in parabiotic mice by permanent ligation of the left anterior descending artery and examined the ability of injury-exposed neutrophils to permeate the parabiotic barrier and induce granulopoiesis in noninfarcted parabionts. Last, using multiple neutrophil adoptive and BM transplant studies, we studied the molecular mechanisms that govern reverse migration and retention of the primed neutrophils, IL-1ß secretion, and granulopoiesis. Cardiac function was assessed by echocardiography. RESULTS: MI promoted greater accumulation of the inflammasome-primed neutrophils in the BM. Introducing a time-dependent parabiotic barrier to the free movement of neutrophils inhibited their ability to stimulate granulopoiesis in the noninfarcted parabionts. Previous priming of the NLRP3 inflammasome is not a prerequisite, but the presence of a functional CXCR4 (C-X-C-motif chemokine receptor 4) on the primed-neutrophils and elevated serum S100A8/A9 levels are necessary for homing and retention of the reverse-migrating neutrophils. In the BM, the primed-neutrophils secrete IL-1ß through formation of gasdermin D pores and promote granulopoiesis. Pharmacological and genetic strategies aimed at the inhibition of neutrophil homing or release of IL-1ß in the BM markedly suppressed MI-induced granulopoiesis and improved cardiac function. CONCLUSIONS: Our data reveal a new paradigm of how circulatory cells establish a direct communication between organs by delivering signaling molecules (eg, IL-1ß) directly at the sites of action rather through systemic release. We suggest that this pathway may exist to limit the off-target effects of systemic IL-1ß release.

The contributions of DNA accessibility and transcription factor occupancy to enhancer activity during cellular differentiation.

During gene regulation, DNA accessibility is thought to limit the availability of transcription factor (TF) binding sites, while TFs can increase DNA accessibility to recruit additional factors that upregulate gene expression. Given this interplay, the causative regulatory events in the modulation of gene expression remain unknown for the vast majority of genes. We utilized deeply sequenced ATAC-Seq data and site-specific knock-in reporter genes to investigate the relationship between the binding-site resolution dynamics of DNA accessibility and the expression dynamics of the enhancers of Cebpa during macrophage-neutrophil differentiation. While the enhancers upregulate reporter expression during the earliest stages of differentiation, there is little corresponding increase in their total accessibility. Conversely, total accessibility peaks during the last stages of differentiation without any increase in enhancer activity. The accessibility of positions neighboring C/EBP-family TF binding sites, which indicates TF occupancy, does increase significantly during early differentiation, showing that the early upregulation of enhancer activity is driven by TF binding. These results imply that a generalized increase in DNA accessibility is not sufficient, and binding by enhancer-specific TFs is necessary, for the upregulation of gene expression. Additionally, high-coverage ATAC-Seq combined with time-series expression data can infer the sequence of regulatory events at binding-site resolution.

LucFlow: A method to measure Luciferase reporter expression in single cells.

Reporter assays, in which the expression of an inert protein is driven by gene regulatory elements such as promoters and enhancers, are a workhorse for investigating gene regulation. Techniques for measuring reporter gene expression vary from single-cell or single-molecule approaches having low throughput to bulk Luciferase assays that have high throughput. We developed a Luciferase Reporter Assay using Flow-Cytometry (LucFlow), which measures reporter expression in single cells immunostained for Luciferase. We optimized and tested LucFlow with a murine cell line that can be differentiated into neutrophils, into which promoter-reporter and enhancer-promoter-reporter constructs have been integrated in a site-specific manner. The single-cell measurements are comparable to bulk ones but we found that dead cells have no detectable Luciferase protein, so that bulk assays underestimate reporter expression. LucFlow is able to achieve a higher accuracy than bulk methods by excluding dead cells during flow cytometry. Prior to fixation and staining, the samples are spiked with stained cells that can be discriminated during flow cytometry and control for tube-to-tube variation in experimental conditions. Computing fold change relative to control cells allows LucFlow to achieve a high level of precision. LucFlow, therefore, enables the accurate and precise measurement of reporter expression in a high throughput manner.

The contributions of DNA accessibility and transcription factor occupancy to enhancer activity during cellular differentiation.

The upregulation of gene expression by enhancers depends upon the interplay between the binding of sequence-specific transcription factors (TFs) and DNA accessibility. DNA accessibility is thought to limit the ability of TFs to bind to their sites, while TFs can increase accessibility to recruit additional factors that upregulate gene expression. Given this interplay, the causative regulatory events underlying the modulation of gene expression during cellular differentiation remain unknown for the vast majority of genes. We investigated the binding-site resolution dynamics of DNA accessibility and the expression dynamics of the enhancers of an important neutrophil gene, Cebpa, during macrophage-neutrophil differentiation. Reporter genes were integrated in a site-specific manner in PUER cells, which are progenitors that can be differentiated into neutrophils or macrophages in vitro by activating the pan-leukocyte TF PU.1. Time series data show that two enhancers upregulate reporter expression during the first 48 hours of neutrophil differentiation. Surprisingly, there is little or no increase in the total accessibility, measured by ATAC-Seq, of the enhancers during the same time period. Conversely, total accessibility peaks 96 hrs after PU.1 activation-consistent with its role as a pioneer-but the enhancers do not upregulate gene expression. Combining deeply sequenced ATAC-Seq data with a new bias-correction method allowed the profiling of accessibility at single-nucleotide resolution and revealed protected regions in the enhancers that match all previously characterized TF binding sites and ChIP-Seq data. Although the accessibility of most positions does not change during early differentiation, that of positions neighboring TF binding sites, an indicator of TF occupancy, did increase significantly. The localized accessibility changes are limited to nucleotides neighboring C/EBP-family TF binding sites, showing that the upregulation of enhancer activity during early differentiation is driven by C/EBP-family TF binding. These results show that increasing the total accessibility of enhancers is not sufficient for upregulating their activity and other events such as TF binding are necessary for upregulation. Also, TF binding can cause upregulation without a perceptible increase in total accessibility. Finally, this study demonstrates the feasibility of comprehensively mapping individual TF binding sites as footprints using high coverage ATAC-Seq and inferring the sequence of events in gene regulation by combining with time-series gene expression data.

TLR-4 Inhibition Attenuates Inflammation, Thrombosis, and Stenosis in Arteriovenous Fistula in Yucatan Miniswine.

Arteriovenous fistula (AVF) is the preferred vascular access in hemodialysis patients; however, it is afflicted with a high failure rate. Chronic inflammation, excessive neointimal hyperplasia (NIH), vessel stenosis, early thrombosis, and failure of outward remodeling are the major causes of AVF maturation failure. Inflammatory mediator toll-like receptor (TLR)-4 plays a critical role in NIH, arterial thrombosis, and stenosis. We investigated the effect of TLR-4 inhibition on early thrombosis. Yucatan miniswine were used to create AVF involving femoral artery and femoral vein and treated with TLR-4 inhibitor TAK-242 with ethanol as the vehicle. The vessels were assessed after 12 weeks using histomorphometry, immunostaining, ultrasound, angiography, and optical coherence tomography. Inhibition of TLR-4 attenuated inflammation and early thrombosis in 50% of animals, and blood flow was present through AVF in 25% of animals. Thus, targeting TLR-4 to attenuate inflammation and early thrombosis might be a therapeutic approach to keep AVF patent and maintain blood flow through the outflow vein.

Novel ligands and modulators of triggering receptor expressed on myeloid cells receptor family: 2015-2020 updates.

Introduction: Triggering receptors expressed on myeloid cells (TREMs) are inflammatory amplifiers with defined pathophysiological role in various infectious diseases, acute and chronic aseptic inflammations, and a variety of cancers, depicting TREMs as prominent therapeutic targets.Areas covered: Herein, updates from 2015 to 2020 are discussed to divulge the TREM ligands, as well as their peptide blockers, claimed to modulate their expression. The article also presents different strategies employed during the last five years to block interactions between TREMs and their ligands to treat various disease conditions by modulating their expression and activity.Expert opinion: There has been significant progress in the discovery of novel ligands and modulators of TREMs in the last five years that mainly revolved around the function of TREM molecules. A few peptides showed encouraging results to modulate the expression and activity of TREMs in preclinical studies, and these peptides are currently under clinical investigation. Based on the findings so far in several careful studies, we expect novel therapeutics in the near future which could have the ability to treat various disease conditions associated with TREM expression.

S100A8/A9 in Myocardial Infarction.

S100A8/A9 represents a novel biomarker and therapeutic target in sterile inflammatory diseases. Among the various S100 proteins, S100A8 and S100A9 have been shown to be the most important of all the damage-associated molecular pattern (DAMP) proteins in sterile inflammatory conditions such as diabetes, cardiovascular disease, autoimmune disorders, etc. We present here methods to quantify S100A8/A9 expression in various tissues in mouse models of myocardial infarction (MI) using flow cytometry (FC), immunofluorescence, quantitative real-time polymerase chain reaction (q-RT-PCR), and enzyme-linked immunosorbent assays (ELISA).