Probes for INS

Abstract

BACKGROUND:

Discoidin domain receptor 1 (DDR1) is a collagen-activated receptor tyrosine kinase extensively implicated in diseases such as cancer, atherosclerosis and fibrosis. Multiple preclinical studies, performed using either a gene deletion or a gene silencing approaches, have shown this receptor being a major driver target of fibrosis and glomerulosclerosis.

METHODS:

The present study investigated the role and relevance of DDR1 in human crescentic glomerulonephritis (GN). Detailed DDR1 expression was first characterized in detail in human GN biopsies using a novel selective anti-DDR1 antibody using immunohistochemistry. Subsequently the protective role of DDR1 was investigated using a highly selective, novel, small molecule inhibitor in a nephrotoxic serum (NTS) GN model in a prophylactic regime and in the NEP25 GN mouse model using a therapeutic intervention regime.

RESULTS:

DDR1 expression was shown to be mainly limited to renal epithelium. In humans, DDR1 is highly induced in injured podocytes, in bridging cells expressing both parietal epithelial cell (PEC) and podocyte markers and in a subset of PECs forming the cellular crescents in human GN. Pharmacological inhibition of DDR1 in NTS improved both renal function and histological parameters. These results, obtained using a prophylactic regime, were confirmed in the NEP25 GN mouse model using a therapeutic intervention regime. Gene expression analysis of NTS showed that pharmacological blockade of DDR1 specifically reverted fibrotic and inflammatory gene networks and modulated expression of the glomerular cell gene signature, further validating DDR1 as a major mediator of cell fate in podocytes and PECs.

CONCLUSIONS:

Together, these results suggest that DDR1 inhibition might be an attractive and promising pharmacological intervention for the treatment of GN, predominantly by targeting the renal epithelium.

Abstract

In response to myocardial infarction (MI), cardiac macrophages regulate inflammation and scar formation. We hypothesized that macrophages undergo polarization state changes over the MI time course and assessed macrophage polarization transcriptomic signatures over the first week of MI. C57BL/6 J male mice (3-6 months old) were subjected to permanent coronary artery ligation to induce MI, and macrophages were isolated from the infarct region at days 1, 3, and 7 post-MI. Day 0, no MI resident cardiac macrophages served as the negative MI control. Whole transcriptome analysis was performed using RNA-sequencing on n = 4 pooled sets for each time. Day 1 macrophages displayed a unique pro-inflammatory, extracellular matrix (ECM)-degrading signature. By flow cytometry, day 0 macrophages were largely F4/80highLy6Clow resident macrophages, whereas day 1 macrophages were largely F4/80lowLy6Chigh infiltrating monocytes. Day 3 macrophages exhibited increased proliferation and phagocytosis, and expression of genes related to mitochondrial function and oxidative phosphorylation, indicative of metabolic reprogramming. Day 7 macrophages displayed a pro-reparative signature enriched for genes involved in ECM remodeling and scar formation. By triple in situ hybridization, day 7 infarct macrophages in vivo expressed collagen I and periostin mRNA. Our results indicate macrophages show distinct gene expression profiles over the first week of MI, with metabolic reprogramming important for polarization. In addition to serving as indirect mediators of ECM remodeling, macrophages are a direct source of ECM components. Our study is the first to report the detailed changes in the macrophage transcriptome over the first week of MI.

Hair growth disorders often carry a major psychological burden. Therefore, more effective human hair growth-modulatory agents urgently need to be developed. Here, we used the hypertrichosis-inducing immunosuppressant, Cyclosporine A (CsA), as a lead compound to identify new hair growth-promoting molecular targets. Through microarray analysis we identified the Wnt inhibitor, secreted frizzled related protein 1 (SFRP1), as being down-regulated in the dermal papilla (DP) of CsA-treated human scalp hair follicles (HFs) ex vivo. Therefore, we further investigated the function of SFRP1 using a pharmacological approach and found that SFRP1 regulates intrafollicular canonical Wnt/β-catenin activity through inhibition of Wnt ligands in the human hair bulb. Conversely, inhibiting SFRP1 activity through the SFRP1 antagonist, WAY-316606, enhanced hair shaft production, hair shaft keratin expression, and inhibited spontaneous HF regression (catagen) ex vivo. Collectively, these data (a) identify Wnt signalling as a novel, non-immune-inhibitory CsA target; (b) introduce SFRP1 as a physiologically important regulator of canonical β-catenin activity in a human (mini-)organ; and (c) demonstrate WAY-316606 to be a promising new promoter of human hair growth. Since inhibiting SFRP1 only facilitates Wnt signalling through ligands that are already present, this 'ligand-limited' therapeutic strategy for promoting human hair growth may circumvent potential oncological risks associated with chronic Wnt over-activation.

Neutrophils require directional cues to navigate through the complex structure of venular walls and into inflamed tissues. Here we applied confocal intravital microscopy to analyze neutrophil emigration in cytokine-stimulated mouse cremaster muscles. We identified differential and non-redundant roles for the chemokines CXCL1 and CXCL2, governed by their distinct cellular sources. CXCL1 was produced mainly by TNF-stimulated endothelial cells (ECs) and pericytes and supported luminal and sub-EC neutrophil crawling. Conversely, neutrophils were the main producers of CXCL2, and this chemokine was critical for correct breaching of endothelial junctions. This pro-migratory activity of CXCL2 depended on the atypical chemokine receptor 1 (ACKR1), which is enriched within endothelial junctions. Transmigrating neutrophils promoted a self-guided migration response through EC junctions, creating a junctional chemokine "depot" in the form of ACKR1-presented CXCL2 that enabled efficient unidirectional luminal-to-abluminal migration. Thus, CXCL1 and CXCL2 act in a sequential manner to guide neutrophils through venular walls as governed by their distinct cellular sources.

During early human pregnancy the uterine mucosa transforms into the decidua, into which the fetal placenta implants and where placental trophoblast cells intermingle and communicate with maternal cells. Trophoblast-decidual interactions underlie common diseases of pregnancy, including pre-eclampsia and stillbirth. Here we profile the transcriptomes of about 70,000 single cells from first-trimester placentas with matched maternal blood and decidual cells. The cellular composition of human decidua reveals subsets of perivascular and stromal cells that are located in distinct decidual layers. There are three major subsets of decidual natural killer cells that have distinctive immunomodulatory and chemokine profiles. We develop a repository of ligand-receptor complexes and a statistical tool to predict the cell-type specificity of cell-cell communication via these molecular interactions. Our data identify many regulatory interactions that prevent harmful innate or adaptive immune responses in this environment. Our single-cell atlas of the maternal-fetal interface reveals the cellular organization of the decidua and placenta, and the interactions that are critical for placentation and reproductive success.

The oral mucosa is one of the most rapidly dividing tissues in the body and serves as a barrier to physical and chemical insults from mastication, food, and microorganisms. Breakdown of this barrier can lead to significant morbidity and potentially life-threatening infections for patients. Determining the identity and organization of oral epithelial progenitor cells (OEPCs) is therefore paramount to understanding their roles in homeostasis and disease. Using lineage tracing and label retention experiments, we show that rapidly dividing OEPCs are located broadly within the basal layer of the mucosa throughout the oral cavity. Quantitative clonal analysis demonstrated that OEPCs undergo population-asymmetrical divisions following neutral drift dynamics and that they respond to chemotherapy-induced damage by altering daughter cell fates. Finally, using single-cell RNA-seq, we establish the basal layer population structure and propose a model that defines the organization of cells within the basal layer.