IL-4 controls activated neutrophil FcγR2b expression and migration into inflamed joints.

Neutrophils are the most abundant immune cells found in actively inflamed joints of patients with rheumatoid arthritis (RA), and most animal models for RA depend on neutrophils for the induction of joint inflammation. Exogenous IL-4 and IL-13 protect mice from antibody-mediated joint inflammation, although the mechanism is not understood. Neutrophils display a very strong basal expression of STAT6, which is responsible for signaling following exposure to IL-4 and IL-13. Still, the role of IL-4 and IL-13 in neutrophil biology has not been well studied. This can be explained by the low neutrophil surface expression of the IL-4 receptor α-chain (IL-4Rα), essential for IL-4- and IL-13-induced STAT6 signaling. Here we identify that colony stimulating factor 3 (CSF3), released during acute inflammation, mediates potent STAT3-dependent neutrophil IL-4Rα up-regulation during sterile inflammatory conditions. We further demonstrate that IL-4 limits neutrophil migration to inflamed joints, and that CSF3 combined with IL-4 or IL-13 results in a prominent neutrophil up-regulation of the inhibitory Fcγ receptor (FcγR2b). Taking these data together, we demonstrate that the IL-4 and CSF3 pathways are linked and play important roles in regulating proinflammatory neutrophil behavior.

Modulating T-cell activation with antisense oligonucleotides targeting lymphocyte cytosolic protein 2.

Dysregulated T-cell activation is a hallmark of several autoimmune diseases such as rheumatoid arthritis (RA) and multiple sclerosis (MS). The lymphocyte cytosolic protein 2 (LCP2), also known as SLP-76, is essential for the development and activation of T cells. Despite the critical role of LCP2 in T-cell activation and the need for developing drugs that modify T-cell activation, no LCP2 inhibitors have been developed. This can be explained by the "undruggable" nature of LCP2, lacking a structure permissive to standard small molecule inhibitor modalities. Here, we explored an alternative drug modality, developing antisense oligonucleotides (ASOs) targeting LCP2 mRNAs, and evaluated its activity in modulating T-cell activation. We identified a set of 3' UTR targeting LCP2 ASOs, which knocked down LCP2 in a human T-cell line and primary human T cells and found that these suppressed T-cell receptor mediated activation. We also found that the ASOs suppressed FcεR1-mediated mast cell activation, in line with the role of LCP2 in mast cells. Taken together, our data provide examples of how immunomodulatory ASOs that interfere with undruggable targets can be developed and propose that such drug modalities can be used to treat autoimmune diseases.

Green listed-a CRISPR screen tool.

Motivation: Genome editing using versions of the bacterial CRISPR/Cas9 system can be used to probe the function of selected genes in any organism. Green Listed is a web-based tool that rapidly designs custom CRISPR screens targeting sets of genes defined by the user. It could thus be used to design screens targeting for example all genes differentially expressed during a specific stimuli or all genes related to a specific pathway or function, as well as to generate targeted secondary screens following a large-scale screen. Availability and Implementation: The software, including a demo function as well as explanatory texts and videos, is available through greenlisted.cmm.ki.se . Contact: fredrik.wermeling@ki.se.

MicroRNA-24 suppression of N-deacetylase/N-sulfotransferase-1 (NDST1) reduces endothelial cell responsiveness to vascular endothelial growth factor A (VEGFA).

Heparan sulfate (HS) proteoglycans, present at the plasma membrane of vascular endothelial cells, bind to the angiogenic growth factor VEGFA to modulate its signaling through VEGFR2. The interactions between VEGFA and proteoglycan co-receptors require sulfated domains in the HS chains. To date, it is essentially unknown how the formation of sulfated protein-binding domains in HS can be regulated by microRNAs. In the present study, we show that microRNA-24 (miR-24) targets NDST1 to reduce HS sulfation and thereby the binding affinity of HS for VEGFA. Elevated levels of miR-24 also resulted in reduced levels of VEGFR2 and blunted VEGFA signaling. Similarly, suppression of NDST1 using siRNA led to a reduction in VEGFR2 expression. Consequently, not only VEGFA binding, but also VEGFR2 protein expression is dependent on NDST1 function. Furthermore, overexpression of miR-24, or siRNA-mediated reduction of NDST1, reduced endothelial cell chemotaxis in response to VEGFA. These findings establish NDST1 as a target of miR-24 and demonstrate how such NDST1 suppression in endothelial cells results in reduced responsiveness to VEGFA.

On the roles and regulation of chondroitin sulfate and heparan sulfate in zebrafish pharyngeal cartilage morphogenesis.

The present study addresses the roles of heparan sulfate (HS) proteoglycans and chondroitin sulfate (CS) proteoglycans in the development of zebrafish pharyngeal cartilage structures. uxs1 and b3gat3 mutants, predicted to have impaired biosynthesis of both HS and CS because of defective formation of the common proteoglycan linkage tetrasaccharide were analyzed along with ext2 and extl3 mutants, predicted to have defective HS polymerization. Notably, the effects on HS and CS biosynthesis in the respective mutant strains were shown to differ from what had been hypothesized. In uxs1 and b3gat3 mutant larvae, biosynthesis of CS was shown to be virtually abolished, whereas these mutants still were capable of synthesizing 50% of the HS produced in control larvae. extl3 and ext2 mutants on the other hand were shown to synthesize reduced amounts of hypersulfated HS. Further, extl3 mutants produced higher levels of CS than control larvae, whereas morpholino-mediated suppression of csgalnact1/csgalnact2 resulted in increased HS biosynthesis. Thus, the balance of the Extl3 and Csgalnact1/Csgalnact2 proteins influences the HS/CS ratio. A characterization of the pharyngeal cartilage element morphologies in the single mutant strains, as well as in ext2;uxs1 double mutants, was conducted. A correlation between HS and CS production and phenotypes was found, such that impaired HS biosynthesis was shown to affect chondrocyte intercalation, whereas impaired CS biosynthesis inhibited formation of the extracellular matrix surrounding chondrocytes.

Functional overlap between chondroitin and heparan sulfate proteoglycans during VEGF-induced sprouting angiogenesis.

OBJECTIVE: Heparan sulfate proteoglycans regulate key steps of blood vessel formation. The present study was undertaken to investigate if there is a functional overlap between heparan sulfate proteoglycans and chondroitin sulfate proteoglycans during sprouting angiogenesis. METHODS AND RESULTS: Using cultures of genetically engineered mouse embryonic stem cells, we show that angiogenic sprouting occurs also in the absence of heparan sulfate biosynthesis. Cells unable to produce heparan sulfate instead increase their production of chondroitin sulfate that binds key angiogenic growth factors such as vascular endothelial growth factor A, transforming growth factor ß, and platelet-derived growth factor B. Lack of heparan sulfate proteoglycan production however leads to increased pericyte numbers and reduced adhesion of pericytes to nascent sprouts, likely due to dysregulation of transforming growth factor ß and platelet-derived growth factor B signal transduction. CONCLUSIONS: The present study provides direct evidence for a previously undefined functional overlap between chondroitin sulfate proteoglycans and heparan sulfate proteoglycans during sprouting angiogenesis. Our findings provide information relevant for potential future drug design efforts that involve targeting of proteoglycans in the vasculature.

Comprehensive biochemical analysis of rare prostacyclin receptor variants: study of association of signaling with coronary artery obstruction.

Currently, pharmacogenetic studies are at an impasse as the low prevalence (<2%) of most variants hinder their pharmacogenetic analysis with population sizes often inadequate for sufficiently powered studies. Grouping rare mutations by functional phenotype rather than mutation site can potentially increase sample size. Using human population-based studies (n = 1,761) to search for dysfunctional human prostacyclin receptor (hIP) variants, we recently discovered 18 non-synonymous mutations, all with frequencies less than 2% in our study cohort. Eight of the 18 had defects in binding, activation, and/or protein stability/folding. Mutations (M113T, L104R, and R279C) in three highly conserved positions demonstrated severe misfolding manifested by impaired binding and activation of cell surface receptors. To assess for association with coronary artery disease, we performed a case-control study comparing coronary angiographic results from patients with reduced cAMP production arising from the non-synonymous mutations (n = 23) with patients with non-synonymous mutations that had no reduction in cAMP (n = 17). Major coronary artery obstruction was significantly increased in the dysfunctional mutation group in comparison with the silent mutations. We then compared the 23 dysfunctional receptor patients with 69 age- and risk factor-matched controls (1:3). This verified the significantly increased coronary disease in the non-synonymous dysfunctional variant cohort. This study demonstrates the potential utility of in vitro functional characterization in predicting clinical phenotypes and represents the most comprehensive characterization of human prostacyclin receptor genetic variants to date.

CRISPR/Cas9-Induced DNA Damage Enriches for Mutations in a p53-Linked Interactome: Implications for CRISPR-Based Therapies.

Inactivating p53 mutations are the most abundant genetic alterations found in cancer. Here we show that CRISPR/Cas9-induced double-stranded DNA breaks enrich for cells deficient in p53 and in genes of a core CRISPR-p53 tumor suppressor interactome. Such enrichment could predispose to cancer development and thus pose a challenge for clinical CRISPR use. Transient p53 inhibition could suppress the enrichment of cells with these mutations. The level of DNA damage response induced by an sgRNA influenced the enrichment of p53-deficient cells and could be a relevant parameter in sgRNA design to limit cellular enrichment. Furthermore, a dataset of >800 human cancer cell lines identified additional factors influencing the enrichment of p53-mutated cells, including strong baseline CDKN1A expression as a predictor for an active CRISPR-p53 axis. Taken together, these data provide details about p53 biology in the context of CRISPR-induced DNA damage and identify strategies to enable safer CRISPR use. SIGNIFICANCE: CRISPR-mediated DNA damage enriches for cells with escape mutations in a core CRISPR-p53 interactome, which can be suppressed by transient inhibition of p53.

A rapid CRISPR competitive assay for in vitro and in vivo discovery of potential drug targets affecting the hematopoietic system.

CRISPR/Cas9 can be used as an experimental tool to inactivate genes in cells. However, a CRISPR-targeted cell population will not show a uniform genotype of the targeted gene. Instead, a mix of genotypes is generated - from wild type to different forms of insertions and deletions. Such mixed genotypes complicate analysis of the role of the targeted gene in the studied cell population. Here, we present a rapid and universal experimental approach to functionally analyze a CRISPR-targeted cell population that does not involve generating clonal lines. As a simple readout, we leverage the CRISPR-induced genetic heterogeneity and use sequencing to identify how different genotypes are enriched or depleted in relation to the studied cellular behavior or phenotype. The approach uses standard PCR, Sanger sequencing, and a simple sequence deconvoluting software, enabling laboratories without specific in-depth experience to perform these experiments. As proof of principle, we present examples studying various aspects related to hematopoietic cells (T cell development in vivo and activation in vitro, differentiation of macrophages and dendritic cells, as well as a leukemia-like phenotype induced by overexpressing a proto-oncogene). In conclusion, we present a rapid experimental approach to identify potential drug targets related to mature immune cells, as well as normal and malignant hematopoiesis.

In vitro and ex vivo functional characterization of human HLA-DRB1∗04 restricted T cell receptors.

Recent advances in single-cell sequencing technologies enable the generation of large-scale data sets of paired TCR sequences from patients with autoimmune disease. Methods to validate and characterize patient-derived TCR data are needed, as well as relevant model systems that can support the development of antigen-specific tolerance inducing drugs. We have generated a pipeline to allow streamlined generation of 'artificial' T cells in a robust and reasonably high throughput manner for in vitro and in vivo studies of antigen-specific and patient-derived immune responses. Hereby chimeric (mouse-human) TCR alpha and beta constructs are re-expressed in three different formats for further studies: (i) transiently in HEK cells for peptide-HLA tetramer validation experiments, (ii) stably in the TCR-negative 58 â€‹T cell line for functional readouts such as IL-2 production and NFAT-signaling, and lastly (iii) in human HLA-transgenic mice for studies of autoimmune disease and therapeutic interventions. As a proof of concept, we have used human HLA-DRB1∗04:01 restricted TCR sequences specific for a type I diabetes-associated GAD peptide, and an influenza-derived HA peptide. We show that the same chimeric TCR constructs can be used in each of the described assays facilitating sequential validation and prioritization steps leading to humanized animal models.

Acceleration of cardiovascular disease by a dysfunctional prostacyclin receptor mutation: potential implications for cyclooxygenase-2 inhibition.

Recent increased adverse cardiovascular events observed with selective cyclooxygenase-2 inhibition led to the withdrawal of rofecoxib (Vioxx) and valdecoxib (Bextra), but the mechanisms underlying these atherothrombotic events remain unclear. Prostacyclin is the major end product of cyclooxygenase-2 in vascular endothelium. Using a naturally occurring mutation in the prostacyclin receptor, we report for the first time that a deficiency in prostacyclin signaling through its G protein-coupled receptor contributes to atherothrombosis in human patients. We report that a prostacyclin receptor variant (R212C) is defective in adenylyl cyclase activation in both patient blood and in an in vitro COS-1 overexpression system. This promotes increased platelet aggregation, a hallmark of atherothrombosis. Our analysis of patients in 3 separate white cohorts reveals that this dysfunctional receptor is not likely an initiating factor in cardiovascular disease but that it accelerates the course of disease in those patients with the greatest risk factors. R212C was associated with cardiovascular disease only in the high cardiovascular risk cohort (n=980), with no association in the low-risk cohort (n=2293). In those at highest cardiovascular risk, both disease severity and adverse cardiovascular events were significantly increased with R212C when compared with age- and risk factor-matched normal allele patients. We conclude that for haploinsufficient mutants, such as the R212C, the enhanced atherothrombotic phenotype is likely dependent on the presence of existing atherosclerosis or injury (high risk factors), analogous to what has been observed in the cyclooxygenase-2 inhibition studies or prostacyclin receptor knockout mice studies. Combining both biochemical and clinical approaches, we conclude that diminished prostacyclin receptor signaling may contribute, in part, to the underlying adverse cardiovascular outcomes observed with cyclooxygenase-2 inhibition.

Designing custom CRISPR libraries for hypothesis-driven drug target discovery.

Over the last decade Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) has been developed into a potent molecular biology tool used to rapidly modify genes or their expression in a multitude of ways. In parallel, CRISPR-based screening approaches have been developed as powerful discovery platforms for dissecting the genetic basis of cellular behavior, as well as for drug target discovery. CRISPR screens can be designed in numerous ways. Here, we give a brief background to CRISPR screens and discuss the pros and cons of different design approaches, including unbiased genome-wide screens that target all known genes, as well as hypothesis-driven custom screens in which selected subsets of genes are targeted (Fig. 1). We provide several suggestions for how a custom screen can be designed, which could broadly serve as inspiration for any experiment that includes candidate gene selection. Finally, we discuss how results from CRISPR screens could be translated into drug development, as well as future trends we foresee in the rapidly evolving CRISPR screen field.

Novel signaling pathways promote a paracrine wave of prostacyclin-induced vascular smooth muscle differentiation.

The important athero-protective role of prostacyclin is becoming increasingly evident as recent studies have revealed adverse cardiovascular effects in mice lacking the prostacyclin receptor, in patients taking selective COX-2 inhibitors, and in patients in the presence of a dysfunctional prostacyclin receptor genetic variant. We have recently reported that this protective mechanism includes the promotion of a quiescent differentiated phenotype in human vascular smooth muscle cells (VSMC). Herein, we address the intriguing question of how localized endothelial release of the very unstable eicosanoid, prostacyclin, exerts a profound effect on the vascular media, often 30 cell layers thick. We report a novel PKA-, Akt-1- and ERK1/2-dependent prostacyclin-induced prostacyclin release that appears to play an important role in propagation of the quiescent, differentiated phenotype through adjacent arterial smooth muscle cells in the vascular media. Treating VSMC with the prostacyclin analog iloprost induced differentiation (contractile protein expression and contractile morphology), and also up-regulated COX-2 expression, leading to prostacyclin release by VSMC. This paracrine prostacyclin release, in turn, promoted differentiation and COX-2 induction in neighboring VSMC that were not exposed to iloprost. Using siRNA and pharmacologic inhibitors, we report that this positive feedback mechanism, prostacyclin-induced prostacyclin release, is mediated by cAMP/PKA signaling, ERK1/2 activation, and a novel prostacyclin receptor signaling pathway, inhibition of Akt-1. Furthermore, these pathways appear to be regulated by the prostacyclin receptor independently of one another. We conclude that prevention of de-differentiation and proliferation through a paracrine positive feedback mechanism is a major cardioprotective function of prostacyclin.

Prostacyclin receptor/thromboxane receptor interactions and cellular responses in human atherothrombotic disease.

Twenty-five years have passed since Vane and colleagues proposed a prostacyclin and thromboxane balance as critical to cardiovascular homeostasis. Prostacyclin prevents platelet aggregation and promotes vasodilatation, opposing the effects of thromboxane. Possible compensation by redundant functions, such as nitric oxide, long prevented appreciation of this balance. Effective use of low-dose aspirin in the secondary prevention of atherothrombosis suggested a clinical importance for the balance. However, it was not until the cyclooxygenase-2 inhibitor rofecoxib was withdrawn because of increased cardiovascular events that this critical balance was confirmed in humans. Moreover, clinical observations are supported by elegant animal receptor knockout experiments and subsequent human genetic variant studies. Combined, these findings provide valuable insight into the roles of these prostanoids in the development of atherothrombosis, emphasizing the need to reevaluate the use of selective prostacyclin- and thromboxane-based therapies in cardiovascular disease.

Ambient pH controls the expression of endopolygalacturonase genes in the necrotrophic fungus Sclerotinia sclerotiorum.

In the necrotrophic fungus Sclerotinia sclerotiorum, secretion of polygalacturonases (PGs) and decrease of the environmental pH via oxalic acid production are considered as the main pathogenicity determinants. In order to evaluate the relationship between these two aspects of the infection process, we analyzed the expression of the endoPG-encoding genes pg1-3. Transcription of pg1-3 was not carbon regulated but was strictly controlled by pH and highly favored in a narrow range of acidic pH. During plant infection, a pH gradient was established in relation to oxalic acid secretion. Transcripts of pg1-3 were localized to the zone of colonization of healthy tissues while transcripts of genes encoding other lytic enzymes were restricted to the more acidic zones of the infected tissues. Our results show that progressive acidification of the ambient medium by the fungus is a major strategy for the sequential expression of pathogenicity factors.

Molecular characterization and in planta detection of Sclerotinia sclerotiorum endopolygalacturonase genes.

Sclerotinia sclerotiorum, a plant pathogenic ascomycete, secretes multiple pectinolytic enzymes that facilitate penetration, colonization, and maceration of the plant tissues. Molecular analysis has previously revealed that the pectinolytic system of the fungus is organized as a multigene family, among which a subfamily of three members encoding for neutral endopolygalacturonase (endoPG) isoforms has been characterized. Here we describe the isolation and characterization of three additional endoPG-encoding genes ( pg5, pg6, and pg7) that belong to distinct phylogenetic groups. Pairwise sequence comparison between the known endoPGs from S. sclerotiorum revealed 43% to 97% identity, and the genomic organization of the pectinolytic system showed a great similarity to that of the related necrotroph Botrytis cinerea. During plant pathogenesis, a sequential expression of the endoPG-encoding genes was shown.

Cloning and sequence analysis of the glyceraldehyde-3-phosphate dehydrogenase gene from the zygomycetes fungus Rhizomucor miehei.

Rhizomucor miehei is important from a biotechnological aspect in consequence of its content of aspartic proteinase, which has high milk-clotting activity. A genomic library of R. miehei NRRL 5901 has been constructed in a phage (Lambda Fix II) vector. The glyceraldehyde-3-phosphate dehydrogenase (gpd) gene was isolated from this library by hybridization of the recombinant phage clones with a gpd-specific gene probe generated by polymerase chain reaction. The complete nucleotide sequence encodes a putative polypeptide chain of 336 amino acids interrupted by 5 introns. The predicted amino acid sequence of this gene shows a high degree of sequence similarity to the glyceraldehyde-3-phosphate dehydrogenase proteins from yeast and filamentous fungi. The promoter region, containing a consensus TATA box, and 246-bp downstream from the putative stop codon were also determined. The possibility of using the gpd promoter in the construction of new transformation vectors is discussed.