Plate Design for and Cherry Picking of Bacterial RNAi Clones for Systematic Error Detection in High-Throughput Caenorhabditis elegans RNAi Screens.

Automated or semi-automated high-throughput RNAi screens are highly prone to systematic errors because of multistep repetitive protocols and extensive use of automated instruments. A well-designed RNAi library can help detect and minimize systematic human and robotic errors. In this unit, we describe how to design an RNAi bacterial library for use in conjunction with the well-studied nematode Caenorhabditis elegans for automated phenotypic screens. We provide strategies to design and assemble RNAi libraries to reduce or eliminate systematic errors. These strategies serve as a good quality-control check and facilitate obtaining high-quality data from a genome-wide and sub-library RNAi screen. © 2018 by John Wiley & Sons, Inc.

RNAi Screening: Automated High-Throughput Liquid RNAi Screening in Caenorhabditis elegans.

RNAi is a powerful reverse genetics tool that has revolutionized genetic studies in model organisms. The bacteriovorous nematode Caenorhabditis elegans can be genetically manipulated by feeding it an Escherichia coli strain that expresses a double-stranded RNA (dsRNA) corresponding to a C. elegans gene, which leads to systemic silencing of the gene. This unit describes protocols for performing an automated high-throughput RNAi screen utilizing a full-genome C. elegans RNAi library. The protocols employ liquid-handling robotics and 96-well plates. © 2018 by John Wiley & Sons, Inc.

Intestinal Epithelial Wnt Signaling Mediates Acetylcholine-Triggered Host Defense against Infection.

Regulated antimicrobial peptide expression in the intestinal epithelium is key to defense against infection and to microbiota homeostasis. Understanding the mechanisms that regulate such expression is necessary for understanding immune homeostasis and inflammatory disease and for developing safe and effective therapies. We used Caenorhabditis elegans in a preclinical approach to discover mechanisms of antimicrobial gene expression control in the intestinal epithelium. We found an unexpected role for the cholinergic nervous system. Infection-induced acetylcholine release from neurons stimulated muscarinic signaling in the epithelium, driving downstream induction of Wnt expression in the same tissue. Wnt induction activated the epithelial canonical Wnt pathway, resulting in the expression of C-type lectin and lysozyme genes that enhanced host defense. Furthermore, the muscarinic and Wnt pathways are linked by conserved transcription factors. These results reveal a tight connection between the nervous system and the intestinal epithelium, with important implications for host defense, immune homeostasis, and cancer.

Quorum-sensing regulator RhlR but not its autoinducer RhlI enables Pseudomonas to evade opsonization.

When Drosophila melanogaster feeds on Pseudomonas aeruginosa, some bacteria cross the intestinal barrier and eventually proliferate in the hemocoel. This process is limited by hemocytes through phagocytosis. P. aeruginosa requires the quorum-sensing regulator RhlR to elude the cellular immune response of the fly. RhlI synthesizes the autoinducer signal that activates RhlR. Here, we show that rhlI mutants are unexpectedly more virulent than rhlR mutants, both in fly and in nematode intestinal infection models, suggesting that RhlR has RhlI-independent functions. We also report that RhlR protects P. aeruginosa from opsonization mediated by the Drosophila thioester-containing protein 4 (Tep4). RhlR mutant bacteria show higher levels of Tep4-mediated opsonization, as compared to rhlI mutants, which prevents lethal bacteremia in the Drosophila hemocoel. In contrast, in a septic model of infection, in which bacteria are introduced directly into the hemocoel, Tep4 mutant flies are more resistant to wild-type P. aeruginosa, but not to the rhlR mutant. Thus, depending on the infection route, the Tep4 opsonin can either be protective or detrimental to host defense.

The Selective Estrogen Receptor Modulator Raloxifene Inhibits Neutrophil Extracellular Trap Formation.

Raloxifene is a selective estrogen receptor modulator typically prescribed for the prevention/treatment of osteoporosis in postmenopausal women. Although raloxifene is known to have anti-inflammatory properties, its effects on human neutrophils, the primary phagocytic leukocytes of the immune system, remain poorly understood. Here, through a screen of pharmacologically active small molecules, we find that raloxifene prevents neutrophil cell death in response to the classical activator phorbol 12-myristate 13-acetate (PMA), a compound known to induce formation of DNA-based neutrophil extracellular traps (NETs). Inhibition of PMA-induced NET production by raloxifene was confirmed using quantitative and imaging-based assays. Human neutrophils from both male and female donors express the nuclear estrogen receptors ERα and ERß, known targets of raloxifene. Similar to raloxifene, selective antagonists of these receptors inhibit PMA-induced NET production. Furthermore, raloxifene inhibited PMA-induced ERK phosphorylation, but not reactive oxygen species production, pathways known to be key modulators of NET production. Finally, we found that raloxifene inhibited PMA-induced, NET-based killing of the leading human bacterial pathogen, methicillin-resistant Staphylococcus aureus. Our results reveal that raloxifene is a potent modulator of neutrophil function and NET production.

Activation of the Raf-MEK-ERK pathway is required for neutrophil extracellular trap formation.

The signaling mechanisms leading to the formation of neutrophil extracellular traps (NETs), relevant in infections, sepsis and autoimmune diseases, are poorly understood. Neutrophils are not amenable to studies with conventional genetic techniques. Using a new chemical genetic analysis we show that the Raf-MEK-ERK pathway is involved in NET formation through activation of NADPH oxidase and upregulation of antiapoptotic proteins. We identify potential targets for drugs addressing NET-associated diseases.

Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps.

Neutrophils release decondensed chromatin termed neutrophil extracellular traps (NETs) to trap and kill pathogens extracellularly. Reactive oxygen species are required to initiate NET formation but the downstream molecular mechanism is unknown. We show that upon activation, neutrophil elastase (NE) escapes from azurophilic granules and translocates to the nucleus, where it partially degrades specific histones, promoting chromatin decondensation. Subsequently, myeloperoxidase synergizes with NE in driving chromatin decondensation independent of its enzymatic activity. Accordingly, NE knockout mice do not form NETs in a pulmonary model of Klebsiella pneumoniae infection, which suggests that this defect may contribute to the immune deficiency of these mice. This mechanism provides for a novel function for serine proteases and highly charged granular proteins in the regulation of chromatin density, and reveals that the oxidative burst induces a selective release of granular proteins into the cytoplasm through an unknown mechanism.

Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis.

Systemic lupus erythematosus (SLE) is an autoimmune disease in which patients develop autoantibodies to DNA, histones, and often to neutrophil proteins. These form immune complexes that are pathogenic and may cause lupus nephritis. In SLE patients, infections can initiate flares and are a major cause of mortality. Neutrophils respond to infections and release extracellular traps (NETs), which are antimicrobial and are made of DNA, histones, and neutrophil proteins. The timely removal of NETs may be crucial for tissue homeostasis to avoid presentation of self-antigens. We tested the hypothesis that SLE patients cannot clear NETs, contributing to the pathogenesis of lupus nephritis. Here we show that serum endonuclease DNase1 is essential for disassembly of NETs. Interestingly, a subset of SLE patients' sera degraded NETs poorly. Two mechanisms caused this impaired NET degradation: (i) the presence of DNase1 inhibitors or (ii) anti-NET antibodies prevented DNase1 access to NETs. Impairment of DNase1 function and failure to dismantle NETs correlated with kidney involvement. Hence, identification of SLE patients who cannot dismantle NETs might be a useful indicator of renal involvement. Moreover, NETs might represent a therapeutic target in SLE.

Restoration of NET formation by gene therapy in CGD controls aspergillosis.

Chronic granulomatous disease (CGD) patients have impaired nicotinamide adenine dinucleotide phosphate (NADPH) oxidase function, resulting in poor antimicrobial activity of neutrophils, including the inability to generate neutrophil extracellular traps (NETs). Invasive aspergillosis is the leading cause of death in patients with CGD; it is unclear how neutrophils control Aspergillus species in healthy persons. The aim of this study was to determine whether gene therapy restores NET formation in CGD by complementation of NADPH oxidase function, and whether NETs have antimicrobial activity against Aspergillus nidulans. Here we show that reconstitution of NET formation by gene therapy in a patient with CGD restores neutrophil elimination of A nidulans conidia and hyphae and is associated with rapid cure of preexisting therapy refractory invasive pulmonary aspergillosis, underlining the role of functional NADPH oxidase in NET formation and antifungal activity.