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High-Throughput CRISPR Screening Identifies Genes Involved in Macrophage Viability and Inflammatory Pathways.

ABSTRACT: Macrophages are critical effector cells of the immune system, and understanding genes involved in their viability and function is essential for gaining insights into immune system dysregulation during disease. We use a high-throughput, pooled-based CRISPR-Cas screening approach to identify essential genes required for macrophage viability. In addition, we target 3' UTRs to gain insights into previously unidentified cis-regulatory regions that control these essential genes. Next, using our recently generated nuclear factor ?B (NF-?B) reporter line, we perform a fluorescence-activated cell sorting (FACS)-based high-throughput genetic screen and discover a number of previously unidentified positive and negative regulators of the NF-?B pathway. We unravel complexities of the TNF signaling cascade, showing that it can function in an autocrine manner in macrophages to negatively regulate the pathway. Utilizing a single complex library design, we are capable of interrogating various aspects of macrophage biology, thus generating a resource for future studies.

SUBMITTER: Covarrubias S

PROVIDER: S-EPMC7901356 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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High-Throughput CRISPR Screening Identifies Genes Involved in Macrophage Viability and Inflammatory Pathways.

Covarrubias Sergio S Vollmers Apple Cortez AC Capili Allyson A Boettcher Michael M Shulkin Aaron A Correa Michele Ramos MR Halasz Haley H Robinson Elektra K EK O'Briain Laura L Vollmers Christopher C Blau James J Katzman Sol S McManus Michael T MT Carpenter Susan S

Cell reports 20201201 13

Macrophages are critical effector cells of the immune system, and understanding genes involved in their viability and function is essential for gaining insights into immune system dysregulation during disease. We use a high-throughput, pooled-based CRISPR-Cas screening approach to identify essential genes required for macrophage viability. In addition, we target 3' UTRs to gain insights into previously unidentified cis-regulatory regions that control these essential genes. Next, using our recent ...[more]

PMID: 33378675

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Project description:Candida albicans, an important fungal pathogen of humans, displays different morphologies, such as yeast, pseudo-hyphae and hyphae, which are recognized unequally by phagocytic cells of the innate immune response. Once C. albicans cells invade host tissues, immune cells such as macrophages are attracted to the site of infection and activated to recognize, engulf and kill the pathogen. We have investigated this fungal cell-macrophage interface by using high-throughput screening of the C. albicans GRACE library to identify genes that can influence this interaction and modify the kinetics of engulfment. Compared with the wild-type (WT) strain, we identified generally faster rates of engulfment for those fungal strains with constitutive pseudo-hyphal and hyphal phenotypes, whereas yeast-form-locked strains showed a reduced and delayed recognition and internalization by macrophages. We identified a number of GRACE strains that showed normal morphological development but exhibited different recognition and engulfment kinetics by cultured macrophages and characterized two mutants that modified interactions with the murine and human-derived macrophages. One mutant inactivated an uncharacterized C. albicans open reading frame that is the ortholog of S. cerevisiae OPY1, the other inactivated CaKRE1. The modified interaction was monitored during a 4 h co-culture. Early in the interaction, both opy1 and kre1 mutant strains showed reduced recognition and engulfment rates by macrophages when compared with WT cells. At fungal germ tube initiation, the engulfment kinetics increased for both mutants and WT cells, however the WT cells still showed a higher internalization by macrophages up to 2 h of interaction. Subsequently, between 2 and 4 h of the interaction, when most macrophages contain engulfed fungal cells, the engulfment kinetics increased for the opy1 mutant and further decreased for the kre1 mutant compared with Ca-WT. It appears that fungal morphology influences macrophage association with C. albicans cells and that both OPY1 and KRE1 play roles in the interaction of the fungal cells with phagocytes.

Dataset Information

High-Throughput CRISPR Screening Identifies Genes Involved in Macrophage Viability and Inflammatory Pathways.

Publications

High-Throughput CRISPR Screening Identifies Genes Involved in Macrophage Viability and Inflammatory Pathways.

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