RNAi Screening in Hematopoietic Cells

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

Abstract

The discovery and development of RNAi and CRISPR/Cas9 genetic screening technologies have provided researchers with invaluable tools for wide-scale and rapid genetic screening. A theme of our research program has been to develop methodology for efficient application of these screening technologies in immune cell lineages, and to implement screens in both human and mouse hematopoietic cells to interrogate the mechanistic basis of immune cell responses to pathogenic stimuli. Our efforts are generally focused on macrophages as they form the first line of defense against numerous bacterial and viral pathogens and characterization of these initial encounters are central to collaborative efforts in the LISB to generate integrated models of host-pathogen interactions. Genetic screen data are susceptible to a myriad of experimental biases, some of which can be mitigated by computational analysis for which we have previously developed sophisticated software tools such as SIGNAL (Selection by Iterative pathway Group and Network Analysis Looping: https://signal.niaid.nih.gov). We have applied the SIGNAL analysis to our previously published screens of the human and mouse macrophage responses to LPS (Sun et al (2017) Sci. Data. 4:170007; Li et al (2017) Sci. Data. 4:170008). Analysis of commonly enriched pathways identified the expected enrichment of innate immune processes, but also a strong enrichment for spliceosome components. In FY2023, we have completed an in-depth analysis of RNA splicing in LPS-challenged macrophages using a combination of long-read (PacBio) and in-depth (Illumina) sequencing. This has revealed an additional regulatory layer that extends beyond the well-known gene transcription changes that are induced by LPS, with a strong pattern of differential isoform usage emerging even among genes whose aggregate gene transcription levels do not change. We are currently comparing these LPS-induced splicing patterns in healthy patient macrophages compared to patients with myelodysplastic syndromes (MDS) to determine whether defects in this PRR-driven splicing response might explain some of the innate immune dysregulation observed in this disease. In FY2023 we have also continued a collaboration with Karin Petersons NIAID lab at the Rocky Mountain Labs to screen for host factors that regulate susceptibility to the La Crosse bunyavirus (LACV). LACV is a leading cause of pediatric encephalitis as only children show susceptibility to this pathogen through blood brain barrier compromise. We used a combination of in-depth transcript profiling and targeted siRNA screening to identify candidate genes whose age-related differential expression might underlie the pediatric susceptibility to LACV. We identified several genes whose expression affected viral pathogenesis in brain capillary endothelial cells (BCECs). Prominent among these were Connexin43 (Cx43/Gja1) and EphrinA2 (Efna2). We demonstrated that pharmacological induction of Cx43 could inhibit neurological disease in weanling mice while deficiency of Efna2 increased disease in adult mice. Thus, our studies illuminate a strategy to identify therapeutic targets that could form the basis of new drug treatments for this dangerous childhood disease.