RNAi Screening in Hematopoietic Cells

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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 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 described screens of the human and mouse macrophage responses to LPS screens (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 FY2022, we have conducted 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 MDS patients to determine whether defects in this PRR-driven splicing response might explain some of the innate immune dysregulation observed in this disease. Beyond our continued study of the TLR4 pathway response to bacterial LPS, we are also extending our studies to interrogate the more recently discovered cytosolic LPS sensing pathway, which activates the non-canonical inflammasome response and the release of IL-1 family inflammatory cytokines. Recent studies have shown this to be a critical component of the broader physiological response to LPS and a major contributor to septic shock outcomes in Gram-negative bacterial infections. We have collaborated with the NIH-NCATS screening facility to complete a genome-scale screen of the IL-1 alpha response to cytosolic LPS and we are actively studying the gene hits that have emerged from this project. In FY 2022, we have continued investigation of an important role for the mitochondria and cellular metabolism in inflammasome activation. Among genes emerging from our screen, we have identified three nucleotide diphosphate kinases, and we have further investigated the role of the Nme4 gene in inflammasome activation. This study reveals an Nme4-dependent mitochondrial fitness checkpoint which supports the macrophage response to infection and cytokine release. Consistent with this model, we find that Nme4-deficient mice resist LPS-induced endotoxic shock, due to a diminished inflammatory cytokine response. We are continuing our investigation of further genes from this screen that may uncover novel regulators of the non-canonical inflammasome. In FY2022 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 are using 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. Our studies, which are ongoing, have the potential to identify therapeutic targets that could form the basis of new drug treatments for this dangerous childhood disease.