RNAi Screening in Hematopoietic Cells

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

Abstract

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. In FY 2021, we have further extended this work to publish the development of a novel bioinformatic method termed SIGNAL (Selection by Iterative pathway Group and Network Analysis Looping). This model applies multiple pathway and network enrichment steps on a screening dataset in an iterative manner, correcting for the biases of individual steps in a complementary fashion. We have developed a sophisticated web-based interface for the SIGNAL application, to permit its use by other investigators (https://signal.niaid.nih.gov). We have applied the SIGNAL analysis method described above 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 proteasome and spliceosome components. In FY2021, our studies suggest that the degradation of inhibitory proteins following PRR activation could be a broad regulatory mechanism setting a pathway activation threshold, and that alternative splicing is an important mechanism to both modulate the macrophage state and propagate the inflammatory response. We have identified specific regulators of splicing required for macrophages to mount a robust response to infectious challenge. 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 towards 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 2021, 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 (NDPK-D protein) in inflammasome activation. This recently published 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.