The discovery of RNA interference (RNAi) and the major advances in the understanding of small RNA biology in the past decade have provided researchers with an invaluable tool for wide-scale and rapid genetic screening. A central goal of our research program has been to develop methodology for efficient application of RNA interference (RNAi) screening technology in hematopoietic cell lineages, and to implement genome-wide RNAi screens in both human and mouse hematopoietic cells to interrogate the mechanistic basis of immune cell responses to pathogenic stimuli. Our current 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 the LSB-wide efforts to generate quantitative models of host-pathogen interactions. We have developed assays in macrophage/monocyte cell lines with both microscopy-based 'high content'single cell readouts, and also bioluminescence-based population assays using luciferase reporters driven by inflammatory gene promoters. While the majority of our screening data has come from using reporters for the NF-κB component p65/relA and for TNFαtranscription, we are also developing reporters for MAPK and IRF activation and for additional inflammatory cytokines. Together, this panel of reporters will provide a comprehensive range of biosensors for evaluation of the macrophage activation profile in response to various pathogenic inputs. Effective delivery of siRNA into hematopoietic cells remains a significant obstacle to the implementation of effective siRNA screens. We have developed highly efficient lipid-based transfection protocols in 384-well format for both mouse (RAW264.7) and human (THP1) cell lines where we can routinely achieve 85-95% knockdown of target protein, and avoid non-specific activation of the macrophage response to dsRNA. We have also confirmed our assays conform to the reproducibility and uniformity criteria established by the NCATS small molecule screening group, widely accepted as best practices in the high throughput screening field. We have implemented two genome wide siRNA screens to identify genes involved in the human and mouse LPS responses. As the best characterized TLR stimulus, data from these screens will provide a valuable comparison of the endotoxin response in mouse and human cells, with important potential clinical relevance in the context of septic shock and endotoxin tolerance. We have previously described the completion of the primary phase of these screens, and the selection of several hundred candidate regulators of the LPS-induced inflammatory response. Hits were selected based on a combination of phenotypic characteristics and low likelihood of off-target effects based on siRNA seed sequence analysis. Functionally enriched hits were identified through connections to the core TLR and NF-κB signaling pathways using a variety of pathway analysis software applications and custom informatics analyses developed in collaboration with the LSB informatics group. In 2013, we completed the experimental phase and informatics analysis of both the human and mouse secondary screens. This has led to the selection of approximately 40 novels regulators of LPS induced TNFαin human macrophages and approximately 80 regulators of NF κB and/or TNFαin mouse macrophages. Experiments are ongoing to establish the mechanisms underlying these genes regulation of the TLR4 pathway. During the analysis of our screening data, we also curated a set of 126 genes comprising a canonical set of TLR signaling components including TLR receptors, proximal signaling components, NF-κB and MAPK pathway proteins, transcription factors, cytokines and negative regulators. During the secondary screens in both human and mouse macrophages, we targeted this canonical TLR gene set with six additional individual siRNAs for every gene, and also tested their effects on the macrophage response not only to LPS (TLR4), but also to P3C (TLR2/1) and R848 (TLR7/8). While confirming the expected specificity for the TLRs and some of the core pathway components, this study has identify some unexpected selectivity and differences in signaling responses through these TLR pathways, and also some particularly striking differences between human and mouse macrophages. We are currently pursuing the findings from these screens to determine if they represent clinically important differences in how humans and mice respond to bacterial stimuli. The signaling pathways and transcription factor responses induced in macrophages upon TLR stimulation are regulated by feedback loops that modulate the kinetics and magnitude of gene transcription. Among these, NF-κB has been a paradigm for a signal- responsive transcription factor that operates in a feedback regulatory network. Despite the considerable literature on NF-κB activation and function, there remains a lack of data on NF-κB single cell dynamics in hematopoietic cells (especially macrophages) responding to pathogenic stimuli. The development of a mouse macrophage cell line expressing GFP tagged p65/relA for the above siRNA screen provides an opportunity for us to address this. Moreover, the coupled TNFαpromoter-driven transcriptional reporter provides a unique secondary readout that allows evaluation of the consequences of NF-κB activation at a single cell level. In collaboration with Mia Sung and Gordon Hager at the NCI, we are using these cells to study how macrophages interpret different LPS doses in the context of NF-κB activation and TNFαtranscriptional output. This year, we made considerable progress in this project. Using live cell imaging, we monitored both NF-κB signaling dynamics and TNFαtranscription in single macrophages exposed to bacterial lipopolysaccharide (LPS). Our analysis revealed a novel positive feedback loop: induction of Rela, encoding the active subunit of NF-κB. This feedback rewires the regulatory network above a distinct dose. Mathematical modeling and experimental validation showed that the positive feedback overrides negative feedback and discriminates LPS doses that elicit an authentic innate immune response. Taking advantage of our genome-wide siRNA screens in the same mouse macrophage cell line, we identified the transcription factor Ikaros as a key component underlying the switch from negative to positive feedback at higher LPS dose. Switching of feedback dominance may be a general mechanism in immune cells for integrating opposing feedback on a key transcriptional regulator and setting a host response threshold. A paper describing these data is currently under review. We are also using the macrophage reporter cells developed for screening as models for single cell infection studies with murine cytomegalovirus (mCMV) in collaboration with Peter Ghazal at the University of Edinburgh, and for Salmonella studies with Clare Bryant at the University of Cambridge. The project with Dr. Ghazals lab led to the publication of a study demonstrating that the immediate-early 1 (ie1) gene of murine cytomegalovirus attenuates TNF alpha activation as a means to alter the host cell inflammatory response during an acute infection.
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