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. We have also confirmed our assays conform to the plate uniformity criteria established by the NCGC small molecule screening group, finding no significant edge effects with either assay which has permitted the use of full 384-well plates in our primary screens. We have identified reproducible positive siRNA controls for a range of TLR ligands, including the TLR receptors, receptor-associated adapter proteins and protein kinases involved in the activation of the MAPK and NF-κB signaling pathways. Considering that the response to dsRNA during pathogen infection is very robust in macrophages, and that a strong subsequent interferon response would be problematic for the interpretation of effects resulting from gene-specific siRNA knockdown, it was important to evaluate our described protocols for any non-specific macrophage response to transfected siRNA. We found no significant elevation in type-I interferon from either the RAW or THP-1 reporter cell lines during our assay window of 48 to 72 hr post-transfection. Last year, we described completion of the experimental phase of primary two genome wide screens to identify genes involved in the human and mouse LPS response. 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. In 2012, we completed the statistical analysis of the primary screen data. We used the freely available CellHTS2 software package to process our primary data and calculate z-scores for the deviation of each gene from the normalized median for each screen readout. We selected approximately 600 primary hits for both human and mouse screens based on a combination of phenotypic strength and connections to the core TLR and NF-κB signaling pathways identified using a variety of pathway analysis software applications. Recent studies have shown that off target effects (driven by miRNA-like targeting of 3UTRs in unintended target genes by the seed sequence of an siRNA) are quite prevalent in RNAi screens. To reduce the propagation of these unwanted effects in our secondary screens, we chose to use six non-pooled siRNAs from alternative vendors (three each from Ambion and Qiagen). Secondary screens are ongoing using this approach. During the analysis of our primary screening data, we curated a set of 128 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. We found that approximately 26 of these targets were strong hits in the human LPS primary screen, while a further 15 were weak hits. This led us to ask whether this hit frequency was reasonable, and whether unexpected negatives are determined more by insufficient KD or by cell-type variability in components required for signaling (since canonical pathways are often derived from studies in a range of model cell types). We screened the LPS response for six additional individual siRNAs for every gene in this set, which we would expect to give effective depletion of the target gene by at least one siRNA. Analysis of the data set identified a small number of core pathway components as strong hits, presumably due to insufficient KD in the primary screen, including TRAM, TRAF6 and TAB1. However, the total number of strong canonical pathway hits in the secondary screen was actually reduced to around 25 genes, suggesting that many components that influence TLR signaling do so in a cell type specific manner. This is consistent with recent informatic analyses of screens done with the same pathogenic stimulus but in different cell systems, which suggest that while common pathways are identified in these parallel screens, the specific genes most sensitive to siRNA KD are often non-overlapping. However, in our assay of LPS induced TNF transcription, we do see a noteworthy pattern where the strongest phenotypes cluster in the initial receptor and adapter proteins and the TNF transcriptional enhanceosome components, while the phenotypes are weaker in the intermediate steps when signaling branches through the NF-κB and MAPK pathways. 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 macrophage cells 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. We have initiated several collaborations using this novel assay platform. We are working with Mia Sung and Gordon Hager at the NCI to study how macrophages interpret different LPS doses in the context of NF-κB activation and TNFαtranscriptional output. We are also using the cells 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.
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