CRISPR/Cas-based screening of long non-coding RNAs (lncRNAs) in macrophages with an NF-κB reporter.

The innate immune system protects against infections by initiating an inducible inflammatory response. NF-κB is one of the critical transcription factors controlling this complex response, but some aspects of its regulation remain unclear. For example, although long non-coding RNAs (lncRNAs) have been shown to critically regulate gene expression, only a fraction of these have been functionally characterized, and the extent to which lncRNAs control NF-κB expression is unknown. Here, we describe the generation of a GFP-based NF-κB reporter system in immortalized murine bone marrow-derived macrophages (iBMDM). Activation of this reporter, using Toll-like receptor ligands, resulted in GFP expression, which could be monitored by flow cytometry. We also established a CRISPR/Cas9 gene deletion system in this NF-κB reporter line, enabling us to screen for genes that regulate NF-κB signaling. Our deletion-based approach identified two long intergenic non-coding(linc)RNAs, lincRNA-Cox2 and lincRNA-AK170409, that control NF-κB signaling. We demonstrate a potential novel role for lincRNA-Cox2 in promoting IκBα degradation in the cytoplasm. For lincRNA-AK170409, we provide evidence that this nuclearly-localized lincRNA regulates a number of inflammation-related genes. In conclusion, we have established an NF-κB-GFP iBMDM reporter cell line and a line that stably expresses Cas9. Our approach enabled the identification of lincRNA-Cox2 and lincRNA-AK170409 as NF-κB regulators, and this tool will be useful for identifying additional genes involved in regulating this transcription factor critical for immune function

Characterizing Long Noncoding RNAs in Innate Immunity

The innate immune system provides a first line of defense against pathogens by initiating a protective inflammatory response. Upon infection or tissue damage, circulating monocytes migrate to the site of inflammation, where they can differentiate into macrophages. Macrophages are critical innate immune cells important for recognizing invading pathogens and/or damage signals through the use of Toll-like receptors (TLRs) that result in the initiation of host defense pathways, including activation of the major transcription factor NF-κB. Careful regulation of the immune response is essential for eliminating pathogens and preventing sustained inflammation and tissue damage, driving autoimmune disease or cancer.Long non-coding RNAs (lncRNAs) have been implicated as critical regulators of gene expression in the innate immune response, controlling the magnitude, duration and resolution of the inflammatory response. The functional characterization of these genes, specifically in pathways that affect immune cell differentiation and/or their respective function, remain largely unexplored and reveals critical gaps in knowledge. The current dissertation focuses on how lncRNAs regulate innate immunity in macrophage cells. In the first chapter, we explore the role of GAPLINC —gastric adenocarcinoma predictive long intergenic noncoding RNA — a lncRNA functionally conserved across humans and mice. RNA-sequencing analysis reveals a specific role for GAPLINC in regulating NF-κB signaling in both primary human and mouse macrophages. In GAPLINC-depleted cells, we observed enhanced expression of immune response genes that are direct NF-κB targets. Interestingly, Gaplinc knockout mice show resistance to LPS-induced endotoxic shock. Further, we find that basal expression of inflammatory genes prevents clot formation to protect against multiorgan failure and death. These findings identify GAPLINC as a negative regulator of immune genes and highlight the role that lncRNAs can play in the treatment of sepsis and the development of new therapies. In the second chapter, we move beyond the characterization of a single gene candidate and utilize high-throughput clustered regularly interspaced short palindromic repeat (CRISPR) screens to identify a whole host of novel NF-κB regulators along with macrophage-specific essential genes and regulatory elements within those genes From our screen, we identify 115 novel regulators of NF-kB and 60 macrophage-specific viability genes. Additionally, this single-screening approach reveals exciting new information on the complex regulatory biology of TNF and builds upon decades of existing research. We provide evidence that membrane-bound TNF can function in an autocrine manner to negatively regulate inflammation, likely through binding of TNF to the p75 receptor in macrophages. Our data show that editing p55 inhibits Il6 production while editing p75 results in increased production of Il6, highlighting the opposing positive and negative regulatory roles of these two receptors. This has profound effects on improving anti-TNF therapy as blocking the proinflammatory p55 Tnf receptor using antibody-mediated approaches can allow TNF to alternatively bind p75 to promote anti-inflammatory signaling. Finally, in the third chapter, we use long-read Nanopore sequencing to interrogate isoform usage in primary human macrophages treated with different inflammatory stimuli. We used a variety of ligands, LPS, Pam3CSK4, R848, and Poly(I:C) to activate TLR4, TLR1/2, TLR7/8, and TLR3, respectively, to provide comprehensive transcriptome scale isoform information so we can better inform and improve the outcome of single gene-focused follow-up studies. We provide a resource detailing isoform usage present before-and-after stimulation, identify new exons in genes, and provide corresponding levels of expression for each gene as well as neighboring genes. In addition, all this information has been made accessible for the user’s convenience through the UCSC Genome browser. We hope this resource can act as a starting point for other researchers who work in this area of innate immunity to conduct more in-depth mechanistic work

Characterizing Long Noncoding RNAs in Innate Immunity

The innate immune system provides a first line of defense against pathogens by initiating a protective inflammatory response. Upon infection or tissue damage, circulating monocytes migrate to the site of inflammation, where they can differentiate into macrophages. Macrophages are critical innate immune cells important for recognizing invading pathogens and/or damage signals through the use of Toll-like receptors (TLRs) that result in the initiation of host defense pathways, including activation of the major transcription factor NF-κB. Careful regulation of the immune response is essential for eliminating pathogens and preventing sustained inflammation and tissue damage, driving autoimmune disease or cancer.Long non-coding RNAs (lncRNAs) have been implicated as critical regulators of gene expression in the innate immune response, controlling the magnitude, duration and resolution of the inflammatory response. The functional characterization of these genes, specifically in pathways that affect immune cell differentiation and/or their respective function, remain largely unexplored and reveals critical gaps in knowledge. The current dissertation focuses on how lncRNAs regulate innate immunity in macrophage cells. In the first chapter, we explore the role of GAPLINC —gastric adenocarcinoma predictive long intergenic noncoding RNA — a lncRNA functionally conserved across humans and mice. RNA-sequencing analysis reveals a specific role for GAPLINC in regulating NF-κB signaling in both primary human and mouse macrophages. In GAPLINC-depleted cells, we observed enhanced expression of immune response genes that are direct NF-κB targets. Interestingly, Gaplinc knockout mice show resistance to LPS-induced endotoxic shock. Further, we find that basal expression of inflammatory genes prevents clot formation to protect against multiorgan failure and death. These findings identify GAPLINC as a negative regulator of immune genes and highlight the role that lncRNAs can play in the treatment of sepsis and the development of new therapies. In the second chapter, we move beyond the characterization of a single gene candidate and utilize high-throughput clustered regularly interspaced short palindromic repeat (CRISPR) screens to identify a whole host of novel NF-κB regulators along with macrophage-specific essential genes and regulatory elements within those genes From our screen, we identify 115 novel regulators of NF-kB and 60 macrophage-specific viability genes. Additionally, this single-screening approach reveals exciting new information on the complex regulatory biology of TNF and builds upon decades of existing research. We provide evidence that membrane-bound TNF can function in an autocrine manner to negatively regulate inflammation, likely through binding of TNF to the p75 receptor in macrophages. Our data show that editing p55 inhibits Il6 production while editing p75 results in increased production of Il6, highlighting the opposing positive and negative regulatory roles of these two receptors. This has profound effects on improving anti-TNF therapy as blocking the proinflammatory p55 Tnf receptor using antibody-mediated approaches can allow TNF to alternatively bind p75 to promote anti-inflammatory signaling. Finally, in the third chapter, we use long-read Nanopore sequencing to interrogate isoform usage in primary human macrophages treated with different inflammatory stimuli. We used a variety of ligands, LPS, Pam3CSK4, R848, and Poly(I:C) to activate TLR4, TLR1/2, TLR7/8, and TLR3, respectively, to provide comprehensive transcriptome scale isoform information so we can better inform and improve the outcome of single gene-focused follow-up studies. We provide a resource detailing isoform usage present before-and-after stimulation, identify new exons in genes, and provide corresponding levels of expression for each gene as well as neighboring genes. In addition, all this information has been made accessible for the user’s convenience through the UCSC Genome browser. We hope this resource can act as a starting point for other researchers who work in this area of innate immunity to conduct more in-depth mechanistic work

Generation of an isoform-level transcriptome atlas of macrophage activation.

RNA-seq is routinely used to measure gene expression changes in response to cell perturbation. Genes upregulated or downregulated following some perturbation are designated as genes of interest, and their most expressed isoform(s) would then be selected for follow-up experimentation. However, because of its need to fragment RNA molecules, RNA-seq is limited in its ability to capture gene isoforms and their expression patterns. This lack of isoform-specific data means that isoforms would be selected based on annotation databases that are incomplete, not tissue specific, or do not provide key information on expression levels. As a result, minority or nonexistent isoforms might be selected for follow-up, leading to loss in valuable resources and time. There is therefore a great need to comprehensively identify gene isoforms along with their corresponding levels of expression. Using the long-read nanopore-based R2C2 method, which does not fragment RNA molecules, we generated an Isoform-level transcriptome Atlas of Macrophage Activation that identifies full-length isoforms in primary human monocyte-derived macrophages. Macrophages are critical innate immune cells important for recognizing pathogens through binding of pathogen-associated molecular patterns to toll-like receptors, culminating in the initiation of host defense pathways. We characterized isoforms for most moderately-to-highly expressed genes in resting and toll-like receptor-activated monocyte-derived macrophages, identified isoforms differentially expressed between conditions, and validated these isoforms by RT-qPCR. We compiled these data into a user-friendly data portal within the UCSC Genome Browser (https://genome.ucsc.edu/s/vollmers/IAMA). Our atlas represents a valuable resource for innate immune research, providing unprecedented isoform information for primary human macrophages

A conserved long noncoding RNA, GAPLINC, modulates the immune response during endotoxic shock.

Recent studies have identified thousands of long noncoding RNAs (lncRNAs) in mammalian genomes that regulate gene expression in different biological processes. Although lncRNAs have been identified in a variety of immune cells and implicated in immune response, the biological function and mechanism of the majority remain unexplored, especially in sepsis. Here, we identify a role for a lncRNA-gastric adenocarcinoma predictive long intergenic noncoding RNA (GAPLINC)-previously characterized for its role in cancer, now in the context of innate immunity, macrophages, and LPS-induced endotoxic shock. Transcriptome analysis of macrophages from humans and mice reveals that GAPLINC is a conserved lncRNA that is highly expressed following macrophage differentiation. Upon inflammatory activation, GAPLINC is rapidly down-regulated. Macrophages depleted of GAPLINC display enhanced expression of inflammatory genes at baseline, while overexpression of GAPLINC suppresses this response. Consistent with GAPLINC-depleted cells, Gaplinc knockout mice display enhanced basal levels of inflammatory genes and show resistance to LPS-induced endotoxic shock. Mechanistically, survival is linked to increased levels of nuclear NF-κB in Gaplinc knockout mice that drives basal expression of target genes typically only activated following inflammatory stimulation. We show that this activation of immune response genes prior to LPS challenge leads to decreased blood clot formation, which protects Gaplinc knockout mice from multiorgan failure and death. Together, our results identify a previously unknown function for GAPLINC as a negative regulator of inflammation and uncover a key role for this lncRNA in modulating endotoxic shock

A conserved long noncoding RNA, GAPLINC, modulates the immune response during endotoxic shock.

Recent studies have identified thousands of long noncoding RNAs (lncRNAs) in mammalian genomes that regulate gene expression in different biological processes. Although lncRNAs have been identified in a variety of immune cells and implicated in immune response, the biological function and mechanism of the majority remain unexplored, especially in sepsis. Here, we identify a role for a lncRNA-gastric adenocarcinoma predictive long intergenic noncoding RNA (GAPLINC)-previously characterized for its role in cancer, now in the context of innate immunity, macrophages, and LPS-induced endotoxic shock. Transcriptome analysis of macrophages from humans and mice reveals that GAPLINC is a conserved lncRNA that is highly expressed following macrophage differentiation. Upon inflammatory activation, GAPLINC is rapidly down-regulated. Macrophages depleted of GAPLINC display enhanced expression of inflammatory genes at baseline, while overexpression of GAPLINC suppresses this response. Consistent with GAPLINC-depleted cells, Gaplinc knockout mice display enhanced basal levels of inflammatory genes and show resistance to LPS-induced endotoxic shock. Mechanistically, survival is linked to increased levels of nuclear NF-κB in Gaplinc knockout mice that drives basal expression of target genes typically only activated following inflammatory stimulation. We show that this activation of immune response genes prior to LPS challenge leads to decreased blood clot formation, which protects Gaplinc knockout mice from multiorgan failure and death. Together, our results identify a previously unknown function for GAPLINC as a negative regulator of inflammation and uncover a key role for this lncRNA in modulating endotoxic shock

High-Throughput CRISPR Screening Identifies Genes Involved in Macrophage Viability and Inflammatory Pathways.

Macrophages are critical effector cells of the immune system, and understanding genes involved in their viability and function is essential for gaining insights into immune system dysregulation during disease. We use a high-throughput, pooled-based CRISPR-Cas screening approach to identify essential genes required for macrophage viability. In addition, we target 3' UTRs to gain insights into previously unidentified cis-regulatory regions that control these essential genes. Next, using our recently generated nuclear factor κB (NF-κB) reporter line, we perform a fluorescence-activated cell sorting (FACS)-based high-throughput genetic screen and discover a number of previously unidentified positive and negative regulators of the NF-κB pathway. We unravel complexities of the TNF signaling cascade, showing that it can function in an autocrine manner in macrophages to negatively regulate the pathway. Utilizing a single complex library design, we are capable of interrogating various aspects of macrophage biology, thus generating a resource for future studies

High-Throughput CRISPR Screening Identifies Genes Involved in Macrophage Viability and Inflammatory Pathways.

Macrophages are critical effector cells of the immune system, and understanding genes involved in their viability and function is essential for gaining insights into immune system dysregulation during disease. We use a high-throughput, pooled-based CRISPR-Cas screening approach to identify essential genes required for macrophage viability. In addition, we target 3' UTRs to gain insights into previously unidentified cis-regulatory regions that control these essential genes. Next, using our recently generated nuclear factor κB (NF-κB) reporter line, we perform a fluorescence-activated cell sorting (FACS)-based high-throughput genetic screen and discover a number of previously unidentified positive and negative regulators of the NF-κB pathway. We unravel complexities of the TNF signaling cascade, showing that it can function in an autocrine manner in macrophages to negatively regulate the pathway. Utilizing a single complex library design, we are capable of interrogating various aspects of macrophage biology, thus generating a resource for future studies