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.
