UC Santa Cruz

Infiltrating and resident macrophages are the critical effector cells of the innate immune system, providing the first line of defense against infection through the induction of inflammation. While an inflammatory response is critical for combating pathogens, excessive or uncontrolled inflammatory responses are drivers of diseases, such as cancer and autoimmune diseases. These pathways have been studied for decades, however, a complete understanding of the molecular mechanisms governing inflammation remains elusive. Recently, the contribution of non-coding RNA regions, such as long non-coding RNAs (lncRNAs), have been implicated as critical regulators of the innate immune response through controlling the magnitude, duration, and resolution of inflammation. The functional characterization of how lncRNAs, other ncRNAs regions of protein-coding genes and alternative splicing regulate innate immunity remains largely unexplored.This thesis focuses on how lncRNAs and untranslated regions (UTRs) of mRNAs regulate innate immunity using both in vitro and in vivo methods, as well as developing a novel cell line necessary for culturing myeloid cells. First, we generated a HEK-293T cell line expressing murine GM-CSF that secretes high levels of GM-CSF (~180ng/ml) into complete media as an alternative to commercial GM-CSF. We assessed the ability of supernatant GM-CSF to culture alveolar macrophages (AM) and bone marrow-derived dendritic cells (BMDC) by flow cytometry, as well as RT-qPCR and ELISA to measure inflammatory responses. Taken together our results show that the crude supernatant GM-CSF yielded a more pure and higher number of BMDCs, as well as maintained inflammatory competency in either the BMDCs or AMs populations, in comparison to commercially available GM-CSF. Second, this thesis focuses on a seminal lincRNA in the field of immunity, lincRNA-Cox2, which is a broad-acting regulatory component functioning to mediate the activation and repression of distinct classes of immune genes in macrophages. LincRNAs function to control gene expression either in cis, where they influence the expression and/or chromatin state of neighboring genes, or in trans where the lincRNA leaves the site of transcription and affects genes on different chromosomes. In this study, utilizing different genetic manipulations in vitro and in vivo we found that lincRNA-Cox2 functions through an enhancer RNA mechanism to regulate its neighboring protein-coding gene Ptgs2 in cis. More importantly, lincRNA-Cox2 also functions in trans, independently of Ptgs2, to regulate critical innate immune genes in vivo. Subsequently, using bulk and single-cell RNA-seq, in addition to fluorescence-activated cell sorting, we show that lincRNA-Cox2 is most highly expressed in alveolar macrophages where it functions to control immune gene expression following acute lung injury. Utilizing a newly generated lincRNA-Cox2 transgenic overexpressing mouse, we show that it can function in trans to control genes including Ccl3 and Ccl4. Finally, using a chronic obstructive pulmonary disease (COPD) mouse model we find lincRNA-Cox2 is induced by cigarette smoke (CS) and functions as a positive and negative regulator in vivo and in vitro. Collectively, this work provides insights into how lincRNA-Cox2 functions in trans to impact inflammatory signaling during acute and chronic inflammatory models. Finally, the contribution of alternative splicing to the regulation of innate immune responses remains poorly studied. Through analysis of differential splicing using both short-read and long-read RNA sequencing of human and mouse macrophages, we identified that alternative first exon (AFE) changes are a prominent widespread event during inflammatory activation. Of these AFE events, we identified 95 unannotated transcription start sites (TSS) in mice using a de novo transcriptome generated by long-read native RNA sequencing, one of which is in the cytosolic receptor for dsDNA and a known inflammatory inducible gene, Aim2. We show that this unannotated, inflammatory-specific, AFE isoform of Aim2 contains an iron-responsive element in its 5′UTR enabling mRNA translation to be regulated by iron levels. This work highlights the importance of examining alternative isoform changes and translational regulation in the innate immune response and uncovers novel regulatory mechanisms of Aim2. The studies presented here identify novel mechanistic details of ncRNA genes or ncRNA regions of protein-coding genes using in vivo and in vitro models, setting in place a new RNA-immune-regulatory layer within innate immunity.