UC Santa Cruz

Inflammation is the host’s natural response to protect against infection and maintain homeostasis. The innate immune system is our first line of defense where immune cells such as dendritic cells, macrophages and neutrophils recognize and initiate responses to the pathogen through pattern recognition receptors (PRRs). Macrophages play an essential role in propagating initial stages of the inflammatory response; hence, it is critical to fully understand their signaling mechanisms to better combat infectious and inflammatory conditions. Macrophages sense danger signals through PRRs -such as TLR4- which recognizes pathogen-associated molecular patterns (PAMPs) on the pathogen surface triggering pro-inflammatory signaling cascades to promote macrophage activation. Proper propagation of this response is essential to mount sufficient inflammatory activation, however, careful regulation is needed to maintain homeostasis. While we are making tremendous strides in understanding inflammatory signaling pathways and their triggers, the factors responsible for maintaining an effective and balanced response are not well known. This dissertation addresses newly discovered functional regulators of the immune response and stresses the need for developing new and specialized tools to better understand the complexity of the regulatory machinery. Unraveling the complexity of signaling pathways that govern the magnitude and intensity of response to stimulus continues to progress under the limitations of the tools we use to study it. Development of NGS has propelled a whole field towards better understanding of gene expression changes under inflammatory conditions. However, this powerful technology is limited in detecting subtle and intricate post transcriptional changes in splicing, editing and modifications. In the first chapter, we highlight the evolving capabilities of NGS that allowed for the study of functional lncRNAs in the immune response as well as splice variants of certain genes that modulate immune activation. We also discuss how long read sequencing allowed for the construction of the complete genome and enabled us to study the role of alternative splicing and RNA modifications in modulating immunity. In the second chapter we discuss a novel role for HNRNPA2B1 in regulating the macrophage inflammatory response. HNRNPA2B1 is an abundant RNA binding protein that’s involved in RNA processing and maturation, in this chapter, we discuss its involvement in promoting IFNG signaling in macrophages to modulate macrophage activation upon exposure to stimulus. HNRNPA2B1 depletion in macrophages resulted in mice being less responsive to endotoxic shock and more sensitive to Salmonella infection due to disruption in the IFNG response signaling causing macrophages to be less efficient in clearing the pathogen. As a result of the disruption in the IFNG response cascade, the macrophage overall inflammatory gene expression was reduced which was represented by lower expression levels of TFs such as JAKs and STATs as well as lower cytokine production in the serum and spleen. Mechanistically, HNRNPA2B1 depletion in macrophages altered splicing outcome of the IFNGR leading to lower levels of the receptor on cell surface. This chapter further highlights the complexity of the immune regulatory machinery represented by the role played by alternative splicing in affecting organism-wide changes in the inflammatory status and disease outcome. In the third chapter we delve into innate immune modulation because of smoke exposure since it’s highly linked to many inflammatory diseases such as chronic obstructive pulmonary disease (COPD). Here, we showcase smoke exposure novel involvement in immune activation, specifically through modulation in gene expression of a large number of coding and non-coding transcripts in macrophages. Most importantly, smoke exposure led to the activation of an immune regulating lncRNA -lincRNACox2- which has been shown to regulate inflammatory gene expression in mice. Through complex mouse models, we show that LincRNACox2 functions in the lung to regulate inflammatory gene expression in mice following smoke exposure. This dissertation showcases significant advances in understanding modes of immune response regulation through an evolving view that extends beyond regulation through gene expression. We highlight non-traditional modes of gene expression regulation through RNA binding proteins, alternative splicing, and non-coding transcripts (e.g., lncRNAs).