Most of our understanding about the cellular response to microbes by innate immune cells is shaped by our work with macrophages in culture. Because there exist such a large number of specialized innate immune cells, it stands to reason that the response to pathogen by different cell types will be specialized, even if the same innate immune receptors are used - my work documents two such incidents. Here we report that TLR2 activation by multiple viruses leads to production of type I interferon (IFN) only in Ly6Chigh inflammatory monocytes. Importantly, TLR2-dependent induction of type I IFN only occurs in response to viruses, not bacterial TLR2 ligands, indicating that TLR2 is capable of discriminating between these pathogen classes. Separately, we demonstrate that bone marrow neutrophils need to be "armed" by GM-CSF before they respond to TLR ligands, unlike other innate immune cells. We then go on to show that certain pro-inflammatory signals in vivo are sufficient to "arm" neutrophils so they could more readily respond to pathogen associated microbial patterns.

UNC93B1, a multipass transmembrane protein required for TLR3, TLR7, TLR9, TLR11, TLR12, and TLR13 function, controls trafficking of TLRs from the endoplasmic reticulum (ER) to endolysosomes. In this dissertation, I introduce the field of innate immunity, endosomal TLRs, and the known roles of UNC93B1. Furthermore, I discuss results from our studies to understand the mechanism by which UNC93B1 mediates its regulatory effects. Our results demonstrate that UNC93B1 enters the secretory pathway and directly controls the packaging of TLRs into COPII vesicles that bud from the ER. Unlike other COPII loading factors, UNC93B1 remains associated with the TLRs through post-Golgi sorting steps. Unexpectedly, these steps are different among endosomal TLRs. TLR9 requires UNC93B1-mediated recruitment of adaptor protein complex 2 (AP-2) for delivery to endolysosomes while TLR7, TLR11, TLR12, and TLR13 utilize alternative trafficking pathways. In addition, we established several assays to determine UNC93B1 function and describe a mouse model to better understand the regulation of an endogenous nucleic acid sensing TLR, TLR9. Our studies describe mechanisms for differential sorting of endosomal TLRs by UNC93B1, which may explain the distinct roles played by these receptors in certain autoimmune diseases.

Toll-like receptors (TLRs) contribute to host resistance to microbial pathogens and drive the evolution of virulence mechanisms. We have examined the relationship between host resistance and pathogen virulence using mice with a functional allele of the Nramp-1 gene and lacking combinations of TLRs. Mice deficient in both TLR2 and TLR4 were highly susceptible to the intracellular bacterial pathogen Salmonella typhimurium, consistent with reduced innate immune function. However, mice lacking additional TLRs involved in S. typhimurium recognition were less susceptible to infection. In these TLR-deficient cells, bacteria failed to upregulate Salmonella pathogenicity island 2 (SPI-2) genes and did not form a replicative compartment. We demonstrate that TLR signaling enhances the rate of acidification of the Salmonella containing phagosome, and inhibition of this acidification prevents SPI-2 induction. Our results indicate that S. typhimurium requires cues from the innate immune system to regulate virulence genes necessary for intracellular survival, growth, and systemic infection.

Mammalian Toll-like receptors (TLRs) 3, 7, 8 and 9 initiate immune responses to infection by recognizing microbial nucleic acids; however, these responses come at the cost of potential autoimmunity due to inappropriate recognition of self nucleic acid. The localization of TLR9 and TLR7 to intracellular compartments appears to play a role in facilitating responses to viral nucleic acids while maintaining tolerance to self nucleic acid, yet the cell biology regulating the trafficking and localization of these receptors remains poorly understood. Here, we define the route by which TLR9 and TLR7 exit the endoplasmic reticulum (ER) and traffic to endolysosomes. Surprisingly, the ectodomains of TLR9 and TLR7 are cleaved in the endolysosome, such that no full-length protein is detectable in the compartment where ligand is recognized. Remarkably, though both forms of TLR9 are capable of binding ligand, only the

processed form of TLR9 recruits MyD88 upon activation, arguing that this truncated receptor, rather than the full-length form, is functional. TLR9 proteolysis is a multi-step process. The first step removes the majority of the ectodomain and can be carried out by asparagine endopeptidase or cathepsin family members. This initial cleavage event is followed by a trimming event that is solely cathepsin mediated but also required for optimal receptor signaling. The dual requirement for asparagine endopeptidase and cathepsins is observed in all cell types analyzed, including mouse macrophages and dendritic cells. Importantly, TLR7 and TLR3 are processed in an analogous manner, suggesting that proteolysis is a regulatory strategy that has been adopted along side the ability to recognize nucleic acids as ligands. Finally, conditions that prevent receptor proteolysis, including forced surface localization, render the receptor non-functional. We propose that ectodomain cleavage represents a strategy to restrict receptor activation to endolysosomal compartments and prevent TLRs from responding to self nucleic acid.

Although apoptotic cells (ACs) contain nucleic acids that can be recognized by Toll-like receptors (TLRs), engulfment of ACs does not initiate inflammation in healthy organisms. In this dissertation I explore the mechanisms that help maintain this homeostasis. I discuss nucleic acid sensing TLRs and their regulation. I also provide an introduction to the clearance of ACs from the body. I discuss how I identified and characterized macrophage populations that continually engulf ACs in several distinct tissues. These macrophages share characteristics compatible with immunologically silent clearance of ACs, including high expression of AC recognition receptors, low expression of TLR9, and reduced TLR responsiveness to nucleic acids. When removed from tissues these macrophages lose many of these characteristics and generate inflammatory responses to AC-derived nucleic acids, suggesting that cues from the tissue microenvironment are required to program macrophages for silent AC clearance. I show that KLF2 and KLF4 control expression of many genes within this AC clearance program. Coordinated expression of AC receptors with genes that limit responses to nucleic acids may represent a central feature of tissue macrophages that ensures maintenance of homeostasis

Abstract

The Regulation of TLR7 and TLR9 in the Prevention of Autoimmunity

By

Zachary Robert Newman

Doctor of Philosophy in Molecular and Cell Biology

University of California, Berkeley

Professor Gregory M Barton, Chair

Recognition of nucleic acids is an evolutionarily conserved trait of the immune system that enables detection of a diverse array of microbial species with relatively few receptors. However, the inappropriate recognition of self-derived nucleic acids is well documented for its role in autoimmune diseases. Two such receptors that recognize nucleic acids, Toll-like receptor (TLR) 7 and TLR9, are highly regulated in order to maintain the proper discrimination between self and non-self. In this dissertation I discuss the background of what is known about TLRs, with an emphasis on nucleic acid sensing TLRs and evidence for their involvement in autoimmune disease. I also provided an introduction to the phenomenon of codon usage bias and discuss the debate surrounding its functional significance. I then present our findings that codon bias may be playing a significant role in the balance of protein levels between TLR7 and TLR9. In an endogenous context, we observe a markedly different measured translational efficiency for TLR7 compared to TLR9, correlating with their degree of codon bias. Codon optimization of TLR7’s coding sequence dramatically increases the translation efficiency and subsequent protein levels in an exogenous setting. Furthermore, by scaling the level of optimization we reveal how the different mechanisms of transcription, mRNA stability, and translation, all of which contribute to overall protein levels, are affected by codon bias. In contrast to the exogenous setting, codon optimization of TLR7 at the endogenous locus did not result in an increase in TLR7 function suggesting additional levels of regulation may be involved. In this dissertation I also introduce a novel method to study the functional significance of misregulated localization of nucleic acid sensing TLRs in an endogenous context. Our studies introduced a conditional mutant allele of TLR9 that is capable of signaling from the cell surface. The mutant is well expressed in a controlled, cell-specific manner with preliminary results suggesting potential autoimmune consequences. Combined, our studies significantly add to the literature by illuminating the importance of two distinct mechanisms regulating nucleic acid sensing TLRs in the prevention of autoimmunity.

Recognition of nucleic acids as a signature of infection by Toll-like receptors (TLRs) 7 and 9 exposes the host to potential self-recognition and autoimmunity. It has been proposed that intracellular compartmentalization is largely responsible for reliable self/non-self discrimination by these receptors. Our lab has previously shown that TLR9 and TLR7 require processing prior to activation, which may further restrict TLR9 and TLR7 compartmentalization and reinforce self/non-self discrimination; although, this possibility has remained untested. We have identified residues within the TLR9 transmembrane (TM) region that confer the requirement for ectodomain proteolysis. TLR9 TM mutants responded to extracellular DNA and mice expressing such receptors died from systemic inflammation and anemia. This inflammatory disease did not require lymphocytes and appears to require recognition of self-DNA by dendritic cells. These results provide the first demonstration that TLR-intrinsic mutations can lead to a break in tolerance and support the hypothesis that ectodomain processing has evolved to reinforce self/non-self discrimination by nucleic acid-sensing TLRs.