Role of CD5L in control of human innate immune function
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Innate immune signaling has numerous ways to sense changes in cellular metabolism to ensure appropriate response to threatening agents. For instance, cholesterol metabolites act as ligands for nuclear receptor transcription factors, such as LXRs, that connect cholesterol homeostasis to immune signaling. LXRs also control expression of genes that serve as additional connection points between immune and metabolic signaling. One of such genes is CD5L, which encodes a scavenger receptor-like molecule. This molecule has recently been shown to control RORγt nuclear receptor-mediated inflammatory signaling by changing cellular metabolism of Th17 cells. Even though CD5L is an abundant circulatory protein, in Th17 cells it regulates the inflammatory state in cell autonomous fashion. The effect of endogenous CD5L expression levels in human immune cells remains largely unexplored. It is therefore important to understand the role of CD5L endogenous expression in human macrophages and identify the mechanism by which CD5L functions. In addition to its intracellular immunomodulatory role, the extracellular CD5L has been shown to interact with factor H, a negative regulator of the complement system. This observation led us to formulate and test a hypothesis that CD5L could control bactericidal activity of the complement. In the current study, our first aim was to characterize the inflammatory role in human macrophages of RORα, a member of the ROR nuclear receptor family. We found that macrophages disrupted for RORA, had a dramatic increase in basal expression of a subset of NF-ҡB-regulated genes, at both transcriptional and translational levels mimicking classically activated M1 macrophages. RNAseq analysis of mutant cells further revealed the hyper-inflammatory state in mutant cells confirming the anti-inflammatory role for RORα in human macrophages. Our second aim was to assess the role of CD5L in defining the inflammatory state in macrophages and to define the mechanism of this function. Following disruption of CD5L gene in human macrophages, we observed a dramatic decrease in basal expression levels of a subset of NF-ҡB-regulated genes when compared to control cell line. In addition, RNAseq analysis provided more evidence supporting the pro-inflammatory role for CD5L in macrophages. This phenotype is opposite to the observed anti-inflammatory role for RORα. Thus, we hypothesized that hypo-inflammatory phenotype caused by deletion of CD5L, is mediated by activation of RORα, similarly to how RORγt family member responds to changes in CD5L levels in Th17 cells. Because CD5L has been shown to remodel the cellular lipidome, we used mass spectrometry-based analysis of intracellular lipid content. This analysis has revealed substantial changes in lipid content of CD5Ldeletion cells compared to control, supporting a possibility that these alterations affect the pool of ligands for RORα nuclear receptor and thus alter its activity. The third aim of the thesis was to test if CD5L is involved in control of bactericidal activity of the complement. We observed that in the presence of free CD5L there is diminished binding of factor H to Neisseria gonorrhoeae. Furthermore, recombinant CD5L changed bactericidal activity of normal human serum, suggesting direct involvement of CD5L in control of the complement activation. Overall, our work demonstrated that CD5L controls the inflammatory state of human macrophages using its lipidome remodeling function and regulation of RORα activity. Thus, we identified a novel connection between inflammatory and metabolic signaling pathways. Moreover, we found a novel aspect of the circulating CD5L function. Our experiments indicate that CD5L changes the dynamics of factor H recruitment by bacterial pathogens, such as Neisseria gonorrhoeae. Furthermore, our ex vivo complement killing assay revealed the effect of CD5L on the viability of the bacteria. These findings offer a potential target for future treatment for the infection.