Innate immune recognition by human toll-like receptor 10 and other members of the toll-like receptor 2 subfamily

摘要

Toll-like receptors (TLRs) are central receptors of the innate immune system which drive host inflammation and adaptive immune responses upon detection of invading microbes. Drugs targeting TLRs are of considerable interest as potential inflammatory regulators, vaccine adjuvants, and novel immunotherapeutics. Among human TLRs, TLR10 is the only remaining family member without a defined agonist or function. Phylogenetic analysis reveals that TLR10 is most related to TLR1 and TLR6, both of which mediate immune responses to a variety of microbial and fungal components in cooperation with TLR2. Knowledge gained of this orphan receptor is useful to fully understand the function of TLR2 subfamily, which comprises TLRs 2, 1, 6 and 10. The primary objective of this doctoral dissertation research is to define the innate immune sensing function of TLR10 and understand the mechanisms responsible for ligand recognition within the TLR2 subfamily.Chapter One introduces the general field of innate immunity and reviews the current knowledge on TLRs, including structure and function, signaling pathways as well as signaling regulation of these receptors with an emphasis on the TLR2 subfamily.Chapter Two describes a high-throughput chemical library screen developed to uncover the ligands of TLR10 and novel TLR2 agonists. A synthetic chemical library of 24,000 compounds was screened using an IL-8 driven-luciferase reporter in cells expressing TLRs 2, 1, 6 and 10. The screen failed to discover TLR10 ligands, but yielded several novel TLR2 dependent activators that utilize TLR1, TLR6, or both as co-receptors. These novel small molecule compounds are aromatic in nature and structurally unrelated to any known TLR2 agonists. Two of the most potent compounds exhibit species specificity and are inactive toward murine peritoneal macrophages. Mutational analysis reveals that while the central extracellular region of TLR1 is required for stimulation, there are subtle differences in the mechanism of stimulation mediated by the synthetic compounds in comparison to natural lipoprotein agonists. The three most potent compounds stimulate TNF-α production form human peripheral blood monocytes. The implication of these results and the potential importance of the novel TLR2 agonists are also discussed (Guan et al. Journal of Biological Chemistry. 2010 July 30, 285: 23755-23762).Chapter Three focuses on the identification of TLR10 ligands and characterization of sensing mechanisms by this receptor. The generation and analysis of chimeric receptors, containing the extracellular recognition domain of TLR10 and the intracellular signaling domain of TLR1, revealed that TLR10, in cooperation with TLR2, senses triacylated lipopeptides and a wide variety of other microbial-derived agonists shared by TLR1, but not TLR6. TLR10 requires TLR2 for innate immune recognition and these receptors colocalize in the phagosome and physically interact in an agonist dependent fashion. Computational modeling and mutational analysis of TLR10 show preservation of the essential TLR2 dimer interface and lipopeptide binding channel found in TLR1. Co-immunoprecipitation experiments indicate that, similar to TLR2/1, TLR2/10 complexes recruit the proximal adaptor MyD88 to the activated receptor complex. However, TLR10, either alone or in cooperation with TLR2, fails to activate typical TLR-induced signaling including NF-κB, IL-8 or IFN-β driven reporters (Guan et al. Journal of Immunology, 2010 May 1;184(9):5094-103). This finding explains why I was unable to discover synthetic ligands of TLR10 in Chapter Two. Interestingly, the novel chemical compounds are agonists recognized only by TLR2/1 and not by TLR2/10. Chapter Four describes the generation of monoclonal antibodies against the extracellular domain of TLR10. A total of 15 hybridoma lines were established that secrete monoclonal antibodies that bind strongly to TLR10, but to neither TLR 1 nor TLR6. The purified anti-TLR10 antibody was used to examine endogenous TLR10 expression in human peripheral blood. I found that granulocytes, natural killer (NK) cells, monocytes and T cells lack detectable expression of TLR10. However human B cells express high cell surface levels of this receptor, suggesting that TLR10 plays a functional role in the B cell lineage.Chapter Five summarizes major findings from the dissertation research, assesses their contribution and potential implications to the TLR field, and explores future research directions for understanding the biological roles of TLR10.