Trafficking of endosomal Toll-like receptors

Abstract

Over the past decade we have learned much about nucleic acid recognition by the innate immune system and in particular by Toll-like receptors (TLRs). These receptors localize to endosomal compartments where they are poised to recognize microbial nucleic acids. Multiple regulatory mechanisms function to limit responses to self DNA or RNA, and breakdowns in these mechanisms can contribute to autoimmune or inflammatory disorders. In this review we discuss our current understanding of the cell biology of TLRs involved in nucleic acid recognition and how localization and trafficking of these receptors regulates their function.

Keywords: AP complex; UNC93B1; autoimmunity; innate immunity; type I interferon.

Figure 1. Overview of Toll-like Receptor Trafficking

TLRs are synthesized in the endoplasmic reticulum (ER), traffic to the Golgi, and ultimately localize to the cell surface or remain intracellular in endosomes or lysosomes. All TLRs have a horseshoe-like ectodomain structure and interact with their ligands as dimers. Surface resident TLRs 1, 2, 4, 5, and 6 recognize microbial ligands such as lipopolysaccharide (LPS), bacterial lipoproteins, and flagellin. Most endosomal TLRs recognize microbial derived nucleic acids. The ectodomains of these nucleic acid sensing TLRs undergo proteolytic processing in endosomes to generate functional receptors capable of signaling upon ligand recognition.

Figure 2. Trafficking of nucleic acid sensing TLRs from ER to endosomes

(A) Regulation at the Endoplasmic Reticulum (ER). ER export is an important mode of regulation for controlling levels of functional TLRs in the cell. TLRs 3, 7, 8, 9, 11, 12, and 13 require UNC93B1 to exit the ER. UNC93B1 is necessary for TLR9 incorporation into COPII vesicles. There is evidence for direct competition between TLR7 and TLR9 for UNC93B1, as the presence of TLR9 inhibits trafficking of TLR7. To what extent other TLRs compete for UNC93B1 is still an open question. For most TLRs, with the exception of TLR3, trafficking from the ER also requires the folding chaperones gp96 and PRAT4A. (B) Trafficking routes from Golgi to endosome. Upon reaching the Golgi, nucleic acid sensing TLRs are sorted to endosomes. UNC93B1 traffics with nucleic acid sensing TLRs to endosomes, but the role for UNC93B1 in mediating TLR trafficking from Golgi to endosomes differs for individual TLRs. While TLR9 requires UNC93B1 to recruit the AP-2 complex at the cell surface to mediate endocytosis of the UNC93B1/TLR9 complex, TLR7 can directly recruit the AP-4 complex and mediate its own trafficking to endosomes. The trafficking pathways for other endosomal TLRs have not been directly investigated. TLR9, and presumably TLR7, are ubiqutinated and recognized by the HRS/ESCRT pathway to maintain receptor stability in lysosomes.

Figure 3. Heterogeneity of nucleic acid sensing TLR signaling compartments

Nucleic acids encounter TLRs in endosomes after uptake from the extracellular milieu. Depending on the method of delivery into the cell (e.g. in an immune complex, as oligonucleotides, or encapsulated in a viral particle), nucleic acids may reach distinct endosomes with unique signaling properties. Early and late endosomes are spatially and temporally distinct from one another. TLR9 signaling from late endosomes leads to activation of NF-κB transcription factors and induction of pro-inflammatory cytokines (TNFα, IL-12p40 and IL-6) (A and B). TLR9 signaling from a distinct population of endosomes leads recruitment of IRF and induction of type I IFN (IFNα, IFNβ). One model suggests that this specialized compartment is simply early endosomes (A). Two other compartments capable of type I IFN induction have been described, and it remains unclear whether these three compartments are, in fact, distinct. The AP-3 complex regulates TLR9 trafficking to mature lysosome related organelles (LROs), compartments distinct from endosomes and lysosomes and capable of initiating type I interferon signaling (B). Second, a compartment for DNA-immune complex (DNA-IC)-dependent IFNα signaling requires components of the autophagy pathway through a mechanism termed LC3 associated phagocytosis (LAP). DNA-IC compartments mature using solely the LAP pathway and not the AP-3 dependent pathway (C). In contrast, CpG also utilizes autophagy pathways (mediated by ATG5 and ATG7) but also requires AP-3 for type I IFN signaling. While much of characterization of these compartments has focused on TLR9, it is likely that TLR7 populates similarly diverse and specialized endosomes.