Cutting edge: the UNC93B1 tyrosine-based motif regulates trafficking and TLR responses via separate mechanisms.

Sensing of nucleic acids by TLRs is crucial in the host defense against viruses and bacteria. Unc-93 homolog B1 (UNC93B1) regulates the trafficking of nucleic acid-sensing TLRs from the endoplasmic reticulum to endolysosomes, where the TLRs encounter their respective ligands and become activated. In this article, we show that a carboxyl-terminal tyrosine-based sorting motif (YxxΦ) in UNC93B1 differentially regulates human nucleic acid-sensing TLRs in a receptor- and ligand-specific manner. Destruction of YxxΦ abolished TLR7, TLR8, and TLR9 activity toward nucleic acids in human B cells and monocytes, whereas TLR8 responses toward small molecules remained intact. YxxΦ in UNC93B1 influenced the subcellular localization of human UNC93B1 via both adapter protein complex (AP)1- and AP2-dependent trafficking pathways. However, loss of AP function was not causal for altered TLR responses, suggesting AP-independent functions of YxxΦ in UNC93B1.

The epithelia-specific membrane trafficking factor AP-1B controls gut immune homeostasis in mice.

BACKGROUND & AIMS: Epithelial cells that cover the intestinal mucosal surface maintain immune homeostasis and tolerance in the gastrointestinal tract. However, little is known about the molecular mechanisms that regulate epithelial immune functions. Epithelial cells are distinct in that they are highly polarized; this polarity is, at least in part, established by the epithelium-specific polarized sorting factor adaptor protein (AP)-1B. We investigated the role of AP-1B-mediated protein sorting in the maintenance of gastrointestinal immune homeostasis. METHODS: The role of AP-1B in intestinal immunity was examined in AP-1B-deficient mice (Ap1m2(-/-)) by monitoring their phenotypes, intestinal morphology, and epithelial barrier functions. AP-1B-mediated protein sorting was examined in polarized epithelial cells from AP-1B knockdown and Ap1m2(-/-) mice. RESULTS: Ap1m2(-/-) mice developed spontaneous chronic colitis, characterized by accumulation of interleukin-17A-producing, T-helper 17 cells. Deficiency of AP-1B caused epithelial immune dysfunction, such as reduced expression of antimicrobial proteins and impaired secretion of immunoglobulin A. These defects promoted intestinal dysbiosis and increased bacterial translocation within the mucosa. Importantly, AP-1B deficiency led to mistargeting of a subset of basolateral cytokine receptors to the apical plasma membrane in a polarized epithelial cell line and in colonic epithelial cells from mice. AP1M2 expression was reduced significantly in colonic epithelium samples from patients with Crohn's disease. CONCLUSIONS: AP-1B is required for proper localization of a subset of cytokine receptors in polarized epithelial cells, which allows them to respond to cytokine signals from underlying lamina propria cells. The AP-1B-mediated protein sorting machinery is required for maintenance of immune homeostasis and prevention of excessive inflammation.

Antibody to AP1B adaptor blocks biosynthetic and recycling routes of basolateral proteins at recycling endosomes.

The epithelial-specific adaptor AP1B sorts basolateral plasma membrane (PM) proteins in both biosynthetic and recycling routes, but the site where it carries out this function remains incompletely defined. Here, we have investigated this topic in Fischer rat thyroid (FRT) epithelial cells using an antibody against the medium subunit micro1B. This antibody was suitable for immunofluorescence and blocked the function of AP1B in these cells. The antibody blocked the basolateral recycling of two basolateral PM markers, Transferrin receptor (TfR) and LDL receptor (LDLR), in a perinuclear compartment with marker and functional characteristics of recycling endosomes (RE). Live imaging experiments demonstrated that in the presence of the antibody two newly synthesized GFP-tagged basolateral proteins (vesicular stomatitis virus G [VSVG] protein and TfR) exited the trans-Golgi network (TGN) normally but became blocked at the RE within 3-5 min. By contrast, the antibody did not block trafficking of green fluorescent protein (GFP)-LDLR from the TGN to the PM but stopped its recycling after internalization into RE in approximately 45 min. Our experiments conclusively demonstrate that 1) AP1B functions exclusively at RE; 2) TGN-to-RE transport is very fast and selective and is mediated by adaptors different from AP1B; and 3) the TGN and AP1B-containing RE cooperate in biosynthetic basolateral sorting.