ABSTRACT
The DENN domain is an evolutionary conserved protein module found in all eukaryotes and serves as an exchange factor for Rab-GTPases to regulate diverse cellular functions. Variants in DENND1B are associated with development of childhood asthma and other immune disorders. To understand how DENND1B may contribute to human disease, Dennd1b(-/-) mice were generated and exhibit hyper-allergic responses following antigen challenge. Dennd1b(-/-) TH2, but not other TH cells, exhibit delayed receptor-induced T cell receptor (TCR) downmodulation, enhanced TCR signaling, and increased production of effector cytokines. As DENND1B interacts with AP-2 and Rab35, TH2 cells deficient in AP-2 or Rab35 also exhibit enhanced TCR-mediated effector functions. Moreover, human TH2 cells carrying asthma-associated DENND1B variants express less DENND1B and phenocopy Dennd1b(-/-) TH2 cells. These results provide a molecular basis for how DENND1B, a previously unrecognized regulator of TCR downmodulation in TH2 cells, contributes to asthma pathogenesis and how DENN-domain-containing proteins may contribute to other human disorders.
Subject(s)
Asthma/immunology , Death Domain Receptor Signaling Adaptor Proteins/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Receptors, Antigen, T-Cell/metabolism , Signal Transduction , Th2 Cells/immunology , Animals , Cytokines/genetics , Cytokines/immunology , Dendritic Cells/immunology , Female , Guanine Nucleotide Exchange Factors/genetics , Humans , Hypersensitivity/immunology , Lymphocyte Activation , Mice , Polymorphism, Single Nucleotide , Th2 Cells/metabolism , rab GTP-Binding Proteins/geneticsABSTRACT
The examination of tissue histology by light microscopy is a fundamental tool for investigating the structure and function of organs under normal and disease states. Many current techniques for tissue sectioning, imaging and analysis are time-consuming, and they present major limitations for 3D tissue reconstruction. The introduction of methods to achieve the optical clearing and subsequent light-sheet laser scanning of entire transparent organs without sectioning represents a major advance in the field. We recently developed a highly reproducible and versatile clearing procedure called 3D imaging of solvent-cleared organs, or 3DISCO, which is applicable to diverse tissues including brain, spinal cord, immune organs and tumors. Here we describe a detailed protocol for performing 3DISCO and present its application to various microscopy techniques, including example results from various mouse tissues. The tissue clearing takes as little as 3 h, and imaging can be completed in â¼45 min. 3DISCO is a powerful technique that offers 3D histological views of tissues in a fraction of the time and labor required to complete standard histology studies.