Spatial transcriptomic characterization of pathologic niches in IPF.

Despite advancements in antifibrotic therapy, idiopathic pulmonary fibrosis (IPF) remains a medical condition with unmet needs. Single-cell RNA sequencing (scRNA-seq) has enhanced our understanding of IPF but lacks the cellular tissue context and gene expression localization that spatial transcriptomics provides. To bridge this gap, we profiled IPF and control patient lung tissue using spatial transcriptomics, integrating the data with an IPF scRNA-seq atlas. We identified three disease-associated niches with unique cellular compositions and localizations. These include a fibrotic niche, consisting of myofibroblasts and aberrant basaloid cells, located around airways and adjacent to an airway macrophage niche in the lumen, containing SPP1+ macrophages. In addition, we identified an immune niche, characterized by distinct lymphoid cell foci in fibrotic tissue, surrounded by remodeled endothelial vessels. This spatial characterization of IPF niches will facilitate the identification of drug targets that disrupt disease-driving niches and aid in the development of disease relevant in vitro models.

Programmed Cell Death-1 (PD-1) anchoring to the GPI-linked co-receptor CD48 reveals a novel mechanism to modulate PD-1-dependent inhibition of human T cells.

Activation of PD-1 by anchoring it to Antigen Receptor (AR) components or associated co-receptors represents an attractive approach to treat autoimmune conditions. In this study, we provide evidence that CD48, a common lipid raft and Src kinase-associated coreceptor, induces significant Src kinase-dependent activation of PD-1 upon crosslinking, while CD71, a receptor excluded from these compartments, does not. Functionally, using bead-conjugated antibodies we demonstrate that CD48-dependent activation of PD-1 inhibits proliferation of AR-induced primary human T cells, and similarly, PD-1 activation using PD-1/CD48 bispecific antibodies inhibits IL-2, enhances IL-10 secretion, and reduces NFAT activation in primary human and Jurkat T cells, respectively. As a whole, CD48-dependent activation of PD-1 represents a novel mechanism to fine tune T cell activation, and by functionally anchoring PD-1 with receptors other than AR, this study provides a conceptual framework for rational development of novel therapies that activate inhibitory checkpoint receptors for treatment of immune-mediated diseases.

Timing of ESCRT-III protein recruitment and membrane scission during HIV-1 assembly.

The Endosomal Sorting Complexes Required for Transport III (ESCRT-III) proteins are critical for cellular membrane scission processes with topologies inverted relative to clathrin-mediated endocytosis. Some viruses appropriate ESCRT-IIIs for their release. By imaging single assembling viral-like particles of HIV-1, we observed that ESCRT-IIIs and the ATPase VPS4 arrive after most of the virion membrane is bent, linger for tens of seconds, and depart ~20 s before scission. These observations suggest that ESCRT-IIIs are recruited by a combination of membrane curvature and the late domains of the HIV-1 Gag protein. ESCRT-IIIs may pull the neck into a narrower form but must leave to allow scission. If scission does not occur within minutes of ESCRT departure, ESCRT-IIIs and VPS4 are recruited again. This mechanistic insight is likely relevant for other ESCRT-dependent scission processes including cell division, endosome tubulation, multivesicular body and nuclear envelope formation, and secretion of exosomes and ectosomes.

Readily Accessible Multiplane Microscopy: 3D Tracking the HIV-1 Genome in Living Cells.

Human immunodeficiency virus (HIV)-1 infection and the associated disease AIDS are a major cause of human death worldwide with no vaccine or cure available. The trafficking of HIV-1 RNAs from sites of synthesis in the nucleus, through the cytoplasm, to sites of assembly at the plasma membrane are critical steps in HIV-1 viral replication, but are not well characterized. Here we present a broadly accessible microscopy method that captures multiple focal planes simultaneously, which allows us to image the trafficking of HIV-1 genomic RNAs with high precision. This method utilizes a customization of a commercial multichannel emission splitter that enables high-resolution 3D imaging with single-macromolecule sensitivity. We show with high temporal and spatial resolution that HIV-1 genomic RNAs are most mobile in the cytosol, and undergo confined mobility at sites along the nuclear envelope and in the nucleus and nucleolus. These provide important insights regarding the mechanism by which the HIV-1 RNA genome is transported to the sites of assembly of nascent virions.

APP intracellular domain-WAVE1 pathway reduces amyloid-ß production.

An increase in amyloid-ß (Aß) production is a major pathogenic mechanism associated with Alzheimer's disease (AD), but little is known about possible homeostatic control of the amyloidogenic pathway. Here we report that the amyloid precursor protein (APP) intracellular domain (AICD) downregulates Wiskott-Aldrich syndrome protein (WASP)-family verprolin homologous protein 1 (WAVE1 or WASF1) as part of a negative feedback mechanism to limit Aß production. The AICD binds to the Wasf1 promoter, negatively regulates its transcription and downregulates Wasf1 mRNA and protein expression in Neuro 2a (N2a) cells. WAVE1 interacts and colocalizes with APP in the Golgi apparatus. Experimentally reducing WAVE1 in N2a cells decreased the budding of APP-containing vesicles and reduced cell-surface APP, thereby reducing the production of Aß. WAVE1 downregulation was observed in mouse models of AD. Reduction of Wasf1 gene expression dramatically reduced Aß levels and restored memory deficits in a mouse model of AD. A decrease in amounts of WASF1 mRNA was also observed in human AD brains, suggesting clinical relevance of the negative feedback circuit involved in homeostatic regulation of Aß production.

Temporal and spatial organization of ESCRT protein recruitment during HIV-1 budding.

HIV-1 virions assemble at the plasma membrane of mammalian cells and recruit the endosomal sorting complex required for transport (ESCRT) machinery to enable particle release. However, little is known about the temporal and spatial organization of ESCRT protein recruitment. Using multiple-color live-cell total internal reflection fluorescence microscopy, we observed that the ESCRT-I protein Tsg101 is recruited together with Gag to the sites of HIV-1 assembly, whereas later-acting ESCRT proteins (Chmp4b and Vps4A) are recruited sequentially, once Gag assembly is completed. Chmp4b, a protein that is required to mediate particle scission, is recruited to HIV-1 assembly sites ∼10 s before the ATPase Vps4A. Using two-color superresolution imaging, we observed that the ESCRT machinery (Tsg101, Alix, and Chmp4b/c proteins) is positioned at the periphery of the nascent virions, with the Tsg101 assemblages positioned closer to the Gag assemblages than Alix, Chmp4b, or Chmp4c. These results are consistent with the notion that the ESCRT machinery is recruited transiently to the neck of the assembling particle and is thus present at the appropriate time and place to mediate fission between the nascent virus and the plasma membrane.