Cary D. Austin

Localization of the AP-3 adaptor complex defines a novel endosomal exit site for lysosomal membrane proteins

The adaptor protein (AP) 3 adaptor complex has been implicated in the transport of lysosomal membrane proteins, but its precise site of action has remained controversial. Here, we show by immuno-electron microscopy that AP-3 is associated with budding profiles evolving from a tubular endosomal compartment that also exhibits budding profiles positive for AP-1. AP-3 colocalizes with clathrin, but to a lesser extent than does AP-1. The AP-3– and AP-1–bearing tubular compartments contain endocytosed transferrin, transferrin receptor, asialoglycoprotein receptor, and low amounts of the cation-independent mannose 6-phosphate receptor and the lysosome-associated membrane proteins (LAMPs) 1 and 2. Quantitative analysis revealed that of these distinct cargo proteins, only LAMP-1 and LAMP-2 are concentrated in the AP-3–positive membrane domains. Moreover, recycling of endocytosed LAMP-1 and CD63 back to the cell surface is greatly increased in AP-3–deficient cells. Based on these data, we propose that AP-3 defines a novel pathway by which lysosomal membrane proteins are transported from tubular sorting endosomes to lysosomes.

In vivo blockade of OX40 ligand inhibits thymic stromal lymphopoietin driven atopic inflammation

Thymic stromal lymphopoietin (TSLP) potently induces deregulation of Th2 responses, a hallmark feature of allergic inflammatory diseases such as asthma, atopic dermatitis, and allergic rhinitis. However, direct downstream in vivo mediators in the TSLP-induced atopic immune cascade have not been identified. In our current study, we have shown that OX40 ligand (OX40L) is a critical in vivo mediator of TSLP-mediated Th2 responses. Treating mice with OX40L-blocking antibodies substantially inhibited immune responses induced by TSLP in the lung and skin, including Th2 inflammatory cell infiltration, cytokine secretion, and IgE production. OX40L-blocking antibodies also inhibited antigen-driven Th2 inflammation in mouse and nonhuman primate models of asthma. This treatment resulted in both blockade of the OX40-OX40L receptor-ligand interaction and depletion of OX40L-positive cells. The use of a blocking, OX40L-specific mAb thus presents a promising strategy for the treatment of allergic diseases associated with pathologic Th2 immune responses.

Increased expression of immunoreactive thymic stromal lymphopoietin in patients with severe asthma.

BACKGROUND Thymic stromal lymphopoietin (TSLP) is a cytokine implicated in the pathophysiology of asthma through 2 distinct pathways: a TSLP-OX40 ligand (OX40L)-T cell axis and a TSLP-mast cell axis. Whether these pathways are active in human asthma is unknown. OBJECTIVE We sought to investigate whether mucosal TSLP protein expression relates to asthma severity and distinct immunologic pathways. METHODS In healthy subjects and patients with mild-to-severe asthma, we immunostained bronchial biopsy specimens for TSLP, OX40, OX40L, T(H)2 cytokines, and inflammatory cell markers. We examined gene expression using RNA microarrays and quantitative RT-PCR. RESULTS There was considerable heterogeneity in the levels of TSLP, IL-13, and IL-4 immunostaining across the cohort of asthmatic patients examined. Overall, TSLP protein expression was significantly increased in airway epithelium and lamina propria of asthmatic patients, particularly in patients with severe asthma. TSLP immunostaining in both compartments correlated with the severity of airflow obstruction. The majority of leukocytes expressing IL-13 were possibly nuocytes. Accounting for intersubject variability, the 55% of asthmatic patients with increased IL-13 immunostaining in the lamina propria also had increased IL-4 and TSLP expression. This was further substantiated by significant correlations between TSLP gene expression, a T(H)2 gene expression signature, and eosinophilic inflammation in bronchial biopsy specimens. Immunostaining for OX40, OX40L, and CD83 was sparse, with no difference between asthmatic patients and healthy subjects. CONCLUSION TSLP expression is increased in a subset of patients with severe asthma in spite of high-dose inhaled or oral corticosteroid therapy. Targeting TSLP might only be efficacious in the subset of asthma characterized by increased TSLP expression and T(H)2 inflammation.

IgE⁺ memory B cells and plasma cells generated through a germinal-center pathway.

Immunoglobulin E (IgE) antibodies are pathogenic in asthma and allergic diseases, but the in vivo biology of IgE-producing (IgE+) cells is poorly understood. A model of the differentiation of IgE+ B cells proposes that IgE+ cells develop through a germinal-center IgG1+ intermediate and that IgE memory resides in the compartment of IgG1+ memory B cells. Here we have used a reporter mouse expressing green fluorescent protein associated with membrane IgE transcripts (IgE-GFP) to assess in vivo IgE responses. In contrast to the IgG1-centered model of IgE switching and memory, we found that IgE+ cells developed through a germinal-center IgE+ intermediate to form IgE+ memory B cells and plasma cells. Our studies delineate a new model for the in vivo biology of IgE switching and memory.

Death-receptor activation halts clathrin-dependent endocytosis

Endocytosis is crucial for various aspects of cell homeostasis. Here, we show that proapoptotic death receptors (DRs) trigger selective destruction of the clathrin-dependent endocytosis machinery. DR stimulation induced rapid, caspase-mediated cleavage of key clathrin-pathway components, halting cellular uptake of the classic cargo protein transferrin. DR-proximal initiator caspases cleaved the clathrin adaptor subunit AP2α between functionally distinct domains, whereas effector caspases processed clathrin’s heavy chain. DR5 underwent ligand-induced, clathrin-mediated endocytosis, suggesting that internalization of DR signaling complexes facilitates clathrin-pathway targeting by caspases. An endocytosis-blocking, temperature-sensitive dynamin-1 mutant attenuated DR internalization, enhanced caspase stimulation downstream of DRs, and increased apoptosis. Thus, DR-triggered caspase activity disrupts clathrin-dependent endocytosis, leading to amplification of programmed cell death.

Therapeutic antibodies reveal Notch control of transdifferentiation in the adult lung

Prevailing dogma holds that cell–cell communication through Notch ligands and receptors determines binary cell fate decisions during progenitor cell divisions, with differentiated lineages remaining fixed. Mucociliary clearance in mammalian respiratory airways depends on secretory cells (club and goblet) and ciliated cells to produce and transport mucus. During development or repair, the closely related Jagged ligands (JAG1 and JAG2) induce Notch signalling to determine the fate of these lineages as they descend from a common proliferating progenitor. In contrast to such situations in which cell fate decisions are made in rapidly dividing populations, cells of the homeostatic adult airway epithelium are long-lived, and little is known about the role of active Notch signalling under such conditions. To disrupt Jagged signalling acutely in adult mammals, here we generate antibody antagonists that selectively target each Jagged paralogue, and determine a crystal structure that explains selectivity. We show that acute Jagged blockade induces a rapid and near-complete loss of club cells, with a concomitant gain in ciliated cells, under homeostatic conditions without increased cell death or division. Fate analyses demonstrate a direct conversion of club cells to ciliated cells without proliferation, meeting a conservative definition of direct transdifferentiation. Jagged inhibition also reversed goblet cell metaplasia in a preclinical asthma model, providing a therapeutic foundation. Our discovery that Jagged antagonism relieves a blockade of cell-to-cell conversion unveils unexpected plasticity, and establishes a model for Notch regulation of transdifferentiation.

Lkb1 regulates organogenesis and early oncogenesis along AMPK-dependent and -independent pathways.

A combination of ex vivo embryonic tissue culture, genetic manipulation, and chemical genetics reveals novel details of Lkb1-mediated regulation of tissue morphogenesis.

Conditional Deletion of NF-κB–Inducing Kinase (NIK) in Adult Mice Disrupts Mature B Cell Survival and Activation

NF-κB–inducing kinase (NIK) is a primary regulator of the noncanonical NF-κB signaling pathway, which plays a vital role downstream of BAFF, CD40L, lymphotoxin, and other inflammatory mediators. Germline deletion or inactivation of NIK in mice results in the defective development of B cells and secondary lymphoid organs, but the role of NIK in adult animals has not been studied. To address this, we generated mice containing a conditional allele of NIK. Deletion of NIK in adult mice results in decreases in B cell populations in lymph nodes and spleen, similar to what is observed upon blockade of BAFF. Consistent with this, B cells from mice in which NIK is acutely deleted fail to respond to BAFF stimulation in vitro and in vivo. In addition, mice with induced NIK deletion exhibit a significant decrease in germinal center B cells and serum IgA, which is indicative of roles for NIK in additional pathways beyond BAFF signaling. Our conditional NIK-knockout mice may be broadly useful for assessing the postdevelopmental and cell-specific roles of NIK and the noncanonical NF-κB pathway in mice.

Characterization of the guinea pig CMV gH/gL/GP129/GP131/GP133 complex in infection and spread

In human cytomegalovirus (HCMV), the UL128-131A locus plays an essential role in cellular tropism and spread. Here, we report the complete annotation of the GP129-133 locus from guinea pig cytomegalovirus (GPCMV) and the discovery of the UL131A homolog, named GP133. We have found that similar to HCMV the GP129-133 proteins form a pentamer complex with the GPCMV glycoproteins gH and gL. In addition, we find that the GP129-133 proteins play a critical role in entry as the GP129-133 deletion mutant shows a defect in both endothelial and fibroblast cell entry. Although the GP129-133 deletion strain can propagate in vitro, we find that the deletion fails to spread in vivo. Interestingly, the wildtype strain can spontaneously give rise to the GP129-133 deletion strain during in vivo spread, suggesting genetic instability at this locus.

Mycobacterium marinum SecA2 Promotes Stable Granulomas and Induces Tumor Necrosis Factor Alpha In Vivo

ABSTRACT SecA2 is an ATPase present in some pathogenic Gram-positive bacteria, is required for translocation of a limited set of proteins across the cytosolic membrane, and plays an important role in virulence in several bacteria, including mycobacteria that cause diseases such as tuberculosis and leprosy. However, the mechanisms by which SecA2 affects virulence are incompletely understood. To investigate whether SecA2 modulates host immune responses in vivo, we studied Mycobacterium marinum infection in two different hosts: an established zebrafish model and a recently described mouse model. Here we show that M. marinum ΔsecA2 was attenuated for virulence in both host species and SecA2 was needed for normal granuloma numbers and for optimal tumor necrosis factor alpha response in both zebrafish and mice. M. marinum ΔsecA2 was more sensitive to SDS and had unique protrusions from its cell envelope when examined by cryo-electron tomography, suggesting that SecA2 is important for bacterial cell wall integrity. These results provide evidence that SecA2 induces granulomas and is required for bacterial modulation of the host response because it affects the mycobacterial cell envelope.