Increased autophagic sequestration in adaptor protein-3 deficient dendritic cells limits inflammasome activity and impairs antibacterial immunity.

Bacterial pathogens that compromise phagosomal membranes stimulate inflammasome assembly in the cytosol, but the molecular mechanisms by which membrane dynamics regulate inflammasome activity are poorly characterized. We show that in murine dendritic cells (DCs), the endosomal adaptor protein AP-3 -which optimizes toll-like receptor signaling from phagosomes-sustains inflammasome activation by particulate stimuli. AP-3 independently regulates inflammasome positioning and autophagy induction, together resulting in delayed inflammasome inactivation by autophagy in response to Salmonella Typhimurium (STm) and other particulate stimuli specifically in DCs. AP-3-deficient DCs, but not macrophages, hyposecrete IL-1ß and IL-18 in response to particulate stimuli in vitro, but caspase-1 and IL-1ß levels are restored by silencing autophagy. Concomitantly, AP-3-deficient mice exhibit higher mortality and produce less IL-1ß, IL-18, and IL-17 than controls upon oral STm infection. Our data identify a novel link between phagocytosis, inflammasome activity and autophagy in DCs, potentially explaining impaired antibacterial immunity in AP-3-deficient patients.

Impairment of dendritic cell functions in patients with adaptor protein-3 complex deficiency.

Hermansky-Pudlak syndrome type 2 (HPS2) is a primary immunodeficiency due to adaptor protein-3 (AP-3) complex deficiency. HPS2 patients present neutropenia, partial albinism, and impaired lysosomal vesicles formation in hematopoietic cells. Given the role of dendritic cells (DCs) in the immune response, we studied monocyte-derived DCs (moDCs) and plasmacytoid DCs (pDCs) in two HPS2 siblings. Mature HPS2 moDCs showed impaired expression of CD83 and DC-lysosome-associated membrane protein (LAMP), low levels of MIP1-ß/CCL4, MIG/CXCL9, and severe defect of interleukin-12 (IL-12) secretion. DCs in lymph-node biopsies from the same patients showed a diffuse cytoplasm reactivity in a large fraction of DC-LAMP(+) cells, instead of the classical dot-like stain. In addition, analysis of pDC-related functions of blood-circulating mononuclear cells revealed reduced interferon-α secretion in response to herpes simplex virus-1 (HSV-1), whereas granzyme-B induction upon IL-3/IL-10 stimulation was normal. Finally, T-cell costimulatory activity, as measured by mixed lymphocyte reaction assay, was lower in patients, suggesting that function and maturation of DCs is abnormal in patients with HPS2.

Defective release of α granule and lysosome contents from platelets in mouse Hermansky-Pudlak syndrome models.

Hermansky-Pudlak syndrome (HPS) is characterized by oculocutaneous albinism, bleeding diathesis, and other variable symptoms. The bleeding diathesis has been attributed to δ storage pool deficiency, reflecting the malformation of platelet dense granules. Here, we analyzed agonist-stimulated secretion from other storage granules in platelets from mouse HPS models that lack adaptor protein (AP)-3 or biogenesis of lysosome-related organelles complex (BLOC)-3 or BLOC-1. We show that α granule secretion elicited by low agonist doses is impaired in all 3 HPS models. High agonist doses or supplemental adenosine 5'-diphosphate (ADP) restored normal α granule secretion, suggesting that the impairment is secondary to absent dense granule content release. Intravital microscopy following laser-induced vascular injury showed that defective hemostatic thrombus formation in HPS mice largely reflected reduced total platelet accumulation and affirmed a reduced area of α granule secretion. Agonist-induced lysosome secretion ex vivo was also impaired in all 3 HPS models but was incompletely rescued by high agonist doses or excess ADP. Our results imply that (1) AP-3, BLOC-1, and BLOC-3 facilitate protein sorting to lysosomes to support ultimate secretion; (2) impaired secretion of α granules in HPS, and to some degree of lysosomes, is secondary to impaired dense granule secretion; and (3) diminished α granule and lysosome secretion might contribute to pathology in HPS.

The risk of hemophagocytic lymphohistiocytosis in Hermansky-Pudlak syndrome type 2.

Genetic disorders of lymphocyte cytotoxicity predispose patients to hemophagocytic lymphohistiocytosis (HLH). Reduced lymphocyte cytotoxicity has been demonstrated in Hermansky-Pudlak syndrome type 2 (HPS2), but only a single patient was reported who developed HLH. Because that patient also carried a potentially contributing heterozygous RAB27A mutation, the risk for HLH in HPS2 remains unclear. We analyzed susceptibility to HLH in the pearl mouse model of HPS2. After infection with lymphocytic choriomeningitis virus, pearl mice developed all key features of HLH, linked to impaired virus control caused by a moderate defect in CTL cytotoxicity in vivo. However, in contrast to perforin-deficient mice, the disease was transient, and all mice fully recovered and controlled the infection. An additional heterozygous Rab27a mutation did not aggravate the cytotoxicity defect or disease parameters. In the largest survey of 22 HPS2 patients covering 234 patient years, we identified only 1 additional patient with HLH and 2 with incomplete transient HLH-like episodes, although cytotoxicity or degranulation was impaired in all 16 patients tested. HPS2 confers a risk for HLH that is lower than in Griscelli or Chediak-Higashi syndrome, probably because of a milder defect in cytotoxicity. Preemptive hematopoietic stem cell transplantation does not appear justified in HPS2.

Defective HIV-1 particle assembly in AP-3-deficient cells derived from patients with Hermansky-Pudlak syndrome type 2.

Adaptor protein complex 3 (AP-3) is a heterotetramer that is involved in signal-mediated protein sorting to endosomal-lysosomal organelles. AP-3 deficiency in humans, induced by mutations in the AP3B1 gene, which encodes the ß3A subunit of the AP-3 complex, results in Hermansky-Pudlak syndrome 2 (HPS2), which is a rare genetic disorder with defective lysosome-related organelles. In a previous study, we identified the AP-3 complex as an important contributor to HIV-1 assembly and release. We hypothesized that cells from patients affected by HPS2 should demonstrate abnormalities of HIV-1 assembly. Here we report that HIV-1 particle assembly and release are indeed diminished in HPS2 fibroblast cultures. Transient or stable expression of the full-length wild-type ß3A subunit in HPS2 fibroblasts restored the impaired virus assembly and release. In contrast, virus-like particle release mediated by MA-deficient Gag mutants lacking the AP-3 binding site was not altered in HPS2 cells, indicating that the MA domain serves as the major viral determinant required for the recruitment of the AP-3 complex. AP-3 deficiency decreased HIV-1 Gag localization at the plasma membrane and late endosomes and increased the accumulation of HIV-1 Gag at an intermediate step between early and late endosomes. Blockage of the clathrin-mediated endocytic pathway in HPS2 cells did not reverse the inhibited virus assembly and release imposed by the AP-3 deficiency. These results demonstrate that the intact and stable AP-3 complex is required for HIV-1 assembly and release, and the involvement of the AP-3 complex in late stages of the HIV-1 replication cycle is independent of clathrin-mediated endocytosis.

Novel insights from adaptor protein 3 complex deficiency.

Hermansky-Pudlak type 2 is an autosomal recessive disorder characterized by oculocutaneous albinism, bleeding disorders, recurrent infections, and moderate/severe neutropenia. The disease is caused by mutations in the AP3B1 gene encoding for the beta3A subunit of the adaptor protein 3 (AP-3) complex. Because the expression of the beta3A subunit is normally ubiquitous, its deficiency leads to a precise phenotype in cells with a large number of intracellular granules, such as neutrophils, natural killer cells, cytotoxic T lymphocytes, platelets, and melanocytes. Given the AP-3 deficiency, the lysosomal membrane proteins are not appropriately sorted to the granules but are delivered to plasma membrane with subsequent effects on cell development and differentiation. Missorting of proteins (eg, tyrosinase) in melanocytes and platelets accounts for oculocutaneous albinism and bleeding disorders, respectively. Absence of AP-3 leads to low intracellular content of neutrophil elastase and consequently to neutropenia. Abnormal movement of lytic granules and reduced perforin content in cytotoxic T lymphocytes and natural killer cells account for their respective defects in cytolytic activity. It is likely that the investigation of the physiopathology of Hermansky-Pudlak type 2 syndrome will reveal nonredundant functions of this adaptor protein in the intracellular trafficking of membrane proteins.

Neuronal and non-neuronal functions of the AP-3 sorting machinery.

Vesicles selectively exchange lipids, membrane proteins and luminal contents between organelles along the exocytic and endocytic routes. The repertoire of membrane proteins present in these vesicles is crucial for their targeting and function. Vesicle composition is determined at the time of their biogenesis by cytosolic coats. The heterotetrameric protein adaptor protein complex 3 (AP-3), a coat component, participates in the generation of a diverse group of secretory organelles and lysosome-related organelles. Recent work has shed light on the mechanisms that regulate AP-3 and the trafficking pathways controlled by this adaptor. Phenotypic analysis of organisms carrying genetic deficiencies in the AP-3 pathway highlight its role regulating the targeting of lysosomal, melanosomal and synaptic vesicle-specific membrane proteins. Synaptic vesicles from AP-3-deficient mice possess altered levels of neurotransmitter and ion transporters, molecules that ultimately define the type and amount of neurotransmitter stored in these vesicles. These findings reveal a complex picture of how AP-3 functions in multiple tissues, including neuronal tissue, and expose potential links between endocytic sorting mechanisms and the pathogenesis of psychiatric disorders such as schizophrenia.

Regulating secretory lysosomes.

Secretory lysosomes are lysosomes which are capable of undergoing regulated secretion in response to external stimuli. Many cells of the immune system use secretory lysosomes to release proteins involved in their specialised effector mechanisms. Precisely how lysosomal secretion is regulated in each of these cell types is now the study of much research as these mechanisms control the ability of each of these cells to function. Studies on a number of human genetic diseases have identified some key proteins in controlling secretory lysosome release, and now many interacting partners have been identified. The different regulatory components seem to vary from one cell type to another, providing a multitude of ways for fine tuning the release of secretory lysosomes.

The endo-lysosomal sorting machinery interacts with the intermediate filament cytoskeleton.

Cytoskeletal networks control organelle subcellular distribution and function. Herein, we describe a previously unsuspected association between intermediate filament proteins and the adaptor complex AP-3. AP-3 and intermediate filament proteins cosedimented and coimmunoprecipitated as a complex free of microtubule and actin binding proteins. Genetic perturbation of the intermediate filament cytoskeleton triggered changes in the subcellular distribution of the adaptor AP-3 and late endocytic/lysosome compartments. Concomitant with these architectural changes, and similarly to AP-3-null mocha cells, fibroblasts lacking vimentin were compromised in their vesicular zinc uptake, their organellar pH, and their total and surface content of AP-3 cargoes. However, the total content and surface levels, as well as the distribution of the transferrin receptor, a membrane protein whose sorting is AP-3 independent, remained unaltered in both AP-3- and vimentin-null cells. Based on the phenotypic convergence between AP-3 and vimentin deficiencies, we predicted and documented a reduced autophagosome content in mocha cells, a phenotype previously reported in cells with disrupted intermediate filament cytoskeletons. Our results reveal a novel role of the intermediate filament cytoskeleton in organelle/adaptor positioning and in regulation of the adaptor complex AP-3.

Adaptor protein 3-dependent microtubule-mediated movement of lytic granules to the immunological synapse.

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disease characterized by platelet defects and oculocutaneous albinism. Individuals with HPS type 2 (HPS2) lack the cytosolic adaptor protein 3 (AP-3) involved in lysosomal sorting, and are also immunodeficient. Here we characterize an HPS2 mutation and demonstrate that AP-3 deficiency leads to a loss of cytotoxic T lymphocyte (CTL)-mediated cytotoxicity. Although the lysosomal protein CD63 was mislocalized to the plasma membrane, perforin and granzymes were correctly localized to the lytic granules in AP-3-deficient CTLs. However, the lytic granules of AP-3-deficient CTLs were enlarged and were unable to move along microtubules and dock within the secretory domain of the immunological synapse. These data show that AP-3 is essential for polarized secretion from CTLs.