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Hermansky-Pudlak syndrome (HPS), particularly types 1 and 4, is characterized by progressive pulmonary fibrosis, a major cause of morbidity and mortality. However, the precise mechanisms driving pulmonary fibrosis in HPS are not fully elucidated. Our previous studies suggested that CHI3L1-driven fibroproliferation may be a notable factor in HPS-associated fibrosis. This study aimed to explore the role of CHI3L1-CRTH2 interaction on type 2 innate lymphoid cells (ILC2s) and explored the potential contribution of ILC2-fibroblast crosstalk in the development of pulmonary fibrosis in HPS. We identified ILC2s in lung tissues from patients with idiopathic pulmonary fibrosis and HPS. Using bleomycin-challenged WT and Hps1-/- mice, we observed that ILC2s were recruited and appeared to contribute to fibrosis development in the Hps1-/- mice, with CRTH2 playing a notable role in ILC2 accumulation. We sorted ILC2s, profiled fibrosis-related genes and mediators, and conducted coculture experiments with primary lung ILC2s and fibroblasts. Our findings suggest that ILC2s may directly stimulate the proliferation and differentiation of primary lung fibroblasts partially through amphiregulin-EGFR-dependent mechanisms. Additionally, specific overexpression of CHI3L1 in the ILC2 population using the IL-7Rcre driver, which was associated with increased fibroproliferation, indicates that ILC2-mediated, CRTH2-dependent mechanisms might contribute to optimal CHI3L1-induced fibroproliferative repair in HPS-associated pulmonary fibrosis.

The immunome (immune cell phenotype, gene expression, and serum cytokines profiling) in pulmonary fibrosis is incompletely defined. Studies focusing on inherited forms of pulmonary fibrosis provide insights into mechanisms of fibrotic lung disease in general. To define the cellular and molecular immunologic phenotype in peripheral blood, high-dimensional flow cytometry and large-scale gene expression of peripheral blood mononuclear cells and serum proteomic multiplex analyses were performed and compared in a cohort with familial pulmonary fibrosis (FPF), an autosomal dominant disorder with incomplete penetrance; Hermansky-Pudlak syndrome pulmonary fibrosis (HPSPF), a rare autosomal recessive disorder; and their unaffected relatives. Our results showed high peripheral blood concentrations of activated central memory helper cells in patients with FPF. Proportions of CD38 + memory CD27 - B-cells, IgA + memory CD27 + B-cells, IgM + and IgD + B-cells, and CD39 + T helper cells were increased whereas those of CD39 - T helper cells were reduced in patients affected with either familial or HPSPF. Gene expression and serum proteomic analyses revealed enrichment of upregulated genes associated with mitosis and cell cycle control in circulating mononuclear cells as well as altered levels of several analytes, including leptin, cytokines, and growth factors. In conclusion, dysregulation of the extra-pulmonary immunome is a phenotypic feature of FPF or HPSPF. Further studies investigating the blood immunome are indicated to determine the role of immune system dysregulation in the pathogenesis of pulmonary fibrosis., Clinical Trial Registration: www.ClinicalTrials.gov, identifiers NCT00968084, NCT01200823, NCT00001456, and NCT00084305.

Hermansky-Pudlak syndrome (HPS) encompasses disorders with abnormal function of lysosomes and lysosome-related organelles, and some patients who develop immunodeficiency. The basic mechanisms contributing to immune dysfunction in HPS are ill-defined. We analysed natural killer (NK) cells from patients diagnosed with HPS-1, HPS-2, HPS-4, and an unreported HPS subtype. NK cells from an HPS-2 and an unreported HPS subtype share a similar cellular phenotype with defective granule release and cytotoxicity, but differ in cytokine exocytosis. Defining NK cell activity in several types of HPS provides insights into cellular defects of the disorder and understanding of mechanisms contributing to HPS pathogenesis., (© 2016 John Wiley & Sons Ltd.)

Alveolar epithelial cell (AEC) dysfunction underlies the pathogenesis of pulmonary fibrosis in Hermansky-Pudlak syndrome (HPS) and other genetic syndromes associated with interstitial lung disease; however, mechanisms linking AEC dysfunction and fibrotic remodeling are incompletely understood. Since increased macrophage recruitment precedes pulmonary fibrosis in HPS, we investigated whether crosstalk between AECs and macrophages determines fibrotic susceptibility. We found that AECs from HPS mice produce excessive MCP-1, which was associated with increased macrophages in the lungs of unchallenged HPS mice. Blocking MCP-1/CCR2 signaling in HPS mice with genetic deficiency of CCR2 or targeted deletion of MCP-1 in AECs normalized macrophage recruitment, decreased AEC apoptosis, and reduced lung fibrosis in these mice following treatment with low-dose bleomycin. We observed increased TGF-β production by HPS macrophages, which was eliminated by CCR2 deletion. Selective deletion of TGF-β in myeloid cells or of TGF-β signaling in AECs through deletion of TGFBR2 protected HPS mice from AEC apoptosis and bleomycin-induced fibrosis. Together, these data reveal a feedback loop in which increased MCP-1 production by dysfunctional AECs results in recruitment and activation of lung macrophages that produce TGF-β, thus amplifying the fibrotic cascade through AEC apoptosis and stimulation of fibrotic remodeling.

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-β/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., (© 2016 by The American Society of Hematology.)

Hermansky Pudlak type 2 syndrome (HPS2) is a rare autosomal recessive primary immune deficiency caused by mutations on β3A gene (AP3B1 gene). The defect results in the impairment of the adaptor protein 3 (AP-3) complex, responsible for protein sorting to secretory lysosomes leading to oculo-cutaneous albinism, bleeding disorders and immunodeficiency. We have studied peripheral blood and lymph node biopsies from two siblings affected by HPS2. Lymph node histology showed a nodular lymphocyte predominance type Hodgkin lymphoma (NLPHL) in both HPS2 siblings. By immunohistochemistry, CD8 T-cells from HPS2 NLPHL contained an increased amount of perforin (Prf) + suggesting a defect in the release of this granules-associated protein. By analyzing peripheral blood immune cells we found a significant reduction of circulating NKT cells and of CD56(bright)CD16(-) Natural Killer (NK) cells subset. Functionally, NK cells were defective in their cytotoxic activity against tumor cell lines including Hodgkin Lymphoma as well as in IFN-γ production. This defect was associated with increased baseline level of CD107a and CD63 at the surface level of unstimulated and IL-2-activated NK cells. In summary, these results suggest that a combined and profound defect of innate and adaptive effector cells might explain the susceptibility to infections and lymphoma in these HPS2 patients.

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.

Patients with Hermansky-Pudlak syndrome type 2 (HPS2) present with oculocutaneous albinism, nystagmus, prolonged bleeding time, and increased susceptibility to infections. Twelve HPS2 patients with mutations in the β3A-subunit of the cytosolic adaptor-related protein complex 3 (AP3B1, also called HPS2) have been described so far. Here, we report on a patient with oculocutaneous albinism who developed a life-threatening bleeding after tonsillectomy. She presented with moderate neutropenia and reduced granulopoiesis. Analyzing patient's impaired platelet function using electron microscopy and flow cytometry led to the diagnosis of HPS2. Flow cytometric analysis of the patient's platelets showed already elevated CD63 expression on resting platelets with no further increase after thrombin stimulation. Natural killer (NK) cell degranulation was partially impaired but target cell lysis of NK cells and cytotoxic T-lymphocytes (CTLs) were normal and the patient did not develop signs of hemophagocytic syndrome. Molecular genetic analyses revealed a novel 2 bp-deletion (c.3222_3223delTG) in the last exon of AP3B1 causing a frameshift and a prolonged altered protein. The location of the deletion at the very C-terminal end may prevent a complete loss of the HPS2 protein leading to a less pronounced severity of immunodeficiency than in other HPS2 patients.

Cytotoxic T lymphocytes, natural killer cells, and NKT cells are effector cells able to kill infected cells. In some inherited human disorders, a defect in selected proteins involved in the cellular cytotoxicity mechanism results in specific clinical syndromes, grouped under the name of familial hemophagocytic lymphohistiocytosis. Recent advances in genetic studies of these patients has allowed the identification of different genetic subsets. Additional genetic immune deficiencies may also induce a similar clinical picture. International cooperation and prospective trials resulted in refining the diagnostic and therapeutic approach to these rare diseases with improved outcome but also with improved knowledge of the mechanisms underlying granule-mediated cellular cytotoxicity in humans.

Rationale: Individuals with Hermansky-Pudlak syndrome type 1 (HPS-1), an autosomal recessive disorder characterized by defective biogenesis of lysosome-related organelles, develop an accelerated form of progressive fibrotic lung disease. The etiology of pulmonary fibrosis associated with HPS-1 is unknown., Objectives: To investigate the potential pathogenesis of pulmonary fibrosis in HPS-1, lung cells and proteins from individuals with HPS-1 were studied., Methods: Forty-one subjects with HPS-1 with and without pulmonary fibrosis were evaluated with pulmonary function tests, high-resolution computed tomography scan, and bronchoscopy. Bronchoalveolar lavage cells and analytes were analyzed., Measurements and Main Results: Concentrations of total bronchoalveolar lavage cells and alveolar macrophages were significantly higher in epithelial lining fluid from subjects with HPS-1 with and without pulmonary fibrosis compared with healthy research volunteers. Concentrations of cytokines and chemokines (i.e., monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha, and granulocyte-macrophage colony-stimulating factor) in alveolar epithelial lining fluid were significantly higher in subjects with HPS-1 with and without pulmonary fibrosis compared with healthy research volunteers (P < 0.001). In vitro, HPS-1 pulmonary fibrosis alveolar macrophages, which did not express HPS1 mRNA, secreted significantly higher concentrations of monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha, and regulated upon activation, normal T cell expressed and secreted (RANTES) protein compared with normal cells (P = 0.001, P = 0.014, and P = 0.011, respectively). Pirfenidone suppressed HPS-1 alveolar macrophage cytokine and chemokine secretion in vitro in a dose-dependent manner., Conclusions: In HPS-1, alveolar inflammation predominantly involves macrophages and is associated with high lung concentrations of cytokines and chemokines. HPS-1 alveolar macrophages provide a model system in which to study the pathogenesis and treatment of HPS pulmonary fibrosis.

We report on the molecular etiology of an unusual clinical phenotype associating congenital neutropenia, thrombocytopenia, developmental delay, and hypopigmentation. Using genetic linkage analysis and targeted gene sequencing, we defined a homozygous genomic deletion in AP3B1, the gene encoding the beta chain of the adaptor protein-3 (AP-3) complex. The mutation leads to in-frame skipping of exon 15 and thus perturbs proper assembly of the heterotetrameric AP-3 complex. Consequently, trafficking of transmembrane lysosomal proteins is aberrant, as shown for CD63. In basal keratinocytes, the incorporated immature melanosomes were rapidly degraded in large phagolysosomes. Despite distinct ultramorphologic changes suggestive of aberrant vesicular maturation, no functional aberrations were detected in neutrophil granulocytes. However, a comprehensive immunologic assessment revealed that natural killer (NK) and NKT-cell numbers were reduced in AP-3-deficient patients. Our findings extend the clinical and molecular phenotype of human AP-3 deficiency (also known as Hermansky-Pudlak syndrome, type 2) and provide further insights into the role of the AP-3 complex for the innate immune system.

Adaptor protein-3 (AP-3) is an ubiquitous cytoplasmic complex that shuttles cargo proteins from the trans-Golgi and a tubular-endosomal compartment to endosome-lysosome-related organelles. Lack of the beta3A subunit of this complex causes Hermansky-Pudlak syndrome type 2, an autosomal recessive disease characterized by partial albinism, prolonged bleeding tendency, and immunodeficiency. To investigate the pathogenesis of immunodeficiency, we studied natural killer (NK) cells and neutrophil functions in 2 previously unreported siblings affected by Hermansky-Pudlak type 2 syndrome. In both patients we observed a dramatic reduction of cytolytic activity of freshly isolated and of IL-2-activated NK cells. Levels of perforin were reduced in unstimulated NK cells, thereby accounting for the impairment of NK cytolitic activity. In addition, analysis of neutrophils in these patients demonstrated that intracellular elastase content was largely reduced while CD63 expression on plasma membrane was substantially increased. Taken together, these observations suggest that type 2 Hermansky-Pudlak syndrome is characterized by defects of innate immunity.

Pulmonary inflammation, abnormalities in alveolar type II cell and macrophage morphology, and pulmonary fibrosis are features of Hermansky-Pudlak Syndrome (HPS). We used the naturally occurring "pearl" HPS2 mouse model to investigate the mechanisms of lung inflammation observed in HPS. Although baseline bronchoalveolar lavage (BAL) cell counts and differentials were similar in pearl and strain-matched wild-type (WT) mice, elevated levels of proinflammatory (MIP1gamma) and counterregulatory (IL-12p40, soluble TNFr1/2) factors, but not TNF-alpha, were detected in BAL from pearl mice. After intranasal LPS challenge, BAL levels of TNF-alpha, MIP1alpha, KC, and MCP-1 were 2- to 3-fold greater in pearl than WT mice. At baseline, cultured pearl alveolar macrophages (AMs) had markedly increased production of inflammatory cytokines. Furthermore, pearl AMs had exaggerated TNF-alpha responses to TLR4, TLR2, and TLR3 ligands, as well as increased IFN-gamma/LPS-induced NO production. After 24 h in culture, pearl AM LPS responses reverted to WT levels, and pearl AMs were appropriately refractory to continuous LPS exposure. In contrast, cultured pearl peritoneal macrophages and peripheral blood monocytes did not produce TNF-alpha at baseline and had LPS responses which were no different from WT controls. Exposure of WT AMs to heat- and protease-labile components of pearl BAL, but not WT BAL, resulted in robust TNF-alpha secretion. Similar abnormalities were identified in AMs and BAL from another HPS model, pale ear HPS1 mice. We conclude that the lungs of HPS mice exhibit hyperresponsiveness to LPS and constitutive and organ-specific macrophage activation.

Hermansky-Pudlak syndrome (HPS) defines a group of at least seven autosomal recessive disorders characterized by albinism and prolonged bleeding due to defects in the lysosome-related organelles, melanosomes and platelet-dense granules, respectively. Most HPS genes, including HPS3, HPS5 and HPS6, encode ubiquitously expressed novel proteins of unknown function. Here, we report the biochemical characterization of a stable protein complex named Biogenesis of Lysosome-related Organelles Complex-2 (BLOC-2), which contains the HPS3, HPS5 and HPS6 proteins as subunits. The endogenous HPS3, HPS5 and HPS6 proteins from human HeLa cells coimmunoprecipitated with each other from crude extracts as well as from fractions resulting from size-exclusion chromatography and density gradient centrifugation. The native molecular mass of BLOC-2 was estimated to be 340 +/- 64 kDa. As inferred from the biochemical properties of the HPS6 subunit, BLOC-2 exists in a soluble pool and associates to membranes as a peripheral membrane protein. Fibroblasts deficient in the BLOC-2 subunits HPS3 or HPS6 displayed normal basal secretion of the lysosomal enzyme beta-hexosaminidase. Our results suggest a common biological basis underlying the pathogenesis of HPS-3, -5 and -6 disease.

Lytic granules harbour many of the dangerous apoptosis-inducing molecules of the immune system, including perforin, granzymes and Fas ligand. Safe transport, storage and release of these lytic components is vital. As a secretory lysosome, the lytic granule is able to accomplish these roles, as well as conferring the lysosomal functions of cytotoxic T lymphocytes and natural killer cells. Secretory lysosomes are common to many other haemopoietic cells and also melanocytes. Many of the proteins used in lysosomal secretion are found in both melanocytes and hemopoietic cells, and are dysfunctional in genetic diseases with defects in these proteins. The genetically heterogeneous Hermansky-Pudlak syndrome represents an excellent model for revealing proteins involved in secretory lysosome functioning. However, studies of this disease reveal differences between the various different types of secretory lysosomes, including lytic granules.

Cytotoxic T lymphocytes and natural killer cells kill their targets by secreting specialized granules that contain potent cytotoxic molecules. Through the study of rare immunodeficiency diseases in which this granule pathway of killing is impaired, proteins such as Rab27a have been identified as components of the secretory machinery of these killer cells. Recent evidence suggests that the destruction of activated lymphocytes through granule-mediated killing may be an important mechanism of immunological homeostasis. Although the process by which this occurs is not yet known, it is possible that events taking place at the immunological synapse may render the killer cell susceptible to fratricidal attack by other killer cells.

The secretory lysosomes found in haemopoietic cells provide a very efficient mechanism for delivering the effector proteins of many immune cells in response to antigen recognition. Although secretion shows some similarities to the secretion of specialized granules in other secretory cell types, some aspects of secretory lysosome release appear to be unique to melanocytes and cells of the haemopoietic lineage. Mast cells and platelets have provided excellent models for studying secretion, but recent advances in characterizing the immunological synapse allow a very fine dissection of the secretory process in T lymphocytes. These studies show that secretory lysosomes are secreted from the centre of the talin ring at the synapse. Proper secretion requires a series of Rab and cytoskeletal elements which play critical roles in the specialized secretion of lysosomes in haemopoietic cells.