Airway mucus forms the structural basis of the local innate immune defense mechanism. It is an integrated, active, viscoelastic gel matrix evolved to protect the exposed lung from physical, chemical, and pathological erosion. Exosomes are biologically active vesicles secreted by different cell types including epithelial, hematopoietic, and some tumor cells. They are also present in some biological fluids such as serum, urine, breast milk, and bronchoalveolar lavage fluid. In this study, we demonstrate for the first time that exosome-like vesicles with antiviral properties are present in human tracheobronchial epithelial (HTBE) cell culture secretions. These vesicles have been isolated by differential centrifugation and are characterized further by mass spectrometry, flow cytometry, immunoblotting, electron microscopy, and light-scattering methods. HTBE vesicles exhibited characteristic exosomal size (30-100 nm) and morphology (cup-shaped) with a buoyant density in sucrose of 1.12-1.18 g/ml. Biochemical characterization further revealed typical surface, cytoskeletal, and cytoplasmic proteins characteristic of exosomes, including the multivesicular and late endosomal membrane markers Tsg101 and CD63. The presence of RNA was also observed. The epithelial mucins MUC1, MUC4, and MUC16 also contributed to the vesicles' structure. Notably, alpha-2,6-linked sialic acid was associated with these mucin molecules and subsequent functional analysis showed that these vesicles have a neutralizing effect on human influenza virus, which is known to bind sialic acid. Taken together, these findings suggest that airway epithelial cells release exosome-like vesicles and that these structures may be involved in diverse physiological processes in airway biology, including innate mucosal defense.
Rationale Primary ciliary dyskinesia (PCD) is an autosomal recessive, genetically heterogeneous disorder characterized by oto-sino-pulmonary disease and situs abnormalities (Kartagener syndrome) due to abnormal structure and/or function of cilia. Most patients currently recognized to have PCD have ultrastructural defects of cilia; however, some patients have clinical manifestations of PCD and low levels of nasal nitric oxide, but normal ultrastructure, including a few patients with biallelic mutations in DNAH11. Objectives In order to test further for mutant DNAH11 as a cause of PCD, we sequenced DNAH11 in patients with a PCD clinical phenotype, but no known genetic etiology. Methods We sequenced 82 exons and intron/exon junctions in DNAH11 in 163 unrelated patients with a clinical phenotype of PCD, including those with normal ciliary ultrastructure (n=58), defects in outer ± inner dynein arms (n=76), radial spoke/central pair defects (n=6), and 23 without definitive ultrastructural results, but who had situs inversus (n=17), or bronchiectasis and/or low nasal nitric oxide (n=6). Additionally, we sequenced DNAH11 in 13 patients with isolated situs abnormalities to see if mutant DNAH11 could cause situs defects without respiratory disease. Results Of the 58 unrelated PCD patients with normal ultrastructure, 13 (22%) had two (biallelic) mutations in DNAH11; plus, 2 PCD patients without ultrastructural analysis had biallelic mutations. All mutations were novel and private. None of the patients with dynein arm or radial spoke/central pair defects, or isolated situs abnormalities, had mutations in DNAH11. Of the 35 identified mutant alleles, 24 (69%) were nonsense, insertion/deletion or Ioss-of-function splice-site mutations. Conclusions Mutations in DNAH11 are a common cause of PCD in patients without ciliary ultrastructural defects; thus, genetic analysis can be used to ascertain the diagnosis of PCD in this challenging group of patients.
Mucus, with its burden of inspired particulates, and pathogens, is cleared from mucosal surfaces of the airways by cilia beating within the periciliary layer (PCL). The PCL is held to be ‘watery’ and free of mucus by thixotropic-like forces arising from beating cilia. With radii of gyration ~250 nm, however, polymeric mucins should reptate readily into the PCL, so we assessed the glycocalyx for barrier functions. The PCL stained negative for MUC5AC and MUC5B, but it was positive for keratan sulfate, a glycosaminoglycan commonly associated with glycoconjugates. Shotgun proteomics showed keratan sulfate-rich fractions from mucus containing abundant tethered mucins, MUC1, MUC4, and MUC16, but no proteoglycans. Immuno-histology by light and electron microscopy localized MUC1 to microvilli, MUC4 and MUC20 to cilia, and MUC16 to goblet cells. Electron and atomic force microscopy revealed molecular lengths of 190–1,500 nm for tethered mucins, and a finely textured glycocalyx matrix filling interciliary spaces. Adenoviral particles were excluded from glycocalyx of the microvilli, while the smaller AAV penetrated, but were trapped within. Hence, tethered mucins organized as a space-filling glycocalyx function as a selective barrier for the PCL, broadening their role in innate lung defense and offering new molecular targets for conventional and gene therapies.
BACKGROUND:Motile cilia dysfunction causes primary ciliary dyskinesia (PCD), situs inversus totalis (SI), and a spectrum of laterality defects, yet the prevalence of laterality defects other than SI in PCD has not been prospectively studied.
Defects of motile cilia cause primary ciliary dyskinesia (PCD), characterized by recurrent respiratory infections and male infertility. Using whole-exome resequencing and high-throughput mutation analysis, we identified recessive biallelic mutations in ZMYND10 in 14 families and mutations in the recently identified LRRC6 in 13 families. We show that ZMYND10 and LRRC6 interact and that certain ZMYND10 and LRRC6 mutations abrogate the interaction between the LRRC6 CS domain and the ZMYND10 C-terminal domain. Additionally, ZMYND10 and LRRC6 colocalize with the centriole markers SAS6 and PCM1. Mutations in ZMYND10 result in the absence of the axonemal protein components DNAH5 and DNALI1 from respiratory cilia. Animal models support the association between ZMYND10 and human PCD, given that zmynd10 knockdown in zebrafish caused ciliary paralysis leading to cystic kidneys and otolith defects and that knockdown in Xenopus interfered with ciliogenesis. Our findings suggest that a cytoplasmic protein complex containing ZMYND10 and LRRC6 is necessary for motile ciliary function.
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