BMI-1 extends proliferative potential of human bronchial epithelial cells while retaining their mucociliary differentiation capacity

Munye, Mustafa M.; Shoemark, Amelia; Hirst, Robert A.; Delhove, Juliette; Sharp, Tyson V.; McKay, Tristan R.; O'Callaghan, C; Baines, Deborah L.; Howe, Steven J.; Hart, Stephen L.

doi:10.1152/ajplung.00471.2016

Cited by 39 publications

(32 citation statements)

References 28 publications

(38 reference statements)

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“…ALI-cell culture, transformation of lymphocytes using EBVs, and generation of human transcriptome profiles were performed as previously described. 41 , 42 Indeed, in agreement with a function of DNAH9 in motile cilia, we observed comparable DNAH9 expression in nasal brushing biopsies and ALI-cultured nasal epithelial cells where hundreds of motile cilia per cell were present, whereas EBV-infected lymphocytes and blood cells lacking any motile cilia showed no or weak expression levels of DNAH9 or other genes encoding ODA proteins ( Figure S5 ). Furthermore, we observed upregulation of DNAH9 expression during in vitro ciliogenesis of respiratory cells between day 3 and day 15, similar to the expression of other genes encoding ODA components ( Figure 2 D).…”

Section: Main Textsupporting

confidence: 85%

Recessive DNAH9 Loss-of-Function Mutations Cause Laterality Defects and Subtle Respiratory Ciliary-Beating Defects

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Antony

Maver

et al. 2018

The American Journal of Human Genetics

106

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Dysfunction of motile monocilia, altering the leftward flow at the embryonic node essential for determination of left-right body asymmetry, is a major cause of laterality defects. Laterality defects are also often associated with reduced mucociliary clearance caused by defective multiple motile cilia of the airway and are responsible for destructive airway disease. Outer dynein arms (ODAs) are essential for ciliary beat generation, and human respiratory cilia contain different ODA heavy chains (HCs): the panaxonemally distributed γ-HC DNAH5, proximally located β-HC DNAH11 (defining ODA type 1), and the distally localized β-HC DNAH9 (defining ODA type 2). Here we report loss-of-function mutations in DNAH9 in five independent families causing situs abnormalities associated with subtle respiratory ciliary dysfunction. Consistent with the observed subtle respiratory phenotype, high-speed video microscopy demonstrates distally impaired ciliary bending in DNAH9 mutant respiratory cilia. DNAH9 -deficient cilia also lack other ODA components such as DNAH5, DNAI1, and DNAI2 from the distal axonemal compartment, demonstrating an essential role of DNAH9 for distal axonemal assembly of ODAs type 2. Yeast two-hybrid and co-immunoprecipitation analyses indicate interaction of DNAH9 with the ODA components DNAH5 and DNAI2 as well as the ODA-docking complex component CCDC114. We further show that during ciliogenesis of respiratory cilia, first proximally located DNAH11 and then distally located DNAH9 is assembled in the axoneme. We propose that the β-HC paralogs DNAH9 and DNAH11 achieved specific functional roles for the distinct axonemal compartments during evolution with human DNAH9 function matching that of ancient β-HCs such as that of the unicellular Chlamydomonas reinhardtii .

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Section: Main Textsupporting

confidence: 85%

Recessive DNAH9 Loss-of-Function Mutations Cause Laterality Defects and Subtle Respiratory Ciliary-Beating Defects

Loges

Antony

Maver

et al. 2018

The American Journal of Human Genetics

106

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“…Expression of αENaC in BMI-1 transduced CF bronchial epithelial (CFBE) cells 33 attained maximal levels after 2–5 days in ALI cultures ( figure 2A ), and so, ALI culture transfections in ongoing experiments were performed after at least 5 days ALI culture. Transfections of CFBE monolayers with 100 nM αENaC siRNA reduced αENaC mRNA by 30% (n=3) compared with control siRNA-treated cultures ( figure 2B ).…”

Section: Resultsmentioning

confidence: 99%

Effective silencing of ENaC by siRNA delivered with epithelial-targeted nanocomplexes in human cystic fibrosis cells and in mouse lung

Tagalakis

Munye

Ivanova

et al. 2018

Thorax

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IntroductionLoss of the cystic fibrosis transmembrane conductance regulator in cystic fibrosis (CF) leads to hyperabsorption of sodium and fluid from the airway due to upregulation of the epithelial sodium channel (ENaC). Thickened mucus and depleted airway surface liquid (ASL) then lead to impaired mucociliary clearance. ENaC regulation is thus a promising target for CF therapy. Our aim was to develop siRNA nanocomplexes that mediate effective silencing of airway epithelial ENaC in vitro and in vivo with functional correction of epithelial ion and fluid transport.MethodsWe investigated translocation of nanocomplexes through mucus and their transfection efficiency in primary CF epithelial cells grown at air–liquid interface (ALI). Short interfering RNA (SiRNA)-mediated silencing was examined by quantitative RT-PCR and western analysis of ENaC. Transepithelial potential (Vt), short circuit current (Isc), ASL depth and ciliary beat frequency (CBF) were measured for functional analysis. Inflammation was analysed by histological analysis of normal mouse lung tissue sections.ResultsNanocomplexes translocated more rapidly than siRNA alone through mucus. Transfections of primary CF epithelial cells with nanocomplexes targeting αENaC siRNA, reduced αENaC and βENaC mRNA by 30%. Transfections reduced Vt, the amiloride-sensitive Isc and mucus protein concentration while increasing ASL depth and CBF to normal levels. A single dose of siRNA in mouse lung silenced ENaC by approximately 30%, which persisted for at least 7 days. Three doses of siRNA increased silencing to approximately 50%.ConclusionNanoparticle-mediated delivery of ENaCsiRNA to ALI cultures corrected aspects of the mucociliary defect in human CF cells and offers effective delivery and silencing in vivo.

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“…Although defective mucociliary clearance in CF make it difficult to eradicate pathogenic bacteria, it might be possible to mitigate the effects of resident pathogens by promoting growth of bacterial taxa whose dominance is associated with better outcomes [11]. Reproducible infection models, such as CF specific air liquid interface cell cultures, might be used to identify paradigms to manage microbiota community structure [36]. Further, combining these paradigms with longitudinal patient studies might elucidate the underlying mechanisms that govern microbial diversity and dominance in the CF lung, and the role played by intensive antibiotic administration in the context of advancing lung disease [11].…”

Section: Discussionmentioning

confidence: 99%

Lung function and microbiota diversity in cystic fibrosis

et al. 2020

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Background: Chronic infection and concomitant airway inflammation is the leading cause of morbidity and mortality for people living with cystic fibrosis (CF). Although chronic infection in CF is undeniably polymicrobial, involving a lung microbiota, infection surveillance and control approaches remain underpinned by classical aerobic culture-based microbiology. How to use microbiomics to direct clinical management of CF airway infections remains a crucial challenge. A pivotal step towards leveraging microbiome approaches in CF clinical care is to understand the ecology of the CF lung microbiome and identify ecological patterns of CF microbiota across a wide spectrum of lung disease. Assessing sputum samples from 299 patients attending 13 CF centres in Europe and the USA, we determined whether the emerging relationship of decreasing microbiota diversity with worsening lung function could be considered a generalised pattern of CF lung microbiota and explored its potential as an informative indicator of lung disease state in CF. Results:We tested and found decreasing microbiota diversity with a reduction in lung function to be a significant ecological pattern. Moreover, the loss of diversity was accompanied by an increase in microbiota dominance. Subsequently, we stratified patients into lung disease categories of increasing disease severity to further investigate relationships between microbiota characteristics and lung function, and the factors contributing to microbiota variance. Core taxa group composition became highly conserved within the severe disease category, while the rarer satellite taxa underpinned the high variability observed in the microbiota diversity. Further, the lung microbiota of individual patient were increasingly dominated by recognised CF pathogens as lung function decreased. Conversely, other bacteria, especially obligate anaerobes, increasingly dominated in those with better lung function. Ordination analyses revealed lung function and antibiotics to be main explanators of compositional variance in the microbiota and the core and satellite taxa. Biogeography was found to influence acquisition of the rarer satellite taxa. (Continued on next page)Conclusions: Our findings demonstrate that microbiota diversity and dominance, as well as the identity of the dominant bacterial species, in combination with measures of lung function, can be used as informative indicators of disease state in CF.

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BMI-1 extends proliferative potential of human bronchial epithelial cells while retaining their mucociliary differentiation capacity

Cited by 39 publications

References 28 publications

Recessive DNAH9 Loss-of-Function Mutations Cause Laterality Defects and Subtle Respiratory Ciliary-Beating Defects

Recessive DNAH9 Loss-of-Function Mutations Cause Laterality Defects and Subtle Respiratory Ciliary-Beating Defects

Effective silencing of ENaC by siRNA delivered with epithelial-targeted nanocomplexes in human cystic fibrosis cells and in mouse lung

Lung function and microbiota diversity in cystic fibrosis

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