Cell Culture Differentiation and Proliferation Conditions Influence the In Vitro Regeneration of the Human Airway Epithelium.

The human airway mucociliary epithelium can be recapitulated in vitro using primary cells cultured in an Air-Liquid Interface (ALI), a reliable surrogate to perform pathophysiological studies. As tremendous variations exist between media used for ALI-cultured human airway epithelial cells, our study aimed to evaluate the impact of several media (BEGMTM, PneumaCultTM, "Half&Half" and "Clancy") on cell type distribution using single-cell RNA sequencing and imaging. Our work revealed the impact of these media on cell composition, gene expression profile, cell signaling and epithelial morphology. We found higher proportions of multiciliated cells in PneumaCultTM-ALI and Half&Half, stronger EGF signaling from basal cells in BEGMTM-ALI, differential expression of the SARS-CoV-2 entry factor ACE2, and distinct secretome transcripts depending on media used. We also established that proliferation in PneumaCultTM-Ex Plus favored secretory cell fate, showing the key influence of proliferation media on late differentiation epithelial characteristics. Altogether, our data offer a comprehensive repertoire for evaluating the effects of culture conditions on airway epithelial differentiation and will help to choose the most relevant medium according to the processes to be investigated such as cilia, mucus biology or viral infection. We detail useful parameters that should be explored to document airway epithelial cell fate and morphology.

The MIR34B/C genomic region contains multiple potential regulators of multiciliogenesis.

The MIR449 genomic locus encompasses several regulators of multiciliated cell (MCC) formation (multiciliogenesis). The miR-449 homologs miR-34b/c represent additional regulators of multiciliogenesis that are transcribed from another locus. Here, we characterized the expression of BTG4, LAYN, and HOATZ, located in the MIR34B/C locus using single-cell RNA-seq and super-resolution microscopy from human, mouse, or pig multiciliogenesis models. BTG4, LAYN, and HOATZ transcripts were expressed in both precursors and mature MCCs. The Layilin/LAYN protein was absent from primary cilia, but it was expressed in apical membrane regions or throughout motile cilia. LAYN silencing altered apical actin cap formation and multiciliogenesis. HOATZ protein was detected in primary cilia or throughout motile cilia. Altogether, our data suggest that the MIR34B/C locus may gather potential actors of multiciliogenesis.

Novel dynamics of human mucociliary differentiation revealed by single-cell RNA sequencing of nasal epithelial cultures.

The upper airway epithelium, which is mainly composed of multiciliated, goblet, club and basal cells, ensures proper mucociliary function and can regenerate in response to assaults. In chronic airway diseases, defective repair leads to tissue remodeling. Delineating key drivers of differentiation dynamics can help understand how normal or pathological regeneration occurs. Using single-cell transcriptomics and lineage inference, we have unraveled trajectories from basal to luminal cells, providing novel markers for specific populations. We report that: (1) a precursor subgroup of multiciliated cells, which we have entitled deuterosomal cells, is defined by specific markers, such as DEUP1, FOXN4, YPEL1, HES6 and CDC20B; (2) goblet cells can be precursors of multiciliated cells, thus explaining the presence of hybrid cells that co-express markers of goblet and multiciliated cells; and (3) a repertoire of molecules involved in the regeneration process, such as keratins or components of the Notch, Wnt or BMP/TGFß pathways, can be identified. Confirmation of our results on fresh human and pig airway samples, and on mouse tracheal cells, extend and confirm our conclusions regarding the molecular and cellular choreography at work during mucociliary epithelial differentiation.

CDC20B is required for deuterosome-mediated centriole production in multiciliated cells.

Multiciliated cells (MCCs) harbor dozens to hundreds of motile cilia, which generate hydrodynamic forces important in animal physiology. In vertebrates, MCC differentiation involves massive centriole production by poorly characterized structures called deuterosomes. Here, single-cell RNA sequencing reveals that human deuterosome stage MCCs are characterized by the expression of many cell cycle-related genes. We further investigated the uncharacterized vertebrate-specific cell division cycle 20B (CDC20B) gene, which hosts microRNA-449abc. We show that CDC20B protein associates to deuterosomes and is required for centriole release and subsequent cilia production in mouse and Xenopus MCCs. CDC20B interacts with PLK1, a kinase known to coordinate centriole disengagement with the protease Separase in mitotic cells. Strikingly, over-expression of Separase rescues centriole disengagement and cilia production in CDC20B-deficient MCCs. This work reveals the shaping of deuterosome-mediated centriole production in vertebrate MCCs, by adaptation of canonical and recently evolved cell cycle-related molecules.

The "one airway, one disease" concept in light of Th2 inflammation.

In line with the pathophysiological continuum described between nose and bronchus in allergic respiratory diseases, we assessed whether nasal epithelium could mirror the Type 2 T-helper cell (Th2) status of bronchial epithelium.Nasal and bronchial cells were collected by brushing from healthy controls (C, n=13), patients with allergic rhinitis and asthma (AR, n=12), and patients with isolated allergic rhinitis (R, n=14). Cellular composition was assessed by flow cytometry, gene expression was analysed by RNA sequencing and Th2, Type 17 T-helper cell (Th17) and interferon (IFN) signatures were derived from the literature.Infiltration by polymorphonuclear neutrophils (PMN) in the nose excluded 30% of the initial cohort. All bronchial samples from the AR group were Th2-high. The gene expression profile of nasal samples from the AR group correctly predicted the paired bronchial sample Th2 status in 71% of cases. Nevertheless, nasal cells did not appear to be a reliable surrogate for the Th2 response, in particular due to a more robust influence of the IFN response in 14 out of 26 nasal samples. The Th2 scores in the nose and bronchi correlated with mast cell count (both p<0.001) and number of sensitisations (p=0.006 and 0.002), while the Th17 scores correlated with PMN count (p=0.006 and 0.003).The large variability in nasal cell composition and type of inflammation restricts its use as a surrogate for assessing bronchial Th2 inflammation in AR patients.

Characterizing isomiR variants within the microRNA-34/449 family.

miR-34/449 microRNAs are conserved regulators of multiciliated cell differentiation. Here, we evidence and characterize expression of two isomiR variant sequences from the miR-34/449 family in human airway epithelial cells. These isomiRs differ from their canonical counterparts miR-34b and miR-449c by one supplemental uridine at their 5'-end, leading to a one-base shift in their seed region. Overexpression of canonical miR-34/449 or 5'-isomiR-34/449 induces distinct gene expression profiles and biological effects. However, some target transcripts and functional activities are shared by both canonical microRNAs and isomiRs. Indeed, both repress important targets that result in cell cycle blockage and Notch pathway inhibition. Our findings suggest that 5'-isomiR-34/449 may represent additional mechanisms by which miR-34/449 family finely controls several pathways to drive multiciliogenesis.

MicroRNAs as key regulators of GTPase-mediated apical actin reorganization in multiciliated epithelia.

Multiciliated cells (MCCs), which are present in specialized vertebrate tissues such as mucociliary epithelia, project hundreds of motile cilia from their apical membrane. Coordinated ciliary beating in MCCs contributes to fluid propulsion in several biological processes. In a previous work, we demonstrated that microRNAs of the miR-34/449 family act as new conserved regulators of MCC differentiation by specifically repressing cell cycle genes and the Notch pathway. Recently, we have shown that miR-34/449 also modulate small GTPase pathways to promote, in a later stage of differentiation, the assembly of the apical actin network, a prerequisite for proper anchoring of centrioles-derived neo-synthesized basal bodies. We characterized several miR-34/449 targets related to small GTPase pathways including R-Ras, which represents a key and conserved regulator during MCC differentiation. Direct RRAS repression by miR-34/449 is necessary for apical actin meshwork assembly, notably by allowing the apical relocalization of the actin binding protein Filamin-A near basal bodies. Our studies establish miR-34/449 as central players that orchestrate several steps of MCC differentiation program by regulating distinct signaling pathways.

miR-34/449 control apical actin network formation during multiciliogenesis through small GTPase pathways.

Vertebrate multiciliated cells (MCCs) contribute to fluid propulsion in several biological processes. We previously showed that microRNAs of the miR-34/449 family trigger MCC differentiation by repressing cell cycle genes and the Notch pathway. Here, using human and Xenopus MCCs, we show that beyond this initial step, miR-34/449 later promote the assembly of an apical actin network, required for proper basal bodies anchoring. Identification of miR-34/449 targets related to small GTPase pathways led us to characterize R-Ras as a key regulator of this process. Protection of RRAS messenger RNA against miR-34/449 binding impairs actin cap formation and multiciliogenesis, despite a still active RhoA. We propose that miR-34/449 also promote relocalization of the actin binding protein Filamin-A, a known RRAS interactor, near basal bodies in MCCs. Our study illustrates the intricate role played by miR-34/449 in coordinating several steps of a complex differentiation programme by regulating distinct signalling pathways.

BMP signalling controls the construction of vertebrate mucociliary epithelia.

Despite the importance of mucociliary epithelia in animal physiology, the mechanisms controlling their establishment are poorly understood. Using the developing Xenopus epidermis and regenerating human upper airways, we reveal the importance of BMP signalling for the construction of vertebrate mucociliary epithelia. In Xenopus, attenuation of BMP activity is necessary for the specification of multiciliated cells (MCCs), ionocytes and small secretory cells (SSCs). Conversely, BMP activity is required for the proper differentiation of goblet cells. Our data suggest that the BMP and Notch pathways interact to control fate choices in the developing epidermis. Unexpectedly, BMP activity is also necessary for the insertion of MCCs, ionocytes and SSCs into the surface epithelium. In human, BMP inhibition also strongly stimulates the formation of MCCs in normal and pathological (cystic fibrosis) airway samples, whereas BMP overactivation has the opposite effect. This work identifies the BMP pathway as a key regulator of vertebrate mucociliary epithelium differentiation and morphogenesis.

miR-199a-5p Is upregulated during fibrogenic response to tissue injury and mediates TGFbeta-induced lung fibroblast activation by targeting caveolin-1.

As miRNAs are associated with normal cellular processes, deregulation of miRNAs is thought to play a causative role in many complex diseases. Nevertheless, the precise contribution of miRNAs in fibrotic lung diseases, especially the idiopathic form (IPF), remains poorly understood. Given the poor response rate of IPF patients to current therapy, new insights into the pathogenic mechanisms controlling lung fibroblasts activation, the key cell type driving the fibrogenic process, are essential to develop new therapeutic strategies for this devastating disease. To identify miRNAs with potential roles in lung fibrogenesis, we performed a genome-wide assessment of miRNA expression in lungs from two different mouse strains known for their distinct susceptibility to develop lung fibrosis after bleomycin exposure. This led to the identification of miR-199a-5p as the best miRNA candidate associated with bleomycin response. Importantly, miR-199a-5p pulmonary expression was also significantly increased in IPF patients (94 IPF versus 83 controls). In particular, levels of miR-199a-5p were selectively increased in myofibroblasts from injured mouse lungs and fibroblastic foci, a histologic feature associated with IPF. Therefore, miR-199a-5p profibrotic effects were further investigated in cultured lung fibroblasts: miR-199a-5p expression was induced upon TGFß exposure, and ectopic expression of miR-199a-5p was sufficient to promote the pathogenic activation of pulmonary fibroblasts including proliferation, migration, invasion, and differentiation into myofibroblasts. In addition, we demonstrated that miR-199a-5p is a key effector of TGFß signaling in lung fibroblasts by regulating CAV1, a critical mediator of pulmonary fibrosis. Remarkably, aberrant expression of miR-199a-5p was also found in unilateral ureteral obstruction mouse model of kidney fibrosis, as well as in both bile duct ligation and CCl4-induced mouse models of liver fibrosis, suggesting that dysregulation of miR-199a-5p represents a general mechanism contributing to the fibrotic process. MiR-199a-5p thus behaves as a major regulator of tissue fibrosis with therapeutic potency to treat fibroproliferative diseases.

Distinct epithelial gene expression phenotypes in childhood respiratory allergy.

Epithelial cell contribution to the natural history of childhood allergic respiratory disease remains poorly understood. Our aims were to identify epithelial pathways that are dysregulated in different phenotypes of respiratory allergy. We established gene expression signatures of nasal brushings from children with dust mite-allergic rhinitis, associated or not associated with controlled or uncontrolled asthma. Supervised learning and unsupervised clustering were used to predict the different subgroups of patients and define altered signalling pathways. These profiles were compared with those of primary cultures of human nasal epithelial cells stimulated with either interleukin (IL)-4, IL-13, interferon (IFN)-α, IFN-ß or IFN-γ, or during in vitro differentiation. A supervised method discriminated children with allergic rhinitis from healthy controls (prediction accuracy 91%), based on 61 transcripts, including 21 T-helper cell (Th) type 2-responsive genes. This method was then applied to predict children with controlled or uncontrolled asthma (prediction accuracy 75%), based on 41 transcripts: nine of them, which were down-regulated in uncontrolled asthma, are directly linked to IFN. This group also included GSDML, which is genetically associated with asthma. Our data revealed a Th2-driven epithelial phenotype common to all children with dust mite allergic rhinitis. It highlights the influence of epithelially expressed molecules on the control of asthma, in association with atopy and impaired viral response.

MicroRNA-based silencing of Delta/Notch signaling promotes multiple cilia formation.

Multiciliated cells lining the surface of some vertebrate epithelia are essential for various physiological processes, such as airway cleansing. Their apical surface is constituted by hundreds of motile cilia, which beat in a coordinated manner to generate directional fluid flow. We recently reported the identification of microRNAs of the miR-449 family as evolutionary conserved key regulators of vertebrate multiciliogenesis. This novel function of miR-449 was established using in vivo and in vitro antisense approaches in two distinct experimental models. miR-449 strongly accumulated in multiciliated cells in human airway epithelium and Xenopus laevis embryonic epidermis, where it triggered centriole multiplication and multiciliogenesis by directly repressing the Delta/Notch pathway. Our data complement previous reports that showed the blocking action of miR-449 on the cell cycle, and unraveled a novel conserved mechanism whereby Notch signaling must undergo microRNA-mediated inhibition to permit differentiation of ciliated cell progenitors. We review here several important questions regarding the links between microRNAs and the Notch pathway in the control of cell fate.

Control of vertebrate multiciliogenesis by miR-449 through direct repression of the Delta/Notch pathway.

Multiciliated cells lining the surface of some vertebrate epithelia are essential for various physiological processes, such as airway cleansing. However, the mechanisms governing motile cilia biosynthesis remain poorly elucidated. We identify miR-449 microRNAs as evolutionarily conserved key regulators of vertebrate multiciliogenesis. In human airway epithelium and Xenopus laevis embryonic epidermis, miR-449 microRNAs strongly accumulated in multiciliated cells. In both models, we show that miR-449 microRNAs promote centriole multiplication and multiciliogenesis by directly repressing the Delta/Notch pathway. We established Notch1 and its ligand Delta-like 1(DLL1) as miR-449 bona fide targets. Human DLL1 and NOTCH1 protein levels were lower in multiciliated cells than in surrounding cells, decreased after miR-449 overexpression and increased after miR-449 inhibition. In frog, miR-449 silencing led to increased Dll1 expression. Consistently, overexpression of Dll1 mRNA lacking miR-449 target sites repressed multiciliogenesis, whereas both Dll1 and Notch1 knockdown rescued multiciliogenesis in miR-449-deficient cells. Antisense-mediated protection of miR-449-binding sites of endogenous human Notch1 or frog Dll1 strongly repressed multiciliogenesis. Our results unravel a conserved mechanism whereby Notch signalling must undergo miR-449-mediated inhibition to permit differentiation of ciliated cell progenitors.

Impact of microRNA in normal and pathological respiratory epithelia.

Extensive sequencing efforts, combined with ad hoc bioinformatics developments, have now led to the identification of 1222 distinct miRNAs in human (derived from 1368 distinct genomic loci) and of many miRNAs in other multicellular organisms. The present chapter is aimed at describing a general experimental strategy to identify specific miRNA expression profiles and to highlight the functional networks operating between them and their mRNA targets, including several miRNAs deregulated in cystic fibrosis and during differentiation of airway epithelial cells.

Identification of keratinocyte growth factor as a target of microRNA-155 in lung fibroblasts: implication in epithelial-mesenchymal interactions.

BACKGROUND: Epithelial-mesenchymal interactions are critical in regulating many aspects of vertebrate embryo development, and for the maintenance of homeostatic equilibrium in adult tissues. The interactions between epithelium and mesenchyme are believed to be mediated by paracrine signals such as cytokines and extracellular matrix components secreted from fibroblasts that affect adjacent epithelia. In this study, we sought to identify the repertoire of microRNAs (miRNAs) in normal lung human fibroblasts and their potential regulation by the cytokines TNF-alpha, IL-1beta and TGF-beta. METHODOLOGY/PRINCIPAL FINDINGS: MiR-155 was significantly induced by inflammatory cytokines TNF-alpha and IL-1beta while it was down-regulated by TGF-beta. Ectopic expression of miR-155 in human fibroblasts induced modulation of a large set of genes related to "cell to cell signalling", "cell morphology" and "cellular movement". This was consistent with an induction of caspase-3 activity and with an increase in cell migration in fibroblasts tranfected with miR-155. Using different miRNA bioinformatic target prediction tools, we found a specific enrichment for miR-155 predicted targets among the population of down-regulated transcripts. Among fibroblast-selective targets, one interesting hit was keratinocyte growth factor (KGF, FGF-7), a member of the fibroblast growth factor (FGF) family, which owns two potential binding sites for miR-155 in its 3'-UTR. Luciferase assays experimentally validated that miR-155 can efficiently target KGF 3'-UTR. Site-directed mutagenesis revealed that only one out of the 2 potential sites was truly functional. Functional in vitro assays experimentally validated that miR-155 can efficiently target KGF 3'-UTR. Furthermore, in vivo experiments using a mouse model of lung fibrosis showed that miR-155 expression level was correlated with the degree of lung fibrosis. CONCLUSIONS/SIGNIFICANCE: Our results strongly suggest a physiological function of miR-155 in lung fibroblasts. Altogether, this study implicates this miRNA in the regulation by mesenchymal cells of surrounding lung epithelium, making it a potential key player during tissue injury.

Extracellular nucleotides and interleukin-8 production by ARPE cells: potential role of danger signals in blood-retinal barrier activation.

PURPOSE: RPE cell activation is an important feature of autoimmune uveitis. This investigation focused on whether extracellular nucleotides could contribute to this activation, and the effects of ATPgammaS, UTP, and UDP on the production of IL-8 by RPE cells was studied in relation to their expression of functional P2Y receptors. METHODS: ARPE-19 cells were cultured with ATPgammaS, UTP, UDP, and TNF. IL-8 gene transcription and protein production were measured by semiquantitative RT-PCR and ELISA. Western blot analysis and RT-PCR were used to investigate ERK 1/2 activation and P2Y expression. Changes in intracellular calcium and cAMP concentration were analyzed by spectrofluorometry and radioimmunoassay. RESULTS: Stimulation of ARPE-19 cells with ATPgammaS, UTP, and UDP induced IL-8 gene transcription and protein secretion. TNFalpha induction of IL-8 secretion was also increased by ATPgammaS, UTP, and UDP. Nucleotide induction of IL-8 production was blocked by PD98059, and all nucleotides stimulated ERK 1/2 phosphorylation. P2Y(2) and P2Y(6) mRNAs were detected in ARPE-19 cells. All tested nucleotides induced a pulse of intracellular calcium. CONCLUSIONS: ATPgammaS, UTP, and UDP stimulate both basal and TNFalpha-induced IL-8 secretion in RPE cells through an ERK 1/2-dependent pathway. The results suggest that those effects are mediated by P2Y(2) and P2Y(6) receptors.

Extracellular nucleotides induce COX-2 up-regulation and prostaglandin E2 production in human A549 alveolar type II epithelial cells.

Extracellular nucleotides regulate ion transport, mucociliary clearance as well as inflammatory properties of the airway epithelium by acting on P2 receptors. Cyclooxygenase-2 (COX-2) is a key enzyme involved in the synthesis of prostaglandins during inflammation. In this study, using calcium imaging, DNA microarray experiments, real-time Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and prostaglandin E2 (PGE2) measurement, we show for the first time that ATP, UTP or INS365 compound (P2Y2 receptor agonists) up-regulate COX-2 expression by approximately 3-fold and enhance the release of PGE2 in human A549 airway epithelial cells. Our data suggest that P2Y receptors may represent putative targets in airway inflammatory diseases.