Functional development of mechanosensitive hair cells in stem cell-derived organoids parallels native vestibular hair cells

Inner ear sensory epithelia contain mechanosensitive hair cells that transmit information to the brain through innervation with bipolar neurons. Mammalian hair cells do not regenerate and are limited in number. Here we investigate the potential to generate mechanosensitive hair cells from mouse embryonic stem cells in a three-dimensional (3D) culture system. The system faithfully recapitulates mouse inner ear induction followed by self-guided development into organoids that morphologically resemble inner ear vestibular organs. We find that organoid hair cells acquire mechanosensitivity equivalent to functionally mature hair cells in postnatal mice. The organoid hair cells also progress through a similar dynamic developmental pattern of ion channel expression, reminiscent of two subtypes of native vestibular hair cells. We conclude that our 3D culture system can generate large numbers of fully functional sensory cells which could be used to investigate mechanisms of inner ear development and disease as well as regenerative mechanisms for inner ear repair

Functional development of mechanosensitive hair cells in stem cell-derived organoids parallels native vestibular hair cells

Inner ear sensory epithelia contain mechanosensitive hair cells that transmit information to the brain through innervation with bipolar neurons. Mammalian hair cells do not regenerate and are limited in number. Here we investigate the potential to generate mechanosensitive hair cells from mouse embryonic stem cells in a three-dimensional (3D) culture system. The system faithfully recapitulates mouse inner ear induction followed by self-guided development into organoids that morphologically resemble inner ear vestibular organs. We find that organoid hair cells acquire mechanosensitivity equivalent to functionally mature hair cells in postnatal mice. The organoid hair cells also progress through a similar dynamic developmental pattern of ion channel expression, reminiscent of two subtypes of native vestibular hair cells. We conclude that our 3D culture system can generate large numbers of fully functional sensory cells which could be used to investigate mechanisms of inner ear development and disease as well as regenerative mechanisms for inner ear repair

Structural and functional characterization of endothelial microparticles released by cigarette smoke

Circulating endothelial microparticles (EMPs) are emerging as biomarkers of chronic obstructive pulmonary disease (COPD) in individuals exposed to cigarette smoke (CS), but their mechanism of release and function remain unknown. We assessed biochemical and functional characteristics of EMPs and circulating microparticles (cMPs) released by CS. CS exposure was sufficient to increase microparticle levels in plasma of humans and mice, and in supernatants of primary human lung microvascular endothelial cells. CS-released EMPs contained predominantly exosomes that were significantly enriched in let-7d, miR-191; miR-126; and miR125a, microRNAs that reciprocally decreased intracellular in CS-exposed endothelium. CS-released EMPs and cMPs were ceramide-rich and required the ceramide-synthesis enzyme acid sphingomyelinase (aSMase) for their release, an enzyme which was found to exhibit significantly higher activity in plasma of COPD patients or of CS-exposed mice. The ex vivo or in vivo engulfment of EMPs or cMPs by peripheral blood monocytes-derived macrophages was associated with significant inhibition of efferocytosis. Our results indicate that CS, via aSMase, releases circulating EMPs with distinct microRNA cargo and that EMPs affect the clearance of apoptotic cells by specialized macrophages. These targetable effects may be important in the pathogenesis of diseases linked to endothelial injury and inflammation in smokers

Down-Regulation of Serum/Glucocorticoid Regulated Kinase 1 in Colorectal Tumours Is Largely Independent of Promoter Hypermethylation

Background: We have previously shown that serum/glucocorticoid regulated kinase 1 (SGK1) is down-regulated in colorectal cancers (CRC) with respect to normal tissue. As hyper-methylation of promoter regions is a well-known mechanism of gene silencing in cancer, we tested whether the SGK1 promoter region was methylated in colonic tumour samples. Methodology/Principal Findings: We investigated the methylation profile of the two CpG islands present in the promoter region of SGK1 in a panel of 5 colorectal cancer cell lines by sequencing clones of bisulphite-treated DNA samples. We further confirmed our findings in a panel of 10 normal and 10 tumour colonic tissue samples of human origin. We observed CpG methylation only in the smaller and more distal CpG island in the promoter region of SGK1 in both normal and tumour samples of colonic origin. We further identified a single nucleotide polymorphism (SNP, rs1743963) which affects methylation of the corresponding CpG. Conclusions/Significance: Our results show that even though partial methylation of the promoter region of SGK1 is present

Alpha-1 antitrypsin supplementation improves alveolar macrophages efferocytosis and phagocytosis following cigarette smoke exposure.

Cigarette smoking (CS), the main risk factor for COPD (chronic obstructive pulmonary disease) in developed countries, decreases alveolar macrophages (AM) clearance of both apoptotic cells and bacterial pathogens. This global deficit of AM engulfment may explain why active smokers have worse outcomes of COPD exacerbations, episodes characterized by airway infection and inflammation that carry high morbidity and healthcare cost. When administered as intravenous supplementation, the acute phase-reactant alpha-1 antitrypsin (A1AT) reduces the severity of COPD exacerbations in A1AT deficient (AATD) individuals and of bacterial pneumonia in murine models, but the effect of A1AT on AM scavenging functions has not been reported. Apoptotic cell clearance (efferocytosis) was measured in human AM isolated from patients with COPD, in primary rat AM or differentiated monocytes exposed to CS ex vivo, and in AM recovered from mice exposed to CS. A1AT (100 μg/mL, 16 h) significantly ameliorated efferocytosis (by ~50%) in AM of active smokers or AM exposed ex vivo to CS. A1AT significantly improved AM global engulfment, including phagocytosis, even when cells were simultaneously challenged with apoptotic and Fc-coated (bacteria-like) targets. The improved efferocytosis in A1AT-treated macrophages was associated with inhibition of tumor necrosis factor-α converting enzyme (TACE) activity, decreased mannose receptor shedding, and markedly increased abundance of efferocytosis receptors (mannose- and phosphatidyl serine receptors and the scavenger receptor B2) on AM plasma membrane. Directed airway A1AT treatment (via inhalation of a nebulized solution) restored in situ airway AM efferocytosis after CS exposure in mice. The amelioration of CS-exposed AM global engulfment may render A1AT as a potential therapy for COPD exacerbations

Rapid clearance of heavy chain-modified hyaluronan during resolving acute lung injury

Abstract Background Several inflammatory lung diseases display abundant presence of hyaluronic acid (HA) bound to heavy chains (HC) of serum protein inter-alpha-inhibitor (IαI) in the extracellular matrix. The HC-HA modification is critical to neutrophil sequestration in liver sinusoids and to survival during experimental lipopolysaccharide (LPS)-induced sepsis. Therefore, the covalent HC-HA binding, which is exclusively mediated by tumor necrosis factor α (TNFα)-stimulated-gene-6 (TSG-6), may play an important role in the onset or the resolution of lung inflammation in acute lung injury (ALI) induced by respiratory infection. Methods Reversible ALI was induced by a single intratracheal instillation of LPS or Pseudomonas aeruginosa in mice and outcomes were studied for up to six days. We measured in the lung or the bronchoalveolar fluid HC-HA formation, HA immunostaining localization and roughness, HA fragment abundance, and markers of lung inflammation and lung injury. We also assessed TSG-6 secretion by TNFα- or LPS-stimulated human alveolar macrophages, lung fibroblast Wi38, and bronchial epithelial BEAS-2B cells. Results Extensive HC-modification of lung HA, localized predominantly in the peri-broncho-vascular extracellular matrix, was notable early during the onset of inflammation and was markedly decreased during its resolution. Whereas human alveolar macrophages secreted functional TSG-6 following both TNFα and LPS stimulation, fibroblasts and bronchial epithelial cells responded to only TNFα. Compared to wild type, TSG-6-KO mice, which lacked HC-modified HA, exhibited modest increases in inflammatory cells in the lung, but no significant differences in markers of lung inflammation or injury, including histopathological lung injury scores. Conclusions Respiratory infection induces rapid HC modification of HA followed by fragmentation and clearance, with kinetics that parallel the onset and resolution phase of ALI, respectively. Alveolar macrophages may be an important source of pulmonary TSG-6 required for HA remodeling. The formation of HC-modified HA had a minor role in the onset, severity, or resolution of experimental reversible ALI induced by respiratory infection with gram-negative bacteria

Native and polymerized A1AT <i>ex-vivo</i> effect on CS-exposed AM efferocytosis.

<p><b>A</b>. Absolute efferocytosis index representing % of cells that engulfed fluorescently labeled apoptotic targets among AM isolated from active smokers (dark circles) compared to those from healthy non-smokers (light gray circles). Native A1AT (Aralast NP, 100 μg/mL, 16 h) significantly increased efferocytosis in AM from smokers (dark gray circles), but not in those from healthy non-smokers (gray squares). 1-way ANOVA, Sidak’s multiple comparisons test, * p<0.05. <b>B-C</b>. Relative efferocytosis index representing % of AM that engulfed fluorescently labeled apoptotic targets following indicated exposures, when compared to those exposed to control media. Primary rat AM (<b>B</b>) or NR8383 AM (<b>C</b>) were exposed <i>ex-vivo</i> to AC or CS (4h, 3% or 10%, respectively, 4 h), native A1AT (Aralast NP, 100 μg/mL, 16 h), and compared to IL-4 (20 ng/mL, 72 h). <b>D</b>. Polymerized A1AT (Aralast NP, 100 μg/mL, 16h) has no effect on CS-exposed NR8383 AM efferocytosis. Data are presented as mean ± SEM, 1-way ANOVA, Sidak’s multiple comparisons test, * p<0.05, ** p<0.001, *** p<0.0001.</p

Native A1AT effect on CS-exposed AM phagocytosis and global scavenging function following concomitant Fc-coated and apoptotic targets exposure.

<p><b>A-B.</b> Relative phagocytosis index representing % of primary rat AM that engulfed fluorescently labeled Fc-coated targets after CS-exposure (10%, 4 h) and A1AT treatment (Aralast NP, 100 μg/mL, 4 h), compared to AM engulfment in control media. 1-way ANOVA, Sidak’s multiple comparisons test, * p<0.05. Note the lack of significant effect on phagocytosis at baseline (AC) or during CS-exposure following 16 h of A1AT treatment. <b>B-C</b>. iNOS expression (<b>B</b>) and TNF-α secretion (<b>C</b>) in primary rat AM exposed to CS (3%, 4 h) and treated with LPS (100 ng/mL, 4 h) and A1AT (Aralast NP, 100 μg/mL, 4 h). <b>D.</b> Higher Fc-coated to apoptotic targets ratio (0:1 to 10:1) dose-dependently inhibits NR8383 AM efferocytosis. <b>E.</b> Higher mouse anti-human CD3⁺ CTG-labeled Jurkat T-cells (Fc-coated targets) to apoptotic targets ratio (0:1 to 5:1) dose-dependently inhibits primary rat AM efferocytosis. <b>F.</b> Higher apoptotic to Fc-coated targets ratio (0:1 to 24:1) dose-dependently inhibits NR8383 AM phagocytosis. <b>G.</b> Overall engulfment [efferocytosis (E), phagocytosis (P), and efferocytosis + phagocytosis (E+P)] of CS-exposed and A1AT-treated (Aralast NP, 100 μg/mL, 16 h) primary rat AM when concomitantly exposed to apoptotic and Fc-coated targets at 1:1 ratio. 1-way ANOVA, Sidak’s multiple comparisons test, * p<0.05. Data are presented as mean ± SEM.</p