scholarly journals Flow cytometric analysis and purification of airway epithelial cell subsets

2020 ◽  
Author(s):  
Luke R. Bonser ◽  
Kyung Duk Koh ◽  
Kristina Johansson ◽  
Semil P. Choksi ◽  
Dan Cheng ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Flow Cytometric Analysis ◽  
Cell Subset ◽  
Airway Epithelial Cells ◽  
Secretory Cells ◽  
Airway Epithelial ◽  
Specific Expression ◽  
Flow Cytometric ◽  
Micro Rnas ◽  
Epithelial Biology

AbstractThe human airway epithelium is essential in homeostasis, and epithelial dysfunction contributes to chronic airway disease. Development of flow cytometric methods to characterize subsets of airway epithelial cells will enable further dissection of airway epithelial biology. Leveraging single cell RNA-sequencing (scRNA-seq) data in combination with known cell type-specific markers, we developed panels of antibodies to characterize and isolate the major airway epithelial subsets (basal, ciliated, and secretory cells) from human bronchial epithelial cell cultures. We also identified molecularly distinct subpopulations of secretory cells and demonstrated cell subset-specific expression of low abundance transcripts and micro-RNAs that are challenging to analyze with current scRNA-seq methods. These new tools will be valuable for analyzing and separating airway epithelial subsets and interrogating airway epithelial biology.

scholarly journals Flow Cytometric Analysis and Purification of Airway Epithelial Cell Subsets

2020 ◽  
Author(s):  
Luke R Bonser ◽  
Kyung Duk Koh ◽  
Kristina Johansson ◽  
Semil P Choksi ◽  
Dan Cheng ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Airway Epithelial Cell ◽  
Flow Cytometric Analysis ◽  
Airway Epithelial ◽  
Flow Cytometric

scholarly journals Infection of bovine well-differentiated airway epithelial cells by Pasteurella multocida: actions and counteractions in the bacteria–host interactions

2020 ◽  
Vol 51 (1) ◽  
Author(s):  
Ang Su ◽  
Jie Tong ◽  
Yuguang Fu ◽  
Sandy Müller ◽  
Yenehiwot Berhanu Weldearegay ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Layer ◽  
Airway Epithelial Cells ◽  
Infection Model ◽  
Target Cells ◽  
Respiratory Disorder ◽  
Airway Epithelial ◽  
Epithelial Cell Layer ◽  
Well Differentiated

AbstractPasteurella (P.) multocida is a zoonotic pathogen, which is able to cause respiratory disorder in different hosts. In cattle, P. multocida is an important microorganism involved in the bovine respiratory disease complex (BRDC) with a huge economic impact. We applied air–liquid interface (ALI) cultures of well-differentiated bovine airway epithelial cells to analyze the interaction of P. multocida with its host target cells. The bacterial pathogen grew readily on the ALI cultures. Infection resulted in a substantial loss of ciliated cells. Nevertheless, the epithelial cell layer maintained its barrier function as indicated by the transepithelial electrical resistance and the inability of dextran to get from the apical to the basolateral compartment via the paracellular route. Analysis by confocal immunofluorescence microscopy confirmed the intactness of the epithelial cell layer though it was not as thick as the uninfected control cells. Finally, we chose the bacterial neuraminidase to show that our infection model is a sustainable tool to analyze virulence factors of P. multocida. Furthermore, we provide an explanation, why this microorganism usually is a commensal and becomes pathogenic only in combination with other factors such as co-infecting microorganisms.

scholarly journals FOXO3a regulates rhinovirus-induced innate immune responses in airway epithelial cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Joao Gimenes-Junior ◽  
Nicole Owuar ◽  
Hymavathi Reddy Vari ◽  
Wuyan Li ◽  
Nathaniel Xander ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Lung Inflammation ◽  
Inflammatory Cytokine ◽  
Airway Epithelial Cell ◽  
Critical Role ◽  
Airway Epithelial Cells ◽  
Type I ◽  
Airway Epithelial ◽  
Antiviral Responses

AbstractForkhead transcription factor class O (FOXO)3a, which plays a critical role in a wide variety of cellular processes, was also found to regulate cell-type-specific antiviral responses. Airway epithelial cells express FOXO3a and play an important role in clearing rhinovirus (RV) by mounting antiviral type I and type III interferon (IFN) responses. To elucidate the role of FOXO3a in regulating antiviral responses, we generated airway epithelial cell-specific Foxo3a knockout (Scga1b1-Foxo3a−/−) mice and a stable FOXO3a knockout human airway epithelial cell line. Compared to wild-type, Scga1b1-Foxo3a−/− mice show reduced IFN-α, IFN-β, IFN-λ2/3 in response to challenge with RV or double-stranded (ds)RNA mimic, Poly Inosinic-polycytidylic acid (Poly I:C) indicating defective dsRNA receptor signaling. RV-infected Scga1b1-Foxo3a−/− mice also show viral persistence, enhanced lung inflammation and elevated pro-inflammatory cytokine levels. FOXO3a K/O airway epithelial cells show attenuated IFN responses to RV infection and this was associated with conformational change in mitochondrial antiviral signaling protein (MAVS) but not with a reduction in the expression of dsRNA receptors under unstimulated conditions. Pretreatment with MitoTEMPO, a mitochondrial-specific antioxidant corrects MAVS conformation and restores antiviral IFN responses to subsequent RV infection in FOXO3a K/O cells. Inhibition of oxidative stress also reduces pro-inflammatory cytokine responses to RV in FOXO3a K/O cells. Together, our results indicate that FOXO3a plays a critical role in regulating antiviral responses as well as limiting pro-inflammatory cytokine expression. Based on these results, we conclude that FOXO3a contributes to optimal viral clearance and prevents excessive lung inflammation following RV infection.

Ibudilast, a nonselective phosphodiesterase inhibitor, regulates Th1/Th2 balance and NKT cell subset in multiple sclerosis

2004 ◽  
Vol 10 (5) ◽  
pp. 494-498 ◽  
Author(s):  
Juan Feng ◽  
Tatsuro Misu ◽  
Kazuo Fujihara ◽  
Saburo Sakoda ◽  
Yuji Nakatsuji ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
Flow Cytometric Analysis ◽  
Cell Subset ◽  
Phosphodiesterase Inhibitor ◽  
Interleukin 4 ◽  
Clinical Effects ◽  
Autoimmune Encephalomyelitis ◽  
Flow Cytometric ◽  
Mrna Ratio ◽  
Killer T Cells

We investigated the immunoregulatory effects of ibudilast, a nonselective phosphodiesterase inhibitor, at a clinically applicable dose (60 mg/day p.o. for four weeks) in multiple sclerosis (MS) patients. Sensitive real-time PCR for quantifying cytokine mRNA in the blood CD4- cells revealed that the ibudilast monotherapy significantly reduced tumour necrosis factor-a and interferon (IFN)-g mRNA and the IFN-g/interleukin-4 mRNA ratio, suggesting a shift in the cytokine profile from Th1 toward Th2 dominancy. In a flow cytometric analysis, natural killer T cells, which have been reported to relate to Th2 responses in MS and its animal model (experimental autoimmune encephalomyelitis), increased significantly after the therapy. None of the significant immunological changes were seen in healthy subjects or untreated MS patients. Ibudilast may be a promising therapy for MS and its clinical effects warrant further study.

Modulation of airway inflammation and bacterial clearance by epithelial cell ICAM-1

2004 ◽  
Vol 287 (3) ◽  
pp. L598-L607 ◽  
Author(s):  
Alicia L. Humlicek ◽  
Liyi Pang ◽  
Dwight C. Look
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Airway Epithelial Cells ◽  
Leukocyte Recruitment ◽  
Intercellular Adhesion Molecule ◽  
Additional Contribution ◽  
Bacterial Clearance ◽  
Airway Epithelial ◽  
Intercellular Adhesion Molecule 1 ◽  
Bacterial Killing

Many cell types in the airway express the adhesive glycoprotein for leukocytes intercellular adhesion molecule-1 (ICAM-1) constitutively and/or in response to inflammatory stimuli. In this study, we identified functions of ICAM-1 on airway epithelial cells in defense against infection with Haemophilus influenzae. Initial experiments using a mouse model of airway infection in which the bacterial inoculum was mixed with agar beads that localize inflammation in airways demonstrated that ICAM-1 expression was required for efficient clearance of H. influenzae. Airway epithelial cell ICAM-1 expression required few or no leukocytes, suggesting that epithelial cells could be activated directly by interaction with bacteria. Specific inhibition of ICAM-1 function on epithelial cells by orotracheal injection of blocking antibodies resulted in decreased leukocyte recruitment and H. influenzae clearance in the airway. Inhibition of endothelial cell ICAM-1 resulted in a similar decrease in leukocyte recruitment but did not affect bacterial clearance, indicating that epithelial cell ICAM-1 had an additional contribution to airway defense independent of effects on leukocyte migration. To assess this possibility, we used an in vitro model of neutrophil phagocytosis of bacteria and observed significantly greater engulfment of bacteria by neutrophils adherent to epithelial cells expressing ICAM-1 compared with nonadherent neutrophils. Furthermore, bacterial phagocytosis and killing by neutrophils after interaction with epithelial cells were decreased when a blocking antibody inhibited ICAM-1 function. The results indicate that epithelial cell ICAM-1 participates in neutrophil recruitment into the airway, but its most important role in clearance of H. influenzae may be assistance with neutrophil-dependent bacterial killing.

Paraoxonase-2 deficiency enhancesPseudomonas aeruginosaquorum sensing in murine tracheal epithelia

2007 ◽  
Vol 292 (4) ◽  
pp. L852-L860 ◽  
Author(s):  
David A. Stoltz ◽  
Egon A. Ozer ◽  
Carey J. Ng ◽  
Janet M. Yu ◽  
Srinivasa T. Reddy ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Quorum Sensing ◽  
Airway Epithelial Cell ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Airway Epithelia ◽  
Tracheal Epithelial Cells ◽  
Human Airway ◽  
Tracheal Epithelial

Pseudomonas aeruginosa is an important cause of nosocomial infections and is frequently present in the airways of cystic fibrosis patients. Quorum sensing mediates P. aeruginosa's virulence and biofilm formation through density-dependent interbacterial signaling with autoinducers. N-3-oxododecanoyl homoserine lactone (3OC12-HSL) is the major autoinducer in P. aeruginosa. We have previously shown that human airway epithelia and paraoxonases (PONs) degrade 3OC12-HSL. This study investigated the role of PON1, PON2, and PON3 in airway epithelial cell inactivation of 3OC12-HSL. All three PONs were present in murine tracheal epithelial cells, with PON2 and PON3 expressed at the highest levels. Lysates of tracheal epithelial cells from PON2, but not PON1 or PON3, knockout mice had impaired 3OC12-HSL inactivation compared with wild-type mice. In contrast, PON1-, PON2-, or PON3-targeted deletions did not affect 3OC12-HSL degradation by intact epithelia. Overexpression of PON2 enhanced 3OC12-HSL degradation by human airway epithelial cell lysates but not by intact epithelia. Finally, using a quorum-sensing reporter strain of P. aeruginosa, we found that quorum sensing was enhanced in PON2-deficient airway epithelia. In summary, these results show that loss of PON2 impairs 3OC12-HSL degradation by airway epithelial cells and suggests that diffusion of 3OC12-HSL into the airway cells can be the rate-limiting step for degradation of the molecule.

Pseudomonas aeruginosapyocyanin directly oxidizes glutathione and decreases its levels in airway epithelial cells

2004 ◽  
Vol 287 (1) ◽  
pp. L94-L103 ◽  
Author(s):  
Yunxia Q. O'Malley ◽  
Krzysztof J. Reszka ◽  
Douglas R. Spitz ◽  
Gerene M. Denning ◽  
Bradley E. Britigan
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Culture Media ◽  
Alveolar Epithelial Cell ◽  
A549 Cells ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Alternative Source ◽  
Cell Functions

Production of pyocyanin enhances Pseudomonas aeruginosa virulence. Many of pyocyanin's in vitro and in vivo cytotoxic effects on human cells appear to result from its ability to redox cycle. Pyocyanin directly accepts electrons from NADH or NADPH with subsequent electron transfer to oxygen, generating reactive oxygen species. Reduced glutathione (GSH) is an important cellular antioxidant, and it contributes to the regulation of redox-sensitive signaling systems. Using the human bronchial epithelial (HBE) and the A549 human type II alveolar epithelial cell lines, we tested the hypothesis that pyocyanin can deplete airway epithelial cells of GSH. Incubation of both cell types with pyocyanin led to a concentration-dependent loss of cellular GSH (up to 50%) and an increase in oxidized GSH (GSSG) in the HBE, but not A549 cells, at 24 h. An increase in total GSH, mostly as GSSG, was detected in the culture media, suggesting export of GSH or GSSG from the pyocyanin-exposed cells. Loss of GSH could be due to pyocyanin-induced H2O2formation. However, overexpression of catalase only partially prevented the pyocyanin-mediated decline in cellular GSH. Cell-free electron paramagnetic resonance studies revealed that pyocyanin directly oxidizes GSH, forming pyocyanin free radical and O2−·. Pyocyanin oxidized other thiol-containing compounds, cysteine and N-acetyl-cysteine, but not methionine. Thus GSH may enhance pyocyanin-induced cytotoxicity by functioning as an alternative source of reducing equivalents for pyocyanin redox cycling. Pyocyanin-mediated alterations in cellular GSH may alter epithelial cell functions by modulating redox sensitive signaling events.

Role of f-box factor foxj1 in differentiation of ciliated airway epithelial cells

2004 ◽  
Vol 286 (4) ◽  
pp. L650-L657 ◽  
Author(s):  
Yingjian You ◽  
Tao Huang ◽  
Edward J. Richer ◽  
Jens-Erik Harboe Schmidt ◽  
Joseph Zabner ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Basal Body ◽  
Ciliated Cell ◽  
Airway Epithelial Cells ◽  
Cell Phenotype ◽  
Basal Bodies ◽  
Airway Epithelial ◽  
Ciliated Cells ◽  
Wild Type

Factors required for commitment of an undifferentiated airway epithelial cell to a ciliated cell are unknown. Cell ultrastructure analysis indicates ciliated cell commitment activates a multistage program involving synthesis of cilia precursor proteins and assembly of macromolecular complexes. Foxj1 is an f-box transcription factor expressed in ciliated cells and shown to be required for cilia formation by gene deletion in a mouse model. To identify a specific role for foxj1 in directing the ciliated cell phenotype, we evaluated the capacity of foxj1 to induce ciliogenesis and direct cilia assembly. In a primary culture model of wild-type mouse airway epithelial cells, foxj1 expression preceded the appearance of cilia and in cultured foxj1 null cells cilia did not develop. Delivery of foxj1 to polarized epithelial cell lines and primary cultured alveolar epithelial cells failed to promote ciliogenesis. Similarly, delivery of foxj1 to wild-type airway epithelial cells did not enhance the total number of ciliated cells. In contrast, delivery of foxj1 to null cells resulted in the appearance of cilia. Analysis revealed that, in the absence of foxj1, null cells contained cilia precursor basal bodies, indicating prior commitment to ciliogenesis. However, the basal bodies were disorganized within the apical compartment and failed to dock with the apical membrane. Reconstitution of foxj1 in null cells restored normal basal body organization, resulting in axoneme growth. Thus foxj1 functions in late-stage ciliogenesis to regulate programs promoting basal body docking and axoneme formation in cells previously committed to the ciliated cell phenotype.

scholarly journals Pseudomonas aeruginosa Acquires Biofilm-Like Properties within Airway Epithelial Cells

2005 ◽  
Vol 73 (12) ◽  
pp. 8298-8305 ◽  
Author(s):  
Raquel Garcia-Medina ◽  
W. Michael Dunne ◽  
Pradeep K. Singh ◽  
Steven L. Brody
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Chronic Infection ◽  
Acute Infection ◽  
Airway Epithelial Cells ◽  
Cell Contact ◽  
Intracellular Bacteria ◽  
Bacterial Survival ◽  
Airway Epithelial

ABSTRACT Pseudomonas aeruginosa can notably cause both acute and chronic infection. While several virulence factors are implicated in the acute phase of infection, advances in understanding bacterial pathogenesis suggest that chronic P. aeruginosa infection is related to biofilm formation. However, the relationship between these two forms of disease is not well understood. Accumulating evidence indicates that, during acute infection, P. aeruginosa enters epithelial cells, a process viewed as either a host-mediated defense response or a pathogenic mechanism to avoid host-mediated killing. We investigated the possibility that epithelial cell entry during early P. aeruginosa-epithelial cell contact favors bacterial survival and is linked to chronic infection. Using electron microscopy and confocal microscopy to analyze primary culture airway epithelial cells infected with P. aeruginosa, we found that epithelial cells developed pod-like clusters of intracellular bacteria with regional variation in protein expression. Extracellular gentamicin added to the medium after acute infection led to the persistence of intracellular P. aeruginosa for at least 3 days. Importantly, compared to bacterial culture under planktonic conditions, the intracellular bacteria were insensitive to growth inhibition or killing by antibiotics that were capable of intraepithelial cell penetration. These findings suggest that P. aeruginosa can use airway epithelial cells as a sanctuary for persistence and develop a reversible antibiotic resistance phenotype characteristic of biofilm physiology that can contribute to development of chronic infection.

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