scholarly journals Selection of reference genes for quantitative PCR: identifying reference genes for airway epithelial cells exposed to Pseudomonas aeruginosa

2020 ◽  
Vol 319 (2) ◽  
pp. L256-L265
Author(s):  
Thomas H. Hampton ◽  
Katja Koeppen ◽  
Laura Bashor ◽  
Bruce A. Stanton
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Epithelial Cells ◽  
Quantitative Pcr ◽  
Reference Gene ◽  
Reference Genes ◽  
Primary Cultures ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Reference Gene Expression

Most quantitative PCR (qPCR) experiments report differential expression relative to the expression of one or more reference genes. Therefore, when experimental conditions alter reference gene expression, qPCR results may be compromised. Little is known about the magnitude of this problem in practice. We found that reference gene responses are common and hard to predict and that their stability should be demonstrated in each experiment. Our reanalysis of 15 airway epithelia microarray data sets retrieved from the National Center for Biotechnology Information (NCBI) identified no common reference gene that was reliable in all 15 studies. Reanalysis of published RNA sequencing (RNA-seq) data in which human bronchial epithelial cells (HBEC) were exposed to Pseudomonas aeruginosa revealed that minor experimental details, including bacterial strain, may alter reference gene responses. Direct measurement of 32 TaqMan reference genes in primary cultures of HBEC exposed to P. aeruginosa (strain PA14) demonstrated that choosing an unstable reference gene could make it impossible to observe statistically significant changes in IL8 gene expression. We found that reference gene instability is a general phenomenon and not limited to studies of airway epithelial cells. In a diverse compendium of 986 human microarray experiments retrieved from the NCBI, reference genes were differentially expressed in 42% of studies. Experimentally induced changes in reference gene expression ranged from 21% to 212%. These results highlight the importance of identifying adequate reference genes for each experimental system and documenting their response to treatment in each experiment. This will enhance experimental rigor and reproducibility in qPCR studies.

scholarly journals Pseudomonas aeruginosa Infection of Airway Epithelial Cells Modulates Expression of Kruppel-Like Factors 2 and 6 via RsmA-Mediated Regulation of Type III Exoenzymes S and Y

2006 ◽  
Vol 74 (10) ◽  
pp. 5893-5902 ◽  
Author(s):  
Eoin P. O'Grady ◽  
Heidi Mulcahy ◽  
Julie O'Callaghan ◽  
Claire Adams ◽  
Fergal O'Gara
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Transcription Factors ◽  
Epithelial Cells ◽  
Airway Epithelial Cells ◽  
Effector Proteins ◽  
Host Cells ◽  
Airway Epithelial ◽  
Type Iii ◽  
Enhanced Expression

ABSTRACT Pseudomonas aeruginosa is an important opportunistic pathogen which is capable of causing both acute and chronic infections in immunocompromised patients. Successful adaptation of the bacterium to its host environment relies on the ability of the organism to tightly regulate gene expression. RsmA, a small RNA-binding protein, controls the expression of a large number of virulence-related genes in P. aeruginosa, including those encoding the type III secretion system and associated effector proteins, with important consequences for epithelial cell morphology and cytotoxicity. In order to examine the influence of RsmA-regulated functions in the pathogen on gene expression in the host, we compared global expression profiles of airway epithelial cells in response to infection with P. aeruginosa PAO1 and an rsmA mutant. The RsmA-dependent response of host cells was characterized by significant changes in the global transcriptional pattern, including the increased expression of two Kruppel-like factors, KLF2 and KLF6. This increased expression was mediated by specific type III effector proteins. ExoS was required for the enhanced expression of KLF2, whereas both ExoS and ExoY were required for the enhanced expression of KLF6. Neither ExoT nor ExoU influenced the expression of the transcription factors. Additionally, the increased gene expression of KLF2 and KLF6 was associated with ExoS-mediated cytotoxicity. Therefore, this study identifies for the first time the human transcription factors KLF2 and KLF6 as targets of the P. aeruginosa type III exoenzymes S and Y, with potential importance in host cell death.

scholarly journals Transcriptome Analysis of Pseudomonas aeruginosa after Interaction with Human Airway Epithelial Cells

2004 ◽  
Vol 72 (9) ◽  
pp. 5433-5438 ◽  
Author(s):  
Anders Frisk ◽  
Jill R. Schurr ◽  
Guoshun Wang ◽  
Donna C. Bertucci ◽  
Luis Marrero ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Epithelial Cells ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Human Airway ◽  
Normal Human ◽  
Human Airway Epithelial Cells

ABSTRACT The transcriptional profile of Pseudomonas aeruginosa after interactions with primary normal human airway epithelial cells was determined using Affymetrix GeneChip technology. Gene expression profiles indicated that various genes involved in phosphate acquisition and iron scavenging were differentially regulated.

NF-κB activation and sustained IL-8 gene expression in primary cultures of cystic fibrosis airway epithelial cells stimulated with Pseudomonas aeruginosa

2005 ◽  
Vol 288 (3) ◽  
pp. L471-L479 ◽  
Author(s):  
Theresa Joseph ◽  
Dwight Look ◽  
Thomas Ferkol
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Epithelial Cells ◽  
Inflammatory Response ◽  
Cell Cultures ◽  
Primary Cultures ◽  
Airway Epithelial Cells ◽  
Mrna Levels ◽  
Airway Epithelial

The progression of lung disease in cystic fibrosis (CF) is characterized by an exuberant inflammatory response mounted by the respiratory epithelium that is further exacerbated by bacterial infection. Recent studies have demonstrated upregulation of nuclear factor-κB (NF-κB) in response to infection in genetically modified cell culture models, which is associated with expression of interleukin (IL)-8. Using human airway epithelial cells grown in primary culture, we examined in vitro activation of NF-κB in cells isolated from five CF (ΔF508/ΔF508) and three non-CF (NCF) patients in response to Pseudomonas aeruginosa. Immunofluorescence, gel-shift, and immunoblot assays demonstrated a rapid translocation of NF-κB subunits (p50 and p65) to the nucleus in both CF and NCF cell cultures. However, nuclear extracts from CF cells both before and following P. aeruginosa stimulation revealed elevated NF-κB activation compared with NCF cells. Additionally, elevated nuclear levels of the NF-κB inhibitor IκBα were detected in nuclei of CF cells after P. aeruginosa stimulation, but this increase was transient. There was no difference in IL-8 mRNA levels between CF and NCF cells early after stimulation, whereas expression was higher and sustained in CF cells at later times. Our results also demonstrated increased baseline translocation of NF-κB to nuclei of primary CF epithelial cell cultures, but intranuclear IκBα may initially block its effects following P. aeruginosa stimulation. Thus, IL-8 mRNA expression was prolonged after P. aeruginosa stimulation in CF epithelial cells, and this sustained IL-8 expression may contribute to the excessive inflammatory response in CF.

scholarly journals Pseudomonas aeruginosa Inhibition of Flagellin-Activated NF-κB and Interleukin-8 by Human Airway Epithelial Cells

2009 ◽  
Vol 77 (7) ◽  
pp. 2857-2865 ◽  
Author(s):  
Jose Pena ◽  
Zhu Fu ◽  
Christian Schwarzer ◽  
Terry E. Machen
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Epithelial Cells ◽  
Conditioned Medium ◽  
Primary Cultures ◽  
Airway Epithelial Cells ◽  
Interleukin 8 ◽  
Airway Epithelial ◽  
Human Airway ◽  
Basolateral Side ◽  
Human Airway Epithelial Cells

ABSTRACT Pseudomonas aeruginosa-induced activation of NF-κB and secretion of proinflammatory cytokines by airway epithelial cells require that the bacteria express flagellin. We tested whether P. aeruginosa and human airway epithelial cells secrete factors that modulated this response. Experiments were performed with both the Calu-3 cell line and primary cultures of tracheal epithelial cells. P. aeruginosa strain PAK ΔfliC (flagellin knockout) did not activate NF-κB or interleukin-8 (IL-8) but inhibited flagellin-activated NF-κB by 40 to 50% and IL-8 secretion by 20 to 25%. PAK ΔfliC also inhibited NF-κB induced by IL-1β and Toll-like receptor 2 agonist Pam3CSK4. Similar inhibitions were observed with strains PAK, PAO1, and PA14. The inhibitory factor was present in conditioned medium isolated from PAK ΔfliC or Calu-3 plus PAK ΔfliC, but it was not present in conditioned medium isolated from Calu-3 cells alone or from PAK ΔfliC that had been heat treated. Inhibition by PAK ΔfliC-conditioned medium was exerted from either the apical or the basolateral side of the epithelium, was enhanced in simple Ringer's solution over that in tissue culture medium, and did not result from altered pH or depletion of glucose. The inhibitory effect of conditioned medium was abolished by boiling and appeared from filtration studies to result from effects of a factor with a molecular mass of <3 kDa. These and further studies with isogenic mutants led to the conclusion that the NF-κB and IL-8 response of airway epithelial cells to P. aeruginosa results from a balance of proinflammatory effects of flagellin and antiinflammatory effects of a small (<3-kDa), heat-sensitive factor(s) that is not lipopolysaccharide, C12 homoserine lactone, alginate, CIF, or exotoxin A, S, T, U, or Y.

scholarly journals Somatic cell hemoglobin modulates nitrogen oxide metabolism in the human airway epithelium

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nadzeya Marozkina ◽  
Laura Smith ◽  
Yi Zhao ◽  
Joe Zein ◽  
James F. Chmiel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Nitrogen Oxides ◽  
Airway Epithelium ◽  
Airflow Obstruction ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Human Airway ◽  
Human Airway Epithelium ◽  
Human Airway Epithelial Cells

AbstractEndothelial hemoglobin (Hb)α regulates endothelial nitric oxide synthase (eNOS) biochemistry. We hypothesized that Hb could also be expressed and biochemically active in the ciliated human airway epithelium. Primary human airway epithelial cells, cultured at air–liquid interface (ALI), were obtained by clinical airway brushings or from explanted lungs. Human airway Hb mRNA data were from publically available databases; or from RT-PCR. Hb proteins were identified by immunoprecipitation, immunoblot, immunohistochemistry, immunofluorescence and liquid chromatography- mass spectrometry. Viral vectors were used to alter Hbβ expression. Heme and nitrogen oxides were measured colorimetrically. Hb mRNA was expressed in human ciliated epithelial cells. Heme proteins (Hbα, β, and δ) were detected in ALI cultures by several methods. Higher levels of airway epithelial Hbβ gene expression were associated with lower FEV1 in asthma. Both Hbβ knockdown and overexpression affected cell morphology. Hbβ and eNOS were apically colocalized. Binding heme with CO decreased extracellular accumulation of nitrogen oxides. Human airway epithelial cells express Hb. Higher levels of Hbβ gene expression were associated with airflow obstruction. Hbβ and eNOS were colocalized in ciliated cells, and heme affected oxidation of the NOS product. Epithelial Hb expression may be relevant to human airways diseases.

scholarly journals Individual Matrix Metalloproteinases Control Distinct Transcriptional Responses in Airway Epithelial Cells Infected with Pseudomonas aeruginosa

2007 ◽  
Vol 75 (12) ◽  
pp. 5640-5650 ◽  
Author(s):  
Sean Y. Kassim ◽  
Sina A. Gharib ◽  
Brigham H. Mecham ◽  
Timothy P. Birkland ◽  
William C. Parks ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Initial Point ◽  
Global Gene Expression ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Transcriptional Responses ◽  
Matrix Metalloproteinase 7 ◽  
Node Network ◽  
Global Gene Expression Analysis

ABSTRACT Airway epithelium is the initial point of host-pathogen interaction in Pseudomonas aeruginosa infection, an important pathogen in cystic fibrosis and nosocomial pneumonia. We used global gene expression analysis to determine airway epithelial transcriptional responses dependent on matrilysin (matrix metalloproteinase 7 [MMP-7]) and stromelysin-2 (MMP-10), two MMPs induced by acute P. aeruginosa pulmonary infection. Extraction of differential gene expression (EDGE) analysis of gene expression changes in P. aeruginosa-infected organotypic tracheal epithelial cell cultures from wild-type, Mmp7 −/−, and Mmp10 −/− mice identified 2,091 matrilysin-dependent and 1,628 stromelysin-2-dependent genes that were differentially expressed. Key node network analysis showed that these MMPs controlled distinct gene expression programs involved in proliferation, cell death, immune responses, and signal transduction, among other host defense processes. Our results demonstrate discrete roles for these MMPs in regulating epithelial responses to Pseudomonas infection and show that a global genomics strategy can be used to assess MMP function.

scholarly journals Pseudomonas aeruginosa LPS or Flagellin Are Sufficient to Activate TLR-Dependent Signaling in Murine Alveolar Macrophages and Airway Epithelial Cells

PLoS ONE ◽  
2009 ◽  
Vol 4 (10) ◽  
pp. e7259 ◽  
Author(s):  
Eloïse Raoust ◽  
Viviane Balloy ◽  
Ignacio Garcia-Verdugo ◽  
Lhousseine Touqui ◽  
Reuben Ramphal ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Epithelial Cells ◽  
Alveolar Macrophages ◽  
Airway Epithelial Cells ◽  
Airway Epithelial

Exploring host-pathogen interactions at the epithelial surface: application of transcriptomics in lung biology

2007 ◽  
Vol 292 (2) ◽  
pp. L367-L377 ◽  
Author(s):  
Joost B. Vos ◽  
Nicole A. Datson ◽  
Klaus F. Rabe ◽  
Pieter S. Hiemstra
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Large Scale ◽  
Brain Research ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Host Pathogen Interactions ◽  
Epithelial Surface ◽  
Complex Interactions ◽  
Host Pathogen

The epithelial surface of the airways is the largest barrier-forming interface between the human body and the outside world. It is now well recognized that, at this strategic position, airway epithelial cells play an eminent role in host defense by recognizing and responding to microbial exposure. Conversely, inhaled microorganisms also respond to contact with epithelial cells. Our understanding of this cross talk is limited, requiring sophisticated experimental approaches to analyze these complex interactions. High-throughput technologies, such as DNA microarray analysis and serial analysis of gene expression (SAGE), have been developed to screen for gene expression levels at large scale within single experiments. Since their introduction, these hypothesis-generating technologies have been widely used in diverse areas such as oncology and brain research. Successful application of these genomics-based technologies has also revealed novel insights in host-pathogen interactions in both the host and pathogen. This review aims to provide an overview of the SAGE and microarray technology illustrated by their application in the analysis of host-pathogen interactions. In particular, the interactions between epithelial cells in the human lungs and clinically relevant microorganisms are the central focus of this review.

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