scholarly journals Expression of the Nontypeable Haemophilus influenzae Type IV Pilus Is Stimulated by Coculture with Host Respiratory Tract Epithelial Cells

2019 ◽  
Vol 87 (12) ◽  
Author(s):  
Elaine M. Mokrzan ◽  
Taylor J. Johnson ◽  
Lauren O. Bakaletz
Keyword(s):  
Epithelial Cells ◽  
Respiratory Tract ◽  
Haemophilus Influenzae ◽  
Promoter Activity ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Type Iv Pilus ◽  
Human Airway ◽  
Type Iv ◽  
Pilin Protein

ABSTRACT The type IV pilus (Tfp) of nontypeable Haemophilus influenzae (NTHI) mediates adherence, colonization, motility, and biofilm formation, and the major protein subunit, PilA, is a promising vaccine candidate. Thus, it is crucial to understand how Tfp expression is regulated within the microenvironments of the human nasopharynx, which NTHI colonizes asymptomatically, and the more distal regions of the respiratory tract where NTHI-induced diseases occur. Here, we examined the effects of coculture of NTHI with human airway epithelial cells and heme availability on Tfp expression at temperatures typical of the human nasopharynx (34°C) or warmer anatomical sites during infection (37°C). Tfp expression was estimated by pilA promoter activity, pilA gene expression, and relative abundances of PilA and pilin protein. The results revealed that at both temperatures, NTHI cocultured with airway epithelial cells demonstrated significantly greater expression of pilA, PilA/pilin protein, and likely, fully assembled Tfp than NTHI cultured on an abiotic surface. Because NTHI is a heme auxotroph, we hypothesized that availability of heme from host cells might be a signal for Tfp expression. Thereby, we cultured NTHI in iron-limited medium, and we observed that supplementation with heme significantly increased pilA promoter activity. Collectively, our data suggested that NTHI Tfp expression was stimulated by soluble factor(s) released by epithelial cells, which are present in all microenvironments of the respiratory tract. The expression of this target antigen under conditions that mimic the human airway strongly supports the rationale for the use of PilA as a vaccine immunogen to prevent NTHI-induced diseases of the respiratory tract.

scholarly journals Nontypeable Haemophilus influenzae Type IV Pilus Mediates Augmented Adherence to Rhinovirus-Infected Human Airway Epithelial Cells

2020 ◽  
Vol 88 (9) ◽  
Author(s):  
Stephen L. Toone ◽  
Michelle Ratkiewicz ◽  
Laura A. Novotny ◽  
Binh L. Phong ◽  
Lauren O. Bakaletz
Keyword(s):  
Epithelial Cells ◽  
Respiratory Tract ◽  
Haemophilus Influenzae ◽  
Host Cell ◽  
Bacterial Load ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Upper Respiratory Tract ◽  
Type Iv ◽  
Upper Respiratory

ABSTRACT Human rhinovirus (hRV) is frequently detected in the upper respiratory tract, and symptomatic infection is associated with an increased nasopharyngeal bacterial load, with subsequent development of secondary bacterial diseases. Nontypeable Haemophilus influenzae (NTHI) is a commensal bacterial species of the human nasopharynx; however, in the context of prior or concurrent upper respiratory tract viral infection, this bacterium commonly causes multiple diseases throughout the upper and lower respiratory tracts. The present study was conducted to determine the mechanism(s) by which hRV infection promotes the development of NTHI-induced diseases. We showed that hRV infection of polarized primary human airway epithelial cells resulted in increased adherence of NTHI, due in part to augmented expression of CEACAM1 and ICAM1, host cell receptors to which NTHI binds via engagement of multiple adhesins. Antibody blockade of these host cell receptors significantly reduced NTHI adherence. With a specific focus on the NTHI type IV pilus (T4P), which we have previously shown binds to ICAM1, an essential adhesin and virulence determinant, we next showed that T4P-directed antibody blockade significantly reduced NTHI adherence to hRV-infected airway cells and, further, that expression of this adhesin was required for the enhanced adherence observed. Collectively, these data provide a mechanism by which “the common cold” promotes diseases due to NTHI, and they add further support for the use of PilA (the majority subunit of T4P) as a vaccine antigen, since antibodies directed against PilA are expected to limit the notably increased bacterial load associated with hRV coinfection and thereby to prevent secondary NTHI-induced diseases of the respiratory tract.

Zn2+-induced IL-8 expression involves AP-1, JNK, and ERK activities in human airway epithelial cells

2006 ◽  
Vol 290 (5) ◽  
pp. L1028-L1035 ◽  
Author(s):  
Yu-Mee Kim ◽  
William Reed ◽  
Weidong Wu ◽  
Philip A. Bromberg ◽  
Lee M. Graves ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Protein Expression ◽  
Promoter Activity ◽  
Fluorescent Protein ◽  
Airway Epithelial Cells ◽  
Epithelial Cell Line ◽  
Airway Epithelial ◽  
Human Airway ◽  
Mrna And Protein Expression ◽  
Human Airway Epithelial Cells

Exposure to zinc-laden particulate matter in ambient and occupational settings has been associated with proinflammatory responses in the lung. IL-8 is an important proinflammatory cytokine in the human lung and is induced in human airway epithelial cells exposed to zinc. In this study, we examined the cellular mechanisms responsible for Zn2+-induced IL-8 expression. Zn2+ stimulation resulted in pronounced increases in both IL-8 mRNA and protein expression in the human airway epithelial cell line (BEAS-2B). IL-8 promoter activity was significantly increased by Zn2+ exposure in BEAS-2B cells, indicating that Zn2+-induced IL-8 expression is transcriptionally mediated. Mutation of the activating protein (AP)-1 response element in an IL-8 promoter-enhanced green fluorescent protein construct reduced Zn2+-induced IL-8 promoter activity. Moreover, Zn2+ exposure of BEAS-2B cells induced the phosphorylation of the AP-1 proteins c-Fos and c-Jun. We observed that Zn2+ exposure induced the phosphorylation of ERK, JNK, and p38 MAPKs, whereas inhibition of ERK or JNK activity blocked IL-8 mRNA and protein expression in BEAS-2B cells treated with Zn2+. In addition, we investigated the role of protein tyrosine phosphatases in the activation of signaling by Zn2+. Zn2+ treatment inhibited ERK- and JNK-directed phosphatase activities in BEAS-2B cells. These results suggested that Zn2+-induced inhibition of phosphatase activity is an initiating event in MAPK and AP-1 activation that leads to enhanced IL-8 expression by human airway epithelial cells.

scholarly journals Nontypeable Haemophilus influenzae Responds to Virus-Infected Cells with a Significant Increase in Type IV Pilus Expression

mSphere ◽  
2020 ◽  
Vol 5 (3) ◽  
Author(s):  
Elaine M. Mokrzan ◽  
Kolapo A. Dairo ◽  
Laura A. Novotny ◽  
Lauren O. Bakaletz
Keyword(s):  
Epithelial Cells ◽  
Virus Infection ◽  
Respiratory Tract ◽  
Haemophilus Influenzae ◽  
Chronic Obstructive ◽  
Upper Respiratory Tract ◽  
Type Iv Pilus ◽  
Content Type ◽  
Type Iv ◽  
Upper Respiratory

ABSTRACT Nontypeable Haemophilus influenzae (NTHI) colonizes the human nasopharynx, but when the host immune response is dysregulated by upper respiratory tract (URT) virus infection, NTHI can gain access to more distal airway sites and cause disease. The NTHI type IV pilus (T4P) facilitates adherence, benign colonization, and infection, and its majority subunit PilA is in clinical trials as a vaccinogen. To further validate the strategy of immunization with PilA against multiple NTHI-induced diseases, it is important to demonstrate T4P expression under microenvironmental conditions that predispose to NTHI infection of the airway. Because URT infection commonly facilitates NTHI-induced diseases, we examined the influence of ongoing virus infection of respiratory tract epithelial cells on NTHI T4P expression in vitro. Polarized primary human airway epithelial cells (HAEs) were sequentially inoculated with one of three common URT viruses, followed by NTHI. Use of a reporter construct revealed that NTHI upregulated pilA promoter activity when cultured with HAEs infected with adenovirus (AV), respiratory syncytial virus (RSV), or rhinovirus (RV) versus that in mock-infected HAEs. Consistent with these results, pilA expression and relative PilA/pilin abundance, as assessed by quantitative reverse transcription-PCR (qRT-PCR) and immunoblot, respectively, were also significantly increased when NTHI was cultured with virus-infected HAEs. Collectively, our data strongly suggest that under conditions of URT virus infection, PilA vaccinogen induction of T4P-directed antibodies is likely to be highly effective against multiple NTHI-induced diseases by interfering with T4P-mediated adherence. We hypothesize that this outcome could thereby limit or prevent the increased load of NTHI in the nasopharynx that characteristically precedes these coinfections. IMPORTANCE Nontypeable Haemophilus influenzae (NTHI) is the predominant bacterial causative agent of many chronic and recurrent diseases of the upper and lower respiratory tracts. NTHI-induced chronic rhinosinusitis, otitis media, and exacerbations of cystic fibrosis and chronic obstructive pulmonary disease often develop during or just after an upper respiratory tract viral infection. We have developed a vaccine candidate immunogen for NTHI-induced diseases that targets the majority subunit (PilA) of the type IV twitching pilus (T4P), which NTHI uses to adhere to respiratory tract epithelial cells and that also plays a role in disease. Here, we showed that NTHI cocultured with virus-infected respiratory tract epithelial cells express significantly more of the vaccine-targeted T4P than NTHI that encounters mock-infected (healthy) cells. These results strongly suggest that a vaccine strategy that targets the NTHI T4P will be effective under the most common predisposing condition: when the human host has a respiratory tract virus infection.

scholarly journals Infection of Primary Human Bronchial Epithelial Cells by Haemophilus influenzae: Macropinocytosis as a Mechanism of Airway Epithelial Cell Entry

1999 ◽  
Vol 67 (8) ◽  
pp. 4161-4170 ◽  
Author(s):  
Margaret R. Ketterer ◽  
Jian Q. Shao ◽  
Douglas B. Hornick ◽  
Ben Buscher ◽  
Venkata K. Bandi ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Haemophilus Influenzae ◽  
Actin Polymerization ◽  
Microscopic Analysis ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Cortical Actin ◽  
Electron Microscopic ◽  
Human Airway ◽  
Human Airway Epithelial Cells

ABSTRACT Nontypeable Haemophilus influenzae is an exclusive human pathogen which infects the respiratory epithelium. We have initiated studies to explore the interaction of the nontypeableH. influenzae strain 2019 with primary human airway epithelial cells by electron and confocal microscopy. Primary human airway cell cultures were established as monolayers on glass collagen-coated coverslips or on semipermeable membranes at an air-fluid interface. Scanning electron microscopy indicated that bacteria adhered to nonciliated cells in the population. The surface of infected cells showed evidence of cytoskeletal rearrangements manifested by microvilli and lamellipodia extending toward and engaging bacteria. Confocal microscopic analysis demonstrated that infection induced actin polymerization with an increase in cortical actin as well as evidence of actin strands around the bacteria. Transmission electron microscopic analysis showed lamellipodia and microvilli surrounding organisms, as well as organisms adherent to the cell surface. These studies also demonstrated the presence of bacteria within vacuoles inside of airway cells. Confocal microscopic studies with Texas red-labeled dextran (molecular weight, 70,000) indicated that H. influenzae cells were entering cells by the process of macropinocytosis. These studies indicate that nontypeable H. influenzae can initiate cytoskeletal rearrangement within human airway epithelium, resulting in internalization of the bacteria within nonciliated human airway epithelial cells by the process of macropinocytosis.

scholarly journals In Vitro Modelling of Respiratory Virus Infections in Human Airway Epithelial Cells – A Systematic Review

2021 ◽  
Vol 12 ◽  
Author(s):  
Laurine C. Rijsbergen ◽  
Laura L. A. van Dijk ◽  
Maarten F. M. Engel ◽  
Rory D. de Vries ◽  
Rik L. de Swart
Keyword(s):  
Cell Culture ◽  
Epithelial Cells ◽  
Respiratory Tract ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Culture Systems ◽  
Human Airway ◽  
Cell Culture Systems ◽  
Immortalized Cell

Respiratory tract infections (RTI) are a major cause of morbidity and mortality in humans. A large number of RTIs is caused by viruses, often resulting in more severe disease in infants, elderly and the immunocompromised. Upon viral infection, most individuals experience common cold-like symptoms associated with an upper RTI. However, in some cases a severe and sometimes life-threatening lower RTI may develop. Reproducible and scalable in vitro culture models that accurately reflect the human respiratory tract are needed to study interactions between respiratory viruses and the host, and to test novel therapeutic interventions. Multiple in vitro respiratory cell culture systems have been described, but the majority of these are based on immortalized cell lines. Although useful for studying certain aspects of viral infections, such monomorphic, unicellular systems fall short in creating an understanding of the processes that occur at an integrated tissue level. Novel in vitro models involving primary human airway epithelial cells and, more recently, human airway organoids, are now in use. In this review, we describe the evolution of in vitro cell culture systems and their characteristics in the context of viral RTIs, starting from advances after immortalized cell cultures to more recently developed organoid systems. Furthermore, we describe how these models are used in studying virus-host interactions, e.g. tropism and receptor studies as well as interactions with the innate immune system. Finally, we provide an outlook for future developments in this field, including co-factors that mimic the microenvironment in the respiratory tract.

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