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Characterization of extended co-culture of non-typeable Haemophilus influenzae with primary human respiratory tissues.

Ren D, Nelson KL, Uchakin PN, Smith AL, Gu XX, Daines DA - Exp. Biol. Med. (Maywood) (2012)

Bottom Line: These infections often recur and can become chronic.Scanning and transmission electron microscopy of tissues confirmed that intact NTHi were persisting paracellularly, while organisms observed in intracellular vacuoles appeared degraded.Furthermore, the apical surface and tight junctions of the infected tissues were undisturbed, with high transepithelial electrical resistances, while the basal cell layer displayed more junctional disorganization and wider intercellular spaces than the uninfected control tissues.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Medical Sciences, Mercer University School of Medicine, 1550 College Street, Macon, GA 31207, USA.

ABSTRACT
Non-typeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that comprise part of the normal flora of the human upper airway, but are also responsible for a number of mucosal infections such as otitis media and bronchitis. These infections often recur and can become chronic. To characterize the effect of long-term co-culture of NTHi with human tissues, we infected primary respiratory epithelial cells grown at the air-liquid interface with three NTHi strains over a range of 1-10 days. Scanning and transmission electron microscopy of tissues confirmed that intact NTHi were persisting paracellularly, while organisms observed in intracellular vacuoles appeared degraded. Furthermore, the apical surface and tight junctions of the infected tissues were undisturbed, with high transepithelial electrical resistances, while the basal cell layer displayed more junctional disorganization and wider intercellular spaces than the uninfected control tissues. Although the tissues elaborated the cytokine profile reported for NTHi-caused otitis media in vivo, there was little change in the dynamics of cytokine secretion over the time points tested. Finally, we report that NTHi strains released outer membrane vesicles (OMVs) during extended co-culture with the tissues, and show that these OMVs directly interact with host cell membranes.

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Related in: MedlinePlus

Cytokine profile of EpiAirway inserts infected with strain 86-028NP over time. ELISA assays were performed to detect the levels of IL-1β, IL-6, TNF-α, GM-CSF and IL-8 in pooled EpiAirway washes (n = 2) during co-culture. Day 0 indicates assays prior to inoculation, and the following days indicate length of time of co-culture. IL, interleukin; GM-CSF, granulocyte/macrophage colony-stimulating factor; ELISA, enzyme-linked immunosorbent assay; TNF, tumor necrosis factor
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EBM-1111-RM-377F4: Cytokine profile of EpiAirway inserts infected with strain 86-028NP over time. ELISA assays were performed to detect the levels of IL-1β, IL-6, TNF-α, GM-CSF and IL-8 in pooled EpiAirway washes (n = 2) during co-culture. Day 0 indicates assays prior to inoculation, and the following days indicate length of time of co-culture. IL, interleukin; GM-CSF, granulocyte/macrophage colony-stimulating factor; ELISA, enzyme-linked immunosorbent assay; TNF, tumor necrosis factor

Mentions: To determine whether the ALI tissues expressed the epithelial cell cytokine profile during long-term infections with NTHi in vitro that has been reported during infections in vivo,17 ELISA assays were performed on the pooled apical washes of EpiAirway tissues co-cultured with strain 86-028NP at day 0 (prior to infection) and at two, four and six days after infection (Figure 4). Interleukin-8 (IL-8) was the predominant cytokine, followed by IL-6, IL-1 and granulocyte/macrophage colony-stimulating factor (GM-CSF). Uninfected EpiAirway tissues produced approximately 200 ng/mL of IL-8 (Figure 4, day 0 time point), as airway epithelium produced IL-8 on a constitutive basis.18 No significant amounts of IL-2, IL-4, IL-10, IL-12p40, IL-17 or interferon-γ were detected in the samples. Further, there were no statistically significant changes in the dynamics of the secretion of tumor necrosis factor-α, IL-1β, IL-6, IL-8, and GM-CSF in the apical washes over the time points tested. This is consistent with a previous study showing that the mRNA levels of proinflammatory cytokines elicited by NTHi infections in vivo peaked at 3–6 hours after infection, and prior to the first clinical signs of otitis media.19Figure 4


Characterization of extended co-culture of non-typeable Haemophilus influenzae with primary human respiratory tissues.

Ren D, Nelson KL, Uchakin PN, Smith AL, Gu XX, Daines DA - Exp. Biol. Med. (Maywood) (2012)

Cytokine profile of EpiAirway inserts infected with strain 86-028NP over time. ELISA assays were performed to detect the levels of IL-1β, IL-6, TNF-α, GM-CSF and IL-8 in pooled EpiAirway washes (n = 2) during co-culture. Day 0 indicates assays prior to inoculation, and the following days indicate length of time of co-culture. IL, interleukin; GM-CSF, granulocyte/macrophage colony-stimulating factor; ELISA, enzyme-linked immunosorbent assay; TNF, tumor necrosis factor
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3369423&req=5

EBM-1111-RM-377F4: Cytokine profile of EpiAirway inserts infected with strain 86-028NP over time. ELISA assays were performed to detect the levels of IL-1β, IL-6, TNF-α, GM-CSF and IL-8 in pooled EpiAirway washes (n = 2) during co-culture. Day 0 indicates assays prior to inoculation, and the following days indicate length of time of co-culture. IL, interleukin; GM-CSF, granulocyte/macrophage colony-stimulating factor; ELISA, enzyme-linked immunosorbent assay; TNF, tumor necrosis factor
Mentions: To determine whether the ALI tissues expressed the epithelial cell cytokine profile during long-term infections with NTHi in vitro that has been reported during infections in vivo,17 ELISA assays were performed on the pooled apical washes of EpiAirway tissues co-cultured with strain 86-028NP at day 0 (prior to infection) and at two, four and six days after infection (Figure 4). Interleukin-8 (IL-8) was the predominant cytokine, followed by IL-6, IL-1 and granulocyte/macrophage colony-stimulating factor (GM-CSF). Uninfected EpiAirway tissues produced approximately 200 ng/mL of IL-8 (Figure 4, day 0 time point), as airway epithelium produced IL-8 on a constitutive basis.18 No significant amounts of IL-2, IL-4, IL-10, IL-12p40, IL-17 or interferon-γ were detected in the samples. Further, there were no statistically significant changes in the dynamics of the secretion of tumor necrosis factor-α, IL-1β, IL-6, IL-8, and GM-CSF in the apical washes over the time points tested. This is consistent with a previous study showing that the mRNA levels of proinflammatory cytokines elicited by NTHi infections in vivo peaked at 3–6 hours after infection, and prior to the first clinical signs of otitis media.19Figure 4

Bottom Line: These infections often recur and can become chronic.Scanning and transmission electron microscopy of tissues confirmed that intact NTHi were persisting paracellularly, while organisms observed in intracellular vacuoles appeared degraded.Furthermore, the apical surface and tight junctions of the infected tissues were undisturbed, with high transepithelial electrical resistances, while the basal cell layer displayed more junctional disorganization and wider intercellular spaces than the uninfected control tissues.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Medical Sciences, Mercer University School of Medicine, 1550 College Street, Macon, GA 31207, USA.

ABSTRACT
Non-typeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that comprise part of the normal flora of the human upper airway, but are also responsible for a number of mucosal infections such as otitis media and bronchitis. These infections often recur and can become chronic. To characterize the effect of long-term co-culture of NTHi with human tissues, we infected primary respiratory epithelial cells grown at the air-liquid interface with three NTHi strains over a range of 1-10 days. Scanning and transmission electron microscopy of tissues confirmed that intact NTHi were persisting paracellularly, while organisms observed in intracellular vacuoles appeared degraded. Furthermore, the apical surface and tight junctions of the infected tissues were undisturbed, with high transepithelial electrical resistances, while the basal cell layer displayed more junctional disorganization and wider intercellular spaces than the uninfected control tissues. Although the tissues elaborated the cytokine profile reported for NTHi-caused otitis media in vivo, there was little change in the dynamics of cytokine secretion over the time points tested. Finally, we report that NTHi strains released outer membrane vesicles (OMVs) during extended co-culture with the tissues, and show that these OMVs directly interact with host cell membranes.

Show MeSH
Related in: MedlinePlus