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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

Transepithelial electrical resistance (TER) of EpiAirway (a) and ALI NHBE (b) cell cultures. (a) EpiAirway cultures were inoculated with strain R2846 and the TER was measured during co-culture. (b) NHBE cell cultures at the ALI were inoculated with strain R2846 and the TER was measured during co-culture. Horizontal axes denote time after inoculation for each reading. Error bars are SD. ALI, air–liquid interface; NHBE, normal human bronchial epithelial
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EBM-1111-RM-377F3: Transepithelial electrical resistance (TER) of EpiAirway (a) and ALI NHBE (b) cell cultures. (a) EpiAirway cultures were inoculated with strain R2846 and the TER was measured during co-culture. (b) NHBE cell cultures at the ALI were inoculated with strain R2846 and the TER was measured during co-culture. Horizontal axes denote time after inoculation for each reading. Error bars are SD. ALI, air–liquid interface; NHBE, normal human bronchial epithelial

Mentions: After infection, the TER, a measure of functional tight junctions, was quantified over time in both EpiAirway tissues (Figure 3a) and ALI NHBE cells (Figure 3b) infected with strain R2846. Average TER values expressed in Ω cm2 are reported. In both models, no significant decrease in TER was observed over the course of the co-culture with NTHi. In a separate experiment, the TER of two ALI NHBE cell cultures was measured prior to infection with strain R2846, and again eight days after inoculation. To disrupt the TER in these infected ALI NHBE cell cultures, 0.2 mL of 0.02% ethylene glycol tetraacetic acid was added to the apical surface of each insert for four hours. The TERs decreased from more than 400 to 20 and 22 Ω cm2, respectively, indicating that the measured electrical resistance was due to functional tight junctions. These data support the SEM and TEM findings and confirm that extended co-culture with NTHi does not disturb epithelial cell tight junction integrity.Figure 3


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)

Transepithelial electrical resistance (TER) of EpiAirway (a) and ALI NHBE (b) cell cultures. (a) EpiAirway cultures were inoculated with strain R2846 and the TER was measured during co-culture. (b) NHBE cell cultures at the ALI were inoculated with strain R2846 and the TER was measured during co-culture. Horizontal axes denote time after inoculation for each reading. Error bars are SD. ALI, air–liquid interface; NHBE, normal human bronchial epithelial
© Copyright Policy - open-access
Related In: Results  -  Collection

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

EBM-1111-RM-377F3: Transepithelial electrical resistance (TER) of EpiAirway (a) and ALI NHBE (b) cell cultures. (a) EpiAirway cultures were inoculated with strain R2846 and the TER was measured during co-culture. (b) NHBE cell cultures at the ALI were inoculated with strain R2846 and the TER was measured during co-culture. Horizontal axes denote time after inoculation for each reading. Error bars are SD. ALI, air–liquid interface; NHBE, normal human bronchial epithelial
Mentions: After infection, the TER, a measure of functional tight junctions, was quantified over time in both EpiAirway tissues (Figure 3a) and ALI NHBE cells (Figure 3b) infected with strain R2846. Average TER values expressed in Ω cm2 are reported. In both models, no significant decrease in TER was observed over the course of the co-culture with NTHi. In a separate experiment, the TER of two ALI NHBE cell cultures was measured prior to infection with strain R2846, and again eight days after inoculation. To disrupt the TER in these infected ALI NHBE cell cultures, 0.2 mL of 0.02% ethylene glycol tetraacetic acid was added to the apical surface of each insert for four hours. The TERs decreased from more than 400 to 20 and 22 Ω cm2, respectively, indicating that the measured electrical resistance was due to functional tight junctions. These data support the SEM and TEM findings and confirm that extended co-culture with NTHi does not disturb epithelial cell tight junction integrity.Figure 3

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