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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

NTHi release outer membrane vesicles while co-cultured with EpiAirway tissues. (a) TEM of strain R2866 after a five-day co-culture with EpiAirway tissues. The bacteria are paracellular and are releasing OMVs (arrows), some of which appear to interact with the host cell membrane. (b) IEM which shows strain 86-028NP co-cultured with EpiAirway tissues for five days releasing OMVs that react with anti-NTHi antiserum (arrows) while located paracellularly. NTHi, non-typeable Haemophilus influenzae; TEM, transmission electron microscopy; IEM, immunoelectron microscopy
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EBM-1111-RM-377F6: NTHi release outer membrane vesicles while co-cultured with EpiAirway tissues. (a) TEM of strain R2866 after a five-day co-culture with EpiAirway tissues. The bacteria are paracellular and are releasing OMVs (arrows), some of which appear to interact with the host cell membrane. (b) IEM which shows strain 86-028NP co-cultured with EpiAirway tissues for five days releasing OMVs that react with anti-NTHi antiserum (arrows) while located paracellularly. NTHi, non-typeable Haemophilus influenzae; TEM, transmission electron microscopy; IEM, immunoelectron microscopy

Mentions: We observed that both strains R2866 and 86-028NP released outer membrane vesicles (OMVs) during long-term co-culture with EpiAirway tissues (Figure 6). The vesicles appeared to be shed when the organisms were paracellular, with some vesicles associating with the lateral membrane of the epithelial cells (Figure 6a). These OMVs were labeled with anti-NTHi antiserum and were identified by IEM, indicating that they originated from the NTHi strains (Figure 5c, arrowhead; Figure 6b, arrows). The role of these OMVs during long-term infection is currently under investigation.Figure 6


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)

NTHi release outer membrane vesicles while co-cultured with EpiAirway tissues. (a) TEM of strain R2866 after a five-day co-culture with EpiAirway tissues. The bacteria are paracellular and are releasing OMVs (arrows), some of which appear to interact with the host cell membrane. (b) IEM which shows strain 86-028NP co-cultured with EpiAirway tissues for five days releasing OMVs that react with anti-NTHi antiserum (arrows) while located paracellularly. NTHi, non-typeable Haemophilus influenzae; TEM, transmission electron microscopy; IEM, immunoelectron microscopy
© Copyright Policy - open-access
Related In: Results  -  Collection

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

EBM-1111-RM-377F6: NTHi release outer membrane vesicles while co-cultured with EpiAirway tissues. (a) TEM of strain R2866 after a five-day co-culture with EpiAirway tissues. The bacteria are paracellular and are releasing OMVs (arrows), some of which appear to interact with the host cell membrane. (b) IEM which shows strain 86-028NP co-cultured with EpiAirway tissues for five days releasing OMVs that react with anti-NTHi antiserum (arrows) while located paracellularly. NTHi, non-typeable Haemophilus influenzae; TEM, transmission electron microscopy; IEM, immunoelectron microscopy
Mentions: We observed that both strains R2866 and 86-028NP released outer membrane vesicles (OMVs) during long-term co-culture with EpiAirway tissues (Figure 6). The vesicles appeared to be shed when the organisms were paracellular, with some vesicles associating with the lateral membrane of the epithelial cells (Figure 6a). These OMVs were labeled with anti-NTHi antiserum and were identified by IEM, indicating that they originated from the NTHi strains (Figure 5c, arrowhead; Figure 6b, arrows). The role of these OMVs during long-term infection is currently under investigation.Figure 6

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