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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

Immunoelectron microscopy of EpiAirway tissues co-cultured for five days with strains 86-028NP or R2866. Following co-culture, most of the intact 86-028NP are paracellular (a, arrows). The bacteria that are intracellular appear to be degraded (boxed area). N = nucleus. (b) Magnified view of the boxed area in (a). Arrows indicate the intracellular and degraded bacteria, which react with the anti-NTHi antiserum. N = nucleus. (c) Paracellular organisms of strain R2866. Arrows indicate intact bacteria; arrowhead indicates an outer membrane vesicle (OMV) that reacts with anti-NTHi antiserum. NTHi, non-typeable Haemophilus influenzae
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EBM-1111-RM-377F5: Immunoelectron microscopy of EpiAirway tissues co-cultured for five days with strains 86-028NP or R2866. Following co-culture, most of the intact 86-028NP are paracellular (a, arrows). The bacteria that are intracellular appear to be degraded (boxed area). N = nucleus. (b) Magnified view of the boxed area in (a). Arrows indicate the intracellular and degraded bacteria, which react with the anti-NTHi antiserum. N = nucleus. (c) Paracellular organisms of strain R2866. Arrows indicate intact bacteria; arrowhead indicates an outer membrane vesicle (OMV) that reacts with anti-NTHi antiserum. NTHi, non-typeable Haemophilus influenzae

Mentions: Immunoelectron microscopy (IEM) with anti-NTHi antiserum followed by gold-labeled secondary antibodies of EpiAirway tissues after five days of infection with strains R2866 and 86-028NP demonstrated that NTHi were located in focal sites within the EpiAirway tissues, rather than existing as a uniform infection throughout the tissues (approximately 80 organisms are located between the cells shown in Figure 5a). The organisms observed within intracellular membrane-bound vacuoles appeared to be degraded, and IEM confirmed that these vacuoles contained NTHi (approximately 21 vacuoles reacted with the anti-NTHi antiserum shown in Figure 5b). The majority of the intact organisms visualized were located between the epithelial cells in the basal cell layers (Figure 5c). The paracellular location of NTHi following infection of immortalized human respiratory epithelial cells has been observed previously,20 and the presence of NTHi within intracellular vacuoles in primary cultures has also been reported.21 However, the duration of the co-culture of NTHi with the human cells for both of these studies was 30 h or less. Our data reveal that, over extended periods of co-culture with these ALI tissues, the location of intact NTHi is primarily paracellular, with intracellular vacuoles containing organisms in various stages of degradation.Figure 5


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)

Immunoelectron microscopy of EpiAirway tissues co-cultured for five days with strains 86-028NP or R2866. Following co-culture, most of the intact 86-028NP are paracellular (a, arrows). The bacteria that are intracellular appear to be degraded (boxed area). N = nucleus. (b) Magnified view of the boxed area in (a). Arrows indicate the intracellular and degraded bacteria, which react with the anti-NTHi antiserum. N = nucleus. (c) Paracellular organisms of strain R2866. Arrows indicate intact bacteria; arrowhead indicates an outer membrane vesicle (OMV) that reacts with anti-NTHi antiserum. NTHi, non-typeable Haemophilus influenzae
© Copyright Policy - open-access
Related In: Results  -  Collection

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

EBM-1111-RM-377F5: Immunoelectron microscopy of EpiAirway tissues co-cultured for five days with strains 86-028NP or R2866. Following co-culture, most of the intact 86-028NP are paracellular (a, arrows). The bacteria that are intracellular appear to be degraded (boxed area). N = nucleus. (b) Magnified view of the boxed area in (a). Arrows indicate the intracellular and degraded bacteria, which react with the anti-NTHi antiserum. N = nucleus. (c) Paracellular organisms of strain R2866. Arrows indicate intact bacteria; arrowhead indicates an outer membrane vesicle (OMV) that reacts with anti-NTHi antiserum. NTHi, non-typeable Haemophilus influenzae
Mentions: Immunoelectron microscopy (IEM) with anti-NTHi antiserum followed by gold-labeled secondary antibodies of EpiAirway tissues after five days of infection with strains R2866 and 86-028NP demonstrated that NTHi were located in focal sites within the EpiAirway tissues, rather than existing as a uniform infection throughout the tissues (approximately 80 organisms are located between the cells shown in Figure 5a). The organisms observed within intracellular membrane-bound vacuoles appeared to be degraded, and IEM confirmed that these vacuoles contained NTHi (approximately 21 vacuoles reacted with the anti-NTHi antiserum shown in Figure 5b). The majority of the intact organisms visualized were located between the epithelial cells in the basal cell layers (Figure 5c). The paracellular location of NTHi following infection of immortalized human respiratory epithelial cells has been observed previously,20 and the presence of NTHi within intracellular vacuoles in primary cultures has also been reported.21 However, the duration of the co-culture of NTHi with the human cells for both of these studies was 30 h or less. Our data reveal that, over extended periods of co-culture with these ALI tissues, the location of intact NTHi is primarily paracellular, with intracellular vacuoles containing organisms in various stages of degradation.Figure 5

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