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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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Survival of NTHi strains over time inside EpiAirway tissues and ALI NHBE cells. (a) Internalized (gentamicin-resistant) bacteria of strain 86-028NP recovered from EpiAirway tissues, 2–10 days after infection (n = 6). (b) Internalized strain R2846 recovered from ALI NHBE cells, 1–5 days after infection (n = 2). (c) Internalized strain R2846 recovered from EpiAirway tissues, 1–8 days after infection (n = 2). Error bars are SD. ALI, air–liquid interface; NHBE, normal human bronchial epithelial; NTHi, non-typeable Haemophilus influenzae
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EBM-1111-RM-377F1: Survival of NTHi strains over time inside EpiAirway tissues and ALI NHBE cells. (a) Internalized (gentamicin-resistant) bacteria of strain 86-028NP recovered from EpiAirway tissues, 2–10 days after infection (n = 6). (b) Internalized strain R2846 recovered from ALI NHBE cells, 1–5 days after infection (n = 2). (c) Internalized strain R2846 recovered from EpiAirway tissues, 1–8 days after infection (n = 2). Error bars are SD. ALI, air–liquid interface; NHBE, normal human bronchial epithelial; NTHi, non-typeable Haemophilus influenzae

Mentions: To determine the length of time that NTHi could be co-cultured with ALI tissues, we tested the ability of various NTHi strains to survive within ALI NHBE cells and EpiAirway tissues over time points ranging from 1 to 10 days (Figure 1). At each desired time point, the tissues were harvested and the gentamicin-resistant (internalized) bacteria were enumerated by plate counts. Figure 1a shows the numbers of bacteria surviving within EpiAirway tissues from long-term co-cultures, expressed as viable gentamicin-resistant CFU recovered per square centimeter of tissue (GmR CFU/cm2), for NTHi strain 86-028NP. These data are consistent with previous results observed for strain R2866.15 Figure 1b illustrates the GmR CFU/cm2 of strain R2846 in ALI cultures of NHBE cells, and Figure 1c shows R2846 recovered from EpiAirway tissues. This confirms that ALI cultures can be used to investigate the long-term interactions of various strains of NTHi with primary human epithelial cells in vitro.Figure 1


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)

Survival of NTHi strains over time inside EpiAirway tissues and ALI NHBE cells. (a) Internalized (gentamicin-resistant) bacteria of strain 86-028NP recovered from EpiAirway tissues, 2–10 days after infection (n = 6). (b) Internalized strain R2846 recovered from ALI NHBE cells, 1–5 days after infection (n = 2). (c) Internalized strain R2846 recovered from EpiAirway tissues, 1–8 days after infection (n = 2). Error bars are SD. ALI, air–liquid interface; NHBE, normal human bronchial epithelial; 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-377F1: Survival of NTHi strains over time inside EpiAirway tissues and ALI NHBE cells. (a) Internalized (gentamicin-resistant) bacteria of strain 86-028NP recovered from EpiAirway tissues, 2–10 days after infection (n = 6). (b) Internalized strain R2846 recovered from ALI NHBE cells, 1–5 days after infection (n = 2). (c) Internalized strain R2846 recovered from EpiAirway tissues, 1–8 days after infection (n = 2). Error bars are SD. ALI, air–liquid interface; NHBE, normal human bronchial epithelial; NTHi, non-typeable Haemophilus influenzae
Mentions: To determine the length of time that NTHi could be co-cultured with ALI tissues, we tested the ability of various NTHi strains to survive within ALI NHBE cells and EpiAirway tissues over time points ranging from 1 to 10 days (Figure 1). At each desired time point, the tissues were harvested and the gentamicin-resistant (internalized) bacteria were enumerated by plate counts. Figure 1a shows the numbers of bacteria surviving within EpiAirway tissues from long-term co-cultures, expressed as viable gentamicin-resistant CFU recovered per square centimeter of tissue (GmR CFU/cm2), for NTHi strain 86-028NP. These data are consistent with previous results observed for strain R2866.15 Figure 1b illustrates the GmR CFU/cm2 of strain R2846 in ALI cultures of NHBE cells, and Figure 1c shows R2846 recovered from EpiAirway tissues. This confirms that ALI cultures can be used to investigate the long-term interactions of various strains of NTHi with primary human epithelial cells in vitro.Figure 1

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