Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 May 4;18(1):84.
doi: 10.1186/s12931-017-0567-0.

Rhinovirus C targets ciliated airway epithelial cells

Theodor F Griggs  1   2   3 , Yury A Bochkov  4 , Sarmila Basnet  4 , Thomas R Pasic  5 , Rebecca A Brockman-Schneider  4 , Ann C Palmenberg  6 , James E Gern  4   7
Affiliations

Rhinovirus C targets ciliated airway epithelial cells

Theodor F Griggs et al. Respir Res. .

Abstract

Background: The Rhinovirus C (RV-C), first identified in 2006, produce high symptom burdens in children and asthmatics, however, their primary target host cell in the airways remains unknown. Our primary hypotheses were that RV-C target ciliated airway epithelial cells (AECs), and that cell specificity is determined by restricted and high expression of the only known RV-C cell-entry factor, cadherin related family member 3 (CDHR3).

Methods: RV-C15 (C15) infection in differentiated human bronchial epithelial cell (HBEC) cultures was assessed using immunofluorescent and time-lapse epifluorescent imaging. Morphology of C15-infected differentiated AECs was assessed by immunohistochemistry.

Results: C15 produced a scattered pattern of infection, and infected cells were shed from the epithelium. The percentage of cells infected with C15 varied from 1.4 to 14.7% depending on cell culture conditions. Infected cells had increased staining for markers of ciliated cells (acetylated-alpha-tubulin [aat], p < 0.001) but not markers of goblet cells (wheat germ agglutinin or Muc5AC, p = ns). CDHR3 expression was increased on ciliated epithelial cells, but not other epithelial cells (p < 0.01). C15 infection caused a 27.4% reduction of ciliated cells expressing CDHR3 (p < 0.01). During differentiation of AECs, CDHR3 expression progressively increased and correlated with both RV-C binding and replication.

Conclusions: The RV-C only replicate in ciliated AECs in vitro, leading to infected cell shedding. CDHR3 expression positively correlates with RV-C binding and replication, and is largely confined to ciliated AECs. Our data imply that factors regulating differentiation and CDHR3 production may be important determinants of RV-C illness severity.

Keywords: Air-liquid interface; Bronchial epithelium; CDHR3; Ciliated cells; Rhinovirus C.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
C15 inoculation of airway epithelial cells causes a speckled pattern of infection and infected cell shedding. HBEC-ALI cultures were inoculated for 18 h with C15 or media alone and imaged by fluorescent microscopy (a and b, respectively). Nuclei stained with Hoechst (blue), C15 capsid stained with monoclonal antibody against VP2 (red). Inoculated cultures were also imaged by confocal microscopy and analyzed by z-stacking (c and d) or apical surface views (e and f). Nuclei stained with Syto-13 (green), secretory cells stained with WGA (blue), and C15 capsid stained with monoclonal antibody (orange). Scale bars indicate 20 μm
Fig. 2
Fig. 2
Analysis of C15 infectivity by immunofluorescent staining. HBEC-ALI cultures were infected for 18 h with C15 and imaged apically and z-stacked orthogonally by confocal microscopy. Secretory and ciliated cells were stained with WGA and rabbit polyclonal antibody against aat, respectively (false color red), C15 capsid stained with monoclonal antibody (false color green), nuclei stained with Syto-13 (false color blue). Scale bars indicate 20 μm
Fig. 3
Fig. 3
Immunohistochemical analysis of C15 infectivity in HBEC-ALI cultures. PCM-differentiated HBEC-ALI cultures were inoculated with a C15 or b media alone, stained for C15 capsid, and imaged by light microscopy. C15 capsid is represented by brown-staining cells. Scale bars indicate 20 μm
Fig. 4
Fig. 4
Single cell analysis of RV-C infection and epithelial cell surface markers. HBEC-ALI cultures infected or mock-infected for 18 h with C15 or BEGM alone, respectively, were labeled with antibodies against C15 capsid (C15), and either a acetylated-alpha-tubulin (aat, Cilia) or b fluorescently labeled-WGA (WGA) and analyzed by flow cytometry. The graphs summarize the percentage of C15+ cells in ciliated and non-ciliated cells (n = 4 independent experiments, two cell donors). Mock, mock-inoculated cultures; C15, C15-inoculated cultures. *** p < 0.001
Fig. 5
Fig. 5
Highly differentiated cells are more susceptible to RV-C infection. Differentiated cultures were incubated for 18 h after inoculation with C15 or BEGM alone, labeled with antibodies against C15 capsid (C15) and either a aat (Cilia) or b Muc5AC and analyzed by flow cytometry. The graphs show the percentage of C15-positive cells by cell type (n = 6). *** p < 0.001
Fig. 6
Fig. 6
CDHR3 is predominantly expressed by ciliated epithelial cells and diminishes following C15 inoculation. PC-differentiated cultures inoculated or mock-inoculated for 18 h with C15 (C15+) or BEGM alone (Mock), respectively, were labeled with antibodies against C15 capsid (C15), CDHR3, and aat (cilia) and analyzed by flow cytometry (n = 6). a Relative median fluorescence intensity (rMFI) of CDHR3 in ciliated (Yes) and nonciliated (No) cell populations of mock-inoculated cultures. b Frequency of CDHR3 positive ciliated cells out of the total ciliated cell population in mock- and C15-inoculated cultures. c rMFI of CDHR3 in ciliated cells negative (C15-) or positive (C15+) for C15 staining. Values are normalized to the double-negative (nonciliated and CDHR3-) population in each experiment. ** p ≤ 0.01, * p ≤ 0.05
Fig. 7
Fig. 7
CDHR3 expression in differentiating bronchial epithelial cells positively correlates with RV-C binding and replication. a CDHR3 protein expression (~100 kDa) and b mRNA expression (n = 8) by differentiating airway epithelial cells in ALI using PCM from day 1 to day 28. c BECs in ALI were infected weekly with C15 and the infected cell samples were collected and analyzed for viral binding (2 hpi) and viral yield (24 hpi). d and e Correlations (linear regression) between cellular CDHR3 mRNA expression and C15 binding and yield
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Fendrick AM, Monto AS, Nightengale B, Sarnes M. The economic burden of non-influenza-related viral respiratory tract infection in the United States. Arch Intern Med. 2003;163(4):487–94. doi: 10.1001/archinte.163.4.487. - DOI - PubMed
    1. Jakab GJ. Mechanisms of bacterial superinfections in viral pneumonias. Schweiz Med Wochenschr. 1985;115(3):75–86. - PubMed
    1. Monto AS, Bryan ER, Ohmit S. Rhinovirus infections in Tecumseh, Michigan: frequency of illness and number of serotypes. J Infect Dis. 1987;156(1):43–9. doi: 10.1093/infdis/156.1.43. - DOI - PubMed
    1. Hament J-M, Kimpen JL, Fleer A, Wolfs TF. Respiratory viral infection predisposing for bacterial disease: a concise review. FEMS Immunol Med Microbiol. 1999;26(3–4):189–95. doi: 10.1111/j.1574-695X.1999.tb01389.x. - DOI - PubMed
    1. Ishizuka S, Yamaya M, Suzuki T, Takahashi H, Ida S, Sasaki T, et al. Effects of rhinovirus infection on the adherence of Streptococcus pneumoniae to cultured human airway epithelial cells. J Infect Dis. 2003;188(12):1928–39. doi: 10.1086/379833. - DOI - PubMed

Supplementary concepts