Comparison of immune response to human rhinovirus C and respiratory syncytial virus in highly differentiated human airway epithelial cells

Abstract

Background: Human rhinovirus C (HRV-C) accounts for a large proportion of HRV-related illnesses, but the immune response to HRV-C infection has not been elucidated. Our objective was to assess the effect of HRV-C on cytokine secretion in human bronchial epithelial (HBE) cells grown at air-liquid interface (ALI) and compare it with that of respiratory syncytial virus (RSV).

Methods: HBE cells were differentiated at ALI culture and the full-length cDNA clones of HRV-C651 and HRV-C15, clinical isolates of HRV-C79 and HRV-C101, and two RSV isolates were inoculated in the HBE cells. The effect of HRV-C on cytokine secretion was assessed and compared with that of RSV.

Results: HRV-Cs infect and propagate in fully differentiated HBE cells and significantly increase the secretion of IFN-λ1, CCL5, IP10, IL-6, IL-8, and MCP-1. The virus loads positively correlated with the levels of the cytokines. HRV-C induced lower secretion of CCL5 (P = 0.048), IL-6 (P = 0.016), MCP-1 (P = 0.008), and IL-8 (P = 0.032), and similar secretion of IP10 (P = 0.214) and IFN-λ1 (P = 0.214) when compared with RSV.

Conclusion: HBE ALI culture system supported HRV-C infection and propagation and HRV-C induced relatively weaker cytokine expression than RSV.

Keywords: Air–liquid interface; Cytokines; Human bronchial epithelial cell; Human rhinovirus C; Immune response; Respiratory syncytial virus.

Fig. 1

Immunofluorescence of differentiated HBE cells. The differentiated HBE cells were fixed, followed by incubation with mouse monoclonal anti-β tubulin antibody (red, A), mouse Muc5AC monoclonal antibody (green, B), or monoclonal anti-ZO-1 antibody (red, C). Confocal images were taken with a magnification of 200× for A and B, and 360× for C. Nuclei were stained with DAPI. Scale bars, 20 μm

Fig. 2

Immunofluorescence of differentiated HBE cells infected with HRV-C651 or HRV-C15. Immunofluorescence with the rabbit polyclonal anti-HRV-C VP1 protein-antibody only (A) and mAbJ2 antibody (D). At 24 h post-inoculation with HRV-C651 or HRV-C15, the HBE cells were fixed, followed by incubation with VP1 protein-antibody (B and C) and mAb12 antibody detecting double-strand RNA (E and F). The red region indicates VP1 protein (B and C) or the double-stranded RNA of HRV-C (E and F). Confocal images were taken with a magnification of 200×. Nuclei were stained with DAPI

Fig. 3

Four HRV-C strains and two RSV strains grew successfully in ALI HBE cells (A and B). Virus RNA load from infected tissue supernatants was measured by real-time RT-PCR collected at the apical surface of ALI HBE cells infected with HRV-C strains (HRV-C 651, HRV-C15, HRV-C 79, or HRV-C 101) and two RSV strains (RSV1 or RSV2). Error bars represent the standard deviation calculated from biological replicates (n = 3)

Fig. 4

Time course of cytokine production induced by HRV-Cs and RSVs. Levels of cytokines in basal medium of HBE cultures were collected at each hour after HRV-Cs or RSVs inoculation in ALI HBE cells

Fig. 5

Comparison cytokine levels (mean ± SEM) between HRV-Cs and RSVs within 48-h PI in ALI HBE cells. Mann–Whitney test for comparisons between two viral groups was used to determine statistical significance. *P < 0.05, **P < 0.01