Figure 1.
HRV induces the release of IL-1α, IL-1β and IL-18 by NHBE cells.
NHBE cells were infected with or without HRV14 (A and B) or HRV1b (C). After 48 h the levels of IL-1α (A), IL-1β (A, B and C) and IL-18 (A) released into the media were measured by ELISA. In (B), some samples were treated with an anti ICAM-1 antibody or an isotype control (both 1.33 μM) for the duration of the infection. Data is expressed as mean ± SEM. An * indicates a significant up-regulation in protein release, a # indicates there was a significant inhibition in protein release (***/# # # p<0.001, **/# # p = 0.001–0.01, */# p = <0.05). N = 8 (A), 7 (B) or 4 (C). Untreated indicates cells were treated with medium alone.
Figure 2.
HRV14 induces the release of the pro- and mature forms of IL-1β and IL-18.
(A) NHBE cells were infected with or without HRV14 and the levels of IL-1β and pro-IL-1β released into the media after 48 hours was measured by ELISA. Untreated indicates cells were incubated with medium alone. Data is expressed as mean ± SEM, an * indicates a significant up-regulation in protein release (IL-1β, p = 0.0168. pro-IL-1β, p = 0.0151.), N = 6. The levels of IL-18 in NHBE lysates (B) or media taken from NHBE cells (C) was measured by ELISA before and after treatment with rCaspase-1 in order to detect the presence of pro-IL-18. The data in (B) is expressed as mean ± SEM and is a representative graph from 3 independent experiments, the data in (C) is mean ± SEM (N = 3).
Figure 3.
IL-1β and IL-18 are released by an active caspase-1 dependent event.
NHBE cells were infected with or without HRV14 (A–C) in the presence of the V5 Bax inhibitory peptide (B) or the caspase-1 inhibitor YVAD (C) at the indicated concentrations. A – indicates treatment with the vehicle control. After 48 hours the level of cell death or cytokine release was measured. In B and C individual experiments were normalised by expressing each replicate as a percentage of the median signal detected in the HRV treated sample. Data shown is the Mean percentage ± SEM (N = 3).
Figure 4.
IL-1 is required for the HRV dependent release of IL-6 and IL-8 but not IP-10 in NHBE cells.
A) NHBE cells were infected with HRV14 in the presence of a control IgG1 antibody (33 nM), IL-18BP-Fc (33 nM) or anakinra (10 nM). A – indicates no inhibitor treatment. The levels of IL-6, IL-8, and IP-10 released into the media after 48 hours was assessed by ELISA (N = 5). Data is expressed as Mean ± SEM, B) Levels of IL-8 mRNA were determined in NHBE cells that were infected with HRV14 for 24 hours in the presence or absence of anakinra (10nM) (N = 3). – indicates no treatment. The data is displayed as Mean (± SEM) fold-expression in relation to the uninfected samples (2−ΔΔCt). An * indicates a significant increase in protein release compared to untreated NHBE cells (*** p<0.001, ** p = 0.001–0.01), a # indicates a significant decrease in protein release/ mRNA expression of HRV14 treated samples compared to the control (### p<0.001, ## p = 0.001–0.01) and ns indicates not significant (p>0.05).
Figure 5.
Anakinra does not inhibit viral entry or replication.
NHBE cells were uninfected, infected with HRV14 or infected with HRV14 in the presence of anakinra (10nM). After the indicated time RNA was extracted and changes in viral copy number was assessed using qRT-PCR. Data is expressed as Mean ± SEM, an ns indicates there was no significant difference (n>0.05) in copy number between samples treated with HRV14 alone or HRV14 in the presence of anakinra.
Table 1.
IL-1 is required for the HRV-dependent release of pro-inflammatory cytokines and neutrophil chemoattractants but not T-cell chemoattractants or members of the IFN family.
Figure 6.
IL-1 is required for the HRV-dependent release of pro-inflammatory cytokines and neutrophil chemoattractants but not T-cell chemoattractants or members of the IFN family.
NHBE cells were uninfected, infected with HRV14 or infected with HRV14 in the presence of anakinra (10 nM). After the indicated time RNA was extracted and changes in mRNA was determined by a microfluidic genecard. The data from CSF2 (A), CXCL2 (B), CXCL11 (C) or IFNB1 (D) are presented and is expressed as Mean (± SEM) fold expression (2−ΔΔCt) in relation to the untreated controls (A–C) or the HRV14 treated samples (D) (N = 3). An * indicates a significant increase in mRNA expression on treatment with HRV14 compared to untreated NHBE cells, a # indicates a significant decrease in mRNA expression when the infected NHBE cells were treated with anakinra. An ns indicates not significant, (***/# # # p<0.001, **/# # p = 0.001–0.01, */# p = <0.05).