Volatile emanations from in vitro airway cells infected with human rhinovirus

Abstract

Respiratory viral infections such as human rhinovirus (HRV) can lead to substantial morbidity and mortality, especially in people with underlying lung diseases such as asthma and COPD. One proposed strategy to detect viral infections non-invasively is by volatile organic compound (VOC) assessment via analysis of exhaled breath. The epithelial cells are one of the most important cell lines affected during respiratory infections as they are the first line of pathogen defense. Efforts to discover infection-specific biomarkers can be significantly aided by understanding the VOC emanations of respiratory epithelial cells. Here we test the hypothesis that VOCs obtained from the headspace of respiratory cell culture will differentiate healthy cells from those infected with HRV. Primary human tracheobronchial cells were cultured and placed in a system designed to trap headspace VOCs. HRV-infected cells were compared to uninfected control cells. In addition, cells treated with heat-killed HRV and poly(I:C), a TLR3 agonist, were compared to controls. The headspace was sampled with solid-phase microextraction fibers and VOCs were analyzed by gas chromatography/mass spectrometry. We determined differential expression of compounds such as aliphatic alcohols, branched hydrocarbons, and dimethyl sulfide by the infected cells, VOCs previously associated with oxidative stress and bacterial infection. We saw no major differences between the killed-HRV, poly(I:C), and control cell VOCs. We postulate that these compounds may serve as biomarkers of HRV infection, and that the production of VOCs is not due to TLR3 stimulation but does require active viral replication. Our novel approach may be used for the in vitro study of other important respiratory viruses, and ultimately it may aid in identifying VOC biomarkers of viral infection for point-of-care diagnostics.

Figure 1

Volatile organic compound (VOC) collection system. Three Transwell culture wells are placed in a jar with the basal ends in contact with ALI+A medium. VOCs accumulate in the headspace (represented by small dots). The top is sealed to prevent VOC-ambient air exchange. A solid-phase microextraction (SPME) fiber (top) is introduced through the lid septum into the headspace to sample the VOCs. ALI+A, air-liquid interface plus vitamin A

Figure 2

Experimental setup. Four main experiments were performed, A through D. Two additional experiments were also performed, third column, “Additional Experiments”: A. medium and empty jar; and, C. Poly(I:C) and heat-killed HRV. Each jar contains three air-liquid interface cultures and 5 mL of ALI+A medium (except in A, additional experiments). Each jar was sampled with two SPME fibers (arrow, noted) at each time point, designated in the last column, “Time Points.” The major groups studied were Control and HRV-infected cells, first two columns, respectively. Experiments B, C & D contain one less jar in each main group because the VOC spectra within each group (e.g. the control group) were identical and therefore did not warrant replication. The 48-h time points were added to the last two experiments, C & D, as the cells were found to be alive and healthy after the first two experiments, A & B. HRV, human rhinovirus; SPME, solid-phase microextraction; ALI+A, air-liquid interface plus vitamin A.

Figure 3

Representative chromatograms of control and HRV-infected TBE cells compared to medium background. The chromatograms are slightly offset for clarity, and this example is from retention time 25 to 32.5 min. Note that the medium (ALI+A) gives little signal, as represented by the low ion count. In this example there is a significant up-regulation of VOC production after infection. TBE, tracheobronchial epithelial cells; HRV, human rhinovirus; VOC, volatile organic compound; ALI+A, air-liquid interface plus vitamin A

Figure 4

Representative Alamar blue measure of TBE cell viability. Both HRV-infected and control cells were assessed at three 1-hour time points after 2 hours incubation with Alamar blue. Optical intensity of the supernatant liquid was measured against a standard 570 nm wavelength. Increasing intensity correlates with cellular metabolic activity, which is a surrogate for viability. As seen, the control (dashed) and HRV-infected (solid) TBE cells have similar increases in optical intensity indicating that both are alive. Error boundaries (dots) are shown around the 3-hour time point, and these are similar for the other time points. This experiment was done on cells at 48-hours post-placement in the jars, and Alamar experiments on 12 and 24-hour cells provided similar results. HRV, human rhinovirus; TBE, tracheobronchial epithelial cells