Fig. 4. Metabolism drives barrier function.
a Metabolism-associated gene profiling of HRV-C15 infected ALI cultures over 24 h, assessed by RT-PCR. Data are represented as boxplots, n = 5 donors. b Representative immunoblot of ALI culture whole-cell lysates evaluating PGC-1α protein during HRV-C15 infection >24 h. GAPDH was used as a loading control. Representative immunoblot was selected from three independent experiments performed in ALI cultures derived from three different lung donors, n = 3 donors. c PPARGC1A and HK2 mRNA expression by three strains of HRV at 24 h post infection. Data are represented as boxplots and analyzed by one-way ANOVA with Holm–Sidak multiple comparisons: PPARGC1A *p = 0.044, **p = 0.004, HK2 *p = 0.049, **p = 0.008, n = 6 donors. d, Left: barrier function permeability assay using FITC-dextran (3–5 kDa) flux 12 h post infection in ALI cultures treated with or without 3 μM oligomycin A. Data are represented by individual human bronchial epithelial (HBE) donor lines and analyzed by one-way ANOVA with Holm–Sidak multiple comparisons: ****p < 0.0001, n = 6 donors. Right: matched intracellular HRV-C15 RNA copy number at 12 h post infection, quantified by RT-PCR. Dashed line represents RT-PCR detection limit. There was no detectable HRV-C15 in noninfected ALI cultures. Data are represented as boxplots and analyzed by two-tailed paired t test: **p = 0.003, n = 6 donors. e, Left: barrier function permeability assay of FITC-dextran (3–5 kDa) flux 12 h post infection in ALI cultures treated with or without 100 μM 2-DG. Data are represented as boxplots and analyzed by one-way ANOVA. 2-DG treatment was not significantly different from HRV-C15 alone, n = 5 donors. Right: matched intracellular HRV-C15 RNA copy number at 12 h post infection, quantified by RT-PCR. Dashed line represents RT-PCR detection limit. There was no detectable HRV-C15 in noninfected ALI cultures. Data are represented as boxplots and analyzed by two-tailed paired t test: *p = 0.04, n = 6 donors. f Graphical representation of metabolic pathways and HRV-C15 replication inhibition to summarize findings from (d, e). g Noninfected ALI cultures were biopsy punched (4 mm) and loaded into Seahorse XFe24 plates and OCR (top) and ECAR (bottom) measurements were performed in technical triplicates per donor prior to and following injection of 3 μM oligomycin A. Baseline OCR and ECAR were determined by the final reading prior to oligomycin injection compared to the maximal reading post-oligomycin injection. Real-time data are graphically represented by individual HBE donor lines, with a dashed line indicating oligomycin A injection event. Baseline OCR and ECAR bar graphs indicate mean ± SD and analyzed by paired t test: OCR *p = 0.012, ECAR ****p < 0.0001, n = 5 donors. h, Left: representative immunofluorescence image of oxidative stress (ROS) in live ALI cultures detected using CellROX (green) and Hoechst 33342 (blue) following a 15-min incubation with either 3 μM oligomycin A or 1 μM H2O2. Scale bar, 100 μm. Representative confocal images and quantification were obtained from three independent experiments performed in ALI cultures derived from three different lung donors and selected from five fields per condition per donor. Data are represented as mean of five fields of view per donor ± SD and analyzed by one-way ANOVA with Holm–Sidak multiple comparisons: left to right **p = 0.008, **p = 0.007, n = 3 donors. Data represented as boxplots indicate the median (center line), upper and lower box bounds (IQR = first and third quartiles), and whiskers (min and max values), with individual donor data points superimposed onto the boxplot.