Fig. 3: Activation of the RIG-I inflammasome impaired RIG-I-dependent interferon signaling in bronchial epithelium of patients with asthma.

a Volcano plots of all (black), significant (red), and significant antiviral (green) genes in bronchial brushings from controls (upper panel) and patients with asthma (lower panel) after in vivo RV-A16 infection (control n = 7, asthma n = 17). b Heatmap of antiviral genes significantly changed four days after in vivo RV-A16 infection in healthy controls (left panel) and/or in patients with asthma (right panel) presented together with the log₂ fold change (FC) (black bars) (control n = 7, asthma n = 17). Yellow and grey left-side color bars represent genes upregulated and downregulated, respectively. c, d RV-A16 virus load in (c) the bronchoalveolar lavage (BAL) fluid and d the nasal lavage (NL) fluid in control individuals and patients with asthma four days after in vivo RV-A16 infection (control n = 9, asthma n = 19). Data presented as viral RNA copies per 1 mL of BAL/NL. e–j in vitro-cultured HBECs from patients with asthma were infected in vitro with RV-A16 in the presence or absence of BX795, a chemical inhibitor of TBK1 and IKKε, or vehicle. mRNA expression of (e) IFNL2/3 (IFNλ) and (f) DDX58 (RIG-I) assessed using RT-PCR and presented as relative quantification (RQ = 2^-ΔΔCt) as compared to the vehicle condition (n = 5). g Secretion of CXCL10, CXCL11, CCL3, and CCL4 proteins into the apical compartment assessed with the Proximity Extension Assay (PEA) targeted proteomics (n = 6). Expression of (h) RV-A16 positive strand (RV-A16 viral RNA) and (i) IL1B (IL-1β) assessed using RT-PCR and presented as relative quantification (RQ = 2^-ΔΔCt) (n = 5). j IL-1β release to the apical compartment of in vitro-cultured HBECs from patients with asthma assessed by ELISA (n = 8). k–n in vitro-cultured HBECs from patients with asthma were infected in vitro with RV-A16 in the presence or absence of YVAD, a caspase-1 inhibitor or vehicle. mRNA expression of (k) IFNB (IFNβ) and (l) DDX58 (RIG-I) (n = 4). Data are demonstrated as the percentage of the expression normalized to the in vitro RV-A16 condition. m Secretion of CXCL10, CXCL11, CCL3, and CCL4 into apical compartment assessed with the PEA proteomics (n = 6). n RIG-I release to the apical compartment assessed by PEA in in vitro-cultured HBECs from patients with asthma (n = 5). o Representative confocal images of RIG-I expression in bronchial biopsies at baseline, scale bars: 20 μm. Quantification based on the mean fluorescence intensity (MFI) x10³: 10 equal epithelial areas from each biopsy (demonstrated as circles, squares, triangles, or diamonds) of control subjects (n = 3) and patients with asthma (n = 3). Fig. 3a–d, (o) presents in vivo RV-A16 infection study, Fig. 3e–n shows in vitro ALI-differentiated HBECs. Patients with asthma/HBECs from patients with asthma are presented in red, control individuals/HBECs from control individuals are presented in blue. (n) indicates the number of biologically independent samples examined over one infection (in vivo RV-A16 infection) or at least three independent experiments (in vitro-cultured HBECs). Heatmap displays normalized gene expression across the groups (row normalization). Transcriptome data analyzed with Bioconductor microarray analysis workflow [https://www.bioconductor.org/packages/release/workflows/vignettes/arrays/inst/doc/arrays.html], raw p-value presented. Asterisks represent statistical significance, p-value: *<0.05; **<0.005; ***<0.0005, ****<0.00005. Bar graph data present mean ± SEM analyzed with one-way ANOVA (Kruskal–Wallis test), RM one-way ANOVA (Friedman test), or mixed-effects model with post-hoc analysis, as appropriate, depending on the data relation and distribution. Proximity Extension Assay (PEA) targeted proteomics data were analyzed by Bioconductor limma package¹⁰⁴, raw p-value presented, and presented as normalized protein expression (NPX). Source data are provided as Source Data files. ALI Air-liquid interface, BAL bronchoalveolar lavage; BX795, TBK1/IKKε inhibitor; HBECs differentiated human bronchial epithelial cells, MFI mean fluorescent intensity, NL nasal lavage, NPX normalized protein expression, RV-A16 rhinovirus A16, YVAD ac-YVAD-cmk (caspase-1 inhibitor).