Panel A: A heatmap showing mRNA expression levels of cytokines in wild-type (WT) and VRAC knockout (KO) bone marrow-derived macrophages (BMDMs) incubated in hypo-osmotic media. The heatmap uses a color scale from white to red, with red indicating higher expression levels. The x-axis represents different conditions (WT and KO), and the y-axis lists various cytokines. Panel B: A heat map displaying mRNA expression levels of interferons under the same conditions as Panel A. Panel C: A heatmap showing mRNA expression levels of interferon-stimulated genes under the same conditions. Panel D: A line graph depicting qRT-PCR analysis of Ifnb expression in WT and VRAC KO BMDMs over time. The x-axis shows time in hours, and the y-axis shows fold expression. Panel E: A line graph showing qRT-PCR analysis of Cxcl10 expression under the same conditions as Panel D. Panel F: A scatter bar plot illustrating qRT-PCR analysis of Rsad2 expression in WT and VRAC KO BMDMs after 6 hours of incubation. The x-axis shows the conditions, and the y-axis shows fold expression. Panel G: A scatter bar plot showing qRT-PCR analysis of Ccl7 expression under the same conditions as Panel F. Panel H: A scatter bar plot displaying qRT-PCR analysis of Il1b expression under the same conditions. Panel I: A western blot image showing viperin protein levels in WT and VRAC KO BMDMs over time. Panel J: A line graph showing densitometry analysis of the western blot in Panel I. The x-axis shows time in hours, and the y-axis shows viperin expression normalized to beta-actin. Panel K: A scatter bar plot depicting qRT-PCR analysis of Cxcl10 expression in WT and IFNAR KO BMDMs after 6 hours of incubation. Panel L: A scatter bar plot showing qRT-PCR analysis of Rsad2 expression under the same conditions as Panel K. Panel M: A western blot image showing viperin and pro-IL-1 protein levels in WT and IFNAR KO BMDMs after 6 hours of incubation. Panel N: A diagram outlining the pipeline for Ingenuity Pathway Analysis (IPA) of RNA-sequencing data. Panel O: A bar graph showing the top 10 predicted upregulated and downregulated transcription factors. Panel P: A bar graph displaying the top 10 predicted upregulated and downregulated kinases. Panel Q: A bar graph illustrating predicted upregulated nucleotide sensing pathways.
Changes in macrophage cell volume promote inflammation through type I Interferon signaling. (A–C) Heatmaps of mRNA expression of cytokines (A), interferons (B), and ISGs (C) from RNA-seq of WT or VRAC KO BMDMs ± incubation in hypo-osmotic media (50% vol/vol H2O in DMEM). (D and E) qRT-PCR analysis of Ifnb and Cxcl10 in WT and VRAC KO BMDMs ± incubation in hypo-osmotic media for the indicated time points (n = 4). (F–H) qRT-PCR analysis of Rsad2 (F), Ccl7 (G), and Il1b (H) in WT and VRAC KO BMDMs ± incubation in hypo-osmotic media for 6 h (n = 6). (I) Western blot for viperin in WT and VRAC KO BMDMs incubated in hypo-osmotic media for the indicated time points (n = 4). (J) Densitometry of (I) (n = 4). (K and L) qRT-PCR analysis of Cxcl10 (K) and Rsad2 (L) in WT and IFNAR KO BMDMs ± incubation in hypo-osmotic media (6 h) (n = 3). (M) Western blot for viperin and pro-IL-1β in WT and IFNAR KO BMDMs incubated in hypo-osmotic media (6 h) (n = 3). (N) Pipeline for IPA of RNA-seq data (from Fig. 1 A) to identify potential differential upstream regulators in WT BMDMs following incubation in hypo-osmotic media. (O and P) Top 10 predicted upregulated and downregulated transcription factors (O) and kinases (P). (Q) Predicted upregulated nucleotide-sensing pathways. *P < 0.05, **P < 0.01, ***P < 0.001, determined by a one-way ANOVA (2D and E) or by a two-way ANOVA (2F–L) with Sidak’s post hoc analysis. Values shown are the mean ± the SEM. IPA, Ingenuity Pathway Analysis. Source data are available for this figure: SourceData F2.