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. 2023 Jan 24;11(1):e005518. doi: 10.1136/jitc-2022-005518

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© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

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Upregulation of GBPs and AIM2 in vivo and during induction of trained immunity in vitro. (A) Protocol for transcriptome analysis of monocyte-enriched cell suspensions before and during intravesical BCG therapy. Note that the cells were not stimulated ex vivo before RNA-sequencing. (B) Gene expression of GBP1, GBP2, GBP4, GBP5 and AIM2 at pre-BCG1, BCG6 and pre-BCG7. Each color indicates an individual patient. The bar represents the mean gene expression of all 6 patients for each time point. For the changes in mean gene expression between pre-BCG1 and BCG6: GBP1 p=0.0001, p_adjust=0.053; GBP2 p=0.0002, p_adjust=0.114; GBP4 p=0.0070, p_adjust=1; GBP5 p=0.0016, p_adjust=0.5051; AIM2 p=0.0027, p_adjust=0.7203. And between pre-BCG1 and pre-BCG7 only GBP1 and AIM2 were significant: GBP1 p=0.0084, p_adjust=1; AIM2 p=0.0427, p_adjust=1. (C) The in vitro trained immunity protocol as described by Domínguez-Andrés et al.59 Monocytes are isolated from the blood, seeded on a cell culture plate and subsequently incubated with a trained immunity-inducing stimulus (BCG), and a control condition (RPMI), for the first 24 hours. After 24 hours, the training stimulus is washed away and the culture medium is refreshed. On day 6, the restimulation stimulus (LPS), or a negative control restimulation (RPMI) is added. After 24 hours of restimulation, the supernatant is collected for cytokine measurements. For this particular experiment, cells were harvested at baseline (d0), After the first 4 hours (4 hour), 24 hours (24 hours), at day 6 before restimulation (d6), and at day 6, 4 hours after restimulation with LPS (d6+4 hour LPS), and RNA was isolated and gene expression was measured. (D) Changes in gene expression for GBP1, GBP2, GBP4, GBP5 and AIM2 are displayed as median (solid line), and 25th and 75th quartiles (shaded) for three conditions: BCG Bulgaria (green), BCG Denmark (blue), and RPMI (gray, negative control). The expression of all genes was significantly increased in D6-BCG-trained macrophages compared with RPMI macrophages (GBP1 p=0.0001, p_adjust=0.03; GBP2 p=7.78^-5, p_adjust=0.02; GBP4 p=2.79^-6, p_adjust=0.0013; GBP5 p=7.40^-8, p_adjust=8.10^-5; AIM2 p=0.00015, p_adjust=0.034). All adjusted p values in this figure were calculated using the Benjamini-Hochberg method.