IS induces trained immunity in human monocytes.

A. Schematic of in vitro experimental model for innate trained immunity. B-C. Human monocytes were treated with the indicated concentration of IS for 24 hr, followed by a subsequent 5-day culture in human serum. On day 6, the cells were restimulated with the indicated concentrations of LPS for 24 hr. TNF-α and IL-6 proteins levels were quantified by ELISA. D. After training with 1,000 μM IS, monocytes were restimulated with 10 μg/ml Pam3cys. TNF-α and IL-6 protein levels were quantified by ELISA. E. After training with 1,000 μM IS, monocytes were restimulated with 10 ng/ml LPS for 24 hr. The mRNA expression of IL-1β, IL-10, and MCP-1 was analyzed by RT-qPCR. F. In vitro experimental scheme of uremic serum-induced trained immunity. G-I. The pooled normal serum (NS) from healthy controls (HCs) or uremic serum (US) from patients with ESRD were used for treatment of monocytes isolated from HCs for 24 hr at 30% (v/v) followed by resting for 5 days. After stimulation with LPS for 24 hr, TNF-α and IL-6 production were analyzed using ELISA (G) and RT-qPCR (H). After stimulation with LPS (10 ng/ml) for 24 hr, mRNA expression of IL-1β and MCP-1 were determined by RT-qPCR (I). Bar graphs show the mean ± SEM. * = p < 0.05, and ** = p < 0.01 by two-tailed paired t-test.

IS-induced trained immunity is linked to metabolic rewiring.

Glycolysis and mitochondrial stress tests were conducted on IS (1,000 μM)-trained macrophages (n = 3∼4) using the Seahorse XF-analyzer. A. ECAR (extracellular acidification rate) levels were measured after sequential treatment with glucose, oligomycin, and 2-DG. B. Cellular glycolysis and glycolytic capacity were analyzed. C. OCR (Oxygen consumption rate) levels were measured after sequential treatment with oligomycin, FCCP, and Rotenone/antimycin A (Ro/AA). D. Basal respiration, maximal respiration, and ATP production were analyzed. E. Monocytes were pretreated with 2DG, followed by IS-training for 6 days. Cells were restimulated with LPS for 24 hr and TNF-α and IL-6 in supernatants were quantified by ELISA (n = 5). Bar graphs show the mean ± SEM. *= p < 0.05, **= p < 0.01, and *** = p< 0.001 by two-tailed paired t-test.

IS-induced trained immunity is accomplished through epigenetic modification.

A. Experimental scheme of ChIP-qPCR for IS (1,000 μM)-trained macrophages. B. On day 6 after IS-training, cells were fixed with 1% formaldehyde, lysed, and sonicated. A ChIP assay was performed using anti-H3K4me3 antibody and enrichment of H3K4me3 in the promoter site of TNFA and IL6 loci was quantified by qPCR. 1% input was used as a normalization control. C. Monocytes were pre-treated with 5’-methylthioadenosine (MTA, a non-selective methyltransferase inhibitor; 200 μM) and then were trained with IS for 6 days, followed by restimulation with LPS for 24 hrs. TNF-α and IL-6 proteins levels were quantified by ELISA. D. A ChIP assay was performed in IS-trained macrophages pre-treated with 2DG. 2% input was used as a normalization control. E. ChIP-Seq analysis was performed with anti-H3K4me3 antibody on chromatin isolated at day 6 from IS-trained and control macrophages. Enriched peaks in ChIP-Seq on H3K4me3 are shown as a volcano plot. (FC > 1.3, p < 0.05) F. Functional annotation of 59 upregulated Differentially regulated peaks (DRPs) on H3K4me3 in IS-trained macrophages were analyzed by Gene Ontology (Go) analysis with Go biological pathway and Reactome gene sets (FC > 1.3, p < 0.05). G. Screen shots of H3K4me3 modification in the promoter regions of IFI16, XRCC5, PQBP1 PSMA1, PSMA3, and OAZ3. *= p< 0.05, **= p < 0.01, and *** = p < 0.001 by two-tailed paired t-test.

IS-induced trained immunity is regulated by AhR.

Monocytes were pretreated with or without GNF351 (AhR antagonist; 10 μM) followed by IS (1,000 μM)-training for 6 days. A. On day 6, nuclear and cytosol fraction were prepared and immunoblotted for AhR protein. Band intensity in immunoblots was quantified by densitometry. β-ACTIN was used as a normalization control. B-D. On day 6, IS-trained cells with or without GNF351 were restimulated with LPS (10 ng/ml), for 24 hr. TNF-α and IL-6 in supernatants were quantified by ELISA (B). Expression of TNF-α and IL-6 (C) and IL-1β, MCP-1, and IL-10 mRNA (D) was analyzed by RT-qPCR. E. Monocytes were transfected with siRNA targeting AhR (siAhR) or negative control (siNC) for 1 day, followed by stimulation with IS for 24 hours. After a resting period of 5 days, cells were re-stimulated with LPS for 24 hours. mRNA expression levels of TNF-α and IL-6 were assessed using RT-qPCR. F. Enrichment of H3K4me3 on promoters of TNFA and IL6 loci was assessed on day 6 after IS-training. 1% input was used as a normalization control. Bar graphs show the mean ± SEM. * = p < 0.05, **= p < 0.01, and *** = p < 0.001 by two-tailed paired t-test.

AhR-dependent induction of the arachidonic acid pathway contributes to IS-induced trained immunity.

A. RNA-Seq analysis was performed on IS (1,000 μM)-trained monocytes. Volcano plots show differentially expressed genes between IS-trained and non-trained macrophages. B. Functional annotation of upregulated or downregulated genes (FC > ±2, p < 0.05) in IS-trained macrophages analyzed by Gene Ontology (GO) analysis with the Reactome Gene Set. C-D. GSEA (C) and heatmap (D) of genes related to the AA metabolism in IS-trained macrophages compared to non-trained cells or compared to IS-trained macrophages with GNF351 (10 μM) treatment were analyzed. E-F. On day 6 after IS-training with or without GNF351, expression of CYP1B1, ALOX5, ALOX5AP, and LTB4R1 mRNAs were quantitated using RT-qPCR (E) and cell lysates were prepared and immunoblotted for ALOX5 and ALOX5AP proteins (F). Band intensity in immunoblots was quantified by densitometry. β-ACTIN was used as a normalization control. G. Monocytes were transfected with siRNA targeting AhR (siAhR) or negative control (siNC) for 1 day, followed by stimulation with IS for 24 hours. After a resting period of 5 days, mRNA expression level of each gene was assessed using RT-qPCR. H. Monocytes were pretreated with zileuton (ALOX5 inhibitor, 100 μM) and trained with IS for 6 days followed by restimulation with LPS (10 ng/ml) for 24 hr. TNF-α and IL-6 in supernatants were quantified by ELISA. I. A ChIP assay was performed in IS-trained macrophages pre-treated with zileuton. 2% input was used as a normalization control. J. The pooled normal serum (NS) from health controls (HCs) or uremic serum (US) from patients with ESRD were used to treat monocytes isolated from HCs for 24 hr at 30% (v/v) followed by resting for 5 days. Expression of ALOX5, ALOX5AP, and LTB4R1 mRNAs were quantitated using RT-qPCR in trained macrophages with NS or US for 6 days. Bar graphs show the mean ± SEM. * = p < 0.05, **= p < 0.01, ***= p< 0.001 by two-tailed paired t-test.

Ex vivo and in vivo validation of IS-induced trained immunity.

A-C. CD14+ monocytes from ESRD patents and age-matched HCs were rested for 6 days and stimulated by LPS (10 ng/ml) for 24 hrs (A). TNF-α and IL-6 in supernatants were quantified by ELISA (B) and mRNA expression of IL-1β and MCP-1 were quantitated using RT-qPCR (C). D-G. ALOX5 and ALOX5AP protein levels in monocytes of (E-F) and in M-CSF-derived HMDM (G-H) of ESRD patients and HCs were analyzed by immunoblot analysis. Band intensity in immunoblots was quantified by densitometry. β-ACTIN was used as a normalization control. I. C57BL/6 mice were injected daily with 200 mg/kg IS for 5 days and rested for another 5 days prior to LPS (5 mg/kg) treatment. Mice were sacrificed at 75 min post-LPS injection. J. TNF-α and IL-6 in serum were quantified by ELISA. K. Before LPS injection, IS-trained mice were sacrificed, and spleens were mechanically separated. Isolated splenic myeloid cells were treated ex vivo with LPS (10 ng/ml) for 24 hr and TNF-α and IL-6 in supernatants were quantified by ELISA. L-M. The level of ALOX5 protein in splenic myeloid cells isolated from IS-trained or control mice was analyzed by western blot. The graph shows the band intensity quantified by the densitometry (M). N. Isolated splenic myeloid cells were treated ex vivo with LPS (10 ng/ml), along with zileuton (100 µM). The levels of TNF-α and IL-6 in the supernatants were quantified using ELISA. The graphs show the median (B-C) or the mean ± SEM (F-N). *= p < 0.05, **= p < 0.01, and *** = p < 0.001 by unpaired non-parametric t-test or by two-tailed paired t-test between zileuton treatment group and no-treatment group (N).

Proposed mechanism of IS-induced trained immunity.

IS-induced trained immunity in human monocytes is mediated by epigenetic reprogramming and metabolic rewiring via histone modification H3K4m3 and increased glycolysis and mitochondrial respiration, respectively. Direct interaction of uremic toxin IS with the AhR in human monocytes activates AhR signaling pathways that are involved in enhanced expression of the arachidonic acid metabolism-related genes ALOX5, ALOX5AP, and LTB4R1 and augmented production of TNF-α and IL-6 upon stimulation with LPS as secondary stimulus via epigenetic regulation. A pivotal role of each pathway or molecule was confirmed by in vitro assay with inhibitors including GNF351 (an AhR antagonist), zileuton (an ALOX5 inhibitor), U75302 (a BLT1 receptor inhibitor), 2DG (a glycolysis inhibitor), and MTA (a non-selective methyltransferase inhibitor). Meanwhile, the AhR-independent mechanism contributes to metabolic rewiring, such as increased glycolysis in IS-trained macrophages, which leads to enhanced proinflammatory responses upon secondary stimulation.