The Effects of Fisetin on Gene Expression Profile and Cellular Metabolism in IFN-γ-Stimulated Macrophage Inflammation

Abstract

Although interferon-gamma (IFN-γ) is known as a critical factor in polarizing macrophages into the pro-inflammatory state for immune response, how dietary flavonoids regulate IFN-γ response for anti-inflammation is incompletely elucidated. This study aims to investigate the effect of fisetin, a typical flavonol, on the inhibition of IFN-γ-induced inflammation by RNA sequencing (RNA-Seq) and cellular metabolism analysis. RAW264 macrophages pretreated with fisetin following IFN-γ stimulation were subjected to RNA-Seq to analyze alterations in gene expression. Cellular signaling and transcription were investigated using enrichment analysis, motif analysis, and transcription factor prediction. Cellular metabolic state was assessed by measuring the oxygen consumption rate (OCR) and lactate level to reflect mitochondrial respiration and glycolysis. Alterations in signaling proteins were confirmed by Western blot. The results revealed that fisetin downregulated the IFN-γ-induced expression of pro-inflammatory genes and M1 marker genes such as Cxcl9, Il6, Cd80, Cd86, and Nos2. In cellular metabolism, fisetin upregulated the oxidative phosphorylation (OXPHOS) pathway, restored impaired OCR, and reduced lactate production induced by IFN-γ. Motif analysis suggested that fisetin suppressed the activation of IFN-regulatory factor 1 (IRF1). Western blot data further confirmed that fisetin inhibited the phosphorylation of Jak1, Jak2, and STAT1, and decreased the nuclear accumulation of phosphorylated STAT1 and IRF1 induced by IFN-γ. Taken together, our data revealed that fisetin is a potent flavonoid that attenuates IFN-γ-stimulated murine macrophage inflammation and ameliorates disrupted cellular metabolism with a possible Jak1/2-STAT1-IRF1 pathway.

Keywords: IFN-γ; IRF1; RNA-Seq; cellular metabolism; fisetin; inflammation; macrophages.

Figure 1

Fisetin altered the gene expression profile in interferon-gamma (IFN)-γ-stimulated macrophages. (A) Principal component analysis (PCA) of RNA sequencing (RNA-Seq) data showed that the gene expression profile with or without fisetin pretreatment in IFN-γ-stimulated macrophages formed distinct clusters. (B) Volcano plot of the differentially expressed genes (DEGs) in IFN-γ-stimulated cells compared to non-treated cells (IFN-γ/CON). (C) Volcano plot of the DEGs in fisetin-pretreated IFN-γ-stimulated cells compared to IFN-γ-stimulated cells (FisIFN-γ/IFN-γ). RAW264 cells were pre-cultured for 21 h and starved in serum-free medium for 2.5 h. The cells were then treated with or without 5 μM fisetin for 30 min and subsequently exposed to 10 ng/mL IFN-γ for 12 h. Total RNA was isolated for RNA-Seq. Fold change (FC) > 1.3 or < 0.77 were identified as DEGs with q-value < 0.05. Three biological replicates were used for each group.

Figure 2

Fisetin inhibited IFN-γ-induced inflammation. (A,B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of upregulated DEGs in IFN-γ-stimulated cells compared to non-treated cells (IFN-γ/CON) (A) and downregulated DEGs in fisetin-pretreated IFN-γ-stimulated cells compared to IFN-γ-stimulated cells (FisIFN-γ/IFN-γ) (B). Red and blue terms represent that the pathway was upregulated by IFN-γ, but downregulated by fisetin pretreatment. (C,D) Gene Set Enrichment Analysis (GSEA) plots of the total altered genes in interferon gamma response gene set in IFN-γ-stimulated cells compared to control without treatment (C) and in fisetin-pretreated IFN-γ-stimulated cells compared to IFN-γ-stimulated cells (D). (E,F) GSEA plots of the total altered genes in inflammatory response gene set in IFN-γ-stimulated cells compared to control without treatment (E) and in fisetin-pretreated IFN-γ-stimulated cells compared to IFN-γ-stimulated cells (F). The gene sets were from mouse-ortholog hallmark gene sets of the molecular signatures database. (G–I) Expression heatmaps of representative genes from interferon gamma response gene set (G), representative pro-inflammatory and anti-inflammatory genes (H), as well as representative genes from macrophages M1 and M2 markers (I). Color gradient reflects row Z-score.

Figure 3

Fisetin modulated metabolism in IFN-γ-stimulated macrophages. (A,B) GSEA plot of oxidative phosphorylation (OXPHOS) gene set in IFN-γ-stimulated cells compared to non-treated cells (A) and in fisetin-pretreated IFN-γ-stimulated cells compared to IFN-γ-stimulated cells (B). The gene set was from mouse-ortholog hallmark gene sets of the molecular signatures database. (C) Expression heatmap of representative genes from OXPHOS gene set. Color gradient reflects row Z-score. (D) Fisetin rescued impaired basal oxygen consumption rate (OCR) in IFN-γ-induced macrophages (n = 6). (E) Fisetin decreased IFN-γ-enhanced lactate production (n = 3). For (D,E), RAW264 cells were pre-cultured for 21 h and starved in serum-free medium for 2.5 h. The cells were then treated with 0–5 μM fisetin for 30 min and subsequently exposed to 10 ng/mL IFN-γ for 12 h. The cells were used for OCR measurement, and the culture medium was collected for lactate determination. Each value represents the mean ± SD; different letters between groups indicate significant differences (p < 0.05). For OCR and lactate determination, at least two separate experiments were performed.

Figure 4

Motif ISRE and IRF1 presented in the promoters of fisetin-downregulated DEGs. (A) Homer known motif enrichment results of IFN-γ-upregulated DEGs compared to non-treated cells (IFN-γ/CON, UP). Top 11 motifs were shown. (B) Homer known motif enrichment results of fisetin-downregulated DEGs compared to IFN-γ-stimulated cells (FisIFN-γ/IFN-γ, DOWN). Top 11 motifs were shown. (C) Homer de novo motif enrichment results of fisetin-downregulated DEGs compared to IFN-γ-stimulated cells (FisIFN-γ/IFN-γ, DOWN). The generated 5 motifs were shown. The motifs colored in red are enriched in both IFN-γ-upregulated and fisetin-downregulated DEGs.

Figure 5

Fisetin-downregulated genes were associated with IFN-regulatory factor1 (IRF1). (A,B) Transcription factor inference by epigenetic landscape in silico deletion analysis (LISA) on the top 500 DEGs that were upregulated (Vertical axis) or downregulated (Horizontal axis) by IFN-γ, compared to non-treated cells (A), and by fisetin pretreatment, compared to IFN-γ-stimulated cells (B). (C–E) Expression heatmaps of the representative genes targeted by STAT1 (C), IRF1 (D), or both STAT1 and IRF1 (E). Color gradient reflects row Z-score.

Figure 6

Fisetin suppressed IFN-γ-induced pro-inflammatory mediators at protein levels. (A) Fisetin repressed IFN-γ-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in a dose-dependent manner. (B) Fisetin dose-dependently inhibited IFN-γ-induced nitric oxide (NO) production (n = 3). RAW264 cells were pre-cultured for 21 h and starved in serum-free medium for 2.5 h. The cells were treated with 0–20 μM fisetin for 30 min and then exposed to 10 ng/mL IFN-γ for 12 h. Whole-cell lysates were harvested for Western blot assay, and the culture medium was collected for NO determination. The relative density was calculated as the intensity of the treatment relative to that of the control normalized to α-tubulin by densitometry. Each value represents the mean ± SD; different letters between groups indicate significant differences (p < 0.05). The blots presented are representatives from at least three independent experiments, using cells derived from at least two separate preparations. For NO determination, at least two separate experiments were performed.

Figure 7

IFN-γ did not activate MAPK pathway. (A,B) Time-course experiment showed that LPS activated MKK4-JNK-AP-1 cascade with no effect on Jak2-STAT1-IRF1 pathway (A), while IFN-γ activated Jak2-STAT1-IRF1 pathway with no effect on MKK4-JNK-AP-1 cascade (B). (C) IFN-γ did not activate AP-1 (p-c-Jun). RAW264 cells were pre-cultured as described above. The cells were then treated with or without 20 μM fisetin for 30 min and subsequently exposed to 40 ng/mL LPS or 10 ng/mL IFN-γ. Whole-cell lysates were harvested after a defined stimulation time (A,B) or 30 min (for c-Jun and p-c-Jun in (C)), and analyzed by Western blot assay. The relative density was calculated as the intensity of the treatment relative to that of the control normalized to α-tubulin or respective total proteins by densitometry. The bands of total proteins for (A,B) and semi-quantitative graphs are given in Supplementary Figure S1. Each value represents the mean ± SD; different letters between groups indicate significant differences (p < 0.05). The blots presented are representatives from at least three independent experiments, using cells derived from at least two separate preparations.

Figure 8

Fisetin inhibited Jak1/2-STAT1-IRF1 pathway. (A) Fisetin inhibited phosphorylation of Jak1, Jak2, and STAT1, and the expression of IRF1 induced by IFN-γ. (B) Fisetin reduced nuclear accumulation of p-STAT1 and IRF1 in a dose-dependent manner. RAW264 cells were pre-cultured as described above. The cells were then treated with 0–20 μM fisetin for 30 min and subsequently exposed to 10 ng/mL IFN-γ. Whole-cell lysates were harvested after 30 min (for p-Jak1, Jak1, p-Jak2, Jak2, p-STAT1, and STAT1 in (A)) or 2 h (for IRF1 in (A,B)), and then analyzed by Western blot assay. Nuclear and cytoplasmic fractionation was performed as described in Materials and Methods. The relative density was calculated as the intensity of the treatment relative to that of the control normalized to respective total proteins, α-tubulin (for cytoplasmic fraction), or TBP (for nuclear fraction) by densitometry. Each value represents the mean ± SD; different letters between groups indicate significant differences (p < 0.05). The blots presented are representatives from at least three independent experiments, using cells derived from at least two separate preparations.