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The transcriptional PPARβ/δ network in human macrophages defines a unique agonist-induced activation state.

Adhikary T, Wortmann A, Schumann T, Finkernagel F, Lieber S, Roth K, Toth PM, Diederich WE, Nist A, Stiewe T, Kleinesudeik L, Reinartz S, Müller-Brüsselbach S, Müller R - Nucleic Acids Res. (2015)

Bottom Line: Surprisingly, bioinformatic analyses also identified immune stimulatory effects.Comparison with published data revealed a significant overlap of the PPARβ/δ transcriptome with coexpression modules characteristic of both anti-inflammatory and pro-inflammatory cytokines.Our findings indicate that PPARβ/δ agonists induce a unique macrophage activation state with strong anti-inflammatory but also specific immune stimulatory components, pointing to a context-dependent function of PPARβ/δ in immune regulation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology and Tumor Research (IMT), Center for Tumor Biology and Immunology (ZTI), Philipps University, 35043 Marburg, Germany.

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Genome-wide identification of PPARβ/δ target genes in macrophages. (A) Overlap of genes induced by L165,041 and repressed by ST247 or PT-S264 in MDMs cultured for 6 days followed by treatment with DMSO or ligands for 24 h. Data are derived from two independent experiments using either R10 (L165,041, ST247) or XV0 (L165,041, PT-S264) medium. Genes with a logFC > 0.7 in one culture condition, a logFC > 0 in both media, an FPKM ≥ 0.3 and a raw tag count of at least 50 were scored as positive. (B) Overlap of genes repressed by L165,041 and activated by ST247 in MDMs (conditions as in (A)). (C) IPA ‘Diseases and Functions Annotation’ of L165,041-regulated genes (examples of functionally different clusters with low P-values and high z-scores). (D) Overlap of L165,041-regulated genes linked to different functions (according to IPA ‘Diseases and Functions Annotation’; all clusters with n > 30 genes). (E) IPA ‘Upstream Regulator Analysis’ of L165,041-regulated genes (top regulators by P-value). (F) RT-qPCR analysis of target gene regulation by the PPARβ/δ agonist GW501516 in BMDMs from wild-type and Ppard  mice differentiated for 6 days in the presence of GM-CSF (sample size: 3 each). The data show the fold change (mean of triplicates) in response to the ligand relative to solvent treated wild-type and Ppard  control cells.
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Figure 2: Genome-wide identification of PPARβ/δ target genes in macrophages. (A) Overlap of genes induced by L165,041 and repressed by ST247 or PT-S264 in MDMs cultured for 6 days followed by treatment with DMSO or ligands for 24 h. Data are derived from two independent experiments using either R10 (L165,041, ST247) or XV0 (L165,041, PT-S264) medium. Genes with a logFC > 0.7 in one culture condition, a logFC > 0 in both media, an FPKM ≥ 0.3 and a raw tag count of at least 50 were scored as positive. (B) Overlap of genes repressed by L165,041 and activated by ST247 in MDMs (conditions as in (A)). (C) IPA ‘Diseases and Functions Annotation’ of L165,041-regulated genes (examples of functionally different clusters with low P-values and high z-scores). (D) Overlap of L165,041-regulated genes linked to different functions (according to IPA ‘Diseases and Functions Annotation’; all clusters with n > 30 genes). (E) IPA ‘Upstream Regulator Analysis’ of L165,041-regulated genes (top regulators by P-value). (F) RT-qPCR analysis of target gene regulation by the PPARβ/δ agonist GW501516 in BMDMs from wild-type and Ppard mice differentiated for 6 days in the presence of GM-CSF (sample size: 3 each). The data show the fold change (mean of triplicates) in response to the ligand relative to solvent treated wild-type and Ppard control cells.

Mentions: We used this experimental system to identify ligand-responsive genes as well as PPARβ/δ and RXR binding sites in macrophages by deep sequencing technologies. RNA-Seq data obtained with MDMs cultured either in R10 or serum-free synthetic X-VIVO 10 medium (XV0) revealed a total of 285 protein-coding genes upregulated by PPARβ/δ agonist L165,041 and 246 genes downregulated by the inverse agonists ST247 or PT-S264; logFC ≥ 0.7; FPKM ≥ 0.3), 29.6% of the latter (n = 73) overlapping with the agonist-induced gene set (Figure 2A; Supplementary Table S2). Our RNA-Seq also identified a large fraction of genes repressed by the agonist L165,041 (n = 388) and upregulated by the inverse agonist ST247 (n = 174), with 40 genes (10.3%) overlapping (Figure 2B; Supplementary Table S2). Diseases and functions annotation of the L165,041-induced gene set showed a strong association with the inhibition of cell death of immune cells and suppression of immune cell functions, including migration, inflammatory response, activation, homing, adhesion, chemotaxis and phagocytosis (Figure 2C; Supplementary Table S3). The gene set representing inflammation clearly overlapped with cell survival, migration/movement, adhesion and recruitment/infiltration/ chemotaxis (Figure 2D), suggesting that these to a large extent represent genes with functions in immune regulation. Interestingly, ‘Inflammation of intestine’ and ‘Colitis’ showed a positive activation z-score (Figure 2C), providing a first hint that the response to L165,041 may not be strictly anti-inflammatory. Likewise, lipid metabolism (‘Concentration of acylglycerol’) was upregulated, consistent with the known metabolic role of PPARβ/δ. Finally, analysis of the known upstream regulators of these genes (signaling molecules and transcription factors) identified two groups: canonically regulated (L165,041-induced) genes known to be activated by PPAR agonists (pirixinic acid, fibrates, glitazones) were upregulated by L165,041, while genes induced by pro-inflammatory signaling via LPS, TNFα, IFNγ, IL-1β, STAT3 or TLR4 were downregulated (inverse target genes).


The transcriptional PPARβ/δ network in human macrophages defines a unique agonist-induced activation state.

Adhikary T, Wortmann A, Schumann T, Finkernagel F, Lieber S, Roth K, Toth PM, Diederich WE, Nist A, Stiewe T, Kleinesudeik L, Reinartz S, Müller-Brüsselbach S, Müller R - Nucleic Acids Res. (2015)

Genome-wide identification of PPARβ/δ target genes in macrophages. (A) Overlap of genes induced by L165,041 and repressed by ST247 or PT-S264 in MDMs cultured for 6 days followed by treatment with DMSO or ligands for 24 h. Data are derived from two independent experiments using either R10 (L165,041, ST247) or XV0 (L165,041, PT-S264) medium. Genes with a logFC > 0.7 in one culture condition, a logFC > 0 in both media, an FPKM ≥ 0.3 and a raw tag count of at least 50 were scored as positive. (B) Overlap of genes repressed by L165,041 and activated by ST247 in MDMs (conditions as in (A)). (C) IPA ‘Diseases and Functions Annotation’ of L165,041-regulated genes (examples of functionally different clusters with low P-values and high z-scores). (D) Overlap of L165,041-regulated genes linked to different functions (according to IPA ‘Diseases and Functions Annotation’; all clusters with n > 30 genes). (E) IPA ‘Upstream Regulator Analysis’ of L165,041-regulated genes (top regulators by P-value). (F) RT-qPCR analysis of target gene regulation by the PPARβ/δ agonist GW501516 in BMDMs from wild-type and Ppard  mice differentiated for 6 days in the presence of GM-CSF (sample size: 3 each). The data show the fold change (mean of triplicates) in response to the ligand relative to solvent treated wild-type and Ppard  control cells.
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Figure 2: Genome-wide identification of PPARβ/δ target genes in macrophages. (A) Overlap of genes induced by L165,041 and repressed by ST247 or PT-S264 in MDMs cultured for 6 days followed by treatment with DMSO or ligands for 24 h. Data are derived from two independent experiments using either R10 (L165,041, ST247) or XV0 (L165,041, PT-S264) medium. Genes with a logFC > 0.7 in one culture condition, a logFC > 0 in both media, an FPKM ≥ 0.3 and a raw tag count of at least 50 were scored as positive. (B) Overlap of genes repressed by L165,041 and activated by ST247 in MDMs (conditions as in (A)). (C) IPA ‘Diseases and Functions Annotation’ of L165,041-regulated genes (examples of functionally different clusters with low P-values and high z-scores). (D) Overlap of L165,041-regulated genes linked to different functions (according to IPA ‘Diseases and Functions Annotation’; all clusters with n > 30 genes). (E) IPA ‘Upstream Regulator Analysis’ of L165,041-regulated genes (top regulators by P-value). (F) RT-qPCR analysis of target gene regulation by the PPARβ/δ agonist GW501516 in BMDMs from wild-type and Ppard mice differentiated for 6 days in the presence of GM-CSF (sample size: 3 each). The data show the fold change (mean of triplicates) in response to the ligand relative to solvent treated wild-type and Ppard control cells.
Mentions: We used this experimental system to identify ligand-responsive genes as well as PPARβ/δ and RXR binding sites in macrophages by deep sequencing technologies. RNA-Seq data obtained with MDMs cultured either in R10 or serum-free synthetic X-VIVO 10 medium (XV0) revealed a total of 285 protein-coding genes upregulated by PPARβ/δ agonist L165,041 and 246 genes downregulated by the inverse agonists ST247 or PT-S264; logFC ≥ 0.7; FPKM ≥ 0.3), 29.6% of the latter (n = 73) overlapping with the agonist-induced gene set (Figure 2A; Supplementary Table S2). Our RNA-Seq also identified a large fraction of genes repressed by the agonist L165,041 (n = 388) and upregulated by the inverse agonist ST247 (n = 174), with 40 genes (10.3%) overlapping (Figure 2B; Supplementary Table S2). Diseases and functions annotation of the L165,041-induced gene set showed a strong association with the inhibition of cell death of immune cells and suppression of immune cell functions, including migration, inflammatory response, activation, homing, adhesion, chemotaxis and phagocytosis (Figure 2C; Supplementary Table S3). The gene set representing inflammation clearly overlapped with cell survival, migration/movement, adhesion and recruitment/infiltration/ chemotaxis (Figure 2D), suggesting that these to a large extent represent genes with functions in immune regulation. Interestingly, ‘Inflammation of intestine’ and ‘Colitis’ showed a positive activation z-score (Figure 2C), providing a first hint that the response to L165,041 may not be strictly anti-inflammatory. Likewise, lipid metabolism (‘Concentration of acylglycerol’) was upregulated, consistent with the known metabolic role of PPARβ/δ. Finally, analysis of the known upstream regulators of these genes (signaling molecules and transcription factors) identified two groups: canonically regulated (L165,041-induced) genes known to be activated by PPAR agonists (pirixinic acid, fibrates, glitazones) were upregulated by L165,041, while genes induced by pro-inflammatory signaling via LPS, TNFα, IFNγ, IL-1β, STAT3 or TLR4 were downregulated (inverse target genes).

Bottom Line: Surprisingly, bioinformatic analyses also identified immune stimulatory effects.Comparison with published data revealed a significant overlap of the PPARβ/δ transcriptome with coexpression modules characteristic of both anti-inflammatory and pro-inflammatory cytokines.Our findings indicate that PPARβ/δ agonists induce a unique macrophage activation state with strong anti-inflammatory but also specific immune stimulatory components, pointing to a context-dependent function of PPARβ/δ in immune regulation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology and Tumor Research (IMT), Center for Tumor Biology and Immunology (ZTI), Philipps University, 35043 Marburg, Germany.

Show MeSH
Related in: MedlinePlus