PPARγ Deficiency Suppresses the Release of IL-1β and IL-1α in Macrophages via a Type 1 IFN-Dependent Mechanism

Abstract

Obesity and diabetes modulate macrophage activation, often leading to prolonged inflammation and dysfunctional tissue repair. Increasing evidence suggests that the NLRP3 inflammasome plays an important role in obesity-associated inflammation. We have previously shown that activation of the lipotoxic inflammasome by excess fatty acids in macrophages occurs via a lysosome-dependent pathway. However, the mechanisms that link cellular lipid metabolism to altered inflammation remain poorly understood. PPARγ is a nuclear receptor transcription factor expressed by macrophages that is known to alter lipid handling, mitochondrial function, and inflammatory cytokine expression. To undercover novel links between metabolic signaling and lipotoxic inflammasome activation, we investigated mouse primary macrophages deficient in PPARγ. Contrary to our expectation, PPARγ knockout (KO) macrophages released significantly less IL-1β and IL-1α in response to lipotoxic stimulation. The suppression occurred at the transcriptional level and was apparent for multiple activators of the NLRP3 inflammasome. RNA sequencing revealed upregulation of IFN-β in activated PPARγKO macrophages, and this was confirmed at the protein level. A blocking Ab against the type 1 IFNR restored the release of IL-1β to wild type levels in PPARγKO cells, confirming the mechanistic link between these events. Conversely, PPARγ activation with rosiglitazone selectively suppressed IFN-β expression in activated macrophages. Loss of PPARγ also resulted in diminished expression of genes involved in sterol biosynthesis, a pathway known to influence IFN production. Together, these findings demonstrate a cross-talk pathway that influences the interplay between metabolism and inflammation in macrophages.

Figure 1.. PPARγ loss of function is associated with impaired IL-1 cytokine release in primary macrophages.

(A-C) Peritoneal macrophages (pMACs) isolated from WT (open bars) or mPPARγKO mice (filled bars) were treated with control (BSA-LPS), palm (250 μM)-LPS (100 ng), or stearate (150 μM)-LPS for 20h and the release of IL-1β (A), (B) IL-1α, and (C) TNFα was determined by ELISA. (D-F) pMACs isolated from WT (open bars) or mPPARγKO mice (filled bars) were treated with LPS and ATP, alum or silica as described in the methods and IL-1β (D), (E) IL-1α, and (F) TNFα was determined by ELISA. WT pMACs were treated with veh (open bars) or T0070907 (T007; gray bars) for 24h after which they received the indicated stimuli. The release of IL-1β (G), (H) IL-1α, and (I) TNFα was determined by ELISA. Bar graphs report the mean ± standard error (SE) for a minimum of 3 experiments, each performed in triplicate. *, p<0.05 for WT vs. mPPARγKO or veh vs. T007

Figure 2.. PPARγ deficiency leads to decreased levels of IL-1 mRNA and protein levels.

(A) WT or mPPARγKO (KO) macrophages were stimulated with BSA-PBS (vehicle) or palm-LPS for 16h and the protein level of pro-IL-1β and pro-IL-1α was assessed by western blotting. Tubulin (tub) is shown as a loading control. (B) pMACs isolated from WT (open bars) or mPPARγKO mice (filled bars) were treated with vehicle or palm-LPS for 8h and mRNA expression of IL-1β, IL-1α, and NLRP3 was assessed by qRT-PCR. (C, D) Kinetic assessment of IL-1β (C) and TNFα (D) mRNA levels following palm-LPS stimulation in WT and mPPARγKO cells. (E) WT or mPPARγ KO cells were treated with palm-LPS for 4h after actinomycin D was added to the culture. The levels of IL-1β mRNA relative to baseline was performed by qRT-PCR. Each genotype was compared to its own baseline which was arbitrarily defined as 1. Bar graphs report the mean ± standard error (SE) for a minimum of 3 experiments, each performed in triplicate. *, p<0.05 for WT vs. mPPARγKO.

Figure 3.. mPPARγKO macrophages produce less IL-1 cytokines in vivo.

(A-C) WT or mPPARγKO mice were injected with thioglycollate to induce macrophage recruitment to the peritoneal cavity. At day 4, when ~90% of the cells in the peritoneum are macrophages, the mice were given an intraperitoneal injection of LPS (10 μg/200 μl) and 16h later IL-1β (A), IL-1α (B), and TNFα (C) levels in the peritoneal fluid was quantified by ELISA. (D) Day 4 peritoneal cell count for WT vs. mPPARγKO mice following thioglycollate injection. Bar graphs report the mean ± standard error (SE) and the individual dots each represent one mouse. The p values for the comparison of WT vs. mPPARγKO mice are shown.

Figure 4.. The suppression of IL-1 release from PPARγ KO macrophages is independent of nitric oxide and IL-10.

(A) iNOS mRNA levels in WT vs. PPARγKO pMACs 8h after the indicated stimulation. (B) WT or mPPARγKO pMACS were treated with vehicle or palm-LPS and nitrite levels were quantified via colorimetric assay. At the same time, WT macrophages were treated with palm-LPS together with IFNβ (500U) in the presence or absence of the iNOS inhibitor L-NIL (10 μM). (C) pMACs from WT or KO mice were treated with palm-LPS in the presence of vehicle or L-NIL and IL-1β release was quantified by ELISA. (D) WT macrophages were treated with palm-LPS for 20h in the presence of vehicle (open bars) or recombinant IL-10 (20 ng/ml; gray bars) ± α-IL-10 receptor blocking antibody (IL-10Rab) and IL-1β release was measured by ELISA. (E) pMACs from WT or mPPARγKO mice were treated with palm-LPS for 20h in the presence of control antibody or IL-10Rab and IL-1β release was quantified by ELISA. Bar graphs report the mean ± standard error (SE) for a minimum of 3 experiments, each performed in triplicate. *, p<0.05 for WT vs. mPPARγKO, #, p<0.05 for veh vs. L-NIL or IL-10Rab. ND, not detected

Figure 5.. RNA sequencing reveals type 1 interferon gene signature in activated PPARγKO macrophages.

(A, B) WT or PPARγKO pMACs were treated with BSA-PBS or palm-LPS for 8h after which RNA was isolated and RNA sequencing was performed. (A) Group summary for pathways related to IFNβ in PPARg vs. WT pMACS treated with LPS. (B) Heatmap expression profile of genes from the cellular response to IFNβ GO biologic processes module. WT and PPARγKO cells are shown under basal conditions (BSA-PBS) and after activation (palm-LPS). The color map for fold change is shown.

Figure 6.. Augmented expression and release of IFNβ in PPARγ deficient macrophages in response to palm-LPS.

(A) pMACs isolated from WT (open bars) or mPPARγKO mice (filled bars) were treated with vehicle or palm-LPS for 8h and mRNA expression of the indicated targets was determined by qRT-PCR. (B) Macrophages were treated with vehicle or palm-LPS for 4h or 8h after which cell lysates were isolated and phopho(P)-STAT1 (Y701) was assessed by western blotting. Total (tot) STAT1 and tubulin are shown as controls. (C, D) pMACs from WT or PPARγ KO mice were stained with an antibody the type 1 interferon receptor (IFNAR) and surface expression was assessed by flow cytometry. A representative histogram (C) and grouped MFI data (D) are shown. (E) mRNA levels of IFNβ were quantified in WT and mPPARγKO cells at 1h after palm-LPS treatment via qRT-PCR. (F) IFNβ release from WT and KO pMACs 6h after palm-LPS stimulation. (G) Protein was isolated from WT or PPARγKO macrophages and TRIF protein expression was assessed by western blotting. Bar graphs report the mean ± standard error (SE) for a minimum of 3 experiments, each performed in triplicate. *, p<0.05 for WT vs. mPPARγKO; ns, non-significant

Figure 7.. Suppression of IL-1 expression occurs via a type 1 interferon dependent mechanism.

(A) WT or PPARγKO pMACs were treated with palm-LPS for 20h in the presence of an IFNAR blocking antibody or control Ig and IL-1β release was quantified by ELISA. (B) WT or IFNAR KO pMACs were treated with veh or the PPARγ antagonist T0070907 24h prior to stimulation with palm-LPS and IL-1β release was quantified by ELISA. (C, D) WT or PPARγKO pMACs were treated with palm-LPS for 8h (mRNA) or 16h (protein) in the presence of an IFNAR blocking antibody or control Ig and IL-1β expression was assessed via qRT-PCR (C) and western blotting (D). (E) WT or KO pMACs were stimulated with palm-LPS or IFNβ (500U) for 4h and P-STAT was assessed by western blotting. (F) pMACs from WT or mPPARγKO mice were treated with IFNβ and mRNA expression of the IFN gene target CXCL10 was assessed via qRT-PCR. Bar graphs report the mean ± standard error (SE) for a minimum of 3 experiments, each performed in triplicate. *, p<0.05 for WT vs. mPPARγKO or; ns, non-significant

Figure 8.. Low dose IFNβ phenocopies PPARγ loss-of-function in primary macrophages.

(A, B) WT pMACs were treated with palm-LPS and increasing concentrations of IFNβ after which IL-1β (A) and TNFα (B) release was quantified by ELISA. (C) Macrophages were treated with vehicle or palm-LPS for 16h in the presence of IFNβ (50U) and pro-IL-1β and NLRP3 protein levels were assessed by western blotting. Tubulin is shown as a loading control. (D) pMACS were treated as indicated and gene expression of IL-1β and CXCL10 was assessed 8h after stimulation via qRT-PCR. (E) pMACS were stimulated with palm-LPS ± IFNβ in the presence of IFNAR blocking ab or control Ig and IL-1β release was determined by ELISA. (F, G) WT (open bar) or IFNAR KO (gray bar) macrophages were stimulated with palm-LPS and IL-1β mRNA (F) or protein (G) levels were assessed at 8h and 16h, respectively. Bar graphs report the mean ± standard error (SE) for a minimum of 3 experiments, each performed in triplicate. *, p<0.05 for veh vs. IFNβ or WT vs. IFNAR KO; ns, non-significant

Figure 9.. PPARγ activation suppresses IFNβ.

(A-F) WT or PPARγ KO macrophages were treated with BSA-PBS/veh (open bars), palm-LPS/veh (filled bars), palm-LPS/rosiglitazone 1 μM (gray bars) for 16h followed by stimulation with palm-LPS for 8h in continued presence of the PPARγ agonist. mRNA was isolated from the macrophages and the expression of IFNβ (A) and several of its gene targets (B-D) was assessed by qRT-PCR. In addition, mRNA expression of the pro-inflammatory cytokines IL-1β (E) and TNFα (F) was also determined. (G-H) WT or KO pMACs were treated with rosiglitazone or vehicle as described above followed by palm-LPS for 16h. The release of IFNβ (G) and IL-1β (H) was quantified by ELISA. Bar graphs report the mean ± standard error (SE) for a minimum of 3 experiments, each performed in triplicate. *, p<0.05 for veh vs. rosiglitazone; ns, non-significant

Figure 10.. Expression of genes involved in sterol biosynthesis are suppressed in PPARγ deficient cells.

(A) Heatmap display of gene expression from sterol biosynthesis pathway obtained from RNA sequencing data macrophages treated with BSA-PBS or palm-LPS for 8h. The brackets indicate genes with particularly low expression in activated PPARγ KO cells (blue). (B) schematic of the metabolic pathway and enzymes (red text) involved in sterol biosynthesis. (C) mRNA expression of the indicated sterol biosynthesis genes was determined via qRT-PCR in WT and PPARγKO cells treated with control or palm-LPS for 8h. (D) pMACs from WT or PPARγ deficient macrophages were treated with 1 μM rosiglitazone (rosi) or vehicle (veh) for 16h after which they were treated with palm-LPS and the expression of sterol biosynthesis genes was assessed by qRT-PCR. (E) pMACs from WT or PPARγ deficient macrophages were treated with BSA-PBS or palm-LPS for 8h after which cells were lysed and free cholesterol, 25-hydroxycholesterol (HC), and 27-HC were measured by GC-MS/MS. Data is presented as normalized ratio an internal standard. Bar graphs report the mean ± standard error (SE) for a minimum of 3 experiments, each performed in triplicate. *, p<0.05 for WT vs. mPPARγKO; #, p<0.05 for veh vs. rosi; ns, non-significant

Figure 11.. PPARγ KO macrophages have enhanced sensitivity to STING ligands.

(A-C) WT or PPARγKO pMACs were stimulated with the STING activator cGAMP (0.5 μg/ml) or palm-LPS and mRNA was harvested 8h later. The expression of type 1 interferon gene targets CXCL10 (A), MX1 (B), and iNOS (C) was assessed via qRT-PCR. (D, E) pMACs from WT or PPARγ deficient macrophages were stimulated with the indicated concentrations of cGAMP and the expression of CXCL10 (D) and iNOS (E) was assessed by qRT-PCR. (F) WT or STING knock-in mice (KI) were treated with PBS or 10 μg/ml of cGAMP and the mRNA expression of CXCL10 was determined at 8h. (G-I) WT or PPARγKO pMACs were treated with cGAMP (0.5 μg/ml) or transfected with PIC for 8h and the expression of the indicated IFN gene targets was assessed by qRT-PCR. Bar graphs report the mean ± standard error (SE) for a minimum of 3 experiments, each performed in triplicate. *, p<0.05 for WT vs. mPPARγKO or STING KI; ns, non-significant