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. 2017 Feb 21;114(8):E1395-E1404.
doi: 10.1073/pnas.1621188114. Epub 2017 Jan 30.

Targeting IRE1 with small molecules counteracts progression of atherosclerosis

Ozlem Tufanli  1   2 , Pelin Telkoparan Akillilar  1   2 , Diego Acosta-Alvear  3   4 , Begum Kocaturk  1   2 , Umut Inci Onat  1   2 , Syed Muhammad Hamid  1   2 , Ismail Çimen  1   2 , Peter Walter  5   4 , Christian Weber  6   7 , Ebru Erbay  8   2
Affiliations

Targeting IRE1 with small molecules counteracts progression of atherosclerosis

Ozlem Tufanli et al. Proc Natl Acad Sci U S A. .

Abstract

Metaflammation, an atypical, metabolically induced, chronic low-grade inflammation, plays an important role in the development of obesity, diabetes, and atherosclerosis. An important primer for metaflammation is the persistent metabolic overloading of the endoplasmic reticulum (ER), leading to its functional impairment. Activation of the unfolded protein response (UPR), a homeostatic regulatory network that responds to ER stress, is a hallmark of all stages of atherosclerotic plaque formation. The most conserved ER-resident UPR regulator, the kinase/endoribonuclease inositol-requiring enzyme 1 (IRE1), is activated in lipid-laden macrophages that infiltrate the atherosclerotic lesions. Using RNA sequencing in macrophages, we discovered that IRE1 regulates the expression of many proatherogenic genes, including several important cytokines and chemokines. We show that IRE1 inhibitors uncouple lipid-induced ER stress from inflammasome activation in both mouse and human macrophages. In vivo, these IRE1 inhibitors led to a significant decrease in hyperlipidemia-induced IL-1β and IL-18 production, lowered T-helper type-1 immune responses, and reduced atherosclerotic plaque size without altering the plasma lipid profiles in apolipoprotein E-deficient mice. These results show that pharmacologic modulation of IRE1 counteracts metaflammation and alleviates atherosclerosis.

Keywords: atherosclerosis; endoplasmic reticulum stress; lipotoxicity; metaflammation; unfolded protein response.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
IRE1 regulates the expression of proatherogenic genes. (A) RNA-seq analysis in BMDMs treated with 60 μM STF-083010 or DMSO (control) for 6 hours. Volcano plot of differentially expressed mRNAs. (B) Analysis of atherosclerosis-related mRNAs using the IPA tool (details are in the text). (C–E) Confirmation of IRE1-dependent atherogenic gene regulation in mouse BMDMs treated with STF-083010 or DMSO (control) by qRT-PCR. (F–H) qRT-PCR analysis of atherogenic gene expression in IRE1−/− MEFs on forced expression of XBP1s or restoring IRE1’s function. Data: mean values ± SEM; n = 3. Student’s t test. Ctrl, control; ns, not significant. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.
Fig. S1.
Fig. S1.
Modulation of IRE1 signaling by various methods: validation and impact on various proatherogenic genes. (A) qRT-PCR analysis of Mmp9 mRNA levels in mouse BMDMs treated with 60 μM STF-083010 or DMSO (control). (B–E) qRT-PCR analysis of Il-1β, Ccl2, S100a8, and Mmp9 mRNA levels in mouse BMDMs treated with 4µ8c (at the indicated doses) or DMSO (control). (F) Validation of results in A and Fig. 1 A–E. IRE1 phosphorylation was detected by Western blot in lysates of PA-stimulated BMDMs treated with STF-083010 or DMSO (control). (G and H) Validation of results in B–E. (G) qRT-PCR analysis of spliced Xbp1 mRNA levels in mouse BMDMs treated with increasing doses of 4µ8C. (H) Western blot analysis of IRE1 phosphorylation in BMDMs after IRE1 inhibition with 4µ8c. (I) Validation of results in Fig. 1 F–H. qRT-PCR analysis of spliced Xbp1 mRNA levels in IRE1−/− MEFs transfected with plasmids encoding IRE1 or XBP1s. Statistics are the same as in Fig. 1. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.
Fig. 2.
Fig. 2.
IRE1 regulates lipid-induced IL-1β secretion in mouse and human macrophages. Il-1β (A) mRNA and (B) protein levels measured from LPS-primed and PA-stimulated mouse BMDMs treated with 150 μM STF-083010 or DMSO (control) by qRT-PCR and ELISA, respectively. Il-1β (C) mRNA and (D) protein levels measured from LPS-primed and PA-stimulated mouse BMDMs transfected with siRNAs against Ire1α or Xbp1 by qRT-PCR and ELISA, respectively. (E) Secreted IL-1β from LPS-primed, PA-stimulated human PBMCs treated or not treated with 100 μM 4μ8c measured by ELISA. (F) Same as E but protein levels were measured by immunoblotting to show the immature and processed forms of the cytokine (representative image of three independent blots). Statistics are the same as in Fig. 1. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.
Fig. S2.
Fig. S2.
Inhibition of IRE1 by various methods in lipid-stressed macrophages: validation of its impact on IL-1β. (A and B) Validation of results in Fig. 2 A and B. (A) qRT-PCR analysis of spliced Xbp1 mRNA levels in LPS-primed, PA-stimulated mouse BMDMs treated with 150 μM STF-083010 or DMSO (control). (B) Western Blot analysis of p-IRE in LPS-primed, PA-stimulated mouse BMDMs treated with 150 μM STF-083010 or DMSO (control). (C–F) Inhibition of IRE1 RNase activity by 4µ8c and its impact on IL-1β. (C) qRT-PCR analysis of Il-1β mRNA levels in LPS-primed, PA-stimulated mouse BMDMs treated with 4µ8c (at the indicated doses). (D) Western blot analysis of secreted mature IL-1β from LPS-primed, PA-stimulated BMDMs treated with 4µ8c (at the indicated doses) or DMSO (control). (E) qRT-PCR analysis of spliced Xbp1 mRNA levels in LPS-primed, PA-stimulated mouse BMDMs treated with 4µ8c (at the indicated doses) or DMSO (control). (F) Western blot analysis of IRE1 phosphorylation in LPS-primed, PA-stimulated mouse BMDMs treated with 4µ8c (at the indicated doses) or DMSO (control). (G and H) Validation of Ire1α and Xbp1 knockdown in Figs. 2 C and D and 3B. qRT-PCR analysis of (G) Ire1α or (H) spliced Xbp1 mRNA levels in LPS-primed, PA-stimulated mouse BMDMs transfected with siRNAs against Ire1α or Xbp1. (I) Validation of results in Fig. 2 E and F. qRT-PCR analysis of spliced Xbp1 mRNA levels in LPS-primed human PBMCs treated with PA or a combination of PA and 4µ8c. Statistics are the same as in Fig. 1. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.
Fig. S3.
Fig. S3.
IRE1 regulates lipid-induced CCL2 production in macrophages. (A) qRT-PCR analysis of Ccl2 mRNA levels in LPS-primed, PA-stimulated BMDMs that were treated with 150 μM STF-083010 or DMSO (control). (B) ELISA measurements of secreted CCL2 from the conditioned medium used to culture the cells in A. (C and D) qRT-PCR analysis of Ccl2 mRNA levels in LPS-primed, PA-stimulated BMDMs that were (C) treated with 4µ8c (at the indicated doses) or DMSO (control) or (D) transfected with siRNAs against Ire1α or Xbp1. (E) ELISA measurements of secreted CCL2 from the cells in D. (F) qRT-PCR analysis of Ccl2 mRNA levels in LPS-primed, PA-stimulated human PBMCs treated with 4µ8c (at the indicated doses) or DMSO (control). Statistics are the same as in Fig. 1. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.
Fig. 3.
Fig. 3.
IRE1 inhibitors block lipid-induced mtROS release and inflammasome activation. (A and B) mtROS production was measured in LPS-primed, PA-stimulated mouse BMDMs after (A) 100 μM 4µ8c or DMSO (control) treatment or (B) transfection with scrambled or Xbp1-specific siRNAs. (C–E) Immunoblots of the levels of the zymogen (p45) and mature (p10) forms of caspase-1 in LPS-primed mouse BMDMs pretreated with 4μ8c (at the indicated doses) or DMSO (control) and stimulated with (C) PA, (D) other NLRP3 agonists (5 nM ATP, 200 μg/mL alum, or 400 μg/mL cholesterol crystals), or (E) specific activators of other inflammasome complexes [5 μg/mL poly(dA:dT) and 1 μg/mL flagellin] according to previously published protocols (described in detail in Experimental Procedures). Blots shown are representative of three independent experiments. Statistics are the same as in Fig. 1. CE, cell extract; SN, supernatant. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.
Fig. S4.
Fig. S4.
IRE1 perpetuates mtROS production and inflammasome activation. (A) Representative confocal microscopy images of mtROS production in BMDMs (Fig. 3A). MitoSOX fluorescence indicator (red) and Mitotracker (green) are shown. mtROS (yellow) levels are quantified and depicted in the graph in Fig. 3A. (Scale bar: 20 µm.) (B and C) PA-stimulated BMDMs treated with the indicated doses of 4μ8c (at the indicated doses) or DMSO (control) were analyzed with (B) qRT-PCR for Txnip mRNA and (C) calcium uptake. (D and E) Western blot analysis of caspase-1 activation in LPS-primed, PA-stimulated mouse BMDMs (D) treated with 150 μM STF-083010 or DMSO (control) or (E) transfected with siRNAs against Ire1α or Xbp1 Procaspase-1 (p45) and cleaved caspase-1 (p10) levels are indicated. Statistics are the same as in Fig. 1. CE, cell extract. SN, supernatant. *P ≤ 0.05; ***P ≤ 0.001.
Fig. 4.
Fig. 4.
IRE1 inhibitors reduce plaque area in a mouse model of atherosclerosis. (A) Experimental design in ApoE−/− mice using STF-083010 (10 mg/kg per day). (B) En face aorta analysis of atherosclerotic plaques in ApoE−/− mice. (Left) Sudan IV staining of atherosclerotic plaques. (Right) Quantification of plaque area (n = 13–14). (C) Analysis of aortic sinus plaque area in the animals in A and B. (Left and Center) Oil Red O staining of aorta cross-sections. (Right) Quantification of plaque area (n = 5–6). (D–F) Experimental design and data for analogous experiments in ApoE−/− mice using 4μ8c (10 mg/kg per day; E, n = 7–9; F, n = 5). Statistics are the same as in Fig. 1. (Scale bar: 350 µm.) *P ≤ 0.05; ***P ≤ 0.001.
Fig. S5.
Fig. S5.
Treatment of ApoE−/− mice with IRE1 inhibitors suppresses Xbp1 mRNA splicing and degradation of canonical RIDD targets. (A) qRT-PCR analysis of spliced Xbp1 mRNA levels from spleen tissue of ApoE−/− mice that were fed a Western diet for 12 weeks and treated with STF-083010 (10 mg/kg per day) or vehicle (control) for 6 weeks. (B and C) qRT-PCR analysis of the mRNA levels of reported RIDD targets in the same tissues as in A. (D) Liver protein lysates from the same mice as in A were analyzed for the phosphorylated form of IRE1 by Western blotting. (E) H&E staining of the livers from the same mice in A. (Scale bar: 50 µm.) (F) Plasma ALT activity measurements from the same mice in A. Statistics are the same as in Fig. 1. ns, Not significant. *P ≤ 0.05.
Fig. S6.
Fig. S6.
Lipid and lipoprotein profiles do not change in animals treated with IRE1 inhibitors. (A) Total plasma triglyceride and lipoprotein [very LDL (VLDL), LDL, and HDL] triglyceride levels in STF-083010– and vehicle-treated groups. (B) Total plasma cholesterol and lipoprotein (VLDL, LDL, and HDL) cholesterol levels in STF-083010– and vehicle-treated groups. (C and D) Lipoprotein profiles in control (blue) and STF-083010–treated (red) ApoE−/− mice. Statistics are the same as in Fig. 1. ns, not significant.
Fig. S7.
Fig. S7.
Suppression of Xbp1 mRNA splicing by IRE1 inhibitors in vivo. (A) qRT-PCR analysis of spliced Xbp1 mRNA levels in spleen of ApoE−/− mice that were fed a Western diet for 12 weeks and treated with 4µ8c (10 mg/kg per day) or vehicle (control) for 4 weeks. (B) Liver protein lysates from the same mice as in A were analyzed for the phosphorylated form of IRE1 by Western blotting. (C) H&E staining of the livers from the same mice in A. (Scale bar: 50 µm.) (D) Plasma ALT activity measurements from the same mice in A. Statistics are the same as in Fig. 1. ns, not significant. *P ≤ 0.05.
Fig. 5.
Fig. 5.
IRE1 inhibitors alter plaque composition and inflammation. Immunohistochemical and TUNEL assay analyses of proximal aorta cryosections from ApoE−/− mice (Fig. 4) treated with an IRE1 inhibitor. In each case, a representative image is shown in Left and Center, and the quantification of the data appears on in Right. (A) MOMA-2. (Scale bar: 100 µm.) (B) TUNEL assay (apoptotic cells are shown with arrowheads). (Scale bar: 50 µm.) (C) IL-1β. (Scale bar: 100 μm.) Statistics are the same as in Fig. 1. ns, not significant. *P ≤ 0.05; **P ≤ 0.01.
Fig. S8.
Fig. S8.
IRE1 inhibitors alter plaque composition and inflammation. Immunohistochemical and immunofluorescence analyses of proximal aorta sections obtained from ApoE−/− mice treated with STF-083010 or controls. In each case, representative images are shown in Left and Center, and the quantification of the data appears in Right. (A) Necrotic area was calculated (from indicated area) in the H&E-stained plaque sections. (Scale bar: 150 µm.) (B) Masson’s Trichrome collagen stain (collagen, blue; cytoplasm and muscle fibers, red; n = 5). (Scale bar: 200 µm.) (C) Fibrous cap thickness calculation from samples in A. (Scale bar: 100 µm.) (D) Anti–α-smooth muscle actin (VSMCs marker). (Scale bar: 200 µm.) (E) Anti-CD3 (T-cell marker). Arrowheads show CD3-positive cells (n = 5). Statistics are the same as in Fig. 1. (Scale bar: 200 µm.) ns, not significant. *P ≤ 0.05.
Fig. 6.
Fig. 6.
IRE1 inhibitors suppress hyperlipidemia-induced Th-1 immune responses and IL-18 cytokine levels. (A) Plasma IL-18 in ApoE−/− mice (Fig. 4) treated with an IRE1 inhibitor was measured by ELISA (n = 7). (B–D) Flow cytometry analysis of IFN-γ, IL-4, and IL-17 in splenocytes from ApoE−/− mice treated with IRE1 inhibitor and activated with PMA/ionomycin (n = 5). Statistics are the same as in Fig. 1. ns, not significant. *P ≤ 0.05; ***P ≤ 0.001.
Fig. S9.
Fig. S9.
IRE1 inhibitors reduce IL-1β levels in tissues and suppress Th1 immune responses. qRT-PCR analysis of Il-1β mRNA levels in (A) bone marrow or (B) spleen tissue of ApoE−/− mice that were fed a Western diet and treated with IRE1 inhibitors or vehicle (control). (C–E) Flow cytometry analysis of (C) IFN-γ, (D) IL-4, and (E) IL-17 levels in splenocytes activated with PMA/ionomycin (n = 5). Statistics are the same as in Fig. 1. *P ≤ 0.05; ***P ≤ 0.001.
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