. 2015 Dec 17;163(7):1716-29.

doi: 10.1016/j.cell.2015.11.045. Epub 2015 Dec 10.

Limiting Cholesterol Biosynthetic Flux Spontaneously Engages Type I IFN Signaling

Autumn G York¹, Kevin J Williams², Joseph P Argus¹, Quan D Zhou¹, Gurpreet Brar¹, Laurent Vergnes³, Elizabeth E Gray⁴, Anjie Zhen⁵, Nicholas C Wu⁶, Douglas H Yamada⁷, Cameron R Cunningham², Elizabeth J Tarling⁸, Moses Q Wilks⁹, David Casero¹⁰, David H Gray¹¹, Amy K Yu¹, Eric S Wang¹, David G Brooks¹², Ren Sun¹, Scott G Kitchen⁵, Ting-Ting Wu¹, Karen Reue¹³, Daniel B Stetson⁴, Steven J Bensinger¹⁴

Affiliations

¹ Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
² Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA.
³ Department of Human Genetics, University of California, Los Angeles, David Geffen School of Medicine, Los Angeles, CA 90095, USA.
⁴ Department of Immunology, University of Washington, 750 Republican Street, Box 358059, Seattle, WA 98109, USA.
⁵ Division of Hematology/Oncology, University of California, Los Angeles, David Geffen School of Medicine, Los Angeles, CA 90095, USA; UCLA AIDS Institute and the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, Los Angeles, CA 90095, USA.
⁶ Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁷ Immuno-Oncology Discovery Research; Janssen Research & Development, LLC, Spring House, PA 19477, USA.
⁸ Division of Cardiology, University of California, Los Angeles, David Geffen School of Medicine, Los Angeles, CA 90095, USA; Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁹ Center for Advanced Medical Imaging Sciences, Department of Radiology, Massachusetts General Hospital, Boston, MA 02114, USA.
¹⁰ Department of Pathology and Laboratory Medicine, University of California, Los Angeles, David Geffen School of Medicine, Los Angeles, CA 90095, USA.
¹¹ Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA; Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹² Princess Margaret Cancer Center, Immune Therapy Program, University Health Network, Toronto, ON M5G 2M9, Canada; Department of Immunology, University of Toronto, Toronto, ON M5S 1A8, Canada.
¹³ Department of Human Genetics, University of California, Los Angeles, David Geffen School of Medicine, Los Angeles, CA 90095, USA; Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹⁴ Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Pathology and Laboratory Medicine, University of California, Los Angeles, David Geffen School of Medicine, Los Angeles, CA 90095, USA. Electronic address: sbensinger@mednet.ucla.edu.

PMID: 26686653
PMCID: PMC4783382
DOI: 10.1016/j.cell.2015.11.045

Limiting Cholesterol Biosynthetic Flux Spontaneously Engages Type I IFN Signaling

Autumn G York et al. Cell. 2015.

. 2015 Dec 17;163(7):1716-29.

doi: 10.1016/j.cell.2015.11.045. Epub 2015 Dec 10.

Authors

Affiliations

¹ Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
² Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA.
³ Department of Human Genetics, University of California, Los Angeles, David Geffen School of Medicine, Los Angeles, CA 90095, USA.
⁴ Department of Immunology, University of Washington, 750 Republican Street, Box 358059, Seattle, WA 98109, USA.
⁵ Division of Hematology/Oncology, University of California, Los Angeles, David Geffen School of Medicine, Los Angeles, CA 90095, USA; UCLA AIDS Institute and the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, Los Angeles, CA 90095, USA.
⁶ Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁷ Immuno-Oncology Discovery Research; Janssen Research & Development, LLC, Spring House, PA 19477, USA.
⁸ Division of Cardiology, University of California, Los Angeles, David Geffen School of Medicine, Los Angeles, CA 90095, USA; Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁹ Center for Advanced Medical Imaging Sciences, Department of Radiology, Massachusetts General Hospital, Boston, MA 02114, USA.
¹⁰ Department of Pathology and Laboratory Medicine, University of California, Los Angeles, David Geffen School of Medicine, Los Angeles, CA 90095, USA.
¹¹ Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA; Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹² Princess Margaret Cancer Center, Immune Therapy Program, University Health Network, Toronto, ON M5G 2M9, Canada; Department of Immunology, University of Toronto, Toronto, ON M5S 1A8, Canada.
¹³ Department of Human Genetics, University of California, Los Angeles, David Geffen School of Medicine, Los Angeles, CA 90095, USA; Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹⁴ Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Pathology and Laboratory Medicine, University of California, Los Angeles, David Geffen School of Medicine, Los Angeles, CA 90095, USA. Electronic address: sbensinger@mednet.ucla.edu.

PMID: 26686653
PMCID: PMC4783382
DOI: 10.1016/j.cell.2015.11.045

Abstract

Cellular lipid requirements are achieved through a combination of biosynthesis and import programs. Using isotope tracer analysis, we show that type I interferon (IFN) signaling shifts the balance of these programs by decreasing synthesis and increasing import of cholesterol and long chain fatty acids. Genetically enforcing this metabolic shift in macrophages is sufficient to render mice resistant to viral challenge, demonstrating the importance of reprogramming the balance of these two metabolic pathways in vivo. Unexpectedly, mechanistic studies reveal that limiting flux through the cholesterol biosynthetic pathway spontaneously engages a type I IFN response in a STING-dependent manner. The upregulation of type I IFNs was traced to a decrease in the pool size of synthesized cholesterol and could be inhibited by replenishing cells with free cholesterol. Taken together, these studies delineate a metabolic-inflammatory circuit that links perturbations in cholesterol biosynthesis with activation of innate immunity.

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Figures

**Figure 1. Type I interferon signaling shifts the balance of lipid synthesis and import**
**(A)** Percent synthesis of palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1) and cholesterol as measured by Isotopic Spectral Analysis (ISA) on C75BL/6 BMDMs with or without interferonβ (1000U/mL IFNβ), Poly:IC (2ug/mL) or MHV-68 (MOI=0.5) +/− 10ug/mL IFNAR neutralizing antibody for 48h. **(B)** Total cellular palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1) and cholesterol from BMDMs stimulated as in (A). **(C)** qPCR analysis of *Fasn*, *Sqle*, and *Msr1* gene expression in BMDMs infected with MHV-68 (MOI=0.5) +/− 10ug/mL IFNAR neutralizing antibody for 48h. **(D)** qPCR analysis of indicated lipid synthesis genes in quiescent LysM Cre^+/− control (designated Control) or LysM Cre^+/− SCAP^fl/fl (SCAP^−/−). **(E)** Percent synthesis of palmitic acid (16:0), stearic acid (18:0) and cholesterol as measured by ISA of Control or SCAP^−/− BMDMs unstimulated or stimulated with IFNβ as above for 24h. **(F)** Total cellular palmitic acid (16:0), stearic acid (18:0) and cholesterol from Control or SCAP^−/− BMDMs unstimulated or stimulated with IFNβ as above for 24h. All mass spec and gene expression experiments are expressed as means ± SD from three independent experiments. *P < 0.05; **P < 0.01, ***P < 0.005 (two-tailed unpaired Student’s t test). See also Figures S1 and S2.

**Figure 2. Genetic inhibition of the lipid biosynthetic program primes antiviral immunity**
**(A)** qPCR analysis of murine gammaherpesvirus-68 (MHV-68) genes in Control or SCAP^−/− BMDMs infected with MHV-68 (MOI=0.5) for 48h. **(B)** qPCR analysis of HIV-1 mRNA (left) and representative immunoblots of HIV-1 p24 protein levels (right) from shControl or shSCAP THP1 macrophages 96h after infection. **(C)** MHV-68 titers from the lungs of LysM Cre^+/− control (Control) or LysM Cre^+/− SCAP^fl/fl (SCAP^−/−) mice on d.7 post intranasal infection (N=7; data shown is the combined results from two separate infection experiments of n=3 per group and n=4 per group). **(D)** qPCR analysis of indicated MHV-68 genes in WT BMDMs pretreated for 4h with Control or SCAP^−/− conditioned media (C.M.) before MHV-68 infection (MOI=0.5) for 48h. C.M. from Control or SCAP^−/− BMDMs was collected on d.7 post-differentiation. **(E)** qPCR analysis of *Ifnb1* and representative interferon stimulated genes (ISGs) in unstimulated control or SCAP^−/− BMDMs on d.8 of differentiation. **(F)** qPCR analysis of ISGs in *ex vivo* alveolar macrophage isolated from bronchoalveolar lavage from LysM Cre^+/− control (Control) or LysM Cre^+/− SCAP^fl/fl (SCAP^−/−) (3 mice/group). **(G)** qPCR analysis of indicated ISGs in unstimulated control or SCAP^−/− BMDMs on d.9 of differentiation +/− 5ug/mL IFNAR blocking antibody for last 48h. **(H)** qPCR analysis of ISGs in WT BMDMs treated for 4h with Control or SCAP^−/− conditioned media (C.M.). **(I)** MHV-68 ORF29 and ORF57 gene expression in WT BMDMs pretreated for 4h with Control or SCAP^−/− conditioned media (CM) + 2ug/mL IFNAR blocking antibody before MHV-68 infection (MOI=0.5) for 48h. **(J)** qPCR analysis of *Ifnb1* and *Il1b* expression in Control or SCAP^−/− BMDMs unstimulated or stimulated with LPS (50ng/mL) or Poly:IC (1ug/mL) for 1h on d.8 of differentiation. All experiments are reported as means ± SD from three independent experiments, unless noted otherwise. *P < 0.05; **P < 0.01, ***P < 0.005 (two-tailed unpaired Student’s t test). See also Figure S3.

**Figure 3. Inhibiting the SREBP2 transcriptional pathway engages a type I IFN inflammatory response**
**(A)** RNA-seq data from unstimulated shControl, shSREBP1 and shSREBP2 THP1 cells 72 h after PMA-differentiation into macrophages. Data shown are biologic replicates of each genotype. **(B)** qPCR analysis of *IFNB1* and *MX1* in unstimulated shControl, shSREBP1 and shSREBP2 primary PBMC-derived human macrophages. **(C)** qPCR analysis of Influenza A RNA from shControl, shSREBP1 or shSREBP2 THP1 cells 72h post infection. **(D)** qPCR analysis of *Ifnb1* and indicated ISGs expression in WT control or SREBP2^−/− MEFs cultured in DMEM containing 1% FBS for 24h. **(E)** qPCR analysis of *Mx2* expression in WT or SREBP2^−/− MEFs cultured in 1% FBS for 24h +/− 5ug/mL IFNAR blocking antibody as indicated **(F)** qPCR analysis of indicated MHV-68 gene expression in WT or SREBP2^−/− MEFs infected with MHV-68 (MOI=1.0) for 24h. **(G)** qPCR analysis of *Mx2* in WT or SREBP2^−/− MEFs +/− MHV infection (MOI=1.0) for 24h. **(H)** qPCR analysis of MHV-68 ORF57 expression in WT or SREBP2^−/− MEFs infected with MHV-68 (MOI=1.0) for 24h +/− 24h pretreatment with 5ug/mL IFNAR blocking antibody. All experiments are reported as means ± SD from three independent experiments. *P < 0.05; **P < 0.01, ***P < 0.005 (two-tailed unpaired Student’s t test). See also Figure S4.

**Figure 4. Limiting flux throught the cholesterol biosynthetic pathway engages a type I IFN response**
**(A)** qPCR analysis of ISG expression in population control (Wildtype) primary human fibroblasts or fibroblasts from an individual with MVK-deficiency (MKD; ~2% MVK activity compared to population controls). **(B)** qPCR analysis of *MVK, IFNB1* and ISG expression in shControl or shMVK THP1 cells after 72h of PMA-differentiation **(C)** qPCR analysis of ISG expression in population control (Wildtype) primary human fibroblasts or fibroblasts from an individual with MVK-deficiency (MKD) primed with 2ug/mL Poly:IC for 24h. **(D)** qPCR analysis of *ABCA1* and *IDOL* expression in shControl, shSREBP2 and shSCAP THP1 macrophages differentiated for 72h. **(E)** qPCR analysis of indicated ISG expression in THP1 shControl or shSREBP2 cells differentiated as above and stimulated with vehicle (DMSO) or LXR ligand (1uM GW3965) for 72h. **(F)** qPCR analysis of indicated ISG expression of differentiated THP1 shControl or shSREBP2 macrophages in media supplemented with 0.1mg/mL methyl beta cyclodextrin (MβCD)-cholesterol (“Chol”) as indicated for 72h. G. qPCR analysis of MHV-68 ORF57 expression in Control or SCAP^−/− BMDMs +/− 0.25mg/mL MβCD-cholesterol (Chol) for 48h, then infected with MHV-68 (MOI=0.5) for 24h. All experiments are reported as means ± SD from three independent experiments. *P < 0.05; **P < 0.01, ***P < 0.005 (two-tailed unpaired Student’s t test). See also Figure S5.

**Figure 5. IRF3/STING link changes in cholesterol biosynthesis with type I IFN production**
**(A)** Left: qPCR analysis of *Ifnb1* in WT or SREBP2^−/− MEFs cultured in 1% FBS for 24h +/− 5ug/mL IFNAR blocking antibody as indicated. Right: qPCR analysis of *Ifnb1* in WT and SREBP2^−/− MEFs treated 0.075mg/mL MβCD-cholesterol (Chol) as indicated for 72h. **(B)** qPCR analysis of *Irf3* and *Irf7* in WT or SREBP2^−/− MEFs transfected with control siRNA, siIRF3 or siIRF7 **(C)** qPCR analysis of *Ifnb1, Mx1, Mx2* and *Ccl2* in WT or SREBP2^−/− MEFs transfected with control siRNA, siIRF3 or siIRF7 **(D)** qPCR analysis of *Ifnb1, Mx1, Mx2* and *Ccl2 in* WT or SREBP2^−/− MEFs transfected with control siRNA, siMAVS or siSTING as indicated **(E)** qPCR of *IFNB1* and *MX1* gene expression from control (Cas9) or CRISPR/Cas9-edited STING (ΔSTING) THP1 cells stably transduced with shControl or shSREBP2. **(F)** qPCR analysis of *Ifnb1* and *Mx1* in WT or SREBP2^−/− MEFs transfected with control siRNA or siRNA to cGAS. All experiments are reported as means ± SD from three independent experiments. *P < 0.05; **P < 0.01, ***P < 0.005 (two-tailed unpaired Student’s t test). See also Figure S6.

**Figure 6. Perturbations in cholesterol homeostasis alters STING sensitivity to di-cyclic nucleotides**
**(A)** Western blot analysis of phospho-TBK1 (pTBK1) and total TBK1 from whole cell lysates in WT or SREBP2^−/− MEFs cultured in 1% FBS for 24h +/− 5ug/mL IFNAR blocking antibody as indicated. **(B)** qPCR analysis of *Tbk1, Ifnb1, Mx1, Mx2* and *Ccl2 in* WT or SREBP2^−/− MEFs transfected with control siRNA, or siTBK1. **(C)** Representative western blot analysis of phospho-TBK1 (pTBK1) and total TBK1 from whole cell lysates of SREBP2^−/− MEFs transfected with control siRNA, siSTING or sicGAS as indicated. **(D)** Western blot analysis of pTBK1 and TBK1 total in Control or SCAP^−/− BMDMs +/− 0.25mg/mL MβCD-cholesterol (Chol) for 48h or 10ug/mL IFNAR neutralizing antibody. **(E)** Western blot analysis of pTBK1 and TBK1 total in Control or SCAP^−/− BMDMs +/− 5ug/mL c-di-GMP (cGMP) for 45 min. **(F)** qPCR analysis of *Ifnb1* expression in Control or SCAP^−/− BMDMs cultured +/− 0.25mg/mL MβCD-cholesterol for 48h, then stimulated with 5ug/mL cGMP for 1h.

See this image and copyright information in PMC

Comment in

How Low Cholesterol Is Good for Anti-viral Immunity.
O'Neill LA. O'Neill LA. Cell. 2015 Dec 17;163(7):1572-4. doi: 10.1016/j.cell.2015.12.004. Cell. 2015. PMID: 26687349
Immunometabolism: The antiviral effect of limiting lipids.
Minton K. Minton K. Nat Rev Immunol. 2016 Feb;16(2):76-7. doi: 10.1038/nri.2016.6. Epub 2016 Jan 19. Nat Rev Immunol. 2016. PMID: 26781936 No abstract available.

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Limiting Cholesterol Biosynthetic Flux Spontaneously Engages Type I IFN Signaling

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Limiting Cholesterol Biosynthetic Flux Spontaneously Engages Type I IFN Signaling

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