1. Volume 44, Issue 6, Pages 1325-1336 (June 2016)
Type 1 Interferons Induce Changes in Core Metabolism that Are Critical for Immune Function 
Duojiao Wu, David E. Sanin, Bart Everts, Qiongyu Chen, Jing Qiu, Michael D. Buck, Annette Patterson, Amber M. Smith, Chih-Hao Chang, Zhiping Liu, Maxim N. Artyomov, Erika L. Pearce, Marina Cella, Edward J. Pearce 
Immunity 
Volume 44, Issue 6, Pages (June 2016)
DOI: /j.immuni
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2. Immunity 2016 44, 1325-1336DOI: (10.1016/j.immuni.2016.06.006)
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3. Figure 1
Stimulation of pDCs by CpGA Results in an Increase in FAO That Is Essential for Cellular Activation
CD11cintB220+Siglec H+ pDCs were sorted from Flt3L-bone marrow cultures at day 8 and stimulated with CpGA (ODN 1585, sequence: 5’- ggGGTCAACGTTGAgggggg -3’), or other TLR agonists.
(A) IFN-α was measured by ELISA at 24 hr post activation.
(B) TNF-α and (C) IL-6 were measured in the same samples using CBA.
(D and E) ECAR of SIRPα+DCs (D) and pDCs (E), following treatment with TLR agonists, were determined by EFA.
(F) Basal OCR and (G) SRC (maximum OCR after the addition of FCCP, calculated as a percentage of baseline OCR), were determined by EFA.
(H–K) Relative basal OCR (H), or cytokine production (I–K) of pDCs activated with CpGA without or with etomoxir.
(L) Bone marrow cells in Flt3L cultures were transduced with retrovirus encoding either shRNA against luciferase (control) or CPT1a (hpCPT1a). pDCs were sorted from these cultures on the basis of retroviral reporter gene expression, and stimulated for 24 h without or with CpGA, after which IFN-α was measured in cell supernatants by ELISA. In (D) and (E), data are mean ± SEM of reads from triplicate samples from one experiment, and are representative of data from three experiments. In the other panels, data are mean ± SEM from at least three independent experiments. All differences are statistically significant (at least p < 0.05, Student’s t test); ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < Please see Figure S1.
Immunity  , DOI: ( /j.immuni )
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4. Figure 2
Stimulation of pDCs with CpGA Results in Type 1 IFN Production that Plays an Essential Role in Subsequent Changes in Cellular Metabolism and Activation
(A) Mitochondrial fitness tests were used to compare OCR of pDCs stimulated with CpGA for times indicated. Data represent mean ± SEM of reads from triplicate samples from one experiment, and are representative of data from three experiments.
(B) RNaseq analysis of type 1 IFNs, Il6 and Tnf expression induced in pDCs by CpGA stimulation for 24 hr. Data from two separate control and three separate CpGA-stimulated cell samples are shown.
(C) IFN-α accumulation in pDC culture supernatant at times indicated post activation with CpGA. Data are mean ± SEM from three independent samples from one experiment, and are representative of data from four experiments.
(D) Mitochondrial fitness tests were used to compare OCR of WT or Ifnar1−/− pDCs 24 hr after stimulation with resting or CpGA-stimulated WT or Ifnar1−/− pDCs. Data are mean ± SEM of reads from triplicate samples from one experiment, and are representative of data from three experiments.
(E) IFN-α production by resting or CpGA-stimulated WT or Ifnar1−/− pDCs. Bars represent mean ± SEM of three independent experiments. Note log scale.
(F) Genes expressed differentially in CpGA-activated versus resting pDCs were identified by analysis of RNaseq data. This dataset was analyzed using INTERFEROME. The subset of ISGs was analyzed for GO biological processes using DAVID. All differences shown are statistically significant (at least p < 0.05, Student’s t test); ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < Please see Figure S2.
Immunity  , DOI: ( /j.immuni )
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5. Figure 3 IFN-α Directly Promotes FA Oxidation
(A) Mitochondrial fitness tests were used to compare OCR of WT and Ifnar1−/− pDCs stimulated with IFN-α, as indicated.
(B) OCR values during a mitochondrial fitness test, (C) basal OCR, and (D) maximal OCR of PDV epithelial cells that had been cultured without (control) or with IFN-α for 24 hr.
(E) OCR values during a mitochondrial fitness test, and (F) basal OCR comparison, of PDV epithelial cells that had been cultured without (control) or with IFN-α or IFN-α plus anti-IFNAR (MAR1-5A3) for 24 hr. Data are mean ± SEM of reads from triplicate samples from one experiment representative of three (A, B, E), or mean values ± SEM from three or more independent experiments (C, D, F). Data in (C) and (F) were collected within the same experiments but are shown separately for clarity. All differences shown are statistically significant (at least p < 0.05, Student’s t test); ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < Please see Figure S3.
Immunity  , DOI: ( /j.immuni )
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6. Figure 4
Viral Infection Induces Type 1 IFN-Dependent Changes in Host Cell FAO that Are Important for Further Type 1 IFN Production and Anti-Viral Effects
(A) OCR values during a mitochondrial fitness test of uninfected PDV epithelial cells versus PDV cells infected with LCMV Arm for 24 hr.
(B) Basal OCR of PDV cells at 24 hr post infection in the absence or presence of blocking anti-IFNAR Ab.
(C and D) C57BL/6J mice were infected with 2 × 105 PFU of LCMV Arm and treated with etomoxir (20 mg/kg) 1 day prior to infection and 1 day afterward. Serum and tissue were collected at 72 hr post-infection and (C) serum IFN-α, and (D) expression of LCMV glycoprotein (LCMV gp) in liver and spleen were measured. Data are mean ± SEM of reads from triplicate samples from one experiment representative of three (A), or mean ± SEM of reads from three independent experiments (B–D). All differences shown are statistically significant (at least p < 0.05, Student’s t test); ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < Please see Figure S4.
Immunity  , DOI: ( /j.immuni )
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7. Figure 5
pDC Activation Is Associated with a Late Increase in Glycolysis and Requires Mitochondrial Pyruvate Import and FAS
Purified pDCs were stimulated with CpGA plus or minus inhibitors for 24 hr.
(A) Relative change in basal ECAR at 24 hr post activation with CpGA.
(B and C) Effect of the MPC inhibitor UK5099 on IFN-α and TNF-α production by CpGA-stimulated pDCs.
(D and E) Effect of the ACC1 inhibitor TOFA on IFN-α and TNF-α production by CpGA-stimulated pDCs.
(F) Relative basal OCR of pDCs activated with CpGA without or with inhibitors.
(G) Relative basal ECAR of resting pDCs or pDCs stimulated with IFN-α for 24 hr.
(H and I) Relative basal OCR of pDCs (H) or basal OCR of PDV cells (I) stimulated with IFN-α in the absence or presence of inhibitors. For all panels, data are mean ± SEM from three independent experiments. Data in (B)–(E) were collected within the same experiments but are shown separately for clarity. All differences shown are statistically significant (at least p < 0.05, Student’s t test); ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < Please see Figure S5.
Immunity  , DOI: ( /j.immuni )
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8. Figure 6
Metabolic Reprogramming Induced by Type 1 IFN Leads to Enhanced ATP Availability
ATP was measured in pDCs that had been stimulated under various conditions for 24 hr: (A) Resting pDCs, or pDCs stimulated with CpGA alone or in the presence of MAR1-5A3 to block IFNAR, or with IFN-α, or (B–D) pDCs cultured alone or with the indicated inhibitors, or with CpGA alone or with the indicated inhibitors. Data are mean ± SEM of reads from triplicate samples from one experiment, representative of three. The statistical significance of differences in ATP, by Student’s t test, are marked as follows: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p <
Immunity  , DOI: ( /j.immuni )
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9. Figure 7
PPARα Responsive Gene Signature Is Present in Response to Stimulation with Type I IFN and Plays a Role in Regulating Changes in FA Metabolism Induced by This Cytokine
Data from RNaseq analysis of resting pDCs versus pDCs stimulated with IFN-α for 24 hr were analyzed.
(A) Differentially expressed genes (>2-fold change) were analyzed to identify metabolic genes defined by RGD genes database ( which were then examined further using DAVID focused on KEGG pathways. Pathways are color coded to match data in (A). This set of differentially expressed metabolic genes was used to develop an interactive network of genes using STRING that was annotated and modified using Cytoscape. Thicker edges represent interactions with higher support form published literature. KEGG pathways are color-highlighted as in (A). Each circle represents an individual gene. Genes with more than one color are components of more than one pathway.
(B) On the basis of the KEGG analysis in (A), we specifically expanded the gene search for PPAR signaling pathways and built a network using the same strategy described in (A). Individual gene names are shown.
(C) Extracts of pDCs, treated as indicated, cDCs, or heart tissue, were separated by SDS-PAGE, transferred to a membrane and probed for PPARα and GAPDH (loading control).
(D) Splenic pDCs were fixed and permeabilized, stained with an anti- PPARα antibody, and analyzed by flow cytometry.
(E and F) pDCs were stimulated with CpG with or without the PPARα antagonist GW6471 for 24 hr, after which (E) IFN-α and (F) basal OCR were measured (cells were cultured at 2.2 × 105 well in 200 μl of medium in these experiments).
(G) pDCs were stimulated with IFN-α with or without GW6471 for 24 hr, after which OCR was measured.
(H) OCR during a mitochondrial fitness test of PDV cells infected with LCMV Arm in the presence or absence of GW6471 for 24 hr.
(I) Basal OCR of PDV cells infected with LCMV Arm in the presence or absence of GW6471 for 24 hr.
(J) PDV cells were infected with LCMV for 24 hr in the presence or absence of IFN-α and/or GW6471, after which LCMV gp expression was measured by real time RT-PCR.
The data in (A) and (B) are from RNA-seq analysis of three samples of stimulated and three samples of unstimulated pDCs. Data in (C) and (D) are from individual experiments representative of two experiments. Data in (H) are mean ± SEM of reads from triplicate samples from one experiment representative of three. Data in (E)–(G), (I), (J) are mean values ± SEM from three independent experiments. All differences shown are statistically significant (at least p < 0.05, Student’s t test); ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < Please see Figure S6.
Immunity  , DOI: ( /j.immuni )
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