Research ArticleSepsis

ALK is a therapeutic target for lethal sepsis

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Science Translational Medicine  18 Oct 2017:
Vol. 9, Issue 412, eaan5689
DOI: 10.1126/scitranslmed.aan5689
  • Fig. 1. Identification of bioactive compounds modulating STING activation.

    (A) Heatmap of STING activity changes based on IFNβ release from iBMDMs after 3′3′-cGAMP (10 μg/ml, 16 hours) stimulation in the absence or presence of 464 bioactive compounds (10 μM). (B) Structure of the compound identified to inhibit (blue) or promote (red) STING activity. (C to E) IFNβ release assayed using enzyme-linked immunosorbent assay (ELISA) from iBMDMs (C), pPMs (D), and pPBMCs (E) treated with 3′3′-cGAMP (10 μg/ml) in the absence or presence of indicated bioactive compounds (10 μM) for 16 hours [n = 3; data are means ± SD; *P < 0.05 versus 3′3′-cGAMP group, analysis of variance (ANOVA) least significant difference (LSD) test]. (F) Heatmap of STING activity changes as judged by IFNβ release from iBMDMs after 3′3′-cGAMP (10 μg/ml, 16 hours) stimulation in the absence or presence of 174 signaling modulating compounds. The top five negative (inhibitory) and positive (agonistic) regulators are noted.

  • Fig. 2. Pharmacologic inhibition of ALK impairs STING activation.

    (A) iBMDMs were stimulated with indicated STING ligands (10 μg/ml) in the absence or presence of LDK378 (10 μM), AP26113 (10 μM), or control vehicle [dimethyl sulfoxide (DMSO)] for 16 hours, and the release of IFNβ was assayed using ELISA (n = 3; data are means ± SD; *P < 0.05 versus DMSO group, ANOVA LSD test). (B) Heatmap of IFNβ release changes in macrophages or monocytes after STING ligand (10 μg/ml) stimulation in combination with LDK378 (10 μM), AP26113 (10 μM), or vehicle (DMSO) for 16 hours. (C) iBMDMs were stimulated with indicated STING ligands (10 μg/ml) in the absence or presence of LDK378 (10 μM), AP26113 (10 μM), or vehicle (DMSO) for 16 hours, and IFNβ mRNA was assayed with quantitative polymerase chain reaction (n = 3; data are means ± SD; *P < 0.05 versus DMSO group, ANOVA LSD test). (D) Heatmap of IFNβ mRNA changes in macrophages or monocytes after STING ligand (10 μg/ml) stimulation in combination with LDK378 (10 μM), AP26113 (10 μM), or vehicle (DMSO) for 16 hours. AU, arbitrary units. (E and F) Western blot analysis of indicated protein expression in iBMDMs (E) or J774A.1 cells (F) after 3′3′-cGAMP (10 μg/ml) stimulation in combination with LDK378 (10 μM), AP26113 (10 μM), or vehicle (DMSO) for 3 to 16 hours. (G and H) Western blot analysis of indicated protein expression in iBMDMs (G) or J774A.1 cells (H) after c-di-AMP (10 μg/ml) or DMXAA (10 μg/ml) stimulation in combination with LDK378 (10 μM), AP26113 (10 μM), or vehicle (DMSO) for 16 hours.

  • Fig. 3. Genetic silencing of ALK limits STING activation.

    (A) Western blot analysis of ALK expression in ALK stable knockdown iBMDMs (n = 3; data are means ± SD; *P < 0.05 versus control shRNA group, t test). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (B to D) Indicated iBMDMs were stimulated with 3′3′-cGAMP (10 μg/ml) or c-di-AMP (10 μg/ml) for 16 hours, and cell morphology (B), viability (C), and cell cycle phase (D) were assayed (scale bars, 200 μm). (E and F) Indicated iBMDMs were stimulated with indicated STING ligands (10 μg/ml) for 16 hours, and IFNβ protein release (E) and IFNβ mRNA (F) were assayed [n = 3; data are means ± SD; *P < 0.05 versus control (Ctrl) shRNA group, ANOVA LSD test]. (G and H) Heatmap of IFNβ protein release (G) and IFNβ mRNA expression (H) changes in indicated ALK-WT (wild-type) and ALK-knockdown macrophages or monocytes after STING ligand (10 μg/ml) stimulation for 16 hours. (I) Western blot analysis of indicated protein expression in ALK-WT and ALK-knockdown iBMDMs after stimulation with 3′3′-cGAMP (10 μg/ml), c-di-AMP (10 μg/ml), or DMXAA (10 μg/ml) for 16 hours.

  • Fig. 4. ALK/EGFR binding triggers AKT-dependent STING activation.

    (A) Western blot analysis of indicated protein expression in iBMDMs and RAW264.7 and THP1 cells after stimulation with 3′3′-cGAMP (10 μg/ml), c-di-AMP (10 μg/ml), or DMXAA (10 μg/ml) for 16 hours. (B) Heatmap of RTKs phosphorylation changes in iBMDMs after 3′3′-cGAMP (10 μg/ml) or c-di-AMP (10 μg/ml) stimulation for 16 hours with or without pharmacologic (LDK378, 10 μM) or genetic inhibition of ALK. (C) Relative EGFR phosphorylation assayed in parallel to (B). (D) Immunoprecipitation (IP) analysis of the interaction between ALK and EGFR in iBMDMs after 3′3′-cGAMP (10 μg/ml) or c-di-AMP (10 μg/ml) stimulation for 16 hours with or without LDK378 (10 μM) or OSI-420 (10 μM). IB, immunoblotting. (E) Western blot analysis of indicated protein expression in iBMDMs after treatment with 3′3′-cGAMP (10 μg/ml) or c-di-AMP (10 μg/ml) for 16 hours with or without LDK378 (10 μM), OSI-420 (10 μM), or GDC-0068 (10 μM). (F) Western blot analysis of EGFR expression in EGFR stable knockdown iBMDMs. (G) Western blot analysis of indicated protein expression in EGFR-WT and EGFR-knockdown iBMDMs after stimulation with 3′3′-cGAMP (10 μg/ml) or c-di-AMP (10 μg/ml) for 16 hours. (H and I) iBMDMs were treated with 3′3′-cGAMP (10 μg/ml) or c-di-AMP (10 μg/ml) for 16 hours with or without LDK378 (10 μM), OSI-420 (10 μM), or GDC-0068 (10 μM), and IFNβ protein release (H) and IFNβ mRNA expression (I) were assayed (n = 3; data are means ± SD; *P < 0.05 versus 3′3′-cGAMP or c-di-AMP group, ANOVA LSD test).

  • Fig. 5. ALK and STING have overlapping and distinct immune functions in immune chemical release.

    (A) Heatmap of immune chemical profile in WT, ALK-knockdown (KD), or STING-knockout (KO) iBMDMs after stimulation with LPS (1 μg/ml), 3′3′-cGAMP (10 μg/ml), or c-di-AMP (10 μg/ml) for 16 hours with or without LDK378 (10 μM). (B) Changes in immune chemical release in WT iBMDMs after LPS, 3′3′-cGAMP, and c-di-AMP treatment. (C) Changes in immune chemical release between ALK-KD and STING-KO iBMDMs in response to 3′3′-cGAMP, c-di-AMP, or LPS.

  • Fig. 6. Inhibition of the ALK-STING pathway protects mice against CLP-induced polymicrobial sepsis.

    (A) Schematic depiction of the CLP model. (B) Administration of LDK378 or depletion of STING in mice prevented CLP (22-gauge needle)–induced animal death (n = 17 mice per group; *P < 0.05, Kaplan-Meier survival analysis). (C to G) In parallel, tissue hematoxylin and eosin staining (day 3; scale bars, 200 μm) (C), serum enzyme activity (days 2 to 7) (D), cytokine mRNA (day 3) (E), serum antibody array (day 3) (F), and heatmap of immune chemical profile (day 3) (G) were assayed (n = 3 to 5 mice per group; each bar represents the mean of the data; *P < 0.05, ANOVA LSD test). The top five down-regulated circulating immune chemical mediators in LDK378 and STING−/− groups compared with control group included IL-10, serpin E1, serpin F1, TIM-1, and CXCL2. High-resolution images related to (C), (F), and (G) are shown in figs. S12 and S13.

  • Fig. 7. Inhibition of the ALK-STING pathway protects mice against LPS-induced endotoxemia.

    (A) Schematic depicting the endotoxemia model. (B) Administration of LDK378 or depletion of STING in mice prevented LPS (10 mg/kg)–induced animal death (n = 18 mice per group; *P < 0.05, Kaplan-Meier survival analysis). (C to G) In parallel, tissue hematoxylin and eosin staining (24 hours; scale bars, 200 μm) (C), serum enzyme activity (12 to 48 hours) (D), cytokine mRNA (24 hours) (E), serum antibody array (24 hours) (F), and heatmap of immune chemical profile (24 hours) (G) were assayed (n = 3 to 5 mice per group; each bar represents the mean of the data; *P < 0.05, ANOVA LSD test). The top five down-regulated circulating immune chemical mediators in LDK378 and STING−/− groups compared with control group included EGF, CD14, CXCL1, endoglin, and CCL22. High-resolution images related to (C), (F), and (G) are shown in figs. S14 and S15.

  • Fig. 8. Gene and protein changes in ALK-dependent STING pathways in human sepsis.

    (A) Box plots comparing measures of ALK, EGFR, STING, TBK1, and IRF3 mRNA in PBMC samples of sepsis patients (n = 16) and healthy controls (n = 16). The mRNAs are presented as median value (black line), interquartile range (box), and minimum and maximum of all data (black line). *P < 0.05 versus control group, t test. (B) Table depicting clinical characteristics of sepsis patients and healthy control individuals. (C) Western blot analysis of indicated protein expression in PBMC samples of sepsis patients and healthy controls.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/412/eaan5689/DC1

    Materials and Methods

    Fig. S1. ALK inhibitors block STING activation.

    Fig. S2. Pharmacologic inhibition of ALK blocks STING ligand–induced IFNβ release and expression.

    Fig. S3. Pharmacologic inhibition of ALK blocks STING activation.

    Fig. S4. Genetic inhibition of ALK limits STING activation.

    Fig. S5. ALK does not bind known STING regulators.

    Fig. S6. Inhibition of ALK limits RTK phosphorylation in STING activation.

    Fig. S7. ALK binds EGFR during STING activation.

    Fig. S8. The ALK-EGFR-AKT pathway mediates STING activation.

    Fig. S9. Knockdown of EGFR inhibits STING activation.

    Fig. S10. The ALK-EGFR-AKT pathway mediates STING ligand–induced IFNβ release and expression.

    Fig. S11. ALK mediates LPS-induced macrophage activation.

    Fig. S12. Histological analysis of tissue injury in CLP-treated mice.

    Fig. S13. Heatmap of circulating immune chemical profile in indicated mice.

    Fig. S14. Histological analysis of tissue injury in LPS-treated mice.

    Fig. S15. Heatmap of circulating immune chemical profile in indicated mice.

    Fig. S16. Effects of targeting the ALK-STING pathway on CLP-induced septic death.

    Fig. S17. Schematic depicting the pathologic role of ALK-dependent STING pathways in lethal sepsis.

    Table S1. Reagent sources.

    Table S2. Individual-level data corresponding to the different figures (provided as an Excel file).

    Reference (70)

  • Supplementary Material for:

    ALK is a therapeutic target for lethal sepsis

    Ling Zeng, Rui Kang, Shan Zhu, Xiao Wang, Lizhi Cao, Haichao Wang, Timothy R. Billiar, Jianxin Jiang,* Daolin Tang*

    *Corresponding author. Email: tangd2{at}upmc.edu (D.T.); jiangjx{at}cta.cq.cn (J.J.)

    Published 18 October 2017, Sci. Transl. Med. 9, eaan5689 (2017)
    10.1126/scitranslmed.aan5689

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. ALK inhibitors block STING activation.
    • Fig. S2. Pharmacologic inhibition of ALK blocks STING ligand–induced IFNβ release and expression.
    • Fig. S3. Pharmacologic inhibition of ALK blocks STING activation.
    • Fig. S4. Genetic inhibition of ALK limits STING activation.
    • Fig. S5. ALK does not bind known STING regulators.
    • Fig. S6. Inhibition of ALK limits RTK phosphorylation in STING activation.
    • Fig. S7. ALK binds EGFR during STING activation.
    • Fig. S8. The ALK-EGFR-AKT pathway mediates STING activation.
    • Fig. S9. Knockdown of EGFR inhibits STING activation.
    • Fig. S10. The ALK-EGFR-AKT pathway mediates STING ligand–induced IFNβ release and expression.
    • Fig. S11. ALK mediates LPS-induced macrophage activation.
    • Fig. S12. Histological analysis of tissue injury in CLP-treated mice.
    • Fig. S13. Heatmap of circulating immune chemical profile in indicated mice.
    • Fig. S14. Histological analysis of tissue injury in LPS-treated mice.
    • Fig. S15. Heatmap of circulating immune chemical profile in indicated mice.
    • Fig. S16. Effects of targeting the ALK-STING pathway on CLP-induced septic death.
    • Fig. S17. Schematic depicting the pathologic role of ALK-dependent STING pathways in lethal sepsis.
    • Table S1. Reagent sources.
    • Reference (70)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S2. Individual-level data corresponding to the different figures (provided as an Excel file).

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