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PNAS 110 (13): 5109-5114

Copyright © 2013 by the National Academy of Sciences.

TLR9 mediates cellular protection by modulating energy metabolism in cardiomyocytes and neurons

Yasunori Shintania,1, Amar Kapoora, Masahiro Kanekoa, Ryszard T. Smolenskib, Fulvio D’Acquistoa, Steven R. Coppena, Narumi Harada-Shojia, Hack Jae Leea, Christoph Thiemermanna, Seiji Takashimac, Kenta Yashiroa, and Ken Suzukia,1

aWilliam Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London EC1M 6BQ, United Kingdom; bDepartment of Biochemistry, Medical University of Gdansk, 80-211 Gdansk, Poland; and cDepartment of Molecular Cardiology, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan


Figure 01
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Fig. 1. TLR9 reduces energy substrates with activation of AMPK independent of MyD88, leading to increased stress tolerance in cardiomyocytes. (A) Administration of CpG-ODN reduced the level of energy substrates and increased the AMP/ATP ratio in ex vivo perfused heart in wild-type (WT) mice, but not in TLR9–/– mice. The hearts (n = 6 for WT, n = 5 for TLR9–/–) were analyzed at 30 min after the administration of CpG-ODN (type B, 0.25 μM) or vehicle (con). (B) CpG-ODN increased the phosphorylation of AMPK and its substrate, ACC, without a decrease in I{kappa}Bα in ex vivo perfused heart in WT mice, but not in TLR9–/– mice. (C) CpG-ODN (type B at 3 μM or type A at 1 μM) activated AMPK 60 min after administration in cultured cardiomyocytes (CM), but not in RAW264.7 macrophages (RAW) or in cardiac fibroblasts (CFb). In contrast, the phosphorylation of p38 MAPK and a decrease in I{kappa}Bα were seen in RAW264.7, but not in cardiomyocytes. The data represent four independent experiments. (D) Time-course of AMPK activation by CpG-ODN in cardiomyocytes. Oligomycin A (10 μg/mL for 60 min) was used as a positive control. The data represent two independent experiments. (E) The change in intracellular ATP levels in cardiomyocytes treated with CpG-ODN using luminescence-based measurement (n = 3). The data represent two independent experiments. (F) Pretreatment with CpG-ODN (30 min before 16 h of hypoxia, n = 6–8) increased the survival of cardiomyocytes against hypoxia. AMPK inhibitor, compound C (1 μM), abrogated the protective effect of CpG-ODN. Cell viability was normalized with normoxia control. The data represent three independent experiments. (G) The CpG-mediated AMPK activation was not affected in the neonatal cardiomyocytes from MyD88–/– mice. The data represent three independent experiments. Values indicate densitometric ratio of pAMPK/AMPK or tubulin in immunoblots, mean ± SEM. Error bars indicate SEM. *P < 0.05 **P < 0.01 ***P < 0.001 compared with control.

 

Figure 02
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Fig. 2. Unc93b1 is a pivotal switch for the distinct TLR9 responses in cardiomyocytes and immune cells. (A) RT-PCR screening of the TLR9 signaling molecules in RAW264.7 (RAW) and mouse neonatal cardiomyocytes (CM). Unc93b1 was far less expressed in cardiomyocytes than in RAW264.7 cells. (B) MyD88 associated with TLR9 in RAW264.7 cells but not in cardiomyocytes. TLR9 was transiently transfected in both cardiomyocytes and RAW264.7 cells. The cell lysates were immunoprecipitated with anti-Flag antibody at 0, 30, or 60 min after CpG stimulation, immunoblotted with MyD88 antibody. A total of 1% of whole cell lysates was used as input. The molecular weight of overexpressed TLR9 in cardiomyocytes matched with the full-length form (arrow), but not to the cleaved form (arrowhead), whereas both forms were found in RAW264.7 cells. (C) The Unc93b1 knockdown transformed the response to CpG-ODN in RAW264.7 cells from the inflammatory TLR9 signaling to the alternative TLR9 signaling that resulted in AMPK activation. The data represent three independent experiments. (D–F) Conversely, the overexpression of Unc93b1 diminished TLR9-induced AMPK activation (D), switched on the inflammatory TLR9 signaling in cardiomyocytes with degradation of I{kappa}Bα (D), appearance of the cleaved form of TLR9 (E), association with MyD88 (E), and subsequent inflammatory cytokine production (F). The cardiomyocytes, which were transfected with the indicated adenoviruses, were stimulated by CpG-ODN for 60 min (D and E) or 16 h (n = 3 for each group) (F). ***P < 0.001. Error bars indicate SEM. The data represent three independent experiments. Values indicate densitometric ratio of pAMPK/tubulin in immunoblots, mean ± SEM *P < 0.05 compared with 0 min.

 

Figure 03
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Fig. 3. Unc93b1 overexpression transformed the trafficking of TLR9 in cardiomyocytes. (A) TLR9 (HA; green) stayed in the ER [sarcoplasmic/endoplsmic reticulum Ca2+-ATPase (SERCA2); red] at any time points of 0, 30, and 180 min after CpG-ODN stimulation in cardiomyocytes. (B) TLR9 did not translocate to the endosome compartment (EEA1; red) in cardiomyocytes regardless of before and after CpG-ODN stimulation. (C) Unc93b1 overexpression transformed the trafficking of TLR9 to EEA1-positive endosomes in cardiomyocytes. Please note that part of TLR9 existed in the endosome compartment before stimulation. The cardiomyocytes, which were transfected with the adenoviruses encoding TLR9-HA-Flag (AC) and Unc93b1 in (C), were stimulated by CpG-ODN. (Scale bars, 10 μm.) Images were obtained with confocal microscopy. Experiments were repeated at least twice and the representative images are shown.

 

Figure 04
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Fig. 4. Endocytosed DNA translocates to the ER via retrograde transport system in cardiomyocytes. (A) Biotin-labeled CpG-ODN (green) localized in the ER (SERCA2; red) 30 min after administration in cardiomyocytes. (B) Retro-2 (100 μM for 30 min pretreatment before administration of CpG-ODN), the inhibitor or retrograde transport, changed the distribution pattern of endocytosed DNA in cardiomyocytes. Endocytosed CpG-ODN accumulated in EEA1-positive endosomes (red). (Scale bars, 10 μm.) Images were obtained with confocal microscopy. Experiments were repeated at least twice and the representative images are shown. (C) Retro-2 inhibited the TLR9-mediated AMPK activation in cardiomyocytes. The data represent four independent experiments. (D) Retro-2 also inhibited the CpG-ODN–induced AMPK activation in the Unc93b1 knocked-down RAW264.7 cells, but not in control RAW cells. The data represent four independent experiments. Values indicate densitometric ratio of pAMPK/tubulin in immunoblots, mean ± SEM *P < 0.05, **P < 0.01 compared with the control.

 

Figure 05
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Fig. 5. The alternative protective TLR9 signaling also operates in differentiated neuronal cells. (A) Differentiated neuronal cells (Right; D) demonstrated a robust and transient increase in phosphorylated AMPK after CpG-ODN stimulation without degradation of I{kappa}Bα, whereas undifferentiated SH-SY5Y cells (Left, UD) showed a subtle increase in phosphorylated AMPK. Typical morphology of the corresponding differentiation status is shown below the blots. The data represent three independent experiments. Values indicate densitometric ratio of pAMPK/tubulin in immunoblots, mean ± SEM *P < 0.05, **P < 0.01 compared with 0 min. (B) Administration of CpG-ODN significantly reduced intracellular ATP levels in the differentiated neuronal cells 60 min after treatment. n = 3 for each group. The data represent two independent experiments. (C) Tlr9 expression significantly increased along with the differentiation status (Upper), whereas the expression level of Unc93b1 remained low regardless of differentiation (Lower). The expression level was normalized to the level of human monocytes/macrophages, U937 cells which were differentiated with 10 nM phorbol 12-miristate 13-acetate (PMA) (n = 4–5 for each group). *P < 0.05 compared with the undifferentiated SH-SY5Y cells. (D) Pretreatment with CpG-ODN significantly decreased cell death (assessed by lactate dehydrogenase release; Left) and increased cell viability (measured by MTT assay; Right) after hydrogen peroxide (25 μM for 24 h, n = 12 for each group) in differentiated neuronal cells. *P < 0.05 compared with the control. *P < 0.05. Error bars indicate SEM. (E) Schematic presentation of two different TLR9 signaling pathways. Cardiomyocytes or neurons with low expression level of Unc93b1 sense released DNA (danger signal) at the ER from damaged cells upon injury, reduce energy metabolism, and consequently increase the stress tolerance through AMPK activation, whereas immune cells with higher expression of Unc93b1 initiate the inflammatory response at the endosome. The alternative and inflammatory TLR9 signaling pathways are interchangeable depending on the expression levels of Unc93b1.

 


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