| Project/Area Number |
19K07606
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Multi-year Fund |
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| Section | 一般 |
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| Review Section |
Basic Section 49070:Immunology-related
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| Research Institution | Osaka University |
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Principal Investigator |
TSAI CHAO・YUAN 大阪大学, 免疫学フロンティア研究センター, 特任研究員(常勤) (10727001)
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| Project Period (FY) |
2019-04-01 – 2022-03-31
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| Project Status |
Granted (Fiscal Year 2019)
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| Budget Amount *help |
¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000)
Fiscal Year 2021: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2020: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Fiscal Year 2019: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
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| Keywords | B cell / Autophagy / Secretory vesicles / IL-4/CD40 signaling / Immune regulation / Autophagosome / Autophagosomes / Extracellular vesicles / intercellular network |
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| Outline of Research at the Start |
Autophagy is known as an intracellular degradation system. Our proposed research may reveal an important role of secretory autophagy in the immune network, which is different from previous reported, such as LC3-associated phagocytosis and autophagy-mediated cytokine maturation and secretion.
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| Outline of Annual Research Achievements |
Cell-derived extracellular vesicles (EVs) are small membrane-enclosed vesicles with the diameter ranging from 30 to 1000 nm including exosomes and microvesicles et al. and act as cargos to deliver the biomolecular messages between cells. Autophagy, which is known as an intracellular degradation system that contributes to cell hemostasis, is also involved in the unconventional secretion of cytosolic proteins. Recent reports have showed that microtubule-associated protein 1A/1B-light chain 3 (LC3)-II-positive vesicles could be found in cell-derived EVs, suggesting that autophagy may play a role during cell-to-cell communication. However, the secretion of autophagy-associated vesicles is still controversial. Whether external stimulation can induce such secretion remains largely unknown. Since a potential source of EVs is the B cell, we study the molecular mechanism and physiological role of the secretion of autophagosome-like vesicles (ALVs) in activated primary B cells. We found that IL-4/CD40 axis specifically induced the ALVs secretion in B cells. Albeit both IL-4/CD40 and IL-4/LPS stimulation induced the autophagy, IL-4/CD40 signaling did not increase the expression of genes involved in the autophagosome-lysosome fusion comparing to IL-4/LPS signaling. Additionally, live-imaging confocal microscopic examination showed that IL-4/CD40 signaling induced lower activity of autophagosome-lysosome fusion than IL-4/LPS signaling. Together our results suggest that ALVs may be secreted through the recycling endosome pathway.
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| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
In the first fiscal year, we focused on the exploring the mechanism of how IL-4/CD40 signaling induced ALVs secretion in B cells. IL-4/CD40 axis is specifically required for ALVs secretion since neither other cytokines, such as IL-6, IL-21 and IFN-γ, nor other receptors, such as TLR4, TLR9 and BCR, induce such secretion. Albeit both IL-4/CD40 and IL-4/LPS signaling induced the autophagy, IL-4/CD40 signaling induced lower expression of LAMP-2 (a marker of lysosome and late endosome) but higher expression of EEA-1 (a marker of early endosome) than IL-4/LPS signaling. Additionally, IL-4/CD40 signaling did not increase the expression of genes involved in the autophagosome-lysosome fusion comparing to IL-4/LPS signaling. Furthermore, IL-4/CD40 signaling induced lower activity of autophagosome-lysosome fusion than IL-4/LPS signaling in GFP-LC3-expressing B cells which was revealed by high-resolution/live-imaging confocal microscopy. Moreover, the LC3-II could be detected in the membrane-bound IgG+ vesicles sorted by magnetic beads. Together our results suggest that ALVs may be secreted through the recycling endosome pathway. Since previous reports indicated that autophagosome size is 200 to 500 nm in diameter, the LC-3-II+ ALVs were also found in the microvesicles fraction (100 to 1000nm in diameter). We also generated several atg7-knockout (KO) A20 B cell lines by CRISPR-Cas9 techniques and will use them for confirming our findings. Finally, the generation of B cell-specific atg7 KO (ATG7 B-KO) mice by crossing atg7Flox/Flox with CD19-Cre or Mb1-Cre mice is ongoing.
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| Strategy for Future Research Activity |
We will continue to clarify the molecular mechanism of IL-4/CD40 signals-induced ALVs secretion. Whether such ALVs are unique or fused with other intercellular vesicles, such as endosomes or lysosomes, will be examined by high-resolution confocal microscopy and transmission electronic microscopy. Given the fact that autophagosomes can be secreted as extracellular vesicles (EVs), which play an important role in cell-to-cell communication, autophagic process may also contribute to the transportation of cytosolic biomolecules including proteins and nuclear acids. We will clarify what kinds of biomolecules are carried by LC3-II-positive EVs. EVs from WT or Atg7-deficient B cells will be isolated and their protein composition will be analyzed by mass spectrometry and RNA composition including messenger RNA (mRNA) and microRNA will be analyzed by RNA-sequencing (RNA-seq). Finally, we will study the physiological function of ALVs. Our study may reveal a novel mechanism that autophagic process contributes to the secretion of extracellular vesicles as well as the intercellular communications.
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