| 研究実績の概要 |
Interactions between the microbiota and the mucosal immune system are absolutely important in shaping mucosal immune responses. It is becoming clear that specific microbes influence specific lymphoid and non-lymphoid populations, for example commensal bacteria survive within dendritic cells in Peyer’s patches and enhance mucosal IgA responses. We showed that Stenotrophomonas maltophilia is constitutively present in vivo in colonic lamina propria macrophages but not in lymphoid-tissue resident macrophages. Clinically isolated S. maltophilia enter bone marrow-derived macrophages (BMDMs) in vitro and persistent colonization increases mitochondrial respiration and IL-10 production. Colonization by S. maltophilia is impaired by IL-10 deficiency. The bacteria secrete a 25-KDa protein encoded by smlt2713. Expression of smlt2713 in BMDMs induces IL-10 production. Bacteria deficient in smlt2713 show reduced colonization and IL-10 production. In vivo, pre-transfer of smlt2713-transduced BMDMs protects against chronic enterocolitis. We thus identify a novel symbiotic network between commensal bacteria and colonic macrophages. During this fiscal year (2021), we studied the molecular basis of host-microbe symbiosis mediated by smlt2713 in the tissue resident macrophages by using LC-MS/MS analysis for the identification of host-derived counterpart molecule against the smlt2713 and bulk RNA sequence analysis of the comprehensive gene expression by smlt2713 exposed BMDMs that is important for the creation of host-microbe symbiosis.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
3: やや遅れている
理由
Because of covid-19 pandemic infection, I had to cancel all the collaboration with National Institute of Biomedical Innovation, Ibaraki-Osaka described for the original experiments 2021. Instead, I did the following experiments to reveal the molecular mechanism for the hos-microbe symbiosis mediated by smlt2713.
1. Ligand identification of smlt2713. To assess the molecular basis of host-microbe symbiosis mediated by smlt2713 in the colonic macrophages, LC-MS/MS analysis of BMDM cellular proteins exclusively absorbed to FG NHS-beads (Tamagawa Seiki) immobilized with highly purified smlt2713 revealed that "perforin-2" encoding by the MPEG1, macrophage expressed gene 1, was a highly confident effector molecule identified strongly bound to smlt2713.
2. RNA-sequence and RT-PCR analysis of smlt2713 exposed BMDMs. To get more comprehensive information regarding appropriate gene expression by tissue resident macrophages which is crucial for the creation of this symbiosis mediated by smlt2713, RNA-sequence analysis of bulk mRNA purified from one million smlt2713 exposed BMDMs (differentiated by M-CSF, ten days after the beginning of the culture) clearly showed that the following genes were remarkably expressed in comparison to mRNA purified from control BMDMs cultured without any stimulants; the genes encoding for IL-1b(interleukin-1beta), SAA3 (serum amyloid A3), Acod1 (aconitate decarboxylase 1), Arg1 (arginase 1)/Arg2 (arginase 2), Lcn2 (lipocalin 2).
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| 今後の研究の推進方策 |
Because of the current covid-19 pandemic, I should do at least the following experiments at Hiroshima, during next fiscal year 2022, for the submission of the scientific original paper regarding this project.
1. We are planning to do several imaging experiments to assess the biological significance of the molecular interaction between smlt2713 and perforin-2 for the symbiotic cohabitation of tissue resident macrophages by S. maltophilia.
1) Microscopic imaging analysis of the perforin-2/smlt2713 interaction for the persistent symbiotic colonization of tissue resident macrophages with S. maltophilia. (1) Microscopic analysis of perforin-2-GFP fusion protein trafficking within BMDMs exposed to smlt2713 in vitro. (2) Microscopic analysis of perforin-2-GFP fusion protein trafficking within BMDMs infected with smlt2713+/- S. maltophilia in vitro.
2. We are also planning to assess the plausibility of unique simultaneous mRNA expression of the following effectors (Acod1, IL-1b, SAA3, Lcn2, Arg1/Arg2) for the creation of smlt2713 mediated host microbe symbiosis, especially focusing on the immune metabolic aspects by using in vitro and in vivo models.
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