Abstract
Tryptophan 2,3-dioxygenase (TDO2) was an initial rate-limiting enzyme of the kynurenine (Kyn) pathway in tryptophan (Trp) metabolism. We undertook this study to determine a comprehensive analysis of TDO2 expression in immune cells and assess the characterization of immune cell phenotype in TDO2 knockout mice. The expression of TDO2 in various tissues of DBA/1 mice was detected by quantitative real-time PCR (qPCR) and immunohistochemistry. Both flow cytometry and immunofluorescence were used to analyze the expression of TDO2 in immune cells. Furthermore, TDO2 knockout (KO) mice were generated by CRISPR/Cas9 technology to detect immune cell phenotype. TDO2 protein level in liver was tested by western blot. High-performance liquid chromatography was used to detect the level of Trp and Kyn. Flow cytometry was used to test the proportions of splenic lymphocyte subsets in wild-type (WT) and TDO2 KO mice. We found that TDO2 was expressed in various tissues and immune cells, and TDO2 staining was mainly observed in the cytoplasm of cells. There was no difference in the development of immune cells between TDO2 KO mice and WT mice, including T cells, B cells, memory B cells, plasma cells, dendritic cells, and natural killer cells. Interestingly, the reduced M1/M2 ratio was observed in the peritoneal macrophages of TDO2 KO mice. Taken together, these findings enriched the known expression profile of TDO2, especially its expression in immune cells. Our study suggested that TDO2-mediated Trp-Kyn metabolism pathway might be involved in the immune response.
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Abbreviations
- Trp:
-
Tryptophan
- Kyn:
-
Kynurenine
- KP:
-
Kynurenine pathway
- IDO1:
-
Indoleamine 2,3-dioxygenase 1
- IDO2:
-
Indoleamine 2,3-dioxygenase 2
- TDO2:
-
Tryptophan 2,3-dioxygenase 2
- KO:
-
Knockout
- WT:
-
Wild-type
- PM:
-
Peritoneal macrophage
- SMC:
-
Spleen mononuclear cell
- MFI:
-
Mean fluorescence intensity
- DC:
-
Dendritic cell
- NK:
-
Natural killer
- Th17:
-
T helper type 17
- Treg:
-
Regulatory T cell
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Acknowledgements
Authors would like to thank Ms. Yue Cui for microinjection and technical guidance.
Funding
This work was financially supported by the National Nature Science Foundation of China (Nos. 81573443, 81673444), the Anhui Province Natural Science Fund (outstanding youth) (No. 170808J10), the Key Projects of Outstanding Young Talent Support Program in Colleges and Universities (No. gxyqZD2016043), the Collaborative innovation project of key scientific research platform in Anhui Universities (GXXT-2020-065) and the Project of Introduction and Cultivation of Leading Talents in Universities and Colleges (4501027105).
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Susu Li, Siyu Li and YC contributed to the conception and experimental design. Susu Li and Siyu Li performed the experiments and collected data. Analysis of the data and preparation of the manuscript was performed by Susu Li and YC. All authors contributed to the interpretation of results. All authors have read and approved the final version of the manuscript and agree to be accountable for all aspects of the work in ensuring the questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.
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All animal experiments were approved by the Animal Experiment Clinical Review Committee of the Institute of Clinical Pharmacology of Anhui Medical University (LLSC20200669). All researches were carried out in accordance with the guidance of the Animal Care and Use Committee and adhered to the principles of the laboratory animal care guidelines. The present study complied with principles and standards for reporting animal experiments in Experimental Physiology (Grundy, 2015).
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Li, S., Li, S., Zhao, Y. et al. A comprehensive analysis of TDO2 expression in immune cells and characterization of immune cell phenotype in TDO2 knockout mice. Transgenic Res 30, 781–797 (2021). https://doi.org/10.1007/s11248-021-00281-8
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DOI: https://doi.org/10.1007/s11248-021-00281-8