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Myd88 knockdown with RNA interference induces in vitro immune hyporesponsiveness in dendritic cells from rhesus monkeys

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Abstract

Immature dendritic cells (imDCs) are activated and mature to initiate an adaptive immune response, resulting in allograft rejection and transplantation failure. Myeloid differentiation factor 88 (Myd88) is a key factor in the Toll-like receptor (TLR) signaling pathway. Here, we investigated the effect of Myd88 silencing on DC function and immune response. CD34 + cells were isolated from the bone marrow of rhesus monkeys by the immunomagnetic bead method and then infected with an adenovirus expressing Myd88-specific short hairpin RNA (sh-Myd88). sh-NC (nontargeting negative control)- or sh-Myd88-infected DCs were treated with lipopolysaccharide (LPS) for another 48 h to induce DCS maturation. The maturation of DCs was identified by immunofluorescence staining for MHCII, CD80, and CD86. DC apoptosis was examined using Annexin V/PI staining. DC-related cytokine levels (IFN-γ and IL-12) were assessed by ELISA. A mixed lymphocyte reaction (MLR) was performed to test the effect of Myd88-silenced DCs on T lymphocytes in vitro. The results showed that compared with control or sh-NC-infected DCs, Myd88-silenced DCs had lower MHCII, CD80, CD86, and DC-related cytokine (IFN-γ and IL-12) levels. Myd88 did not affect the apoptosis of DCs. MLR demonstrated that Myd88 silencing could effectively block LPS-activated T cell proliferation in vitro. These data were consistent with the characteristics of tolerogenic DCs. In conclusion, our data indicated that Myd88 silencing could inhibit the maturation of imDCs and alleviate immune rejection, which provides a reference for immune tolerance in clinical liver transplantation.

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Acknowledgements

We thank the Central Cell Laboratory of Kunming Institute of Zoology, Chinese Academy of Science for cell culture work.

Funding

This study was supported by the National Natural Science Foundation of China (No. 81160069), Yunnan Provincial Science and Technology (No. 2018FE001-170), Applied Basic Research Foundation of Yunnan Province (No. 2016FB133), and National Natural Science Foundation of China (No. 81860124).

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Author notes

  1. Qiuhong Wang and Dongyun Cun contributed equally to this work.

Authors and Affiliations

  1. Department of Hepatopancreatobiliary Surgery, The Second Affiliated Hospital of Kunming Medical University, No. 112, Kunrui Road, Kunming, 650101, Yunnan, People’s Republic of China

    Qiuhong Wang, Dongyun Cun, Peng Chen & Mingdao Hu

  2. Department of General Surgery, The First People’s Hospital of Qujing City, Yunnan Province, Qujing City, 655000, People’s Republic of China

    Desong Xu

  3. Department of Hepatobiliary Surgery, Fujian Province, The First Hospital of Putian City, No.389 Longdejing Street, Putian, 351100, People’s Republic of China

    Liang Lin

  4. Department of Gastrointestinal Surgery, Handan Central Hospital, Handan, 056001, Hebei Province, People’s Republic of China

    Jian Jiao

  5. Department of Hepatopancreatobiliary, The First People’s Hospital of Yunnan Province, The Affiliated Hospital of Kunming University of Science and Technology Kunming, Yunnan Province, Kunming, People’s Republic of China

    Li Zhang

  6. Department of Gastrointestinal Surgery, First Affiliated Hospital of Kunming Medical University, Kunming, Yunnan, 650032, People’s Republic of China

    Cheng Xi

  7. Emergency Department, The Central People’s Hospital of Yichang City, Hubei Province, Yichang, 443000, People’s Republic of China

    Wei Li

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  1. Qiuhong Wang
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  2. Dongyun Cun
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  6. Li Zhang
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  8. Wei Li
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  9. Peng Chen
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  10. Mingdao Hu
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Contributions

All authors contributed to the study conception and design. Peng Chen and Mingdao Hu designed the study. Material preparation, data collection, and analysis were performed by Qiuhong Wang, Dongyun Cun, Desong Xu, Liang Lin, Jian Jiao, Li Zhang, Cheng Xi, and Wei Li. The first draft of the manuscript was written by Qiuhong Wang and Dongyun Cun. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Peng Chen or Mingdao Hu.

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Wang, Q., Cun, D., Xu, D. et al. Myd88 knockdown with RNA interference induces in vitro immune hyporesponsiveness in dendritic cells from rhesus monkeys. Immunogenetics 74, 303–312 (2022). https://doi.org/10.1007/s00251-022-01260-x

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  • DOI: https://doi.org/10.1007/s00251-022-01260-x

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