Reverse genetics in humanized mice reveals CARD8-mediated pyroptosis causing pancytopenia in human DPP9 deficiency
Tianli Xiao, J. Richard Brewer, Maximillian Carlino, Ailin Han, Yamato J. Takabe, Chia-Yi Lee, Fengrui Zhang, Mi Chen, Holly Nicole Blackburn, Amin H. Nassar, Qiankun Wang, Kristen Brennand, Liang Shan, Esen Sefik, Diane S. Krause, Richard A. Flavell
Tianli Xiao, J. Richard Brewer, Maximillian Carlino, Ailin Han, Yamato J. Takabe, Chia-Yi Lee, Fengrui Zhang, Mi Chen, Holly Nicole Blackburn, Amin H. Nassar, Qiankun Wang, Kristen Brennand, Liang Shan, Esen Sefik, Diane S. Krause, Richard A. Flavell
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Research Article Hematology Immunology

Reverse genetics in humanized mice reveals CARD8-mediated pyroptosis causing pancytopenia in human DPP9 deficiency

Abstract

Loss-of-function mutation in the human gene dipeptidyl peptidase 9 (DPP9) causes Hatipoglu syndrome leading to severe inflammasomopathy. A key feature of the disease is pancytopenia, and patients require bone marrow transplantation, but the mechanism of cell loss is unclear, since Dpp9-mutant mice have normal hematopoiesis, suggesting that a distinct mechanism of disease occurs in humans. Here, we present a model of human DPP9 deficiency leveraging reverse genetics in the MISTRG6 humanized mice. We found that CRISPR editing of human CD34+ hematopoietic stem and progenitor cells (HSPCs) led to very efficient and persistent gene deletion in vivo. Human DPP9 deletion recapitulated cytopenia in peripheral blood and in the bone marrow, and cell loss was cell intrinsic. However, DPP9 deletion led to few transcriptional changes suggesting posttranscriptional regulation in human HSPCs. Mechanistically, DPP9 deficiency led to activation of the CARD8 inflammasome, resulting in HSPC pyroptosis, whereas NLRP1 was dispensable for cell death. Thus, our results reveal a unique human mechanism of disease and offer therapeutic insight for this inflammasomopathy.

Authors

Tianli Xiao, J. Richard Brewer, Maximillian Carlino, Ailin Han, Yamato J. Takabe, Chia-Yi Lee, Fengrui Zhang, Mi Chen, Holly Nicole Blackburn, Amin H. Nassar, Qiankun Wang, Kristen Brennand, Liang Shan, Esen Sefik, Diane S. Krause, Richard A. Flavell

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Figure 1

Efficient and persistent gene KO in human CD34+ HSPCs in the MISTRG6 humanized mouse model.

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Efficient and persistent gene KO in human CD34+ HSPCs in the MISTRG6 hum...
(A) Schematic of gene editing followed by engraftment in the MISTRG6 humanized mouse model. MISTRG6 pups were injected intrahepatically in the first 3 days after birth without preconditioning. (B and C) TRAC–/– and control CD34+ HSPCs were engrafted into MISTRG6 mice, and the mice were assessed 12-16 weeks after engraftment. Data were pooled from 2 experiments. (B) Representative flow plot from splenic cells. (C) Number of human T cells in tissues. (D–F) CSF1R–/– and control CD34+ HSPCs were engrafted into MISTRG6 mice and assessed 9 weeks after engraftment. (D) Expression of CSF1R on human CD33+ myeloid cells in the blood 9 weeks after engraftment. Representative histogram and summary data are shown. (E) Number of CD14+CD16+ and CD16+CD14– monocytes in the blood. (F) Number of CD68+ macrophages in the liver. Experiments are representative of results from mice engrafted with cells from 2 human donors. **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001, by 2-tailed Student’s t test. Data shown as mean ± SD.