Persistent DNA damage–induced NLRP12 improves hematopoietic stem cell function
Qiqi Lin, Limei Wu, Zhilin Ma, Fabliha Ahmed Chowdhury,1, Habibul Hasan Mazumder, Wei Du
Qiqi Lin, Limei Wu, Zhilin Ma, Fabliha Ahmed Chowdhury,1, Habibul Hasan Mazumder, Wei Du
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Research Article Aging Hematology

Persistent DNA damage–induced NLRP12 improves hematopoietic stem cell function

Abstract

NOD-like receptor 12 (NLRP12) is a member of the nucleotide-binding domain and leucine-rich repeat containing receptor inflammasome family that plays a central role in innate immunity. We previously showed that DNA damage upregulated NLRP12 in hematopoietic stem cells (HSCs) of mice deficient in the DNA repair gene Fanca. However, the role of NLRP12 in HSC maintenance is not known. Here, we show that persistent DNA damage–induced NLRP12 improves HSC function in both mouse and human models of DNA repair deficiency and aging. Specifically, treatment of Fanca–/– mice with the DNA cross-linker mitomycin C or ionizing radiation induces NLRP12 upregulation in phenotypic HSCs. NLRP12 expression is specifically induced by persistent DNA damage. Functionally, knockdown of NLRP12 exacerbates the repopulation defect of Fanca–/– HSCs. Persistent DNA damage–induced NLRP12 was also observed in the HSCs from aged mice, and depletion of NLRP12 in these aged HSCs compromised their self-renewal and hematopoietic recovery. Consistently, overexpression of NLRP12 substantially improved the long-term repopulating function of Fanca–/– and aged HSCs. Finally, persistent DNA damage–induced NLRP12 maintains the function of HSCs from patients with FA or aged donors. These results reveal a potentially novel role of NLRP12 in HSC maintenance and suggest that NLRP12 targeting has therapeutic potential in DNA repair disorders and aging.

Authors

Qiqi Lin, Limei Wu, Zhilin Ma, Fabliha Ahmed Chowdhury,1, Habibul Hasan Mazumder, Wei Du

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Figure 1

Persistent DNA damage induces Nlrp12 upregulation in Fanca–/– HSCs.

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Persistent DNA damage induces Nlrp12 upregulation in Fanca–/– HSCs. (A) ...
(A) Gating strategy for FACS for LSK (Lin–Sca-1+c-Kit+), SLAM (Lin–Sca-1+c-Kit+CD150+CD48–), MPP (multipotent progenitor; Lin–Sca-1+c-Kit+CD150–CD48–), and HPC (hematopoietic progenitor cell; Lin–c-Kit+Sca-1+CD150–CD48+ and Lin–c-Kit+Sca-1+CD150+CD48+) cell fractions isolated from Fanca–/– mice and their WT littermates. (B and C) Persistent DNA damage in Fanca–/– HSCs. Fanca–/– mice or their WT littermates were i.p. injected with a single dose of MMC (0.75 mg/kg) followed by flow cytometry analysis for γ-H2AX and Comet assay for DNA strand breaks at different time points. Representative flow plots (B, upper) and MFI kinetics (B, lower) and comet images at 8 hours post–MMC treatment (C, left) and olive tail moment (right) are shown. Original magnification, ×100. 0h, untreated control. (D) Kinetics of DNA damage–induced Nlrp12 expression in HSCs. Fanca–/– mice or their WT littermates were i.p. injected with a single dose of MMC (0.75 mg/kg) followed by cell sorting for SLAM cells at different time points. RNAs were then extracted from such cells followed by qPCR analysis for Nlrp12 expression using primers listed in Supplemental Table 1. Samples were normalized to the level of GAPDH mRNA (n = 6–9 per group). (E) Persistent DNA damage induces Nlrp12 upregulation specifically in Fanca–/– HSCs. Whole BM cells (WBMCs) from mice described in D at 0 hours (–MMC) and 16 hours (+MMC) were subjected to cell sorting for SLAM (LSK CD150+CD48–), MPP (LSK CD150–CD48–), or HPC (LSK CD150–CD48+ and LSK CD150+CD48+) cell fractions. RNAs were then extracted from such cells followed by qPCR analysis for Nlrp12 expression. Results are shown as means ± SD of 3 independent experiments (n = 6–9 per group). (F) Increased NLRP12 proteins in Fanca–/– HSCs. The cell fractions of HSCs, MPPs, and HPCs described in E were subjected to intracellular NLRP12 staining and flow cytometry analysis. Representative histogram (left) and quantification of MFI (right) are shown. (n = 6–9 per group). *P < 0.05; **P < 0.01; ***P < 0.001 MMC versus untreated control (0h). Paired or unpaired 2-tailed Student’s t test was used for 2-group comparison and 1-way ANOVA for comparison of more than 2 groups.