Page 133 - Haematologica Vol. 109 - July 2024

ARTICLE - Ppm1b regulates HSC homeostasis
HN252, indicating that Ppm1b inhibition led to the prolif- erative suppression of HSC (Figure 1B). Compared to the freshly isolated LSK from BM (uncultured), the frequency of HSC at the G0-phase (Ki67-) was obviously decreased, which represents quiescent cells, while active cycling cells (Ki67+) were dramatically increased. This is consistent with previous findings that HSC undergoes active cell cycling stimulated by supplemented hematopoietic cytokines in in vitro culture.31,32 Notably, HN252 treatment led to a significant increase in G0 cells coupled with the reduced G1 and G2/M-phase frequency in LSK, suggesting that Ppm1b is required for HSC to switch from quiescence to an active state (Figure 1C, D).
In line with the findings of HN252 treatment, Ppm1b knock- down significantly inhibited the proliferation of LSK in in vitro culture, accompanied by a remarkable increase in cell death in LSK (Figure 1E, F). These data indicate that Ppm1b plays functional roles in the proliferation of HSC.
Ppm1b deficiency mice exhibited B-cell leukocytopenia
To explore the role of Ppm1b in the hematopoietic system, we generated hematopoietic-specific Ppm1b KO mice (conditional KO, CKO) by crossing mice bearing Ppm1b allele with loxP-flanked exon 2 with Vav-Cre transgenic mice (Online Supplementary Figure S2A). The ablation efficiency of Ppm1b was confirmed in mononuclear cells of BM and PB by qRT-PCR (Online Supplementary Figure S2B). CKO mice exhibited a dominant defect in white blood cells (WBC) that was mainly due to the reduction of lympho- cytes in PB, whereas red blood cells (RBC) and platelets (PLT) were unaffected (Figure 2A, Online Supplementary Figure S2C). Interestingly, the low lymphocyte attributed to the significantly reduced B cells (B220+) but not T cells (CD3+) in PB from CKO mice compared to that of control mice (Online Supplementary Figure S2D). Similar defective B lymphocytes were also observed in CKO BM (Figure 2B). We next examined the B-cell compartment in the BM ac- cording to the well-established surface markers (Online Supplementary Figure S2E), which is characterized by pre-pro B cell, pro-B cell, pre-B cell, immature B cell, and mature B cell.33 Loss of Ppm1b resulted in a remarkable decrease in cell number in all subpopulations of B lym- phocytes compared to that of WT controls, although there was no difference in their relative frequencies between the two groups of mice (Online Supplementary Figure S2E-I). Furthermore, common lymphoid progenitor (CLP) cells that commit into B-lineage progenitor cells also ex- hibited a decrease in CKO mice (Figure 2C, D). However, loss of Ppm1b did not disrupt the T lymphogenesis that also starts with the commitment of CLP, evidenced by the equal thymus and subpopulations of T lymphocytes (Online Supplementary Figure S2J-L). These findings suggest that Ppm1b is required for the commitment and proliferation of B-biased lymphoid progenitor cells from CLP, which is further supported by the markedly reduced colony number
Z. Lu et al. and size from CKO lineage-negative BM cells supplemented
with IL-7 (Figure 2E, F, Online Supplementary Figure S2M).
Loss of Ppm1b led to the suppression of phenotypic hematopoietic stem cell expansion in vivo
Ppm1bCKO mice exhibited a significantly diminished fre- quency and absolute number of HSC (lineage-Sca1+c-Kit+, LSK) in BM (Figure 3A, Online Supplementary Figure S3A), while the committed HPC (lineage-Sca1-c-Kit+, LK) were comparable between these two groups of mice (Online Supplementary Figure S3B). To further explore the impact of Ppm1b deficiency on the HSC, we examined its sub- populations according to the different identifications.8,34 Interestingly, Ppm1bCKO mice exhibited the obviously de- creased long-term HSC (CD34−CD135−LSK, LT-HSC) and short-term HSC (CD34+CD135−LSK, ST-HSC) (Figure 3B, Online Supplementary Figure S3C). A similar reduction was also observed in SLAM-HSC (CD150+CD48−LSK) represent- ing the quiescent HSC in Ppm1bCKO mice (Online Supple- mentary Figure S3D).35 To further determine the number of functional HSC of Ppm1bCKO mice in the BM, we performed limiting dilution assays and found that the frequency of competitive repopulation units (CRU) in Ppm1bCKO mice was significantly lower than that of control mice (Figure 3C). Indeed, Ppm1bCKO HSC showed comparable homing activity to WT ones (Online Supplementary Figure S3E). Moreover, Ppm1b deletion had an undetectable effect on the HSC survival (data not shown). Cell cycle analysis by Ki67 staining demonstrated that Ppm1b-deficient HSC showed more quiescence than that of WT control (Figure 3D). This is confirmed by the low cell division of ex vivo cultured Ppm1b-deficient LSK indicated by the relatively high CFSE staining (Online Supplementary Figure S3F). These findings indicate that loss of Ppm1b impaired the active proliferation of HSC.
To confirm the regulatory role of Ppm1b in functional HSC, we challenged CKO mice with 5-fluorouracil (5-FU) that eliminates cycling cells and triggers the hematopoietic repopulation.36 All the Ppm1bCKO mice died at 20 days of continuous 5-FU injection, whereas 40% of WT mice sur- vived after 3 rounds of administration (Figure 3E), indicat- ing that Ppm1b is required for the stressed reconstitution of HSC. Consistently, the frequencies of HSC at the G0 and G1 phase in CKO mice also showed a more dramatic decrease after 5-FU administration compared with the steady state (Online Supplementary Figure S3G), suggesting that Ppm1b deletion impaired the transition between G0 to G1 phase under stress hematopoiesis.
To further assess the function of Ppm1b-deficient HSC, we performed competitive serial transplantation assays (Online Supplementary Figure S3H). Ppm1bCKO-derived cells showed a successive reduction in the primary recipients and subsequent recipients (Online Supplementary Figure S3 I), indicating the impaired reconstitution of CKO HSC. Notably, the frequencies of LSK and SLAM-LSK derived
Haematologica | 109 July 2024
2147