Signal-Transducing Adaptor Protein-2 Blocks B Cell Recovery Under Hematological Stress at Pre-B Stage Via TLR4 Signaling

All lineages of blood cell production from hematopoietic stem cells (HSC) are maintained for an entire lifetime. That is consistent under homeostatic condition, while hematopoiesis is changed in response to various types of stress, including infections and aging. It has been reported that hematopoietic stem and progenitor cells (HSPC) are targets of pathogen products and danger signals. After the exposure to Gram-negative lipopolysaccharide (LPS) that is the ligand of Toll-like receptor (TLR) 4, HSC enter cell-cycle and differentiate into myeloid lineage cells while B lymphopoiesis is suppressed. Although the regulation of hematopoiesis by HSC is well studied, many questions still remain regarding how progenitors contribute the response to hematopoietic demands. Signal-transducing adaptor protein-2 (STAP-2) was cloned as a c-fms/M-CSFR interacting protein in 2003, and the interaction with a variety of signaling or transcriptional molecules such as STAT5, MyD88 and IκB kinase (IKK) in macrophages or T cells has been reported. The function as an adaptor protein in various cell types and signaling pathways, and its ubiquitous expression promoted us to investigate the effect of STAP-2 on HSPC under hematopoietic stress using gene-modified mice.

We previously reported that hematopoiesis in STAP-2 knock out mice (KO) is normal under homeostatic condition. First in this study, competitive bone marrow (BM) transplantation experiment was conducted using KO. Four months after intravenous injection of Lineage^- Sca1⁺ cKit^high (LSK) HSPC (CD45.2⁺) derived from KO or wild-type mice (WT) with competitor BM mononuclear cells (CD45.1⁺), the engraftment and lineage distribution were comparable, indicating that STAP-2 does not affect the function of long-term HSC. Interestingly, we found that chimerism in peripheral B cells at 1 month after transplantation in KO LSK transplanted mice was higher than control with statistical significance. In BM, STAP-2 deficiency increased pre-B and immature B cell cells, while there were no differences in numbers of LSK HSPC or Lin^- Sca1⁺ cKit^low Flk2^high common lymphoid progenitor (CLP) between WT and KO donor. To evaluate the role of STAP-2 in early B lymphopoiesis, we generated transgenic mice (Tg) that overexpress STAP-2 under the control of Eμ enhancer and Lck proximal promoter. The promoter could drive expression of the inserted cDNA in lymphoid lineage cells from CLP stage. In Tg, we observed significant decrease of B progenitors compared to WT BM. When HSPC (LSK or CLP) derived from Tg were cultured with OP9 stromal cells under B cell condition in the presence of IL-7, Flt3-ligand and SCF, the production of B220⁺ B cells were significantly suppressed. The same was true in stromal-cell free cultures. In colony-forming unit (CFU) assays, we found that STAP-2 overexpression decreased the ability to generate pre-B colonies, while the deficiency promoted high recovery with statistical significance (recovered colony number; 40.0 ± 1.4 in WT, 7.0 ± 1.4 in Tg, and 89.0 ± 5.7 in KO). To elucidate the detailed mechanisms, RNA-seq experiment with WT and Tg under steady-state as well as WT and KO 1 month after transplantation was conducted. Data among them were compared, and bioinformative approach with Ingenuity Pathway Analysis revealed that STAP-2 upregulates TLR4 signaling in pre-B cells during stress hematopoiesis. Consistent with the results, when LPS was added into CFU cultures, the recovered pre-B colony was suppressed. Moreover, deletion of STAP-2 partially cancelled the inhibitory effect of LPS on pre-B cell proliferation.

In summary, we found that STAP-2 regulates the early B lymphopoiesis under hematopoietic stress. While STAP-2 did not influence stemness of HSC or homeostatic hematopoiesis, short-term recovery of B cells after transplantation was promoted by STAP-2 deficiency. This effect was induced by the activated proliferation of pre-B cells. Analyses with Tg mice confirmed the inhibitory effect of STAP-2 on early B lymphopoiesis. RNA-seq analyses have shown that activation of TLR4 signaling at pre-B stage occurs 1 month after transplantation as well as in Tg mice, and STAP-2 deficiency partially cancelled the inhibitory effects of LPS in CFU pre-B assays. Our study unveiled the critical role of lineage progenitors in short-term response to hematological demands and developmental stage-specific regulation of TLR4-STAP-2 pathway.