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M. Ichii et al.
When the levels of p38 MAPK, ERK1/2 and STAT5 phos- phorylation in B progenitors were evaluated under steady state, there were no differences between WT and Tg (Figure 6A). After mice were irradiated, the phosphoryla- tion was activated. We found that STAP-2 overexpression exaggerated p38 phosphorylation significantly, compared to WT B progenitors (Figure 6B).
While several inflammatory molecules including LPS are known to be activators of B cells at the maturation stage following export to the periphery, recent studies showed that the TLR4 signaling pathway inhibits proliferation of pro-B and pre-B cells, and promotes the differentiation to IgM+ immature B cells in BM.28,29 Consistent with previous studies, the suppression of B-cell differentiation in BM was observed 3 days following sub-lethal dosing of LPS treatment (1.0 mg/kg) (Figure 6C). When Tg mice were treated, the actual B-cell number in peripheral blood decreased and a significant reduction of B progenitors in BM was observed (Figure 6C and D). The proliferation and apoptosis analyses, including BrdU and Annexin V staining methods, revealed a similar pattern to those after sub-lethal irradiation in vivo, indicating that the same mechanism is employed under these hematologic stres- sors (Figure 6E). Treatment of LPS-induced septic Tg mice with the p38 MAPK inhibitor SB203580 increased the number of B progenitors in BM, but this increase was not statistically significant (Online Supplementary Figure S3).
To study the specific interaction between each signal and STAP-2, pre-B-culture systems were used. This made it possible to eliminate the influence of HSPC and other lineage cells, which are also targets of the same signals such as IFN, TNFα, IL-1β, MAPK, and TLRs.5-10 When LPS was added to CFU pre-B cultures, we found that deletion of STAP-2 attenuated the inhibitory effect of LPS (Figure 6F). The cancelation, although incomplete, was statistical- ly significant, and the same was true in stroma-free liquid cultures (Figure 6G). Similar to reports following LPS treat- ment in vivo, apoptosis was induced (Figure 6H); however, BrdU analysis revealed that differences were not observed with or without treatment (data not shown). Moreover, STAP-2 deficiency led to a statistically significant reduc- tion in the effects (Figure 6H). Regarding effects on differ- entiation, the expression of IgM after one week of culture was identical between WT and KO pre-B-cell cultures (Figure 6I). The limited effects on TLR4 signaling indicates the existence of other pathways involved with STAP-2. The addition of IFNγ, TNFα, IL-1β and IL-6 into pre-B-cell cultures did not induce statistically significant differences between WT and KO cultures (data not shown).
Our previous studies showed that Stap-2 mRNA is induced in murine hepatocytes in response to stimulation by LPS and IL-6.14 However, the mRNA level in pre-B or pro-B cells did not change after LPS treatment in vivo, indi- cating the sustained protein level is sufficient to serve as the suppressor of B-progenitor proliferation (Figure 7A). In contrast, we found the expression level varied after sub- lethal irradiation (Figure 7B). When 13 mice were irradiat- ed, the expression in pre-B cells was upregulated in five mice (38.5%) while in the same number of mice that was completely lost. The same was observed in pro-B cells. Interestingly, Stap-2 expression in lineage negative cells was increased approximately four times compared to con- trols. The changes recovered to normal levels one month after irradiation (Figure 7C). These results indicated that Stap-2 mRNA expression may be regulated via the direct
effects of several hematologic stress factors, as well as indirectly by the affected microenvironment.
Lastly, we sought to determine if the upregulated inflammation during hematologic stress could alter STAP- 2 expression in pre-B cells. Several cytokines such as IFNγ, IL-6, IL-1β, TNFα, and LPS were added to pre-B-cell cul- tures, and the expression of Stap-2 mRNA was analyzed after 24 hours of stimulation (Figure 7D). Interestingly, only TNF significantly increased Stap-2 expression. These results indicate that inflammatory signals during hemato- logic stress may use STAP-2 for negative regulation of B lymphopoiesis.
Discussion
Here we demonstrate that STAP-2 governs early B lym- phopoiesis in BM during hematopoietic stress. While STAP-2 did not influence stemness of HSC or homeostatic hematopoiesis, short-term recovery of B cells was impaired by STAP-2 following transplantation. This effect was due to the inhibited proliferation of pre-B cells. RNA- Seq analyses showed that activation of several inflamma- tory signals occurs one month after transplantation in Tg mice. Culture experiments indicated developmental stage- specific inhibitory regulation of the TLR4-STAP-2 path- way as one of the mechanisms. Our study unveiled the critical role of STAP-2 in B-cell lineage progenitors in short-term responses to hematologic stress (Figure 8).
Previously, we and others reported that STAP-2 functions as an adaptor protein in a variety of signaling pathways, including immune responses and tumorigenesis. STAP-2 regulates cell growth, integrin-mediated adhesion, migra- tion induced by CXCL12, and apoptosis in T cells.17,30,31 The production of cytokines in macrophages is also affected by STAP-2. In tumorigenesis, STAP-2 binds to BCR-ABL in chronic myeloid leukemia cells, and also inter- acts with Brk, which mediates STAT5 activation in breast cancers.21,22 Recent studies reported that STAP-2 has a signif- icant impact on disease severity in mouse models of IgE allergy and inflammatory bowel disease.18,24 In this study, we show a novel function of STAP-2 during stress hematopoiesis. Consistent with previous reports, STAP-2 modulates inflammatory signals such as TLR4 in pre-B cells.
Sustained hematopoiesis is regulated by various mecha- nisms, including cell-intrinsic and -extrinsic factors, and the role of HSC in hematologic stress has been intensively studied.1-3,5,9 Unlike HSC, lineage-committed progenitors lose multipotency, yet retain the ability to proliferate dur- ing differentiation. In murine erythropoiesis, it is reported that robust expansion of erythroid progenitors in BM and spleen occurs to counteract anemic stress using signals dif- ferent from those in homeostatic conditions.32,33 In B lym- phopoiesis, various factors such as infection, aging and G- CSF administration, are known suppressors of BM B lym- phopoiesis.34-38 However, some studies focused on the immune functions of B lymphocytes,39 and the effects of G-CSF on B progenitors in BM are indirect.36,37 How B pro- genitors in BM respond to stress is not well understood. In the current study, we show that STAP-2 impairs pre-B- stage proliferation and delays the recovery of B cells dur- ing hematologic stress, such as transplantation and irradi- ation. The proliferation of lineage-committed progenitors appears to be more important in hematologic regeneration than previously believed.
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haematologica | 2021; 106(2)