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Effects of STAP-2 on B-cell recovery
Figure 8. STAP-2 attenuates B-cell recovery under hematologic stress. A graphic depiction of hemato- logic stresses that variably regulate STAP-2 mRNA expression via the direct effects of several hemato- logic stress factors, such as TNFα, as well as indirect effects imposed by the affected microenvironment. STAP-2 impairs B-cell recovery under hematologic stress.
proliferation of pre-B cells leading to compromised B-cell recovery during hematologic stress.
Inflammatory signals upregulated following hematopoietic stem cell transplantation are exaggerated by STAP-2
To determine the molecular basis for the inefficient B lymphopoiesis in STAP-2 Tg BM, transcriptome analysis was used to identify the pathways regulated by STAP-2 under hematologic stress. We evaluated global gene expression profiles of pre-B cells derived from WT and Tg BM under steady-state as well as WT and KO BM one month post transplantation by RNA-sequencing (RNA- Seq) experiments (Figure 5). The number of significantly regulated transcripts is shown in Figure 5A.
First we compared pre-B cells isolated one month after transplantation with cells under homeostasis. It was expected that pathways related to hematologic develop- ment and genes with lymphoid signatures would be sig- nificantly regulated (Figure 5B). Furthermore, a bioinfor- matic approach with Ingenuity Pathway Analysis revealed that groups of genes involved in inflammatory responses were strongly upregulated during stress hematopoiesis. Differential display between WT and KO after transplan- tation showed that the deletion of STAP-2 prevented inflammatory changes induced by stress. STAP-2 overex- pression activated these pathways even under steady- state conditions.
Next, we investigated which signals were commonly
regulated among the following three pairs: WT with or without transplantation, WT or KO with transplantation, and WT or Tg under steady state (Figure 5C and D). As a result, pathways related to cytokines (IFN, TNF, IL-1β and IL-6), innate immune system (TLR and complement sys- tem), NFκB and p38 MAPK were extracted. Among them, the pathway activated to the greatest degree by STAP-2 and regenerative stress triggered receptor expression on myeloid cells type-1 (TREM-1) signaling (Figure 5C). This molecule has recently been shown to play an important role in controlling the intensity of innate immune respons- es.25-27 Upregulation of TREM-1 was verified by quantita- tive PCR and flow cytometry (Online Supplementary Figure S2A-C). Similar to macrophages, the activation of TLR4 signaling in Baf-3 pre-B cells upregulated the expression of TREM-1 (Online Supplementary Figure S2D). There were no shared pathways involved in B-cell development, and pro- tein expression of IL7R, and mRNA expression of E2a, Ebf1, Pax5, Rag1 and Rag2 were not regulated under these conditions (Figure 5E and F).
Inflammatory network regulates stress B lymphopoiesis in bone marrow
Our findings from RNA-Seq experiments indicate STAP-2 modulation with inflammatory signals delays B- cell recovery after hematologic stress. This was also con- firmed with the evaluation of phosphorylated forms of the target proteins of inflammatory signaling pathways, such as JAK/STAT and MAPK in B progenitors in vivo.
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