GADD45b Plays an Essential Role in the G-CSF Triggered Granulocytic Differentiation of Human Hematopoietic Cells

The mechanism of maturation arrest of bone marrow myeloid progenitors in severe congenital neutropenia (CN) patients is not fully elucidated. We found, that treatment of healthy individuals with G-CSF induces mRNA expression of GADD45b (Growth Arrest and DNA-Damage-inducible, beta) in CD33⁺ bone marrow myeloid progenitors. However, the expression of GADD45b was not activated in CD33⁺ cells of G-CSF treated CN patients. GADD45b functions as stress sensor downstream of G-CSF signaling and is essential in stress-induced murine myelopoiesis. Less is known about the function of GADD45b in the myeloid differentiation of human HSPCs. We hypothesized, that the inability of G-CSF to induce GADD45b expression might be a cause of diminished granulopoiesis in CN patients. To test this hypothesis, we inhibited GADD45b expression in CD34⁺ cells and iPSCs of healthy donors by introducing indels in exon 1 of the GADD45B gene using specific CRISPR/Cas9-gRNA ribonucleoprotein (RNP). We evaluated G-CSF-triggered myeloid differentiation of GADD45b-deficient iPSCs using embryoid body (EB)-based method and found that iPSCs cells present with severely diminished granulocytic differentiation upon GADD45b knockout, as assessed by FACS, CFU assay and morphological examination of cytospin slides. We also observed reduced G-CSF-mediated granulocytic differentiation of GADD45b-deficient CD34⁺ cells of healthy individuals in colony-forming units (CFU) assay and liquid culture differentiation followed by FACS analysis on day 7 and day 14.

Importantly, rescue of GADD45b in HSPCs of one CN patient by lentivirus-based transduction of GADD45B cDNA restored defective granulocytic differentiation, as compared to control transduced cells. These data strongly support the essential role of GADD45b in G-CSF-mediated granulocytic differentiation. GADD45b rescue analysis of additional CN patients cells is ongoing.

To study the mechanism of GADD45b activation upon G-CSF stimulation of hematopoietic cells, we performed in silico analysis of GADD45B promoter and found putative binding sites for G-CSF responsive hematopoietic transcription factors, including CEBPA, CEBPB, KLF4, STAT3 and STAT5. Using the dual luciferase reporter assay with 1.6 kb region of the GADD45B gene promoter, we found that KLF4, STAT5, CEBPA and CEBPB activate GADD45b expression in a dose-dependent manner. Intriguingly, CEBPA expression is severely diminished in myeloid cells of CN patients (Skokowa et al., 2006) and we assumed that G-CSF is not able to activate GADD45b expression in CN patients because of defective CEBPA. To study the mechanism by which GADD45b mediates myeloid differentiation, we performed RNA sequencing of WT or GADD45b-deficient CD34⁺ HSPCs treated or not with G-CSF. Interestingly, in GADD45b-deficient cells, G-CSF failed to induce mRNA expression of several genes essential for granulocytic differentiation and granulocyte functions including GLI1, CAMP/LL37, MMP8, CD16, LCN2, OLFM4, CX3CR1, SIGLEC5, as compared to WT cells. Reactome and Gene Set Enrichment Analysis (GSEA) of RNA-Seq data sets also revealed deregulation of the "myeloid CEBPA network", "GLI proteins pathway" and "neutrophil degranulation pathway" in G-CSF-treated GADD45b-deficient CD34⁺ cells, as compared to control G-CSF-exposed cells. Of note, severely diminished expression of plasma CAMP/LL37 levels is a unique feature of CN patients (Y. Ye et al. 2015).

In summary, our data suggest that GADD45b plays an essential role in granulocytic differentiation of human hematopoietic cells and inability of G-CSF to induce GADD45b expression in myeloid cells of CN patients may be a reason for the defective granulopoiesis.