A Lack of Secretory Leukocyte Protease Inhibitor (SLPI) Causes Defects in Myeloid Differentiation

Abstract 10

Previously, we demonstrated severe diminished mRNA and protein levels of secretory leukocyte protease inhibitor (SLPI) in CD33⁺ myeloid cells in PMNs and in plasma of patients with severe congenital neutropenia (CN), as compared to G-CSF treated healthy individuals. We further analysed whether diminished levels of SLPI are associated with the “maturation arrest“ of myeloid cells seen in CN patients. We inhibited SLPI in the CD34⁺ bone marrow hematopoietic progenitor cells and in the acute myelid leukemia cell line NB4 using lentivirus-based transduction with SLPI-specific shRNA and analysed G-CSF- or ATRA-triggered myeloid differentiation, respectively. We found that G-CSF-triggered myeloid differentiation of CD34⁺ cells transduced with SLPI-specific shRNA was significantly diminished, in comparison to ctrl shRNA transduced cells (on day 14 of differentiation 53.8 % of CD11b⁺ cells in SLPI shRNA group vs 86.3 % in the ctrl shRNA group (ttest: p = 0.005); 13.1 % of CD16⁺ cells in SLPI shRNA group vs 48.8 % in the ctrl shRNA group (ttest: p = 0.0006)). This was accompanied by G₀/G₁ cell cycle arrest and elevated apoptosis of SLPI shRNA transduced cells: on day 7 of differentiation 83.9 % of SLPI shRNA transduced cells and 66.6 % of the ctrl shRNA transduced cells were in G₀/G₁ phase (ttest: p = 0.03); 11 % of annexin V positive cells in SLPI shRNA transduced group vs 6.5 % in ctrl shRNA group (ttest: p = 0.004). Similar effects on the ATRA-induced myeloid differentiation were observed after inhibition of SLPI in the NB4 cell line.

We next analysed the mechanisms of diminished myeloid differentiation due to diminished SLPI levels. SLPI is a cationic serine protease inhibitor with antiprotease, primarily anti-neutrophil elastase, activities. Previously we showed regulation of neutrophil elastase by SLPI in myeloid cells. However, SLPI is also known to modulate intracellular signal transduction pathways such as NF-kB and Erk. SLPI is involved in the phosphorylation of Erk1/2 protein. Erk1/2 protein plays a role in myeloid differentiation. We found that ATRA treatment of NB4 cells transduced with ctrl shRNA led to the transient time-dependent phosphorylation of Erk1/2 on day 2 of culture with subsequent decline on day 4 of treatment. Interestingly, Erk1/2 phosphorylation was almost completely abolished on day 2 of ATRA treatment of NB4 cells transduced with SLPI shRNA. Recently, we demonstrated an essential role of LEF-1 in the granulocytic differentiation and severe diminished LEF-1 expression levels in myeloid cells of CN patients. It has been shown that Erk1/2 phosphorylates LEF-1 transcription factor. In line with defective phosphorylation of Erk1/2 we measured severe diminished levels of phosphorylated LEF-1 in SLPI shRNA transduced cells, in comparison to ctrl shRNA samples. Therefore, we concluded that SLPI regulates myeloid differentiation by regulation of Erk1/2 dependent LEF-1 phosphorylation and autoregulation.

To evaluate additional intracellular signaling pathways which are affected during G-CSF-triggered myeloid differentiation if SLPI is downregulated, we performed microarray analysis of RNA from CD34⁺ cells transduced with SLPI or ctrl shRNA and subsequently treated with G-CSF for 24 hours. Analysis of the microarray data using Ingenuity Systems Pathway Analysis (IPA) revealed severe significant defects in the signaling cascades involved in the differentiation (p-Value = 0.0093), proliferation (p-Value = 0.015), survival (p-Value = 0.012) and chemotaxis (p-Value = 0.0093) of myeloid progenitor cells and neutrophils which was correlated with significant downregulation of FPR2, MMP8, XCL1, S100A9, IL2 and OLFM4 mRNA expression. Moreover, signaling systems regulating DNA replication checkpoint and arrest in G_o/G₁ phase were also significantly affected (p-Value = 0.0009 and 0.0065, respectively) due to downregulation of mRNA expression of CHEK1, CCNE2, CDC6, MYBL1 and MYC.

Taken together, SLPI is a new candidate factor playing an important role in myelopoiesis by controlling proliferation, differentiation and cell cycle of myeloid cells not only by inhibition of neutrophil elastase but also by regulation of intracellular signaling pathways.