Key Points• The natural inhibitor of neutrophil elastase, SLPI, is severely reduced in severe congenital neutropenia patients.• SLPI controls myeloid differentiation by regulation of NFkB, ERK1/2:LEF-1, and c-myc activation.We identified diminished levels of the natural inhibitor of neutrophil elastase (NE), secretory leukocyte protease inhibitor (SLPI), in myeloid cells and plasma of patients with severe congenital neutropenia (CN). We further found that downregulation of SLPI in CD34 1 bone marrow (BM) hematopoietic progenitors from healthy individuals resulted in markedly reduced in vitro myeloid differentiation accompanied by cell-cycle arrest and elevated apoptosis. Reciprocal regulation of SLPI by NE is well documented, and we previously demonstrated diminished NE levels in CN patients. Here, we found that transduction of myeloid cells with wild-type NE or treatment with exogenous NE increased SLPI messenger RNA and protein levels, whereas transduction of mutant forms of NE or inhibition of NE resulted in downregulation of SLPI. An analysis of the mechanisms underlying the diminished myeloid differentiation caused by reduced SLPI levels revealed that downregulation of SLPI with short hairpin RNA (shRNA) upregulated nuclear factor kB levels and reduced phospho-extracellular signal-regulated kinase (ERK1/2)-mediated phosphorylation and activation of the transcription factor lymphoid enhancer-binding factor-1 (LEF-1). Notably, microarray analyses revealed severe defects in signaling cascades regulating the cell cycle, including c-Myc-downstream signaling, in myeloid cells transduced with SLPI shRNA. Taken together, these results indicate that SLPI controls the proliferation, differentiation, and cell cycle of myeloid cells. (Blood. 2014;123(8):1239-1249

2165 Secretory Leukocyte Protease Inhibitor (SLPI) is a cationic serine protease inhibitor with antiprotease, primarily anti-Neutrophil ELastase (NE), activities. Moreover, SLPI modulates intracellular signal transduction pathways such as NF-kB and Erk. The molecular interaction and the balance between NE and SLPI is tightly regulated. On the one side, NE upregulates the SLPI expression and at the other hand SLPI inhibits the NE-induced degradation of proteins. We identified severe diminished levels of SLPI mRNA in CD33+ myeloid cells and in PMNs of patients with severe congenital neutropenia (CN) harbouring either ELANE or HAX1 mutations, as compared to patients with cyclic neutropenia (CyN) and to healthy individuals. SLPI protein levels in plasma of CN patients were also significantly reduced. We further analysed whether diminished levels of SLPI are associated with the „maturation arrest“ of myeloid cells seen in CN patients. We inhibited SLPI using lentivirus-based transduction of the myeloid cell line NB4 with SLPI-specific shRNA and analysed ATRA-triggered myeloid differentiation. Indeed, myeloid differentiation was severely affected in NB4 cells transduced with SLPI-specific shRNA, as compared to control shRNA transduced cells. Further, we analysed the mechanisms leading to SLPI downregulation. Previously, we identified severely reduced mRNA and protein levels of NE in myeloid cells and in plasma of CN patients with either ELANE or HAX1 mutations, as compared to healthy individuals. Knowing that NE induces SLPI expression, we assumed that diminished NE levels may be responsible for the low SLPI expression in CN patients. Indeed, inhibition of NE in the myeloid cell line NB4 using NE-specific shRNAs led to diminished expression of SLPI mRNA, as compared to ctrl shRNA transduced cells. At the same time, we also found that transduction of the myeloid cell line NB4 with wild type (WT) NE resulted in the increased expression of SLPI mRNA but mutated (MUT) forms of NE as found in CN patients were not able to induce SLPI mRNA, as compared to ctrl transduced cells. Taken together, both diminished NE levels and mutations in ELANE gene may cause downregulation of SLPI. In summary, SLPI is severely downregulated in CN patients due to defective NE protein levels and ELANE mutations. As a consequence, the anti-microbial and antiinflammatory activities of SLPI are diminished in CN patients. Disclosures: No relevant conflicts of interest to declare.

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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 Go/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. Disclosures: No relevant conflicts of interest to declare.