Julia Skokowa

Neutrophils: Between host defence, immune modulation, and tissue injury.

Neutrophils, the most abundant human immune cells, are rapidly recruited to sites of infection, where they fulfill their life-saving antimicrobial functions. While traditionally regarded as short-lived phagocytes, recent findings on long-term survival, neutrophil extracellular trap (NET) formation, heterogeneity and plasticity, suppressive functions, and tissue injury have expanded our understanding of their diverse role in infection and inflammation. This review summarises our current understanding of neutrophils in host-pathogen interactions and disease involvement, illustrating the versatility and plasticity of the neutrophil, moving between host defence, immune modulation, and tissue damage.

The role of sirtuin 2 activation by nicotinamide phosphoribosyltransferase in the aberrant proliferation and survival of myeloid leukemia cells

Background Inhibitors of nicotinamide phosphoribosyltransferase have recently been validated as therapeutic targets in leukemia, but the mechanism of leukemogenic transformation downstream of this enzyme is unclear. Design and Methods Here, we evaluated whether nicotinamide phosphoribosyltransferase’s effects on aberrant proliferation and survival of myeloid leukemic cells are dependent on sirtuin and delineated the downstream signaling pathways operating during this process. Results We identified significant upregulation of sirtuin 2 and nicotinamide phosphoribosyltransferase levels in primary acute myeloid leukemia blasts compared to in hematopoietic progenitor cells from healthy individuals. Importantly, specific inhibition of nicotinamide phosphoribosyltransferase or sirtuin 2 significantly reduced proliferation and induced apoptosis in human acute myeloid leukemia cell lines and primary blasts. Intriguingly, we found that protein kinase B/AKT could be deacetylated by nicotinamide phosphoribosyltransferase and sirtuin 2. The anti-leukemic effects of the inhibition of nicotinamide phosphoribosyltransferase or sirtuin 2 were accompanied by acetylation of protein kinase B/AKT with subsequent inhibition by dephosphorylation. This leads to activation of glycogen synthase kinase-3 β via diminished phosphorylation and, ultimately, inactivation of β-catenin by phosphorylation. Conclusions Our results provide strong evidence that nicotinamide phosphoribosyltransferase and sirtuin 2 participate in the aberrant proliferation and survival of leukemic cells, and suggest that the protein kinase B/AKT/ glycogen synthase kinase-3 β/β-catenin pathway is a target for inhibition of nicotinamide phosphoribosyltransferase or sirtuin 2 and, thereby, leukemia cell proliferation.

Severe congenital neutropenia: inheritance and pathophysiology.

Purpose of reviewSevere congenital neutropenia is a heterogeneous disorder of hematopoiesis characterized by a maturation arrest of granulopoiesis at the level of promyelocytes with peripheral blood absolute neutrophil counts below 0.5 × 109/l. In this review we summarize our current knowledge on inheritance and pathophysiolgy of congenital neutropenia. Recent findingsThere are two major subtypes of congenital neutropenia as judged by inheritance: autosomal dominant trait defined by neutrophil elastase mutations consisting of 60% of patients and autosomal recessive trait comprising approximately 30% of patients. This genetic heterogeneity suggests that several pathologic mechanisms may lead to the same phenotype due to downregulation of common myeloid transcription factors. Lymphoid enhancer-binding factor 1 is the most promising candidate, as its abrogation together with downregulation of lymphoid enhancer-binding factor 1 target genes is compatible with this phenotype. Congenital neutropenia is considered as a preleukemic syndrome, since after 10 years of observation the cumulative incidence for leukemia is 21%. Acquired granulocyte colony-stimulating factor receptor mutations are detected in approximately 80% of congenital neutropenia patients who developed acute myeloid leukemia. SummaryCongenital neutropenia is a congenital disorder of hematopoiesis inherited by autosomal dominant or recessive traits. Downregulation of lymphoid enhancer-binding factor 1 is involved in the pathophysiology of all congenital neutropenia patients. Congenital neutropenia patients with acquired granulocyte colony-stimulating factor receptor mutations define a group with high risk for development of leukemia.

G-csf receptor mutations in patients with congenital neutropenia

Purpose of reviewIn this review, we summarize our current knowledge on the acquisition of granulocyte-colony stimulating factor receptor (G-CSFR) gene (CSF3R) mutations in patients with congenital neutropenia and their role in leukemogenesis. Congenital neutropenia is a heterogeneous disorder of hematopoiesis characterized by a maturation arrest of granulopoiesis at the level of promyelocytes with peripheral blood absolute neutrophil counts below 0.5 × 109/l. Recent findingsThere are two major subtypes of congenital neutropenia as judged by inheritance, comprising the majority of congenital neutropenia patients: autosomal dominant trait defined by neutrophil elastase mutations consisting of 60% of patients and autosomal recessive trait comprising approximately 30% of patients, both presenting with the same clinical and morphological phenotype. Congenital neutropenia is considered as a preleukemic syndrome, as the cumulative incidence for leukemia is more than 25% after 20 years of observation. Acquired CSF3R mutations are detected in approximately 80% of congenital neutropenia patients who developed acute myeloid leukemia suggesting that these mutations are involved in leukemogenesis. One possible pathomechanism causing leukemia is that clones of cells harboring acquired CSF3R mutations have a growth advantage over wild type cells in vivo during granulocyte-colony stimulating factor treatment due to activation of STAT5 and ß-catenin, both known to be involved in leukemogenesis. SummaryCongenital neutropenia patients with acquired CSF3R mutations define a group with high risk for development of leukemia.

The diversity of mutations and clinical outcomes for ELANE-associated neutropenia.

Purpose of reviewMutations in the gene for neutrophil elastase, ELANE, cause cyclic neutropenia (CyN) and severe congenital neutropenia (SCN). This study summarized data from the Severe Chronic Neutropenia International Registry (SCNIR) on genotype–phenotype relationships of ELANE mutations to important clinical outcomes. We also summarize findings for ELANE mutations not observed in SCNIR patients. Recent findingsThere were 307 SCNIR patients with 104 distinctive ELANE mutations who were followed longitudinally for up to 27 years. The ELANE mutations were diverse; there were 65 single amino acid substitutions; 61 of these mutations (94%) were ‘probably’ or ‘possibly damaging’ by PolyPhen-2 analysis, and one of the ‘benign’ mutations was associated with two cases of acute myeloid leukemia (AML). All frame-shift mutations (19/19) were associated with the SCN. The pattern of mutations in the SCN versus CyN was significantly different (P < 10–4), but some mutations were observed in both groups (overlapping mutations). The cumulative incidence of severe adverse events, that is, myelodysplasia, AML, stem cell transplantation, or deaths was significantly greater for patients with SCN versus those with CyN or overlapping mutations. Specific mutations (i.e. G214R or C151Y) had a high risk for evolution to AML. SummarySequencing is useful for predicting outcomes of ELANE-associated neutropenia.

Interactions among HCLS1, HAX1 and LEF-1 proteins are essential for G-CSF-triggered granulopoiesis.

We found that hematopoietic cell–specific Lyn substrate 1 (HCLS1 or HS1) is highly expressed in human myeloid cells and that stimulation with granulocyte colony-stimulating factor (G-CSF) leads to HCLS1 phosphorylation. HCLS1 binds the transcription factor lymphoid-enhancer binding factor 1 (LEF-1), transporting LEF-1 into the nucleus upon G-CSF stimulation and inducing LEF-1 autoregulation. In patients with severe congenital neutropenia, inherited mutations in the gene encoding HCLS1-associated protein X-1 (HAX1) lead to profound defects in G-CSF–triggered phosphorylation of HCLS1 and subsequently to reduced autoregulation and expression of LEF-1. Consistent with these results, HCLS1-deficient mice are neutropenic. In bone marrow biopsies of the majority of tested patients with acute myeloid leukemia, HCLS1 protein expression is substantially elevated, associated with high levels of G-CSF synthesis and, in some individuals, a four-residue insertion in a proline-rich region of HCLS1 protein known to accelerate intracellular signaling. These data demonstrate the importance of HCLS1 in myelopoiesis in vitro and in vivo.

A lack of secretory leukocyte protease inhibitor (SLPI) causes defects in granulocytic differentiation

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(+) 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 κB 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.

LEF-1 is a decisive transcription factor in neutrophil granulopoiesis.

Abstract:  We found that lymphoid enhancer‐binding factor 1 (LEF‐1) is a decisive transcription factor in granulopoiesis controlling proliferation, proper lineage commitment, and granulocytic differentiation via regulation of its target genes C/EBP‐α, cyclin D1, c‐myc, and survivin. Myeloid progenitor cells of patients with severe congenital neutropenia (CN) showed a severe downregulation of LEF‐1 and its target genes expression. Expression of neutrophil elastase (NE) is also severely reduced in CN myeloid progenitors. Intriguingly, ELA2 gene promoter is positively regulated by direct binding of LEF‐1 or LEF‐1 target gene C/EBP‐α. In summary we demonstrated that LEF‐1 is not only crucial in lymphopoiesis, but also in myelopoiesis, documenting new functions of LEF‐1.

Severe congenital neutropenias

Severe congenital neutropenias are a heterogeneous group of rare haematological diseases characterized by impaired maturation of neutrophil granulocytes. Patients with severe congenital neutropenia are prone to recurrent, often life-threatening infections beginning in their first months of life. The most frequent pathogenic defects are autosomal dominant mutations in ELANE, which encodes neutrophil elastase, and autosomal recessive mutations in HAX1, whose product contributes to the activation of the granulocyte colony-stimulating factor (G-CSF) signalling pathway. The pathophysiological mechanisms of these conditions are the object of extensive research and are not fully understood. Furthermore, severe congenital neutropenias may predispose to myelodysplastic syndromes or acute myeloid leukaemia. Molecular events in the malignant progression include acquired mutations in CSF3R (encoding G-CSF receptor) and subsequently in other leukaemia-associated genes (such as RUNX1) in a majority of patients. Diagnosis is based on clinical manifestations, blood neutrophil count, bone marrow examination and genetic and immunological analyses. Daily subcutaneous G-CSF administration is the treatment of choice and leads to a substantial increase in blood neutrophil count, reduction of infections and drastic improvement of quality of life. Haematopoietic stem cell transplantation is the alternative treatment. Regular clinical assessments (including yearly bone marrow examinations) to monitor treatment course and detect chromosomal abnormalities (for example, monosomy 7 and trisomy 21) as well as somatic pre-leukaemic mutations are recommended.

Low platelet counts after induction therapy for childhood acute lymphoblastic leukemia are strongly associated with poor early response to treatment as measured by minimal residual disease and are prognostic for treatment outcome

Background Numerous reports have been published on the association between kinetics of leukemic cells during early treatment of childhood acute lymphoblastic leukemia and therapeutic outcome. In contrast, little is known about the prognostic relevance of normal blood counts in this setting. Design and Methods Normal hematopoiesis during and after induction treatment (days 8, 15 and 33) was correlated with therapeutic outcome in a cohort of 256 children with acute lymphoblastic leukemia treated in one of three consecutive ALL-BFM trials at a single institute. Replication analysis of positive findings was performed in an independent cohort of 475 patients from the ALL-BFM 2000 multicenter trial. Results A platelet count in the first quartile on treatment day 33 and a neutrophil count above the median on day 8 were significantly associated with treatment outcome, conferring multivariate risk ratios for an event of 3.27 (95% confidence interval 1.60–6.69) and 2.26 (95% confidence interval 1.23–4.29), respectively. Replication analysis confirmed the prognostic effect of platelet count on treatment day 33 and demonstrated a strong association with minimal residual disease-based risk group distribution (P<0.00001). Conclusions Platelet counts after induction treatment may improve treatment stratification for patients with childhood acute lymphoblastic leukemia and be of particular interest in non-minimal residual disease-based trials. (ALL-BFM 2000 is registered at: ClinicalTrials.gov: NCT00430118. National Cancer Institute: Protocol ID 68529)