Alternatively spliced CSF3R isoforms in SRSF2 P95H mutated myeloid neoplasms.

Alternatively spliced colony stimulating factor 3 receptor (CSF3R) isoforms Class III and Class IV are observed in myelodysplastic syndromes (MDS), but their roles in disease remain unclear. We report that the MDS-associated splicing factor SRSF2 affects the expression of Class III and Class IV isoforms and perturbs granulopoiesis. Add-back of the Class IV isoform in Csf3r-null mouse progenitor cells increased granulocyte progenitors with impaired neutrophil differentiation, while add-back of the Class III produced dysmorphic neutrophils in fewer numbers. These CSF3R isoforms were elevated in patients with myeloid neoplasms harboring SRSF2 mutations. Using in vitro splicing assays, we confirmed increased Class III and Class IV transcripts when SRSF2 P95 mutations were co-expressed with the CSF3R minigene in K562 cells. Since SRSF2 regulates splicing partly by recognizing exonic splicing enhancer (ESE) sequences on pre-mRNA, deletion of either ESE motifs within CSF3R exon 17 decreased Class IV transcript levels without affecting Class III. CD34+ cells expressing SRSF2 P95H showed impaired neutrophil differentiation in response to G-CSF and was accompanied by increased levels of Class IV. Our findings suggest that SRSF2 P95H promotes Class IV splicing by binding to key ESE sequences in CSF3R exon 17, and that SRSF2, when mutated, contributes to dysgranulopoiesis.

G-CSF, the guardian of granulopoiesis.

A considerable amount of continuous proliferation and differentiation is required to produce daily a billion new neutrophils in an adult human. Of the few cytokines and factors known to control neutrophil production, G-CSF is the guardian of granulopoiesis. G-CSF/CSF3R signaling involves the recruitment of non-receptor protein tyrosine kinases and their dependent signaling pathways of serine/threonine kinases, tyrosine phosphatases, and lipid second messengers. These pathways converge to activate the families of STAT and C/EBP transcription factors. CSF3R mutations are associated with human disorders of neutrophil production, including severe congenital neutropenia, neutrophilia, and myeloid malignancies. More than three decades after their identification, cloning, and characterization of G-CSF and G-CSF receptor, fundamental questions remain about their physiology.

Inducible expression of a disease-associated ELANE mutation impairs granulocytic differentiation, without eliciting an unfolded protein response.

Severe congenital neutropenia (SCN) is characterized by a near absence of neutrophils, rendering individuals with this disorder vulnerable to recurrent life-threatening infections. The majority of SCN cases arise because of germline mutations in the gene elastase, neutrophil-expressed (ELANE) encoding the neutrophil granule serine protease neutrophil elastase. Treatment with a high dose of granulocyte colony-stimulating factor increases neutrophil production and reduces infection risk. How ELANE mutations produce SCN remains unknown. The currently proposed mechanism is that ELANE mutations promote protein misfolding, resulting in endoplasmic reticulum stress and activation of the unfolded protein response (UPR), triggering death of neutrophil precursors and resulting in neutropenia. Here we studied the ELANE mutation p.G185R, often associated with greater clinical severity (e.g. decreased responsiveness to granulocyte colony-stimulating factor and increased leukemogenesis). Using an inducible expression system, we observed that this ELANE mutation diminishes enzymatic activity and granulocytic differentiation without significantly affecting cell proliferation, cell death, or UPR induction in murine myeloblast 32D and human promyelocytic NB4 cells. Impaired differentiation was associated with decreased expression of genes encoding critical hematopoietic transcription factors (Gfi1, Cebpd, Cebpe, and Spi1), cell surface proteins (Csf3r and Gr1), and neutrophil granule proteins (Mpo and Elane). Together, these findings challenge the currently prevailing model that SCN results from mutant ELANE, which triggers endoplasmic reticulum stress, UPR, and apoptosis.

G-CSF and GM-CSF in Neutropenia.

G-CSF and GM-CSF are used widely to promote the production of granulocytes or APCs. The U.S. Food and Drug Administration approved G-CSF (filgrastim) for the treatment of congenital and acquired neutropenias and for mobilization of peripheral hematopoietic progenitor cells for stem cell transplantation. A polyethylene glycol-modified form of G-CSF is approved for the treatment of neutropenias. Clinically significant neutropenia, rendering an individual immunocompromised, occurs when their number is <1500/µl. Current guidelines recommend their use when the risk for febrile neutropenia is >20%. GM-CSF (sargramostim) is approved for neutropenia associated with stem cell transplantation. Because of its promotion of APC function, GM-CSF is being evaluated as an immunostimulatory adjuvant in a number of clinical trials. More than 20 million persons have benefited worldwide, and >$5 billion in sales occur annually in the United States.

Systems approach to phagocyte production and activation: neutrophils and monocytes.

Granulocyte differentiation and immune response function is a dynamic process governed by a highly coordinated transcriptional program that regulates cellular fate and function, often in a context-dependent manner. Advances in high-throughput technologies and bioinformatics have allowed us to better understand complex biological processes at the genomic and proteomic levels. Components of the environmental milieu, along with the molecular mechanisms that drive the development, activation, and regulation of granulocytes, have since been elucidated. In this chapter, we present the intricate network in which these elements come together and influence one another. In particular, we describe the critical roles of transcription factors like PU.1, CCAAT/enhancer-binding protein (C/EBPα; alpha), C/EBPε (epsilon), and growth factor independent-1 (Gfi-1). We also review granulocyte colony-stimulating factor (G-CSF) receptor-induced signal transduction pathways, their influence on proliferation and differentiation, and the cooperativity of cytokines and chemokines in this process.

Comparison of nerve growth factor receptor binding models using heterodimeric muteins.

Nerve growth factor (NGF) is a homodimer that binds to two distinct receptor types, TrkA and p75, to support survival and differentiation of neurons. The high-affinity binding on the cell surface is believed to involve a heteroreceptor complex, but its exact nature is unclear. We developed a heterodimer (heteromutein) of two NGF muteins that can bind p75 and TrkA on opposite sides of the heterodimer, but not two TrkA receptors. Previously described muteins are Δ9/13 that is TrkA negative and 7-84-103 that is signal selective through TrkA. The heteromutein (Htm1) was used to study the heteroreceptor complex formation and function, in the putative absence of NGF-induced TrkA dimerization. Cellular binding assays indicated that Htm1 does not bind TrkA as efficiently as wild-type (wt) NGF but has better affinity than either homodimeric mutein. Htm1, 7-84-103, and Δ9/13 were each able to compete for cold-temperature, cold-chase stable binding on PC12 cells, indicating that binding to p75 was required for a portion of this high-affinity binding. Survival, neurite outgrowth, and MAPK signaling in PC12 cells also showed a reduced response for Htm1, compared with wtNGF, but was better than the parent muteins in the order wtNGF > Htm1 > 7-84-103 >> Δ9/13. Htm1 and 7-84-103 demonstrated similar levels of survival on cells expressing only TrkA. In the longstanding debate on the NGF receptor binding mechanism, our data support the ligand passing of NGF from p75 to TrkA involving a transient heteroreceptor complex of p75-NGF-TrkA.

Two SHIPs passing in the middle of the immune system.

Immunity requires a complex, multiscale system of molecules, cells, and cytokines. In this issue of the European Journal of Immunology, Collazo et al. [Eur. J. Immunol. 2012. 42: 1785-1796] provide evidence that links the lipid phosphatase SHIP1 with the coordination of interactions between regulatory T (Treg) cells and myeloid-derived suppressor cells (MDSCs). Using conditional knockouts of SHIP1 in either the myeloid or T-cell-lineage of mice, the authors show that the regulated development of Treg cells is controlled directly by cell-intrinsic SHIP1, and indirectly by extrinsic SHIP1 control of an unknown myeloid cell. Regulation of MDSCs is also determined by SHIP1 in an extrinsic manner, again via an as-yet-unknown myeloid cell. Furthermore, this extrinsic control of Treg cells and MDSCs is mediated in part by increased production of G-CSF, a growth factor critical for the production of neutrophils, in SHIP1-deficient mice. Thus, a physiologically important implication of this report is the collaboration between the innate and adaptive immune systems in fine tuning of Treg cells as discussed in this commentary.

Chronic and acute models of retinal neurodegeneration TrkA activity are neuroprotective whereas p75NTR activity is neurotoxic through a paracrine mechanism.

In normal adult retinas, NGF receptor TrkA is expressed in retinal ganglion cells (RGC), whereas glia express p75(NTR). During retinal injury, endogenous NGF, TrkA, and p75(NTR) are up-regulated. Paradoxically, neither endogenous NGF nor exogenous administration of wild type NGF can protect degenerating RGCs, even when administered at high frequency. Here we elucidate the relative contribution of NGF and each of its receptors to RGC degeneration in vivo. During retinal degeneration due to glaucoma or optic nerve transection, treatment with a mutant NGF that only activates TrkA, or with a biological response modifier that prevents endogenous NGF and pro-NGF from binding to p75(NTR) affords significant neuroprotection. Treatment of normal eyes with an NGF mutant-selective p75(NTR) agonist causes progressive RGC death, and in injured eyes it accelerates RGC death. The mechanism of p75(NTR) action during retinal degeneration due to glaucoma is paracrine, by increasing production of neurotoxic proteins TNF-α and α(2)-macroglobulin. Antagonists of p75(NTR) inhibit TNF-α and α(2)-macroglobulin up-regulation during disease, and afford neuroprotection. These data reveal a balance of neuroprotective and neurotoxic mechanisms in normal and diseased retinas, and validate each neurotrophin receptor as a pharmacological target for neuroprotection.