Sandra Mifsud

PU.1 regulates the commitment of adult hematopoietic progenitors and restricts granulopoiesis.

Although the transcription factor PU.1 is essential for fetal lymphomyelopoiesis, we unexpectedly found that elimination of the gene in adult mice allowed disturbed hematopoiesis, dominated by granulocyte production. Impaired production of lymphocytes was evident in PU.1-deficient bone marrow (BM), but myelocytes and clonogenic granulocytic progenitors that are responsive to granulocyte colony-stimulating factor or interleukin-3 increased dramatically. No identifiable common lymphoid or myeloid progenitor populations were discernable by flow cytometry; however, clonogenic assays suggested an overall increased frequency of blast colony-forming cells and BM chimeras revealed existence of long-term self-renewing PU.1-deficient cells that required PU.1 for lymphoid, but not granulocyte, generation. PU.1 deletion in granulocyte-macrophage progenitors, but not in common myeloid progenitors, resulted in excess granulocyte production; this suggested specific roles of PU.1 at different stages of myeloid development. These findings emphasize the distinct nature of adult hematopoiesis and reveal that PU.1 regulates the specification of the multipotent lymphoid and myeloid compartments and restrains, rather than promotes, granulopoiesis.

RIPK1 Regulates RIPK3-MLKL-Driven Systemic Inflammation and Emergency Hematopoiesis

Upon ligand binding, RIPK1 is recruited to tumor necrosis factor receptor superfamily (TNFRSF) and Toll-like receptor (TLR) complexes promoting prosurvival and inflammatory signaling. RIPK1 also directly regulates caspase-8-mediated apoptosis or, if caspase-8 activity is blocked, RIPK3-MLKL-dependent necroptosis. We show that C57BL/6 Ripk1(-/-) mice die at birth of systemic inflammation that was not transferable by the hematopoietic compartment. However, Ripk1(-/-) progenitors failed to engraft lethally irradiated hosts properly. Blocking TNF reversed this defect in emergency hematopoiesis but, surprisingly, Tnfr1 deficiency did not prevent inflammation in Ripk1(-/-) neonates. Deletion of Ripk3 or Mlkl, but not Casp8, prevented extracellular release of the necroptotic DAMP, IL-33, and reduced Myd88-dependent inflammation. Reduced inflammation in the Ripk1(-/-)Ripk3(-/-), Ripk1(-/-)Mlkl(-/-), and Ripk1(-/-)Myd88(-/-) mice prevented neonatal lethality, but only Ripk1(-/-)Ripk3(-/-)Casp8(-/-) mice survived past weaning. These results reveal a key function for RIPK1 in inhibiting necroptosis and, thereby, a role in limiting, not only promoting, inflammation.

SOCS3 Is a Critical Physiological Negative Regulator of G-CSF Signaling and Emergency Granulopoiesis

To determine the importance of suppressor of cytokine signaling-3 (SOCS3) in the regulation of hematopoietic growth factor signaling generally, and of G-CSF-induced cellular responses specifically, we created mice in which the Socs3 gene was deleted in all hematopoietic cells. Although normal until young adulthood, these mice then developed neutrophilia and a spectrum of inflammatory pathologies. When stimulated with G-CSF in vitro, SOCS3-deficient cells of the neutrophilic granulocyte lineage exhibited prolonged STAT3 activation and enhanced cellular responses to G-CSF, including an increase in cloning frequency, survival, and proliferative capacity. Consistent with the in vitro findings, mutant mice injected with G-CSF displayed enhanced neutrophilia, progenitor cell mobilization, and splenomegaly, but unexpectedly also developed inflammatory neutrophil infiltration into multiple tissues and consequent hind-leg paresis. We conclude that SOCS3 is a key negative regulator of G-CSF signaling in myeloid cells and that this is of particular significance during G-CSF-driven emergency granulopoiesis.

Suppressors of cytokine signaling (SOCS): negative regulators of signal transduction.

SOCS‐1 was originally identified as an inhibitor of interleukin‐6 signal transduction and is a member of a family of proteins (SOCS‐1 to SOCS‐7 and CIS) that contain an SH2 domain and a conserved carboxyl‐terminal SOCS box motif. Mutation studies have established that critical contributions from both the amino‐terminal and SH2 domains are essential for SOCS‐1 and SOCS‐3 to inhibit cytokine signaling. Inhibition of cytokine‐dependent activation of STAT3 occurred in cells expressing either SOCS‐1 or SOCS‐3, but unlike SOCS‐1, SOCS‐3 did not directly interact with or inhibit the activity of JAK kinases. Although the conserved SOCS box motif appeared to be dispensable for SOCS‐1 and SOCS‐3 action when over‐expressed, this domain interacts with elongin proteins and may be important in regulating protein turnover. In gene knockout studies, SOCS‐1−/− mice were born but failed to thrive and died within 3 weeks of age with fatty degeneration of the liver and hemopoietic infiltration of several organs. The thymus in SOCS‐1−/− mice was small, the animals were lymphopenic, and deficiencies in B lymphocytes were evident within hemopoietic organs. We propose that the absence of SOCS‐1 in these mice prevents lymphocytes and liver cells from appropriately controlling signals from cytokines with cytotoxic side effects. J. Leukoc. Biol. 66: 588–592; 1999.

Polycystic kidneys and chronic inflammatory lesions are the delayed consequences of loss of the suppressor of cytokine signaling-1 (SOCS-1)

Mice with inactivation of the gene encoding the suppressor of cytokine signaling-1 (SOCS-1) die in neonatal life with an IFN-γ-dependent inflammatory disease dominated by fatty degeneration and necrosis of the liver. To establish the long-term pathological consequences of loss of SOCS-1 in mice, where initial survival was made possible by also deleting the IFN-γ gene, a comparison was made of the lifespan of groups of SOCS-1−/− IFN-γ−/−, SOCS-1+/+ IFN-γ−/− and SOCS-1+/+ IFN-γ+/+ mice. Mice lacking the genes for both SOCS-1 and IFN-γ exhibited an accelerated death rate compared with control groups. Disease states developing selectively in SOCS-1−/− IFN-γ−/− mice were polycystic kidneys, pneumonia, chronic skin ulcers, and chronic granulomas in the gut and various other organs. Mice of all three groups developed cataracts, but disease development was accelerated in the groups lacking IFN-γ. SOCS-1−/− IFN-γ−/− mice exhibited a slightly increased predisposition to the development of T lymphoid leukemia, either spontaneous or radiation-induced. The development of polycystic kidneys may be caused by a developmental defect in renal-tubule organization noted in neonatal SOCS-1−/− mice. The chronic infections and granulomas of SOCS-1−/− IFN-γ−/− mice may be based on autoaggression of SOCS-1−/− T lymphoid and related cells or a functional deficiency of these cells when lacking SOCS-1.

Inactivation of PU.1 in adult mice leads to the development of myeloid leukemia

Genetically primed adult C57BL mice were deleted of exon 5 of the gene encoding the transcription factor PU.1 by IFN activation of Cre recombinase. After a 13-week delay, conditionally deleted (PU.1-/-) mice began dying of myeloid leukemia, and 95% of the mice surviving from early postinduction death developed transplantable myeloid leukemia whose cells were deleted of PU.1 and uniformly Gr-1 positive. The leukemic cells formed autonomous colonies in semisolid culture with varying clonal efficiency, but colony formation was enhanced by IL-3 and sometimes by granulocyte-macrophage colony-stimulating factor. Nine of 13 tumors analyzed had developed a capacity for autocrine IL-3 or granulocyte-macrophage colony-stimulating factor production, and there was evidence of rearrangement of the IL-3 gene. Acquisition of autocrine growth-factor production and autonomous growth appeared to be major events in the transformation of conditionally deleted PU.1-/- cells to fully developed myeloid leukemic populations.

Aberrant hematopoiesis in mice with inactivation of the gene encoding SOCS-1

Mice with homozygous inactivation of the gene encoding the suppressor of cytokine signaling-1 (SOCS-1) protein die within 21 days of birth with low body weight, fatty degeneration and necrosis of the liver, infiltration of the lung, pancreas, heart and skin by macrophages and granulocytes and a profound depletion of T- and B-lymphocytes. In the present study, SOCS-1 −/− mice were found to have a moderate neutrophilia, and reduced platelet and hematocrit levels. Replacement of the SOCS-1 gene by a lac-Z reporter gene allowed documentation by FACS sorting that at least a proportion of granulocyte–macrophage progenitor cells transcribe SOCS-1. Most hematopoietic progenitor cell frequencies were normal in −/− marrow as were the size and cellular content of colonies formed by −/− progenitor cells in response to various stimulating factors. However, there was an increased frequency of macrophage progenitor cells in −/− mice and, abnormally, one quarter of all progenitor cells were located in the liver. Progenitor cells from −/− mice were hyper-responsive to stimulation by GM-CSF but not by M-CSF or Multi-CSF (IL-3). Progenitor cells from −/− mice were also hypersensitive to inhibition by interferon-gamma (IFN-γ), the degree of inhibition varying markedly with the stimulating factor used. The suppressive effects of IFN-γ therefore appear to involve interactions with particular growth factor-initiated signals in −/− cells – interactions that are strongly modulated by the action of the SOCS-1 protein.

Functional Analysis of Asb-1 Using Genetic Modification in Mice

ABSTRACT The Asbs are a family of ankyrin repeat proteins that, along with four other protein families, contain a C-terminal SOCS box motif, which was first identified in the suppressor of cytokine signaling (SOCS) proteins. While it is clear that the SOCS proteins are involved in the negative regulation of cytokine signaling, the biological roles of the other SOCS box-containing families are unknown. We have investigated Asb-1 function by generating mice that lack this protein, as well as mice that overexpress full-length or truncated Asb-1 in a wide range of tissues. Although Asb-1 is expressed in multiple organs, including the hematopoietic compartment in wild-type mice, Asb-1−/−mice develop normally and exhibit no anomalies of mature blood cells or their progenitors. While most organs in these mice appear normal, the testes of Asb-1−/− mice display a diminution of spermatogenesis with less complete filling of seminiferous tubules. In contrast, the widespread overexpression of Asb-1 in the mouse has no apparent deleterious effects.

Affinity Maturation of Leukemia Inhibitory Factor and Conversion to Potent Antagonists of Signaling

Leukemia inhibitory factor (LIF)-induced cell signaling occurs following sequential binding to the LIF receptor α-chain (LIFR), then to the gp130 co-receptor used by all members of the interleukin-6 family of cytokines. By monovalently displaying human LIF on the surface of M13 phage and randomizing clusters of residues in regions predicted to be important for human LIFR binding, we have identified mutations, which lead to significant increases in affinity for binding to LIFR. Six libraries were constructed in which regions of 4–6 amino acids were randomized then panned against LIFR. Mutations identified in three distinct clusters, residues 53–57, 102–103, and 150–155, gave rise to proteins with significantly increased affinity for binding to both human and mouse LIFR. Combining the mutations for each of these regions further increased the affinity, such that the best mutants bound to human LIFR with >1000-fold higher affinity than wild-type human LIF. NMR analysis indicated that the mutations did not alter the overall structure of the molecule relative to the native protein, although some local changes occurred in the vicinity of the substituted residues. Despite increases in LIFR binding affinity, these mutants did not show any increase in activity as agonists of LIF-induced proliferation of Ba/F3 cells expressing human LIFR and gp130 compared with wild-type LIF. Incorporation of two additional mutations (Q29A and G124R), which were found to abrogate cell signaling, led to the generation of highly potent antagonists of both human and murine LIF-induced bioactivity.

The lethal effects of transplantation of Socs1-/- bone marrow cells into irradiated adult syngeneic recipients

Injection of neonatal bone marrow cells from mice lacking the gene encoding suppressor of cytokine signaling 1 (SOCS1) into irradiated syngeneic 129/Sv or C57BL/6 mice led to a decreased survival, more rapidly occurring in 129/Sv than in C57BL/6 mice. Moribund mice did not exhibit the acute or chronic diseases developed by Socs1-/- mice but developed a pathology characteristic of graft-versus-host disease with typical chronic inflammatory lesions in the liver, skin, lungs, and gut. The results indicate that cells derived from the Socs1-/- bone marrow are autoaggressive but did not identify the cell types involved. Failure of the engrafted Socs1-/- marrow cells to reproduce the tissue damage typical of Socs1-/- disease indicates that loss of SOCS1 from target tissues may also be required for the development of the Socs1-/- diseases, such as fatty degeneration of the liver, polymyositis, or corneal inflammation.