The E3 ligase Itch is a negative regulator of the homeostasis and function of hematopoietic stem cells.

Although hematopoietic stem cells (HSCs) are the most thoroughly characterized type of adult stem cell, the intricate molecular machinery that regulates their self-renewal properties remains elusive. Here we showed that the E3 ubiquitin ligase Itch negatively regulated the development and function of HSCs. Itch(-/-) mice had HSCs with enhanced frequency, competence and long-term repopulating activity. Itch-deficient HSCs showed accelerated proliferation rates and sustained progenitor properties, as well as more signaling by the transcription factor Notch1, due to more accumulation of activated Notch1. Knockdown of Notch1 in Itch-mutant HSCs resulted in reversion of the phenotype. Thus, we identify Itch as a previously unknown negative regulator of HSC homeostasis and function.

The E3 ligase Itch and deubiquitinase Cyld act together to regulate Tak1 and inflammation.

Chronic inflammation has been strongly associated with tumor progression, but the underlying mechanisms remain elusive. Here we demonstrate that E3 ligase Itch and deubiquitinase Cyld formed a complex via interaction through 'WW-PPXY' motifs. The Itch-Cyld complex sequentially cleaved Lys63-linked ubiquitin chains and catalyzed Lys48-linked ubiquitination on the kinase Tak1 to terminate inflammatory signaling via tumor necrosis factor. Reconstitution of wild-type Cyld but not the mutant Cyld(Y485A), which cannot associate with Itch, blocked sustained Tak1 activation and proinflammatory cytokine production by Cyld(-/-) bone marrow-derived macrophages. Deficiency in Itch or Cyld led to chronic production of tumor-promoting cytokines by tumor-associated macrophages and aggressive growth of lung carcinoma. Thus, we have identified an Itch-Cyld-mediated regulatory mechanism in innate inflammatory cells.

HAX1 deficiency causes autosomal recessive severe congenital neutropenia (Kostmann disease).

Autosomal recessive severe congenital neutropenia (SCN) constitutes a primary immunodeficiency syndrome associated with increased apoptosis in myeloid cells, yet the underlying genetic defect remains unknown. Using a positional cloning approach and candidate gene evaluation, we identified a recurrent homozygous germline mutation in HAX1 in three pedigrees. After further molecular screening of individuals with SCN, we identified 19 additional affected individuals with homozygous HAX1 mutations, including three belonging to the original pedigree described by Kostmann. HAX1 encodes the mitochondrial protein HAX1, which has been assigned functions in signal transduction and cytoskeletal control. Here, we show that HAX1 is critical for maintaining the inner mitochondrial membrane potential and protecting against apoptosis in myeloid cells. Our findings suggest that HAX1 is a major regulator of myeloid homeostasis and underline the significance of genetic control of apoptosis in neutrophil development.

Pancreatic islet expression of chemokine CCL2 suppresses autoimmune diabetes via tolerogenic CD11c+ CD11b+ dendritic cells.

Development of type 1 diabetes in the nonobese diabetic (NOD) mouse is preceded by an immune cell infiltrate in the pancreatic islets. The exact role of the attracted cells is still poorly understood. Chemokine CCL2/MCP-1 is known to attract CCR2(+) monocytes and dendritic cells (DCs). We have previously shown that transgenic expression of CCL2 in pancreatic islets via the rat insulin promoter induces nondestructive insulitis on a nonautoimmune background. We report here an unexpected reduction of diabetes development on the NOD background despite an increased islet cell infiltrate with markedly increased numbers of CD11c(+) CD11b(+) DCs. These DCs exhibited a hypoactive phenotype with low CD40, MHC II, CD80/CD86 expression, and reduced TNF-α but elevated IL-10 secretions. They failed to induce proliferation of diabetogenic CD4(+) T cells in vitro. Pancreatic lymph node CD4(+) T cells were down-regulated ex vivo and expressed the anergy marker Grail. By using an in vivo transfer system, we show that CD11c(+) CD11b(+) DCs from rat insulin promoter-CCL2 transgenic NOD mice were the most potent cells suppressing diabetes development. These findings support an unexpected beneficial role for CCL2 in type 1 diabetes with implications for current strategies interfering with the CCL2/CCR2 axis in humans, and for dendritic cell biology in autoimmunity.

Anti-serpin antibody-mediated regulation of proteases in autoimmune diabetes.

Secretion of anti-serpin B13 autoantibodies in young diabetes-prone nonobese diabetic mice is associated with reduced inflammation in pancreatic islets and a slower progression to autoimmune diabetes. Injection of these mice with a monoclonal antibody (mAb) against serpin B13 also leads to fewer inflammatory cells in the islets and more rapid recovery from recent-onset diabetes. The exact mechanism by which anti-serpin activity is protective remains unclear. We found that serpin B13 is expressed in the exocrine component of the mouse pancreas, including the ductal cells. We also found that anti-serpin B13 mAb blocked the inhibitory activity of serpin B13, thereby allowing partial preservation of the function of its target protease. Consistent with the hypothesis that anti-clade B serpin activity blocks the serpin from binding, exposure to exogenous anti-serpin B13 mAb or endogenous anti-serpin B13 autoantibodies resulted in cleavage of the surface molecules CD4 and CD19 in lymphocytes that accumulated in the pancreatic islets and pancreatic lymph nodes but not in the inguinal lymph nodes. This cleavage was inhibited by an E64 protease inhibitor. Consequently, T cells with the truncated form of CD4 secreted reduced levels of interferon-γ. We conclude that anti-serpin antibodies prevent serpin B13 from neutralizing proteases, thereby impairing leukocyte function and reducing the severity of autoimmune inflammation.

Telomere dysfunction induces environmental alterations limiting hematopoietic stem cell function and engraftment.

Cell-intrinsic checkpoints limit the proliferative capacity of primary cells in response to telomere dysfunction. It is not known, however, whether telomere dysfunction contributes to cell-extrinsic alterations that impair stem cell function and organ homeostasis. Here we show that telomere dysfunction provokes defects of the hematopoietic environment that impair B lymphopoiesis but increase myeloid proliferation in aging telomerase knockout (Terc(-/-)) mice. Moreover, the dysfunctional environment limited the engraftment of transplanted wild-type hematopoietic stem cells (HSCs). Dysfunction of the hematopoietic environment was age dependent and correlated with progressive telomere shortening in bone marrow stromal cells. Telomere dysfunction impaired mesenchymal progenitor cell function, reduced the capacity of bone marrow stromal cells to maintain functional HSCs, and increased the expression of various cytokines, including granulocyte colony-stimulating factor (G-CSF), in the plasma of aging mice. Administration of G-CSF to wild-type mice mimicked some of the defects seen in aging Terc(-/-) mice, including impairment of B lymphopoiesis and HSC engraftment. Conversely, inhibition of G-CSF improved HSC engraftment in aged Terc(-/-) mice. Taken together, these results show that telomere dysfunction induces alterations of the environment that can have implications for organismal aging and cell transplantation therapies.

A novel human primary immunodeficiency syndrome caused by deficiency of the endosomal adaptor protein p14.

Lysosome-related organelles have versatile functions, including protein and lipid degradation, signal transduction and protein secretion. The molecular elucidation of rare congenital diseases affecting endosomal-lysosomal biogenesis has given insights into physiological functions of the innate and adaptive immune system. Here, we describe a previously unknown human primary immunodeficiency disorder and provide evidence that the endosomal adaptor protein p14, previously characterized as confining mitogen-activated protein kinase (MAPK) signaling to late endosomes, is crucial for the function of neutrophils, B cells, cytotoxic T cells and melanocytes. Combining genetic linkage studies and transcriptional profiling analysis, we identified a homozygous point mutation in the 3' untranslated region (UTR) of p14 (also known as MAPBPIP), resulting in decreased protein expression. In p14-deficient cells, the distribution of late endosomes was severely perturbed, suggesting a previously unknown role for p14 in endosomal biogenesis. These findings have implications for understanding endosomal membrane dynamics, compartmentalization of cell signal cascades, and their role in immunity.

Transgenic expression of human signal regulatory protein alpha in Rag2-/-gamma(c)-/- mice improves engraftment of human hematopoietic cells in humanized mice.

Transplantation of human hematopoietic stem cells into severely immunocompromised newborn mice allows the development of a human hematopoietic and immune system in vivo. NOD/scid/γ(c)(-/-) (NSG) and BALB/c Rag2(-/-)γ(c)(-/-) mice are the most commonly used mouse strains for this purpose and a number of studies have demonstrated the high value of these model systems in areas spanning from basic to translational research. However, limited cross-reactivity of many murine cytokines on human cells and residual host immune function against the xenogeneic grafts results in defective development and maintenance of human cells in vivo. Whereas NSG mice have higher levels of absolute human engraftment than similar mice on a BALB/c background, they have a shorter lifespan and NOD ES cells are unsuitable for the complex genetic engineering that is required to improve human hematopoiesis and immune responses by transgenesis or knockin of human genes. We have generated mice that faithfully express a transgene of human signal regulatory protein alpha (SIRPa), a receptor that negatively regulates phagocytosis, in Rag2(-/-)γ(c)(-/-) mice on a mixed 129/BALB/c background, which can easily be genetically engineered. These mice allow significantly increased engraftment and maintenance of human hematopoietic cells reaching levels comparable to NSG mice. Furthermore, we found improved functionality of the human immune system in these mice. In summary, hSIRPa-transgenic Rag2(-/-)γ(c)(-/-) mice represent a unique mouse strain supporting high levels of human cell engraftment, which can easily be genetically manipulated.

Human thrombopoietin knockin mice efficiently support human hematopoiesis in vivo.

Hematopoietic stem cells (HSCs) both self-renew and give rise to all blood cells for the lifetime of an individual. Xenogeneic mouse models are broadly used to study human hematopoietic stem and progenitor cell biology in vivo. However, maintenance, differentiation, and function of human hematopoietic cells are suboptimal in these hosts. Thrombopoietin (TPO) has been demonstrated as a crucial cytokine supporting maintenance and self-renewal of HSCs. We generated RAG2(-/-)γ(c)(-/-) mice in which we replaced the gene encoding mouse TPO by its human homolog. Homozygous humanization of TPO led to increased levels of human engraftment in the bone marrow of the hosts, and multilineage differentiation of hematopoietic cells was improved, with an increased ratio of myelomonocytic verus lymphoid lineages. Moreover, maintenance of human stem and progenitor cells was improved, as demonstrated by serial transplantation. Therefore, RAG2(-/-)γ(c)(-/-) TPO-humanized mice represent a useful model to study human hematopoiesis in vivo.

MAPKAP kinase MK2 maintains self-renewal capacity of haematopoietic stem cells.

The structurally related MAPK-activated protein kinases (MAPKAPKs or MKs) MK2, MK3 and MK5 are involved in multiple cellular functions, including cell-cycle control and cellular differentiation. Here, we show that after deregulation of cell-cycle progression, haematopoietic stem cells (HSCs) in MK2-deficient mice are reduced in number and show an impaired ability for competitive repopulation in vivo. To understand the underlying molecular mechanism, we dissected the role of MK2 in association with the polycomb group complex (PcG) and generated a MK2 mutant, which is no longer able to bind to PcG. The reduced ability for repopulation is rescued by re-introduction of MK2, but not by the Edr2-non-binding mutant of MK2. Thus, MK2 emerges as a regulator of HSC homeostasis, which could act through chromatin remodelling by the PcG complex.

Efficient differentiation and function of human macrophages in humanized CSF-1 mice.

Humanized mouse models are useful tools to understand pathophysiology and to develop therapies for human diseases. While significant progress has been made in generating immunocompromised mice with a human hematopoietic system, there are still several shortcomings, one of which is poor human myelopoiesis. Here, we report that human CSF-1 knockin mice show augmented frequencies and functions of human myeloid cells. Insertion of human CSF1 into the corresponding mouse locus of Balb/c Rag2(-/-) γc(-/-) mice through VELOCIGENE technology resulted in faithful expression of human CSF-1 in these mice both qualitatively and quantitatively. Intra-hepatic transfer of human fetal liver derived hematopoietic stem and progenitor cells (CD34(+)) in humanized CSF-1 (CSF1(h/h)) newborn mice resulted in more efficient differentiation and enhanced frequencies of human monocytes/macrophages in the bone marrow, spleens, peripheral blood, lungs, liver and peritoneal cavity. Human monocytes/macrophages obtained from the humanized CSF-1 mice show augmented functional properties including migration, phagocytosis, activation and responses to LPS. Thus, humanized mice engineered to express human cytokines will significantly help to overcome the current technical challenges in the field. In addition, humanized CSF-1 mice will be a valuable experimental model to study human myeloid cell biology.

c-Cbl deficiency leads to diminished lymphocyte development and functions in an age-dependent manner.

Aging is broadly defined as a progressive decline of tissue and organ functions due to deregulation of various cell intrinsic and extrinsic factors. In the immune system, aging preferentially affects lymphopoiesis and thus results in the reduced competence of the adaptive immune system in the elderly. Despite recent discoveries that shed light on the molecular basis of aging, pathways that lead to diminished lymphoid development in aging individuals remain largely unknown. In the present study, we document that a deficiency of the E3 ubiquitin ligase c-Cbl in lymphocytes results in an age-dependent lymphopenia. c-Cbl-deficient mice show normal frequencies of lymphocytes at 12 weeks of age; however, their development and functions were remarkably diminished at 24 weeks after birth. Intriguingly, c-Cbl mutant lymphocytes displayed increased responses to IL7 in vitro and failed to down-regulate surface levels of IL7Ralpha. Further, our biochemical studies have identified an interaction of c-Cbl with IL7Ralpha and have unraveled the involvement of c-Cbl in the ubiquitylation of IL7Ralpha. In essence, our studies demonstrate that a lack of signaling events mediated by c-Cbl might result in diminished lymphocyte development and functions, particularly, at the later stages of life.

E3 ubiquitin ligase GRAIL controls primary T cell activation and oral tolerance.

T cell unresponsiveness or anergy is one of the mechanisms that maintain inactivity of self-reactive lymphocytes. E3 ubiquitin ligases are important mediators of the anergic state. The RING finger E3 ligase GRAIL is thought to selectively function in anergic T cells but its mechanism of action and its role in vivo are largely unknown. We show here that genetic deletion of Grail in mice leads not only to loss of an anergic phenotype in various models but also to hyperactivation of primary CD4(+) T cells. Grail(-/-) CD4(+) T cells hyperproliferate in vitro to TCR stimulation alone or with concomitant anti-CD28 costimulation, with transient increased survival. In vitro differentiated T helper 1 cells show slight but significant hypersecretion of IFN-gamma in Grail(-/-) mice whereas Th2 and Th17 cytokine secretions are unchanged. Consistent with defective in vitro anergy, oral tolerance is abolished in vivo in OT-II TCR transgenic Grail(-/-) mice fed with ovalbumin. In experimental allergic encephalitis, a model of organ-specific autoimmunity, oral tolerization with myelin basic protein was abrogated as well in Grail(-/-) mice. On the protein level, Grail(-/-) naïve T cells show no significant differences of total and phosphorylated levels of ZAP70, phospholipase Cgamma1, and MAP kinases p38 and JNK but elevated baseline levels of MAP kinase ERK1/2. In summary, we define a role for GRAIL in primary T cell activation, survival, and differentiation. In addition, we formally prove an indispensable role for GRAIL in T cell anergy and oral tolerance-a promising, antigen-specific strategy to treat autoimmune diseases.

Transcription factor Gfi1 restricts B cell-mediated autoimmunity.

The zinc finger transcription factor Gfi1 (growth factor-independent-1) has been involved in various cellular differentiation processes. Gfi1 acts as a transcriptional repressor and splicing control factor upon binding to cognate binding sites in regulatory elements of its target genes. In this study, we report that Gfi1-deficient mice develop autoimmunity. Gfi1-deficient peripheral B cells show a hyperproliferative phenotype leading to expansion of plasma cells, increased levels of nuclear autoantibodies, and Ig deposition in brain and kidneys. Dysregulation of multiple transcription factors and cell cycle control elements may contribute to B cell-dependent autoimmunity. Gfi1 thus emerges as a novel master regulator restricting autoimmunity.

Establishment of immortalized multipotent hematopoietic progenitor cell lines by retroviral-mediated gene transfer of beta-catenin.

OBJECTIVE: Hematopoietic stem cells (HSCs) are characterized by their ability to give rise to all mature blood lineages and to renew themselves. In the present study, we show that beta-catenin plays an essential role in the immortalization of hematopoietic progenitor cells (HPCs). MATERIALS AND METHODS: Cellular, molecular, and cytogenetic properties of the immortalized HPCs were determined using flow cytometry (immunophenotyping), microscopy (telomere length, spectral karyotyping), telomere repeat amplification protocol assay (telomerase activity), real-time polymerase chain reaction (expression studies), and in vitro and in vivo differentiation assays. RESULTS: Retroviral-mediated overexpression of human beta-catenin in lin(-)- and lin(-)Sca-1+c-kit+ hematopoietic cells led to generation of novel, murine interleukin-3-, and stem cell factor-dependent hematopoietic multipotent progenitor cell lines (DK1mix and DK2mix) with stable surface antigen expression of the stem cell markers Sca-1 and c-kit (>92%) in long-term culture. Further, immunophenotypic characterization revealed a CD244+CD150(-)CD48(-) phenotype of DKmix cells, which is consistent with the expression profile of multipotential hematopoietic progenitors. Upon exposure to selective hematopoietic cytokines in vitro, and upon transplantation into irradiated congenic recipients, DKmix cells generated progenies of lymphoid and myeloid lineages. Continuous in vitro proliferation and expansion of DKmix cells were associated with stabilized telomere length and consistent telomerase activity. Interestingly, constitutive expression of beta-catenin was not required for sustained long-term viability and proliferation of immortalized DKmix cells. CONCLUSION: In summary, our findings define beta-catenin as an important regulator in HPC self-renewal and function. Further, our results distinguish the importance of beta-catenin in the immortalization process of HPCs, from its dispensable role in their maintenance.

Memory/effector (CD45RB(lo)) CD4 T cells are controlled directly by IL-10 and cause IL-22-dependent intestinal pathology.

The role of direct IL-10 signaling in different T cell subsets is not well understood. To address this, we generated transgenic mice expressing a dominant-negative IL-10 receptor specifically in T cells (CD4dnIL-10Rα). We found that Foxp3-depleted CD45RB(lo) (regulatory T cell [T(reg) cell]-depleted CD45RB(lo)) but not CD45RB(hi) CD4(+) T cells are controlled directly by IL-10 upon transfer into Rag1 knockout (KO) mice. Furthermore, the colitis induced by transfer of T(reg) cell-depleted CD45RB(lo) CD4(+) T cells into Rag1 KO mice was characterized by reduced Th1 and increased Th17 cytokine messenger RNA levels in the colon as compared with the colitis induced by transfer of CD45RB(hi) T cells. In contrast to the CD45RB(hi) transfer colitis model, in which IL-22 is protective, we found that T cell-derived IL-22 was pathogenic upon transfer of T(reg) cell-depleted CD45RB(lo) T cells into Rag1 KO mice. Our results highlight characteristic differences between colitis induced by naive (CD45RB(hi)) and memory/effector (T(reg) cell-depleted CD45RB(lo)) cells and different ways that IL-22 impacts inflammatory bowel disease.

Myeloid leukemia development in c-Cbl RING finger mutant mice is dependent on FLT3 signaling.

Although myeloid leukemias are primarily caused by leukemic stem cells, the molecular basis of their transformation remains largely unknown. Here, by analyzing mice with a mutation in the RING finger domain of c-Cbl, we show that the E3 ubiquitin ligase activity of c-Cbl is required to restrict myeloid leukemia development. These mice develop a myeloproliferative disease which progresses to leukemia and involves hematopoietic progenitors that exhibit augmented FLT3 signaling. Suppressing this signaling through matings with FLT3 ligand knockout mice prevents leukemia development. We also observe enhanced c-Kit, Akt and Erk activity, and deregulated expression of leukemia-associated transcription factors in hematopoietic progenitors. The characterization of these perturbations provides direction for therapeutics that may aid the treatment of patients with c-Cbl mutations.

Humanized mice for modeling human infectious disease: challenges, progress, and outlook.

Over 800 million people worldwide are infected with hepatitis viruses, human immunodeficiency virus (HIV), and malaria, resulting in more than 5 million deaths annually. Here we discuss the potential and challenges of humanized mouse models for developing effective and affordable therapies and vaccines, which are desperately needed to combat these diseases.