Cristina Panaroni

WASP regulates suppressor activity of human and murine CD4(+)CD25(+)FOXP3(+) natural regulatory T cells.

A large proportion of Wiskott-Aldrich syndrome (WAS) patients develop autoimmunity and allergy. CD4+CD25+FOXP3+ natural regulatory T (nTreg) cells play a key role in peripheral tolerance to prevent immune responses to self-antigens and allergens. Therefore, we investigated the effect of WAS protein (WASP) deficiency on the distribution and suppressor function of nTreg cells. In WAS−/− mice, the steady-state distribution and phenotype of nTreg cells in the thymus and spleen were normal. However, WAS−/− nTreg cells engrafted poorly in immunized mice, indicating perturbed homeostasis. Moreover, WAS−/− nTreg cells failed to proliferate and to produce transforming growth factor β upon T cell receptor (TCR)/CD28 triggering. WASP-dependent F-actin polarization to the site of TCR triggering might not be involved in WAS−/− nTreg cell defects because this process was also inefficient in wild-type (WT) nTreg cells. Compared with WT nTreg cells, WAS−/− nTreg cells showed reduced in vitro suppressor activity on both WT and WAS−/− effector T cells. Similarly, peripheral nTreg cells were present at normal levels in WAS patients but failed to suppress proliferation of autologous and allogeneic CD4+ effector T cells in vitro. Thus, WASP appears to play an important role in the activation and suppressor function of nTreg cells, and a dysfunction or incorrect localization of nTreg cells may contribute to the development of autoimmunity in WAS patients.

Myelopoiesis is regulated by osteocytes through Gsα-dependent signaling

Hematopoietic progenitors are regulated in their respective niches by cells of the bone marrow microenvironment. The bone marrow microenvironment is composed of a variety of cell types, and the relative contribution of each of these cells for hematopoietic lineage maintenance has remained largely unclear. Osteocytes, the most abundant yet least understood cells in bone, are thought to initiate adaptive bone remodeling responses via osteoblasts and osteoclasts. Here we report that these cells regulate hematopoiesis, constraining myelopoiesis through a Gsα-mediated mechanism that affects G-CSF production. Mice lacking Gsα in osteocytes showed a dramatic increase in myeloid cells in bone marrow, spleen, and peripheral blood. This hematopoietic phenomenon was neither intrinsic to the hematopoietic cells nor dependent on osteoblasts but was a consequence of an altered bone marrow microenvironment imposed by Gsα deficiency in osteocytes. Conditioned media from osteocyte-enriched bone explants significantly increased myeloid colony formation in vitro, which was blocked by G-CSF–neutralizing antibody, indicating a critical role of osteocyte-derived G-CSF in the myeloid expansion.

In utero transplantation of adult bone marrow decreases perinatal lethality and rescues the bone phenotype in the knockin murine model for classical, dominant osteogenesis imperfecta

Autosomal dominant osteogenesis imperfecta (OI) caused by glycine substitutions in type I collagen is a paradigmatic disorder for stem cell therapy. Bone marrow transplantation in OI children has produced a low engraftment rate, but surprisingly encouraging symptomatic improvements. In utero transplantation (IUT) may hold even more promise. However, systematic studies of both methods have so far been limited to a recessive mouse model. In this study, we evaluated intrauterine transplantation of adult bone marrow into heterozygous BrtlIV mice. Brtl is a knockin mouse with a classical glycine substitution in type I collagen [alpha1(I)-Gly349Cys], dominant trait transmission, and a phenotype resembling moderately severe and lethal OI. Adult bone marrow donor cells from enhanced green fluorescent protein (eGFP) transgenic mice engrafted in hematopoietic and nonhematopoietic tissues differentiated to trabecular and cortical bone cells and synthesized up to 20% of all type I collagen in the host bone. The transplantation eliminated the perinatal lethality of heterozygous BrtlIV mice. At 2 months of age, femora of treated Brtl mice had significant improvement in geometric parameters (P < .05) versus untreated Brtl mice, and their mechanical properties attained wild-type values. Our results suggest that the engrafted cells form bone with higher efficiency than the endogenous cells, supporting IUT as a promising approach for the treatment of genetic bone diseases.

Evidence for Long-term Efficacy and Safety of Gene Therapy for Wiskott–Aldrich Syndrome in Preclinical Models

Wiskott-Aldrich Syndrome (WAS) is a life-threatening X-linked disease characterized by immunodeficiency, thrombocytopenia, autoimmunity, and malignancies. Gene therapy could represent a therapeutic option for patients lacking a suitable bone marrow (BM) donor. In this study, we analyzed the long-term outcome of WAS gene therapy mediated by a clinically compatible lentiviral vector (LV) in a large cohort of was(null) mice. We demonstrated stable and full donor engraftment and Wiskott-Aldrich Syndrome protein (WASP) expression in various hematopoietic lineages, up to 12 months after gene therapy. Importantly, we observed a selective advantage for T and B lymphocytes expressing transgenic WASP. T-cell receptor (TCR)-driven T-cell activation, as well as B-cells ability to migrate in response to CXCL13, was fully restored. Safety was evaluated throughout the long-term follow-up of primary and secondary recipients of WAS gene therapy. WAS gene therapy did not affect the lifespan of treated animals. Both hematopoietic and nonhematopoietic tumors arose, but we excluded the association with gene therapy in all cases. Demonstration of long-term efficacy and safety of WAS gene therapy mediated by a clinically applicable LV is a key step toward the implementation of a gene therapy clinical trial for WAS.

Impaired osteoblastogenesis in a murine model of dominant osteogenesis imperfecta: A new target for osteogenesis imperfecta pharmacological therapy

The molecular basis underlying the clinical phenotype in bone diseases is customarily associated with abnormal extracellular matrix structure and/or properties. More recently, cellular malfunction has been identified as a concomitant causative factor and increased attention has focused on stem cells differentiation. Classic osteogenesis imperfecta (OI) is a prototype for heritable bone dysplasias: it has dominant genetic transmission and is caused by mutations in the genes coding for collagen I, the most abundant protein in bone. Using the Brtl mouse, a well‐characterized knockin model for moderately severe dominant OI, we demonstrated an impairment in the differentiation of bone marrow progenitor cells toward osteoblasts. In mutant mesenchymal stem cells (MSCs), the expression of early (Runx2 and Sp7) and late (Col1a1 and Ibsp) osteoblastic markers was significantly reduced with respect to wild type (WT). Conversely, mutant MSCs generated more colony‐forming unit‐adipocytes compared to WT, with more adipocytes per colony, and increased number and size of triglyceride drops per cell. Autophagy upregulation was also demonstrated in mutant adult MSCs differentiating toward osteogenic lineage as consequence of endoplasmic reticulum stress due to mutant collagen retention. Treatment of the Brtl mice with the proteasome inhibitor Bortezomib ameliorated both osteoblast differentiation in vitro and bone properties in vivo as demonstrated by colony‐forming unit‐osteoblasts assay and peripheral quantitative computed tomography analysis on long bones, respectively. This is the first report of impaired MSC differentiation to osteoblasts in OI, and it identifies a new potential target for the pharmacological treatment of the disorder. STEM CELLS2012;30:1465–1476

Interactions Between B Lymphocytes and the Osteoblast Lineage in Bone Marrow

The regulatory effects of the immune system on the skeleton during homeostasis and activation have been appreciated for years. In the past decade it has become evident that bone tissue can also regulate immune cell development. In the bone marrow, the differentiation of hematopoietic progenitors requires specific microenvironments, called “niches,” provided by various subsets of stromal cells, many of which are of mesenchymal origin. Among these stromal cell populations, cells of the osteoblast lineage serve a supportive function in the maintenance of normal hematopoiesis, and B lymphopoiesis in particular. Within the osteoblast lineage, distinct differentiation stages exert differential regulatory effects on hematopoietic development. In this review we will highlight the critical role of osteoblast progenitors in the perivascular B lymphocyte niche.

Mesenchymal Progenitors and the Osteoblast Lineage in Bone Marrow Hematopoietic Niches

The bone marrow cavity is essential for the proper development of the hematopoietic system. In the last few decades, it has become clear that mesenchymal stem/progenitor cells as well as cells of the osteoblast lineage, besides maintaining bone homeostasis, are also fundamental regulators of bone marrow hematopoiesis. Several studies have demonstrated the direct involvement of mesenchymal and osteoblast lineage cells in the maintenance and regulation of supportive microenvironments necessary for quiescence, self-renewal and differentiation of hematopoietic stem cells. In addition, specific niches have also been identified within the bone marrow for maturing hematopoietic cells. Here we will review recent findings that have highlighted the roles of mesenchymal progenitors and cells of the osteoblast lineage in regulating distinct stages of hematopoiesis.

PTH Signaling in Osteoprogenitors Is Essential for B-Lymphocyte Differentiation and Mobilization

Cells of the osteoblast lineage provide critical support for B lymphopoiesis in the bone marrow (BM). Parathyroid hormone (PTH) signaling in osteoblastic cells through its receptor (PPR) is an important regulator of hematopoietic stem cells; however, its role in regulation of B lymphopoiesis is not clear. Here we demonstrate that deletion of PPR in osteoprogenitors results in a significant loss of trabecular and cortical bone. PPR signaling in osteoprogenitors, but not in mature osteoblasts or osteocytes, is critical for B‐cell precursor differentiation via IL‐7 production. Interestingly, despite a severe reduction in B‐cell progenitors in BM, mature B‐lymphocytes were increased 3.5‐fold in the BM of mice lacking PPR in osteoprogenitors. This retention of mature IgD+ B cells in the BM was associated with increased expression of vascular cell adhesion molecule 1 (VCAM1) by PPR‐deficient osteoprogenitors, and treatment with VCAM1 neutralizing antibody increased mobilization of B lymphocytes from mutant BM. Our results demonstrate that PPR signaling in early osteoblasts is necessary for B‐cell differentiation via IL‐7 secretion and for B‐lymphocyte mobilization via VCAM1.

Characterization of a novel Alu-Alu recombination-mediated genomic deletion in the TCIRG1 gene in five osteopetrotic patients.

Human malignant autosomal recessive osteopetrosis (ARO) is a genetically heterogeneous disorder caused by reduced bone resorption by osteoclasts. Biallelic mutations in the TCIRG1 gene, encoding the a3 subunit of the vacuolar proton pump, are responsible for more than one half of ARO patients. However, a few patients with monoallelic mutations have been described, raising the possibility of a dominant‐like TCIRG1‐dependent osteopetrosis, of a digenic disease, or of peculiar mutations difficult to detect with standard methods. We describe here a novel genomic deletion in the TCIRG1 gene explaining why, in some patients, mutations in only one allele have previously been found. The analysis of a proband from a consanguineous Turkish family allowed us to define the deletion boundaries encompassing introns 10 and 13 and occurring within AluSx repeat sequences, suggesting Alu‐mediated homologous recombination as a mechanism. An identical genomic deletion at the heterozygous level was found in four unrelated Italian families in whom only a single mutated allele has previously been found. TCIRG1 haplotype analysis in these five families suggests a possible common ancestral origin for this large deletion. In summary, we describe the identification of a novel genomic deletion in the TCIRG1 gene that is of clinical relevance, especially in prenatal diagnosis.

Preclinical activity of CPI-0610, a novel small-molecule bromodomain and extra-terminal protein inhibitor in the therapy of multiple myeloma

Inhibition of the bromodomain and extra-terminal (BET) proteins is a promising therapeutic strategy for various hematologic cancers. Previous studies suggest that BET inhibitors constrain tumor cell proliferation and survival mainly through the suppression of MYC transcription and activity. However, suppression of the transcription of additional genes also contributes to the antitumor activity of BET inhibitors but is less well understood. Here we examined the therapeutic potential of CPI-0610, a potent BET inhibitor currently undergoing phase I clinical testing, in multiple myeloma (MM). CPI-0610 displays potent cytotoxicity against MM cell lines and patient-derived MM cells through G1 cell cycle arrest and caspase-dependent apoptosis. CPI-0610-mediated BET inhibition overcomes the protective effects conferred by cytokines and bone marrow stromal cells. We also confirmed the in vivo efficacy of CPI-0610 in a MM xenograft mouse model. Our study found IKZF1 and IRF4 to be among the primary targets of CPI-0610, along with MYC. Given that immunomodulatory drugs (IMiDs) stabilize cereblon and facilitate Ikaros degradation in MM cells, we combined it with CPI-0610. Combination studies of CPI-0610 with IMiDs show in vitro synergism, in part due to concomitant suppression of IKZF1, IRF4 and MYC, providing a rationale for clinical testing of this drug combination in MM patients.