Genetic background affects neutrophil activity and determines the severity of autoinflammatory osteomyelitis in mice.

The knowledge about the contribution of the innate immune system to health and disease is expanding. However, to obtain reliable results, it is critical to select appropriate mouse models for in vivo studies. Data on genetic and phenotypic changes associated with different mouse strains can assist in this task. Such data can also facilitate our understanding of how specific polymorphisms and genetic alterations affect gene function, phenotypes, and disease outcomes. Extensive information is available on genetic changes in all major mouse strains. However, comparatively little is known about their impact on immune response and in particular on innate immunity. Here, we analyzed a mouse model of chronic multifocal osteomyelitis (CMO), an autoinflammatory disease driven exclusively by the innate immune system, which is caused by an inactivating mutation in the Pstpip2 gene. We investigated how the genetic background of BALB/c, C57BL/6J, and C57BL/6NCrl strains alters the molecular mechanisms controlling disease progression. While all mice developed the disease, symptoms were significantly milder in BALB/c and partially also in C57BL/6J when compared to C57BL/6NCrl. Disease severity correlated with the number of infiltrating neutrophils and monocytes and with the production of chemokines attracting these cells to the site of inflammation. It also correlated with increased expression of genes associated with autoinflammation, rheumatoid arthritis, neutrophil activation, and degranulation, resulting in altered neutrophil activation in vivo. Together, our data demonstrate striking effects of genetic background on multiple parameters of neutrophil function and activity influencing the onset and course of the CMO disease.

Apoptosis induced by JAK2 inhibition is mediated by Bim and enhanced by the BH3 mimetic ABT-737 in JAK2 mutant human erythroid cells.

The activating mutation JAK2 V617F plays a central role in the pathogenesis of polycythemia vera, essential thrombocythemia, and primary myelofibrosis. Inhibition of JAK2 activity leads to growth inhibition and apoptosis in cells with mutated JAK2. However, the proapoptotic proteins involved in JAK2 inhibition-induced apoptosis remain unclear. In this study, we show that JAK2 inhibition-induced apoptosis correlated with up-regulation of the nonphosphorylated form of the BH3-only protein Bim in hematopoietic cell lines bearing JAK2 mutations. Knockdown of Bim dramatically inhibited apoptosis induced by JAK2 inhibition, which was reversed by the BH3 mimetic agent ABT-737. In addition, ABT-737 enhanced the apoptosis induced by JAK2 inhibition in JAK2 V617F(+) HEL and SET-2 cells. The combination of JAK inhibitor I and ABT-737 reduced the number of erythroid colonies derived from CD34(+) cells isolated from JAK2 V617F(+) polycythemia vera patients more efficiently than either drug alone. These data suggest that Bim is a key effector molecule in JAK2 inhibition-induced apoptosis and that targeting this apoptotic pathway could be a novel therapeutic strategy for patients with activating JAK2 mutations.

Endocannabinoids are expressed in bone marrow stromal niches and play a role in interactions of hematopoietic stem and progenitor cells with the bone marrow microenvironment.

Endocannabinoids are lipid signaling molecules that act via G-coupled receptors, CB(1) and CB(2). The endocannabinoid system is capable of activation of distinct signaling pathways on demand in response to pathogenic events or stimuli, hereby enhancing cell survival and promoting tissue repair. However, the role of endocannabinoids in hematopoietic stem and progenitor cells (HSPCs) and their interaction with hematopoietic stem cells (HSC) niches is not known. HSPCs are maintained in the quiescent state in bone marrow (BM) niches by intrinsic and extrinsic signaling. We report that HSPCs express the CB(1) receptors and that BM stromal cells secrete endocannabinoids, anandamide (AEA) (35 pg/10(7) cells), and 2-AG (75.2 ng/10(7) cells). In response to the endotoxin lipopolysaccharide (LPS), elevated levels of AEA (75.6 pg/10(7) cells) and 2-AG (98.8 ng/10(7) cells) were secreted from BM stromal cells, resulting in migration and trafficking of HSPCs from the BM niches to the peripheral blood. Furthermore, administration of exogenous cannabinoid CB(1) agonists in vivo induced chemotaxis, migration, and mobilization of human and murine HSPCs. Cannabinoid receptor knock-out mice Cnr1(-/-) showed a decrease in side population (SP) cells, whereas fatty acid amide hydrolase (FAAH)(-/-) mice, which have elevated levels of AEA, yielded increased colony formation as compared with WT mice. In addition, G-CSF-induced mobilization in vivo was modulated by endocannabinoids and was inhibited by specific cannabinoid antagonists as well as impaired in cannabinoid receptor knock-out mice Cnr1(-/-), as compared with WT mice. Thus, we propose a novel function of the endocannabinoid system, as a regulator of HSPC interactions with their BM niches, where endocannabinoids are expressed in HSC niches and under stress conditions, endocannabinoid expression levels are enhanced to induce HSPC migration for proper hematopoiesis.

Leukemic predisposition of pSca-1/Cb2 transgenic mice.

OBJECTIVE: The gene encoding the peripheral cannabinoid receptor Cb2 is located in the common virus integration site Evi11 and is associated with hematopoietic malignancies in mice. To determine the effect of Cb2 overexpression on hematopoietic development in vivo, Cb2 transgenic mice were generated. MATERIALS AND METHODS: A Cb2 expression vector was constructed containing a Cb2 cDNA fragment cloned into the 14kb Sca-1 (Ly-6E.1) gene. Two transgenic lines in which Cb2 expression is controlled by the Sca-1 promoter were generated, and the effect on hematopoietic development was studied. Expression of Cb2 mRNA or protein was studied by RNase protection analysis and ligand binding assays, respectively. Leukemic predisposition was investigated by injecting newborn transgenic as well as control animals with Cas-Br-M murine leukemia virus (Cas-Br-M MuLV). RESULTS: Although increased expression of the Cb2 gene was observed in hematopoietic tissues, follow-up of more than 1 year did not reveal any hematologic defect. Interestingly, infection of newborn pSca-1/Cb2 transgenic mice with Cas-Br-M MuLV revealed that significantly more transgenic mice developed leukemia than virus-treated control littermates. Because these studies provide evidence for the cooperative potential of Cb2 in leukemia progression, we wished to identify genes that may collaborate with Cb2 in leukemic transformation. Our study suggests that Evi1, another common target for proviral integration in mouse leukemias, may be overexpressed in virus-induced leukemias in pSca-1/Cb2 transgenic mice. CONCLUSIONS: The data indicate that hematopoietic precursor cells that express high levels of Cb2 possess increased susceptibility for leukemia development and that Cb2 and Evi1 might collaborate in leukemogenesis.

Identification, characterization, and function of a novel oncogene: the peripheral cannabinoid receptor Cb2.

By means of retroviral insertional mutagenesis, we identified a novel common virus integration site (cVIS) (Evi11) in murine leukemias, and demonstrated that Cb2, encoding the peripheral cannabinoid receptor, is the potential proto-oncogene located in that region. Cb2 is a 7-transmembrane G protein-coupled receptor (GPCR), which is normally expressed on B lymphocytes. Using transwell assays we observed strong migration of Cb2-expressing cells upon stimulation with 2-arachidonoylglycerol (2-AG), a potent endocannabinoid. Overexpression of Cb2 receptor on murine myeloid precursor cells causes a block of neutrophilic differentiation, a major characteristic of myeloid leukemia. Intriguingly, we could not detect functional Cb2 receptors on normal murine bone marrow precursor cells. Furthermore, analysis of human acute myeloid leukemia (AML) samples revealed the presence of CB2 mRNA transcripts in several cases. Furthermore, migration could be induced by 2-AG when analyzed in one of the patient samples. Our data suggest that the initially identified cVIS, Evi11, encodes for a murine onco-protein and that human CB2 may be involved in certain cases of human AML as well.

PU.1 expression is modulated by the balance of functional sense and antisense RNAs regulated by a shared cis-regulatory element.

The transcription factor PU.1 is an important regulator of hematopoiesis; precise expression levels are critical for normal hematopoietic development and suppression of leukemia. We show here that noncoding antisense RNAs are important modulators of proper dosages of PU.1. Antisense and sense RNAs are regulated by shared evolutionarily conserved cis-regulatory elements, and we can show that antisense RNAs inhibit PU.1 expression by modulating mRNA translation. We propose that such antisense RNAs will likely be important in the regulation of many genes and may be the reason for the large number of overlapping complementary transcripts with so far unknown function.

Distinct gene expression profiles of acute myeloid/T-lymphoid leukemia with silenced CEBPA and mutations in NOTCH1.

Gene expression profiling of acute myeloid leukemia (AML) allows the discovery of previously unrecognized molecular entities. Here, we identified a specific subgroup of AML, defined by an expression profile resembling that of AMLs with mutations in the myeloid transcription factor CCAAT/enhancer-binding protein alpha (C/EBPalpha), while lacking such mutations. We found that in these leukemias, the CEBPA gene was silenced, which was associated with frequent promoter hypermethylation. The leukemias phenotypically showed aberrant expression of T-cell genes, of which CD7 was most consistent. We identified 2 mechanisms that may contribute to this phenotype. First, absence of Cebpa led to up-regulation of specific T-cell transcripts (ie, Cd7 and Lck) in hematopoietic stem cells isolated from conditional Cebpa knockout mice. Second, the enhanced expression of TRIB2, which we identify here as a direct target of the T-cell commitment factor NOTCH1, suggested aberrantly activated Notch signaling. Putatively activating NOTCH1 mutations were found in several specimens of the newly identified subgroup, while a large set of control AMLs was mutation negative. A gene expression prediction signature allowed the detection of similar cases of leukemia in independent series of AML.

The peripheral cannabinoid receptor Cb2, a novel oncoprotein, induces a reversible block in neutrophilic differentiation.

We previously identified a novel common virus integration site, Evi11, by means of retroviral insertional mutagenesis. We demonstrated that the gene encoding the peripheral cannabinoid receptor (Cb2) is the potential target, suggesting that Cb2 is a proto-oncogene. To elucidate a role for this G protein-coupled receptor (GPCR) in leukemic transformation we generated a Cb2-EGFP cDNA construct that was introduced into 32D/G-CSF-R cells. These cells require interleukin 3 (IL-3) to proliferate in vitro, whereas in the presence of granulocyte-colony-stimulating factor (G-CSF) they differentiate toward mature neutrophils. We demonstrate that 32D/G-CSF-R/Cb2-EGFP cells migrate in a transwell assay in reponse to the Cb2 ligand 2-arachidonoylglycerol (2-AG), indicating that the fusion protein was functional. When cultured in the presence of G-CSF neutrophilic differentiation of Cb2-EGFP-expressing 32D/G-CSF-R cells was completely blocked. Moreover, a Cb2-specific antagonist fully recovered the G-CSF-induced neutrophilic differentiation of 32D/G-CSF-R/Cb2-EGFP cells. To investigate which signal transduction pathway(s) may be involved in the block of neutrophilic maturation, differentiation experiments were carried out using specific inhibitors of signaling routes. Interestingly, full rescue of G-CSF-induced neutrophilic differentiation was observed when cells were cultured with the mitogen-induced extracellular kinase (MEK) inhibitors, PD98059 or U0126, and partial recovery was detected with the phosphoinositide 3-kinase (PI3-K) inhibitor LY-294002. These studies demonstrate that the Cb2 receptor is an oncoprotein that blocks neutrophilic differentiation when overexpressed in myeloid precursor cells. Cb2 appears to mediate its activity through MEK/extracellular signal-related kinase (ERK) and PI3-K pathways.

Hematopoietic cells expressing the peripheral cannabinoid receptor migrate in response to the endocannabinoid 2-arachidonoylglycerol.

Cb2 is a novel protooncogene encoding the peripheral cannabinoid receptor. Previous studies demonstrated that 2 distinct noncoding first exons exist: exon-1A and exon-1B, which both splice to protein-coding exon-2. We demonstrate that in retrovirally induced murine myeloid leukemia cells with proviral insertion in Cb2, exon-1B/exon-2 Cb2 messenger RNA levels have been increased, resulting in high receptor numbers. In myeloid leukemia cells without virus insertion in this locus, low levels of only exon-1A/exon-2 Cb2 transcripts were present and receptors could not be detected. To elucidate the function of Cb2 in myeloid leukemia cells, a set of in vitro experiments was carried out using 32D/G-CSF-R (granulocyte colony-stimulating factor receptor) cells transfected with exon-1B/exon-2 Cb2 complementary DNA and a myeloid cell line carrying a virus insertion in Cb2 (ie, NFS 78). We demonstrate that a major function of the Cb2 receptor is stimulation of migration as determined in a transwell assay. Exposure of Cb2-expressing cells to different cannabinoids showed that the true ligand for Cb2 is 2-arachidonoylglycerol (2-AG), which may act as chemoattractant and as a chemokinetic agent. Furthermore, we observed a significant synergistic activity between 2-AG and interleukin-3 or G-CSF, suggesting cross-talk between the different receptor systems. Radioactive-ligand binding studies revealed significant numbers of Cb2 receptors in normal spleen. Transwell experiments carried out with normal mouse spleen cells showed 2-AG-induced migration of B220-, CD19-, immunoglobulin M-, and immunoglobulin D-expressing B lymphocytes. Our study demonstrates that a major function of Cb2 receptor expressed on myeloid leukemia cells or normal splenocytes is stimulation of migration.