Interleukin-6 Receptor Expression Research Articles

Systematic analysis of blood cell transcriptome in end-stage chronic respiratory diseases.

BackgroundEnd-stage chronic respiratory diseases (CRD) have systemic consequences, such as weight loss and susceptibility to infection. However the mechanisms of such dysfunctions are as yet poorly explained. We hypothesized that the genes putatively involved in these mechanisms would emerge from a systematic analysis of blood mRNA profiles from pre-transplant patients with cystic fibrosis (CF), pulmonary hypertension (PAH), and chronic obstructive pulmonary disease (COPD).MethodsWhole blood was first collected from 13 patients with PAH, 23 patients with CF, and 28 Healthy Controls (HC). Microarray results were validated by quantitative PCR on a second and independent group (7PAH, 9CF, and 11HC). Twelve pre-transplant COPD patients were added to validate the common signature shared by patients with CRD for all causes. To further clarify a role for hypoxia in the candidate gene dysregulation, peripheral blood mononuclear cells from HC were analysed for their mRNA profile under hypoxia.ResultsUnsupervised hierarchical clustering allowed the identification of 3 gene signatures related to CRD. One was common to CF and PAH, another specific to CF, and the final one was specific to PAH. With the common signature, we validated T-Cell Factor 7 (TCF-7) and Interleukin 7 Receptor (IL-7R), two genes related to T lymphocyte activation, as being under-expressed. We showed a strong impact of the hypoxia on modulation of TCF-7 and IL-7R expression in PBMCs from HC under hypoxia or PBMCs from CRD. In addition, we identified and validated genes upregulated in PAH or CF, including Lectin Galactoside-binding Soluble 3 and Toll Like Receptor 4, respectively.ConclusionsSystematic analysis of blood cell transcriptome in CRD patients identified common and specific signatures relevant to the systemic pathologies. TCF-7 and IL-7R were downregulated whatever the cause of CRD and this could play a role in the higher susceptibility to infection of these patients.

Abstract 4507: SL-501, a next-generation targeted therapy directed to IL-3R, inhibits the growth of tyrosine kinase inhibitor-resistant CML cells

Abstract Although imatinib and other tyrosine kinase inhibitors (TKIs) achieve high response rates in patients with chronic myeloid leukemia (CML), patients can fail these therapies or develop specific mutations that render TKIs ineffective. Moreover, TKIs have limited effectiveness against CML cancer stem cells (CSCs). It was previously shown that SL-401, a novel targeted therapy directed to the interleukin 3 receptor (IL-3R), inhibits the growth of CML tumor bulk and CSCs, both of which over express IL-3R. SL-501 is a next-generation targeted therapy also directed to IL-3R. SL-501 has approximately 4-fold increased binding affinity for IL-3R and a more than 30-fold increase in potency against acute myeloid leukemia (AML) cells compared to SL-401. In this study, we assessed the activity of SL-501 against a panel of TKI-sensitive and TKI-resistant CML cell lines. KBM5, KBM5-STI, K562, and BV173 cells were treated with SL-501 (range: 5 - 21,000 pg) for 24 or 48 hours and assessed for viability and apoptosis. We found that SL-501 reduced the absolute cell number of all cell lines tested in a dose-dependent manner with IC50s of 20 pM, 21 pM, 31.5 nM, and 3.9 nM, respectively; these concentrations are readily achieved at tolerable doses in the clinic. Moreover, SL-501 significantly reduced the number of imatinib-resistant KBM5-STI viable cells by more than 5-fold after 48 hours. Using caspase and lactate dehydrogenase (LDH) release assays, we also found that SL-501 alone induced apoptosis of all cell lines tested by more than 40% after 24 hours. The combination of SL-501 with TKIs (imatinib, dasatinib, or nilotinib) had an enhanced effect on apoptosis in KBM5, K562, and BV173 cells 24 hours post-treatment. Taken together, these findings indicate that SL-501, a novel next-generation IL-3-targeted therapeutic, may be effective at treating TKI-resistant CML. These promising results together with the IL-3R expression pattern on CML blasts and CSCs warrant further development of SL-501 in CML and, in particular, TKI-resistant CML. Citation Format: Christopher Brooks, Kenneth Hoberman, Ivan Bergstein, Eric Rowinsky. SL-501, a next-generation targeted therapy directed to IL-3R, inhibits the growth of tyrosine kinase inhibitor-resistant CML cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4507. doi:10.1158/1538-7445.AM2014-4507

Abstract 74: The Inflammatory Phenotype Of Mesenchymal Fibroblasts And Its Role In Aging Dependent Cardiac Fibrosis- A Target For Statins?

In the aging mouse (C57BL/6) myocardium fibrosis steadily increases after 14 months of age and is accompanied by elevated numbers of myeloid derived fibroblasts. Recently, we proposed a mechanism by which inflammatory mesenchymal fibroblasts (IMF) derived from mesenchymal stem cells secrete monocyte chemoattractant protein-1 (MCP-1) necessary for myeloid fibroblast induction in the aging heart. The current study extends the characterization of this inflammatory phenotype by describing elevated interleukin-6 (IL-6) secretion and increased expression of IL-6 receptor (IL-6R) in IMF. Since IL-6R lacks an intracellular domain it requires a co-receptor gp130 (generally expressed) to induce an intracellular signal. Thus, generation of an IL-6R soluble receptor allows IL-6 signaling on cells that do not express IL-6R (or expression is low), such as endothelial cells. We investigate the function of IL-6 and IL-6R in the promotion of transendothelial migration of monocytes through cardiac endothelium and their maturation into myeloid fibroblasts in in vitro assay. Treatments with IL-6 and more extensively IL-6+IL-6R resulted in a 3-5 fold increase (above the control level) in myeloid cell migration and maturation into myeloid fibroblasts. Thus IMF can contribute both IL-6 and IL-6R to endothelial cells and facilitate myeloid cell transendothelial migration. In agreement with these data, analysis of the aged mouse heart revealed the presence of fibroblasts expressing IL-6 (procollagen type I + IL-6 + cells), M1 macrophages (CD86 + cells) and M2 macrophages (CD301 + procollagen type I + cells) that were absent in hearts from young mice. The mechanisms by which expression of these factors is upregulated in IMF are being investigated; our data suggest that MCP-1 and IL-6 expression are controlled by the farnesyltransferase (FTase)-Ras-Erk1/2 pathway. Interestingly, since atorvastatin interferes with farnesyl synthesis it also reduced MCP-1 and IL-6 expression in IMF. These data may introduce a new use of this class of drugs in the prevention of the age-related fibrosis.

Interleukin-22 in the pathogenesis of psoriasis

As a critical cytokine expressed by the newly discovered CD4+ T cell subsets (including T helper type 17 (Th17) and Th22 cells),interleukin-22 (IL-22) acts as a molecular bridge between immunocytes and keratinocytes in the pathogenesis of psoriasis.The specific expression of IL-22 receptor determines the selective effect of IL-22 on keratinocytes.IL-22 may take part of the formation of lesions at the late stage by binding its receptor and activating the intracellular signal transducer and activator of transcription (STAT) pathway.Further more,IL-22 has a potential immunomodulatory effect by inducing the specific expression of cytokeratin 17 and CCL20 by keratinocytes. Key words: Psoriasis; Interleukin-22; Receptors, interleukin-22; Signaling pathway

Expression of monocyte subsets and angiogenic markers in relation to carotid plaque neovascularization in patients with pre-existing coronary artery disease and carotid stenosis

Aim. To characterize blood monocyte subsets in patients with different degrees of carotid atherosclerosis and pathological carotid plaque neovascularization.Methods. Assessment of carotid plaque neovascularization using contrast ultrasonography and flow cytometric quantification of monocyte subsets and their receptors involved in inflammation, angiogenesis, and tissue repair was done in 40 patients with carotid stenosis ≥ 50% and CAD (CS > 50), 40 patients with carotid stenosis < 50% and documented CAD (CS < 50), 40 hypercholesterolaemic controls (HC group), and 40 normocholesterolaemic controls (NC).Results. CS > 50 and CS < 50 groups had increased counts of Mon1 (‘classical’ CD14++ CD16-CCR2 + cells) compared to HCs (P = 0.03, and P = 0.009). Mon3 (‘non-classical’ CD14 + CD16++ CCR2- cells) were only increased in CS < 50 compared with HCs (P < 0.01). Both CS>50 and CS < 50 groups showed increased expression of proinflammatory interleukin-6 receptor on Mon1 and Mon2 (‘intermediate’ CD14++ CD16 + CCR2+ cells); TLR4, proangiogenic Tie2 on all subsets (P < 0.01 for all). In multivariate regression analysis only high Mon1 count was a significant predictor of carotid stenosis (P = 0.04) and intima-media thickness (P = 0.02). In multivariate regression analysis only the Mon1 subset was significantly associated with severe, grade 2 neovascularization (P = 0.034).Conclusion. In this pilot study classical monocytes (Mon1) represent the only monocyte subset predictive of the severity of carotid and systemic atherosclerosis, such as carotid intima-media thickness, degree of carotid stenosis, and presence of carotid intraplaque neovascularization.

Upregulated interleukin-21 receptor on B cells associated with the downregulation of IgE in patients with allergic rhinitis.

Allergic diseases, such as allergic rhinitis, caused by an immunoglobulin E (IgE)-mediated reaction to specific allergens are a common chronic condition worldwide. Interleukin-21 (IL-21), a type I cytokine that is produced by T cells, exerts regulatory effects on a variety of immune cells. In our previous study, we found that serum levels of IL-21 were significantly decreased in patients with severe atopic dermatitis, suggesting that IL-21 might play a role in allergic reactions. In this study, we investigated the role of IL-21/IL-21 receptor (IL-21R) in patients with allergic rhinitis. Our results demonstrated that there was no difference in IL-21 serum levels between allergic rhinitis patients and controls. However, allergic patients had significantly increased expression of IL-21R on naive and memory B cells. IL-21R was upregulated through stimulation by the combination of CD40 ligand (CD40L) and IL-4. IL-21 alone neither induced nor inhibited IgE secretion from CD40L-stimulated B cells. However, IL-21 inhibited IgE secretion of B cells that were induced by the combination of CD40L and IL-4 in allergic patients. Moreover, a negative correlation between the expression of IL-21R and serum levels of IgE was found in patients with allergy. These results suggest that the role of IL-21 in an ongoing allergic reaction is to downregulate the IgE level by binding to IL-21R on B cells, which increases the expression in allergic patients.

AB0058 The Disease Modifying Osteoarthritis Drug Diacerein is Able to Antagonize PRO Inflammatory State of Chondrocytes under Mild Mechanical Stimuli

BackgroundThe medical application of disease modifying osteoarthritis drugs (DMOADs) in OA therapy emerged to preserve normal joint function, to reduce disease's intensity and symptoms and to restrain the progression rate...

Clinical effects of soluble interleukin-6 receptor detection on autoimmune rheumatic disease patients.

Objective : This study aims to explore the effects of soluble interleukin-6 receptor (IL-6R) on the clinical diagnosis and treatment of autoimmune rheumatic disease (ARD) patients. Sixty-eight ARD patients enrolled in our hospital recently were selected, the IL-6R levels of whom were detected by double-antibody sandwich ELISA method and compared with the IL-6 levels of normal subjects. The expressions of IL-6R in joint tissues were detected by section staining. The mechanism regarding the effects of IL-6R was postulated by administering the patients with blocking agent. The blood IL-6R level of ARD patients was 2-3 times higher than the IL-6 level of normal subjects with significant difference. The detection results of C-reactive protein and erythrocyte sedimentation rate show that both the indicators were significantly decreased (P=0.0098 and 0.0097 respectively). IL-6R was associated with autoimmunity based on the considerable expression in tissue sections, which was also verified by the alleviated symptoms after blocking IL-6R expression. Detecting soluble IL-6R is able to determine the patient's conditions and treatment effects. Meanwhile, soluble IL-6R detection can also serve as the inflammatory responses of ARD, as well as the determination index for abnormal immune responses and generated antibodies number, which is crucial for early diagnosis.

The disease modifying osteoarthritis drug diacerein is able to antagonize pro inflammatory state of chondrocytes under mild mechanical stimuli

To investigate the combination of mild mechanical stimuli and a disease modifying osteoarthritis drug (DMOAD) in inflammatory activated chondrocytes and to study the combination of drug and mechanical tension on the cellular level as a model for an integrated biophysical approach for osteoarthritis (OA) treatments. Interleukin-1beta (IL-1β) stimulated C28/I2 cells underwent mild mechanically treatment while cultured in the presence of the DMOAD diacerein. The pharmacological input of diacerein was evaluated by cell viability and cell proliferation measurements. Inflammation and treatment induced changes in key regulatory proteins and components of the extracellular matrix (ECM) were characterized by quantitative real-time PCR (qPCR). The effects on metalloproteinase-1 (MMP-1) activity and glycosaminoglycan (GAG) concentration in cell supernatants of treated cells were investigated. C28/I2 cells demonstrated significant changes in expression of inflammatory and cartilage destructive proteins in response to IL-1β stimulation. The chondroprotective action of diacerein in mechanically stimulated cells was mediated by a decrease in interleukin-8 (IL-8), fibronectin-1 (FN-1), collagen type I (Col 1) and MMP-1 expression levels, respectively. Augmented expression of interleukin-6 receptor (IL-6R) and the fibroblast growth factor receptors (FGFRs) by diacerein was not abolished by mechanical treatment. The observed effects were accompanied by a reduced cell proliferation rate, attenuated cell viability and extenuated MMP-1 activity. Diacerein diversely regulates the expression of main regulatory proteins as well as components important to regenerate and set up ECM. Mechanical stimulation does not negatively influence the chondroprotective effect induced by diacerein treatment in immortalized human C28/I2 chondrocytes.

Interleukin-17 receptor expression on vascular endothelial cells of masses of skeletal extramedullary disease in myeloma patients

The goal of the study was to investigate the expression of interleukin-17 (IL-17) and IL-17 receptor (IL-17R) in patients with myeloma bone diseases (MBD) and skeletal extramedullary disease (skeletal EMD). The levels of IL-17 were determined using ELISA. The expression of IL-17R on vascular endothelial cells of bone marrow (BM) and masses of skeletal EMD was detected using immunohistochemistry. The results showed an elevated IL-17 level in BM of BMD and skeletal EMD patients. The microvessel density (MVD) was significantly increased in the masses of skeletal EMD. IL-17R was almost exclusively expressed by endothelial cells, not by myeloma cells in the masses of skeletal EMD patients. We concluded that EMD masses showed increased angiogenesis mediated by IL-17 pathway and in part this may help in myeloma cell-growth under these conditions.

Interleukin-6-induced epithelial-mesenchymal transition through signal transducer and activator of transcription 3 in human cervical carcinoma.

Epithelial-mesenchymal transition (EMT) is an important process in the invasion and metastasis of human cervical carcinoma. The pro-inflammatory cytokine interleukin-6 (IL-6) has been shown as an EMT inducer in multiple carcinomas. However, whether the EMT program can be induced by IL-6 and the mechanisms underlying the IL-6-induced EMT in human cervical carcinoma remain to be determined. In this study, we show that IL-6 receptor (IL-6R) and signal transducer and activator of transcription3 (Stat3) were highly expressed in human cervical squamous cell carcinoma (CSCC) tissues, and the expression of EMT markers was reversed in well-differentiated and poorly-differentiated human CSCC. Additional experiments showed that IL-6 exposure in cervical carcinoma cell lines induced IL-6R and Stat3 expression, markedly promoted cell growth, and altered cell morphology. The treatment of cervical carcinoma cell lines with IL-6 resulted in downregulation of E-Cadherin and upregulation of Vimentin. Importantly, knockdown of Stat3 significantly reversed the IL-6-induced EMT program, suggesting that Stat3 is necessary for IL-6-induced EMT in the progression of human cervical carcinoma. Moreover, Slug, a member of the Snail family of EMT regulators, was observed to be associated with the expression of Stat3. We concluded that IL-6 plays an important role through Stat3 in the EMT induction and can be a potential therapeutic target and biomarker for human cervical carcinoma.

Macrophage-Restricted Interleukin-10 Receptor Deficiency, but Not IL-10 Deficiency, Causes Severe Spontaneous Colitis

Interleukin-10 (IL-10) is a pleiotropic anti-inflammatory cytokine produced and sensed by most hematopoietic cells. Genome-wide association studies and experimental animal models point at a central role of the IL-10 axis in inflammatory bowel diseases. Here we investigated the importance of intestinal macrophage production of IL-10 and their IL-10 exposure, as well as the existence of an IL-10-based autocrine regulatory loop in the gut. Specifically, we generated mice harboring IL-10 or IL-10 receptor (IL-10Rα) mutations in intestinal lamina propria-resident chemokine receptor CX3CR1-expressing macrophages. We found macrophage-derived IL-10 dispensable for gut homeostasis and maintenance of colonic T regulatory cells. In contrast, loss of IL-10 receptor expression impaired the critical conditioning of these monocyte-derived macrophages and resulted in spontaneous development of severe colitis. Collectively, our results highlight IL-10 as a critical homeostatic macrophage-conditioning agent in the colon and define intestinal CX3CR1(hi) macrophages as a decisive factor that determines gut health or inflammation.

Impaired neutrophil extracellular trap formation: a novel defect in the innate immune system of aged individuals.

Neutrophil extracellular traps (NETs) are a recently discovered addition to the defensive armamentarium of neutrophils, assisting in the immune response against rapidly dividing bacteria. Although older adults are more susceptible to such infections, no study has examined whether aging in humans influences NET formation. We report that TNF-α-primed neutrophils generate significantly more NETs than unprimed neutrophils and that lipopolysaccharide (LPS)- and interleukin-8 (IL-8)-induced NET formation exhibits a significant age-related decline. NET formation requires generation of reactive oxygen species (ROS), and this was also reduced in neutrophils from older donors identifying a mechanism for reduced NET formation. Expression of IL-8 receptors (CXCR1 and CXCR2) and the LPS receptor TLR4 was similar on neutrophils from young and old subjects, and neutrophils challenged with phorbol-12-myristate-13-acetate (PMA) showed no age-associated differences in ROS or NET production. Taken together, these data suggest a defect in proximal signalling underlies the age-related decline in NET and ROS generation. TNF-α priming involves signalling through p38 MAP kinase, but activation kinetics were comparable in neutrophils from young and old donors. In a clinical setting, we assessed the capacity of neutrophils from young and older patients with chronic periodontitis to generate NETs in response to PMA and hypochlorous acid (HOCL). Neutrophil extracellular trap generation to HOCL, but not PMA, was lower in older periodontitis patients but not in comparison with age-matched controls. Impaired NET formation is thus a novel defect of innate immunity in older adults but does not appear to contribute to the increased incidence of periodontitis in older adults.

Vitamin D3 treatment fails to restore glucocorticoid-induced down-regulation of interleukin-10 receptor expression on CD4+ T cells

Glucocorticoids (GC) exert some of their anti-inflammatory actions via enhancement of interleukin-10 (IL-10) production by immune cells. Interestingly, however, we previously demonstrated that oral administration of GC to asthmatic patients resulted in down-regulation of interleukin-10 receptor (IL-10R) expression on peripheral blood leukocytes. Here we wished to investigate whether addition of vitamin D3 (vit. D3) could abrogate these down-regulating actions of GC. We performed a comparative analysis of the effects of in vitro stimulation with GC and/or vit. D3 on surface expression of IL-10R on the CD4+ T cell pool. As expected, GC stimulation led to significant reduction of IL-10R. In contrast, culture in the presence of vit. D3 did not result in any significant changes of IL-10R expression. Moreover, addition of vit. D3 to GC culture did not alter GC-induced down-regulation of IL-10R expression. Thus, we conclude that anti-inflammatory actions of vit. D3 are not dependent on modification of IL-10R expression.

Interleukin-8 promotes canine hemangiosarcoma growth by regulating the tumor microenvironment

Interleukin-8 (IL-8) gene expression is highly up-regulated in canine hemangiosarcoma (HSA); however, its role in the pathogenesis of this disease is unknown. We investigated the expression of IL-8 in canine HSA tissues and cell lines, as well and the effects of IL-8 on canine HSA in vitro, and in vivo using a mouse xenograft model for the latter. Constitutive expression of IL-8 mRNA, IL-8 protein, and IL-8 receptor were variable among different tumor samples and cell lines, but they showed stable steady states in each cell line. Upon the addition of IL-8, HSA cells showed transient intracellular calcium fluxes, suggesting that their IL-8 receptors are functional and that IL-8 binding activates relevant signaling pathways. Yet, neither addition of exogenous IL-8 nor blockade of endogenous IL-8 by neutralizing anti-IL-8 antibody (α-IL-8 Ab) affected HSA cell proliferation or survival in vitro. To assess potential effects of IL-8 in other tumor constituents, we stratified HSA cell lines and whole tumor samples into “IL-8 high” and “IL-8 low” groups. Genome-wide gene expression profiling showed that samples in the “IL-8 high” tumor group were enriched for genes associated with a “reactive microenvironment,” including activation of coagulation, inflammation, and fibrosis networks. Based on these findings, we hypothesized that the effects of IL-8 on these tumors were mostly indirect, regulating interactions with the microenvironment. This hypothesis was supported by in vivo xenograft experiments where survival and engraftment of tumor cells was inhibited by administration of neutralizing α-IL-8 Ab. Together, our results suggest that IL-8 contributes to establishing a permissive microenvironment during the early stages of tumorigenesis in HSA.

Histone Deacetylase Inhibitors Modulate Interleukin 6-dependent CD4+ T Cell Polarization in Vitro and in Vivo

Histone deacetylase (HDAC) inhibitors have been associated primarily with an anti-proliferative effect in vitro and in vivo. Recent data provide evidence for an anti-inflammatory potency of HDAC inhibitors in models of experimental colitis. Because the balance of T cell subpopulations is critical for the balance of the mucosal immune system, this study explores the regulatory potency of HDAC inhibitors on T cell polarization as a mechanistic explanation for the observed anti-inflammatory effects. Although HDAC inhibition suppressed the polarization toward the pro-inflammatory T helper 17 (Th17) cells, it enhanced forkhead box P3 (FoxP3)(+) regulatory T cell polarization in vitro and in vivo at the site of inflammation in the lamina propria. This was paralleled by a down-regulation of the interleukin 6 receptor (IL-6R) on naïve CD4(+) T cells on the mRNA as well as on the protein level and changes in the chromatin acetylation at the IL6R gene and its promoter. Downstream of the IL-6R, HDAC inhibition was followed by a decrease in STAT3 phosphorylation as well as retinoic acid receptor-related orphan receptor γT (RORγT) expression, thus identifying the IL-6/STAT3/IL-17 pathway as an important target of HDAC inhibitors. These results directly translated to experimental colitis, where IL-6R expression was suppressed in naïve T cells, paralleled by a significant reduction of Th17 cells in the lamina propria of ITF2357-treated animals, resulting in the amelioration of disease. This study indicates that, in experimental colitis, inhibition of HDAC exerts an anti-inflammatory potency by directing T helper cell polarization via targeting the IL-6 pathway.

Delta-1 provides pleasant stem cell environs

In this issue of Blood, Csaszar et al show that in ex vivo cultures of CD341 cells, simultaneous reduction of interleukin-6 receptor (IL-6R) expression on myeloid progenitor cells by Delta-1 treatment combined with removal of soluble IL-6R using a dynamically fed culture system, reduces mature myeloid cell production, leading to enhanced expansion of primitive hematopoietic stem cells.

Interleukin-17 produced by tumor microenvironment promotes self-renewal of CD133+ cancer stem-like cells in ovarian cancer

Inflammatory cytokines, components of cancer stem cells (CSCs) niche, could affect the characteristics of CSCs such as self-renewal and metastasis. Interleukin-17 (IL-17) is a new pro-inflammatory cytokine mainly produced by T-helper (Th17) cells and macrophages. The effects of IL-17 on the characteristics of CSCs remain to be explored. Here we first demonstrated a role of IL-17 in promoting the self-renewal of ovarian CD133(+) cancer stem-like cells (CSLCs). We detected IL-17-producing cells (CD4(+) cells and CD68(+) macrophages) in the niche of CD133(+)CSLCs. Meanwhile, there was IL-17 receptor expression on CD133(+)CSLCs derived from A2780 cell line and primary ovarian cancer tissues. By recombinant human IL-17 stimulation and IL-17 transfection, the growth and sphere formation capacities of ovarian CD133(+)CSLCs were significantly enhanced in a dose-dependent manner. Moreover, ovarian CD133(+)CSLCs transfected with IL-17 showed greater tumorigenesis capacity in nude mice. These data suggest that IL-17 promoted the self-renewal of ovarian CD133(+)CSLCs. Further investigation through gene profiling revealed that the stimulation function of IL-17 on self-renewal of ovarian CD133(+)CSLCs might be mediated by the nuclear factor (NF)-κB and p38 mitogen-activated protein kinases (MAPK) signaling pathway. NF-κB and p38 MAPK were activated by IL-17. More importantly, IL-17-promoted self-renewal was inhibited by specific inhibitors of NF-κB and p38 MAPK. Taken together, our data indicate that IL-17 contributed to ovarian cancer malignancy through promoting the self-renewal of CD133(+)CSLCs and that IL-17 and its signaling pathway might serve as therapeutic targets for the treatment of ovarian cancer.

PU.1 and Spi-B Oppose Transformation Of Pre-B Cells Through Activation Of Key Genes Involved In B Cell Receptor Signalling

B cell development is controlled by stage-specific expression of transcription factors. Aberrant expression of such factors can lead to B cell acute lymphoblastic leukemia (B-ALL). Deletion of genes encoding the E26 transformation-specific (ETS) transcription factors, PU.1 and Spi-B, in B cells (CD19+/CreSfpi1lox/loxSpib-/- mice, abbreviated to CD19-CreΔPB) leads to B-ALL at 100% incidence and with a median survival of 21 weeks. However, little is known about the target genes of PU.1 and Spi-B that explain leukemic transformation in these mice. In the current study, we investigated the developmental origins and mechanisms of leukemogenesis in CD19-CreΔPB mice. We found that B-ALL cells in CD19-CreΔPB mice had frequently rearranged both their heavy and light chain genes, but retained cell surface expression of interleukin-7 receptor (IL-7R), suggesting aberrant pre-B cell differentiation. Preleukemic CD19-CreΔPB mice had increased frequencies of pre-B cells compared to wild type mice. Pre-B cells, but not mature B cells, purified from the bone marrow of preleukemic CD19-CreΔPB mice could rapidly transfer disease to transplanted recipient mice. B-ALL cells from established tumors had uniform expression of markers indicating a pre-B cell phenotype and contained a high-frequency of leukemia-initiating cells as measured by transplantation assays. Genome-wide analysis of gene expression showed that B cell receptor signalling was the top impaired pathway in B-ALL cells from CD19-CreΔPB mice. Bone marrow cells from CD19-CreΔPB mice had increased responsiveness to IL-7R signalling and could be cultured as IL-7-dependent cell lines. Preleukemic or leukemic cells from CD19-CreΔPB mice expressed reduced levels of the gene encoding Bruton's tyrosine kinase (Btk), which we show is a target gene of PU.1 and/or Spi-B that in combination with reduced BLNK is sufficient to explain increased IL-7R responsiveness. We conclude that mutation of PU.1 and Spi-B predispose developing B cells to leukemogenesis by impairing expression of key genes, such as Btk, that are required for BCR signalling and are involved in attenuation of IL-7 receptor signaling. Disclosures:No relevant conflicts of interest to declare.

SL-401, a Targeted Therapy Directed To The Interleukin-3 Receptor (IL-3R), Possesses Preclinical Cytotoxic Activity Against Chronic Eosinophilic Leukemia

BackgroundHypereosinophilic syndrome (HES), a rare myeloproliferative disorder characterized by a persistently elevated eosinophil count, results in damage to the heart, lungs, peripheral nervous system, and other organs. An acquired (non-familial) form of HES is particularly aggressive and debilitating. Acquired forms of HES are subclassified as secondary (reactive), idiopathic, and clonal HES, the latter often transitioning into chronic eosinophilic leukemia (CEL), or hypereosinophilic leukemia, which can result in myocardial fibrosis and congestive heart failure. Patients may respond to corticosteroids, the monoclonal antibodies mepolizumab and alemtuzumab, and the tyrosine kinase inhibitor imatinib, which is registered for HES patients who express the FIP1L1-PDGFRA fusion protein. However, several of the aforementioned agents also induce severe toxicity. Since eosinophils ubiquitously express the IL-3R, SL-401, a novel IL-3R-targeted therapeutic, which is comprised of IL-3 conjugated to a truncated diphtheria toxin, represents a rational and selective agent for development in hypereosinophilic disorders. MethodsExpression of the IL-3R (CD123) on a human eosinophilic leukemia cell line (EOL-1) was determined by flow cytometry. Cells were washed with phosphate-buffered saline (PBS) and stained with anti-CD123 PE (BD Biosciences) and control IgG for 20 minutes at 4oC and then washed with PBS again. Stained cells were acquired using the LSR II (BD) cytometer and data were analyzed using Flow Jo (Tree Star). To analyze patient samples, PBMCs were prepared and stained using a similar method and then analyzed using Cytopaint software. The sensitivity of the cells to SL-401 was assessed using a CellTiter Glo in vitro cytotoxicity assay. A CD123 positive leukemia cell line (TF-1), which is known to be sensitive to SL-401, was used as a positive control. The cells were cultured in the presence of absence of SL-401 for 48 hours and assessed for viability at concentrations ranging from 5x10-6 to 21 µg/ml (8.7x10-5 to 368 nM). ResultsPrimary eosinophils, elevated in number and CD16 negative, were harvested from a patient with HES and found to be positive for IL-3R. EOL-1 cells, similarly, were found to express high levels of IL-3R, with greater than 98% of cells expressing the receptor compared to isotype control. Based on the IL-3R expression pattern, the sensitivity of EOL-1 cells to SL-401 was then tested. Cells were shown to be highly sensitive to SL-401 after 48-hour incubation in a concentration-dependent manner, with IC50 values of 1, 0.6, and 0.47 pM from triplicate experiments (mean: 0.69 pM). Cell viability was also reduced by 96% at 48 hours post-treatment with a picomolar concentration of SL-401 (350 pM). ConclusionThese results indicate SL-401 possesses potent in vitro anti-cancer activity against CEL, which expresses high levels of the IL-3R. Importantly, SL-401 exhibited potent activity against these cells at concentrations that were significantly lower (>10-fold) than peak plasma concentrations achieved in clinical studies. These findings, together with the need for novel clinical strategies to treat CEL and HES, warrant further exploration of SL-401 for the treatment of patients with hypereosinophilic diseases. Disclosures:Brooks: Stemline Therapeutics: Employment, Equity Ownership. Macri:Stemline Therapeutics, Inc., New York, NY USA : Employment. Bergstein:Stemline Therapeutics: Employment, Equity Ownership, Patents & Royalties. Rowinsky:Stemline Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.