Group 2 innate lymphoid cells are elevated and activated in chronic rhinosinusitis with nasal polyps.

BACKGROUND: Chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) is characterized by type 2 inflammation with high levels of Th2 cytokines. Although T helper cytokines are released from T cells, innate lymphoid cells (ILC) are also known to produce high levels of the same cytokines. However, the presence of various types of ILC in CRS is poorly understood. OBJECTIVE: The objective of this study was to fully characterize the presence of all ILC subsets in CRS and to identify phenotypical differences of group 2 ILC (ILC2) in CRSwNP compared to ILC2 from non-type 2 inflamed areas. METHODS: We investigated the presence of ILC subsets in peripheral blood mononuclear cells (PBMC) from healthy subjects, tonsil tissue, ethmoid tissue from control subjects and patients with non-polypoid CRS (CRSsNP) and CRSwNP, as well as nasal polyp (NP) tissue from CRSwNP by flow cytometry. Sorted ILC2 were cultured in the presence and absence of IL-33 and production of IL-5 and IL-13 was assessed by Luminex. RESULTS: We found that all ILC subsets were present in NP but ILC2 were dominant and significantly elevated compared to PBMC, tonsil, CRSsNP, and normal sinus tissue. We also found that inducible T-cell co-stimulator (ICOS) and side scatter were increased and CD127 was down-regulated in ILC2 from NP compared to blood or tonsil ILC2. Thymic stromal lymphopoietin, IL-7, and IL-33 were able to down-regulate expression of CD127 and increase side scatter in blood ILC2. Furthermore, sorted NP ILC2 but not blood ILC2 spontaneously released type 2 cytokines including IL-5 and IL-13. CONCLUSIONS AND CLINICAL RELEVANCE: These results suggest that ILC2 are not only elevated but also activated in CRSwNP in vivo and that ILC2 may play important roles in the type 2 inflammation in CRSwNP.

Reflections on microRNAs in chronic pulmonary disease: looking into the miR-ror and crystal ball.

Chronic respiratory diseases are a significant health problem requiring novel approaches to both complement existing therapies and provide breakthrough medicines. Recent clinical advances in understanding the behavior of inhaled oligonucleotides provide the impetus for application of this technology to microRNA therapeutics. MicroRNAs are evolutionarily conserved small regulatory RNA molecules involved in tuning gene networks controlling biological and pathological processes. Deletion or overexpression of microRNAs results in phenotypic changes in animal models of disease such as cancer, fibrosis, diabetes, and inflammation. Inhibition of microRNAs in preclinical models of asthma, cystic fibrosis, and idiopathic pulmonary fibrosis has shown therapeutic promise. In animals, inhibitors of microRNAs directly delivered to the airway at doses suitable for nebulizers or hand-held inhalers up-regulate expression of cohorts of genes containing complementary "seed" sequences for specific and directed microRNA binding within their mRNA untranslated regions. These observations suggest the opportunity to exploit intervention in microRNA biology to create new therapies for chronic pulmonary disorders.

STAT3 and STAT1 mediate IL-11-dependent and inflammation-associated gastric tumorigenesis in gp130 receptor mutant mice.

Deregulated activation of STAT3 is frequently associated with many human hematological and epithelial malignancies, including gastric cancer. While exaggerated STAT3 signaling facilitates an antiapoptotic, proangiogenic, and proproliferative environment for neoplastic cells, the molecular mechanisms leading to STAT3 hyperactivation remain poorly understood. Using the gp130(Y757F/Y757F) mouse model of gastric cancer, which carries a mutated gp130 cytokine receptor signaling subunit that cannot bind the negative regulator of cytokine signaling SOCS3 and is characterized by hyperactivation of the signaling molecules STAT1 and STAT3, we have provided genetic evidence that IL-11 promotes chronic gastric inflammation and associated tumorigenesis. Expression of IL-11 was increased in gastric tumors in gp130(Y757F/Y757F) mice, when compared with unaffected gastric tissue in wild-type mice, while gp130(Y757F/Y757F) mice lacking the IL-11 ligand-binding receptor subunit (IL-11Ralpha) showed normal gastric STAT3 activation and IL-11 expression and failed to develop gastric tumors. Furthermore, reducing STAT3 activity in gp130(Y757F/Y757F) mice, either genetically or by therapeutic administration of STAT3 antisense oligonucleotides, normalized gastric IL-11 expression and alleviated gastric tumor burden. Surprisingly, the genetic reduction of STAT1 expression also reduced gastric tumorigenesis in gp130(Y757F/Y757F) mice and coincided with reduced gastric inflammation and IL-11 expression. Collectively, our data have identified IL-11 as a crucial cytokine promoting chronic gastric inflammation and associated tumorigenesis mediated by excessive activation of STAT3 and STAT1.

Interferon-gamma induces X-linked inhibitor of apoptosis-associated factor-1 and Noxa expression and potentiates human vascular smooth muscle cell apoptosis by STAT3 activation.

Interferon (IFN)-gamma actions on the vessel wall play an important role in the pathogenesis of arteriosclerosis, yet the contribution of different IFN-gamma signaling pathways to the phenotypic modulation of vascular smooth muscle cells (VSMCs) are poorly understood. We investigated the effects of IFN-gamma on VSMCs and arteries through interactions involving signal transducer and activator of transcription (STAT) proteins. In addition to STAT1 activation, IFN-gamma consistently phosphorylated STAT3 in human VSMCs but weakly or not at all in human endothelial cells or mouse VSMCs. STAT3 activation resulted in nuclear translocation of this transcription factor. By selectively inhibiting STAT3 and not STAT1 signaling, we identified a number of candidate IFN-gamma-inducible, STAT3-dependent gene products by microarray analysis. Results for selected genes, including the pro-apoptotic molecules X-linked inhibitor of apoptosis associated factor-1 (XAF1) and Noxa, were verified by real time quantitative reverse transcription-PCR and immunoblot analyses. IFN-gamma-induced STAT3 and STAT1 signaling in VSMCs demonstrated reciprocal inhibition. STAT3 activation by IFN-gamma sensitized VSMCs to apoptosis triggered by both death receptor- and mitochondrial-mediated pathways. Knock down of XAF1 and Noxa expression inhibited the priming of VSMCs to apoptotic stimuli by IFN-gamma. Finally, we confirmed the in vivo relevance of our observations using a chimeric animal model of immunodeficient mice bearing human coronary artery grafts in which the expression of XAF1 and Noxa as well as the pro-apoptotic effects induced by IFN-gamma were dependent on STAT3. The data suggest STAT1-independent signaling by IFN-gamma via STAT3 that promotes the death of human VSMCs.

Anti-inflammatory activity of inhaled IL-4 receptor-alpha antisense oligonucleotide in mice.

The Th2 cytokines IL-4 and IL-13 mediate allergic pulmonary inflammation and airways hyperreactivity (AHR) in asthma models through signaling dependent upon the IL-4 receptor-alpha chain (IL-4Ralpha). IL-13 has been further implicated in the overproduction of mucus by the airway epithelium and in lung remodeling that commonly accompanies chronic inflammation. IL-4Ralpha-deficient mice are resistant to allergen-induced asthma, highlighting the therapeutic promise of selective molecular inhibitors of IL-4Ralpha. We designed a chemically modified IL-4Ralpha antisense oligonucleotide (IL-4Ralpha ASO) that specifically inhibits IL-4Ralpha protein expression in lung eosinophils, macrophages, dendritic cells, and airway epithelium after inhalation in allergen-challenged mice. Inhalation of IL-4Ralpha ASO attenuated allergen-induced AHR, suppressed airway eosinophilia and neutrophilia, and inhibited production of airway Th2 cytokines and chemokines in previously allergen-primed and -challenged mice. Histologic analysis of lungs from these animals demonstrated reduced goblet cell metaplasia and mucus staining that correlated with inhibition of Muc5AC gene expression in lung tissue. Therapeutic administration of inhaled IL-4Ralpha ASO in chronically allergen-challenged mice produced a spectrum of anti-inflammatory activity similar to that of systemically administered Dexamethasone with the added benefit of reduced airway neutrophilia. These data support the potential utility of a dual IL-4 and IL-13 oligonucleotide inhibitor in allergy/asthma, and suggest that local inhibition of IL-4Ralpha in the lung is sufficient to suppress allergen-induced pulmonary inflammation and AHR.

Inhibition of growth and metastasis of human hepatocellular carcinoma by antisense oligonucleotide targeting signal transducer and activator of transcription 3.

PURPOSE: Hepatocellular carcinoma (HCC) is an aggressive malignancy and is a devastating clinical complication of chronic liver disease. Therapeutic options are limited mainly because the genetic and biochemical understanding of this disease remains fragmented. We intended to study the role of signal transducer and activator of transcription 3 (STAT3) aberrant signaling in HCC malignancy, and the therapeutic potential of inhibition of STAT3 expression for HCC. EXPERIMENTAL DESIGN: A 2'-O-methoxyethylribose-modified phosphorothioate antisense oligonucleotide (ASO) was used to knock down STAT3 expression in different human HCC cell lines, including the highly metastatic HCCLM3 derived from orthotopic implantation and subsequent lung metastasis in athymic mice. The effects of STAT3 ASO treatment on HCC cells, metastasis, and animal survival following HCCLM3 orthotopic implantation were evaluated. RESULTS: Specific suppression of phosphorylated STAT3 reduced its DNA-binding activity, inhibited the expression of vascular endothelial growth factor, survivin, matrix metalloproteinases 2 and 9, reduced cell proliferation and migratory potential, induced apoptosis in vitro, and inhibited intradermal angiogenesis and s.c. tumorigenesis upon injection in mice. In mice bearing orthotopically implanted HCCLM3, STAT3 inhibition following therapeutic treatment with STAT3 ASO reduced circulating vascular endothelial growth factor and basic fibroblast growth factor, decreased intratumor CD34-positive microvessel density, intrahepatic and intraperitoneal transmission, and lung metastasis. HCC tumor volume and weight were reduced and the survival time of mice bearing orthotopically xenografted HCC was approximately doubled in STAT3 ASO-treated mice (P < 0.05). CONCLUSIONS: Constitutively activated STAT3 is essential for the growth, survival, and metastasis of HCC, suggesting that STAT3-targeted therapy may have utility for HCC.

Stat3 is required for ALK-mediated lymphomagenesis and provides a possible therapeutic target.

Anaplastic large cell lymphomas (ALCLs) are caused by chromosomal translocations that juxtapose the anaplastic lymphoma kinase (ALK) proto-oncogene to a dimerization partner, resulting in constitutive expression of ALK and ALK tyrosine kinase activity. One substrate of activated ALK in human ALCLs is the transcription factor Stat3, and its phosphorylation is accurately recapitulated in a new nucleophosmin (NPM)-ALK transgenic mouse model of lymphomagenesis. Here we show by gene targeting that Stat3 is required for the transformation of mouse embryonic fibroblasts in vitro, for the development of B-cell lymphoma in transgenic mice and for the growth and survival of both human and mouse NPM-ALK-transformed B and T cells. Ablation of Stat3 expression by antisense oligonucleotides significantly (P < 0.0001) impaired the growth of human and mouse NPM-ALK tumors in vivo. Pharmacological ablation of Stat3 represents a new candidate approach for the treatment of human lymphoma

Signal transducer and activator of transcription 3 (STAT3) activation in prostate cancer: Direct STAT3 inhibition induces apoptosis in prostate cancer lines.

Signal transducers and activators of transcription (STAT) were originally discovered as components of cytokine signal transduction pathways. Persistent activation of one STAT, STAT3, is a common feature of prostate cancer. Activated STAT3 was found in pathology specimens obtained from prostatectomy in the cancerous areas but not in the normal margins. Because the activation of STAT3 is mediated by the action of an upstream Janus kinase (JAK) kinase, usually JAK1 or JAK2, the activation step for STAT3 might itself be a target for therapy in prostate cancer. However, the redundancy of upstream kinases may make this strategy unreliable for therapy. To develop molecular targets for prostate cancer treatment, JAK kinase and STAT3 inhibition of two prostate cancer lines were compared. DU145 and NRP-154 cells were treated with JAK kinase inhibitors, analyzed for onset of apoptosis, and measured by annexin V binding and propidium iodide uptake. Activation of caspases in the cells was determined by measuring cleaved caspase-3 following treatment. For determining the effect on mitochondrial membrane depolarization that accompanies apoptosis, the fluorescent dye JC-1 was used. STAT3 was specifically inhibited by transfecting either a dominant-negative (DN) STAT3 plasmid or antisense STAT3 oligonucleotides into the cells. To look for reduction in STAT3 levels within cells, fixed and permeabilized prostate cancer cells were stained with antibody to STAT3. We found that more than one JAK kinase is involved in STAT3 activation in prostate cancer lines. AG490 (JAK2 specific) induced apoptosis in DU145 but not in NRP-154 prostate cancer lines, whereas piceatannol (JAK1 specific) induced apoptosis in NRP-154 but not in DU145 cells. Next, we demonstrated efficacy of specific STAT3 inhibitors in prostate cancer lines. Both induction of apoptosis and reduction in intracellular STAT3 protein were observed following treatment with antisense STAT3 oligonucleotides, while transfection of a DN-STAT3 plasmid into both prostate cancer cell lines resulted in loss of viability and onset of apoptosis. We conclude that STAT3-specific inhibitors, rather than JAK kinase-specific inhibitors, should be more useful therapeutically in treating androgen-resistant prostate cancer and that STAT3 is an appropriate target in the treatment of prostate cancer.

Activation of Stat3 by receptor tyrosine kinases and cytokines regulates survival in human non-small cell carcinoma cells.

Overexpression of receptor tyrosine kinases including the epidermal growth factor receptor (EGF-R) as well as nonreceptor tyrosine kinases, such as Src, have been implicated in the formation of human lung cancers. In addition, cytokines like interleukin-6 (IL-6) have been demonstrated to modulate lung cancer cell growth and elevated levels of IL-6 have been shown to be an adverse prognostic factor for patients with lung cancer. Despite a large body of evidence pointing to their potential importance, few direct studies into the role of signal transducers and activators of transcription (STAT) pathways in human lung cancer have been undertaken. Here we demonstrate that multiple nonsmall cell lung cancer cell lines demonstrate constitutive Stat3 DNA-binding activity. Stat3 DNA-binding activity is specifically upregulated by the addition of epidermal growth factor (EGF), IL-6, and hepatocyte-derived growth factor (HGF). Furthermore, the stimulation of Stat3 DNA-binding activity by EGF requires the activity of EGF-R tyrosine kinase as well as Src-kinase, while the upregulation of Stat3 activity by IL-6 or HGF requires only Src-kinase activity. Treatment of A549 lung cancer cells with PD180970 or SU6656, both pharmacological inhibitors of Src-kinase, resulted in reduced Src and Stat3 activity, cell cycle arrest in G2, and reduced viability of cells accompanied by induction of apoptosis. Treatment of Stat3-positive A549 and H358 cells with antisense Stat3 oligonucleotides results in complete loss of Stat3 DNA-binding activity and apoptosis, while Stat3-positive H1299 cells remained healthy. Finally, an adenoviral vector expressing a dominant-negative Stat3 isoform results in loss of Stat3 DNA-binding activity, apoptosis, and reduced cellular viability. These results demonstrate a role of Stat3 in transducing survival signals downstream of tyrosine kinases such as Src, EGF-R, and c-Met, as well as cytokines such as IL-6, in human nonsmall cell lung cancers.

Protection of CLL B cells by a follicular dendritic cell line is dependent on induction of Mcl-1.

Chronic lymphocytic leukemia (CLL) B cells have defects in apoptosis pathways and therefore accumulate in vivo. However, when removed from the patient and cultured in vitro, these malignant cells rapidly undergo apoptosis. Recent studies suggest that leukemia cell survival is influenced by interactions with nonleukemia cells in the microenvironment of lymph nodes, marrow, and other tissues. To model such cell-cell interactions in vitro, we cultured freshly isolated CLL B cells with a follicular dendritic cell line, HK. CLL B cells cocultured with HK cells were protected from apoptosis, either spontaneous or induced by treatment with anticancer drugs. Protection against spontaneous apoptosis could also be induced by coculturing the CLL B cells with normal dendritic cells (DCs) or with a CD40-ligand (CD154)-expressing fibroblast cell line. Examination of the expression of several apoptosis-regulatory proteins revealed that coculture with HK cells or DCs induced up-regulation of the antiapoptotic Bcl-2 family protein Mcl-1 in CLL B cells, whereas CD40 ligation increased expression of Bcl-X(L). Cell-cell contact was required for HK-induced protection, and introducing neutralizing antibodies against various adhesion molecules showed that CD44 was involved in HK-mediated survival, whereas CD40, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were not. Anti-CD44 antibodies also blocked Mcl-1 induction by HK cells. Mcl-1 antisense oligonucleotides reduced leukemia cell expression of Mcl-1, and significantly suppressed HK-induced protection against apoptosis, whereas control oligonucleotides had no effect. Thus, HK cells protect CLL B cells against apoptosis, at least in part through a CD44-dependent mechanism involving up-regulation of Mcl-1, and this mechanism is distinct from that achieved by CD40 ligation. Consequently, the particular antiapoptotic proteins important for CLL survival may vary depending on the microenvironment.