Gabor Jarai

Inhibition of collagen-induced discoidin domain receptor 1 and 2 activation by imatinib, nilotinib and dasatinib

Imatinib, nilotinib and dasatinib are protein kinase inhibitors which target the tyrosine kinase activity of the Breakpoint Cluster Region-Abelson kinase (BCR-ABL) and are used to treat chronic myelogenous leukemia. Recently, using a chemical proteomics approach another tyrosine kinase, the collagen receptor Discoidin Domain Receptor1 (DDR1) has also been identified as a potential target of these compounds. To further investigate the interaction of imatinib, nilotinib and dasatinib with DDR1 kinase we cloned and expressed human DDR1 and developed biochemical and cellular functional assays to assess their activity against DDR1 and the related receptor tyrosine kinase Discoidin Domain Receptor2 (DDR2). Our studies demonstrate that all 3 compounds are potent inhibitors of the kinase activity of both DDR1 and DDR2. In order to investigate the question of selectivity among DDR1, DDR2 and other tyrosine kinases we have aligned DDR1 and DDR2 protein sequences to other closely related members of the receptor tyrosine kinase family such as Muscle Specific Kinase (MUSK), insulin receptor (INSR), Abelson kinase (c-ABL), and the stem cell factor receptor (c-KIT) and have built homology models for the DDR1 and DDR2 kinase domains. In spite of high similarity among these kinases we show that there are differences within the ATP-phosphate binding loop (P-loop), which could be exploited to obtain kinase selective compounds. Furthermore, the potent DDR1 and DDR2 inhibitory activity of imatinib, nilotinib and dasatinib may have therapeutic implications in a number of inflammatory, fibrotic and neoplastic diseases.

Epithelial-mesenchymal transition in primary human bronchial epithelial cells is Smad-dependent and enhanced by fibronectin and TNF-α

BackgroundDefective epithelial repair, excess fibroblasts and myofibroblasts, collagen overproduction and fibrosis occur in a number of respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD) and pulmonary fibrosis. Pathological conversion of epithelial cells into fibroblasts (epithelial-mesenchymal transition, EMT) has been proposed as a mechanism for the increased fibroblast numbers and has been demonstrated to occur in lung alveolar epithelial cells. Whether other airway cell types also have the capability to undergo EMT has been less explored so far. A better understanding of the full extent of EMT in airways, and the underlying mechanisms, can provide important insights into airway disease pathology and enable the development of new therapies. The main aim of this study was to test whether primary human bronchial epithelial cells are able to undergo EMT in vitro and to investigate the effect of various profibrotic factors in the process.ResultsOur data demonstrate that primary human bronchial epithelial cells (HBECs) are able to undergo EMT in response to transforming growth factor-beta 1 (TGF-β1), as revealed by typical morphological alterations and EMT marker progression at the RNA level by real-time quantitative polymerase chain reaction and, at the protein level, by western blot. By using pharmacological inhibitors we show that this is a Smad-dependent mechanism and is independent of extracellular signal-related kinase pathway activation. Additional cytokines and growth factors such as tumour necrosis factor-alpha (TNF-α), interleukin-1 beta (IL1β) and connective tissue growth factor (CTGF) were also tested, alone or in combination with TGF-β1. TNF-α markedly enhances the effect of TGF-β1 on EMT, whereas IL1β shows only a very weak effect and CTGF has no significant effect. We have also found that cell-matrix contact, in particular to fibronectin, an ECM component upregulated in fibrotic lesions, potentiates EMT in both human alveolar epithelial cells and HBECs. Furthermore, we also show that the collagen discoidin domain receptor 1 (DDR1), generally expressed in epithelial cells, is downregulated during the EMT of bronchial epithelium whereas DDR2 is unaffected. Our results also suggest that bone morphogenetic protein-4 is likely to have a context dependent effect during the EMT of HBECs, being able to induce the expression of EMT markers and, at the same time, to inhibit TGF-β induced epithelial transdifferentiation.ConclusionsThe results presented in this study provide additional insights into EMT, a potentially very important mechanism in fibrogenesis. We show that, in addition to alveolar epithelial type II cells, primary HBECs are also able to undergo EMT in vitro upon TGF-β1 stimulation via a primarily Smad 2/3 dependent mechanism. The effect of TGF-β1 is potentiated on fibronectin matrix and in the presence of TNF-α, representing a millieu reminiscent of fibrotic lesions. Our results can contribute to a better understanding of lung fibrosis and to the development of new therapeutic approaches.

A novel murine model of severe pulmonary arterial hypertension

RATIONALE The complex pathologies associated with severe pulmonary arterial hypertension (PAH) in humans have been a challenge to reproduce in mice due to the subtle phenotype displayed to PAH stimuli. OBJECTIVES Here we aim to develop a novel murine model of PAH that recapitulates more of the pathologic processes, such as complex vascular remodeling and cardiac indices, that are not characteristic of alternative mouse models. METHODS Inhibition of vascular endothelial growth factor receptor (VEGFR) with SU5416 combined with 3 weeks of chronic hypoxia was investigated. Hemodynamics, cardiac function, histological assessment of pulmonary vasculature, and molecular pathway analysis gauged the extent of PAH pathology development. MEASUREMENTS AND MAIN RESULTS The combination of VEGFR inhibition with chronic hypoxia profoundly exacerbated all measures of PAH-like pathology when compared with hypoxia alone (> 45 mm Hg right ventricular pressure, > 0.35 right ventricular hypertrophy). The changes in pulmonary vascular remodeling in response to hypoxia were further enhanced on SU5416 treatment. Furthermore, hypoxia/SU5416 treatment steadily decreased cardiac output, indicating incipient heart failure. Molecular analysis showed a dysregulated transforming growth factor-β/bone morphogenetic protein/Smad axis in SU5416- and/or hypoxia-treated mice as well as augmented induction of IL-6 and Hif-1α levels. These changes were observed in accordance with up-regulation of Tph1 and Pdgfr gene transcripts as well as a rise in platelet-rich serotonin. Biomarker analysis in response to VEGFR inhibition and/or hypoxia revealed distinct signatures that correlate with cytokine profiles of patients with idiopathic PAH. CONCLUSIONS These data describe a novel murine model of PAH, which displays many of the hallmarks of the human disease, thus opening new avenues of investigation to better understand PAH pathophysiology.

The Sphingosine 1-Phosphate Receptor Agonist FTY720 Differentially Affects the Sequestration of CD4+/CD25+ T-Regulatory Cells and Enhances Their Functional Activity

The sphingosine 1-phosphate (S1P) receptor agonist FTY720 is well known for its immunomodulatory activity, sequestering lymphocytes from blood and spleen into secondary lymphoid organs and thereby preventing their migration to sites of inflammation. Because inflammation is critically dependent on a balance between Ag-specific Th/effector cells and T-regulatory cells, we investigated the effect of FTY720 on T-regulatory cell trafficking and functional activity. An increased number of CD4+/CD25+ T cells was found in blood and spleens of FTY720-treated mice, and transfer of these cells resulted in a significantly more pronounced accumulation in spleens but not lymph nodes after treatment, suggesting that this compound differentially affects the homing properties of T-regulatory cells compared with other T cell subsets. Indeed, CD4+/CD25+ T cells express lower levels of S1P1 and S1P4 receptors and demonstrate a reduced chemotactic response to S1P. Moreover, analysis of the functional response of FTY720-treated CD4+/CD25+ T cells revealed an increased suppressive activity in an in vitro Ag-specific proliferation assay. This correlated with enhanced function in vivo, with T-regulatory cells obtained from FTY720-treated mice being able to suppress OVA-induced airway inflammation. Thus, FTY720 differentially affects the sequestration of T-regulatory cells and importantly, increases the functional activity of T-regulatory cells, suggesting that it may have disease-modifying potential in inflammatory disorders.

IL-10 expression profiling in human monocytes

Interleukin‐10 (IL‐10) is a potent anti‐inflammatory cytokine with numerous immunomodulatory effects, including the inhibition of proinflammatory cytokine production. The mechanisms by which IL‐10 exerts these effects still remain largely unknown. As there is evidence that suggests IL‐10‐mediated cytokine suppression requires the induction of an intermediate gene, we have used gene‐chip technology to identify IL‐10‐inducible genes in human monocytes. We have been able to identify a total of 19 genes that are up‐regulated in response to IL‐10. Three of these genes had been identified previously: IL‐1ra, suppressors of cytokine signaling‐3, and CD163; however, the other 16 represent newly identified IL‐10‐responsive genes. Further analysis of the regulation of eight of these genes showed a remarkable specificity to regulation by lipopolysaccharides (LPS) and IL‐10, but not by other anti‐inflammatory mediators such as IL‐4 and transforming growth factor‐β, suggesting that two diverse stimuli such as IL‐10 and LPS may engage common signaling mechanisms.

TLR9 Differentiates Rapidly from Slowly Progressing Forms of Idiopathic Pulmonary Fibrosis

Compared to slow progressors, patients with rapidly progressive idiopathic pulmonary fibrosis express more TLR9, which recognizes unmethylated CpG DNA and stimulates the fibrotic process. Taking a Toll on Breathing Despite the incredible rate of advances being made in medical science, the exact causes of many diseases remain unknown. These diseases are classified as idiopathic—“a disease of its own kind.” But like a thief who leaves clues at a crime scene that disclose his or her identity, diseases can spur aberrant biological processes that hint at the condition’s cause. Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive form of lung disease of unknown origin characterized by the excess production of fibrous connective tissue (fibrosis) in the supporting framework (interstitium) of the lungs. These changes cause the hardening and/or scarring of lung tissue due to excess collagen, resulting in shortness of breath, a chronic dry cough, fatigue, weakness, chest discomfort, loss of appetite, and rapid weight loss. Patients with IPF have a poor prognosis and are usually expected to live only an average of 4 to 6 years after diagnosis; however, IPF displays a very heterogeneous path, with disease progressing rapidly in some patients and more slowly in others. Thus far, physicians have been unable to predict the speed of disease progression in patients newly diagnosed with IPF. Now, Trujillo et al. have identified a marker that differentiates these two patient groups and that may also mediate rapid progression of this disease. Toll-like receptor 9 (TLR9) is an innate immune molecule that recognizes a particular type of DNA frequently found in bacteria and viruses—unmethylated CpG DNA. Signaling through TLR9 promotes the differentiation of lung fibroblasts taken from IPF patients into myofibroblasts—cells that resemble both smooth muscle and fibroblasts—a key process in fibrosis. Trujillo et al. hypothesized that TLR9 may contribute to rapidly progressing IPF. Indeed, they found higher amounts of TLR9 in rapidly progressing IPF patients compared to slow progressing patients and normal controls. Moreover, in a xenograft mouse model of IPF, fibroblasts from rapid progressors induced more severe fibrosis in response to TLR9 activation than those from slow progressors. The presence of CpG also induced epithelial to mesenchymal transition—another hallmark of fibrosis—in a lung epithelial cell line in vitro. Together, these results suggest that TLR9 may serve as a marker for IPF rapid progressors and that TLR9 targeting may be a new therapeutic strategy for treating IPF. Thus, although the cause(s) of IPF remains unknown, the new data offer hope for an improvement in the prognosis and possibly treatment of this devastating disease. Idiopathic pulmonary fibrosis is characterized by diffuse alveolar damage and severe fibrosis, resulting in a steady worsening of lung function and gas exchange. Because idiopathic pulmonary fibrosis is a generally progressive disorder with highly heterogeneous disease progression, we classified affected patients as either rapid or slow progressors over the first year of follow-up and then identified differences between the two groups to investigate the mechanism governing rapid progression. Previous work from our laboratory has demonstrated that Toll-like receptor 9 (TLR9), a pathogen recognition receptor that recognizes unmethylated CpG motifs in bacterial and viral DNA, promotes myofibroblast differentiation in lung fibroblasts cultured from biopsies of patients with idiopathic pulmonary fibrosis. Therefore, we hypothesized that TLR9 functions as both a sensor of pathogenic molecules and a profibrotic signal in rapidly progressive idiopathic pulmonary fibrosis. Indeed, TLR9 was present at higher concentrations in surgical lung biopsies from rapidly progressive patients than in tissue from slowly progressing patients. Moreover, fibroblasts from rapid progressors were more responsive to the TLR9 agonist, CpG DNA, than were fibroblasts from slowly progressing patients. Using a humanized severe combined immunodeficient mouse, we then demonstrated increased fibrosis in murine lungs receiving human lung fibroblasts from rapid progressors compared with mice receiving fibroblasts from slowly progressing patients. This fibrosis was exacerbated by intranasal CpG challenges. Furthermore, CpG induced the differentiation of blood monocytes into fibrocytes and the epithelial-to-mesenchymal transition of A549 lung epithelial cells. These data suggest that TLR9 may drive the pathogenesis of rapidly progressive idiopathic pulmonary fibrosis and may serve as a potential indicator for this subset of the disease.

Nitrogen, carbon, and pH regulation of extracellular acidic proteases of Aspergillus niger

Aspergillus niger secretes a number of enzymes, including proteases, into its culture fluid. The regulation of the two major acidic extracellular proteases, pepA and pepB, was investigated using Northern analyses. Our data suggest that the regulation of pepA and pepB expression occurs predominantly at the level of mRNA content and that, while they are regulated in a similar manner, differences are also clear in their expression. Both genes were found to be under complex regulatory control. The expression of the two genes could be turned off by the presence of good nitrogen or carbon sources in the media, and external protein sources did not induce expression of either gene under conditions of carbon and nitrogen repression. The pH of the medium also played a major role in their regulation as the expression of both genes was completely turned off under alkaline conditions, even when grown in media lacking good nitrogen and carbon sources but containing proteins. We isolated clones containing 5′ non-coding sequences of the pepA gene from a λ genomic library with a pepA specific probe. Analysis and comparison of the promoter sequences of the pepA and pepB genes revealed that both contain several putative AREA- and CREA-binding sites and they also share an 18-bp-long sequence which is 83% identical in these two genes.

Cytokine Induced Phenotypic and Epigenetic Signatures Are Key to Establishing Specific Macrophage Phenotypes

Macrophages (MΦ) play an essential role in innate immune responses and can either display a pro-inflammatory, classically activated phenotype (M1) or undergo an alternative activation program (M2) promoting immune regulation. M-CSF is used to differentiate monocytes into MΦ and IFN-γ or IL-4+IL-13 to further polarize these cells towards M1 or M2, respectively. Recently, differentiation using only GM-CSF or M-CSF has been described to induce a M1- or M2-like phenotype, respectively. In this study, we combined both approaches by differentiating human MΦ in GM-CSF or M-CSF followed by polarization with either IFN-γ or IL-4+IL-13. We describe the phenotypic differences between CD14hi CD163hi CD206int FOLR2-expressing M-CSF MΦ and CD14lo CD163lo CD206hi GM-CSF MΦ but show that both macrophage populations reacted similarly to further polarization with IFN-γ or IL-4+IL-13 with up- and down-regulation of common M1 and M2 marker genes. We also show that high expression of the mannose receptor (CD206), a marker of alternative activation, is a distinct feature of GM-CSF MΦ. Changes of the chromatin structure carried out by chromatin modification enzymes (CME) have been shown to regulate myeloid differentiation. We analyzed the expression patterns of CME during MΦ polarization and show that M1 up-regulate the histone methyltransferase MLL and demethylase KDM6B, while resting and M2 MΦ were characterized by DNA methyltransferases and histone deacetylases. We demonstrate that MLL regulates CXCL10 expression and that this effect could be abrogated using a MLL-Menin inhibitor. Taken together we describe the distinct phenotypic differences of GM-CSF or M-CSF MΦ and demonstrate that MΦ polarization is regulated by specific epigenetic mechanisms. In addition, we describe a novel role for MLL as marker for classical activation. Our findings provide new insights into MΦ polarization that could be helpful to distinguish MΦ activation states.

Cloning and expression analysis of a novel G-protein-coupled receptor selectively expressed on granulocytes

The migration of neutrophils into sites of acute and chronicinflammation is mediated by chemokines. We used degenerate‐primerreverse transcriptase‐polymerase chain reaction (RT‐PCR) to analyzechemokine receptor expression in neutrophils and identify novelreceptors. RNA was isolated from human peripheral blood neutrophils andfrom neutrophils that had been stimulated for 5 h withgranulocyte‐macrophage colony‐stimulating factor or by coculturing withprimary human bronchial epithelial cells. Amplification products werecloned, and clone redundancy was determined. Seven knownG‐protein‐coupled receptors were identified among 38 clones—CCR1,CCR4, CXCR1, CXCR2, CXCR4, HM63, and FPR1—as well as a novel gene,EX33. The full‐length EX33 clone was obtained, and an insilico approach was used to identify the putative murine homologue. TheEX33 gene encodes a 396‐amino‐acid protein with limitedsequence identity to known receptors. Expression studies of severalknown chemokine receptors and EX33 revealed that resting neutrophilsexpressed higher levels of CXCRs and EX33 compared with activatedneutrophils. Northern blot experiments revealed that EX33 is expressedmainly in bone marrow, lung, and peripheral blood leukocytes. UsingRT‐PCR analysis, we showed more abundant expression of EX33 inneutrophils and eosinophils, in comparison with that in T‐ or B‐lymphocytes, indicating cell‐specific expression amongleukocytes.

An inhibitor of NADPH oxidase-4 attenuates established pulmonary fibrosis in a rodent disease model.

Idiopathic pulmonary fibrosis is a chronic progressive disease of increasing prevalence for which there is no effective therapy. Increased oxidative stress associated with an oxidant-antioxidant imbalance is thought to contribute to disease progression. NADPH oxidases (Nox) are a primary source of reactive oxygen species within the lung and cardiovascular system. We demonstrate that the Nox4 isoform is up-regulated in the lungs of patients with IPF and in a rodent model of bleomycin-induced pulmonary fibrosis and vascular remodeling. Nox4 is constitutively active, and therefore increased expression levels are likely to contribute to disease pathology. Using a small molecule Nox4/Nox1 inhibitor, we demonstrate that targeting Nox4 results in attenuation of an established fibrotic response, with reductions in gene transcripts for the extracellular matrix components collagen 1α1, collagen 3α1, and fibronectin and in principle pathway components associated with pulmonary fibrosis and hypoxia-mediated vascular remodeling: transforming growth factor (TGF)-β1, plasminogen activator inhibitor-1, hypoxia-inducible factor, and Nox4. TGF-β1 is a principle fibrotic mediator responsible for inducing up-regulation of profibrotic pathways associated with disease pathology. Using normal human lung-derived primary fibroblasts, we demonstrate that inhibition of Nox4 activity using a small molecule antagonist attenuates TGF-β1-mediated up-regulation in expression of profibrotic genes and inhibits the differentiation of fibroblast to myofibroblasts, that is associated with up-regulation in smooth muscle actin and acquisition of a contractile phenotype. These studies support the view that targeting Nox4 may provide a therapeutic approach for attenuating pulmonary fibrosis.