Peter J. Wermuth

Induction of the expression of profibrotic cytokines and growth factors in normal human peripheral blood monocytes by gadolinium contrast agents

OBJECTIVE Nephrogenic systemic fibrosis (NSF) is a severe fibrosing disorder occurring in patients with renal insufficiency. The majority of patients with this disorder have documented exposure to magnetic resonance imaging contrast agents containing Gd. The purpose of this study was to examine the effects of gadolinium diethylenetriaminepentaacetic acid bismethylamide (Gd[DTPA-BMA]; Omniscan) as compared with Gd-DTPA and GdCl3 on the expression and production of cytokines and growth factors by normal human peripheral blood monocytes in vitro and to examine whether conditioned media from Gd-exposed peripheral blood monocytes could induce a profibrotic phenotype in dermal fibroblasts. METHODS Normal human peripheral blood monocytes isolated by Ficoll-Hypaque gradient centrifugation and plastic adherence were incubated with various concentrations of Gd[DTPA-BMA], Gd-DTPA, or GdCl3. Gene expression of interleukins 4, 6, and 13, interferon-gamma, tumor necrosis factor alpha, transforming growth factor beta, connective tissue growth factor, and vascular endothelial growth factor were assessed by real-time polymerase chain reaction (PCR) analysis. Production and secretion of cytokines and growth factors by Gd compound-exposed monocytes was quantified by enzyme-linked immunosorbent assay proteome multiplex arrays. The effects of conditioned media from the Gd compound-exposed monocytes on the phenotype of normal human dermal fibroblasts were examined by real-time PCR and Western blotting. RESULTS The 3 Gd-containing compounds stimulated the expression and production of numerous cytokines and growth factors by normal human peripheral blood monocytes. Conditioned media from these cells induced a profibrotic phenotype in normal human dermal fibroblasts. CONCLUSION The 3 Gd-containing compounds studied induce potent cellular responses in normal human peripheral blood monocytes, which may participate in the development of tissue fibrosis in NSF.

The significance of macrophage polarization subtypes for animal models of tissue fibrosis and human fibrotic diseases.

The systemic and organ-specific human fibrotic disorders collectively represent one of the most serious health problems world-wide causing a large proportion of the total world population mortality. The molecular pathways involved in their pathogenesis are complex and despite intensive investigations have not been fully elucidated. Whereas chronic inflammatory cell infiltration is universally present in fibrotic lesions, the central role of monocytes and macrophages as regulators of inflammation and fibrosis has only recently become apparent. However, the precise mechanisms involved in the contribution of monocytes/macrophages to the initiation, establishment, or progression of the fibrotic process remain largely unknown. Several monocyte and macrophage subpopulations have been identified, with certain phenotypes promoting inflammation whereas others display profibrotic effects. Given the unmet need for effective treatments for fibroproliferative diseases and the crucial regulatory role of monocyte/macrophage subpopulations in fibrogenesis, the development of therapeutic strategies that target specific monocyte/macrophage subpopulations has become increasingly attractive. We will provide here an overview of the current understanding of the role of monocyte/macrophage phenotype subpopulations in animal models of tissue fibrosis and in various systemic and organ-specific human fibrotic diseases. Furthermore, we will discuss recent approaches to the design of effective anti-fibrotic therapeutic interventions by targeting the phenotypic differences identified between the various monocyte and macrophage subpopulations.

Gadolinium compounds signaling through TLR4 and TLR7 in normal human macrophages: establishment of a proinflammatory phenotype and implications for the pathogenesis of nephrogenic systemic fibrosis.

Nephrogenic systemic sibrosis is a progressive disorder occurring in some renal insufficiency patients exposed to gadolinium-based contrast agents (GdBCA). Previous studies demonstrated that the GdBCA Omniscan upregulated several innate immunity pathways in normal differentiated human macrophages, induced rapid nuclear localization of the transcription factor NF-κB, and increased the expression and production of numerous profibrotic/proinflammatory cytokines, chemokines, and growth factors. To further examine GdBCA stimulation of the innate immune system, cultured human embryonic kidney 293 cells expressing one of seven different human TLRs or one of two human nucleotide-binding oligomerization domain-like receptors were exposed in vitro for 24 h to various GdBCA. The signaling activity of each compound was evaluated by its ability to activate an NF-κB–inducible reporter gene. Omniscan and gadodiamide induced strong TLR4- and TLR7-mediated reporter gene activation. The other Gd compounds examined failed to induce reporter gene activation. TLR pathway inhibition using chloroquine or an inhibitor of IL-1R–associated kinases 1 and 4 in normal differentiated human macrophages abrogated Omniscan-induced gene expression. Omniscan and gadodiamide signaling via TLRs 4 and 7 resulted in increased production and expression of numerous proinflammatory/profibrotic cytokines, chemokines, and growth factors, including CXCL10, CCL2, CCL8, CXCL12, IL-4, IL-6, TGF-β, and vascular endothelial growth factor. These observations suggest that TLR activation by environmental stimuli may participate in the pathogenesis of nephrogenic systemic fibrosis and of other fibrotic disorders including systemic sclerosis.

NFκB activation and stimulation of chemokine production in normal human macrophages by the gadolinium-based magnetic resonance contrast agent Omniscan: possible role in the pathogenesis of nephrogenic systemic fibrosis

Objective Nephrogenic systemic fibrosis (NSF) is a generalised fibrotic disorder occurring in certain individuals with renal insufficiency exposed to gadolinium-based contrast agents (GdBCA) for MRI. Histopathological examination of affected tissues shows increased numbers of activated macrophages. To elucidate the mechanisms responsible for macrophage activation, the effects of the GdBCA Omniscan on normal human macrophage global gene expression, chemokine production and nuclear factor κB (NFκB) activation was examined. Methods Normal human monocyte-derived macrophages were incubated with Omniscan (50 mM) and their gene expression analysed by microarrays and real-time PCR. Macrophage chemokine production was assayed by multiplex ELISA. NFκB activation was assessed by NFκB nuclear localisation and quantitation of intracellular levels of inducible nitric oxide synthase (iNOS) protein. A specific cell-permeable NFκB peptide inhibitor was used to abrogate NFκB stimulation of chemokine and iNOS protein levels. CCL8/MCP-2 in affected skin of patients with NSF was examined by indirect immunofluorescence. Results Omniscan caused a profound change in the transcriptome of differentiated human normal macrophages in vitro, including a large increase in the expression of genes encoding CC and CXC chemokines. It induced rapid nuclear localisation of NFκB and stimulation of iNOS protein levels and chemokine production which were blocked by an NFκB inhibitory peptide. CCL8/MCP-2, the most upregulated chemokine following in vitro macrophage exposure to Omniscan, was strongly increased in NSF-affected skin. Conclusion The GdBCA Omniscan induces potent stimulation of macrophage gene expression, NFκB activation and increased NFκB-mediated production of CC and CXC chemokines and iNOS. These alterations may play a crucial role in the pathogenesis of NSF.

Persistent activation of dermal fibroblasts from patients with gadolinium-associated nephrogenic systemic fibrosis

Background Nephrogenic systemic fibrosis (NSF) is a systemic fibrotic disorder occurring in some patients with renal insufficiency after exposure to gadolinium-based contrast agents (GdBCA). Objectives To examine cultured NSF dermal fibroblast production and expression of collagens I and III, fibronectin, hyaluronic acid and α-smooth muscle actin (α-SMA) during serial passages and the effects of two GdBCA on collagen gene expression and production by normal dermal fibroblasts. Methods NSF fibroblasts were analysed for expression and production of types I and III collagen, fibronectin, hyaluronic acid and α-SMA. Collagen, type I, α1 (COL1A1) promoter transcription was examined in transient transfections. Nuclear extracts were assayed for binding activity of 108 transcription factors, and specific transcription factor binding was examined by electrophoretic gel mobility assays. Normal fibroblasts were cultured with GdBCA, and collagen expression assessed by real-time PCR and western blots. Results NSF fibroblasts displayed a marked increase in collagens I and III, fibronectin and hyaluronic acid production, which was maintained for 9–11 subpassages in vitro. NSF fibroblasts also showed a marked increase in α-SMA expression, twofold higher transcriptional activity of the COL1A1 promoter and increased cREL binding in nuclear extracts compared with normal fibroblasts. GdBCA induced a dose-dependent stimulation of COL1A1 expression and production of type I collagen in normal fibroblasts. Conclusions Fibroblasts from patients with NSF displayed a markedly profibrotic phenotype, which was maintained for several passages in culture. Elevated COL1A1 expression was mediated by transcriptional activation of its promoter associated with increased cREL binding activity. GdBCA stimulated cultured normal fibroblasts to produce increased amounts of collagen.

Stimulation of Transforming Growth Factor-β1-Induced Endothelial-To-Mesenchymal Transition and Tissue Fibrosis by Endothelin-1 (ET-1): A Novel Profibrotic Effect of ET-1.

TGF-β-induced endothelial-to-mesenchymal transition (EndoMT) is a newly recognized source of profibrotic activated myofibroblasts and has been suggested to play a role in the pathogenesis of various fibrotic processes. Endothelin-1 (ET-1) has been implicated in the development of tissue fibrosis but its participation in TGF-β-induced EndoMT has not been studied. Here we evaluated the role of ET-1 on TGF-β1-induced EndoMT in immunopurified CD31+/CD102+ murine lung microvascular endothelial cells. The expression levels of α-smooth muscle actin (α-SMA), of relevant profibrotic genes, and of various transcription factors involved in the EndoMT process were assessed employing quantitative RT-PCR, immunofluorescence histology and Western blot analysis. TGF-β1 caused potent induction of EndoMT whereas ET-1 alone had a minimal effect. However, ET-1 potentiated TGF-β1-induced EndoMT and TGF-β1-stimulated expression of mesenchymal cell specific and profibrotic genes and proteins. ET-1 also induced expression of the TGF-β receptor 1 and 2 genes, suggesting a plausible autocrine mechanism to potentiate TGF-β-mediated EndoMT and fibrosis. Stimulation of TGF-β1-induced skin and lung fibrosis by ET-1 was confirmed in vivo in an animal model of TGF-β1-induced tissue fibrosis. These results suggest a novel role for ET-1 in the establishment and progression of tissue fibrosis.

Induction of a type I interferon signature in normal human monocytes by gadolinium-based contrast agents: comparison of linear and macrocyclic agents

The gadolinium‐based contrast agent (GdBCA) Omniscan activates human macrophages through Toll‐like receptor (TLR)‐4 and TLR‐7 signalling. To explore the mechanisms responsible we compared the ability of linear and macrocyclic GdBCA to induce a type I interferon signature and a proinflammatory/profibrotic phenotype in normal human monocytes in vitro. Expression of genes associated with type I interferon signalling and inflammation and production of their corresponding proteins were determined. Both linear and macrocyclic GdBCA stimulated expression of multiple type I interferon‐regulated genes and the expression of numerous chemokines, cytokines and growth factors in normal human peripheral blood monocytes. There was no correlation between the magnitude of the measured response and the Gd chelate used. To explore the mechanisms responsible for GdBCA induction of fibrosis in nephrogenic systemic fibrosis (NSF) in vitro, normal human dermal fibroblasts were incubated with GdBCA‐treated monocyte culture supernatants and the effects on profibrotic gene expression were examined. Supernatants from monocytes exposed to all GdBCA stimulated types I and III collagen, fibronectin and α‐smooth muscle actin (α‐SMA) expression in normal dermal fibroblasts. The results indicate that the monocyte activation induced by GdBCA may be the initial step in the development of GdBCA associated fibrosis in NSF.

Effect of protein kinase C delta (PKC-δ) inhibition on the transcriptome of normal and systemic sclerosis human dermal fibroblasts in vitro.

Previous studies demonstrated that protein kinase C- δ (PKC-δ) inhibition with the selective inhibitor, rottlerin, resulted in potent downregulation of type I collagen expression and production in normal human dermal fibroblasts and abrogated the exaggerated type I collagen production and expression in fibroblasts cultured from affected skin from patients with the fibrosing disorder systemic sclerosis (SSc). To elucidate the mechanisms involved in the ability of PKC-δ to regulate collagen production in fibroblasts, we examined the effects of PKC-δ inhibition on the transcriptome of normal and SSc human dermal fibroblasts. Normal and SSc human dermal fibroblasts were incubated with rottlerin (5 µM), and their gene expression was analyzed by microarrays. Pathway analysis and gene ontology analysis of differentially expressed genes in each comparison were performed. Identification of significantly overrepresented transcriptional regulatory elements (TREs) was performed using the Promoter Analysis and Interaction Network Toolset (PAINT) program. PKC-δ activity was also inhibited using RNA interference (siRNA) and by treating fibroblasts with a specific PKC-δ inhibitory cell permeable peptide. Differential gene expression of 20 genes was confirmed using real time PCR. PKC-δ inhibition caused a profound change in the transcriptome of normal and SSc human dermal fibroblasts in vitro. Pathway and gene ontology analysis identified multiple cellular and organismal pathways affected by PKC-δ inhibition. Furthermore, both pathway and PAINT analyses indicated that the transcription factor NFκB played an important role in the transcriptome changes induced by PKC-δ inhibition. Multiple genes involved in the degradation of the extracellular matrix components were significantly reduced in SSc fibroblasts and their expression was increased by PKC-δ inhibition. These results indicate that isoform-specific inhibition of PKC-δ profibrotic effects may represent a novel therapeutic approach for SSc and other fibrotic diseases.

Prevalence of mental disorders among detained asylum seekers in deportation arrest in Switzerland and validation of the Brief Jail Mental Health Screen BJMHS

BACKGROUND Though slowly growing, knowledge about prisoners detained for having violated an Alien Act is still marginal and most studies involve detained asylum seekers in the USA and Australia. Little is known about prevalence rates of mental health disorders in such a population. The Brief Jail Mental Health Screening BJMHS has been demonstrated in other prison populations as a valid screening for serious mental illness. AIM The aims of this study were to describe prevalence rates for mental disorders according to ICD-10 and to validate the BJMHS for this population. METHODS 80 inmates at a detention center for prisoners having violated the Swiss Aliens Act were surveyed using the BJMHS at their admission. The results were cross validated with the WHO Composite International Diagnostic Interview (CIDI). RESULTS When omitting disorders caused by smoking tobacco, 76% of the prisoners suffered from at least one mental disorder according to CIDI. Whereas the rates for disorders due to psychoactive substance use as well as schizophrenic and affective disorders were comparable with other prison populations, we found a specific increased reporting of phobic (14%) and post-traumatic stress disorders (23%). The BJMHS detected serious mental illness defined as schizophrenic or affective disorders with a sensitivity of 81.0% and a specificity of 74.6%. DISCUSSION As in other prison populations prevalence rates for mental disorders were markedly above the general population. The specific pattern with high rates of phobic as well as post-traumatic stress disorders may reflect the very often traumatic backgrounds of this population. Whereas the results for the validation of the BJMHS were even better than in other similar studies and the instrument proved to be practicable and helpful to detect serious mental illness, sensitivity for a screening tool of around 80% is still too low. Additionally the fact that other serious mental disorders are not covered emphasizes the importance of other elements in the screening process, including the need to have well-trained staff, and to have a low threshold for psychiatric examination.

Endothelial cell-specific activation of transforming growth factor- β signaling in mice induces cutaneous, visceral, and microvascular fibrosis

In this study, we tested the hypothesis that constitutive endothelial cell-specific activation of TGF-β signaling induces tissue fibrosis and vasculopathy resembling the characteristic fibrotic and vascular alterations of systemic sclerosis. Transgenic mice with inducible expression of a constitutively active TGF-β receptor I specifically in endothelial cells were generated by intercrossing mice harboring a constitutively active TGF-β receptor I with a mouse strain containing the endothelial cell-specific Cdh5 gene promoter directing the tamoxifen-inducible expression of the Cre-ERT2 cassette. Administration of tamoxifen to these mice would result in constitutive TGF-β activation and signaling confined to endothelial lineage cells. The effects of constitutive TGF-β endothelial cell activation were assessed by histopathological examination of skin and various internal organs, tissue hydroxyproline analysis, and assessment of expression of myofibroblast differentiation and TGF-β signaling genes employing real-time PCR and immunohistochemical staining of lung vessels for endothelial- and myofibroblast-specific proteins. Constitutive TGFβ-1 signaling in endothelial cells resulted in cutaneous and visceral fibrosis with prominent fibrotic involvement of the lungs and severe perivascular and subendothelial fibrosis of small arterioles. A marked increase in the expression of fibrosis-associated genes and of genes indicative of myofibroblast activation was also found. Confocal microscopy of lung vessels showed evidence consistent with the induction of endothelial-to-mesenchymal transition (EndoMT). Taken together, our data indicate that transgenic mice with constitutive endothelial cell-specific activation of TGF-β signaling display severe cutaneous, pulmonary, and microvascular fibrosis resembling the fibrotic and microvascular alterations characteristic of systemic sclerosis.