Maximilian B. MacPherson

Assessing nanotoxicity in cells in vitro

Nanomaterials are commonly defined as particles or fibers of less than 1 microm in diameter. For these reasons, they may be respirable in humans and have the potential, based upon their geometry, composition, size, and transport or durability in the body, to cause adverse effects on human health, especially if they are inhaled at high concentrations. Rodent inhalation models to predict the toxicity and pathogenicity of nanomaterials are prohibitive in terms of time and expense. For these reasons, a panel of in vitro assays is described below. These include cell culture assays for cytotoxicity (altered metabolism, decreased growth, lytic or apoptotic cell death), proliferation, genotoxicity, and altered gene expression. The choice of cell type for these assays may be dictated by the procedure or endpoint selected. Most of these assays have been standardized in our laboratory using pathogenic minerals (asbestos and silica) and non-pathogenic particles (fine titanium dioxide or glass beads) as negative controls. The results of these in vitro assays should predict whether testing of selected nanomaterials should be pursued in animal inhalation models that simulate physiologic exposure to inhaled nanomaterials. Conversely, intrathoracic or intrapleural injection of nanomaterials into rodents can be misleading because they bypass normal clearance mechanisms, and non-pathogenic fibers and particles can test positively in these assays.

Asbestos and erionite prime and activate the NLRP3 inflammasome that stimulates autocrine cytokine release in human mesothelial cells

BackgroundPleural fibrosis and malignant mesotheliomas (MM) occur after exposures to pathogenic fibers, yet the mechanisms initiating these diseases are unclear.ResultsWe document priming and activation of the NLRP3 inflammasome in human mesothelial cells by asbestos and erionite that is causally related to release of IL-1β, IL-6, IL-8, and Vascular Endothelial Growth Factor (VEGF). Transcription and release of these proteins are inhibited in vitro using Anakinra, an IL-1 receptor antagonist that reduces these cytokines in a human peritoneal MM mouse xenograft model.ConclusionsThese novel data show that asbestos-induced priming and activation of the NLRP3 inflammasome triggers an autocrine feedback loop modulated via the IL-1 receptor in mesothelial cell type targeted in pleural infection, fibrosis, and carcinogenesis.

Inflammation precedes the development of human malignant mesotheliomas in a SCID mouse xenograft model

Asbestos fibers cause chronic inflammation that may be critical to the development of malignant mesothelioma (MM). Two human MM cell lines (Hmeso, PPM Mill) were used in a SCID mouse xenograft model to assess time‐dependent patterns of inflammation and tumor formation. After intraperitoneal (IP) injection of MM cells, mice were euthanized at 7, 14, and 30 days, and peritoneal lavage fluid (PLF) was examined for immune cell profiles and human and mouse cytokines. Increases in human MM‐derived IL‐6, IL‐8, bFGF, and VEGF were observed in mice at 7 days postinjection of either MM line, and a striking neutrophilia was observed at all time points. Free‐floating tumor spheroids developed in mice at 14 days, and both spheroids and adherent MM tumor masses occurred in all mice at 30 days. Results suggest that inflammation and cytokine production precede and may be critical to the development of MMs.

Alterations in Gene Expression in Human Mesothelial Cells Correlate with Mineral Pathogenicity

Human mesothelial cells (LP9/TERT-1) were exposed to low and high (15 and 75 microm(2)/cm(2) dish) equal surface area concentrations of crocidolite asbestos, nonfibrous talc, fine titanium dioxide (TiO2), or glass beads for 8 or 24 hours. RNA was then isolated for Affymetrix microarrays, GeneSifter analysis and QRT-PCR. Gene changes by asbestos were concentration- and time-dependent. At low nontoxic concentrations, asbestos caused significant changes in mRNA expression of 29 genes at 8 hours and of 205 genes at 24 hours, whereas changes in mRNA levels of 236 genes occurred in cells exposed to high concentrations of asbestos for 8 hours. Human primary pleural mesothelial cells also showed the same patterns of increased gene expression by asbestos. Nonfibrous talc at low concentrations in LP9/TERT-1 mesothelial cells caused increased expression of 1 gene Activating Transcription Factor 3 (ATF3) at 8 hours and no changes at 24 hours, whereas expression levels of 30 genes were elevated at 8 hours at high talc concentrations. Fine TiO2 or glass beads caused no changes in gene expression. In human ovarian epithelial (IOSE) cells, asbestos at high concentrations elevated expression of two genes (NR4A2, MIP2) at 8 hours and 16 genes at 24 hours that were distinct from those elevated in mesothelial cells. Since ATF3 was the most highly expressed gene by asbestos, its functional importance in cytokine production by LP9/TERT-1 cells was assessed using siRNA approaches. Results reveal that ATF3 modulates production of inflammatory cytokines (IL-1 beta, IL-13, G-CSF) and growth factors (VEGF and PDGF-BB) in human mesothelial cells.

Blocking of ERK1 and ERK2 sensitizes human mesothelioma cells to doxorubicin

BackgroundMalignant mesotheliomas (MM) have a poor prognosis, largely because of their chemoresistance to anti-cancer drugs such as doxorubicin (Dox). Here we show using human MM lines that Dox activates extracellular signal-regulated kinases (ERK1 and 2), causally linked to increased expression of ABC transporter genes, decreased accumulation of Dox, and enhanced MM growth. Using the MEK1/2 inhibitor, U0126 and stably transfected shERK1 and shERK2 MM cell lines, we show that inhibition of both ERK1 and 2 sensitizes MM cells to Dox.ResultsU0126 significantly modulated endogenous expression of several important drug resistance (BCL2, ABCB1, ABCC3), prosurvival (BCL2), DNA repair (BRCA1, BRCA2), hormone receptor (AR, ESR2, PPARγ) and drug metabolism (CYP3A4) genes newly identified in MM cells. In comparison to shControl lines, MM cell lines stably transfected with shERK1 or shERK2 exhibited significant increases in intracellular accumulation of Dox and decreases in cell viability. Affymetrix microarray analysis on stable shERK1 and shERK2 MM lines showed more than 2-fold inhibition (p ≤ 0.05) of expression of ATP binding cassette genes (ABCG1, ABCA5, ABCA2, MDR/TAP, ABCA1, ABCA8, ABCC2) in comparison to shControl lines. Moreover, injection of human MM lines into SCID mice showed that stable shERK1 or shERK2 lines had significantly slower tumor growth rates in comparison to shControl lines after Dox treatment.ConclusionsThese studies suggest that blocking ERK1 and 2, which play critical roles in multi-drug resistance and survival, may be beneficial in combination with chemotherapeutic drugs in the treatment of MMs and other tumors.

Osteopontin Modulates Inflammation, Mucin Production, and Gene Expression Signatures After Inhalation of Asbestos in a Murine Model of Fibrosis

Inflammation and lung remodeling are hallmarks of asbestos-induced fibrosis, but the molecular mechanisms that control these events are unclear. Using laser capture microdissection (LCM) of distal bronchioles in a murine asbestos inhalation model, we show that osteopontin (OPN) is up-regulated by bronchiolar epithelial cells after chrysotile asbestos exposures. In contrast to OPN wild-type mice (OPN(+/+)) inhaling asbestos, OPN null mice (OPN(-/-)) exposed to asbestos showed less eosinophilia in bronchoalveolar lavage fluids, diminished lung inflammation, and decreased mucin production. Bronchoalveolar lavage fluid concentrations of inflammatory cytokines (IL-1β, IL-4, IL-6, IL-12 subunit p40, MIP1α, MIP1β, and eotaxin) also were significantly less in asbestos-exposed OPN(-/-) mice. Microarrays performed on lung tissues from asbestos-exposed OPN(+/+) and OPN(-/-) mice showed that OPN modulated the expression of a number of genes (Col1a2, Timp1, Tnc, Eln, and Col3a1) linked to fibrosis via initiation and cross talk between IL-1β and epidermal growth factor receptor-related signaling pathways. Novel targets of OPN identified include genes involved in cell signaling, immune system/defense, extracellular matrix remodeling, and cell cycle regulation. Although it is unclear whether the present findings are specific to chrysotile asbestos or would be observed after inhalation of other fibers in general, these results highlight new potential mechanisms and therapeutic targets for asbestosis and other diseases (asthma, smoking-related interstitial lung diseases) linked to OPN overexpression.

Airway Epithelial NF-κB Activation Modulates Asbestos-Induced Inflammation and Mucin Production In Vivo

To investigate the role of bronchiolar epithelial NF-κB activity in the development of inflammation and fibrogenesis in a murine model of asbestos inhalation, we used transgenic (Tg) mice expressing an IκBα mutant (IκBαsr) resistant to phosphorylation-induced degradation and targeted to bronchial epithelium using the CC10 promoter. Sham and chrysotile asbestos-exposed CC10-IκBαsr Tg+ and Tg− mice were examined for altered epithelial cell proliferation and differentiation, cytokine profiles, lung inflammation, and fibrogenesis at 3, 9, and 40 days. KC, IL-6 and IL-1β were increased (p ≤ 0.05) in bronchoalveolar lavage fluid (BALF) from asbestos-exposed mice, but to a lesser extent (p ≤ 0.05) in Tg+ vs Tg− mice. Asbestos also caused increases in IL-4, MIP-1β, and MCP-1 in BALF that were more elevated (p ≤ 0.05) in Tg+ mice at 9 days. Differential cell counts revealed eosinophils in BALF that increased (p ≤ 0.05) in Tg+ mice at 9 days, a time point corresponding with significantly increased numbers of bronchiolar epithelial cells staining positively for mucus production. At all time points, asbestos caused increased numbers of distal bronchiolar epithelial cells and peribronchiolar cells incorporating the proliferation marker, Ki-67. However, bronchiolar epithelial cell and interstitial cell labeling was diminished at 40 days (p ≤ 0.05) in Tg+ vs Tg− mice. Our findings demonstrate that airway epithelial NF-κB activity plays a role in orchestrating the inflammatory response as well as cell proliferation in response to asbestos.

Activated cAMP Response Element Binding Protein Is Overexpressed in Human Mesotheliomas and Inhibits Apoptosis

Little is known about the cellular mechanisms contributing to the development and chemoresistance of malignant mesothelioma (MM), an aggressive asbestos-associated tumor. A human mesothelial cell line (LP9/TERT-1) and isolated human pleural mesothelial cells showed rapid and protracted asbestos-induced cAMP response element binding protein (CREB1) phosphorylation, which was inhibited in LP9/TERT-1 cells by small molecule inhibitors of epidermal growth factor receptor phosphorylation and protein kinase A. Asbestos increased expression of several CREB target genes (c-FOS, EGR-1, MKP1, BCL2, and MMP13) and apoptosis, which was enhanced using small interfering CREB. Human MM tissue arrays showed elevated endogenous levels of phosphorylated nuclear CREB1 as compared with reactive mesothelial hyperplasias and normal lung tissue. Significantly increased phosphorylated CREB1 and mRNA levels of BCL2, c-FOS, MMP9, and MMP13 were also observed in MM cells in vitro, which were further augmented after addition of Doxorubicin (Dox). Small interfering CREB inhibited migration of MMs, increased apoptosis by Dox, and decreased BCL2 and BCL-xL expression, suggesting a role for these molecules in CREB-induced MM survival. These data indicate that CREB1 and its target genes are up-regulated in asbestos-exposed human mesothelial cells through an epidermal growth factor receptor/protein kinase A pathway. Since activated CREB1 also is increased endogenously in human MM and modifies migration and resistance to Dox-induced apoptosis, inhibition of CREB1 may be a new strategy for MM therapy.

Asbestos modulates thioredoxin-thioredoxin interacting protein interaction to regulate inflammasome activation

BackgroundAsbestos exposure is related to various diseases including asbestosis and malignant mesothelioma (MM). Among the pathogenic mechanisms proposed by which asbestos can cause diseases involving epithelial and mesothelial cells, the most widely accepted one is the generation of reactive oxygen species and/or depletion of antioxidants like glutathione. It has also been demonstrated that asbestos can induce inflammation, perhaps due to activation of inflammasomes.MethodsThe oxidation state of thioredoxin was analyzed by redox Western blot analysis and ROS generation was assessed spectrophotometrically as a read-out of solubilized formazan produced by the reduction of nitrotetrazolium blue (NTB) by superoxide. Quantitative real time PCR was used to assess changes in gene transcription.ResultsHere we demonstrate that crocidolite asbestos fibers oxidize the pool of the antioxidant, Thioredoxin-1 (Trx1), which results in release of Thioredoxin Interacting Protein (TXNIP) and subsequent activation of inflammasomes in human mesothelial cells. Exposure to crocidolite asbestos resulted in the depletion of reduced Trx1 in human peritoneal mesothelial (LP9/hTERT) cells. Pretreatment with the antioxidant dehydroascorbic acid (a reactive oxygen species (ROS) scavenger) reduced the level of crocidolite asbestos-induced Trx1 oxidation as well as the depletion of reduced Trx1. Increasing Trx1 expression levels using a Trx1 over-expression vector, reduced the extent of Trx1 oxidation and generation of ROS by crocidolite asbestos, and increased cell survival. In addition, knockdown of TXNIP expression by siRNA attenuated crocidolite asbestos-induced activation of the inflammasome.ConclusionOur novel findings suggest that extensive Trx1 oxidation and TXNIP dissociation may be one of the mechanisms by which crocidolite asbestos activates the inflammasome and helps in development of MM.

ERK2 is essential for the growth of human epithelioid malignant mesotheliomas

Members of the extracellular signal‐regulated kinase (ERK) family may have distinct roles in the development of cell injury and repair, differentiation and carcinogenesis. Here, we show, using a synthetic small‐molecule MEK1/2 inhibitor (U0126) and RNA silencing of ERK1 and 2, comparatively, that ERK2 is critical to transformation and homeostasis of human epithelioid malignant mesotheliomas (MMs), asbestos‐induced tumors with a poor prognosis. Although MM cell (HMESO) lines stably transfected with shERK1 or shERK2 both exhibited significant decreases in cell proliferation in vitro, injection of shERK2 cells, and not shERK1 cells, into immunocompromised severe combined immunodeficiency (SCID) mice showed significant attenuated tumor growth in comparison to shControl (shCon) cells. Inhibition of migration, invasion and colony formation occurred in shERK2 MM cells in vitro, suggesting multiple roles of ERK2 in neoplasia. Microarray and quantitative real‐time PCR analyses revealed gene expression that was significantly increased (CASP1, TRAF1 and FAS) or decreased (SEMA3E, RPS6KA2, EGF and BCL2L1) in shERK2‐transfected MM cells in contrast to shCon‐transfected MM cells. Most striking decreases were observed in mRNA levels of Semaphorin 3 (SEMA3E), a candidate tumor suppressor gene linked to inhibition of angiogenesis. These studies demonstrate a key role of ERK2 in novel gene expression critical to the development of epithelioid MMs. After injection of sarcomatoid human MM (PPMMill) cells into SCID mice, both shERK1 and shERK2 lines showed significant decreased tumor growth, suggesting heterogeneous effects of ERKs in individual MMs.