ABSTRACT
SHIPKOWSKI, KELLY ANNE. Innate Immune Responses to Engineered Nanomaterials During Allergic Airway Inflammation. (Under the direction of Dr. James C. Bonner).
The field of nanotechnology is continually advancing, and increasing amounts of consumer goods are being produced using engineered nanomaterials (ENMs). The health risks of occupational and/or consumer exposure to ENMs are not completely understood, although significant research indicates that pulmonary exposure to nanomaterials induces toxic effects in the lungs of exposed animals. Multi-walled carbon nanotubes (MWCNTs) are a specific category of ENMs and consist of sheets of graphene rolled into cylinders that are multiple layers thick in order to strengthen their rigidity. MWCNTs have a fiber-like shape, similar to that of asbestos, which allows for a high aspect ratio and makes them difficult to clear from the lung. Studies with rodent models have demonstrated that pulmonary exposure to ENMs, in particular MWCNTs, results in acute lung inflammation and the subsequent development of chronic fibrosis, suggesting a potential human health risk to individuals involved in the manufacturing of products utilizing these nanomaterials. Induction of IL-1β secretion via activation of the inflammasome is a prime mechanism of MWCNT-induced inflammation. The inflammasome is a multi-protein scaffold found in a variety of cell types that forms in response to a variety of immune signals, including particulates.
an exceptionally prominent human disease, and therefore the goal of this research was to better understand how pre-existing allergic airway disease would modulate the innate immune response to MWCNTs. We hypothesized that Th2 cytokines and the allergic asthmatic microenvironment would alter MWCNT-induced inflammasome activation and IL-1β secretion both in vitro and in vivo.
In vitro, THP-1 cells, a human monocytic cell line, were differentiated into
macrophages and exposed to MWCNTs and or recombinant Th2 cytokines, specifically IL-4 and/or IL-13. Exposure of THP-1 cells to MWCNTs alone caused dose-dependent secretion of IL-1β, while co-exposure to IL-4 and/or IL-13 suppressed MWCNT-induced IL-1β. Further analysis determined that IL-4 and IL-13 were phosphorylating the protein signal transducer and activator of transcription 6 (STAT6) and subsequently inhibiting inflammasome activation and function through suppression of caspase-1, a cysteine protease responsible for cleavage of pro-IL-1β into an active, secretable form.
In vivo, wild-type C57BL6 mice were sensitized intranasally with HDM allergen and
upregulated STAT3 mRNA expression in the lungs, liver, and spleen of exposed animals, and at the same induced mixed T helper (Th) responses in the different tissues.
Collectively, these data suggest that the allergic microenvironment induced during asthma can modulate the innate inflammatory response to MWCNTs through inhibition of caspase-1 and inflammasome activation in the lung and through alteration of the transcription factors involved in the T helper immune responses systemically.
Innate Immune Responses to Engineered Nanomaterials During Allergic Airway Inflammation
by
Kelly Anne Shipkowski
A dissertation submitted to the Graduate Faculty of North Carolina State University
in partial fulfillment of the requirements for the Degree of
Doctor of Philosophy
Toxicology
Raleigh, North Carolina 2015
APPROVED BY:
_______________________________ _______________________________
James C. Bonner Robert C. Smart
Committee Chair
DEDICATION
I would like to dedicate this dissertation to my parents, who have continually been my source
of support and pride. I am eternally grateful for every moment that they reminded me that I
BIOGRAPHY
Kelly Anne Shipkowski was born on November 18, 1988, in Newport News, VA, and became interested in science at the ripe age of 2 after catching her first firefly. After getting a jumpstart on education by starting kindergarten a year early, Kelly and her family moved to Cary, North Carolina in time for her to start second grade. As a child, Kelly was always curious and constantly asking questions about how things worked, and her parents were never exasperated with her incessant queries regarding why turtles have shells and what makes acorns bounce. While Kelly was always a shy, quiet child, her competitive nature slowly began to appear in elementary school, as demonstrated by her victories in the geography bee, school science fair, and, most importantly, in the class dodgeball competitions.
ACKNOWLEDGMENTS
First and foremost, I would like to thank my mentor, Dr. James Bonner. During my time in his laboratory he has helped me grow and develop not only as a scientist, but as a person as well. Dr. Bonner has taught me anything and everything about nanotoxicology, but has also expanded my knowledge on all aspects of life outside of work. He has continually provided opportunities for the advancement of my work and career while also offering support and encouragement whenever I needed it. I am incredibly lucky to have ended up with a mentor like Dr. Bonner, and will forever be grateful for the experience I have had working in his lab.
I would also like to thank the members of my committee, Dr. Robert Smart, Dr. Jun Ninomiya-Tsuji, and Dr. Philip Sannes, for their support and guidance throughout my time at NC State. I greatly appreciate the time and effort they have put in to help me make my graduate career a successful one. Furthermore, I would like to thank the members of the Bonner lab, both past and present, Dr. Ellen Baker, Dr. Brian Sayers, Dr. Elizabeth Thompson, Alexia Taylor, Katie Duke, Mark Ihrie, and Erinn Dandley. They have been constant sources of support in both life and in the lab, and made my time at NC State all the more delightful. I also want to thank Janet Roe, Jeanne Burr, and Jackie Broughton for not only helping to make sure I filled out all of the right paperwork on time, but also for generously offering a helping hand whenever needed.
many fumbling presentation practices and, at this point, can most likely explain my research better than I can. I am eternally grateful for his support and encouragement.
TABLE OF CONTENTS
LIST OF FIGURES ... vii
GENERAL INTRODUCTION ... 1
GENERAL RESEARCH HYPOTHESIS ... 29
CHAPTER 1: An Allergic Lung Microenvironment Suppresses Carbon Nanotube-Induced Inflammasome Activation via STAT6-Dependent Inhibition of Caspase-1 ... 31
ABSTRACT ... 32
INTRODUCTION ... 34
MATERIALS AND METHODS ... 38
RESULTS ... 48
DISCUSSION ... 54
CONCLUSIONS ... 63
REFERENCES ... 64
FIGURE LEGENDS ... 70
SUPPORTING INFORMATION ... 76
FIGURES ... 78
CHAPTER 2: Exposure to Multi-walled Carbon Nanotubes Causes Unique Immunotoxic Profiles in the Lung, Liver, and Spleen of Mice Sensitized to House Dust Mite Allergen ... 90
ABSTRACT ... 91
INTRODUCTION ... 93
MATERIALS AND METHODS ... 99
RESULTS ... 103
DISCUSSION ... 107
CONCLUSIONS ... 112
REFERENCES ... 113
FIGURES ... 117
GENERAL DISCUSSION ... 123
CHAPTER 1 ... 125
CHAPTER 2 ... 133
GENERAL CONCLUSIONS ... 140
GENERAL REFERENCES ... 141
APPENDICES ... 165
Appendix A ... 166
LIST OF FIGURES
CHAPTER 1: An Allergic Lung Microenvironment Suppresses Carbon Nanotube-Induced Inflammasome Activation via STAT6-Dependent Inhibition of Caspase-1
CHAPTER 2: Exposure to Multi-walled Carbon Nanotubes Causes Distinct Immunotoxic Profiles in the Lung, Liver, and Spleen of Mice Sensitized to House Dust Mite Allergen
Figure 1: Histopathology and Cytoviva enhanced darkfield microscopy of mouse lung tissue following HDM sensitization and MWCNT exposure ... 117 Figure 2: Exposure to MWCNTs following HDM allergen sensitization induces differential expression of the STAT family of transcription factors in the lungs, liver, and spleen of mice ... 118 Figure 3: Sensitization with HDM allergen combined with MWCNT exposure alters the development of T-helper responses in the lungs, liver, and spleen of mice ... 119 Figure 4: Sensitization with HDM allergen induces a liver-specific increase in mRNA expression of cytokines involved in the STAT1 signaling pathway ... 120 Figure 5: Exposure to MWCNTs following allergen challenge induces a mixed Th2 and T regulatory response in the lungs of mice while exaggerating the T regulatory responses in the spleens of mice ... 121 Figure 6: Schematic illustration demonstrating the systemic immunotixc effects of MWCNT exposure following HDM sensitization ... 122
APPENDICES
Appendix A
GENERAL INTRODUCTION Nanotechnology
Nanotechnology, as defined by the National Nanotechnology Initiative (NNI), is simply “science, engineering, and technology conducted at the nanoscale”, which ranges from 1-100 nanometers [1]. The introduction of nanotechnology and the advancement of nanoscale manipulation have continued to revolutionize a variety of industries, including structural engineering, electronics and biosensors, consumer goods, and medicine [2,3]. Consumer goods such as cosmetics, sunscreens, food, and sporting equipment are manufactured utilizing nanotechnology, and the use of nanoscale production has advanced areas of medicine such as imaging technologies and drug delivery [2,3]. Although there are significant benefits to nanotechnology, there are risks of occupational, and possibly consumer, exposure to the nanoscale materials used in the manufacturing of goods via this technology [2,3].
indicates that not enough research is being performed on the safety and potential health risks of manufacturing using nanotechnology.
Engineered Nanomaterials
The majority of industrial nanotechnology applications utilize engineered nanomaterials (ENMs), in particular ultrafine particles having at least one dimension measuring less than 100 nanometers, be it length, width, or diameter [5-7]. ENMs exist in many different sizes, shapes, and compositions, all of which are properties that affect their potential toxicity [8-11]. In addition to ENMs, ultrafine nanoscale particulates commonly originate from sources of combustion as a by-product of industrial applications and contribute to air pollution [12,13].
Spherical ENMs exist in a variety of chemical compositions, such as carbon black (CB), titanium dioxide (TiO2), zinc oxide (ZnO), and silica dioxide (SiO2) [2,3,14]. Carbon
nanotubes (CNTs) are fiber-like ENMs made of concentric sheets of graphene rolled into cylinders, and CNTs can be found in both single-walled (SWCNTs) and multi-walled (MWCNTs) forms [14]. CNTs have a diameter that falls within the nanoscale but can have a length that is significantly longer, within the micron range, very similar to that of asbestos. This high aspect (length to width) ratio makes CNTs toxic by promoting production of reactive oxygen species (ROS), inducing cellular toxicity, and increasing persistence in exposed tissues, in particular the lung [2,3,14,15]. Many ENMs also induce an influx of intracellular Ca++, causing oxidative stress and production of inflammatory cytokines such as
and physical properties that make them ideal for manufacturing and industrial use. These properties include increased strength and durability as well as high conductivity, reactivity, and optical sensitivity [2,3]. ENMs also have an increased surface area per unit mass, making them ideal for thin film coating applications but also increasing their reactivity [2,3,14,15].
Modification of MWCNTs is a common technique in the manufacturing of ENMs, as functionalization can improve the conductive properties that make ENMs useful for imaging technologies and drug delivery techniques [16-18]. Atomic layer deposition (ALD) is a specific form of thin film coating in which chemical vapor deposition (CVD) techniques are used to coat MWCNTs with metals, oxides, and/or hybrid metals/organics in order to improve electronic and mechanical properties and allow the addition of certain biomolecules to the MWCNTs [16,19-22]. ALD coating with aluminum oxide (Al2O3) or titanium oxide
(TiO2) enhances the addition of biomolecules to MWCNTs by increasing their hydrophilic
properties, while coating with zinc oxide (ZnO) or TiO2 improves the catalytic properties of
MWCNTs by enhancing photosensitivity [16,20,21]. Our laboratory utilized ALD coating of MWCNTs to examine how metal oxide modification would alter the toxicity of MWCNTs, and, interestingly, demonstrated that Al2O3 coating of MWCNTs suppressed production of
capable of altering biological response at both the molecular and subcellular level and inducing pulmonary inflammation and fibrosis following inhalation exposure [2,3, 23-26].
Pulmonary Exposure to Engineered Nanomaterials
The respiratory tract comes into contact with a variety of environmental contaminants on a daily basis, including airborne particles, gases, and infectious microbes. Due to this extensive exposure to toxicants, the respiratory tract has developed unique and complex mechanisms to defend against pulmonary insult [27]. There are three steps involved in the response to pulmonary toxicants: 1) identification of the foreign agents, 2) an acute inflammatory response, and 3) normal or abnormal tissue alterations. Normal tissue remodeling occurs when the acute inflammatory response is resolved, while abnormal tissue remodeling is the result of a lack of resolution and can lead to the development of fibrosis [27]. Pulmonary levels of cytokines, growth factors, lipid mediators, and proteinases all must remain balanced in order for normal tissue repair to occur.
proteins, such as elastins, in the lung, which can lead to emphysema, while overexpression of growth factors can lead to excessive proliferation of fibroblasts and production of collagen, leading to the development of fibrosis [27].
It has been well documented that exposure to airborne particulate matter (PM) causes a wide variety of negative respiratory effects, including, but not limited to, decreased lung function, increased susceptibility to microbial infections, and inhibited mucociliary clearance [2]. The most realistic risk for exposure to ENMs is through occupational exposure, as individuals working with ENMs in an industrial setting would be the most likely to be exposed to dispersed ENMs through the air [2,3]. Environmental and consumer exposure represent smaller, albeit still relevant, human health risks, as ENMs can be released from manufactured products through combustion or degradation [28]. Analysis of the combustion output of common methane, propane, and natural gas stoves has showed that aggregates of MWCNTs and other small carbon-based nanomaterials are present, and MWCNTs have also been determined to be present in a majority of indoor and outdoor PM samples [12].
Oberdörster et al proposed that the pulmonary toxicity of an ENM, be it spherical or fiber-like, is dependent on three different factors: retention, biopersistence, and biodurability [29]. Biopersistence refers to the manifestation of particles in vivo, and can vary depending on the particle dose and the area of the lung in which the particles deposit [29]. For example, many TiO2 nanoparticles are insoluble due to their crystalline structures, so therefore their
is used to define the in vivo chemical actions occurring in the lung that influence biopersistence [29].
Inhalation exposure to well-dispersed ENMs, in particular CNTs, results in deposition at the alveolar duct bifurcations and epithelial surfaces in the distal regions of the lung in rodents [26,29,30]. Inhalation of ENMs also induces ROS production in the lungs due to the increased surface area per unit mass of the particles as well as the presence of residual metal contaminants from the catalytic processes utilized during manufacturing [2]. Inhalation represents the most likely route of human exposure to CNTs, although oral or dermal exposure may be important routes of exposure for other ENMs [2,3]. Deposition in the lung and subsequent toxicity is dependent on a variety of factors such as particle size and shape, electrostatic charge, aggregation state, and dispersion pattern [26,29,30]. Particle size is believed to play the largest role in determination of pulmonary toxicity, as size correlates directly to deposition patterns in the lung [31]. Exposure of rats to ultrafine TiO2 particles
(~21 nm diameter) or fine TiO2 particles (~250 nm diameter) via inhalation demonstrated
that ultrafine particles travel further into the pulmonary interstitium and are retained longer in the lung than fine particles, and this trend was similar in rats exposed to TiO2 (12, 21, 230, or
250 nm diameter) via intratracheal instillation as well [32]. Translocation of TiO2 particles
Rodent studies have demonstrated that exposure to ENMs, in particular CNTs, induces lung inflammation independent of the method of exposure (inhalation, intratracheal instillation, or oropharyngeal aspiration), and inflammation is believed to be the primary reason for the toxic effects of ENM exposure [13,14,34]. Exposure to ENMs induces an influx of inflammatory cells, including neutrophils, macrophages, and lymphocytes, all of which are capable of secreting pro-inflammatory cytokines such as interleukin-(IL)-1β [13,35]. Activated macrophages and epithelial cells secrete IL-1β in response to injury, and IL-1β functions to induce production of other chemokines such as TNF-α, IL-6, and matrix metalloproteinases (MMPs), that recruit neutrophils and fibroblasts to the site(s) of injury [36-39]. IL-1β has been implicated as a fundamental factor in the development of acute pulmonary inflammation and chronic fibrosis, as bleomycin-induced lung inflammation requires IL-1β signaling through the IL-1 receptor (IL-1R) [36]. Well-dispersed MWCNTs worsen the development of interstitial fibrosis, and this correlates with increased levels of inflammatory cytokines, especially IL-1β [40]. Cultured human lung fibroblasts exposed to MWCNTs have increased levels of fibrogenic markers such as osteopontin (OPN), pro-collagen 1 (PC1), tenascin-C (TN-C), and TIMP metalloproteinase inhibitor-1 (TIMP-1), and these responses are suppressed following exposure to IL-1β neutralizing antibodies [41].
particles is dependent more on the volume of the particles, not the mass, and while macrophages are considered to be the primary cell type involved in clearance of particles, Ferin et al showed that particles not cleared by alveolar macrophages are phagocytized by type I alveolar epithelial cells and trafficked to the interstitium [29,32,43].
Inhalation of MWCNTs can also result in the development of pulmonary fibrosis, even a mere 7 days post-exposure [2,3,23-26]. Inhaled MWCNTs have also been shown to reach the subpleural tissue of exposed animals, where they can persist and react with the mesothelial lining, possibly inducing mesothelioma [26,44.45]. The progression and development of fibrosis is dependent on a variety of different factors, in particular the levels of growth factors and pro-fibrotic factors, such as TGF-β, PDGF, and chemokine (C-C motif) ligand 2 (CCL2), secreted by fibroblasts and other cell types in the lung [27,46,47]. The TGF-β family of growth factors consists of cytokines involved in many aspects of fibrosis, including proliferation and survival of airway smooth muscle cells and fibroblasts, repair of the airway epithelium, and structural maintenance of the extracellular matrix (ECM) [48]. There are three members in the TGF-β family, TGF-β1, TGF-β2, and TGF-β3, all of which exert their function by signaling through two trans-membrane receptors, TGF-β receptor I and II (TGF-βRI and TGF-βRII) [49,50]. Levels of TGF-β1, a specific member of the TGF-β family, are upregulated by eosinophils in the airways of asthmatic patients, leading to the development of fibrosis [48,51]. TGF-β2 has also been implicated in cases of severe asthma [48,52].
little is currently known about their function [46]. PDGF signals through two different receptors, either PDGF-receptor α or β (PDGF-Rα or PDGF-Rβ), and levels of these receptors are increased during fibrogenesis. PDGF is capable of acting alone to promote the production of extracellular matrix proteins required in fibrotic responses, but PDGF can also act through a TGF-β-dependent pathway as well [46]. PDGF has also been implicated in the development of cancer and pleural disease [45-47,53]. Mice exposed to MWCNTs with a nickel (Ni) catalyst have higher levels of PDGF in their lungs, and, in vitro, macrophages exposed to Ni have increased expression of PDGF [45-47,53,54].
Nickel nanoparticles (NiNP) have been implicated in the exacerbation of pleural disease, and in vitro research has shown that PDGF exposure enhances NiNP-induced levels of monocyte chemoattractant protein-1 (MCP-1 or CCL2) and interferon-inducible CXC chemokine (CXCL10) [47,55-57]. CCL2 is secreted by many different cell types, including alveolar macrophages, epithelial cells, and T cells, and acts as a chemoattractant for monocytes [48,58,59]. CCL2 functions as a profibrogenic and proangiogenic factor and can induce fibroblasts to generate collagen and TGF-β1 [47,60]. CXCL10 opposes CCL2 action and acts as an antifibrotic factor by suppressing angiogenesis and regulating infiltration of T cells and natural killer (NK) cells [47,61,62]. CXCL10 is also believed to be responsible for promotion of mast cell infiltration in asthma [48,63].
Inflammasomes
production [64-67]. Inflammasomes are protein scaffolds found in a variety of different cell types, including macrophages and epithelial cells [68-70]. Fourteen different inflammasomes are known to exist in humans, while many more variants are present in mice, but very little information is known about many of the different types [69,70]. Each inflammasome is defined by the unique NOD-like receptor (NLR) protein they contain, which acts as the central piece around which the rest of the inflammasome forms [69,70].
The family of NLR proteins is described as having three main domains: 1) a centralized NACHT domain that functions in nucleotide binding and oligimerization, 2) a C-terminal domain of leucine-rich repeats (LRR), and 3) an N-C-terminal pyrin domain (PYD) or caspase recruitment domain (CARD) [69]. The LRRs function as ligand sensors and control self-regulation, while the CARD and PYD domains regulate signaling downstream of the NLR protein via control of protein-protein interactions [69]. The NACHT domain is the only domain present in all of the different NLR members, and it functions to activate the inflammasome signaling cascades through ATP-induced oligimerization [69]. There are three subpopulations within the family of NLR proteins: the nucleotide-binding oligimerization domain (NOD) family, the NLR pyrin domain containing (NLRP) family, and the NLR caspase recruitment domain containing (NLRC) family [69].
and CARD domain containing protein (PYCARD), and acts as an adaptor between the NLRP3 protein and pro-caspase-1 [69,72,73]. Caspase-1 functions as a pro-inflammatory cysteine protease with the primary responsibility of cleaving immature inflammatory cytokines (i.e. IL-1β and IL-18) into mature forms that can be secreted by the cell involved [69,72,73]. During inflammasome activation, the CARD domain of ASC interacts with the CARD domain of pro-caspase-1, inducing cleavage of pro-caspase-1 into a p10/p20 tetramer that is mature, active caspase-1. Active caspase-1 can then cleave pro-IL-1β and, to a lesser extent, pro-IL-18, into active inflammatory cytokines that are then secreted from the cell [69,72,73]. Activation of the NLRP3 inflammasome requires two steps: 1) binding of a pathogen-associated molecular pattern (PAMP) to a pattern recognition receptor (PRR) on the NLRP3 protein itself, and, 2) release of the lysosomal contents to trigger actual inflammasome assembly [67,74].
Particulate Activation of the Inflammasome
induces phosphorylation of nuclear factor (NF)-κB. Once activated, NF-κB translocates into the nucleus and stimulates transcription of pro-IL-1β mRNA [67,74]. MWCNTs and similar fiber-like particles tend to be too large for macrophages to take up fully, resulting in frustrated phagocytosis, cellular damage, and, ultimately, disruption of the lysosome. Disruption of the lysosome causes release of the lysosomal contents, which consists of cysteine proteases such as cathepsin B, into the cytoplasm [67,74]. Pattern recognition receptors (PRRs) on the NLR protein recognize these proteases, activating NLRP3 assembly and inducing cleavage of pro-caspase-1 into an active form that can cleave pro-IL-1β (previously transcribed following LPS priming) into a mature form that is secreted by the cell [67,74]. It is currently unclear as to what acts as a TLR4 primer in vivo, but high-mobility group box 1 (HMGB1) has recently been implicated [75]. Inflammasome activation by MWCNTs is also not specific to macrophages, as it was recently demonstrated that cultured human airway epithelial cells exposed to MWCNTs had increased levels of inflammasome activation [41].
T helper Differentiation and Responses
viruses, and allergens, dendritic cells are activated to process specific antigens and present peptides to naïve CD4+ T cells [81]. In the lung specifically, dendritic cells test allergens on the luminal surface of the airway epithelium and then transport peptides from these allergens to nearby lymph nodes, where naïve CD4+ T cells reside [82,83]. Once in the lymph nodes, dendritic cells present antigens specific for the initial insult (allergen, bacteria, virus, etc) to naïve T cells in order to induce differentiation into distinct Th subpopulations. Differentiation of naïve T cells is further aided by the unique cytokine microenvironment specific to different immune reactions [76]. The entire process of differentiation requires detailed cross-talk between members of the signal transducer and activator of transcription (STAT) family of transcription factors, other transcription factors specific to each Th response, and cytokines unique to individual Th responses [76,84].
suppressed. This implicates Tbet as more important in suppressing GATA-binding protein 3 (GATA3), a transcription factor necessary for Th2 differentiation [76,90]. Tbet also plays a role in the immune capabilities of other cell types, including NK cells, NKT cells, B cells, and dendritic cells [76,91].
STAT1 is activated by IFN-γ, and it is through that activation that Tbet is stimulated to promote Th1 differentiation and further production of IFN-γ [76,92.93]. This positive feedback loop of STAT1-dependent IFN-γ activation implicates STAT1 as a necessary factor for Th1 promotion [76,94]. IFN-γ can activate STAT1 and stimulate Tbet in a variety of cell types other than T cells, including dendritic cells, monocytes, macrophages, and B cells [76,94]. Stat1-/- mice exposed to Toxoplasma gondii have inhibited Tbet expression in response to infection but, interestingly, have IFN-γ levels that are similar to infected wild-type mice [76,94]. STAT2, another member of the STAT1 family, can form heterodimers with STAT1 following activation by type I IFNs, and Stat2-/- mice are less resistant to viral infections due to a lack of IFN responses [76,95]. STAT4 is activated by IL-12 and is critical for in vitro Th1 reactions and in vivo clearance of viral infections [76,96]. The expression of STAT4 is controlled positively and negatively by IFN-γ and IL-4/GATA3, respectively, and, once activated, STAT4 can stimulate IFN-γ and Tbet [76,90,96-100].
factor discovered to be a “master regulator” of Th differentiation, and functions by promoting Th2 differentiation while inhibiting Th1 differentiation [76,101-103]. All naïve CD4 T cells express basal levels of GATA3, as it is required for the maturation of CD4 T cells into a form that can be differentiated [76,104]. Reduction of GATA3 in T cells blocks production of Th2 cytokines in vitro and inhibits promotion of airway hypersensitivity in vivo, and mice lacking Gata3 expression in CD4 T cells have a complete loss of Th2 differentiation [76,105-107].
Interestingly, deletion of Gata3 from mature Th2 cells fully inhibits secretion of IL-13 and IL-5, but only has minor effects on IL-4. This effect appears to be due to GATA3 binding to the IL-13 and IL-5 promoters, while only binding to the IL-4 enhancers [76,108-111].
STAT6 is required for induction of GATA3 and is also necessary for IL-4 dependent Th2 activation [76,112-116]. It is believed that STAT6 does not directly induce transcription of IL-4, but instead controls the Il4/Il13 locus as a whole [76,117]. STAT6 also plays a role in the maintenance of late stage Th2 responses and the production of permanent, memory Th2 cells [76,118]. Research has shown that while STAT6 is needed for in vitro Th2 differentiation, it is possible to induce Th2 responses in vivo independent of STAT6, although these STAT6-independent Th2 responses still require GATA3 activation [76,118-122]. STAT5 is involved in many cell growth and survival responses, but also plays a specific role in Th2 differentiation [76,123-125]. STAT5 binds to specific sites on the Il4 locus in Th2 cells, and cells lacking STAT5 are more apt to differentiate into Th1 cells [76,125,126].
fungi [76]. Th17 induction is dependent on cross-talk between STAT3 and RAR-related orphan receptor gamma t (RORγt) [76,85]. Further differentiation of Th17 cells involves TGF-β combined with either IL-21, IL-23, or IL-6 [76,84]. Unlike the other Th cell subsets, Th17 cells do not have any basal expression of either Tbet or GATA3 and solely express RORγt [76,127-129]. Naïve T cells begin expressing RORγt 8 hours after exposure to either IL-6 or TGF-β, and RORγt is required for production of IL-17 from mature Th17 cells [76,130]. Inhibition of Rorγt in T cells in vitro suppresses IL-17 secretion, and Rorγt-/- mice are less susceptible to the development of experimental autoimmune encephalomyelitis (EAE) [76,130].
STAT3 functions by binding Il17 and/or Il21 and activating RORγt and the IL-23R [76,131-133]. STAT3 can be activated by a variety of different cytokines involved in Th17 cell differentiation and the subsequent Th17 immune response, including 6, 21, and IL-23 [76,131,132,134-137]. Stat3-/-mice and humans with Stat3-/- gene mutations lack CD4 T cells capable of producing IL-17 [76,131-133,138-141]. In vitro, ablation of Stat3 in T cells pre-programmed for a Th17 phenotype results in increased levels of forkhead box P3 (FoxP3) [76,133]. Activation of STAT3 by IL-6 specifically results in suppression of FoxP3 and inhibition of Treg differentiation, implicating an important role for IL-6 in the balance of Th17 and Treg subsets [76,130,133,135,142].
Treg (nTreg) cells [76,85,143]. The primary cytokine involved with Treg promotion is TGF-β, which is required for differentiation and maintenance of nTregs [76,84,144-147]. Mutations in Foxp3 and a lack of functional nTreg cells have been implicated in human autoimmune diseases such as IPEX (immunodeficiency, polyendocrinopathy, and enteropathy, X-linked syndrome) and rodent diseases such as scurfy [76,148-150]. Overexpression of Foxp3 in mature T cells induces a Treg phenotype, including a loss of cytokine production and an increase in inhibitory capabilities [76,151]. On the other hand, inhibition of Foxp3 causes T cells already programmed to become Tregs to differentiate into Th2-like cells instead [76,152]. IL-21, secreted by Th17 cells, has been shown to inhibit FoxP3, implicating IL-21 as one of many different regulators of Treg development [48,132].
STAT5 has been shown to function in regulation of all different Th subsets, most likely due to variable binding of STAT5 to the different genes involved in Th differentiation [76]. Increased activation of STAT5 inhibits differentiation of Th1 and Th17 cells, but enhances differentiation of Th2 and Treg cells [76]. New research has demonstrated that STAT5 controls the Ifng locus and subsequent Th1 differentiation, while also playing a dual role in both the enhancement and inhibition of Th17 differentiation [76,140,153,154]. STAT5 binds to the promoter region of FoxP3 to initiate Treg differentiation, and activation of STAT5 by IL-2 is necessary for differentiation of Treg cells [76,155-158].
effector cytokines can modulate T cell differentiation, including IL-18 for Th1, IL-33 for Th2, and IL-1 for Th17 [76]. IL-12 activation of STAT4 in conjunction with IL-18 production can stimulate IFN-γ expression independent of any T cell receptor (TCR) [76,160.161].
Cytokine Regulation of Asthma and Allergic Inflammation
Lymphokines are secreted primarily from activated T cells, which, in the case of asthma, tend to be predominantly Th2 cells [48,166]. Th2 cells secrete IL-4, IL-5, IL-13, and IL-9, all of which are increased in cases of asthma [48,165]. IL-4 and IL-13 function through binding of the IL-4/IL-13 receptor (IL-4/IL-13R) and phosphorylating STAT6 [48,167,168]. STAT6 regulates many different characteristics of the allergic airway inflammatory response during asthma, such as mucous cell metaplasia, eosinophilic infiltration, airway hyperresponsiveness (AHR), and the progression of airway fibrosis [48,167,168].
IL-4 is required for differentiation of naïve T cells into Th2 cells and is also involved in the first steps of allergen sensitization [48,76]. IL-4 also mediates production of IgE and activation of B cells [15,167,169]. IL-13 is also involved in IgE production and physical alterations in asthmatic airways, but it cannot induce Th2 differentiation [48,170]. IL-13 has been implicated in increased AHR in animal models of acute asthma, and also plays a role in the exacerbation of chronic asthma by inducing goblet cell hyperplasia, airway smooth muscle growth, and fibrosis development [48,170]. IL-13 also acts as a chemokine, and can induce secretion of eotaxin, an eosinophilic chemoattractant [48]. Following allergen sensitization, there is an acute increase in IL-4 levels in bronchoalveolar lavage fluid (BALF), while levels of IL-13 secretion remain persistent over time, most likely due to the accumulation of eosinophils in the airway as the asthmatic responses progresses [48,171].
[48,172,173]. The role of IL-9 in asthma and the Th2 response is not as well-understood, although experimental analysis in mice in vivo has demonstrated that increased IL-9 exacerbates the inflammatory response induced by eosinophils, goblet cell hyperplasia, IgE, and AHR [48,174]. Suppression of IL-9 levels in vivo subsequently decreases eosinophil counts, AHR, and goblet cell hyperplasia following allergen sensitization [48,174]. IL-9 appears to exert most of its effects through IL-13 secretion, although IL-9 does have direct effects on both mast cell and B cell proliferation and maintenance [48,175].
IL-4, IL-13, and IL-5 in mice in vivo, subsequently inducing eosinophilic inflammation, IgE secretion, and AHR [48,180]. In vitro, IL-25 also increases the production of Th2 cytokines from human Th2 cells [48,181].
Th2-related lymphokines are not solely responsible for the development of inflammation during asthma, as many different pro-inflammatory cytokines act to promote and sustain airway inflammatory responses [48]. TNF-α, IL-1β, and IL-6 are all upregulated in sputum and BALF samples from asthmatic patients, and these cytokines function through activation of the transcription factor NF-κB to induce transcription of proinflammatory genes [48]. TNF-α is secreted by a variety of different cell types, including T cells, mast cells, epithelial cells, macrophages, and airway smooth muscle cells, and administration of TNF-α to healthy patients induces neutrophilic airway inflammation [48,182]. TNF-α induces contractions of airway smooth muscle in response to certain stimuli, and inhibition of TNF-α suppresses AHR in asthmatic patients [48,183-185]. IL-1β induces expression of a variety of inflammatory genes and is upregulated in the airways of asthma patients [48]. Allergen-induced AHR can be inhibited in mice with an IL-1 receptor antagonist (IL-1Ra), but this method is not effective in humans [48,186]. IL-6 functions in conjunction with other cytokine families and is enhanced in the sputum of individuals with asthma [48,187]. IL-6 has been implicated in promotion of both Th2 and Th17 subpopulations, suggesting a prominent role for IL-6 in the exacerbation of airway inflammation during asthma [48].
although primarily a pro-fibrotic growth factor, has some immunoregulatory capabilities and can suppress activation of CD4+ T cells [48,188]. IL-10 is the most important of the anti-inflammatory cytokines, and has the ability to suppress production of a variety of pro-inflammatory factors, including TNF-α, IL-5, and granulocyte macrophage-colony stimulating factor (GM-CSF) [48,189]. IL-10 is secreted by Treg cells as well as macrophages, and human asthmatic patients have decreased macrophage IL-10 expression [48,190,191].
beyond the lung, as monocytes collected from patients with atopic dermatitis were shown to have impaired caspase-1-dependent IL-1β secretion relative to healthy controls [192].
Exacerbation of Asthma by Engineered Nanomaterials
Exposure to ENMs, in particular MWCNTs, can cause interstitial fibrotic lesions in the absence of any allergen challenge [198,199]. While many ENMs are toxic on their own, they are also capable of exacerbating airway inflammation in susceptible individuals with pre-existing pulmonary diseases such as asthma and chronic obstructive pulmonary disease (COPD) [2,23,25]. Allergic effects induced by ENMs on the lung include eosinophilic infiltration, goblet cell hyperplasia, and increased AHR [48,200-202]. Interestingly, repeated exposures to MWCNTs can induce allergic airway inflammation and polarize T cells towards a Th2 phenotype in the absence of any allergen [15,26]. Similarly, increases in B cell activation and IgE production have been observed in mice following a single instillation of MWCNTs [15]. The Th2 response following MWCNT exposure alone can be accompanied by an increase in blood and BALF levels of IL-4 and IL-5, both Th2-specific cytokines, and IL-10, an immunoregulatory cytokine [15].
HDM and MWCNTs exacerbated acute airway inflammatory reactions at 1 day post-MWCNT exposure and promoted fibrogenesis at 21 days post-post-MWCNT exposure [193]. The acute inflammatory response was accompanied by a mixed eosinophilic/neutrophilic response as well as an increase in inflammatory cell numbers in the BALF, while the chronic fibrogenic response correlated with increases in pro-fibrogenic growth factors such as TGF-β1, PDGF-A and B, and CCL2 [193].
extent [203]. These data implicated Tbet as a prominent gene with a role in susceptibility to both NiNP and MWCNT exposure [203].
Sayers et al utilized a transgenic mouse model lacking the COX-2 gene to examine its role in the exacerbation of allergic airway remodeling following exposure to MWCNTs, as COX-2 is considered to play a protective role against the development of asthma [204,208]. Airway epithelial cells from asthma patients have suppressed levels of COX-2 mRNA expression, while bronchial epithelial cells from asthma patients have decreased COX-2 levels in response to IL-13 exposure [204,209,210]. MWCNT exposure significantly exacerbated pulmonary inflammation and mucous cell metaplasia in Cox2-/- mice sensitized with OVA relative to wild-type mice [204]. Interestingly, levels of allergen-related cytokines, including IL-13 and IL-5, were also increased by MWCNT exposure in Cox2-/- animals in the
absence of any OVA exposure, implicating COX-2 as necessary for protection against the development of allergic airway disease even in the absence of any allergen challenge [204]. Related research in vitro has also shown that MWCNTs induce COX-2 expression in immortalized mouse macrophages (RAW264.7 cells) [211].
Mice lacking the gene for STAT1, a transcription factor involved in the differentiation of naïve T cells to Th1 cells, were employed in a study done by Thompson et al [205,212]. STAT1 is also involved in the control of both growth arrest and apoptosis, and
increases in many pro-fibrogenic factors, including TGF-β1, OPN, and TNF-α, and fibroblasts isolated from Stat1-/-mice had increased levels of collagen mRNA in response to TGF-β1 exposure [205]. This trend has been seen previously, as Walters et al reported that Stat1-/- mice are more vulnerable to bleomycin-induced pulmonary fibrosis [216]. These combined studies all implicate different, unique genes as incredibly important in the exacerbation of allergic airway disease and asthma following MWCNT exposure.
Systemic Effects of Exposure to Engineered Nanomaterials
Translocation of ENMs throughout the bloodstream and systemic circulation is not well understood, in particular because movement of ENMs is dependent on a variety of factors such as size, shape, composition, and surface coating [2,217,218]. Non-cationic ENMs with a diameter smaller than 34 nm can reach the regional lymph nodes of rats merely 30 minutes after intratracheal instillation, and ENMs with a diameter less than 6 nm can quickly enter the bloodstream [9,219]. More specifically, intratracheal instillation of MWCNTs into the lungs of rats results in MWCNT deposition in local lymph nodes, and these MWCNTs accumulate in the lymph nodes in a time-dependent manner [220]. This was true of intratracheal instillation of TiO2 in rats, as rats exposed to ultrafine TiO2 had large
that pulmonary exposure to MWCNTs specifically resulted in deposition of singlet MWCNTs in the kidney, liver, and brain of exposed mice [223].
ENMs through the lymphatic system and into circulation could be the primary pathway for these particles to exit the lung [223].
GENERAL RESEARCH HYPOTHESIS
The field of nanotechnology is continually expanding, and the market for consumer products produced using this technology is expected to reach $2.4 trillion by the end of 2015 [1-3]. Nanotechnology utilizes engineered nanomaterials (ENMs), which are ultrafine particulates with at least one dimension less than 100 nm [5-7]. ENMs have a variety of chemical and physical properties that make them ideal for use in many industrial settings, including an increased surface area per unit mass, heightened strength and durability, and exceptional conductivity [2,3]. Many of the features that make ENMs useful in industry also make them potentially toxic, making it imperative to study the possible risks of ENM exposure, particularly in terms of human health [2,3]. Multi-walled carbon nanotubes (MWCNTs), are a specific class of ENMs consisting of concentric sheets of graphene rolled into cylinders [14]. MWCNTs have a longer, fiber-like shape, very similar to that of asbestos, which increases the risk of pulmonary toxicity following exposure [2,3,14,15]. MWCNTs are believed to exert their inflammatory effects through activation of the NLRP3 inflammasome and induction of IL-1β secretion [40,41].
CHAPTER 1
An Allergic Lung Microenvironment Suppresses Carbon Nanotube-Induced Inflammasome Activation via STAT6-Dependent Inhibition of Caspase-1
Kelly A. Shipkowski1, Alexia J. Taylor1, Elizabeth A. Thompson1, Ellen E. Glista-Baker1, Brian C. Sayers1, Zachary J. Messenger1, Rebecca N. Bauer2, Ilona Jaspers2, and James C.
Bonner1
1Department of Biological Sciences, Environmental and Molecular Toxicology Program
North Carolina State University, Raleigh, North Carolina, United States of America
2Center for Environmental Medicine, Asthma, and Lung Biology, School of Medicine,
University of North Carolina, Chapel Hill, North Carolina, United States of America
Correspondence: James C. Bonner, Ph.D.
Department of Biological Sciences
Environmental and Molecular Toxicology Program North Carolina State University
Campus Box 7633
Raleigh, NC 27695-7633 USA Phone: (919) 515-8615
Fax: (919) 515-7169
Email: [email protected]
PUBLISHED IN: PLoS ONE. 2015;10(6): e0128888 (doi: 10.1371/journal.pone.0128888).
Funding: This work was funded by NIEHS grants RO1-ESO20897 (JCB) and RC2- ESO18772 (JCB). ZJM is supported by NIEHS training grant T32-ES007046.
Abbreviations: MWCNTs, multi-walled carbon nanotubes; HDM, house dust mite; Th2, T-helper 2; AAM, alternatively-activated macrophage; CAM, classically-activated
ABSTRACT
numbers of neutrophils and IL-1β in BALF as well as reduced pro-caspase-1 in lung tissue. Despite reduced IL-1β mice exposed to MWCNTs after HDM developed more severe airway fibrosis by 21 days and had increased pro-fibrogenic cytokine mRNAs. Conclusions: These data indicate that Th2 cytokines suppress MWCNT-induced inflammasome activation via STAT6-dependent down-regulation of pro-caspase-1 and suggest that suppression of inflammasome activation and IL-1β by an allergic lung microenvironment is a mechanism through which MWCNTs exacerbate allergen-induced airway fibrosis.
INTRODUCTION
Carbon nanotubes (CNTs) are a product of the emerging nanotechnology industry and have numerous potential applications in structural engineering, electronics, and medicine [1,2]. Despite these benefits, CNTs represent an impending risk to human health as it has been shown that mice exposed to CNTs develop pulmonary inflammation and fibrosis following inhalation exposure [1-6]. Structurally, CNTs are graphene sheets rolled into cylinders that are one (“single-walled”, SWCNT) or multiple (“multi-walled”, MWCNT) layers thick. MWCNTs have unique physical and chemical properties that make them particularly hazardous, including a fiber-like shape with increased rigidity reinforced by multiple concentric layers and residual metal catalyst from the manufacturing process [1,2,7,8]. MWCNTs also have a high surface area per unit mass that allows for increased potential for ROS production and subsequent cellular damage [1,2,7,8].
models indicate that specific genes (e.g., COX-2, T-bet) regulate susceptibility to MWCNT-induced exacerbation of allergic airway disease [16,17]. Collectively, these studies suggest that individuals with asthma would be more susceptible to the adverse respiratory effects of inhaled MWCNTs.
Macrophages play a key role in the lung by engulfing inhaled MWCNTs via phagocytosis and removing them from the lungs through the mucociliary escalator or lymphatic drainage [7]. Macrophage phenotype is modified by Th1 and Th2 immune microenvironments that induce classically-activated and alternatively-activated macrophage (CAM and AAM) phenotypes, respectively. Interferon-gamma (IFN-γ), increased in a Th1 microenvironment, induces a CAM phenotype, which is primarily involved in the innate pro-inflammatory immune response and microbial killing. Th2 cytokines, including IL-4 and IL-13, induce an AAM phenotype, which is involved in parasite killing, wound healing, allergy, susceptibility to pathogens, and the pathogenesis of fibrosis [18,19].
inflammatory cytokines such as IL-1β and IL-18 into mature forms that are capable of being secreted. The ASC adaptor functions as a link between the NLRP3 protein and pro-caspase-1. Once NLRP3 is activated, oligimerization occurs, allowing NLRP3 to interact with ASC. The caspase-recruitment domain (CARD) of ASC can then interact with the CARD domain of pro-caspase-1, resulting in cleavage of pro-caspase-1 into active caspase-1, a p10/p20 tetramer. Once active, caspase-1 cleaves pro-IL-1β into a mature form that is subsequently secreted from the cell [25-27]. Functional caspase-1 is essential for cleavage of pro-IL-1β and subsequent secretion of mature IL-1β.
The impact of the asthma microenvironment on inflammasome activation by MWCNTs or any other agent has not been investigated. However, there is evidence that the Th2 cytokines that characterize the asthmatic microenvironment can modulate caspase-1. For example, Th2 cytokines, in particular IL-13, have been shown to inhibit caspase-1 activity. Niebuhr et al demonstrated that treatment of primary human monocytes with IL-13 suppressed caspase-1 activity, while Cihakova et al showed that IL-13-/- mice have increased levels of caspase-1 activation [28,29]. The transcriptional profile of IL-13-treated human monocytes was also thoroughly examined by Scotton et al, who showed significant down-regulation of pro-caspase-1 mRNA following 8 hours of IL-13 exposure [30].
MATERIALS AND METHODS
Cell Culture. Human monocyte cells (THP-1) were purchased from ATCC (Manassas, VA) (Cat#: TIB-202). Cells were cultured in suspension in RPMI-1640 Medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA) at 37ºC and 5% CO2. The THP-1s were grown to confluence and differentiated into
macrophage-like cells using 150 nM of 1α,25-Dihydroxy-Vitamin D3 (EMD Millipore,
Billerica, MA) for 24 hours. Once the cells were semi-adherent, 160 nM of phorbol 12-myristate 13-acatate (PMA) (LC Laboratories, Woburn, MA) in sterile dimethyl sulfoxide (DMSO) (Sigma-Aldrich) was applied to the cells for 30 minutes to initiate maturation from monocyte to macrophage [33]. Following cell maturation, 2 µg/mL lipopolysaccharide (LPS) (Sigma-Aldrich) was added to the cells, following by 10 ng/mL recombinant human IL-13 (#213-IL/CF) or IL-4 (#204-IL/CF) (R&D Systems, Inc., Minneapolis, MN) and/or MWCNTs. Once dosed, cell mixtures were aliquoted into 96 or 48-well plates (Thermo Fisher Scientific, Waltham, MA). Cell supernatants, RNA, and whole cell protein were collected 24 hours post-MWCNT exposure.
Animals and Experimental Design. Pathogen-free, 6 to 8 week-old, male wild type (WT) C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were sensitized intranasally with house dust mite (HDM) allergen (0.5 mg/mL) (Greer Laboratories, Lenoir, NC) or sterile saline under an isoflurane anesthetic five days a week for two weeks. Following allergen sensitization, mice were exposed to MWCNTs (2 mg/kg) or sterile saline via oropharyngeal aspiration under an isoflurane anesthetic. Mice were euthanized 1 day or 21 days after MWCNT exposure via intraperitoneal injection of 0.3 mL pentobarbital (Fatal Plus) (Vortech Pharmaceuticals, Dearborn, MI).
transferred to 70% ethanol, and embedded in paraffin. Three cross-sectional sections of tissue were cut and processed for histopathology with a hematoxylin and eosin (H&E), Alcian blue/periodic acid-Schiff (AB/PAS), and/or Masson’s trichrome stain. Whole blood was collected from the jugular vein and allowed to coagulate for approximately 15 minutes in Serum Separator Tubes (BD Microtainer, Franklin Lakes, NJ) prior to centrifugation for serum collection. Serum was then stored at -80°C.
Semi-Quantitative Morphometric Analysis. Lung Inflammation: Three sections of formalin-fixed lung tissue from each 1 day animal were analyzed under light microscopy and scored for inflammation in a blinded fashion. Lungs were scored on a scale of 1-5 based on inflammatory cell infiltration, alveolar wall thickening, and the manifestation of extracellular matrix. Animals were scored with 1 representing the saline control animals, 2 representing very minimal inflammation, 3 representing mild inflammation, 4 representing moderate inflammation, and 5 representing very severe inflammation [16]. Data was shown as the mean value ± SEM for 11-14 animals per treatment group.
Airway Fibrosis: Quantification of the thickness of collagen surrounding airways was
performed according to a previously published airway intersect method [17]. Briefly, photomicrographs of trichrome-stained sections of lung tissue containing circular to oval-shaped small or medium-sized airways were captured using a 10X objective on an Olympus BX41 microscope (Olympus America Inc., Center Valley, PA) and digitized. The thickness of the collagen layer surrounding the airways was measured using the ruler tool in Adobe Photoshop CS3 extended program (Adobe Systems, Inc., San Jose, CA) at eight equidistant points and averaged. To validate the airway intersect measurements, a second independent method similar to a previously published method [34] was used to measure the airway collagen area corrected for length of basement membrane (i.e., area/perimeter ratio). Briefly, the lasso tool in Adobe Photoshop was used to surround the trichrome-positive collagen around an airway (outer area). A second measurement was made by surrounding the basement membrane of the same airway (inner area) and the length of the airway circumference (i.e., perimeter) was also derived from this measurement. The difference between the outer and inner area was defined as the ‘area’ and divided by the ‘perimeter’ to derive area/perimeter measurements. Both methods were performed in a blinded manner, where the treatment group was unknown to the observer scoring the sections. Five airways per animal were analyzed in a random, blinded manner, and the data were expressed as the mean ± SEM of five animals per treatment group per time point.
culture conditioned medium collected 24 hours post-MWCNT and IL-13 or IL-4 exposure was used for the assay. Cell culture samples were diluted 100-fold in DPBS for IL-1β analysis and assayed following the manufacturer’s protocol. Mouse BALF was left undiluted and assayed for IL-1β following the manufacturer’s protocol. Mouse serum was diluted 1:10 in sterile DPBS and IgE was analyzed using a mouse IgE ELISA kit (#557079) (BD Pharmingen, San Jose, CA). Absorbance was measured at 450nm by the Multiskan EX microplate spectrophotometer (ThermoFisher Scientific) with a correction wavelength of 540nm. IL-1β and IgE concentrations were interpolated from a standard curve using linear regression analysis. Values were expressed as mean±SEM.
Quantitative Real-Time RT-PCR. Taqman Quantitative RT-PCR (qRT-PCR) was utilized to measure mRNA levels in THP-1 monocytes and whole mouse lungs. Cell culture RNA was collected 24 hours post-MWCNT exposure using the Zymo Research Quick-RNA MiniPrep Kit (Genesee Scientific, San Diego, CA) and following the manufacturer’s protocol. 20 µL or 40 µL lysis buffer was added to each well in the 96-well or 48-well plate, respectively, and columns of wells were scraped and combined to form one sample. Right cranial and caudal lobes from each mouse lung were homogenized and whole lung RNA extracted and purified using the Zymo Research Quick-RNA MiniPrep Kit as well. RNA concentrations for each sample were quantified using a Nanodrop 2000 Spectrophotometer (ThermoFisher Scientific) and each sample was normalized to a final concentration of 25ng/µL in RNAse-free H2O. qRT-PCR was performed utilizing reagents from the
StepOne Plus instrument (Applied Biosystems, Foster City, CA). A comparative CT
technique was used to quantify target gene expression. Primers for human pro-IL-1β (Hs01555410_m1) and pro-caspase-1 (Hs00354836_m1) were utilized to analyze THP-1 mRNA levels. Primers for mouse pro-IL-1β (Mm01336189_m1), IL-13 (Mm00434204_m1), IL-4 (Mm00445259_m1), IL-5 (Mm00439646_m1), CXCL1 (Mm04207460_m1), CXCL2 (Mm00436450_m1), CCL2 (Mm00441242_m1), PDGF-A (Mm00833533_m1), PDGF-B (Mm01298578_m1), and TGF-β1 (Mm03024053_m1) were utilized to analyze whole mouse lung mRNA. THP-1 and mouse lung mRNA expression were normalized against the endogenous control β-2-microglobulin for either human (Hs00984230_m1) or mouse (Mm00437762_m1), respectively, and measured relative to vehicle-treated controls for both cell culture and mouse samples. All qRT-PCR primers were purchased from Life Technologies. Each sample was analyzed in duplicate and the StepOne Plus software used to calculate relative quantitation values and express them as fold-change over controls. Fold change was expressed as mean ± SEM.
membranes. Membranes were blocked in 5% nonfat milk in TBS-T (20 mM Tris, 137 mM NaCl, and 0.1% Tween-20) and incubated in primary antibody (1:1000 dilution) overnight at 4°C. Primary antibody incubation was followed by incubation in horseradish peroxidase-conjugated secondary antibody (1:2500 dilution). Immunoblot signals were identified using enhanced chemiluminescence (ECL) (ThermoFisher Scientific). Densitometry was performed to quantify Western blotting signals as previously described [17]. Mouse monoclonal pro-caspase-1 (sc-56036) and rabbit polyclonal pro-IL-1β (sc-7884) primary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX). Polyclonal rabbit total STAT-6 (#9362), polyclonal rabbit phosphorylated STAT-6 (#9361), and polyclonal rabbit β-actin (#4967) primary antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-rabbit (#7074) and anti-mouse (#7076) secondary antibodies were purchased from Cell Signaling.
Caspase-1 Activity Assay. THP-1 cells were grown to confluence and differentiated into macrophage-like cells using 150 nM of 1α,25-Dihydroxy-Vitamin D3 for 24 hours. Once the
removed and cells washed with sterile DPBS. After washing, THP-1 cells were exposed to 50mJ UVB to induce apoptosis. Following UVB treatment, fresh media was added and cells were allowed to incubate at 37°C for 16 hours. Apoptotic cells were then collected and lysed, and analysis of caspase-1 activity was performed using the Caspase-1 Colorimetric Assay Kit from R&D Systems (#BF14100) and following the manufacturer’s protocol.
Pro-Caspase-1 Immunohistochemical Staining. Lung sections were fixed in 10% neutral buffered formalin for 24 hours followed by 70% ethanol before being embedded in paraffin. Tissue sections (5 µM) were deparaffinized, hydrated, treated with 3% H2O2, and subjected
to antigen retrieval with a citrate buffer (pH 6.0) using a 2100-Retriever purchased from Electron Microscopy Sciences (Hatfield, PA). Sections were blocked with 10% normal horse serum for 1 hour at room temperature before being incubated with mouse monoclonal anti-pro-caspase-1 antibody (sc-56036; 1:2000) (Santa Cruz Biotechnology, Inc.) at 4°C for 20 hours. Sections were then incubated with biotinylated anti-mouse IgG for 30 minutes at room temperature and staining was detected using the mouse Vectastain Elite ABC kit (PK-6102) (Vector Laboratories, Burlingame, CA) and 3,3’-diaminobenzidine (DAB) (BioGenex, Fremont, CA) following the manufacturer’s protocol. The sections were counterstained with Mayer’s Hematoxylin (Sigma-Aldrich), dehydrated, and mounted. No pro-caspase-1 staining was observed when the primary antibody was omitted and the control normal horse serum was applied. CASP-1-/- mice, purchased from Jackson Laboratories and utilized by Bauer, et al, acted as a negative control [37]. Pro-caspase-1 staining was quantified utilizing ImageJ
stained cells and epithelium were analyzed by a de-convolution module using a threshold method and then standardized for measurement [34]. Final results were calculated as percent area of stained tissue relative to the total area of the image used. Three airways were quantified per lung section for statistical analysis.
Statistical Analysis. All data was collected and transformed into graphs, and statistical analysis was performed using GraphPad Prism software version 5.00 (GraphPad Software Inc., San Diego, CA). One-way ANOVA with a post hoc Tukey or unpaired Student’s t-test were used to determine significant differences between controls and treatments, and two-way ANOVA with a post-Bonferroni test was used to determine significant differences between treatment groups. Significance was set at p < 0.05 unless otherwise indicated. All 1 day animal data is representative of three replicate experiments, while all 21 day animal data is representative of two replicate experiments.
RESULTS
MWCNT-induced IL-1β secretion by LPS-primed THP-1 cells is inhibited by Th2 cytokines in vitro. THP-1 monocytes were differentiated to macrophages with Vitamin D3 and PMA, primed with LPS, and exposed to MWCNTs in the absence or presence of IL-4 or IL-13 for 24 hours prior to collection of supernatants for measurement of IL-1β by ELISA. Transmission electron microscopy showed that THP-1 cells exhibited a macrophage-like morphology and avidly engulfed MWCNTs (Fig. 1A). Aggregates of MWCNTs and singlet MWCNTs in cells visualized by TEM were consistent with the sizes reported in Methods and described previously [32]. Treatment of differentiated, LPS-primed THP-1 cells with MWCNTs caused a dose-dependent increase in secreted IL-1β in cell supernatants, and co-treatment with IL-4 or IL-13 (10 ng/ml) resulted in significant suppression of MWCNT-induced IL-1β secretion (Fig. 1B). IL-4 was more potent than IL-13 in suppressing MWCNT-induced IL-1β secretion. While MWCNTs were observed in THP-1 cells pre-treated with IL-4 or IL-13, we cannot completely rule out that differences in uptake of MWCNTs could partially account for reduced inflammasome activation in cells pre-treated with these Th2 cytokines.
LPS-primed THP-1 cells with MWCNTs further increased mRNA levels of pro-caspase-1 (Fig. 2B). Co-treatment with IL-4 or IL-4 and IL-13, but not IL-13 alone, reduced MWCNT-induced pro-caspase-1 mRNA levels. Treatment of THP-1 cells with MWCNTs and/or IL-13 or IL-4 did not alter mRNA levels of NLRP3 or PYCARD, two other key components of the NLRP3 inflammasome (data not shown). LPS priming alone did not increase protein levels of pro-caspase-1 as determined by Western blotting (Fig. 2C and E). Treatment with MWCNTs alone increased levels of STAT6, but not phospho-STAT6, as exposure to IL-4 and/or IL-13 was necessary for phosphorylation of STAT6 (Fig. 2C). However, both IL-4 and IL-13 suppressed protein levels of pro-caspase-1 as determined by densitometric evaluation of Western blots (N=3) for pro-caspase-1 that were normalized against β-actin (Fig. 2E).
Inhibition of STAT6 in THP-1 cells increases caspase-1 activity. Treatment of LPS-primed THP-1 cells with either Leflunomide, a STAT6–specific inhibitor, or JAK Inhibitor I (JAKI), a broad JAK/STAT inhibitor, blocked the suppression of caspase-1 activity by IL-4/IL-13 treatment (Fig. 3). Moreover, Leflunomide or JAKI increased levels of caspase-1 activity significantly above controls.
for collection of BALF and lung tissue. Mice exposed to the combination of HDM and MWCNTs had significantly increased total cell counts in BALF at 1 day post-MWCNT exposure compared to mice that received vehicle control or mice that received HDM alone (Fig. 4B). The increase in the total number of inflammatory cells returned to control levels by 21 days post-MWCNT exposure. Cytospins of BALF from mice treated with HDM and MWCNTs showed a mixed neutrophilic/eosinophilic inflammatory response at 1 day compared to the eosinophilic response observed in mice sensitized to HDM alone or the neutrophilic response seen in mice exposed to MWCNTs alone (Fig. 4C and D). BALF cell differential counts at 1 day showed a relative decrease in macrophage counts compared to an increase in the percentage of neutrophils in mice exposed to MWCNTs and an increase in the percentage of eosinophils in mice sensitized to HDM (Fig. 4D). Interestingly, the relative percentage of neutrophils induced by MWCNTs was reduced by approximately half in mice pre-sensitized with HDM, while the relative percentages of eosinophils was approximately the same in mice treated with HDM or HDM and MWCNTs. Lymphocyte counts were not notably different between any treatment groups (Fig. 4D). These inflammatory cell changes in BALF returned to nearly control levels by 21 days post-MWCNT exposure.
decidedly more inflammation than mice exposed to HDM or MWCNTs alone (Fig. 5A and B). Staining of lung sections with an Alcian blue and Periodic acid-Schiff (AB/PAS) combination stain demonstrated that mice sensitized with HDM allergen had notably increased goblet cell hyperplasia and mucin production, but that effect was not exacerbated by MWCNT exposure (S1 Fig). BALF collected from the lungs of mice was analyzed for secreted IL-1β after exposure to MWCNTs with or without HDM sensitization. IL-1β in the BALF was significantly increased at 1 day post-MWCNT exposure and remained elevated at 21 days compared to vehicle control, while sensitization of mice to HDM prior to MWCNT exposure almost completely inhibited IL-1β protein levels in BALF (Fig. 5C). However, pro-IL-1β mRNA was increased in the lungs of mice at 1 day in all treatment groups compared to control and was most pronounced in mice exposed to HDM and MWCNTs (Fig. 5D).
sensitized to HDM had increased serum IgE at 1 day compared to control, and exposure to MWCNTs did not change HDM-induced IgE levels (Fig. 6D). Interestingly, MWCNTs alone caused a modest increase in serum IgE levels. No distinct increases in serum IgE were seen in any treatment groups at 21 days.
HDM sensitization increases MWCNT-induced lung mRNA levels of the monocyte chemoattractant, CCL2, but does not alter mRNAs encoding neutrophil chemoattractants CXCL1 and CXCL2. MWCNTs increased the expression of CCL2 mRNA, and the combination of HDM sensitization followed by MWCNT exposure further increased CCL2 mRNA (Fig. 7A). MWCNTs significantly increased lung mRNA expression of CXCL1 (Fig. 7B) and CXCL2 (Fig. 7C) compared to control animals. While there was a trend for MWCNT-induced CXCL1 and CXCL2 mRNA levels to be suppressed by HDM pre-sensitization, these chemokine mRNA levels were not significantly different between MWCNT and HDM/MWCNT groups. HDM sensitization alone did not affect CXCL1 or CXCL2 mRNA levels.
for pro-caspase-1 immunohistochemistry. Mice that were sensitized to HDM and then exposed to MWCNTs had weaker immuno-staining for pro-caspase-1 in airway epithelium and alveolar macrophages as compared to mice that were exposed to MWCNTs alone (Fig. 8A and B). Quantification of pro-capsase-1 IHC staining further supported the qualitative results observed, demonstrating a significant increase in pro-caspase-1 staining with MWCNTs alone, and that increase being suppressed with the combination of HDM and MWCNTs (Fig. 8C).