Forrest Jessop

Innate immune processes are sufficient for driving silicosis in mice

The lung is constantly exposed to potentially pathogenic particles and microorganisms. It has become evident recently that not only innate but also adaptive immune responses to particulates, such as SiO2 entering the respiratory tract, are complex and dynamic events. Although the cellular mechanisms and anatomical consequences involved in the development of silicosis have been studied extensively, they still remain poorly understood. Based on their capacity for immune regulation, lymphocytes may play a key role in the respiratory response to environmental challenge by SiO2. The objective of this study was to characterize the impact of SiO2 exposure on respiratory immune processes, with particular emphasis on evaluating the importance of lymphocytes in the murine silicosis model. Therefore, lymphopenic mice, including NK‐deficient, Rag1−/−, or a combination (Rag1−/− NK‐depleted), were used and demonstrated that SiO2‐induced fibrosis and inflammation can occur independently of T, B, NK T, and NK cells. Studies in Rag1−/− mice suggest further that lymphocytes may participate in the regulation of SiO2‐induced inflammation through modulation of the Nalp3 inflammasome. This observation may have clinical relevance in the treatment of inflammatory and fibrotic lung diseases that are refractory or respond suboptimally to current therapeutics.

The IL-4Rα pathway in macrophages and its potential role in silica-induced pulmonary fibrosis

Crystalline silica exposure can result in pulmonary fibrosis, where the pulmonary macrophage is key as a result of its ability to react to silica particles. In the mouse silicosis model, there is initial Th1‐type inflammation, characterized by TNF‐α and IFN‐γ. Previous studies determined that Th2 mediators (i.e., IL‐13) are vital to development of pulmonary fibrosis. The present study, using in vivo and in vitro techniques, compares silica exposures between Balb/c and Th2‐deficient mice in an effort to determine the link between Th2 immunity and silicosis. In long‐term experiments, a significant increase in fibrosis and activated interstitial macrophages was observed in Balb/c but not IL‐4Rα−/− mice. Additionally, a significant increase in Ym1 mRNA levels, a promoter of Th2 immunity, was determined in the interstitial leukocyte population of silica‐exposed Balb/c mice. To elucidate the effects of silica on macrophage function, bone marrow‐derived macrophages (BMdM) were exposed to particles and assayed for T cell (TC) stimulation activity. As a control, Ym1 mRNA expression in Balb/c BMdM was determined using IL‐4 stimulation. In the in vitro assay, a significant increase in TC activation, as defined by surface markers and cytokines, was observed in the cultures containing the silica‐exposed macrophages in wild‐type and IL‐4Rα−/− mice, with one exception: IL‐4Rα−/− BMdM were unable to induce an increase in IL‐13. These results suggest that crystalline silica alters cellular functions of macrophages, including activation of TC, and that the increase in Th2 immunity associated with silicosis is via the IL‐4Rα‐Ym1 pathway.

Urinary Levoglucosan as a Biomarker of Wood Smoke Exposure: Observations in a Mouse Model and in Children

Background Biomass smoke is an important source of particulate matter (PM), and much remains to be discovered with respect to the human health effects associated with this specific PM source. Exposure to biomass smoke can occur in one of two main categories: short-term exposures consist of periodic, seasonal exposures typified by communities near forest fires or intentional agricultural burning, and long-term exposures are chronic and typified by the use of biomass materials for cooking or heating. Levoglucosan (LG), a sugar anhydride released by combustion of cellulose-containing materials, is an attractive candidate as a biomarker of wood smoke exposure. Objectives In the present study, Balb/c mice and children were assessed for LG in urine to determine its feasibility as a biomarker. Methods We performed urinary detection of LG by gas chromatography/mass spectrometry after intranasal instillations of LG or concentrated PM (mice) or biomass exposure (mice or humans). Results After instillation, we recovered most of the LG within the first 4 hr. Experiments using glucose instillation proved the specificity of our system, and instillation of concentrated PM from wood smoke, ambient air, and diesel exhaust supported a connection between wood smoke and LG. In addition, LG was detected in the urine of mice exposed to wood smoke. Finally, a pilot human study proved our ability to detect LG in urine of children. Conclusions These results demonstrate that LG in the lungs is detectable in the urine of both mice and humans and that it is a good candidate as a biomarker of exposure to biomass smoke.

Extracellular HMGB1 regulates multi-walled carbon nanotube-induced inflammation in vivo

Abstract Endotoxin is often used to activate NF-κB in vitro when assessing NLRP3 inflammasome activation by various exogenous particles including nanoparticles. However, the endogenous source of this signal 1 is unknown. High-mobility group box 1 (HMGB1) is known to play a critical role in acute lung injury, however the potential contribution of the alarmin HMGB1 to NLRP3 Inflammasome activation has not been determined in response to nanoparticles in vivo. In this study, the ability of multi-walled carbon nanotubes (MWCNT) to cause release of HMGB1 in vitro and in vivo, as well as the potential of HMGB1 to function as signal 1 in vitro and in vivo, was determined. HMGB1 activity in vivo was assessed by administration of HMGB1 neutralization antibodies following MWCNT exposure. Caspase-1−/− mice were utilized to elucidate the dependence of HMGB1 secretion on NLRP3 inflammasome activity. MWCNT exposure increased extracellular HMGB1 levels in primary alveolar macrophages from C57Bl/6 mice and C10 mouse epithelial cell culture supernatants, and in C57Bl/6 mouse lung lavage fluid. MWCNT-induced HMGB1 secretion was dependent upon caspase-1. HMGB1 increased MWCNT-induced IL-1β release from macrophages in vitro, and neutralization of extracellular HMGB1 reduced MWCNT-induced IL-1β secretion in vivo. HMGB1 neutralization was accompanied with overall decreased inflammation. In summary, this study suggests extracellular HMGB1 participates in NLRP3 inflammasome activity and regulates IL-1β associated sterile inflammation induced by MWCNT.

Murine Pulmonary Inflammation Model: A Comparative Study of Anesthesia and Instillation Methods

Various techniques have been utilized historically to generate acute pulmonary inflammation in the murine system. Crystalline silica exposure results in acute inflammation followed by pulmonary fibrosis. Methods of exposure are varied in their techniques, as well as types of anesthesia. Therefore, the current study sought to compare the effects of two major anesthesia (isoflurane and ketamine) and three routes of instillation, intranasal (IN), intratracheal (IT), and trans-oral (TO), on markers of inflammation. Mice were anesthetized with isoflurane or ketamine and instilled IN with silica or phosphate-buffered saline (PBS). Mice were sacrificed and lavaged after 3 days. To assess inflammation, alveolar cells were assessed by cytospin and lavage fluid was analyzed for inflammatory cytokines and total protein. While all parameters were increased in silica-exposed groups, regardless of anesthesia type, there were significant increases in neutrophils and total protein in mice anesthetized with ketamine, compared to isoflurane. In comparing instillation techniques, mice were anesthetized with isoflurane and instilled IN, IT, or TO with silica. Increases were observed in all parameters, except tumor necrosis factor-α, following IT silica instillation as compared to the IN and TO instillation groups. In addition, fluorescent microsphere uptake by alveolar macrophages supported the notion that all methods of instillation were uniform, but IT had significantly greater dispersion. Taken together, these data show that each method of exposure tested generated significant inflammation among the silica groups, and any differences in parameters or techniques should be taken into consideration when developing an animal model to study pulmonary diseases.

Autophagy deficiency in macrophages enhances NLRP3 inflammasome activity and chronic lung disease following silica exposure.

Autophagy is an important metabolic mechanism that can promote cellular survival following injury. The specific contribution of autophagy to silica-induced inflammation and disease is not known. The objective of these studies was to determine the effects of silica exposure on the autophagic pathway in macrophages, as well as the general contribution of autophagy in macrophages to inflammation and disease. Silica exposure enhanced autophagic activity in vitro in Bone Marrow derived Macrophages and in vivo in Alveolar Macrophages isolated from silica-exposed mice. Impairment of autophagy in myeloid cells in vivo using Atg5(fl/fl)LysM-Cre(+) mice resulted in enhanced cytotoxicity and inflammation after silica exposure compared to littermate controls, including elevated IL-18 and the alarmin HMGB1 in the whole lavage fluid. Autophagy deficiency caused some spontaneous inflammation and disease. Greater silica-induced acute inflammation in Atg5(fl/fl)LysM-Cre(+) mice correlated with increased fibrosis and chronic lung disease. These studies demonstrate a critical role for autophagy in suppressing silica-induced cytotoxicity and inflammation in disease development. Furthermore, this data highlights the importance of basal autophagy in macrophages and other myeloid cells in maintaining lung homeostasis.

Phagolysosome acidification is required for silica and engineered nanoparticle-induced lysosome membrane permeabilization and resultant NLRP3 inflammasome activity

&NA; NLRP3 inflammasome activation occurs in response to hazardous particle exposures and is critical for the development of particle‐induced lung disease. Mechanisms of Lysosome Membrane Permeabilization (LMP), a central pathway for activation of the NLRP3 inflammasome by inhaled particles, are not fully understood. We demonstrate that the lysosomal vATPases inhibitor Bafilomycin A1 blocked LMP in vitro and ex vivo in primary murine macrophages following exposure to silica, multi‐walled carbon nanotubes, and titanium nanobelts. Bafilomycin A1 treatment of particle‐exposed macrophages also resulted in decreased active cathepsin L in the cytosol, a surrogate measure for leaked cathepsin B, which was associated with less NLRP3 inflammasome activity. Silica‐induced LMP was partially dependent upon lysosomal cathepsins B and L, whereas nanoparticle‐induced LMP occurred independent of cathepsin activity. Furthermore, inhibition of lysosomal cathepsin activity with CA‐074‐Me decreased the release of High Mobility Group Box 1. Together, these data support the notion that lysosome acidification is a prerequisite for particle‐induced LMP, and the resultant leak of lysosome cathepsins is a primary regulator of ongoing NLRP3 inflammasome activity and release of HMGB1. Graphical abstract Figure. No caption available. HighlightsSilica and nanoparticles cause LMP in macrophages in vitro and in vivo.Phagolysosome acidification is required for particle‐induced LMP.Cathepsin B and L are not required for nanoparticle‐induced LMP.Cathepsin B/L regulate the secretion of HMGB1 with particle exposure.

Approaching a Unified Theory for Particle-Induced Inflammation

Particles such as silica, asbestos, and engineered nanomaterials (ENM) that fall within a relatively small size range (<100 nm in at least one dimension) are known to have serious health consequences following exposure. Studies aimed at determining the mechanisms of toxicity for environmental particles have been ongoing for decades. However, the recent explosion of ENM into the market has resulted in the emergence of many recent studies aimed at determining the mechanisms underlying the pathologies associated with certain forms of ENM. In this chapter, we propose that many of the principles that have guided the toxicity studies of environmental particles may also apply to bioactive ENM. In fact, the initiating event for all downstream pathologies caused by exposure to both bioactive ENM and environmental particles appears to be lysosomal membrane permeabilization (LMP). Therefore, focusing on LMP as the “unifying” principle for particle toxicity studies may allow the field to advance at an increased pace.

Imipramine blocks acute silicosis in a mouse model

BackgroundInhalation of crystalline silica is associated with pulmonary inflammation and silicosis. Although silicosis remains a prevalent health problem throughout the world, effective treatment choices are limited. Imipramine (IMP) is a FDA approved tricyclic antidepressant drug with lysosomotropic characteristics. The aim of this study was to evaluate the potential for IMP to reduce silicosis and block phagolysosome membrane permeabilization.MethodsC57BL/6 alveolar macrophages (AM) exposed to crystalline silica ± IMP in vitro were assessed for IL-1β release, cytotoxicity, particle uptake, lysosomal stability, and acid sphingomyelinase activity. Short term (24xa0h) in vivo studies in mice instilled with silica (± IMP) evaluated inflammation and cytokine release, in addition to cytokine release from ex vivo cultured AM. Long term (six to ten weeks) in vivo studies in mice instilled with silica (± IMP) evaluated histopathology, lung damage, and hydroxyproline content as an indicator of collagen accumulation.ResultsIMP significantly attenuated silica-induced cytotoxicity and release of mature IL-1β from AM in vitro. IMP treatment in vivo reduced silica-induced inflammation in a short-term model. Furthermore, IMP was effective in blocking silica-induced lung damage and collagen deposition in a long-term model. The mechanism by which IMP reduces inflammation was explored by assessing cellular processes such as particle uptake and acid sphingomyelinase activity.ConclusionsTaken together, IMP was anti-inflammatory against silica exposure in vitro and in vivo. The results were consistent with IMP blocking silica-induced phagolysosomal lysis, thereby preventing cell death and IL-1β release. Thus, IMP could be therapeutic for silica-induced inflammation and subsequent disease progression as well as other diseases involving phagolysosomal lysis.

Acute Exposure to Crystalline Silica Reduces Macrophage Activation in Response to Bacterial Lipoproteins

Numerous studies have examined the relationship between alveolar macrophages (AMs) and crystalline silica (SiO2) using in vitro and in vivo immunotoxicity models; however, exactly how exposure to SiO2 alters the functionality of AM and the potential consequences for immunity to respiratory pathogens remains largely unknown. Because recognition and clearance of inhaled particulates and microbes are largely mediated by pattern recognition receptors (PRRs) on the surface of AM, we hypothesized that exposure to SiO2 limits the ability of AM to respond to bacterial challenge by altering PRR expression. Alveolar and bone marrow-derived macrophages downregulate TLR2 expression following acute SiO2 exposure (e.g., 4u2009h). Interestingly, these responses were dependent on interactions between SiO2 and the class A scavenger receptor CD204, but not MARCO. Furthermore, SiO2 exposure decreased uptake of fluorescently labeled Pam2CSK4 and Pam3CSK4, resulting in reduced secretion of IL-1β, but not IL-6. Collectively, our data suggest that SiO2 exposure alters AM phenotype, which in turn affects their ability to uptake and respond to bacterial lipoproteins.