The PINK1/Parkin pathway of mitophagy exerts a protective effect during prion disease.

The PINK1/Parkin pathway of mitophagy has been implicated in the pathogenesis of Parkinson's disease. In prion diseases, a transmissible neurodegenerative disease caused by the misfolded and infectious prion protein (PrPSc), expression of both PINK1 and Parkin are elevated, suggesting that PINK1/Parkin mediated mitophagy may also play a role in prion pathogenesis. Using mice in which expression of either PINK1 (PINK1KO) or Parkin (ParkinKO) has been ablated, we analyzed the potential role of PINK1 and Parkin in prion pathogenesis. Prion infected PINK1KO and ParkinKO mice succumbed to disease more rapidly (153 and 150 days, respectively) than wild-type control C57Bl/6 mice (161 days). Faster incubation times in PINK1KO and ParkinKO mice did not correlate with altered prion pathology in the brain, altered expression of proteins associated with mitochondrial dynamics, or prion-related changes in mitochondrial respiration. However, the expression level of mitochondrial respiration Complex I, a major site for the formation of reactive oxygen species (ROS), was higher in prion infected PINK1KO and ParkinKO mice when compared to prion infected control mice. Our results demonstrate a protective role for PINK1/Parkin mitophagy during prion disease, likely by helping to minimize ROS formation via Complex I, leading to slower prion disease progression.

Route of Francisella tularensis infection informs spatiotemporal metabolic reprogramming and inflammation in mice.

Route of exposure to pathogens can inform divergent disease pathogenesis and mortality rates. However, the features that contribute to these differences are not well established. Host metabolism has emerged as a critical element governing susceptibility and the metabolism of tissue exposure sites are unique. Therefore, specific metabolic niches may contribute to the course and outcome of infection depending on route of infection. In the current study, we utilized a combination of imaging and systems metabolomics to map the spatiotemporal dynamics of the host response to intranasal (i.n.) or intradermal (i.d.) infection of mice using the bacterium Francisella tularensis subsp tularensis (FTT). FTT causes lethal disease through these infection routes with similar inoculation doses and replication kinetics, which allowed for isolation of host outcomes independent of bacterial burden. We observed metabolic modifications that were both route dependent and independent. Specifically, i.d. infection resulted in early metabolic reprogramming at the site of infection and draining lymph nodes, whereas the lungs and associated draining lymph nodes were refractory to metabolic reprogramming following i.n. infection. Irrespective of exposure route, FTT promoted metabolic changes in systemic organs prior to colonization, and caused massive dysregulation of host metabolism in these tissues prior to onset of morbidity. Preconditioning infection sites towards a more glycolytic and pro-inflammatory state prior to infection exacerbated FTT replication within the lungs but not intradermal tissue. This enhancement of replication in the lungs was associated with the ability of FTT to limit redox imbalance and alter the pentose phosphate pathway. Together, these studies identify central metabolic features of the lung and dermal compartments that contribute to disease progression and identify potential tissue specific targets that may be exploited for novel therapeutic approaches.

Targeting 2-Oxoglutarate-Dependent Dioxygenases Promotes Metabolic Reprogramming That Protects against Lethal SARS-CoV-2 Infection in the K18-hACE2 Transgenic Mouse Model.

Dysregulation of host metabolism is a feature of lethal SARS-CoV-2 infection. Perturbations in α-ketoglutarate levels can elicit metabolic reprogramming through 2-oxoglutarate-dependent dioxygenases (2-ODDGs), leading to stabilization of the transcription factor HIF-1α. HIF1-α activation has been reported to promote antiviral mechanisms against SARS-CoV-2 through direct regulation of ACE2 expression (a receptor required for viral entry). However, given the numerous pathways HIF-1α serves to regulate it is possible that there are other undefined metabolic mechanisms contributing to the pathogenesis of SARS-CoV-2 independent of ACE2 downregulation. In this study, we used in vitro and in vivo models in which HIF-1α modulation of ACE2 expression was negated, allowing for isolated characterization of the host metabolic response within SARS-CoV-2 disease pathogenesis. We demonstrated that SARS-CoV-2 infection limited stabilization of HIF-1α and associated mitochondrial metabolic reprogramming by maintaining activity of the 2-ODDG prolyl hydroxylases. Inhibition of 2-ODDGs with dimethyloxalylglycine promoted HIF-1α stabilization following SARS-CoV-2 infection, and significantly increased survival among SARS-CoV-2-infected mice compared with vehicle controls. However, unlike previous reports, the mechanism by which activation of HIF-1α responses contributed to survival was not through impairment of viral replication. Rather, dimethyloxalylglycine treatment facilitated direct effects on host metabolism including increased glycolysis and resolution of dysregulated pools of metabolites, which correlated with reduced morbidity. Taken together, these data identify (to our knowledge) a novel function of α-ketoglutarate-sensing platforms, including those responsible for HIF-1α stabilization, in the resolution of SARS-CoV-2 infection and support targeting these metabolic nodes as a viable therapeutic strategy to limit disease severity during infection.

Effective inhibition of HCoV-OC43 and SARS-CoV-2 by phytochemicals in vitro and in vivo.

OBJECTIVE: Several coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human coronavirus OC43 (HCoV-OC43), can cause respiratory infections in humans. To address the need for reliable anti-coronavirus therapeutics, we screened 16 active phytochemicals selected from medicinal plants used in traditional applications for respiratory-related illnesses. METHODS: An initial screen was completed using HCoV-OC43 to identify compounds that inhibit virus-induced cytopathic effect (CPE) and cell death inhibition. Then the top hits were validated in vitro against both HCoV-OC43 and SARS-CoV-2 by determining virus titer in cell supernatant and virus-induced cell death. Finally, the most active phytochemical was validated in vivo in the SARS-CoV-2-infected B6.Cg-Tg(K18-ACE2)2Prlmn/J mouse model. RESULTS: The phytochemicals lycorine (LYC), capsaicin, rottlerin (RTL), piperine and chebulinic acid (CHU) inhibited HCoV-OC43-induced cytopathic effect and reduced viral titres by up to 4 log. LYC, RTL and CHU also suppressed virus replication and cell death following SARS-CoV-2 infection. In vivo, RTL significantly reduced SARS-CoV-2-induced mortality by ∼40% in human angiotensin-converting enzyme 2 (ACE2)-expressing K18 mice. CONCLUSION: Collectively, these studies indicate that RTL and other phytochemicals have therapeutic potential to reduce SARS-CoV-2 and HCoV-OC43 infections.

Contribution of Lipid Mediators in Divergent Outcomes following Acute Bacterial and Viral Lung Infections in the Obese Host.

Obesity is considered an important comorbidity for a range of noninfectious and infectious disease states including those that originate in the lung, yet the mechanisms that contribute to this susceptibility are not well defined. In this study, we used the diet-induced obesity (DIO) mouse model and two models of acute pulmonary infection, Francisella tularensis subspecies tularensis strain SchuS4 and SARS-CoV-2, to uncover the contribution of obesity in bacterial and viral disease. Whereas DIO mice were more resistant to infection with SchuS4, DIO animals were more susceptible to SARS-CoV-2 infection compared with regular weight mice. In both models, neither survival nor morbidity correlated with differences in pathogen load, overall cellularity, or influx of inflammatory cells in target organs of DIO and regular weight animals. Increased susceptibility was also not associated with exacerbated production of cytokines and chemokines in either model. Rather, we observed pathogen-specific dysregulation of the host lipidome that was associated with vulnerability to infection. Inhibition of specific pathways required for generation of lipid mediators reversed resistance to both bacterial and viral infection. Taken together, our data demonstrate disparity among obese individuals for control of lethal bacterial and viral infection and suggest that dysregulation of the host lipidome contributes to increased susceptibility to viral infection in the obese host.

Lack of the immune adaptor molecule SARM1 accelerates disease in prion infected mice and is associated with increased mitochondrial respiration and decreased expression of NRF2.

Prion diseases are a group of fatal, transmissible neurodegenerative diseases of mammals. In the brain, axonal loss and neuronal death are prominent in prion infection, but the mechanisms remain poorly understood. Sterile alpha and heat/Armadillo motif 1 (SARM1) is a protein expressed in neurons of the brain that plays a critical role in axonal degeneration. Following damage to axons, it acquires an NADase activity that helps to regulate mitochondrial health by breaking down NAD+, a molecule critical for mitochondrial respiration. SARM1 has been proposed to have a protective effect in prion disease, and we hypothesized that it its role in regulating mitochondrial energetics may be involved. We therefore analyzed mitochondrial respiration in SARM1 knockout mice (SARM1KO) and wild-type mice inoculated either with prions or normal brain homogenate. Pathologically, disease was similar in both strains of mice, suggesting that SARM1 mediated axonal degradation is not the sole mechanism of axonal loss during prion disease. However, mitochondrial respiration was significantly increased and disease incubation time accelerated in prion infected SARM1KO mice when compared to wild-type mice. Increased levels of mitochondrial complexes II and IV and decreased levels of NRF2, a potent regulator of reactive oxygen species, were also apparent in the brains of SARM1KO mice when compared to wild-type mice. Our data suggest that SARM1 slows prion disease progression, likely by regulating mitochondrial respiration, which may help to mitigate oxidative stress via NRF2.

Impairing RAGE signaling promotes survival and limits disease pathogenesis following SARS-CoV-2 infection in mice.

Cellular and molecular mechanisms driving morbidity following SARS-CoV-2 infection have not been well defined. The receptor for advanced glycation end products (RAGE) is a central mediator of tissue injury and contributes to SARS-CoV-2 disease pathogenesis. In this study, we temporally delineated key cell and molecular events leading to lung injury in mice following SARS-CoV-2 infection and assessed efficacy of therapeutically targeting RAGE to improve survival. Early following infection, SARS-CoV-2 replicated to high titers within the lungs and evaded triggering inflammation and cell death. However, a significant necrotic cell death event in CD45- populations, corresponding with peak viral loads, was observed on day 2 after infection. Metabolic reprogramming and inflammation were initiated following this cell death event and corresponded with increased lung interstitial pneumonia, perivascular inflammation, and endothelial hyperplasia together with decreased oxygen saturation. Therapeutic treatment with the RAGE antagonist FPS-ZM1 improved survival in infected mice and limited inflammation and associated perivascular pathology. Together, these results provide critical characterization of disease pathogenesis in the mouse model and implicate a role for RAGE signaling as a therapeutic target to improve outcomes following SARS-CoV-2 infection.

Mitophagy antagonism by ZIKV reveals Ajuba as a regulator of PINK1 signaling, PKR-dependent inflammation, and viral invasion of tissues.

Dysregulated inflammation dominated by chemokine expression is a key feature of disease following infection with the globally important human pathogens Zika virus (ZIKV) and dengue virus, but a mechanistic understanding of how pro-inflammatory responses are initiated is lacking. Mitophagy is a quality-control mechanism that regulates innate immune signaling and cytokine production through selective degradation of damaged mitochondria. Here, we demonstrate that ZIKV nonstructural protein 5 (NS5) antagonizes mitophagy by binding to the host protein Ajuba and preventing its translocation to depolarized mitochondria where it is required for PINK1 activation and downstream signaling. Consequent mitophagy suppression amplifies the production of pro-inflammatory chemokines through protein kinase R (PKR) sensing of mitochondrial RNA. In Ajuba-/- mice, ZIKV induces early expression of pro-inflammatory chemokines associated with significantly enhanced dissemination to tissues. This work identifies Ajuba as a critical regulator of mitophagy and demonstrates a role for mitophagy in limiting systemic inflammation following infection by globally important human viruses.

Cutting Edge: Lung-Resident T Cells Elicited by SARS-CoV-2 Do Not Mediate Protection against Secondary Infection.

Immunity to pulmonary infection typically requires elicitation of lung-resident T cells that subsequently confer protection against secondary infection. The presence of tissue-resident T cells in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) convalescent patients is unknown. Using a sublethal mouse model of coronavirus disease 2019, we determined if SARS-CoV-2 infection potentiated Ag-specific pulmonary resident CD4+ and CD8+ T cell responses and if these cells mediated protection against secondary infection. S protein-specific T cells were present in resident and circulating populations. However, M and N protein-specific T cells were detected only in the resident T cell pool. Using an adoptive transfer strategy, we found that T cells from SARS-CoV-2 immune animals did not protect naive mice. These data indicate that resident T cells are elicited by SARS-CoV-2 infection but are not sufficient for protective immunity.

Interferon Gamma Reprograms Host Mitochondrial Metabolism through Inhibition of Complex II To Control Intracellular Bacterial Replication.

The mechanisms by which interferon gamma (IFN-γ) controls the replication of cytosolic pathogens independent of responses, such as the generation of reactive oxygen species/reactive nitrogen species (ROS/RNS), have not been fully elucidated. In the current study, we developed a model using Francisella tularensis, the causative agent of tularemia, in which pathways triggered by IFN-γ commonly associated with bacterial control were not required. Using this model, we demonstrated that IFN-γ-mediated production of itaconate and its ability to impair host mitochondrial function, independent of activity on the pathogen, were central for the restriction of bacterial replication in vitro and in vivo We then demonstrate that IFN-γ-driven itaconate production was dispensable, as directly targeting complex II using cell membrane-permeable metabolites also controlled infection. Together, these findings show that while reprogramming of mitochondrial metabolism is a key factor in IFN-γ control of intracellular bacteria, the development of antimicrobial strategies based on targeting host mitochondrial metabolism independent of this cytokine may be an effective therapeutic approach.

Temporal Manipulation of Mitochondrial Function by Virulent Francisella tularensis To Limit Inflammation and Control Cell Death.

Francisella tularensis subsp. tularensis is a highly pathogenic intracellular bacterium that suppresses host inflammation by impairing the metabolic shift from oxidative phosphorylation to glycolysis. Decreased mitochondrial metabolism is central to initiating a metabolic shift to glycolysis and regulating inflammation, but F. tularensis subsp. tularensis manipulation of host mitochondrial function has not been explored. We demonstrate, using extracellular flux analysis, that F. tularensis subsp. tularensis infection initially improves host macrophage mitochondrial bioenergetics in a capsule-dependent manner. Enhancement of mitochondrial function by F. tularensis subsp. tularensis allowed for modest replication and inhibition of apoptosis early after infection. However, using live cell imaging, we found that F. tularensis subsp. tularensis facilitated the loss of mitochondrial function at later time points during infection in a capsule-independent fashion. This loss of function was paired with oncosis and rapid bacterial replication. Inhibition of oncosis reduced intracellular bacterial numbers, underscoring the requirement for this process during F. tularensis subsp. tularensis infection. These findings establish that temporal mitochondrial manipulation by F. tularensis subsp. tularensis is critical for maintenance of a noninflammatory environment and subsequently aids in optimal replication and dissemination of this pathogenic organism.

Imipramine blocks acute silicosis in a mouse model.

BACKGROUND: Inhalation 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. METHODS: C57BL/6 alveolar macrophages (AM) exposed to crystalline silica ± IMP in vitro were assessed for IL-1ß release, cytotoxicity, particle uptake, lysosomal stability, and acid sphingomyelinase activity. Short term (24 h) 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. RESULTS: IMP significantly attenuated silica-induced cytotoxicity and release of mature IL-1ß 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. CONCLUSIONS: Taken 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-1ß release. Thus, IMP could be therapeutic for silica-induced inflammation and subsequent disease progression as well as other diseases involving phagolysosomal lysis.

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

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.

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.

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., 4 h). 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-1ß, 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.

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

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-1ß release from macrophages in vitro, and neutralization of extracellular HMGB1 reduced MWCNT-induced IL-1ß 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-1ß associated sterile inflammation induced by MWCNT.

Adverse effects of wood smoke PM(2.5) exposure on macrophage functions.

Epidemiological studies have shown a correlation between chronic biomass smoke exposure and increased respiratory infection. Pulmonary macrophages are instrumental in both the innate and the adaptive immune responses to respiratory infection. In the present study, in vitro systems were utilized where alveolar macrophages (AM) and bone marrow-derived macrophages (BMdM) were exposed to concentrated wood smoke-derived particulate matter (WS-PM) and mice were exposed in vivo to either concentrated WS-PM or inhaled WS. In vivo studies demonstrated that WS-exposed mice inoculated with Streptococcus pneumoniae had a higher bacterial load 24 h post-exposure, and corresponding AM were found to have decreased lymphocyte activation activity. Additionally, while classic markers of inflammation (cellular infiltration, total protein, neutrophils) were not affected, there were changes in pulmonary macrophages populations, including significant decreases in macrophages expressing markers of activation in WS-exposed mice. The lymphocyte activation activity of WS-PM-exposed AM was significantly suppressed, but the phagocytic activity appeared unchanged. In an effort to determine a pathway for WS-induced suppression, RelB activation, assessed by nuclear translocation, was observed in AM exposed to either inhaled WS or instilled WS-PM. Finally, an analysis of WS-PM fractions determined the presence of 4-5 polycyclic aromatic hydrocarbons (PAHs), and preliminary work suggests a potential role for these PAHs to alter macrophage functions. These studies show a decreased ability of WS-exposed pulmonary macrophages to effectively mount a defense against infection, the effect lasts at least a week post-exposure, and appears to be mediated via RelB activation.

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 SiO(2) 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 SiO(2). The objective of this study was to characterize the impact of SiO(2) 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 SiO(2)-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 SiO(2)-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.

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-alpha, 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.

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.