Damage associated molecular patterns (DAMPs) are molecules released from dead/dying cells following toxicant and/or environmental exposures that activate the immune response through binding of ...pattern recognition receptors (PRRs). Excessive production of DAMPs or failed clearance leads to chronic inflammation and delayed inflammation resolution. One category of DAMPs are oxidized phospholipids (oxPLs) produced upon exposure to high levels of oxidative stress, such as following ozone (O3) induced inflammation. OxPLs are bound by multiple classes of PRRs that include scavenger receptors (SRs) such as SR class B-1 (SR-BI) and toll-like receptors (TLRs). Interactions between oxPLs and PRRs appear to regulate inflammation; however, the role of SR-BI in oxPL-induced lung inflammation has not been defined. Therefore, we hypothesize that SR-BI is critical in protecting the lung from oxPL-induced pulmonary inflammation/injury. To test this hypothesis, C57BL/6J (WT) female mice were dosed with oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (oxPAPC) by oropharyngeal aspiration which increased pulmonary SR-BI expression. Following oxPAPC exposure, SR-BI deficient (SR-BI−/−) mice exhibited increased lung pathology and inflammatory cytokine/chemokine production. Lipidomic analysis revealed that SR-BI−/− mice had an altered pulmonary lipidome prior to and following oxPAPC exposure, which correlated with increased oxidized phosphatidylcholines (PCs). Finally, we characterized TLR4-mediated activation of NF-κB following oxPAPC exposure and discovered that SR-BI−/− mice had increased TLR4 mRNA expression in lung tissue and macrophages, increased nuclear p65, and decreased cytoplasmic IκBα. Overall, we conclude that SR-BI is required for limiting oxPAPC-induced lung pathology by maintaining lipid homeostasis, reducing oxidized PCs, and attenuating TLR4-NF-κB activation, thereby preventing excessive and persistent inflammation.
•These studies demonstrate that scavenger receptor BI (SR-BI) limits the lung pathology and inflammation following oxidized phospholipids (oxPL) exposure.•These findings indicate that if SR-BI is dysfunctional, there is a lack of oxPL clearance and enhance TLR4 signalling in the lung.•SR-BI is also crucial for maintaining lipid metabolism in the lung at homeostasis and during inflammation
Electrostatic Dust Collectors (EDCs) are in use for passive sampling of bioaerosols, but particular aspects of their performance have not yet been evaluated. This study investigated the effect of ...mailing EDCs on endotoxin loading and the effect of EDC deployment in front of, and away from, heated ventilation on endotoxin sampling. Endotoxin sampling efficiency of heated and unheated EDC cloths was also evaluated. Cross-country express mailing of dust-spiked EDCs yielded no significant changes in endotoxin concentrations compared to dust-only samples for both high-spiked EDCs (p = 0.30) and low-spiked EDCs (p = 0.36). EDCs were also deployed in 20 identical apartments with one EDC placed in front of the univent heater in each apartment and contemporaneous EDC placed on the built-in bookshelf in each apartment. The endotoxin concentrations were significantly different (p = 0.049) indicating that the placement of EDC does impact endotoxin sampling. Heated and unheated EDCs were deployed for 7 days in pairs in farm homes. There was a significant difference between endotoxin concentrations (p = 0.027) indicating that heating EDCs may diminish their electrostatic capabilities and impact endotoxin sampling. The last study investigated the electrostatic charge of 12 heated and 12 unheated EDC cloths. There was a significant difference in charge (p = 0.009) which suggests that heating EDC cloths may make them less effective for sampling. In conclusion, EDCs can be mailed to and from deployment sites, EDC placement in relationship to ventilation is crucial, and heating EDCs reduces their electrostatic charge which may diminish their endotoxin sampling capabilities.
Abstract
Asthma is a heterogeneous airway disease that has increased significantly in recent decades. Clinical studies have shown a positive correlation between serum large high-density lipoprotein ...(HDL) levels and airway protective effects in asthma. However, how cholesterol communicates with the lung during asthma is still not well understood. Scavenger receptor class B type I (SR-BI) is an HDL receptor that has also been shown to regulate glucocorticoid production. SR-BI has also been shown to regulate lymphocyte apoptosis in models of sepsis and atherosclerosis. Recently, we reported that SR-BI dampens the pulmonary innate immune response, but the role of SR-BI in asthma is unknown. Therefore, SR-BI+/+ and SR-BI−/− mice were sensitized with 10 μg house dust mite (HDM) or PBS by oropharyngeal aspiration on day 0, 7, and challenged with 2 μg HDM on day 14, 15, 16. Airway inflammation, cytokines, and serum corticosterone levels were quantified on day 17. SR-BI−/−mice had increased pulmonary neutrophils and lymphocytes after HDM challenge when compared to SR-BI+/+ mice. SR-BI−/− mice had consistently lower levels of serum corticosterone after HDM or PBS exposure. To determine the role of glucocorticoid production in asthma, SR-BI+/+ and SR-BI−/− mice were supplemented with corticosterone in their drinking water during HDM exposure. SR-BI−/− mice with corticosterone treatment had a decrease in pulmonary lymphocytes after HDM exposure. The observed decrease in lymphocytes may be due to glucocorticoid induced apoptosis which was absent in SR-BI−/− mice not treated with glucocorticoids. Our study has shed new light on how cholesterol receptors such as SR-BI play an important role in glucocorticoid mediated lymphocyte apoptosis in asthma.
Abstract
Asthma impacts over 24 million people and costs about $56 billion annually in the United States. Airway neutrophil accumulation and high levels of IL-17 are characteristic of severe asthma. ...However, mechanisms underlying IL-17/neutrophilic inflammation in severe asthma remain poorly defined. Scavenger receptor class B type I (SR-BI) is a multi-recognition receptor that regulates cholesterol trafficking. We previously reported SR-BI dampens pulmonary innate immune responses during bacterial pneumonia, but the role of SR-BI in asthma is unknown. We hypothesize pulmonary SR-BI expression is protective against house dust mite (HDM)-induced IL-17-dependent neutrophilic asthma. SR-BI+/+ and SR-BI−/− mice were sensitized with 10 μg HDM by oropharyngeal aspiration on day 0 and 7, and challenged with 2 μg HDM on day 14, 15, 16. Airway inflammation and cytokine production were quantified on day 17. SR-BI−/− mice had increased neutrophils but decreased eosinophils in bronchoalveolar lavage (BAL) after HDM challenge compared to SR-BI+/+ mice. BAL levels of neutrophil chemoattractants CXCL1, CXCL2 and CXCL5 were not significantly different between SR-BI+/+ and SR-BI−/− mice. SR-BI−/− mice had increased pulmonary IL-17A and GM-CSF production and decreased IL-5 production. To identify cellular sources of airspace IL-17, single cells isolated from lung were labeled with antibodies for flow cytometry. IL-17A increased in neutrophils (CD45+Ly6G+) and macrophages (CD45+CD64+CD11b−CD11c+) but not T cells (CD45+CD4+) of asthmatic SR-BI−/− mice suggesting myeloid cells rather than Th17 cells as sources of IL-17. Overall, SR-BI expression in the lung suppresses neutrophilic inflammation and IL-17 production in HDM-induced asthma model.
Influenza-Mediated Lung Infection Models McGee, Charles E; Sample, Christopher J; Kilburg-Basnyat, Brita ...
Methods in molecular biology,
2019, Letnik:
1960
Journal Article
Odprti dostop
Laboratory rodent influenza infection models have been and continue to be a critical tool for understanding virus-host interactions during infection. The incidence of seasonal influenza infections ...coupled with the need for novel therapeutics and universal vaccines highlights the need to uncover novel mechanisms of pathogenesis and protection. Mouse models are extremely useful for the evaluation of influenza vaccines and provide an invaluable tool to probe the immune response. This chapter describes the technique of intranasal inoculation of male C57BL/6J mice with an H1N1 strain of influenza (A/Puerto Rico/8/1934) and methods for assessing the optimum dose for infection, viral titers in lung tissue, and severity of disease.
Increases in ambient levels of ozone (O
), a criteria air pollutant, have been associated with increased susceptibility and exacerbations of chronic pulmonary diseases through lung injury and ...inflammation. O
induces pulmonary inflammation, in part by generating damage-associated molecular patterns (DAMPs), which are recognized by pattern recognition receptors (PRRs), such as toll-like receptors (TLRs) and scavenger receptors (SRs). This inflammatory response is mediated in part by alveolar macrophages (AMs), which highly express PRRs, including scavenger receptor BI (SR-BI). Once pulmonary inflammation has been induced, an active process of resolution occurs in order to prevent secondary necrosis and to restore tissue homeostasis. The processes known to promote the resolution of inflammation include the clearance by macrophages of apoptotic cells, known as efferocytosis, and the production of specialized pro-resolving mediators (SPMs). Impaired efferocytosis and production of SPMs have been associated with the pathogenesis of chronic lung diseases; however, these impairments have yet to be linked with exposure to air pollutants.
The primary goals of this study were: Aim 1 - to define the role of SR-BI in O
-derived pulmonary inflammation and resolution of injury; and Aim 2 - to determine if O
exposure alters pulmonary production of SPMs and processes known to promote the resolution of pulmonary inflammation and injury.
To address Aim 1, female wild-type (WT) and SR-BI-deficient, or knock-out (SR-BI KO), mice were exposed to either O
or filtered air. In one set of experiments mice were instilled with an oxidized phospholipid (oxPL). Bronchoalveolar lavage fluid (BALF) and lung tissue were collected for the analyses of inflammatory and injury markers and oxPL. To estimate efferocytosis, mice were administered apoptotic cells (derived from the Jurkat T cell line) after O
or filtered air exposure.
To address Aim 2, male WT mice were exposed to either O
or filtered air, and levels of SPMs were assessed in the lung, as well as markers of inflammation and injury in BALF. In some experiments SPMs were administered before exposure to O
or filtered air, to determine whether SPMs could mitigate inflammatory or resolution responses. Efferocytosis was measured as in Aim 1.
For Aim 1, SR-BI protein levels increased in the lung tissue of mice exposed to O
, compared with mice exposed to filtered air. Compared with WT controls, SR-BI KO mice had a significant increase in the number of neutrophils in their airspace 24 hours post O
exposure. The oxPL levels increased in the airspace of both WT and SR-BI KO mice after O
exposure, compared with filtered air controls. Four hours after instillation of an oxPL, SR-BI KO mice had an increase in BALF neutrophils and total protein, and a nonsignificant increase in macrophages compared with WT controls. O
exposure decreased efferocytosis in both WT and SR-BI KO female mice.
For Aim 2, mice given SPM supplementation before O
exposure showed significantly increased AM efferocytosis when compared with the O
exposure control mice and also showed some mitigation of the effects of O
on inflammation and injury. Several SPMs and their precursors were measured in lung tissue using reverse-phase high performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS). At 24 hours after O
exposure 14R-hydroxydocosahexaenoic acid (HDHA) and 10,17-dihydroxydocosahexaenoic acid (diHDoHE) were significantly decreased in lung tissue, but at 6 hours after exposure, levels of these SPMs increased.
Our findings identify novel mechanisms by which O
may induce pulmonary inflammation and also increase susceptibility to and exacerbations of chronic lung diseases.
Electrostatic Dust Collectors (EDCs) are a passive sampling method that has not yet been fully validated. Our first study investigated the effect of EDC mailing and EDC deployment in front of and ...away from heated ventilation on endotoxin concentrations. Endotoxin sampling efficiency of heated and unheated EDC cloths was evaluated. EDCs express mailed cross-country yielded no significant changes in endotoxin concentrations when dust-only samples were compared to high quality control (QC) spiked-EDCs (p=0.21) and low QC spiked-EDCs (p=0.16). EDCs were deployed in 20 apartments with one EDC placed in front of the univent heater and another EDC placed on a built-in bookshelf. Endotoxin concentrations were significantly different (p=0.049) indicating that EDC placement impacts endotoxin sampling. Heated and unheated EDCs were deployed for 7 days in farm homes. There was a significant difference between endotoxin concentrations ( p=0.027). The electrostatic charge of 12 heated and 12 unheated EDC cloths were significantly different (p=0.009). These studies suggest that heating cloths may diminish their electrostatic charge and endotoxin sampling capabilities. The EDC sampling time needed to achieve detectable and reproducible loading for bioaerosols has not been systematically evaluated. In our second study, EDCs were deployed in 15 Iowa farm homes for 7-, 14-, and 28-day sampling periods to determine if endotoxin and allergens could be quantified and if loading rates were uniform (i.e. doubling from 7 to 14 days and 14 to 28 days and quadrupling from 7 to 28 days). Loadings between left and right paired EDC cloths were not significantly different and were highly correlated for endotoxin, total protein, and cat (Fel d1), dog ( Can f1) and mouse (Mus m1) allergens (p <0.001). EDC endotoxin sampling had close agreement between paired samples (Pearson r=0.96, p<0.001). EDC endotoxin loading doubled from 7 to 14-day deployments but the loading rate decreased from 14 to 28 days of sampling with only a 1.38 fold increase. Allergen exposure assessment using EDCs was less satisfactory. Paired EDCs and daily Button aerosol samplers (BS) were used in our third study to concurrently sample endotoxin in 10 farm homes during 7 day periods in summer and winter. Winter sampling included an optical particle counter (OPC) for particulate size and number concentration data. OPC particulate matter (PM) data were divided into PM2.5 and PM10-2.5. Summer sampling yielded geometric mean and geometric standard deviation values of 0.82 EU/m3 (2.7) for inhalable aerosol BS and 737 EU/m 2 (1.9) for EDCs. Winter values were 0.52 EU/m3 (3.1) for BS and 538 EU/m2 (3.0) for EDCs. Seven day endotoxin values of EDCs were significantly and highly correlated with the 7-day BS sampling averages (r=0.70; p<0.001). An Analysis of Variance indicated a 2.37-fold increase in EDC endotoxin concentrations for each unit increase of the ratio of PM2.5 to PM10-2.5. A 10-fold increase in BS endotoxin concentrations was associated with a 12.2-fold increase in EDC endotoxin concentrations. Our fourth study established QC protocols use of EDCs in large field studies. QCs were developed for endotoxin, peptidoglycan, and glucan for analysis alongside the Agricultural Lung Health study EDC samples. The coefficient of variation percentage (CV) for each QC was used to determine variability. For each QC, 20 EDC cloths were analyzed to establish an acceptable range (mean ± 3 standard deviations). Two QCs were established for endotoxin analysis. The high QCs were dust-spiked EDCs with a CV of 29.7%. The low QCs were spiked with E. coli standard and had a CV of 15.6%. One QC was established for peptidoglycan analysis using dust-spiked EDC extracts. Two glucan QCs were established using dust-spiked EDCs with a high CV (51.7%) and yeast-spiked EDCs with a CV of 26.0%. Endotoxin and glucan concentrations of AGLH EDC samples were found to be significantly correlated (r=0.71; p<0.0001). In conclusion, EDCs are an effective passive sampling method for endotoxin exposure assessment in farm homes.
The electrostatic dust collector (EDC) is a passive dust sampling device for exposure assessment of airborne endotoxin and possibly allergens. EDCs consist of a non-conducting plastic folder holding ...two or four electrostatic cloths of defined area. The sampling time needed to achieve detectable and reproducible loading for bioaerosols has not been systematically evaluated. Thus, in 15 Iowa farm homes EDCs were deployed for 7-, 14-, and 28-day sampling periods to determine if endotoxin and allergens could be quantified and if loading rates were uniform over time, i.e. if loads doubled from 7 to 14 days or 14 to 28 days and quadrupled from 7 to 28 days. Loadings between left and right paired EDC cloths were not significantly different and were highly correlated for endotoxin, total protein, and cat (Fel d1), dog (Can f1), and mouse (Mus m1) allergens (P < 0.001). EDCs performed especially well for endotoxin sampling with close agreement between paired samples (Pearson r = 0.96, P < 0.001). Endotoxin loading of the EDCs doubled from 7- to 14-day deployments as hypothesized although the loading rate decreased from 14 to 28 days of sampling with only a 1.38-fold increase. Allergen exposure assessment using EDCs was overall less satisfactory. Although there was reasonable agreement between paired samples, only exposures to cat, dog, and mouse allergens were reliable and these only at the longer deployment times.
Electrostatic Dust Collectors (EDCs) are a passive sampling method that has not yet been fully validated. Our first study investigated the effect of EDC mailing and EDC deployment in front of and ...away from heated ventilation on endotoxin concentrations. Endotoxin sampling efficiency of heated and unheated EDC cloths was evaluated. EDCs express mailed cross-country yielded no significant changes in endotoxin concentrations when dust-only samples were compared to high quality control (QC) spiked-EDCs (p=0.21) and low QC spiked-EDCs (p=0.16). EDCs were deployed in 20 apartments with one EDC placed in front of the univent heater and another EDC placed on a built-in bookshelf. Endotoxin concentrations were significantly different (p=0.049) indicating that EDC placement impacts endotoxin sampling. Heated and unheated EDCs were deployed for 7 days in farm homes. There was a significant difference between endotoxin concentrations (p=0.027). The electrostatic charge of 12 heated and 12 unheated EDC cloths were significantly different (p=0.009). These studies suggest that heating cloths may diminish their electrostatic charge and endotoxin sampling capabilities.
The EDC sampling time needed to achieve detectable and reproducible loading for bioaerosols has not been systematically evaluated. In our second study, EDCs were deployed in 15 Iowa farm homes for 7-, 14-, and 28-day sampling periods to determine if endotoxin and allergens could be quantified and if loading rates were uniform (i.e. doubling from 7 to 14 days and 14 to 28 days and quadrupling from 7 to 28 days). Loadings between left and right paired EDC cloths were not significantly different and were highly correlated for endotoxin, total protein, and cat (Fel d1), dog (Can f1) and mouse (Mus m1) allergens (p<0.001). EDC endotoxin sampling had close agreement between paired samples (Pearson p=0.96, p<0.001). EDC endotoxin loading doubled from 7 to 14-day deployments but the loading rate decreased from 14 to 28 days of sampling with only a 1.38 fold increase. Allergen exposure assessment using EDCs was less satisfactory.
Paired EDCs and daily Button aerosol samplers (BS) were used in our third study to concurrently sample endotoxin in 10 farm homes during 7 day periods in summer and winter. Winter sampling included an optical particle counter (OPC) for particulate size and number concentration data. OPC particulate matter (PM) data were divided into PM2.5 and PM10-2.5. Summer sampling yielded geometric mean and geometric standard deviation values of 0.82 EU/m3 (2.7) for inhalable aerosol BS and 737 EU/m2 (1.9) for EDCs. Winter values were 0.52 EU/m3 (3.1) for BS and 538 EU/m2 (3.0) for EDCs. Seven day endotoxin values of EDCs were significantly and highly correlated with the 7-day BS sampling averages (p=0.70; p<0.001). An Analysis of Variance indicated a 2.37-fold increase in EDC endotoxin concentrations for each unit increase of the ratio of PM2.5 to PM10-2.5. A 10-fold increase in BS endotoxin concentrations was associated with a 12.2-fold increase in EDC endotoxin concentrations.
Our fourth study established QC protocols use of EDCs in large field studies. QCs were developed for endotoxin, peptidoglycan, and glucan for analysis alongside the Agricultural Lung Health study EDC samples. The coefficient of variation percentage (CV) for each QC was used to determine variability. For each QC, 20 EDC cloths were analyzed to establish an acceptable range (mean ± 3 standard deviations). Two QCs were established for endotoxin analysis. The high QCs were dust-spiked EDCs with a CV of 29.7%. The low QCs were spiked with E. coli standard and had a CV of 15.6%. One QC was established for peptidoglycan analysis using dust-spiked EDC extracts. Two glucan QCs were established using dust-spiked EDCs with a high CV (51.7%) and yeast-spiked EDCs with a CV of 26.0%. Endotoxin and glucan concentrations of AGLH EDC samples were found to be significantly correlated (p=0.71; p<0.0001). In conclusion, EDCs are an effective passive sampling method for endotoxin exposure assessment in farm homes.
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