Accurate and reproducible assessments of experimental lung injury and inflammation are critical for basic and translational research. In particular, investigators use various methods for BAL and ...euthanasia; however, the impact of these methods on assessments of injury and inflammation is unknown. To define potential effects, we compared methods of lavage and euthanasia in uninjured mice and after a mild lung injury model (ozone). C57BL/6J male mice (8-10 weeks old) underwent BAL after euthanasia with ketamine/xylazine, carbon dioxide (CO
), or isoflurane. BAL methods included 800 μl of isotonic solution instilled and withdrawn three times, and one or three passive fills and drainage to 20 cm H
O. Parallel experiments were performed 24 hours after 3 hours of ozone (O
) exposure at 2 ppm. BAL total cell counts/differentials and total protein/albumin were determined. Lung histology was evaluated for lung inflammation or injury. BAL cells were cultured and stimulated with PBS, PMA, or LPS for 4 hours and supernatants were evaluated for cytokine content. In uninjured mice, we observed differences due to the lavage and euthanasia methods used. The lavage method increased total cells and total protein/albumin in uninjured and O
-exposed mice, with the 800-μl instillation having the highest values. Isoflurane increased total BAL cells, whereas CO
euthanasia increased the total protein/albumin levels in uninjured mice. These effects limited our ability to detect differences in BAL injury measures after O
exposure. In conclusion, the method used for lavage and euthanasia affects measures of lung inflammation/injury and should be considered a variable in model assessments.
Abstract
Ozone (O3) is a criteria air pollutant known to increase the morbidity and mortality of cardiopulmonary diseases. This occurs through a pulmonary inflammatory response characterized by ...increased recruitment of immune cells into the airspace, pro-inflammatory cytokines, and pro-inflammatory lipid mediators. Recent evidence has demonstrated sex-dependent differences in the O3-induced pulmonary inflammatory response. However, it is unknown if this dimorphic response is evident in pulmonary lipid mediator metabolism. We hypothesized that there are sex-dependent differences in lipid mediator production following acute O3 exposure. Male and female C57BL/6J mice were exposed to 1 part per million O3 for 3 h and were necropsied at 6 or 24 h following exposure. Lung lavage was collected for cell differential and total protein analysis, and lung tissue was collected for mRNA analysis, metabololipidomics, and immunohistochemistry. Compared with males, O3-exposed female mice had increases in airspace neutrophilia, neutrophil chemokine mRNA, pro-inflammatory eicosanoids such as prostaglandin E2, and specialized pro-resolving mediators (SPMs), such as resolvin D5 in lung tissue. Likewise, precursor fatty acids (arachidonic and docosahexaenoic acid; DHA) were increased in female lung tissue following O3 exposure compared with males. Experiments with ovariectomized females revealed that loss of ovarian hormones exacerbates pulmonary inflammation and injury. However, eicosanoid and SPM production were not altered by ovariectomy despite depleted pulmonary DHA concentrations. Taken together, these data indicate that O3 drives an increased pulmonary inflammatory and bioactive lipid mediator response in females. Furthermore, ovariectomy increases susceptibility to O3-induced pulmonary inflammation and injury, as well as decreases pulmonary DHA concentrations.
We describe here the gross and microscopic lesions in 18 experimentally induced and 120 natural Campylobacter abortions. In natural Campylobacter abortions, gross lesions were reported infrequently; ...placentitis was recorded in 6% and hepatic lesions in 4% of our field cases. Placentitis was the microscopic lesion identified most consistently in natural abortions (93%) and was often observed in association with abundant bacterial colonies in chorionic villi (54%) and less often with placental vasculitis (13%). In natural abortions, suppurative fetal pneumonia (48%), necrosuppurative hepatitis (16%), and purulent meningitis (7%) were also observed. The better-preserved specimens from experimentally induced abortions were utilized to define placental changes more precisely. Placentitis was identified in all 18 experimentally induced abortions and was observed most consistently in the chorionic villus stroma (100%), often accompanied by suppurative surface exudate (89%). An inflammatory infiltrate was less commonly identified in the cotyledonary hilus (39%) and intercotyledonary placenta (22%). Bacteria were visualized in H&E-stained sections in 89% of placentas from experimentally infected ewes, primarily as well-demarcated bacterial colonies within subtrophoblastic, sinusoidal capillaries (89%), in the cotyledonary villus stroma (89%), and within the cytoplasm of trophoblasts (22%). Transmission electron microscopy and immunohistochemistry confirmed that the vast majority of the well-demarcated bacterial colonies characteristic of Campylobacter abortion were within subtrophoblastic sinusoidal capillaries. The most characteristic microscopic lesions identified in cases of Campylobacter abortion in sheep were placentitis with placental bacterial colonies, placental vasculitis, and fetal pneumonia.
The Ikaros zinc-finger transcription factor Eos has largely been associated with sustaining the immunosuppressive functions of regulatory T cells. Paradoxically, Eos has more recently been implicated ...in promoting proinflammatory responses in the dysregulated setting of autoimmunity. However, the precise role of Eos in regulating the differentiation and function of effector CD4+ T cell subsets remains unclear. In this study, we find that Eos is a positive regulator of the differentiation of murine CD4+ TH2 cells, an effector population that has been implicated in both immunity against helminthic parasites and the induction of allergic asthma. Using murine in vitro TH2 polarization and an in vivo house dust mite asthma model, we find that EosKO T cells exhibit reduced expression of key TH2 transcription factors, effector cytokines, and cytokine receptors. Mechanistically, we find that the IL-2/STAT5 axis and its downstream TH2 gene targets are one of the most significantly downregulated pathways in Eos-deficient cells. Consistent with these observations, we find that Eos forms, to our knowledge, a novel complex with and supports the tyrosine phosphorylation of STAT5. Collectively, these data define a regulatory mechanism whereby Eos propagates STAT5 activity to facilitate TH2 cell differentiation.
Lung inflammation, caused by acute exposure to ozone (O
), one of the six criteria air pollutants, is a significant source of morbidity in susceptible individuals. Alveolar macrophages (AMØs) are the ...most abundant immune cells in the normal lung, and their number increases after O
exposure. However, the role of AMØs in promoting or limiting O
-induced lung inflammation has not been clearly defined. In this study, we used a mouse model of acute O
exposure, lineage tracing, genetic knockouts, and data from O
-exposed human volunteers to define the role and ontogeny of AMØs during acute O
exposure. Lineage-tracing experiments showed that 12, 24, and 72 hours after exposure to O
(2 ppm) for 3 hours, all AMØs were of tissue-resident origin. Similarly, in humans exposed to filtered air and O
(200 ppb) for 135 minutes, we did not observe at ∼21 hours postexposure an increase in monocyte-derived AMØs by flow cytometry. Highlighting a role for tissue-resident AMØs, we demonstrate that depletion of tissue-resident AMØs with clodronate-loaded liposomes led to persistence of neutrophils in the alveolar space after O
exposure, suggesting that impaired neutrophil clearance (i.e., efferocytosis) leads to prolonged lung inflammation. Moreover, depletion of tissue-resident AMØs demonstrated reduced clearance of intratracheally instilled apoptotic Jurkat cells, consistent with reduced efferocytosis. Genetic ablation of MerTK (MER proto-oncogene, tyrosine kinase), a key receptor involved in efferocytosis, also resulted in impaired clearance of apoptotic neutrophils after O
exposure. Overall, these findings underscore the pivotal role of tissue-resident AMØs in resolving O
-induced inflammation via MerTK-mediated efferocytosis.
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
The purpose of this study was to evaluate the pharmacokinetics of tulathromycin in the plasma and maternal and fetal tissues of pregnant ewes when administered within 24 hours of a single, IV ...Campylobacter jejuni (C. jejuni) challenge. Twelve, pregnant ewes between 72-92 days of gestation were challenged IV with C. jejuni IA3902 and then treated with 1.1 ml/45.36 kg of tulathromycin subcutaneously 18 hours post-challenge. Ewes were bled at predetermined time points and euthanized either at a predetermined time point or following the observation of vaginal bleeding or abortion. Following euthanasia, tissues were collected for bacterial culture, pharmacokinetics and histologic examination. The maximum (geometric) mean tulathromycin plasma concentration was estimated at 0.302 μg/mL, with a peak level observed at around 1.2 hours. The apparent systemic clearance of tulathromycin was estimated at 16.6 L/h (or 0.28 L/kg/h) with an elimination half-life estimated at approximately 22 hours. The mean tissue concentrations were highest in the uterus (2.464 μg/g) and placentome (0.484 μg/g), and were lowest in fetal liver (0.11 μg/g) and fetal lung (0.03 μg/g). Compared to previous reports, results of this study demonstrate that prior IV administration of C. jejuni appeared to substantially alter the pharmacokinetics of tulathromycin, reducing both the peak plasma concentrations and elimination half-life. However, additional controlled trials are required to confirm those observations.
The aim of this study was to assess whether pregnant mice represent a useful model to study the reproductive pathology of Campylobacter jejuni IA3902 using the end point of positive microbial culture ...of the organism from the fetoplacental unit. Pregnant BALB/c and CD-1 mice (14 days’ gestation) were inoculated orally and intraperitoneally (IP) with 1 × 109 colony-forming units/ml of C. jejuni IA3902. The organism was recovered by microbial culture from the fetoplacental unit in 10 of 10 BALB/c and 10 of 10 CD-1 IP-inoculated pregnant mice and in 29% (2/7) BALB/c and 38% (3/8) CD-1 orally inoculated pregnant mice. Gross reproductive lesions included necrosuppurative placentitis, fetal resorption, intrauterine fetal death, stillborn pups (dead neonates), and multifocal hepatitis. Histological changes consisted of locally extensive neutrophilic and necrotizing placentitis with intralesional bacterial colonies of C. jejuni, ulcerative endometritis, random multifocal hepatitis, and rare cholecystitis. Immunohistochemistry for the major outer membrane protein of C. jejuni revealed moderate to large numbers of the organism at the periphery of the placental discs, within trophoblasts and extracellularly, with invasion into the placental disc largely via the vascular network. The organism is trophic for neutral mucin, iron, and L-fucose within the murine placenta. C. jejuni IA3902 has affinity for the murine reproductive tract, specifically the fetoplacental unit, where it results in a necrotizing placentitis with positive microbial recovery after both IP and oral challenge in BALB/c and CD-1 pregnant mice.
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