The Effects of Pregnancy on the Pulmonary Immune Response in a Mouse Model of LPS-Induced Acute Lung Injury.

OBJECTIVE: This study evaluated the effect of pregnancy on the pulmonary innate immune response in a mouse model of acute lung injury (ALI) using nebulized lipopolysaccharide (LPS). STUDY DESIGN: Pregnant (day 14) C57BL/6NCRL mice and nonpregnant controls received nebulized LPS for 15 minutes. Twenty-four hours later, mice were euthanized for tissue harvest. Analysis included blood and bronchoalveolar lavage fluid (BALF) differential cell counts, whole-lung inflammatory cytokine transcription levels by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), and whole-lung vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and BALF albumin by western blot. Mature bone marrow neutrophils from uninjured pregnant and nonpregnant mice were examined for chemotactic response using a Boyden chamber and for cytokine response to LPS by RT-qPCR. RESULTS: In LPS-induced ALI, pregnant mice had higher BALF total cell (p < 0.001) and neutrophil counts (p < 0.001) as well as higher peripheral blood neutrophils (p < 0.01) than nonpregnant mice, but a similar increase (as compared with unexposed mice) in airspace albumin levels. Whole-lung expression of interleukin 6, tumor necrosis factor-α (TNF-α), and keratinocyte chemoattractant (CXCL1) was also similar. In vitro, marrow-derived neutrophils from pregnant and nonpregnant mice had similar chemotaxis to CXCL1 and N-formylmethionine-leucyl-phenylalanine, but neutrophils from pregnant mice expressed lower levels of TNF (p < 0.001) and CXCL1 (p < 0.01) after LPS stimulation. In uninjured mice, VCAM-1 was higher in lungs from pregnant versus nonpregnant mice (p < 0.05). CONCLUSION: In this model, pregnancy is associated with an augmented lung neutrophil response to ALI without increased capillary leak or whole-lung cytokine levels relative to the nonpregnant state. This may stem from increased peripheral blood neutrophil response and intrinsically increased expression of pulmonary vascular endothelial adhesion molecules. Differences in lung innate cell homeostasis may affect the response to inflammatory stimuli and explain severe lung disease in respiratory infection during pregnancy. KEY POINTS: · Inhalation of LPS in midgestation versus virgin mice is associated with increased neutrophilia.. · This occurs without a comparative increase in cytokine expression.. · This may be explained by pregnancy-enhanced pre-exposure expression of VCAM-1 and ICAM-1..

Long Noncoding RNA U90926 Is Induced in Activated Macrophages, Is Protective in Endotoxic Shock, and Encodes a Novel Secreted Protein.

Thousands of long noncoding RNAs are encoded in mammalian genomes, yet most remain uncharacterized. In this study, we functionally characterized a mouse long noncoding RNA named U90926. Analysis of U90926 RNA levels revealed minimal expression across multiple tissues at steady state. However, the expression of this gene was highly induced in macrophages and dendritic cells by TLR activation, in a p38 MAPK- and MyD88-dependent manner. To study the function of U90926, we generated U90926-deficient (U9-KO) mice. Surprisingly, we found minimal effects of U90926 deficiency in cultured macrophages. Given the lack of macrophage-intrinsic effect, we investigated the subcellular localization of U90926 transcript and its protein-coding potential. We found that U90926 RNA localizes to the cytosol, associates with ribosomes, and contains an open reading frame that encodes a novel glycosylated protein (termed U9-ORF), which is secreted from the cell. An in vivo model of endotoxic shock revealed that, in comparison with wild type mice, U9-KO mice exhibited increased sickness responses and mortality. Mechanistically, serum levels of IL-6 were elevated in U9-KO mice, and IL-6 neutralization improved endotoxemia outcomes in U9-KO mice. Taken together, these results suggest that U90926 expression is protective during endotoxic shock, potentially mediated by the paracrine and/or endocrine actions of the novel U9-ORF protein secreted by activated myeloid cells.

Skeletal muscle macrophage ablation in mice.

Macrophages are important mediators of skeletal muscle function in both healthy and diseased states. In vivo specific depletion of macrophages provides an experimental method to understand physiological and pathophysiological effects of macrophages. Systemic depletion of macrophages can deplete skeletal muscle macrophages but also alters systemic inflammatory responses and metabolism, which confounds the muscle specific effects of macrophage depletion. The primary aim of this manuscript is to evaluate two methods of murine intramuscular macrophage depletion in an acute lung injury-associated indirect skeletal muscle wasting mouse model. Adult C57BL/6 (WT) and Macrophage Fas-Induced Apoptosis (MaFIA, C57BL/6-Tg) mice received clodronate liposomes or the dimerization drug AP20187 through intramuscular injection of the tibialis anterior muscle compartment, respectively. Vehicle control was injected in the contralateral muscle. We demonstrate intramuscular AP20187 in the MaFIA mouse depletes macrophages but causes an infiltration of CD45 intermediate neutrophils. In contrast, intramuscular clodronate liposomes successfully depletes macrophages without an associated increase in CD45 intermediate cells. In conclusion, intramuscular clodronate is effective for selective depletion of muscle macrophages without eliciting acute inflammation seen with AP20187 in MaFIA mice. This technique is an important tool to study the functional roles of macrophages in skeletal muscle.

An open-source, lockable mouse wheel for the accessible implementation of time- and distance-limited elective exercise.

Current methods of small animal exercise involve either voluntary (wheel running) or forced (treadmill running) protocols. Although commonly used, each have several drawbacks which cause hesitancy to adopt these methods. While mice will instinctively run on a wheel, the distance and time spent running can vary widely. Forced exercise, while controllable, puts animals in stressful environments in which they are confined and often shocked for "encouragement." Additionally, both methods require expensive equipment and software, which limit these experiments to well-funded laboratories. To counter these issues, we developed a non-invasive mouse running device aimed to reduce handler-induced stress, provide time- and distance-based stopping conditions, and enable investigators with limited resources to easily produce and use the device. The Lockable Open-Source Training-Wheel (LOST-Wheel) was designed to be 3D printed on any standard entry-level printer and assembled using a few common tools for around 20 USD. It features an on-board screen and is capable of tracking distances, running time, and velocities of mice. The LOST-Wheel overcomes the largest drawback to voluntary exercise, which is the inability to control when and how long mice run, using a servo driven mechanism that locks and unlocks the running surface according to the protocol of the investigator. While the LOST-Wheel can be used without a computer connection, we designed an accompanying application to provide scientists with additional analyses. The LOST-Wheel Logger, an R-based application, displays milestones and plots on a user-friendly dashboard. Using the LOST-Wheel, we implemented a timed running experiment that showed distance-dependent decreases in serum myostatin as well as IL-6 gene upregulation in muscle. To make this device accessible, we are releasing the designs, application, and manual in an open-source format. The implementation of the LOST-Wheel and future iterations will improve upon existing murine exercise equipment and research.

Storage conditions of high-fat diets affect pulmonary inflammation.

Obesity alters the risks and outcomes of inflammatory lung diseases. It is important to accurately recapitulate the obese state in animal models to understand these effects on the pathogenesis of disease. Diet-induced obesity is a commonly used model of obesity, but when applied to other disease models like acute respiratory distress syndrome, pneumonia, and asthma, it yields widely divergent. We hypothesized high-fat chow storage conditions would affect lipid oxidation and inflammatory response in the lungs of lipopolysaccharide (LPS)-challenged mice. For 6 weeks, C57BL/6crl mice were fed either a 10% (low-fat diet, LFD) or 60% (high-fat diet, HFD) stored at room temperature (RT, 23°C) for up to 7, 14, 21, or 42 days. Mice were treated with nebulized LPS to induce lung inflammation, and neutrophil levels in bronchoalveolar lavage were determined 24 h later. Lipid oxidation (malondialdehyde, MDA) was assayed by thiobarbituric acid reactive substances in chow and mouse plasma. Concentrations of MDA in chow and plasma rose in proportion to the duration of RT chow storage. Mice fed a HFD stored <2 weeks at RT had an attenuated response 24 h after LPS compared with mice fed an LFD. This effect was reversed after 2 weeks of chow storage at RT. Chow stored above freezing underwent lipid oxidation associated with significant alterations in the LPS-induced pulmonary inflammatory response. Our data show that storage conditions affect lipid peroxidation, which in turn affects pulmonary inflammatory responses in a mouse model of disease. It also suggests changes in the microbiome, although not significantly different suggests decreased variety and richness of bacteria in the gut, a large aspect of the immune system. Dietary composition and storage of chow may also affect pulmonary inflammation and the gut microbiome in humans.

Macrophages augment the skeletal muscle proinflammatory response through TNFα following LPS-induced acute lung injury.

Muscle may contribute to the systemic inflammatory environment during critical illness, but leukocyte interaction and cytokine influence on muscle and its response has not been fully explored in this context. Using an in vivo model of intratracheal lipopolysaccharide (IT LPS)-induced acute lung injury, we show that skeletal muscle rapidly responds with expression of proinflammatory genes, which may be explained by migration of LPS into the circulation. Treatment of mature C2C12 myotubes with LPS at a level achieved in the circulation following IT LPS elicited a proinflammatory cytokine expression profile similar to that of in vivo murine muscle following IT LPS. Stimulation with toll-like receptor (TLR) 2 and 3 agonists provoked comparable responses in C2C12 myotubes. Additionally, co-cultures of C2C12 myotubes and bone marrow-derived macrophages (BMDM) identified the capacity of macrophages to increase myotube proinflammatory gene expression, with tumor necrosis factor-α (TNFα) gene and protein expression largely attributable to BMDM. To investigate the contribution of TNFα in the synergy of the co-culture environment, C2C12 myotubes were treated with recombinant TNFα, co-cultures were established using TNF-deficient BMDM, and co-cultures were also depleted of TNFα using antibodies. To determine whether the in vitro observations were relevant in vivo, mice received intramuscular administration of LPS ± TNFα or TNFα-neutralizing antibodies and showed that TNFα is both sufficient and necessary to induce synergistic cytokine release from muscle. Taken together, these data demonstrate how skeletal muscle tissue may contribute proinflammatory cytokines following acute endotoxin injury and the potential of leukocytes to augment this response via TNFα secretion.

Electrical stimulation prevents doxorubicin-induced atrophy and mitochondrial loss in cultured myotubes.

Muscle contraction may protect against the effects of chemotherapy to cause skeletal muscle atrophy, but the mechanisms underlying these benefits are unclear. To address this question, we utilized in vitro modeling of contraction and mechanotransduction in C2C12 myotubes treated with doxorubicin (DOX; 0.2 µM for 3 days). Myotubes expressed contractile proteins and organized these into functional myofilaments, as electrical field stimulation (STIM) induced intracellular calcium (Ca2+) transients and contractions, both of which were prevented by inhibition of membrane depolarization. DOX treatment reduced myotube myosin content, protein synthesis, and Akt (S308) and forkhead box O3a (FoxO3a; S253) phosphorylation and increased muscle RING finger 1 (MuRF1) expression. STIM (1 h/day) prevented DOX-induced reductions in myotube myosin content and Akt and FoxO3a phosphorylation, as well as increases in MuRF1 expression, but did not prevent DOX-induced reductions in protein synthesis. Inhibition of myosin-actin interaction during STIM prevented contraction and the antiatrophic effects of STIM without affecting Ca2+ cycling, suggesting that the beneficial effect of STIM derives from mechanotransductive pathways. Further supporting this conclusion, mechanical stretch of myotubes recapitulated the effects of STIM to prevent DOX suppression of FoxO3a phosphorylation and upregulation of MuRF1. DOX also increased reactive oxygen species (ROS) production, which led to a decrease in mitochondrial content. Although STIM did not alter DOX-induced ROS production, peroxisome proliferator-activated receptor-γ coactivator-1α and antioxidant enzyme expression were upregulated, and mitochondrial loss was prevented. Our results suggest that the activation of mechanotransductive pathways that downregulate proteolysis and preserve mitochondrial content protects against the atrophic effects of chemotherapeutics.

Bacterial Lipoproteins Constitute the TLR2-Stimulating Activity of Serum Amyloid A.

Studies comparing endogenous and recombinant serum amyloid A (SAA) have generated conflicting data on the proinflammatory function of these proteins. In exploring this discrepancy, we found that in contrast to commercially sourced recombinant human SAA1 (hSAA1) proteins produced in Escherichia coli, hSAA1 produced from eukaryotic cells did not promote proinflammatory cytokine production from human or mouse cells, induce Th17 differentiation, or stimulate TLR2. Proteomic analysis of E. coli-derived hSAA1 revealed the presence of numerous bacterial proteins, with several being reported or probable lipoproteins. Treatment of hSAA1 with lipoprotein lipase or addition of a lipopeptide to eukaryotic cell-derived hSAA1 inhibited or induced the production of TNF-α from macrophages, respectively. Our results suggest that a function of SAA is in the binding of TLR2-stimulating bacterial proteins, including lipoproteins, and demand that future studies of SAA employ a recombinant protein derived from eukaryotic cells.

Induction of cellular and molecular Immunomodulatory pathways by vitamin E and vitamin C.

INTRODUCTION: Vitamins E and C are well known small molecules that have been used to maintain health for decades. Recent studies of the cellular and molecular pathways leading to immunomodulation by these molecules have been of interest, as have their anti-oxidant properties and signal transduction pathways for curing or improving infectious diseases and cancer. Areas covered: Herein, the authors provide a definition and the structural classification of vitamins E and C and how these molecules influence cellular function. The studies include in vitro, ex vivo and in vivo studies in animal models as well as clinical trials. The authors give particular focus to the scientifically factual and putative roles of these molecules in innate and adaptive immunomodulation and prevention or cure of diseases. Expert opinion: The antioxidant properties of vitamins E and C are well studied. However, whether there is a link between their antioxidant and immunomodulation properties is unclear. In addition, there is a strong, albeit putative, prevailing notion that vitamin C can prevent or cure infectious diseases or cancer. Presently, while there is proven evidence that vitamin E possesses immunomodulatory properties that may play a positive role in disease outcomes, this evidence is less available for vitamin C.

Vitamin A or E and a catechin synergize as vaccine adjuvant to enhance immune responses in mice by induction of early interleukin-15 but not interleukin-1ß responses.

Vitamins A and E and select flavonoids in the family of catechins are well-defined small molecules that, if proven to possess immunomodulatory properties, hold promise as vaccine adjuvants and various therapies. In an effort to determine the in vivo immunomodulatory properties of these molecules, we found that although mucosal and systemic vaccinations with a recombinant HIV-1BaL gp120 with either a catechin, epigallo catechin gallate (EGCG) or pro-vitamin A (retinyl palmitate) alone in a vegetable-oil-in-water emulsion (OWE) suppressed antigen-specific responses, the combination of EGCG and vitamin A or E in OWE (Nutritive Immune-enhancing Delivery System, NIDS) synergistically enhanced adaptive B-cell, and CD4(+) and CD8(+) T-cell responses, following induction of relatively low local and systemic innate tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-17, but relatively high levels of early systemic IL-15 responses. For induction of adaptive interferon-γ and TNF-α responses by CD4(+) and CD8(+) T cells, the adjuvant effect of NIDS was dependent on both IL-15 and its receptor. In addition, the anti-oxidant activity of NIDS correlated positively with higher expression of the superoxide dismutase 1, an enzyme involved in reactive oxygen species elimination but negatively with secretion of IL-1ß. This suggests that the mechanism of action of NIDS is dependent on anti-oxidant activity and IL-15, but independent of IL-1ß and inflammasome formation. These data show that this approach in nutritive vaccine adjuvant design holds promise for the development of potentially safer effective vaccines.