•Role of prohibitins (PHBs) in macrophage signaling was studied using PHB knockdown.•PHB knockdown decreases expression and signaling of lipid raft-dependent receptors.•Plasma membrane packing and ...raft formation are altered in PHB knockdown macrophages.•PHB controls the composition of cellular fatty acids.•PHBs impact macrophage signaling by regulating lipid rafts.
Prohibitins (PHB1 and PHB2) are ubiquitously expressed proteins which play critical roles in multiple biological processes, and together form the ring-like PHB complex found in phospholipid-rich cellular compartments including lipid rafts. Recent studies have implicated PHB1 as a mediator of fatty acid transport as well as a membrane scaffold mediating B lymphocyte and mast cell signal transduction. However, the specific role of PHBs in the macrophage have not been characterized, including their role in fatty acid uptake and lipid raft-mediated inflammatory signaling. We hypothesized that the PHB complex regulates macrophage inflammatory signaling through the formation of lipid rafts. To evaluate our hypothesis, RAW 264.7 macrophages were transduced with shRNA against PHB1, PHB2, or scrambled control (Scr), and then stimulated with lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-α), which activate lipid raft-dependent receptor signaling (CD14/TLR4 and TNFR1, respectively). PHB1 knockdown was lethal, whereas PHB2 knockdown (PHB2kd), which also resulted in decreased PHB1 expression, led to attenuated nuclear factor-kappa-B (NF-κB) activation and subsequent cytokine and chemokine production. PHB2kd macrophages also had decreased cell surface TNFR1, CD14, TLR4, and lipid raft marker ganglioside GM1 at baseline and post-stimuli. Post-LPS, PHB2kd macrophages did not increase the concentration of cellular saturated, monounsaturated, and polyunsaturated fatty acids. This was accompanied by decreased lipid raft formation and modified plasma membrane molecular packing, further supporting the PHB complex's importance in lipid raft formation. Taken together, these data suggest a critical role for PHBs in regulating macrophage inflammatory signaling via maintenance of fatty acid composition and lipid raft structure.
Prohibitins are proteins found in phospholipid-rich cellular compartments, including lipid rafts, that play important roles in signaling, transcription, and multiple other cell functions. Macrophages are key cells in the innate immune response and the presence of membrane lipid rafts is integral to signal transduction, but the role of prohibitins in macrophage lipid rafts and associated signaling is unknown. To address this question, prohibitin knockdown macrophages were generated and responses to lipopolysaccharide and tumor necrosis factor-alpha, which act through lipid raft-dependent receptors, were analyzed. Prohibitin knockdown macrophages had significantly decreased cytokine and chemokine production, transcription factor activation, receptor expression, lipid raft assembly and membrane packing, and altered fatty acid remodeling. These data indicate a novel role for prohibitins in macrophage inflammatory signaling through regulation of fatty acid composition and lipid raft formation.
Background In sepsis, circulating cytokines and lipopolysaccharide elicit mitochondrial dysfunction and cardiomyopathy, a major cause of morbidity and mortality with this condition. Emerging research ...places the PHB1 (lipid raft protein prohibitin-1) at the nexus of inflammation, metabolism, and oxidative stress. PHB1 has also been reported in circulation, though its function in this compartment is completely unknown. Methods and Results Using a wide-ranging approach across multiple in vitro and in vivo models, we interrogated the functional role of intracellular and circulating PHB1 in the heart during sepsis, and elucidated some of the mechanisms involved. Upon endotoxin challenge or sepsis induction in rodent models, PHB1 translocates from mitochondria to nucleus in cardiomyocytes and is secreted into the circulation from the liver in a manner dependent on nuclear factor (erythroid-derived 2)-like 2, a key transcriptional regulator of the antioxidant response. Overexpression or treatment with recombinant human PHB1 enhances the antioxidant/anti-inflammatory response and protects HL-1 cardiomyocytes from mitochondrial dysfunction and toxicity from cytokine stress. Importantly, administration of recombinant human PHB1 blunted inflammation and restored cardiac contractility and ATP production in mice following lipopolysaccharide challenge. This cardioprotective, anti-inflammatory effect of recombinant human PHB1 was determined to be independent of nuclear factor (erythroid-derived 2)-like 2, but partially dependent on PI3K/AKT signaling in the heart. Conclusions These findings reveal a previously unknown cardioprotective effect of PHB1 during sepsis, and illustrate a pro-survival, protective role for PHB1 in the circulation. Exploitation of circulating PHB1 as a biomarker and/or therapeutic could have widespread benefit in the clinical management of sepsis and other severe inflammatory disorders.
Estrogen receptor-α (ERα) is a nuclear receptor family member thought to substantially contribute to the metabolic regulation of skeletal muscle. However, previous mouse models utilized to assess the ...necessity of ERα signaling in skeletal muscle were confounded by altered developmental programming and/or influenced by secondary effects, making it difficult to assign a causal role for ERα. The objective of this study was to determine the role of skeletal muscle ERα in regulating metabolism in the absence of confounding factors of development.
A novel mouse model was developed allowing for induced deletion of ERα in adult female skeletal muscle (ERαKOism). ERαshRNA was also used to knockdown ERα (ERαKD) in human myotubes cultured from primary human skeletal muscle cells isolated from muscle biopsies from healthy and obese insulin-resistant women.
Twelve weeks of HFD exposure had no differential effects on body composition, VO2, VCO2, RER, energy expenditure, and activity counts across genotypes. Although ERαKOism mice exhibited greater glucose intolerance than wild-type (WT) mice after chronic HFD, ex vivo skeletal muscle glucose uptake was not impaired in the ERαKOism mice. Expression of pro-inflammatory genes was altered in the skeletal muscle of the ERαKOism, but the concentrations of these inflammatory markers in the systemic circulation were either lower or remained similar to the WT mice. Finally, skeletal muscle mitochondrial respiratory capacity, oxidative phosphorylation efficiency, and H2O2 emission potential was not affected in the ERαKOism mice. ERαKD in human skeletal muscle cells neither altered differentiation capacity nor caused severe deficits in mitochondrial respiratory capacity.
Collectively, these results suggest that ERα function is superfluous in protecting against HFD-induced skeletal muscle metabolic derangements after postnatal development is complete.
•Induced skeletal muscle specific ERαKO (ERαKOism) examines the role of ERα without confounding factors of development.•Skeletal muscle glucose uptake is not impaired in ERαKOism.•Skeletal muscle mitochondrial function is not impaired in ERαKOism.•ERαKD in human myotubes does not severely affect mitochondrial respiratory capacity.
The pro-inflammatory potency and causal relationship with asthma of inhaled endotoxins have underscored the importance of accurately assessing the endotoxin content of organic dusts. The Limulus ...amebocyte lysate (LAL) assay has emerged as the preferred assay, but its ability to measure endotoxin in intact bacteria and organic dusts with similar sensitivity as purified endotoxin is unknown. We used metabolically radiolabeled Neisseria meningitidis and both rough and smooth Escherichia coli to compare dose-dependent activation in the LAL with purified endotoxin from these bacteria and shed outer membrane (OM) blebs. Labeled 14C-3-OH-fatty acids were used to quantify the endotoxin content of the samples. Purified meningococcal and E. coli endotoxins and OM blebs displayed similar specific activity in the LAL assay to the purified LPS standard. In contrast, intact bacteria exhibited fivefold lower specific activity in the LAL assay but showed similar MD-2-dependent potency as purified endotoxin in inducing acute airway inflammation in mice. Pre-treatment of intact bacteria and organic dusts with 0.1 M Tris-HCl/10 mM EDTA increased by fivefold the release of endotoxin. These findings demonstrate that house dust and other organic dusts should be extracted with Tris/EDTA to more accurately assess the endotoxin content and pro-inflammatory potential of these environmental samples.
Abstract
Air pollution levels across the globe continue to rise despite government regulations. The increase in global air pollution levels drives detrimental human health effects, including 7 ...million premature deaths every year. Many of these deaths are attributable to increased incidence of respiratory infections. Considering the COVID-19 pandemic, an unprecedented public health crisis that has claimed the lives of over 6.5 million people globally, respiratory infections as a driver of human mortality is a pressing concern. Therefore, it is more important than ever to understand the relationship between air pollution and respiratory infections so that public health measures can be implemented to ameliorate further morbidity and mortality. This article aims to review the current epidemiologic and basic science research on interactions between air pollution exposure and respiratory infections. The first section will present epidemiologic studies organized by pathogen, followed by a review of basic science research investigating the mechanisms of infection, and then conclude with a discussion of areas that require future investigation.
Paired electrostatic dust collectors (EDCs) and daily, inhalable button samplers (BS) were used concurrently to sample endotoxin in 10 farm homes during 7‐day periods in summer and winter. Winter ...sampling included an optical particle counter (OPC) to measure PM2.5 and PM2.5–10. Electrostatic dust collectors and BS filters were analyzed for endotoxin using the kinetic chromogenic Limulus amebocyte lysate assay. Optical particle counter particulate matter (PM) data were divided into two PM categories. In summer, geometric mean (geometric standard deviation) endotoxin concentrations were 0.82 EU/m3 (2.7) measured with the BS and 737 EU/m2 (1.9) measured with the EDC. 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 highly correlated with the 7‐day BS sampling averages (r = 0.70; P < 0.001). Analysis of variance indicated a 2.4‐fold increase in EDC endotoxin concentrations for each unit increase of the ratio of PM2.5 to PM2.5–10. There was also a significant correlation between BS and EDCs endotoxin concentrations for winter (r = 0.67; P < 0.05) and summer (r = 0.75; P < 0.05). Thus, EDCs sample comparable endotoxin concentrations to BS, making EDCs a feasible, easy to use alternative to BS for endotoxin sampling.
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.
Abstract
Exposure to ozone (O3) induces lung injury, pulmonary inflammation, and alters lipid metabolism. During tissue inflammation, specialized pro-resolving lipid mediators (SPMs) facilitate the ...resolution of inflammation. SPMs regulate the pulmonary immune response during infection and allergic asthma; however, the role of SPMs in O3-induced pulmonary injury and inflammation is unknown. We hypothesize that O3 exposure induces pulmonary inflammation by reducing SPMs. To evaluate this, male C57Bl/6J mice were exposed to filtered air (FA) or 1 ppm O3 for 3 h and necropsied 24 h after exposure. Pulmonary injury/inflammation was determined by bronchoalveolar lavage (BAL) differentials, protein, and lung tissue cytokine expression. SPMs were quantified by liquid chromatography tandem mass spectrometry and SPM receptors leukotriene B4 receptor 1 (BLT-1), formyl peptide receptor 2 (ALX/FPR2), chemokine-like receptor 1 (ChemR23), and SPM-generating enzyme (5-LOX and 12/15-LOX) expression were measured by real time PCR. 24 h post-O3 exposure, BAL PMNs and protein content were significantly increased compared to FA controls. O3-induced lung inflammation was associated with significant decreases in pulmonary SPM precursors (14-HDHA, 17-HDHA), the SPM PDX, and in pulmonary ALX/FPR2, ChemR23, and 12/15-LOX expression. Exogenous administration of 14-HDHA, 17-HDHA, and PDX 1 h prior to O3 exposure rescued pulmonary SPM precursors/SPMs, decreased proinflammatory cytokine and chemokine expression, and decreased BAL macrophages and PMNs. Taken together, these data indicate that O3-mediated SPM reductions may drive O3-induced pulmonary inflammation.
Asthma is a common respiratory disease currently affecting more than 300 million worldwide and is characterized by airway inflammation, hyperreactivity, and remodeling. It is a heterogeneous disease ...consisting of corticosteroid-sensitive T-helper cell type 2-driven eosinophilic and corticosteroid-resistant, T-helper cell type 17-driven neutrophilic phenotypes. One pathway recently described to regulate asthma pathogenesis is cholesterol trafficking. Scavenger receptors, in particular SR-BI (scavenger receptor class B type I), are known to direct cellular cholesterol uptake and efflux. We recently defined SR-BI functions in pulmonary host defense; however, the function of SR-BI in asthma pathogenesis is unknown. To elucidate the role of SR-BI in allergic asthma, SR-BI-sufficient (SR-BI
) and SR-BI-deficient (SR-BI
) mice were sensitized (Days 0 and 7) and then challenged (Days 14, 15, and 16) with a house dust mite (HDM) preparation administered through oropharyngeal aspiration. Airway inflammation and cytokine production were quantified on Day 17. When compared with SR-BI
mice, the HDM-challenged SR-BI
mice had increased neutrophils and pulmonary IL-17A production in BAL fluid. This augmented IL-17A production in SR-BI
mice originated from a non-T-cell source that included neutrophils and alveolar macrophages. Given that SR-BI regulates adrenal steroid hormone production, we tested whether the changes in SR-BI
mice were glucocorticoid dependent. Indeed, SR-BI
mice were adrenally insufficient during the HDM challenge, and corticosterone replacement decreased pulmonary neutrophilia and IL-17A production in SR-BI
mice. Taken together, these data indicate that SR-BI dampens pulmonary neutrophilic inflammation and IL-17A production in allergic asthma at least in part by maintaining adrenal function.
Abstract
Prohibitins (PHB1 and PHB2) are proteins that assemble in hetero-oligomeric complexes within the mitochondrial inner membrane and in the plasma membrane lipid rafts where they are at the ...nexus of metabolic and pro-survival decisions including inflammation. We have recently observed that mice intraperitoneal (i.p.) injected with lipopolysaccharide (LPS) have increased serum prohibitin (PHB), and injection of recombinant PHB1 (rPHB1) post LPS rescues cardiac function. However, the role of PHB in the innate immune response to LPS is currently unknown. Using an in vivo model of sepsis, we injected C57Bl/6J mice i.p. with LPS and 3 subsequent doses of rPHB1. Blood was analyzed for immune cell characterization by flow cytometry, and liver, kidney and lung tissue were harvested for pro-inflammatory cytokine expression. Systemic LPS increased number of blood neutrophils (PMNs), PMN expression of the adhesion molecule CD11b, and tissue expression of TNF-α, IL-6, and IL-1β. Treatment with rPHB1 decreased blood PMN CD11b expression and expression of IL-6 in liver, kidney and lung tissues. To better characterize the role of PHB in immune cells, we knocked down PHB1 or PHB2 in RAW 264.7 macrophages. PHB1- or PHB2-deficient macrophages were then stimulated with LPS, and cell supernatant and lysate were collected for cytokine production. Cell lines deficient in PHB2 had decreased TNF-α compared to macrophages sufficient in PHBs. These data suggest PHB1 and -2 have differential and complex effects on the innate immune response. Cell-associated PHB is required for acute pro-inflammatory signaling responses to LPS that initiate the sepsis response, but extracellular PHB, released in the later phase of sepsis, may feedback to dampen the response.
PDF
