•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.
Alterations to branched-chain keto acid (BCKA) oxidation have been implicated in a wide variety of human diseases, ranging from diabetes to cancer. Although global shifts in BCKA metabolism-evident ...by gene transcription, metabolite profiling, and
flux analyses have been documented across various pathological conditions, the underlying biochemical mechanism(s) within the mitochondrion remain largely unknown.
experiments using isolated mitochondria represent a powerful biochemical tool for elucidating the role of the mitochondrion in driving disease. Such analyses have routinely been utilized across disciplines to shed valuable insight into mitochondrial-linked pathologies. That said, few studies have attempted to model
BCKA oxidation in isolated organelles. The impetus for the present study stemmed from the knowledge that complete oxidation of each of the three BCKAs involves a reaction dependent upon bicarbonate and ATP, both of which are not typically included in respiration experiments. Based on this, it was hypothesized that the inclusion of exogenous bicarbonate and stimulation of respiration using physiological shifts in ATP-free energy, rather than excess ADP, would allow for maximal BCKA-supported respiratory flux in isolated mitochondria. This hypothesis was confirmed in mitochondria from several mouse tissues, including heart, liver and skeletal muscle. What follows is a thorough characterization and validation of a novel biochemical tool for investigating BCKA metabolism in isolated mitochondria.
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.
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.
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.
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.
Inflammation is a complex mechanism primarily driven by the immune system to eradicate pathogens/foreign substances and restore tissue homeostasis. Despite the beneficial effects that inflammation ...employs, signaling can often become dysregulated leading to uncontrolled systemic inflammation and irreversible host tissue damage. Therefore, regulating the cellular and physiological mechanisms of inflammation constitutes a viable avenue of research to mitigate inflammatory disease progression. Herein, we evaluated prohibitins (PHB1 and PHB2), pleiotropic homologous proteins with known anti-inflammatory and antioxidant capabilities, in the context of systemic inflammation as well as macrophage-specific inflammatory signaling. Using two in vivo models of systemic inflammation, we found that PHB1 levels were increased in serum, suggesting a potential signaling role for PHB. Moreover, recombinant PHB1 treatment mitigated systemic inflammation and tissue/organ injury and modulated the phenotype of circulating immune cells. When investigating the role of PHB specifically in monocytes/macrophages, we found that PHB not only increased populations of pro-inflammatory monocytes in vivo but also regulated vital macrophage inflammatory signaling (as shown in vitro). We determined that PHB is a scaffold protein important for macrophage lipid raft formation and subsequent receptor trafficking. PHB modulation of macrophages influenced cell surface display of lipid-raft-dependent receptors and downstream inflammatory signaling cascades. To our knowledge, these are the first data to reveal PHB’s pro-inflammatory effects in macrophages and its mechanistic operation of lipid-raft-dependent signal transduction in macrophages. In this report, we provide insight into the diverse yet complementary roles of PHB in regulating various aspects of immune-driven inflammatory processes.
Background
The Frontal Sinus Masterclass (FSMC) is an effective method for teaching frontal sinus anatomy. A third party developed new software using the same 3-dimensional building block concept. ...The authors sought to determine whether the use of the software for the educational module yielded similar results to the original FSMC, which used wooden blocks.
Methods
The study was performed at a sinus course for residents. A precourse test assessed the ability to decipher frontal sinus anatomy prior to the course. Computed tomography (CT) scans of the sinuses were evaluated in triplanar view, and participants attempted to reconstruct the anatomy using the new software. Subsequently, the course instructor explained the anatomy and showed a short corresponding surgical video of the frontal sinusotomy. Cases progressed in anatomical difficulty and inflammatory load. A postcourse test determined knowledge after the course.
Results
In sum, 50 residents completed the pre- and postcourse tests. Overall scores increased from 60.5% to 65.2% (P = .004). Subanalysis also demonstrated improved ability to locate the frontal drainage pathway from 32.5% to 46% (P = .011) and label the frontal recess cell structures from 64% to 67.6% (P = .045).
Conclusion
There is minimal literature on proven methods for teaching frontal sinus anatomy. Objectively, participants of the modified FSMC simulation training using new software improved their ability to recognize cells of the frontal recess on CT scans. They especially exhibited better localization of the frontal sinus drainage pathway. Subjectively, participants reported benefit from the course and felt they would be better surgeons.
Mechanisms involved in the individual susceptibility to atherosclerotic coronary artery disease (CAD) beyond traditional risk factors are poorly understood. Here, we describe the utility of cultured ...patient-derived endothelial colony-forming cells (ECFCs) in examining novel mechanisms of CAD susceptibility, particularly the role of dysregulated redox signalling. ECFCs were selectively cultured from peripheral blood mononuclear cells from 828 patients from the BioHEART-CT cohort, each with corresponding demographic, clinical and CT coronary angiographic imaging data. Spontaneous growth occurred in 178 (21.5%) patients and was more common in patients with hypertension (OR 1.45 (95% CI 1.03–2.02), p = 0.031), and less likely in patients with obesity (OR 0.62 95% CI 0.40–0.95, p = 0.027) or obstructive CAD (stenosis > 50%) (OR 0.60 95% CI 0.38–0.95, p = 0.027). ECFCs from patients with CAD had higher mitochondrial production of superoxide (O2−–MitoSOX assay). The latter was strongly correlated with the severity of CAD as measured by either coronary artery calcium score (R2 = 0.46; p = 0.0051) or Gensini Score (R2 = 0.67; p = 0.0002). Patient-derived ECFCs were successfully cultured in 3D culture pulsatile mini-vessels. Patient-derived ECFCs can provide a novel resource for discovering mechanisms of CAD disease susceptibility, particularly in relation to mitochondrial redox signalling.
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