Low concentration of polyethylene glycol facilitates separation of extracellular vesicles from bronchoalveolar lavage fluid.

Extracellular vesicles (EVs) in bodily fluids play an essential role in cell-cell cross talk and potentially serve as novel biomarkers in "liquid biopsy." It is crucial to have a consistent, efficient, and reliable method to separate EVs from bodily fluids. Currently, there is no universally accepted, "best" method to separate EVs. Besides differential ultracentrifugation (UC), polyethylene glycol (PEG) is among the commonly used methods for EV separation from bodily fluids. However, the optimal concentration of PEG to be used remains inadequately addressed. We initially observed that the concentration of PEG has a significant impact on the amount of separated EVs and EV-cargos, which are recovered from bronchoalveolar lavage fluid (BALF). To determine the optimal PEG concentration to be used in EV separation from BALF, we first separated the BALF and serum from wild-type C57BL/6 mice. Next, various concentrations of PEG (5%, 10%, and 15% PEG), a commercial kit, and UC were used to obtain EVs from BALF and serum. EVs were characterized, and EV-cargo protein, RNA, and miRNA levels were determined. We found that high concentration of PEG (10% and 15%) altered various EV parameters that are frequently used in EV studies, including EV yield, purity, and morphology. Using miR-15a, miR-142, and miR-223 as examples, we found that 10% and 15% PEG robustly reduced the detected levels of EV-cargo miRNAs compared with those in the EVs separated using UC or 5% PEG. Collectively, low concentration of PEG facilitates the optimal BALF EV separation.

Single-step RT-qPCR for detection of extracellular vesicle microRNAs in vivo: a time- and cost-effective method.

Emerging evidence suggests that extracellular vesicle (EV)-associated microRNAs (miRNAs) are a potential diagnostic tool for liquid biopsy in various human diseases. However, the experimental procedure for the detection of EV-associated miRNAs (EV-miRNAs) from body fluids is relatively complex and not cost-effective. Due to the limited amount of EVs and EV-RNAs, a column-based RNA purification, which is an expensive approach, is often used to detect EV-miRNAs via reverse transcription-quantitative real-time PCR (RT-qPCR). Here, we developed and validated a simple and cost-effective method (single-step RT-qPCR) in which we directly detect EV-miRNAs without RNA purification from the EVs. We validated this protocol using the EVs isolated from mouse broncho-alveolar lavage fluid (BALF) and serum. The obtained EVs were first lysed in the EV-lysis buffer, followed by RT-qPCR without isolation and purification of RNAs. We successfully detected the designated miRNAs from lysed EVs; 106 to 107 EVs were optimal to detect the EV-miRNAs using the single-step RT-qPCR. In our previously published work, using the conventional RT-qPCR method, we have reported that miR-142 and -223 are dramatically upregulated in both BALF and serum EVs after lung infection. Hence, we reassessed and confirmed the level of EV-miR-142/223 using the newly developed single-step RT-qPCR. Notably, inhibition of RNase activity in the lysed EVs remains crucial for the detection of EV-miRNAs. Moreover, repeated freeze-thaw cycling significantly interferes the EV-miRNA quantification. Collectively, the single-step RT-qPCR for the detection of EV-miRNAs in vivo will potentially provide a fast, accurate, and convenient way to quantify circulating and/or body fluid-derived EV-miRNAs. This method may potentially be applied to the diagnostic blood testing used in the medical centers or research laboratories.

Functional Evidence of Pulmonary Extracellular Vesicles in Infectious and Noninfectious Lung Inflammation.

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a highly complex process that can be triggered by both noninfectious (sterile) and infectious stimuli. Inflammatory lung responses are one of the key features in the pathogenesis of this devastating syndrome. How ALI/ARDS-associated inflammation develops remains incompletely understood, particularly after exposure to sterile stimuli. Emerging evidence suggests that extracellular vesicles (EVs) regulate intercellular communication and inflammatory responses in various diseases. In this study, we characterized the generation and function of pulmonary EVs in the setting of ALI/ARDS, induced by sterile stimuli (oxidative stress or acid aspiration) and infection (LPS/Gram-negative bacteria) in mice. EVs detected in bronchoalveolar lavage fluid (BALF) were markedly increased after exposure of animals to both types of stimuli. After sterile stimuli, alveolar type-І epithelial cells were the main source of the BALF EVs. In contrast, infectious stimuli-induced BALF EVs were mainly derived from alveolar macrophages (AMs). Functionally, BALF EVs generated in both the noninfectious and infectious ALI models promoted the recruitment of macrophages in in vivo mouse models. Furthermore, BALF EVs differentially regulated AM production of cytokines and inflammatory mediators, as well as TLR expression in AMs in vivo. Regardless of their origin, BALF EVs contributed significantly to the development of lung inflammation in both the sterile and infectious ALI. Collectively, our results provide novel insights into the mechanisms by which EVs regulate the development of lung inflammation in response to diverse stimuli, potentially providing novel therapeutic and diagnostic targets for ALI/ARDS.

A potential role of microvesicle-containing miR-223/142 in lung inflammation.

BACKGROUND: Uncontrolled lung inflammation is one of the prominent features in the pathogenesis of lung infection- associated acute lung injury (ALI). Microvesicles (MVs) are extracellular nanovesicles that are generated via direct membrane budding. METHODS: Bronchoalveolar lavage fluid (BALF) samples were collected from mice with or without intratracheal lipopolysaccharide (LPS) instillation. BALF MVs were characterised and MV-containing microRNA (miRNA) profiles were assessed and confirmed. Secretion and function of MV-containing miR-223/142 (MV-miR-223/142) were analysed in vivo. RESULTS: In BALF, MVs are mainly derived from macrophages in response to LPS. After intratracheal instillation (i.t.) of LPS or Klebsiella pneumoniae, MV-containing miR-223/142 are dramatically induced in both BALF and serum. Mechanistically, miRNA 3' end uridylation mediates the packing of miR-223/142 into MVs. To investigate the functional role of MV-miR-223/142, we loaded miR-223/142 mimics into unstimulated MVs and delivered them into the murine lungs via i.t. The miR-223/142 mimics-enriched MVs selectively targeted lung macrophages and suppressed the inflammatory lung responses that were triggered by LPS or K. pneumoniae. Mechanistically, miR-223 and miR-142 synergistically suppress Nlrp3 inflammasome activation in macrophages via inhibition of Nlrp3 and Asc, respectively. CONCLUSIONS: In the pathogenesis of lung macrophage-mediated inflammatory responses, MV-miR-223/142 secretion is robustly enhanced and detectable in BALF and serum. Furthermore, restoration of intracellular miR-223/142 via vesicle-mediated delivery suppresses macrophage activation and lung inflammation via inhibition of Nlrp3 inflammasome activation.

Isolation and characterization of extracellular vesicles from Broncho-alveolar lavage fluid: a review and comparison of different methods.

Extracellular vesicles (EVs) are cell-derived membranous vesicles secreted by cells into the extracellular space, which play a role in cell to cell communication. EVs are categorized into 3 groups depending on their size, surface marker, and method of release from the host cell. Recently, EVs have become of interest in the study of multiple disease etiologies and are believed to be potential biomarkers for many diseases. Multiple different methods have been developed to isolate EVs from different samples such as cell culture medium, serum, blood, and urine. Once isolated, EVs can be characterized by technology such as nanotracking analysis, dynamic light scattering, and nanoscale flow cytometry. In this review, we summarize the current methods of EV isolation, provide details into the three methods of EV characterization, and provide insight into which isolation approaches are most suitable for EV isolation from bronchoalveolar lavage fluid (BALF).

Lung Epithelial Cell-Derived Microvesicles Regulate Macrophage Migration via MicroRNA-17/221-Induced Integrin ß1 Recycling.

Robust lung inflammation is one of the prominent features in the pathogenesis of acute lung injury (ALI). Macrophage migration and recruitment are often seen at the early stage of lung inflammatory responses to noxious stimuli. Using an acid inhalation-induced lung injury model, we explored the mechanisms by which acid exposure initiates macrophage recruitment and migration during development of ALI. The lung epithelium comprises a large surface area and functions as a first-line defense against noxious insults. We found that acid exposure induced a remarkable microvesicle (MV) release from lung epithelium as detected in bronchoalveolar lavage fluid. Significantly elevated RNA, rather than protein, was found in these epithelium-derived MVs after acid and included several highly elevated microRNAs, including microRNA (miR)-17 and miR-221. Acid-induced epithelial MV release promoted macrophage migration in vitro and recruitment into the lung in vivo and required, in part, MV shuttling of miR-17 and/or miR-221. Mechanistically, acid-induced epithelial MV miR-17/221 promoted ß1 integrin recycling and presentation back onto the surface of macrophages, in part via a Rab11-mediated pathway. Integrin ß1 is known to play an essential role in regulating macrophage migration. Taken together, acid-induced ALI results in epithelial MV shuttling of miR-17/221 that in turn modulates macrophage ß1 integrin recycling, promoting macrophage recruitment and ultimately contributing to lung inflammation.

Exosome-Mediated Small RNA Delivery: A Novel Therapeutic Approach for Inflammatory Lung Responses.

Exosomes (EXOs) are a type of extracellular nanovesicles released from living cells. Accumulating evidence suggests that EXOs are involved in the pathogenesis of human diseases, including lung conditions. In recent years, the potential of EXO-mediated drug delivery has gained increasing interest. In this report, we investigated whether inhaled EXOs serve as an efficient and practical delivery vehicle to activate or inhibit alveolar macrophages (AMs), subsequently modulating pulmonary immune responses. We first identified the recipient cells of the inhaled EXOs, which were labeled with PKH26. We found that only lung macrophages efficiently take up intratracheally instilled EXOs in vivo. Using modified calcium chloride-mediated transformation, we manipulated small RNA molecules in serum-derived EXOs, including siRNAs, microRNA (miRNA) mimics, and miRNA inhibitors. Via intratracheal instillation, we successfully delivered siRNA and miRNA mimics or inhibitors into lung macrophages using the serum-derived EXOs as vehicles. Furthermore, EXO siRNA or miRNA molecules are functional in modulating LPS-induced lung inflammation in vivo. Beneficially, serum-derived EXOs themselves do not trigger lung immune responses, adding more favorable features to serve as drug delivery agents. Collectively, we developed a novel protocol using serum-derived EXOs to deliver designated small RNA molecules into lung macrophages in vivo, potentially shedding light on future gene therapy of human lung diseases.

Long noncoding RNA FOXD3-AS1 regulates oxidative stress-induced apoptosis via sponging microRNA-150.

The function of most human long noncoding RNAs (lncRNAs) remains unclear. Our studies identified a highly up-regulated mammalian lncRNA, FOXD3-AS1, known as linc1623 in mice, in the setting of hyperoxia/reactive oxygen species (ROS)-induced lung injury. We found that ROS induced a robust expression of FOXD3-AS1 in mouse lung tissue. Functionally, FOXD3-AS1 promoted oxidative stress-induced lung epithelial cell death. In human lung epithelial cells, the microRNA-150 (miR-150) was identified to interact with FOXD3-AS1; this finding was confirmed using the luciferase reporter assays. Consistently, mutation on the miR-150 pairing sequence in FOXD3-AS1 abolished the interactions between FOXD3-AS1 and miR-150. Additionally, miR-150 mimics suppressed the level of FOXD3-AS1. The antisense oligos of FOXD3-AS1 significantly augmented the intracellular level of miR-150, supporting the theory of sponging effects of FOXD3-AS1 on miR-150. We further investigated the cellular function of miR-150 in our lung injury models. MiR-150 conferred a cytoprotective role in lung epithelial cells after oxidative stress, whereas FOXD3-AS1 promoted cell death. Taken together, our studies indicated that FOXD3-AS1 serves as a sponge or as a competing endogenous noncoding RNA for miR-150, restricting its capability to promote cell growth and thereby exaggerating hyperoxia-induced lung epithelial cell death.-Zhang, D., Lee, H., Haspel, J. A., Jin, Y. Long noncoding RNA FOXD3-AS1 regulates oxidative stress-induced apoptosis via sponging microRNA-150.

Enrichment of selective miRNAs in exosomes and delivery of exosomal miRNAs in vitro and in vivo.

Exosomes are nanovesicles secreted by cells and contain various molecules including protein, lipid, and DNA/RNA. They are crucial mediators of the intercellular communication and serve as promising vehicles for drug delivery and gene therapy. Recently, accumulating evidence suggests that microRNAs (miRNAs) may serve as new and potentially powerful targets for therapeutic interventions against various human diseases. However, steadily and effectively delivering miRNA mimics or inhibitors to target cells remains a major obstacle. To enhance the efficacy of exosome-mediated delivery of miRNA molecules, it is crucial to develop a convenient and efficient method to enrich specific miRNAs or antisense oligos in isolated exosomes. Here we report a novel method to prepare specific miRNA molecule-loaded exosomes. Using a modified calcium chloride-mediated transfection method, we successfully enhanced the designated miRNA mimics or inhibitors in isolated exosomes directly, instead of transfecting their mother cells. We also compared this method with direct transfection of exosomes using electroporation. Both methods confirmed that exosomes can serve as cargos to deliver a robustly increased amount of selected miRNA mimic(s) or inhibitor(s) to the recipient cells. Delivery of these miRNA molecule enriched-exosomes subsequently results in highly efficient overexpression or deletion of the designated miRNAs in the recipient cells both in vivo and in vitro. Additionally, we confirmed that exosome-delivered miRNA mimics or inhibitors are functional in the recipient cells. Collectively, we developed a novel protocol to conveniently manipulate exosomal miRNAs with high efficiency and successfully deliver the exosomal miRNA molecules to recipient cells.

Mesencephalic Astrocyte-Derived Neurotrophic Factor as a Urine Biomarker for Endoplasmic Reticulum Stress-Related Kidney Diseases.

Endoplasmic reticulum (ER) stress and disrupted proteostasis contribute to the pathogenesis of a variety of glomerular and tubular diseases. Thus, it is imperative to develop noninvasive biomarkers for detecting ER stress in podocytes or tubular cells in the incipient stage of disease, when a kidney biopsy is not yet clinically indicated. Mesencephalic astrocyte-derived neurotrophic factor (MANF) localizes to the ER lumen and is secreted in response to ER stress in several cell types. Here, using mouse models of human nephrotic syndrome caused by mutant laminin ß2 protein-induced podocyte ER stress and AKI triggered by tunicamycin- or ischemia-reperfusion-induced tubular ER stress, we examined MANF as a potential urine biomarker for detecting ER stress in podocytes or renal tubular cells. ER stress upregulated MANF expression in podocytes and tubular cells. Notably, urinary MANF excretion concurrent with podocyte or tubular cell ER stress preceded clinical or histologic manifestations of the corresponding disease. Thus, MANF can potentially serve as a urine diagnostic or prognostic biomarker in ER stress-related kidney diseases to help stratify disease risk, predict disease progression, monitor treatment response, and identify subgroups of patients who can be treated with ER stress modulators in a highly targeted manner.

miR-185 mediates lung epithelial cell death after oxidative stress.

Lung epithelial cell death is a prominent feature involved in the development of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Hyperoxia-induced ALI is an established animal model mimicking human ARDS. Small noncoding RNAs such as microRNAs (miRNAs) have potent physiological and pathological functions involving multiple disease processes. Emerging interests focus on the potential of miRNAs to serve as novel therapeutic targets and diagnostic biomarkers. We found that hyperoxia highly induces miR-185 and its precursor in human lung epithelial cells in a time-dependent manner, and this observation is confirmed using mouse primary lung epithelial cells. The hyperoxia-induced miR-185 is mediated by reactive oxygen species. Furthermore, histone deacetylase 4 (HDAC4) locates in the promoter region of miR-185. We found that hyperoxia suppresses HDAC4 specifically in a time-dependent manner and subsequently affects histone deacetylation, resulting in an elevated miR-185 transcription. Using MC1586, an inhibitor of class IIa HDACs, we showed that inhibition of class IIa HDACs upregulates the expression of miR-185, mimicking the effects of hyperoxia. Functionally, miR-185 promotes hyperoxia-induced lung epithelial cell death through inducing DNA damage. We confirmed functional roles of miR-185 using both the loss- and gain-of-function approaches. Moreover, multiple 14-3-3δ pathway proteins are highly attenuated by miR-185 in the presence of hyperoxia. Taken together, hyperoxia-induced miR-185 in lung epithelial cells contributes to oxidative stress-associated epithelial cell death through enhanced DNA damage and modulation of 14-3-3δ pathways.

MiR-15a/16 Regulates Apoptosis of Lung Epithelial Cells after Oxidative Stress.

Lung epithelial cell apoptosis is an important feature of hyperoxia-induced lung injury. Death receptor-associated extrinsic pathway and mitochondria-associated intrinsic pathway both mediate the development of lung epithelial cell apoptosis. Despite decades of research, molecular mechanisms of hyperoxia-induced epithelial cell apoptosis remain incompletely understood. Here we report a novel regulatory paradigm in response to hyperoxia-associated oxidative stress. Hyperoxia markedly up-regulated miR-15a/16 levels in lung epithelial cells, broncho-alveolar lavage fluid (BALF) and lung tissue. This effect was mediated by hyperoxia-induced reactive oxygen species (ROS). Functionally, miR-15a/16 inhibitors induced caspase 3-mediated lung epithelial cell apoptosis, in the presence of hyperoxia. MiR-15a/16 inhibitors robustly enhanced FADD level and down-regulated Bcl-2 expression. Consistently, cleaved caspase 8 and 9 were highly induced in the miR-15a/16 deficient cells, after hyperoxia. Using airway epithelial cell specific, miR-15a/16-/- mice, we found that Bcl-2 significantly reduced in lung epithelial cells in vivo after hyperoxia. In contrast, caspase 3, 8 and Bcl-2 associated death promoter (BAD) were highly elevated in the miR-15a/16-/- epithelial cells in vivo. Interestingly, in lung epithelial malignant cells, rather than benign cells, deletion of miR-15a/16 prevented apoptosis. Furthermore, deletion of miR-15a/16 in macrophages also prohibited apoptosis, opposite to what we have found in normal lung epithelial cells. Taken together, our data suggested that miR-15a/16 may exert differential roles in different cell types. MiR-15a/16 deficiency result in lung epithelial cell apoptosis in response to hyperoxia, via modulating both intrinsic and extrinsic apoptosis pathways.

Altered circular RNA expressions in extracellular vesicles from bronchoalveolar lavage fluids in mice after bacterial infections.

Circular RNAs (circRNAs) are a class of transcripts that often are generated by back-splicing that covalently connects the 3'end of the exon to the 5'end. CircRNAs are more resistant to nuclease and more stable than their linear counterparts. One of the well-recognized roles of circRNAs is the miRNA sponging effects that potentially lead to the regulation of downstream proteins. Despite that circRNAs have been reported to be involved in a wide range of human diseases, including cancers, cardiovascular, and neurological diseases, they have not been studied in inflammatory lung responses. Here, we analyzed the circRNA profiles detected in extracellular vesicles (EVs) obtained from the broncho-alveolar lavage fluids (BALF) in response to LPS or acid instillation in mice. Next, we validated two specific circRNAs in the BALF-EVs and BALF cells in response to endotoxin by RT-qPCR, using specific primers targeting the circular form of RNAs rather than the linear host RNAs. The expression of these selected circRNAs in the BALF inflammatory cells, alveolar macrophages (AMs), neutrophils, and lung tissue were analyzed. We further predicted the potential miRNAs that interact with these circRNAs. Our study is the first report to show that circRNAs are detectable in BALF EVs obtained from mice. The EV-cargo circRNAs are significantly altered by the noxious stimuli. The circRNAs identified using microarrays may be validated by RT-qPCR using primers specific to the circular but not the linear form. Future studies to investigate circRNA expression and function including miRNA sponging in lung inflammation potentially uncover novel strategies to develop diagnostic/therapeutic targets.

Emerging Roles of Circular RNA in Macrophage Activation and Inflammatory Lung Responses.

Circular RNA (circRNA) is a type of single-stranded RNA that forms a covalently closed continuous loop, unlike linear RNA. The expression of circRNAs in mammals is often conserved across species and shows tissue and cell specificity. Some circRNA serve as gene regulators. However, the biological function of most circRNAs is unclear. CircRNA does not have 5' or 3' ends. The unique structure of circRNAs provides them with a much longer half-life and more resistance to RNase R than linear RNAs. Inflammatory lung responses occur in the pathogenesis and recovery of many lung diseases. Macrophages form the first line of host defense/innate immune responses and initiate/mediate lung inflammation. For example, in bacterial pneumonia, upon pro-inflammatory activation, they release early response cytokines/chemokines that recruit neutrophils, macrophages, and lymphocytes to sites of infection and clear pathogens. The functional effects and mechanisms by which circRNAs exert physiological or pathological roles in macrophage activation and lung inflammation remain poorly understood. In this article, we will review the current understanding and progress of circRNA biogenesis, regulation, secretion, and degradation. Furthermore, we will review the current reports on the role of circRNAs in macrophage activation and polarization, as well as in the process of inflammatory lung responses.

Protocol to isolate and characterize pulmonary-specific extracellular vesicles in mice.

Extracellular vesicles (EVs) are membranous nanoparticles classified based on their size and surface markers, which can be specific to various cell origins. Here, we present a protocol for the isolation of pulmonary-specific EVs in mice. We describe steps for differential centrifugation, density gradient centrifugation, and commercially available polyethylene glycol(PEG)-based precipitation, employing pulmonary-specific EV-bound chemicals and antibodies. We then detail procedures for the characterization of these EVs through nanoparticle tracking analysis, flow cytometry, scanning electron microscopy, and transmission electron microscopy. For complete details on the use and execution of this protocol, please refer to Lee et al.1,2,3,4.

Beta-lapachone ameliorates the progression of primary sclerosing cholangitis pathogenesis in rodent models.

AIMS: Primary sclerosing cholangitis (PSC) is a rare cholestatic liver disease characterized by chronic inflammation and severe fibrosis for which effective treatment options are currently lacking. In this study, we explored the potential of beta-lapachone (ßL) as a drug candidate for PSC therapy. MATERIALS AND METHODS: We employed an animal model fed a diet containing 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) to assess the preventive and therapeutic effects of ßL. The beneficial effects of ßL on PSC pathogenic characteristics, including blood biomarkers, inflammation, and fibrosis, were determined by assessing relevant parameters. Differential gene expression between each group was analyzed by RNA sequencing of liver tissues. Mdr2-/- mice were utilized to explore the involvement of Abcb4 in the ßL-induced improvement of PSC pathogenesis. KEY FINDINGS: ßL effectively inhibited key features of PSC pathogenesis, as demonstrated by reduced blood biomarkers and improved pathogenic characteristics. Treatment with ßL significantly mitigated DDC-induced apoptosis, cell proliferation, inflammation, and fibrosis. Analysis of differential gene expression confirmed a new insight that ßL could stimulate the expression of genes related to NAD synthesis and Abcb4. Indeed, ßL-induced NAD exhibited effective functioning, as evidenced by enhanced sirt1/3 and acetyl-lysine levels, leading to improved mitochondrial stability. The role of Abcb4 in response to ßL was confirmed in Mdr2/Abcb4 KO mice, where the beneficial effects of ßL were abolished. SIGNIFICANCE: This study provided a new concept for PSC treatment, suggesting that pharmacological stimulation of the NAD synthetic pathway and Abcb4 via ßL ameliorates PSC pathogenesis.

The mechanism underlying correlation of particulate matter-induced ferroptosis with inflammasome activation and iron accumulation in macrophages.

Particulate matter (PM) is a global environmental hazard, which affects human health through free radical production, cell death induction, and immune responses. PM activates inflammasomes leading to excessive inflammatory responses and induces ferroptosis, a type of cell death. Despite ongoing research on the correlation among PM-induced ferroptosis, immune response, and inflammasomes, the underlying mechanism of this relationship has not been elucidated. In this study, we demonstrated the levels of PM-induced cell death and immune responses in murine macrophages, J774A.1 and RAW264.7, depending on the size and composition of particulate matter. PM2.5, with extraction ions, induced significant levels of cell death and immune responses; it induces lipid peroxidation, iron accumulation, and reactive oxygen species (ROS) production, which characterize ferroptosis. In addition, inflammasome-mediated cell death occurred owing to the excessive activation of inflammatory responses. PM-induced iron accumulation activates ferroptosis and inflammasome formation through ROS production; similar results were observed in vivo. These results suggest that the link between ferroptosis and inflammasome formation induced by PM, especially PM2.5 with extraction ions, is established through the iron-ROS axis. Moreover, this study can effectively facilitate the development of a new therapeutic strategy for PM-induced immune and respiratory diseases.

Anti-Inflammatory Effects of Idebenone Attenuate LPS-Induced Systemic Inflammatory Diseases by Suppressing NF-κB Activation.

Inflammation is a natural protective process through which the immune system responds to injury, infection, or irritation. However, hyperinflammation or long-term inflammatory responses can cause various inflammatory diseases. Although idebenone was initially developed for the treatment of cognitive impairment and dementia, it is currently used to treat various diseases. However, its anti-inflammatory effects and regulatory functions in inflammatory diseases are yet to be elucidated. Therefore, this study aimed to investigate the anti-inflammatory effects of idebenone in cecal ligation puncture-induced sepsis and lipopolysaccharide-induced systemic inflammation. Murine models of cecal ligation puncture-induced sepsis and lipopolysaccharide-induced systemic inflammation were generated, followed by treatment with various concentrations of idebenone. Additionally, lipopolysaccharide-stimulated macrophages were treated with idebenone to elucidate its anti-inflammatory effects at the cellular level. Idebenone treatment significantly improved survival rate, protected against tissue damage, and decreased the expression of inflammatory enzymes and cytokines in mice models of sepsis and systemic inflammation. Additionally, idebenone treatment suppressed inflammatory responses in macrophages, inhibited the NF-κB signaling pathway, reduced reactive oxygen species and lipid peroxidation, and normalized the activities of antioxidant enzyme. Idebenone possesses potential therapeutic application as a novel anti-inflammatory agent in systemic inflammatory diseases and sepsis.

Caveolin-1 regulates OMV-induced macrophage pro-inflammatory activation and multiple Toll-like receptors.

Macrophages (MФ), the primary cell of the innate immune system, serves as the first line of defense. During bacterial infection, Gram-negative (G-) bacteria release nanosized outer membrane vesicles (OMVs), facilitating the crosstalk between the microbe and the host. The underlying mechanisms by which OMVs induced pro-inflammatory (M1) activation are still unknown. Our study shows that OMVs caused M1 activation via modulating various toll-like receptor (TLR) expressions as they contain LPS, LTA, bacterial DNAs, and flagellins. Also, we found that caveolin-1 (cav-1), a 21-kDa scaffolding protein of caveolae and lipid rafts, plays a significant role in OMV-induced pro-inflammatory response in regulating various TLR signaling pathways. Specifically, cav-1 deletion increased the expression of OMV-induced TLRs, pro-inflammatory cytokine secretions (TNF-α and IL-1ß), and the reactive oxygen species (ROS) production in MФs. Further, we examined the interaction between Cav-1 and TLR4 by immunoprecipitation, colocalization, and computational models, providing future direction to explore the role of cav-1 in OMV-induced other TLR signaling. Altogether, Cav-1 is a key regulator in OMV-induced multiple TLRs response. This study promotes future research to develop drugs by targeting the specific motif of cav-1 or TLRs against bacterial infection and macrophage-mediated inflammation.

Specific upregulation of extracellular miR-6238 in particulate matter-induced acute lung injury and its immunomodulation.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are life-threatening diseases characterized by a severe inflammatory response and the destruction of alveolar epithelium and endothelium. ALI/ARDS is caused by pathogens and toxic environmental stimuli, such as particulate matter (PM). However, the general symptoms of ALI/ARDS are similar, and determining the cause of lung injury is often challenging. In this study, we investigated whether there is a critical miRNA that characterizes PM-induced ALI. We found that the expression of miR-6238 is specifically upregulated in lung tissue and lung-derived extracellular vesicles (EVs) in response to PM exposure. Notably, bacterial endotoxin (Lipopolysaccharide; LPS or peptidoglycan; PTG) does not induce the expression of miR-6238 in the lung. Instead, the expression of miR-155 is dramatically increased in LPS-induced ALI. We further demonstrated that human lung epithelial cells and macrophages predominantly produce miR-6238 and miR-155, respectively. Mechanistically, EV-miR-6238 is effectively internalized into alveolar macrophages (AMs) and regulates inflammatory responses in vivo. CXCL3 is a main target of miR-6238 in AMs and modulates neutrophil infiltration into the lung alveoli. Collectively, our findings suggest that miR-6238 is a novel regulator of pulmonary inflammation and a putative biomarker that distinguishes PM-induced ALI from endotoxin (LPS/PTG)-mediated ALI.