Extracellular vesicles: Their emerging roles in the pathogenesis of respiratory diseases

Alveoli are the basic structure of the lungs, consisting of various types of parenchymal and bone marrow-derived cells including alveolar macrophages. These various types of cells have several important functions; thus, communication between these cells plays an important role in homeostasis as well as in the pathophysiology of diseases in the lungs. For a better understanding of the pathophysiology of lung diseases, researchers have isolated each type of lung cell to investigate the changes in their gene expressions, including their humoral factor or adhesion molecules, to reveal the intercellular communication among these cells. In particular, investigations during the past decade have focused on extracellular vesicles, which are lipid bilayer delimited vesicles released from a cell that can move among various cells and transfer substances, including microRNAs, mRNAs and proteins, thus, functioning as intercellular messengers. Extracellular vesicles can be classified into three general groups: apoptotic bodies, exosomes, and microparticles. Extracellular vesicles, especially exosomes and microparticles, are attracting increasing attention from pulmonologists as tools for understanding pathogenesis and disease diagnosis. Here, we review studies, including our own, on exosomes and microparticles and their roles in both lung homeostasis and the pathogenesis of lung diseases such as idiopathic pulmonary fibrosis, chronic obstructive lung diseases, and acute respiratory distress syndrome. This review also addresses the roles of extracellular vesicles in COVID-19, the current global public health crisis.     

The endogenous inflammatory reflex inhibits the inflammatory response to different immune challenges in mice

The splanchnic anti-inflammatory pathway, the efferent arm of the endogenous inflammatory reflex, has been shown to suppress the acute inflammatory response of rats to systemic lipopolysaccharide (LPS). Here we show for the first time that this applies also to mice, and that the reflex may be engaged by a range of inflammatory stimuli. Experiments were performed on mice under deep anaesthesia. Half the animals were subjected to bilateral section of the splanchnic sympathetic nerves, to disconnect the splanchnic anti-inflammatory pathway, while the remainder underwent a sham operation. Mice were then challenged intravenously with one of three inflammatory stimuli: the toll-like receptor (TLR)-4 agonist, LPS (60 µg/kg), the TLR-3 agonist Polyinosinic:polycytidylic acid (Poly I:C, 1 mg/kg) or the TLR-2 and -6 agonist dipalmitoyl-S-glyceryl cysteine (Pam2cys, 34 µg/kg). Ninety minutes later, blood was sampled by cardiac puncture for serum cytokine analysis. The splanchnic anti-inflammatory reflex action was assessed by comparing cytokine levels between animals with cut versus those with intact splanchnic nerves. A consistent pattern emerged: Tumor necrosis factor (TNF) levels in response to all three challenges were raised by prior splanchnic nerve section, while levels of the anti-inflammatory cytokine interleukin 10 (IL-10) were reduced. The raised TNF:IL-10 ratio after splanchnic nerve section indicates an enhanced inflammatory state when the reflex is disabled. These findings show for the first time that the inflammatory reflex drives a coordinated anti-inflammatory action also in mice, and demonstrate that its anti-inflammatory action is engaged, in similar fashion, by inflammatory stimuli mimicking a range of bacterial and viral infections.     

PFKFB3参与脂多糖诱导肺成纤维细胞及肺组织有氧糖酵解的观察性研究

目的:观察脂多糖(lipopolysaccharide,LPS)诱导肺成纤维细胞及肺组织有氧糖酵解关键酶6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶3(6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3,PFKFB3)表达及其与有氧糖酵解的关系,探讨在LPS诱导肺纤维化过程中肺成纤维细胞和肺组织有氧糖酵解的潜在机制。方法:将人胚肺成纤维细胞MRC-5细胞系采用随机数字表法分为PBS对照组(PBS组)和LPS组(每组3孔),Western blot检测LPS刺激细胞6h后PFKFB3表达情况,同时免疫荧光显示PFKFB3在细胞内的定位情况;于LPS刺激后48 h采用海马细胞能量代谢仪检测细胞耗氧率(oxygen consumption rate,OCR)和产酸率(extracellular acidification rate,ECAR),并采用比色法检测有氧糖酵解产物乳酸产生情况,同时Western blot检测LPS刺激48 h后Ⅰ型胶原蛋白合成情况。将24只C57BL/6小鼠按随机数字表法分为生理盐水对照组(C组)、LPS组(L组),每组12只,L组、C组连续5d分别腹腔注射5 mg/kg LPS、等容量生理盐水;每组各6只于造模后第7天无痛处死小鼠,取血浆和肺组织,Western blot和免疫荧光检测各组肺组织中PFKFB3表达情况,比色法检测各组小鼠血浆中乳酸的含量;剩余小鼠于造模后第28天取肺组织,一侧肺通过Western blot检测肺组织Ⅰ型胶原蛋白合成情况,另一侧肺做石蜡切片进行病理学检测。结果:与PBS组比较,LPS刺激细胞6h后PFKFB3表达明显升高(P<0.05);LPS刺激细胞48 h后,与PBS组比较,LPS组细胞耗氧率降低、产酸率增加,代谢产物乳酸含量明显升高(P<0.05),同时细胞Ⅰ型胶原蛋白合成显著增加(P<0.05)。与C组比较,L组小鼠腹腔注射LPS 7 d后肺组织中PFKFB3表达明显升高(P<0.05),血浆乳酸含量明显升高(P<0.05);LPS注射28 d后,L组小鼠Ⅰ型胶原蛋白表达明显升高(P<0.05),肺组织出现明显纤维化。结论:在LPS诱导的肺纤维化过程中,LPS可诱导肺成纤维细胞和肺组织中PFKFB3蛋白表达,该过程与其有氧糖酵解过程相关,PFKFB3的表达上调可能是LPS诱导肺成纤维细胞和肺组织有氧糖酵解和肺纤维化的关键环节。     

活血清胰汤灌肠辅助手术治疗对急性重症胰腺炎患者的疗效观察

目的：探讨活血清胰汤灌肠辅助手术治疗急性重症胰腺炎对患者AMS、UAMY、LPS水平及免疫功能的影响。方法：选取2019年3月至2020年3月陕西省宝鸡市中医医院收治的急性重症胰腺炎患者70例作为研究对象，按随机数字表法将其分为对照组和观察组，每组35例。2组均采用经内镜逆行性胰胆管造影术进行治疗，对照组术后给予常规治疗，观察组术后在对照组的基础上加用活血清胰汤灌肠治疗，2组均治疗1周。比较2组治疗1周后的临床疗效；比较2组治疗前、治疗1周后的胰腺炎标志物水平、细胞免疫指标及体液免疫指标水平。结果：治疗1周后，观察组总有效率为94.29%，高于对照组（77.14%，P<0.05）。与治疗前比较，治疗1周后，2组血清AMS、LPS、UAMY及CD8+水平均降低，且观察组低于对照组（P<0.05）；2组血清CD3+、CD4+、IgG、IgM、IgA水平及CD4+/CD8+比值均升高，且观察组高于对照组（P<0.05）。结论：活血清胰汤灌肠辅助手术治疗急性重症胰腺炎，可通过降低患者血清AMS、LPS及UAMY水平，缓解炎性反应，同时可改善免疫功能，进而有助于提高疗效。     

Protective efficacy afforded by live Pasteurella multocida vaccines in chickens is independent of lipopolysaccharide outer core structure

Pasteurella multocida is a major animal pathogen that causes a range of diseases including fowl cholera. P. multocida infections result in considerable losses to layer and breeder flocks in poultry industries worldwide. Both killed whole-cell and live-attenuated vaccines are available; these vaccines vary in their protective efficacy, particularly against heterologous strains. Moreover, until recently there was no knowledge of P. multocida LPS genetics and structure to determine precisely how LPS structure affects the protective capacity of these vaccines. In this study we show that defined lipopolysaccharide (LPS) mutants presented as killed whole-cell vaccines elicited solid protective immunity only against P. multocida challenge strains expressing highly similar or identical LPS structures. This finding indicates that vaccination of commercial flocks with P. multocida killed cell formulations will not protect against strains producing an LPS structure different to that produced by strains included in the vaccine formulation. Conversely, protective immunity conferred by vaccination with live P. multocida strains was found to be largely independent of LPS structure. Birds vaccinated with a range of live mutants belonging to the L1 and L3 LPS genotypes, each expressing a specific truncated LPS structure, were protected against challenge with the parent strain. Moreover, birds vaccinated with any of the five LPS mutants belonging to the L1 LPS genotype were also protected against challenge with an unrelated strain and two of the five groups vaccinated with live LPS mutants belonging to the L3 genotype were protected against challenge with an unrelated strain. In summary, vaccination with live P. multocida aroA mutants producing full-length L1 or L3 LPS or vaccination with live strains producing shortened L1 LPS elicited strong protective immunity against both homologous and heterologous challenge.     

Interleukin-35 pretreatment attenuates lipopolysaccharide-induced heart injury by inhibition of inflammation,apoptosis and fibrotic reactions

Previous studies have demonstrated that targeting inflammation is a promising strategy for treating lipopolysaccharide (LPS)-induced sepsis and related heart injury. Interleukin-35 (IL-35), which consists of two subunits, Epstein–Barr virus-induced gene 3 (EBI3) and p35, is an immunosuppressive cytokine of the IL-12 family and exhibits strong anti-inflammatory activity. However, the role of IL-35 in LPS-induced heart injury reains obscure. In this study, we explored the role of IL-35 in heart injury induced by LPS and its potential mechanisms. Mice were treated with a plasmid encoding IL-35 (pIL-35) and then injected intraperitoneally (ip) with LPS (10 mg/kg). Cardiac function was assessed by echocardiography 12 h later. LPS apparently decreased the expression of EBI3 and p35 and caused cardiac dysfunction and pathological changes, which were significantly improved by pIL-35 pretreatment. Moreover, pIL-35 pretreatment significantly decreased the levels of cardiac proinflammatory cytokines including TNF-α, IL-6, and IL-1β, and the NLRP3 inflammasome. Furthermore, decreased number of apoptotic myocardial cells, increased BCL-2 levels and decreased BAX levels inhibited apoptosis, and LPS-induced upregulation of the expression of cardiac pro-fibrotic genes (MMP2 and MMP9) and fibrotic factor (Collagen type I) was inhibited. Further investigation indicated that pIL-35 pretreatment might suppressed the activation of the cardiac NF-κBp65 and TGF-β1/Smad2/3 signaling pathways in LPS-treated mice. Similar cardioprotective effects of IL-35 pretreatment were observed in mouse myocardial fibroblasts challenged with LPS in vitro. In summary, IL-35 pretreatment can attenuate cardiac inflammation, apoptosis, and fibrotic reactions induced by LPS, implicating IL-35 as a promising therapeutic target in sepsis-related cardiac injury.     

Effects of Long-time Exposure to Lipopolysaccharide on Intestinal Lymph Node Immune Cells and Antibodies Level in Mice

Background: Endotoxin, widely present in the living environment of humans and animals, leads to endotoxemia during a short period. However, the long-term effects of endotoxin on immune function are unclear. Objective: To determine the importance of long-term endotoxin treatment on function of immune system. Methods: The mice were treated with different doses of lipopolysaccharide (LPS) for a month; the collected samples were then analyzed in terms of value changes in hematological parameters, lymphocyte subtypes, and immunoglobulins level. Results: The number of monocytes (MONO) and neutrophils (NEU) in the three treatment groups was significantly lower than the control after 30 days. However, the proportion of CD8+ T lymphocytes showed a rising trend in the mesenteric lymph nodes (MLNs) and Peyer's patches (PPs) while the CD4+ T cell was reduced. At the same time, a decrease was observed in the percentage of CD19+CD38+ B lymphocytes. Interestingly, the change of lymphocytes in PPs was more significant than that in MLNs, suggesting that immune response in the PPs occurred before the MLNs. Consistent with the changes in B cells, the content of IgA and IgG showed a downward trend. Conclusion: Long-term exposure to low-dose endotoxin had little or no effect on the immune function of the body, suggesting that the endotoxin can be rapidly eliminated by the immune system. Nonetheless, the number of immune cells was reduced in the high-dose group. T- and B-lymphocytes were significantly reduced, resulting in a decrease in immunoglobulin level, and showing a significant immune suppression state.     

Impact of acute inflammation on the extinction of aversive gut memories

Impaired extinction of pain-related fear memories can lead to persistent or resurging fear of pain, contributing to the development and maintenance of chronic pain conditions. The mechanisms underlying maladaptive pain-related learning and memory processes remain incompletely understood, particularly in the context of interoceptive, visceral pain. Inflammation is known to interfere with learning and memory, but its effects on the extinction of pain-related fear memories have never been tested. In a randomized, double-blind, placebo-controlled study, we assessed the impact of experimental acute inflammation on the extinction and reinstatement of conditioned visceral pain-related fear. Forty healthy male volunteers underwent differential fear conditioning with visceral pain as clinically relevant unconditioned stimulus (US). Participants then received an intravenous injection of either 0.8 ng/kg lipopolysaccharide (LPS) as inflammatory stimulus or physiological saline as placebo, and extinction training was conducted at the peak of the inflammatory response. Extinction recall and reinstatement test were performed after overnight consolidation. Results showed that visceral pain represents an effective US, eliciting pronounced conditioned pain-related fear responses. Repeated unreinforced presentation of the pain-predictive cue during extinction training resulted in full extinction of the conditioned behavioral response. However, unexpected re-exposure to the US during reinstatement test resulted in return of fear. Despite pronounced LPS-induced effects on inflammatory markers, cortisol, and negative affect, we did not find evidence that acute inflammation resulted in altered fear extinction. The findings support the notion that visceral pain-related fear learning establishes a robust aversive memory trace that remains preserved during inhibitory learning, leaving a latent vulnerability for the return of fear. Inflammation during inhibitory learning did neither weaken nor further amplify this aversive memory trace, suggesting that it is rather resistant to acute inflammation-induced effects, at least in healthy individuals with no additional vulnerability factors.     

腺病毒介导的白介素-24转移对脂多糖诱导的大鼠肾小球系膜细胞凋亡和周期调节蛋白p21、p27及CyclinE的影响

目的 探讨腺病毒介导的IL-24转移对脂多糖（LPS）诱导的大鼠肾小球系膜细胞（GMCs）和细胞周期调节蛋白p21、p27及CyclinE的影响。方法 采用10%FBS/DMEM培养293细胞,扩增腺病毒载体, GMCs培养4~6代细胞后用于分析。①观察IL-24对LPS诱导的GMCs凋亡影响：分为对照组、Ad.IL-24组、LPS组和LPS+ Ad.IL-24组,其中对照组和LPS组GMCs不转染携带IL-24腺病毒（Ad.IL-24）,Ad.IL-24组和LPS+Ad.IL-24组以Ad.IL-24转染GMCs,LPS组和LPS+Ad.IL-24组加LPS培养,以流式细胞术检测培养后24和48 h的GMCs凋亡率;②考察IL-24对GMCs周期蛋白影响：对照组为5%FBS/DMEM培养GMCs,Ad-GFP组采用携带绿色荧光蛋白的对照载体（Ad.GFP）转染GMCs,Ad.IL-24+LPS组以Ad.IL-24转染GMCs,采用Western-blot检测p21、p27及CyclinE表达。结果 ①GMCs在培养后24和48 h细胞凋亡率：对照组分别为(0.86±0.15)%和(0.98±0.4)%;IL-24组分别为(1.02±0.22)%和(1.43±0.31)%;LPS组分别为(2.19±0.81)%和(2.49±0.12)%,LPS+Ad.IL-24组分别为(18.01±1.17)%、(26.82±5.01)%;2个观察时间点细胞凋亡率对照组与IL-24组差异无统计学意义, LPS组显著高于对照组（P＜0.05）,LPS+ Ad.IL-24组细胞凋亡率最高（P＜0.05）。②Ad-GFP组p21、p27表达显著低于对照组（P<0.05）,CyclinE表达显著高于对照组（P<0.05）;Ad.IL-24+LPS组p21、p27表达较Ad-GFP组显著上调（P<0.05）,CyclinE表达较Ad-GFP组下调。结论 IL-24腺病毒载体对正常GMCs无影响,但可使LPS诱导增生的GMCs凋亡增加,其可能机制是上调关键细胞周期负调控蛋白p21、p27及下调CyclinE。