Interleukin-33 facilitates neutrophil recruitment and bacterial clearance in S. aureus-caused peritonitis

Interleukin (IL)-33, a newly recognized member of IL-1 family of cytokines, plays an important role in polarizing Th2-associated immunity. Recently growing evidence indicates that IL-33 also represents a crucial mediator of antimicrobial infection. In this study, we investigated the effect of IL-33 on antibacterial response using an acute Staphylococcus aureus peritoneal infection model. Our results showed that IL-33 administration induced a rapid bacterial clearance and markedly reduced the S. aureus infection-related mortality. IL-33-treated mice displayed increased neutrophil influx into the focus of infection and higher concentrations of chemokine CXCL2 in the peritoneum than untreated mice. The beneficial effect of IL-33 priming was related to reversal of the S. aureus-induced reduction of CXCR2 expression on the surface of neutrophils. Furthermore, conditioning of neutrophils by IL-33 led to the enhancement of complement receptor 3 expression induced by S. aureus, which in turn facilitates the phagocytosis of opsonized S. aureus. Finally, neutrophils primed by IL-33 upregulated the production of reactive oxygen species and the subsequent killing activity for S. aureus. All together, these findings suggest that IL-33, through regulating multiple steps of neutrophil-mediated bactericidal function, provides a profound effect in host antimicrobial defense response.     

Neutrophil extracellular traps in bacterial infections: Strategies for escaping from killing

Neutrophils are among the first responders to virulent factors. They kill microbes by phagocytosis, oxidative burst, and as neutrophil extracellular traps (NETs). NETs production leads to unique cell death depending on, inter alia, reactive oxygen species (ROS). Recently a number of studies highlight the mechanism of bacterial escape from extracellular traps; the process that may influence the outcome of bacterial infections.     

Role of mast cell leukotrienes in neutrophil recruitment and bacterial clearance in infectious peritonitis

Stimulated mast cells release a variety of chemotactic factors such as tumor necrosis factor alpha (TNF-alpha) and leukotriene B4. Recent studies have shown that mast cell-derived TNF-alpha plays a critical role in host defense against Gram negative bacterial infections by the recruitment of neutrophils to the sites of infection. In the present study, we sought to investigate if mast cells release leukotriene (LT) B4 in response to bacteria and, if so, to establish its in vivo relevance. We show that mast cells release significant amounts of LTB4 and LTC4 in response to exposure to FimH-expressing type 1 fimbriated Escherichia coli in vitro. To test the functional significance of mast cell-derived LTs during an E. coli infection in vivo, we examined the effect of a LT-synthesis inhibitor, A-63162, on bacterial clearance and neutrophil influx in an infectious peritonitis model in mast cell-deficient mice (WBB6F1-W/WV) and their normal congenic control (WBB6F1-+/+) mice. Our results show that a treatment with A-63162 reduced neutrophil influx and bacterial clearance in the peritoneal cavities of mast cell-sufficient but not -deficient mice. Thus, mast cell-derived LTs contribute to host defense by mediating early neutrophil influx and bacterial clearance at sites of infection.     

Beyond ROS clearance: Peroxiredoxins in stress signaling and aging

Antioxidants were long predicted to have lifespan-promoting effects, but in general this prediction has not been well supported. While some antioxidants do seem to have a clear effect on longevity, this may not be primarily as a result of their role in the removal of reactive oxygen species, but rather mediated by other mechanisms such as the modulation of intracellular signaling. In this review we discuss peroxiredoxins, a class of proteinaceous antioxidants with redox signaling and chaperone functions, and their involvement in regulating longevity and stress resistance. Peroxiredoxins have a clear role in the regulation of lifespan and survival of many model organisms, including the mouse, Caenorhabditis elegans and Drosophila melanogaster. Recent research on peroxiredoxins − in these models and beyond − has revealed surprising new insights regarding the interplay between peroxiredoxins and longevity signaling, which will be discussed here in detail. As redox signaling is emerging as a potentially important player in the regulation of longevity and aging, increased knowledge of these fascinating antioxidants and their mode(s) of action is paramount.     

Chemokine receptor CXCR2 mediates bacterial clearance rather than neutrophil recruitment in a murine model of pneumonic plague

Pulmonary infection by Yersinia pestis causes pneumonic plague, a necrotic bronchopneumonia that is rapidly lethal and highly contagious. Acute pneumonic plague accompanies the up-regulation of pro-inflammatory cytokines and chemokines, suggesting that the host innate immune response may contribute to the development of disease. To address this possibility, we sought to understand the consequences of neutrophil recruitment during pneumonic plague, and we studied the susceptibility of C3H-HeN mice lacking the CXC chemokine KC or its receptor CXC receptor 2 (CXCR2) to pulmonary Y. pestis infection. We found that without Kc or Cxcr2, disease progression was accelerated both in bacterial growth and development of primary bronchopneumonia. When examined in an antibody clearance model, Cxcr2(-/-) mice were not protected by neutralizing Y. pestis antibodies, yet bacterial growth in the lungs was delayed in a manner associated with a neutrophil-mediated inflammatory response. After this initial delay, however, robust neutrophil recruitment in Cxcr2(-/-) mice correlated with bacterial growth and the development of fulminant pneumonic and septicemic plague. In contrast, attenuated Y. pestis lacking the conserved pigmentation locus could be cleared from the lungs in the absence of Cxcr2 indicating virulence factors within this locus may inhibit CXCR2-independent pathways of bacterial killing. Together, the data suggest CXCR2 uniquely induces host defense mechanisms that are effective against virulent Y. pestis, raising new insight into the activation of neutrophils during infection.     

中性粒细胞小GTP结合蛋白的表达与冠心病发生关系的研究

目的：探讨中性粒细胞活性氧的产生及小GTP结合蛋白Rac2和RhoGDI的差异性表达在冠心病(GHD)发生发展中的作用。方法：采用化学发光法检测活性氧，适时定量RT-PCR检测调控中性粒细胞活性氧产生的两种小GTP结合蛋白Rac2和RhoGDI mRNA表达量的变化。结果：冠心病组中性粒细胞产生活性氧显著增多，冠心病人血清中各种炎性因子有促进中性粒细胞产生活性氧的作用；Rac2mRNA在冠心病组表达量显著性高于正常对照组，而RhoGDI mRNA的表达量在两组中元显著区别，Rac2 mRNA和RhoGDImRNA表达量的比值与中性粒细胞产生活性氧的峰值呈正相关。结论：中性粒细胞通过产生活性氧参与冠心病的发生发展，冠心病病人中性粒细胞Rac2和RhoGDImRNA表达量比例失调是导致活性氧产生增多的重要因素。     

The role and regulation of catalase in respiratory tract opportunistic bacterial pathogens

Respiratory tract bacterial pathogens are the etiologic agents of a variety of illnesses. The ability of these bacteria to cause disease is imparted through survival within the host and avoidance of pathogen clearance by the immune system. Respiratory tract pathogens are continually bombarded by reactive oxygen species (ROS), which may be produced by competing bacteria, normal metabolic function, or host immunological responses. In order to survive and proliferate, bacteria have adapted defense mechanisms to circumvent the effects of ROS. Bacteria employ the use of anti-oxidant enzymes, catalases and catalase-peroxidases, to relieve the effects of the oxidative stressors to which they are continually exposed. The decomposition of ROS has been shown to provide favorable conditions in which respiratory tract opportunistic bacterial pathogens such as Haemophilus influenzae, Mycobacterium tuberculosis, Legionella pneumophila, and Neisseria meningitidis are able to withstand exposure to highly reactive molecules and yet survive. Bacteria possessing mutations in the catalase gene have a decreased survival rate, yet may be able to compensate for the lack of catalatic activity if peroxidatic activity is present. An incomplete knowledge of the mechanisms by which catalase and catalase-peroxidases are regulated still persists, however, in some bacterial species, a regulatory factor known as OxyR has been shown to either up-regulate or down-regulate catalase gene expression. Yet, more research is still needed to increase the knowledge base in relation to this enzyme class. As with this review, we focus on major respiratory tract opportunistic bacterial pathogens in order to elucidate the function and regulation of catalases. The importance of the research could lead to the development of novel treatments against respiratory bacterial infections.     

The disruption of bacterial membrane integrity through ROS generation induced by nanohybrids of silver and clay

Nanohybrids, synthesized via silver nitrate reduction in the presence of silicate clay, exhibit a high potency against bacterial growth. The plate-like clay, due to its anionic surface charges and a large surface area, serves as the support for the formation of silver nanoparticles (AgNPs) 30 nm in diameter. The nanohybrid consisting of Ag/silicate at a 7/93 weight ratio inhibited the growth of dermal pathogens including Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa and Streptococcus pyrogens, as well as the methicillin- and oxacillin-resistant S. aureus (MRSA and ORSA). Scanning electron microscope revealed that these nanohybrids were adherent on the surface of individual bacteria. The thin silicate plates provide a surface for immobilizing AgNPs in one highly concentrated area but prevent them from entering the cell membrane. Subsequent cytotoxicity studies indicated that surface contact with the reduced AgNPs on clay is sufficient to initiate cell death. This toxicity is related to a loss in membrane integrity due to reactive oxygen species (ROS) generation. The hybridization of AgNPs on clay surface is viable for generating a new class of nanohybrids exhibiting mild cytotoxicity but high efficacy for battling drug-resistant bacteria.     

Carrageenan‐induced inflammation promotes ROS generation and neutrophil extracellular trap formation in a mouse model of peritonitis

Neutrophil extracellular traps (NETs) are a combination of DNA fibers and granular proteins, such as neutrophil elastase (NE). NETs are released in the extracellular space in response to different stimuli. Carrageenan is a sulfated polysaccharide extracted from Chondrus crispus, a marine algae, used for decades in research for its potential to induce inflammation in different animal models. In this study, we show for the first time that carrageenan injection can induce NET release in a mouse model of acute peritonitis. Carrageenan induced NET release by viable neutrophils with NE and myeloperoxidase (MPO) expressed on DNA fibers. Furthermore, although this polysaccharide was able to stimulate reactive oxygen species (ROS) generation by peritoneal neutrophils, NADPH oxidase derived ROS were dispensable for NET formation by carrageenan. In conclusion, our results show that carrageenan‐induced inflammation in the peritoneum of mice can induce NET formation in an ROS‐independent manner. These results may add important information to the field of inflammation and potentially lead to novel anti‐inflammatory agents targeting the production of NETs.     

Innate activation of human neutrophils and neutrophil-like cells by the pro-inflammatory bacterial metabolite ADP-heptose and Helicobacter pylori

Lipopolysaccharide inner core heptose metabolites, including ADP-heptose, play a substantial role in the activation of cell-autonomous innate immune responses in eukaryotic cells, via the ALPK1-TIFA signaling pathway, as demonstrated for various pathogenic bacteria. The important role of LPS heptose metabolites during Helicobacter pylori infection of the human gastric niche has been demonstrated for gastric epithelial cells and macrophages, while the role of heptose metabolites on human neutrophils has not been investigated. In this study, we aimed to gain a better understanding of the activation potential of bacterial heptose metabolites for human neutrophil cells. To do so, we used pure ADP-heptose and, as a bacterial model, H. pylori, which can transport heptose metabolites into the human host cell via the Cag Type 4 Secretion System (CagT4SS). Main questions were how bacterial heptose metabolites impact on the pro-inflammatory activation, alone and in the bacterial context, and how they influence maturation of human neutrophils. Results of the present study demonstrated that neutrophils respond with high sensitivity to pure heptose metabolites, and that global regulation networks and neutrophil maturation are influenced by heptose exposure. Furthermore, activation of human neutrophils by live H. pylori is strongly impacted by the presence of LPS heptose metabolites and the functionality of its CagT4SS. Similar activities were determined in cell culture neutrophils of different maturation states and in human primary neutrophils. In conclusion, we demonstrated that specific heptose metabolites or bacteria producing heptoses exhibit a strong activity on cell-autonomous innate responses of human neutrophils.     

Induced CNS expression of CXCL1 augments neurologic disease in a murine model of multiple sclerosis via enhanced neutrophil recruitment

Increasing evidence points to an important role for neutrophils in participating in the pathogenesis of the human demyelinating disease MS and the animal model EAE. Therefore, a better understanding of the signals controlling migration of neutrophils as well as evaluating the role of these cells in demyelination is important to define cellular components that contribute to disease in MS patients. In this study, we examined the functional role of the chemokine CXCL1 in contributing to neuroinflammation and demyelination in EAE. Using transgenic mice in which expression of CXCL1 is under the control of a tetracycline‐inducible promoter active within glial fibrillary acidic protein‐positive cells, we have shown that sustained CXCL1 expression within the CNS increased the severity of clinical and histologic disease that was independent of an increase in the frequency of encephalitogenic Th1 and Th17 cells. Rather, disease was associated with enhanced recruitment of CD11b⁺Ly6G⁺ neutrophils into the spinal cord. Targeting neutrophils resulted in a reduction in demyelination arguing for a role for these cells in myelin damage. Collectively, these findings emphasize that CXCL1‐mediated attraction of neutrophils into the CNS augments demyelination suggesting that this signaling pathway may offer new targets for therapeutic intervention.     

Properdin: New roles in pattern recognition and target clearance

Properdin was first described over 50 years ago by Louis Pillemer and his collaborators as a vital component of an antibody-independent complement activation pathway. In the 1970s properdin was shown to be a stabilizing component of the alternative pathway convertases, the central enzymes of the complement cascade. Recently we have reported that properdin can also bind to target cells and microbes, provide a platform for convertase assembly and function, and promote target phagocytosis. Evidence is emerging that suggests that properdin interacts with a network of target ligands, phagocyte receptors, and serum regulators. Here we review the new findings and their possible implications.     

Kinetics of pulmonary neutrophil recruitment and clearance in a natural and spontaneously resolving model of airway inflammation

BACKGROUND: Neutrophil apoptosis and phagocytic clearance have been proposed as key determinants affecting the resolution of airway inflammation. Objective To determine the kinetics of neutrophil priming, recruitment, activation and subsequent clearance in a naturally occurring equine disease model of neutrophilic pulmonary inflammation. METHODS AND RESULTS: A 5 h mouldy hay/straw challenge in hypersensitive horses induced transient pulmonary dysfunction lasting 4 days. At 24 h circulating neutrophils were primed and displayed delayed rates of spontaneous apoptosis in vitro. Neutrophil numbers in the airspaces peaked at 5 h and then fell abruptly, returning to pre-challenge levels by 4 days. Airspace neutrophils demonstrated increased respiratory burst activity compared with circulating cells and equine neutrophil elastase 2A concentrations increased in parallel with neutrophil numbers indicating in vivo priming and degranulation. The number of apoptotic neutrophils and proportion of alveolar macrophages containing phagocytosed apoptotic neutrophils increased significantly at 24 h and 4 days post-challenge corresponding to the period of most rapid neutrophil clearance. CONCLUSION: This is the first demonstration of spontaneous neutrophil apoptosis and phagocytic removal in a natural disease model of airway inflammation and provides critical kinetic data to support the hypothesis that this clearance pathway plays a central role in the resolution of neutrophilic inflammation.     

活性氧促进中性粒细胞与骨髓基质细胞黏附及机制初探

目的:探讨活性氧对中性粒细胞与骨髓基质细胞(BMSCs)黏附的影响及其机制。方法:采用密度梯度离心法从小鼠胫骨和股骨分离骨髓中性粒细胞,并用DMSO诱导HL60细胞分化为成熟中性粒细胞(d HL60细胞)。利用分光光度法检测CFDA-SE标记的小鼠骨髓中性粒细胞和d HL60细胞在H_2O_2刺激下与BMSCs的黏附。利用荧光显微成像技术和Western blot检测携带去谷胱甘肽化酶谷氧还蛋白1(Grx1)表达载体的慢病毒感染的d HL60细胞中Grx1的表达。PCR检测Grx1敲除小鼠的基因型。结果:(1) Diff-Quick染色显示,分离的骨髓中性粒细胞纯度高于90%; H_2O_2处理后,中性粒细胞与BMSCs黏附率显著增加(P 0. 01)。(2)荧光显微镜观察和Western blot结果表明,Grx1稳转株d HL60细胞中Grx1的表达水平较空载体稳转株细胞显著增加;体外黏附实验表明过表达Grx1的d HL60细胞较对照组d HL60细胞,在相同H_2O_2浓度下,黏附到BMSCs程度显著下降(P 0. 01)。(3)经PCR鉴定,实验所用基因敲除小鼠在全基因水平上未见Grx1;且敲除Grx1的小鼠较野生型小鼠,在相同浓度H_2O_2刺激下,其骨髓中性粒细胞与BMSCs黏附率显著升高。(4)血管细胞黏附分子1(VCAM-1)抗体预处理BMSCs后,由H_2O_2引起的d HL60与BMSCs之间的黏附增强回复到了静息水平。结论:活性氧促进骨髓中中性粒细胞与BMSCs的黏附,其可能的机制是通过诱导VCAM-1黏附信号的S-谷胱甘肽化。     

The effect of reactive oxidant generation in acute exercise-induced proteinuria in trained and untrained rats

Exercise-induced proteinuria is a common consequence of physical activity, although its mechanism is not clear. We investigated whether free radicals generated during exercise play a role in post-exercise proteinuria in sedentary and treadmill-running trained rats, separately. Sedentary and trained rats were randomly divided into four sub-groups: control, antioxidant treatment, exhaustive exercise and an exhaustive exercise plus antioxidant treatment group. Antioxidant therapy was applied by intragastric catheter for 4 weeks with vitamin C (ascorbic acid, 50 mg·kg^–1·day^–1) and vitamin E (-tocopherol, 20 mg·kg^–1·day^–1). Twenty-four-hour urine samples were used for measuring protein levels and protein electrophoresis. Thiobarbituric acid (TBARS) and glutathione (GSH) levels, superoxide dismutase (SOD) and catalase (CAT) activities were assayed in blood and tissues. Increased urinary protein levels and mixed type proteinuria in electrophoresis were identified after exhaustive exercise in sedentary rats. Erythrocyte, kidney and muscle TBARS levels were significantly elevated in this group. Antioxidant treatment prevented the increase in urinary protein levels, TBARS levels and the occurrence of mixed type proteinuria after exhaustive exercise in sedentary rats. Exhaustive exercise in trained rats resulted in elevation of urine protein levels and mixed type proteinuria although kidney TBARS levels were not changed compared to those of the trained controls. Antioxidant therapy in trained and exhausted-trained animals resulted in decreased TBARS levels in the kidney but it did not affect urinary-increased protein levels or electrophoresis in exhausted animals. This findings suggest that the exercise-induced oxidant stress may contribute to post-exercise proteinuria in sedentary rats. However, this mechanism may not be responsible for proteinuria in trained rats.     

Effect of acute exercise on some haematological parameters and neutrophil functions in active and inactive subjects

In this work we studied the possible effects of acute exercise on some haematological parameters and on some functions of neutrophils in seven active and six inactive subjects. Physical exercise (10 min on a cycle ergometer at a heart rate of 150 beats · min^–1) induced a significant increase in total leucocyte, lymphocyte and neutrophil concentrations in active subjects; serum iron and ferritin concentrations were lower in active compared to inactive subjects. Cellular adhesion, bactericidal activity and superoxide anion production did not change after exercise, while we also observed some differences between active and inactive subjects before exercise. In particular, the neutrophils from active subjects showed a significantly higher percentage of adhesion, higher bactericidal activity and lower superoxide anion production. In conclusion, the training induced changes in some neutrophil functions, while acute exercise influenced, overall, leucocyte concentrations.     

氧化应激在乙醛引起的心肌细胞凋亡中的作用

目的： 探讨活性氧（ROS）的氧化损伤机制在经乙醛诱导心肌细胞凋亡过程中的作用，阐明氧化应激水平增高导致细胞凋亡可能是乙醇性心肌病（AHMD）的主要原因之一。 方法: 体外培养大鼠心肌细胞，分别用乙醇（100 μmol/L）和乙醛（100 μmol/L）干预24 h，比较细胞凋亡程度；检测48 h内细胞胞内ROS水平变化、胞外分泌SOD活性变化；并用Western blotting技术检测ROS介导MAPK信号途径相关蛋白磷酸化水平变化；并与氧化剂H2O2处理组和预先加入抗氧化剂乙酰半胱氨酸（NAC）的乙醛处理组比较。 结果: 乙醇和乙醛分别干预心肌细胞24 h后，凋亡途径激活因子caspase 3均被激活(P<0.05)，乙醛较乙醇具有更强的诱导凋亡作用（P<0.05）。乙醛诱导心肌细胞ROS水平增高呈时间依赖性，相应抗氧自由基SOD酶活性也随之增高，分别于18-24 h达到峰值。同时，ROS通过JNK和ERK磷酸化水平增高而激活MAPK途径，诱导心肌细胞凋亡，这种效应可以被NAC逆转，等浓度乙醛弱于H2O2的诱导作用。结论: 乙醛通过增高胞内ROS水平损伤心肌细胞，激活ROS介导JNK途径诱导细胞凋亡。其作用弱于H2O2等强氧化剂，提示降低细胞ROS 水平，阻断凋亡信号通路可有效抑制乙醛诱导的心肌凋亡，对于AHMD临床预防和治疗具有指导意义。     

胆红素对大鼠急性肺损伤形成过程中氧自由基以及caspase-3表达的影响

目的： 探讨胆红素对抗急性肺损伤（ALI）形成的可能机制。方法: 健康雌性Wistar大鼠(190-210 g) 30只，随机分为生理盐水对照组、脂多糖（LPS）致ALI模型组、胆红素干预组。检测肺组织匀浆中羟自由基（OH^-）、过氧化氢(H₂O₂)和超氧阴离子自由基（O₂^·）含量以及肺组织中caspase-3表达的变化。结果: ①ALI模型组肺组织匀浆OH^-、H₂O₂、O₂^·含量及肺组织中caspase-3表达显著高于生理盐水对照组（均P<0.05）。②胆红素干预组肺组织匀浆OH^-、H₂O₂、O₂^·及肺组织中caspase-3表达明显高于ALI正常大鼠（均P<0.05），但少于ALI模型组（均P<0.05）。结论: ①胆红素能在一定程度上减少肺内凋亡细胞数量。②胆红素能减少ALI大鼠肺组织OH^-、H₂O₂、O₂^·水平。③ Caspase-3表达的变化有促脂多糖性肺损伤细胞凋亡作用。     

Value of urinary neopterin in the differential diagnosis of bacterial and viral infections

Summary Neopterin is released by stimulated macrophages. In this study we analyzed the diagnostic potential of urinary neopterin concentrations in patients with bacterial and viral infection. All but one of 17 patients with viral infection had increased urinary neopterin concentrations. Patients with bacterial urinary tract infection also showed increased neopterin concentrations, whereas patients with bacterial pneumonia had significantly lower neopterin levels. In addition, patients with acute bacterial pneumonia had lower neopterin levels than patients with protracted infection. A significant inverse correlation between urinary neopterin and hemoglobin concentrations was found. Neopterin concentrations could serve as a helpful additional marker of infectious diseases. Combined with other clinical and laboratory parameters it is a useful parameter for distinguishing between viral and bacterial origins of infection, as was shown by multivariate stepwise linear discriminant analysis.Abbreviations ESR erythrocyte sedimentation rate     

大鼠星形胶质细胞与运动神经元VSC4.1共培养体系中活性氧产生水平的变化

目的： 探讨野生型星形胶质细胞（ASC）和运动神经元VSC4.1（VSC）共培养时两者产生活性氧（ROS）的相互影响。方法: MTT法测定比较ASC和VSC在单独培养和共培养时受到兴奋性刺激后生长抑制率;ASC与Hoechst 33342标记的VSC共培养或分别单独培养时,激光共聚焦显微镜实时观察2种情况下两者产生ROS的能力。结果: 较高浓度谷氨酸刺激可使共培养的混合细胞抑制率（IRVSC+ASC）明显高于星形胶质细胞抑制率（IRASC）,而较低浓度时IRVSC+ASC高于IRVSC。激光共聚焦显微镜实时观察发现共培养时VSC产生ROS水平明显低于单独培养的VSC,且前者在15 min时出现明显升高,而单独培养的VSC产生的ROS在5 min内达到峰值后即逐渐降低;共培养时ASC产生的ROS在10 min时出现明显升高。结论: ASC和VSC共培养时ASC可降低VSC静息时的ROS水平,在受到兴奋性刺激后ASC延长VSC产生高水平ROS的时程。