Neutrophil Bactericidal Activity Research Articles

Opposing roles of resident and infiltrating immune cells in the defense against Legionella longbeachae via IL-18R/IFN-γ/ROS axis in mice

The immune response against Legionella longbeachae, a causative agent of the often-fatal Legionnaires’ pneumonia, is poorly understood. Here we investigated the specific roles of tissue-resident alveolar macrophages (AM) and infiltrating phagocytes during infection with this pathogen. AM were the predominant cell type that internalized bacteria one day after infection. Three and five days after infection, AM numbers were greatly reduced while there was an influx of neutrophils and later monocyte-derived cells (MC) into lung tissue. AM carried greater numbers of viable L.longbeachae than neutrophils and MC, which correlated with a higher capacity of L.longbeachae to translocate bacterial effector proteins required for bacterial replication into the AM cytosol. Cell ablation experiments demonstrated that AM promoted infection whereas neutrophils and MC were required for efficient bacterial clearance. IL-18 was important for IFN-γ production by IL-18R+ NK cells and T cells which, in turn, stimulated ROS-mediated bactericidal activity in neutrophils resulting in restriction of L.longbeachae infection. Ciliated bronchiolar epithelial cells also expressed IL-18R but did not play a role in IL-18-mediated L.longbeachae clearance. Our results have identified opposing innate functions of tissue-resident and infiltrating immune cells during L.longbeachae infection that may be manipulated to improve protective responses.

Saikosaponin a promotes neutrophil extracellular trap formation and bactericidal activity

This study aimed to investigate the effects of SSa, one of the major triterpenoid saponins extracted from Radix bupleuri, on neutrophil extracellular trap (NET) formation and the mechanism associated with this process. Using Sytox green and immunofluorescence assays, we found SSa rapidly induced NET formation, which depended on NADPH oxidase (NOX)-independent ROS production and autophagy. Pharmacologic inhibitor studies indicated that ERK and PI3K/AKT signalling were also required for SSa-induced NET formation, whereas protein arginine deiminase 4 (PAD4) was not required. Furthermore, we found that SSa promoted neutrophil bactericidal activity mainly through NET formation. Based on flow cytometry and the Cell Counting Kit-8 (CCK-8) assays, the results demonstrated that SSa-induced NET formation occurred without neutrophil death. Taken together, these findings indicated that SSa could be a potential natural product to boost innate immune defense against pathogen attack via NET formation.

Mechanochemically Reprogrammed Interface Orchestrates Neutrophil Bactericidal Activity and Apoptosis for Preventing Implant-Associated Infection.

The onset of implant-associated infection (IAI) triggers a cascade of immune responses, which are initially dominated by neutrophils. Bacterial aggregate formation and hypoxic microenvironment, which occur shortly after implantation, may be two major risk factors that impair neutrophil function and lead to IAI. Here, the implant surface with phytic acid-Zn2+ coordinated TiO2 nanopillar arrays (PA-Zn@TiNPs) and oxygen self-supporting CaO2 nanoparticles, named as CPZTs, is mechanochemically reprogrammed. The engineered CPZTs interface integrates multiple properties to inhibit the formation of nascent biofilm, encompassing antibacterial adhesion, mechanobactericidal effect, and chemobiocidal effect. Meanwhile, continuous oxygenation fuels the neutrophils with reactive oxygen species (ROS) for efficient bacterial elimination on the implant surface and inside the neutrophils. Furthermore, this surface modulation strategy accelerates neutrophil apoptosis and promotes M2 macrophage-mediated osteogenesis both in vitro and in a rat model of IAI. In conclusion, targeting neutrophils for immunomodulation is a practical and effective strategy to prevent IAI and promote bone-implant integration.

Enhancement of Human Neutrophil Bactericidal Activity through ROS Regulation

Antimicrobial agents are vital for treating infectious diseases, but their widespread use has led to a concerning rise in drug-resistant bacteria. The development and overuse of new antimicrobial agents have paradoxically given rise to even more drug-resistant strains. If current trends persist, drug-resistant infections may become the leading global cause of mortality by 2050. However, current measures such as restricting antimicrobial use and promoting appropriate usage have not provided a definitive solution. Therefore, exploring alternative treatment approaches is essential. In this study, our research was based on a novel strategy that shifts the focus from targeting specific bacterial species to leveraging the defense mechanisms of human neutrophils. Neutrophils play a critical role in innate immunity by engulfing bacteria and generating reactive oxygen species (ROS) in phagosomes. The primary ROS producer in neutrophils is the NADPH oxidase (NOX) complex centered on gp91 phox (NOX2), a membrane protein of the NOX family. However, excessive ROS are harmful, necessitating negative regulation. Neutrophils contain a negative regulator of reactive oxygen species (NRROS), which regulates ROS production by competing with p22 phox for binding to NOX2 when inflammation go into ending. NRROS knockout mice have shown increased ROS production and bactericidal activity during bacterial infection, resulting in significantly prolonged survival compared to wild-type mice. If this mechanism could be applied to human cells to enhance bactericidal activity, it would eliminate the need to target specific bacterial species and could revolutionize the treatment of infectious diseases. We used human induced pluripotent stem cells (iPSC) as a model to validate the NRROS knockout effect in human cells, overcoming challenges in isolating peripheral blood neutrophils due to their limited lifespan, poor in vitro culture tolerance, and genetic editing complexities. We successfully generated NRROS-heterozygous knockout (hetero-KO) and NRROS-homozygous knockout (homo-KO) iPSC clones using genome editing techniques. Additionally, using the PiggyBac system, we established doxycycline-inducible NRROS expression clones (homo-KO+Dox) based on homo-KO clones. These iPSC were differentiated into neutrophils and evaluated for function. The isolated neutrophil fraction exhibited characteristic segmented nuclei in all clones. We measured ROS production and examined changes induced by NRROS knockout after stimulation. Immediate superoxide production showed no difference between wild-type and NRROS-KO clones. However, after 2 hours of stimulation, the NRROS-KO clones exhibited significantly elevated hydrogen peroxide production, indicating increased total ROS production, possibly due to delayed NOX complex degradation. Furthermore, we assessed the bactericidal capacity of iPSC-derived neutrophils against multidrug-resistant bacteria, revealing significantly enhanced bactericidal activity in NRROS-KO clones compared to wild-type clones. In conclusion, NRROS knockout in human neutrophils enhances bactericidal capacity through heightened ROS production, offering promising prospects for innovative infectious disease treatments. If successfully harnessed, this innovative treatment modality could revolutionize infectious disease management, combating drug-resistant infections, and improving global health outcomes.

Neutrophil bactericidal activity and host defenses in cystic fibrosis: a narrative review.

Cystic fibrosis (CF) is a disorder that affects the cystic fibrosis transmembrane conductance regulator (CFTR). Without properly functioning CFTR channels, chloride does not exit respiratory epithelial cells, and consequently the mucus lining the surface of the cells becomes thick. This viscous mucus accumulates and causes abnormal function of the mucociliary apparatus, which can lead to bacterial colonization, infections with Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa), and eventually lung damage. Recent studies have shown that the increased susceptibility to respiratory infections in CF patients may also be due to defects in neutrophil function, but the exact mechanism is uncertain. The PubMed database was searched on February 10, 2023 and again on July 23, 2023 to compile a comprehensive list of clinical and experimental studies to evaluate neutrophil function in CF. The first search included a combination of MeSH terms: "cystic fibrosis" and "neutrophils/physiology". A separate second search included a combination of the MeSH terms: "neutrophils" and "cystic fibrosis transmembrane conductance regulator". Neutrophils from patients with CF have decreased transfer of chloride into phagolysosomes after bacterial ingestion and have dysregulated degranulation. This reduces the production of toxic oxidative radicals, especially hypochlorous acid (HOCl), and reduces bactericidal activity. CFTR potentiators correct the dysregulated degranulation in patients with CF and increased neutrophil killing activity. A reduced concentration of chloride in in vitro assays also reduces neutrophil killing activity; these observations are relevant to the reduced chloride concentrations in respiratory secretions in patients with CF. This literature review summarizes studies that demonstrate that an important defect in CF neutrophils lies in the oxygen-dependent pathway in phagolysosomes and studies with ivacaftor demonstrate that this drug corrects CF neutrophil function. These studies demonstrate the potential utility of using easily available neutrophils to study drug effects in CF patients.

Infection-experienced HSPCs protect against infections by generating neutrophils with enhanced mitochondrial bactericidal activity.

Hematopoietic stem and progenitor cells (HSPCs) respond to infection by proliferating and generating in-demand neutrophils through a process called emergency granulopoiesis (EG). Recently, infection-induced changes in HSPCs have also been shown to underpin the longevity of trained immunity, where they generate innate immune cells with enhanced responses to subsequent microbial threats. Using larval zebrafish to live image neutrophils and HSPCs, we show that infection-experienced HSPCs generate neutrophils with enhanced bactericidal functions. Transcriptomic analysis of EG neutrophils uncovered a previously unknown function for mitochondrial reactive oxygen species in elevating neutrophil bactericidal activity. We also reveal that driving expression of zebrafish C/EBPβ within infection-naïve HSPCs is sufficient to generate neutrophils with similarly enhanced bactericidal capacity. Our work suggests that this demand-adapted source of neutrophils contributes to trained immunity by providing enhanced protection toward subsequent infections. Manipulating demand-driven granulopoiesis may provide a therapeutic strategy to boost neutrophil function and treat infectious disease.

Detailed Mechanisms Underlying Neutrophil Bactericidal Activity against Streptococcus pneumoniae.

Neutrophils are an essential cellular component of innate immunity and control bacterial infections through a combination of intracellular and extracellular killing methods. Although the importance of neutrophils has been established, the exact methods used to handle particular bacterial challenges and the efficiency of bacterial killing remain not well understood. In this study, we addressed how neutrophils eliminate Streptococcus pneumoniae (Spn), a leading cause of community acquired and post-influenza bacterial pneumonia. We analyzed killing methods with variable bacterial:neutrophil concentrations and following priming with PAM3CSK4 (P3CSK), an agonist for Toll-like-receptor 2 (TLR2). Our results show that murine neutrophils display surprisingly weak bactericidal activity against Spn, employing a predominantly extracellular mode of killing at lower concentrations of bacteria, whereas challenges with higher bacterial numbers induce both extracellular and intracellular elimination modes but require TLR2 activation. TLR2 activation increased reactive oxygen species (ROS) and neutrophil extracellular trap (NET) formation in response to Spn. Despite this, supernatants from P3CSK-stimulated neutrophils failed to independently alter bacterial replication. Our study reveals that unstimulated neutrophils are capable of eliminating bacteria only at lower concentrations via extracellular killing methods, whereas TLR2 activation primes neutrophil-mediated killing using both intracellular and extracellular methods under higher bacterial burdens.

Enhanced glycolysis by ATPIF1 gene inactivation increased the anti-bacterial activities of neutrophils through induction of ROS and lactic acid

ATP synthase inhibitory factor 1 (ATPIF1) is a mitochondrial protein that regulates the activity of FoF1-ATP synthase. Mice lacking ATPIF1 throughout their bodies (Atpif1−/−) exhibit a reduction in the number of neutrophils. However, it remains unclear whether the inactivation of ATPIF1 impairs the antibacterial function of mice, this study aimed to evaluate it using a mouse peritonitis model. Mice were intraperitoneally injected with E. coli to induce peritonitis, and after 24 h, the colonies of E. coli were counted in agarose plates containing mice peritoneal lavage fluids (PLF) or extract from the liver. Neutrophils were analyzed for glucose metabolism in glycolysis following LPS stimulation. Reactive oxygen species (ROS) and lactic acid (LA) levels in neutrophils were measured using flow cytometry and Seahorse analysis, respectively. N-Acetylcysteine (NAC) and 2-Deoxy-d-glucose (2-DG) were employed to assess the role of ROS and LA in neutrophil bactericidal activity. RNA-seq analysis was conducted in neutrophils to investigate potential mechanisms. In ATPIF1−/− neutrophils, bactericidal activity was enhanced, accompanied by increased levels of ROS and LA compared to wildtype neutrophils. The augmented bactericidal activity of ATPIF1−/− neutrophils was reversed by pretreatment with NAC or 2-DG. RNA-seq analysis revealed downregulation of multiple genes involved in glutathione metabolism, pyruvate oxidation, and heme synthesis, along with increased expression of inflammatory and apoptotic genes. This study suggests that the inactivation of the Atpif1 gene enhances glucose metabolism in neutrophils, resulting in increased bactericidal activity mediated by elevated levels of ROS and LA. Inhibiting ATPIF1 may be a potential approach to enhance antibacterial immunity.

Gp05, a Prophage-Encoded Virulence Factor, Contributes to Persistent Methicillin-Resistant Staphylococcus aureus Endovascular Infection.

Persistent methicillin-resistant Staphylococcus aureus (MRSA) endovascular infections represent a serious public health threat. We recently demonstrated that the presence of a novel prophage ϕSA169 was associated with vancomycin (VAN) treatment failure in experimental MRSA endocarditis. In this study, we assessed the role of a ϕSA169 gene, ϕ80α_gp05 (gp05), in VAN-persistent outcome using gp05 isogenic MRSA strain sets. Of note, Gp05 significantly influences the intersection of MRSA virulence factors, host immune responses, and antibiotic treatment efficacy, including the following: (i) activity of the significant energy-yielding metabolic pathway (e.g., tricarboxylic acid cycle); (ii) carotenoid pigment production; (iii) (p)ppGpp (guanosine tetra- and pentaphosphate) production, which activates the stringent response and subsequent downstream functional factors (e.g., phenol-soluble modulins and polymorphonuclear neutrophil bactericidal activity); and (iv) persistence to VAN treatment in an experimental infective endocarditis model. These data suggest that Gp05 is a significant virulence factor which contributes to the persistent outcomes in MRSA endovascular infection by multiple pathways. IMPORTANCE Persistent endovascular infections are often caused by MRSA strains that are susceptible to anti-MRSA antibiotics in vitro by CLSI breakpoints. Thus, the persistent outcome represents a unique variant of traditional antibiotic resistance mechanisms and a significant therapeutic challenge. Prophage, a critical mobile genetic element carried by most MRSA isolates, provides their bacterial host with metabolic advantages and resistance mechanisms. However, how prophage-encoded virulence factors interact with the host defense system and antibiotics, driving the persistent outcome, is not well known. In the current study, we demonstrated that a novel prophage gene, gp05, significantly impacts tricarboxylic acid cycle activity, stringent response, and pigmentation, as well as vancomycin treatment outcome in an experimental endocarditis model using isogenic gp05 overexpression and chromosomal deletion mutant MRSA strain sets. The findings significantly advance our understanding of the role of Gp05 in persistent MRSA endovascular infection and provide a potential target for development of novel drugs against these life-threatening infections.

Prenylcysteine oxidase 1 like protein is required for neutrophil bactericidal activities

The bactericidal function of neutrophils is dependent on a myriad of intrinsic and extrinsic stimuli. Using systems immunology approaches we identify microbiome- and infection-induced changes in neutrophils. We focus on investigating the Prenylcysteine oxidase 1 like (Pcyox1l) protein function. Murine and human Pcyox1l proteins share ninety four percent aminoacid homology revealing significant evolutionary conservation and implicating Pcyox1l in mediating important biological functions. Here we show that the loss of Pcyox1l protein results in significant reductions in the mevalonate pathway impacting autophagy and cellular viability under homeostatic conditions. Concurrently, Pcyox1l CRISPRed-out neutrophils exhibit deficient bactericidal properties. Pcyox1l knock-out mice demonstrate significant susceptibility to infection with the gram-negative pathogen Psuedomonas aeruginosa exemplified through increased neutrophil infiltrates, hemorrhaging, and reduced bactericidal functionality. Cumulatively, we ascribe a function to Pcyox1l protein in modulation of the prenylation pathway and suggest connections beween metabolic responses and neutrophil functionality.

IL-10 production by granulocytes promotes Staphylococcus aureus craniotomy infection

BackgroundTreatment of brain tumors, epilepsy, or hemodynamic abnormalities requires a craniotomy to access the brain. Nearly 1 million craniotomies are performed in the US annually, which increase to ~ 14 million worldwide and despite prophylaxis, infectious complications after craniotomy range from 1 to 3%. Approximately half are caused by Staphylococcus aureus (S. aureus), which forms a biofilm on the bone flap that is recalcitrant to antibiotics and immune-mediated clearance. However, the mechanisms responsible for the persistence of craniotomy infection remain largely unknown. The current study examined the role of IL-10 in promoting bacterial survival.MethodsA mouse model of S. aureus craniotomy infection was used with wild type (WT), IL-10 knockout (KO), and IL-10 conditional KO mice where IL-10 was absent in microglia and monocytes/macrophages (CX3CR1CreIL-10 fl/fl) or neutrophils and granulocytic myeloid-derived suppressor cells (G-MDSCs; Mrp8CreIL-10 fl/fl), the major immune cell populations in the infected brain vs. subcutaneous galea, respectively. Mice were examined at various intervals post-infection to quantify bacterial burden, leukocyte recruitment, and inflammatory mediator production in the brain and galea to assess the role of IL-10 in craniotomy persistence. In addition, the role of G-MDSC-derived IL-10 on neutrophil activity was examined.ResultsGranulocytes (neutrophils and G-MDSCs) were the major producers of IL-10 during craniotomy infection. Bacterial burden was significantly reduced in IL-10 KO mice in the brain and galea at day 14 post-infection compared to WT animals, concomitant with increased CD4+ and γδ T cell recruitment and cytokine/chemokine production, indicative of a heightened proinflammatory response. S. aureus burden was reduced in Mrp8CreIL-10 fl/fl but not CX3CR1CreIL-10 fl/fl mice that was reversed following treatment with exogenous IL-10, suggesting that granulocyte-derived IL-10 was important for promoting S. aureus craniotomy infection. This was likely due, in part, to IL-10 production by G-MDSCs that inhibited neutrophil bactericidal activity and TNF production.ConclusionCollectively, these findings reveal a novel role for granulocyte-derived IL-10 in suppressing S. aureus clearance during craniotomy infection, which is one mechanism to account for biofilm persistence.

Bestatin as a treatment modality in experimental periodontitis.

Chronic periodontitis (CP), the most prevalent dysbiotic bacteria-driven chronic inflammatory disease, is an underestimated global health problem in itself, and due to a causative relationship with other disorders such as cardiovascular diseases or Alzheimer disease. The CP pathogenesis is primarily driven by Porphyromonas gingivalis in humans, and Porphyromonas gulae in dogs. These microorganisms initiate a pathogenic shift in the composition of the tooth-surface microflora. Our objective was to evaluate antimicrobial effects of bestatin, a potential CP drug candidate. We evaluated bestatin bacteriostatic efficiency against periodontopathogens in planktonic cultures via microplate assay, and mono- and multispecies oral biofilm models. Neutrophil bactericidal activities, such as phagocytosis, were investigated in vitro using granulocytes isolated from the peripheral blood. The therapeutic efficacy and the immunomodulatory function of bestatin was assessed in a murine model of CP. Bestatin exhibited bacteriostatic activity against both P. gingivalis and P. gulae, and controlled the formation and species composition of the biofilm. We demonstrated that bestatin promotes the phagocytosis of periodontopathogens by neutrophils. Finally, we found that providing bestatin in the animal feed prevented alveolar bone resorption. We show that in a murine model of CP bestatin not only shifted the biofilm species composition from pathogenic to a commensal one, but also promoted bacteria clearance by immune cells and alleviated inflammation. Taken together, these results suggest that bestatin is a promising drug choice for the treatment and/or prevention of periodontitis and clinical trials are required to fully evaluate its potency.

Effects of Dietary Blend of Algae Extract Supplementation on Growth, Biochemical, Haemato-Immunological Response, and Immune Gene Expression in Labeo rohita with Aeromonas hydrophila Post-Challenges

In this study, the effects of a mixed algal blend (Chlorella vulgaris, Euglena viridis, and Spirulina platensis) at different levels were evaluated on growth, hematological immune responses, and expression of immune genes in Labeo rohita against post-challenges of Aeromonas hydrophila. Fish samples were fed a diet containing different levels of mixed blend algal (0, 0.01, 0.02, 0.04 and 0.08% of basal diet). At the end of the feeding period, the fish were challenged with A. hydrophila and fish mortality was recorded over a 14-days period. To evaluate the serum biochemical (albumin, globulin), hematological parameters (Hb, RBC and WBC) and immune parameters (neutrophil activity, lysozyme activity, myeloperoxidase activity, antiprotease activity, ceruloplasmin activity, and bactericidal activity), as well as the expression of immune genes (NKEF-B, Lysozyme C and G, TNF α, TLR22, β2M, and β-actin), fish were sampled on Day 7, 14, 21 and 28. Fish were challenged with virulent A. hydrophila 30 days post-feeding and mortalities were recorded over 30 days post-infection. Results demonstrate that fish fed with a mixed algal blend showed that total body weight gain, specific growth rate, total serum protein, globulin, total hemoglobin content, white blood cells, neutrophil, lysozyme, bactericidal, myeloperoxidase, and antiprotease activity in dietary algae blended application was higher than in the control (p < 0.05). According to the results, relative expression of target genes showed significant increases of 0.02 to 0.04% in the treatment group compared to the control group (p < 0.05). At the end of the 30-day exposure to A. hydrophila, the fish that received the mixed algal blend had a significantly higher rate of survival than the control group, with the highest survival rate recorded in the 0.02% mixed algal blend (p < 0.05). According to the effective results of the mixed algal blend on stimulating the immune system and increasing fish resistance to A. hydrophila, it is recommended to use 0.02 to 0.04% of this mixed algal blend in rohu, L. rohita diets.

Immunomosuppressive Effect of Ziziphus Lotus L. (Desf.) Fruit’s Extract on Neutrophil Bactericidal Activity and on in Vivo Humoral Immune Response in Mice.

Background: The fruit of Ziziphus Lotus L. (ZL) is rich in bioactive components. It is known for its high content in polyphenols which gives it its immunomodulatory, antioxidant, and antimicrobial properties. Objective: The intent of the current study was to evaluate, in vivo, the effect of the aqueous extract of ZL fruit’s pulp on humoral immune response as well as its effect on neutrophils’ bactericidal activities, hemolytic and antioxidant and activities. Methods: The antioxidant activity of ZL’s aqueous extract’s was evaluated using DPPH. Hemmagglutination titer assay was used to evaluate the effect of the extract on humoral immune response. ZL extract’s hemolytic activity was assessed by enumerating hemoglobin rates. The effect of ZL extract on the bactericidal activity of Neutrophils was evaluated using MTT colorimetric assay. Results / Discussion: A significant (P<0.05) immunosuppressive effect on humoral immunity (6-fold) was observed. Significant suppression (P<0.05) of the bactericidal activity of neutrophils treated with 0.5 and 1 g/ml of the extract was observed compared to untreated neutrophils. The extract exhibited a high antioxidant activity determined by DPPH test with an IC50 value 10-fold higher (P<0.05) than the IC50 of ascorbic acid. The highest hemolytic activity was found with the lowest concentration of the extract while the higher concentrations tested seem to have an anti-hemolytic activity with a dose dependent effect. Conclusion: The aqueous extract of ZL’s fruit pulp possesses an immunosuppressive activity on both the innate and adaptive immunity responses. Our results demonstrate an anti-oxidative activity as well as an ability to decrease neutrophil bactericidal hemolytic activities as well as humoral immune responses.

Phytochemical characterization and immunomodulatory effects of aqueous, ethanolic extracts and essential oil of Syzygium aromaticum L. on human neutrophils

In Morocco, the flower buds of Syzygium aromaticum L (Clove) from Myrtaceae are essential in traditional medicine; they are used in many forms (infusion, maceration, and essential oil) and are suggested to mitigate inflammatory conditions such as muscle and dental pain as well as rheumatic diseases. This study aims to chemically characterize the aqueous and ethanolic extracts as well as the essential oil from cloves; also, we aimed to evaluate their effects on the bactericidal activity of human neutrophils compared with eugenol. The chemical composition of extracts was evaluated via qualitative phytochemical screening followed by quantitative screening using spectrophotometry and HPLC technique. The essential oil was analyzed by the GC-MS technique. The PMNs bactericidal activity of extracts, essential oil, and eugenol was carried out by MTT assay. The screening of extracts showed the presence of phenols, flavonoids, flavones aglycones, coumarins, and tannins. HPLC analysis revealed the presence of numerous phenolic compounds such as gallic acid, rutin, and quercetin while the GC-MS analysis of essential oil showed that the main components are eugenol (78.67%), eugenyl acetate (11.77%), and caryophyllene (6.85%). The aqueous, ethanolic extracts and essential oil showed an immunomodulatory activity by exerting a significant inhibition of neutrophil bactericidal activity in a dose dependent-manner reaching maximal inhibition at the concentration of 200 µg/ml with only 29.92%, 32.24%, and 48.15%, respectively (p < 0.001).Our study showed the immunomodulatory virtues of cloves as a natural anti-inflammatory agent. The strength of this effect is related to the presence of eugenol and the extraction forms used.

2-Undecanone derived from Pseudomonas aeruginosa modulates the neutrophil activity

Pseudomonas aeruginosa (P. aeruginosa) is a well-known Gram-negative opportunistic pathogen. Neutrophils play key roles in mediating host defense against P. aeruginosa infection. In this study, we identified a metabolite derived from P. aeruginosa that regulates neutrophil activities. Using gas chromatography-mass spectrometry, a markedly increased level of 2-undecanone was identified in the peritoneal fluid of P. aeruginosa-infected mice. 2-Undecanone elicited the activation of neutrophils in a Gai-phospholipase C pathway. However, 2-undecanone strongly inhibited responses to lipopolysaccharide and bactericidal activity of neutrophils against P. aeruginosa by inducing apoptosis. Our results demonstrate that 2-undecanone from P. aeruginosa limits the innate defense activity of neutrophils, suggesting that the production of inhibitory metabolites is a strategy of P. aeruginosa for escaping the host immune system.

Glycine induces enhancement of bactericidal activity of neutrophils

Severe bacterial infections are frequently accompanied by depressed neutrophil functions. Thus, agents that increase the microbicidal activity of neutrophils could add to a direct antimicrobial therapy. Lysophosphatidylcholine augments neutrophil bactericidal activity via the glycine (Gly)/glycine receptor (GlyR) α2/TRPM2/p38 mitogen-activated protein kinase (MAPK) pathway. However, the direct effect of glycine on neutrophil bactericidal activity was not reported. In this study, the effect of glycine on neutrophil bactericidal activity was examined. Glycine augmented bactericidal activity of human neutrophils (EC50 = 238 μM) in a strychnine (a GlyR antagonist)-sensitive manner. Glycine augmented bacterial clearance in mice, which was also blocked by strychnine (0.4 mg/kg, s.c.). Glycine enhanced NADPH oxidase-mediated reactive oxygen species (ROS) production and TRPM2-mediated [Ca2+]i increase in neutrophils that had taken up E. coli. Glycine augmented Lucifer yellow uptake (fluid-phase pinocytosis) and azurophil granule-phagosome fusion in neutrophils that had taken up E. coli in an SB203580 (a p38 MAPK inhibitor)-sensitive manner. These findings indicate that glycine augments neutrophil microbicidal activity by enhancing azurophil granule-phagosome fusion via the GlyRα2/ROS/calcium/p38 MAPK pathway. We suggest that glycine could be a useful agent for increasing neutrophil bacterial clearance.

Mitochondrial Calcium Uniporter Affects Neutrophil Bactericidal Activity during Staphylococcus aureus Infection.

Neutrophils simultaneously restrict Staphylococcus aureus dissemination and facilitate bactericidal activity during infection through the formation of neutrophil extracellular traps (NETs). Neutrophils that produce higher levels of mitochondrial superoxide undergo enhanced terminal NET formation (suicidal NETosis) in response to S. aureus; however, mechanisms regulating mitochondrial homeostasis upstream of neutrophil antibacterial processes are not fully resolved. Here, we demonstrate that mitochondrial calcium uptake 1 (MICU1)-deficient (MICU1-/-) neutrophils accumulate higher levels of calcium and iron within the mitochondria in a mitochondrial calcium uniporter (MCU)-dependent manner. Corresponding with increased ion flux through the MCU, mitochondrial superoxide production is elevated, thereby increasing the propensity for MICU1-/- neutrophils to undergo suicidal NETosis rather than primary degranulation in response to S. aureus. Increased NET formation augments macrophage killing of bacterial pathogens. Similarly, MICU1-/- neutrophils alone are not more antibacterial toward S. aureus, but rather, enhanced suicidal NETosis by MICU1-/- neutrophils facilitates increased bactericidal activity in the presence of macrophages. Similarly, mice with a deficiency in MICU1 restricted to cells expressing LysM exhibit lower bacterial burdens in the heart with increased survival during systemic S. aureus infection. Coinciding with the decrease in S. aureus burdens, MICU1-/- neutrophils in the heart produce higher levels of mitochondrial superoxide and undergo enhanced suicidal NETosis. These results demonstrate that ion flux by the MCU affects the antibacterial function of neutrophils during S. aureus infection.

Neutrophil function and bactericidal activity against Staphylococcus aureus after cardiac surgery with cardiopulmonary bypass.

Staphylococcus aureus is the main bacterial pathogen encountered in mediastinitis after cardiac surgical procedures; it remains a devastating complication with a high mortality rate. As neutrophils have a primordial role in the defense against staphylococcus infection and cardiopulmonary bypass (CPB) is known to induce immunosuppression, the aim of this study was to investigate CPB impact on neutrophil functions. Patients without known immunosuppression scheduled for cardiac surgery with CPB were included. Bone marrow and blood samples were harvested before, during, and after surgery. Neutrophil phenotypic maturation and functions (migration, adhesion, neutrophil extracellular trap [NET] release, reactive oxygen species (ROS) production, phagocytosis, and bacteria killing) were investigated. Two types of Staphylococcus aureus strains (one from asymptomatic nasal carriage and another from mediastinitis infected tissues) were used to assess in vitro bacterial direct impact on neutrophils. We found that CPB induced a systemic inflammation with an increase in circulating mature neutrophils after surgery. Bone marrow sample analysis did not reveal any modification of neutrophil maturation during CPB. Neutrophil lifespan was significantly increased and functions such as NET release and ROS production were enhanced after CPB whereas bacteria killing and phagocytosis were not impacted. Results were similar with the two different isolates of Staphylococcus aureus. These data suggest that CPB induces a recruitment of mature neutrophils via a demargination process rather than impacting their maturation in the bone marrow. In addition, neutrophils are fully efficient after CPB and do not contribute to postoperative immunosuppression.

Characterization of the blood and neutrophil-specific microbiomes and exploration of potential bacterial biomarkers for sepsis in surgical patients.

IntroductionRecent studies have demonstrated the presence of a circulating microbiome in the blood of healthy subjects and chronic inflammatory patients. However, our knowledge regarding the blood microbiome and its potential roles in surgical patients remains very limited. The objective of this study was to determine the blood microbial landscape in surgical patients and to explore its potential associations with postoperative sepsis.Materials and Methods2825 patients who underwent surgical treatments were screened for enrollment and 204 cases were recruited in this study. The patients were sub‐grouped into noninfected, infected, sepsis, and septic shock according to postoperative clinical manifestations. A total of 222 blood samples were obtained for neutrophil isolation, DNA extraction and high‐throughput sequencing, quantitative proteomics analysis, and flow cytometric analyses.ResultsBlood and neutrophils in surgical patients and healthy controls contained highly diverse microbiomes, mainly comprising Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. The majority (80.7%–91.5%) of the microbiomes were composed of gut‐associated bacteria. The microbiomes in septic patients were significantly distinct from those of healthy controls, and marked differences in microbiome composition were observed between sepsis and septic shock groups. Several specific bacterial genera, including Flavobacterium, Agrococcus, Polynucleobacter, and Acidovorax, could distinguish patients with septic shock from those with sepsis, with higher area under curve values. Moreover, Agrococcus, Polynucleobacter, and Acidovorax were positively associated with the sequential (sepsis‐related) organ failure assessment scores and/or acute physiology and chronic health examination scores in septic shock patients. The proteins involved in bactericidal activities of neutrophils were downregulated in septic patients.ConclusionsWe present evidence identifying significant changes of blood and neutrophil‐specific microbiomes across various stages of sepsis, which might be associated with the progression of sepsis after surgical treatments. Several certain bacterial genera in blood microbiome could have potential as microbial markers for early detection of sepsis.