Bactericidal Activity Of Human Neutrophils Research Articles

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.

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.

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.

Lysophosphatidylcholine induces azurophil granule translocation via Rho/Rho kinase/F-actin polymerization in human neutrophils.

Translocation of azurophil granules is pivotal for bactericidal activity of neutrophils, the first-line defense cells against pathogens. Previously, we reported that lysophosphatidylcholine (LPC), an endogenous lipid, enhances bactericidal activity of human neutrophils via increasing translocation of azurophil granules. However, the precise mechanism of LPC-induced azurophil granule translocation was not fully understood. Treatment of neutrophil with LPC significantly increased CD63 (an azurophil granule marker) surface expression. Interestingly, cytochalasin B, an inhibitor of action polymerization, blocked LPC-induced CD63 surface expression. LPC increased F-actin polymerization. LPC-induced CD63 surface expression was inhibited by both a Rho specific inhibitor, Tat-C3 exoenzyme, and a Rho kinase (ROCK) inhibitor, Y27632 which also inhibited LPC-induced F-actin polymerization. LPC induced Rho-GTP activation. NSC23766, a Rac inhibitor, however, did not affect LPC-induced CD63 surface expression. Theses results suggest a novel regulatory mechanism for azurophil granule translocation where LPC induces translocation of azurophil granules via Rho/ROCK/F-actin polymerization pathway.

An ACE inhibitor reduces bactericidal activity of human neutrophils in vitro and impairs mouse neutrophil activity in vivo.

Angiotensin-converting enzyme inhibitors (ACEIs) are used by millions of patients to treat hypertension, diabetic kidney disease, and heart failure. However, these patients are often at increased risk of infection. To evaluate the impact of ACEIs on immune responses to infection, we compared the effect of an ACEI versus an angiotensin receptor blocker (ARB) on neutrophil antibacterial activity. ACEI exposure reduced the ability of murine neutrophils to kill methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Klebsiella pneumoniae in vitro. In vivo, ACEI-treated mice infected with MRSA had increased bacteremia and tissue bacteria counts compared to mice treated with an ARB or with no drug. Similarly, ACEIs, but not ARBs, increased the incidence of MRSA-induced infective endocarditis in mice with aortic valve injury. Neutrophils from ACE knockout (KO) mice or mice treated with an ACEI produced less leukotriene B4 (LTB4) upon stimulation with MRSA or lipopolysaccharide, whereas neutrophils overexpressing ACE produced more LTB4 compared to wild-type neutrophils. As a result of reduced LTB4 production, ACE KO neutrophils showed decreased survival signaling and increased apoptosis. In contrast, neutrophils overexpressing ACE had an enhanced survival phenotype. Last, in a cohort of human volunteers receiving the ACEI ramipril for 1 week, ACEI administration reduced neutrophil superoxide and reactive oxygen species production and neutrophils isolated from volunteers during ramipril treatment had reduced bactericidal activity. Together, these data demonstrate that ACEI treatment, but not ARB treatment, can reduce the bacterial killing ability of neutrophils.

Damage-Associated Molecular Pattern S100A9 Increases Bactericidal Activity of Human Neutrophils by Enhancing Phagocytosis

The damage-associated molecular-pattern S100A9 is found at inflammatory sites in infections and various autoimmune diseases. It is released at very high concentrations in the extracellular milieu by activated neutrophils and monocytes in response to various agents. This proinflammatory protein is found in infected mucosae and tissue abscesses where it acts notably as a potent neutrophil activator. In this study, we examined the role of S100A9 in the control of infections. S100A9 was found to increase human neutrophil bactericidal activity toward Escherichia coli. Although S100A9 induced the accumulation of reactive oxygen species over time through the activation of NADPH oxidase, its antimicrobial activity was mediated mainly by enhancing the efficiency of neutrophil phagocytosis. Interestingly, S100A9 did not act by increasing cell surface expression of CD16, CD32, or CD64 in neutrophils, indicating that its biological effect in FcR-mediated phagocytosis is independent of upregulation of FcγR levels. However, S100A9-induced phagocytic activity required the phosphorylation of Erk1/2, Akt, and Syk. Taken together, our results demonstrate that S100A9 stimulates neutrophil microbicidal activity by promoting phagocytosis.

Involvement of nitric oxide donor compounds in the bactericidal activity of human neutrophils in vitro.

The bactericidal activity of human neutrophils against extracellular and facultatively intracellular bacteria was studied in the presence of the nitric oxide (NO) donors sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1), a molsidomine metabolite. SNP and molsidomine are drugs commonly used as nitrovasodilators in coronary heart disease. It is demonstrated here that the NO donor compounds themselves did not affect the viability and survival of the bacterial strains tested. Neither SNP nor SIN-1 had any effect on the process of bacteria ingestion. In contrast, NO donors enhanced the ability of neutrophils to kill Escherichia coli, Proteus vulgaris and Salmonella Anatum. However, strains differed in their susceptibility to SNP- and SIN-1-mediated killing by neutrophils. Removal of the superoxide anion reduced the bactericidal activity of SNP- and SIN-1-treated neutrophils against E. coli and S. Anatum. This suggests that the NO derivatives formed in the reaction of NO generated from donors with the reactive oxygen species released by phagocytosed neutrophils potentiate the bactericidal activity of human neutrophils in vitro. The above original observation discussed here suggests clinical significance for the treatment of patients with nitrovasodilators in the course of coronary heart disease therapy.

6-Formylpterin Intracellularly Generates Hydrogen Peroxide and Restores the Impaired Bactericidal Activity of Human Neutrophils

The effects of 6-formylpterin on the impaired bactericidal activity of human neutrophils were examined ex vivo. When neutrophils isolated from fresh blood were incubated with 6-formylpterin, the intracellular production of hydrogen peroxide (H2O2) occurred. The H2O2 generation by 6-formylpterin in neutrophils occurred in the presence of diphenyleneiodonium (DPI), an inhibitor of NADPH-oxidase. When neutrophils were incubated with DPI, the killing rate of catalase-positive bacteria, Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), significantly decreased. This impaired bactericidal activity of the DPI-treated neutrophils was a mimic for chronic granulomatous disease (CGD). However, the killing rate of the DPI-treated neutrophils against E. coli and S. aureus significantly increased when 6-formylpterin was administered. Since 6-formylpterin intracellularly generates H2O2 independent from the NADPH-oxidase, it was considered to improve the impaired bactericidal activity of the DPI-treated neutrophils. The use of 6-formylpterin may serve as an option of therapy for CGD.

The effect of extracellular polysaccharides from Streptococcus mutans on the bactericidal activity of human neutrophils

Extracellular polysaccharides (PS) synthesized by oral bacteria constitute one of their major virulence factors. The PS, synthesized from sucrose, facilitate adhesion and colonization by bacteria to tooth surfaces. The study was designed to test the effect of in situ production of extracellular PS by Streptococcus mutans on the bactericidal activity of human neutrophils. These effects were tested on bacteria pre-exposed to sucrose (PS-positive Strep. mutans) and compared to bacteria not exposed to sucrose (PS-negative Strep. mutans). The interactions between neutrophils and Strep. mutans were tested in suspension and on bacteria in an experimental model of dental plaque. Viability of Strep. mutans was measured by [ 3H]-thymidine incorporation into the bacteria. Degranulation of neutrophils was evaluated by the release of lysozyme, and the production of reactive oxygen products was measured by chemiluminescence. When neutrophils were incubated with suspended bacteria, the viability of PS-negative Strep. mutans was 20% of that of bacteria not incubated with neutrophils (control), while the viability of PS-positive Strep. mutans was 40% of the control. In the experimental dental-plaque model, 50% of the PS-negative Strep. mutans were killed by neutrophils while the viability of PS-positive Strep. mutans was not different than of the control. Degranulation of neutrophils was not affected by the presence of extracellular PS of Strep. mutans. Artificial stimulation of neutrophils with phorbol myristate acetate also did not enhance the bactericidal effect of neutrophils on PS-positive Strep. mutans. However, PS-positive Strep. mutans elicited oxygen-reactive products from neutrophils, 2-fold less than with PS-negative Strep. mutans. The results indicate that in situ production of bacterial extracellular polysaccharides might be a major virulence factor of Strep. mutans, enabling PS-positive Strep. mutans in the dental-plaque biofilm to evade killing by human neutrophils.

A mechanism of pathogenicity of Streptococcus milleri group" in pulmonary infection: synergy with an anaerobe

The relationship between Streptococcus constellatus, one of the species of the "Streptococcus milleri group", and Prevotella intermedia was studied in a model of pneumonia in mice and in vitro to elucidate mechanisms of pathogenicity in "S. milleri group"-associated pulmonary infection. Acute pneumonia with or without empyema and lung abscess in mice with mixed infection resulted in 60% mortality rate, but there was only 10% mortality and mild pneumonia in each separate infection. Bacterial clearance of organisms, especially S. constellatus, in mixed infection was delayed. Enhancement of growth of S. constellatus was demonstrated when cultured with P. intermedia; growth was also stimulated by a culture filtrate of P. intermedia which also inhibited bactericidal activity of human neutrophils. In an examination of infectivity and bacterial clearance of S. constellatus with P. intermedia culture filtrate in vivo, there was 20% mortality and delayed clearance of S. constellatus, although the infection was not as severe as that produced by the combination of both organisms. These results suggest that P. intermedia may act with S. constellatus in the production of pulmonary infections by stimulating its growth and suppressing bactericidal activity of the host.

Stimulation of bactericidal activity of human neutrophils by interleukin-2-activated lymphoid cells: Effect of proinflammatory cytokines

Stimulation of bactericidal activity of human neutrophils by interleukin-2-activated lymphoid cells: Effect of proinflammatory cytokines

Effect of a factor released by K562 malignant cells in culture on human neutrophil bactericidal activity

We have previously demonstrated that k562 malignant cells in culture contain and release a low-molecular-mass (8-kDa) factor that inhibits adherence-related functions of neutrophils but does not alter fMet-Leu-Phe- or phorbol ester-induced oxidative burst (M. Amar, N. Amit, T. Pham Huu, S. Chollet-Martin, M.T. Labro, M.A. Gougerot-Pocidalo, and J. Hakim, J. Immunol. 144:4749-4756, 1990). In this study, we investigated the effects of this factor, referred to as inhibitory factor 1 (IF1), on the bactericidal activity of human polymorphonuclear cells (PMNs) on Staphylococcus aureus opsonized in various ways. S. aureus was used either nonopsonized or opsonized with heat-inactivated serum or normal serum containing complement factors. The bactericidal activity of PMNs preincubated with IF1-treated or control medium was examined by counting the surviving bacteria. The ability of IF1-treated PMNs to kill bacteria was diminished when they were opsonized with normal serum. When S. aureus was not opsonized or was opsonized with heat-inactivated serum, the bactericidal activity of IF1-treated PMNs was similar to that of controls. Likewise, the phagocytosis of IF1-treated PMNs was diminished when S. aureus was opsonized with normal serum but was not altered when S. aureus was not opsonized or was opsonized with heat-inactivated serum. These results suggest that the decrease in killing might be due to defective ingestion. The chemiluminescence response of IF1-treated PMNs was inhibited when S. aureus was not opsonized or was opsonized with normal serum. No effect on chemiluminescence was observed when S. aureus was opsonized with heat-inactivated serum. These results suggest that IF1 interferes not only with S. aureus stimulation of PMNs via complement receptors but also with oxygen-dependent bactericidal activity.

Variable effect of toxic shock toxins from different sources on neutrophil function in vitro.

Toxic shock syndrome toxins (TSST) are 23-30 kD proteins that have been isolated from incubation media of strains of Staphylococcus aureus cultured from patients with toxic shock syndrome (TSS). Injection of TSST into animals produces many of the symptoms that characterize TSS including shock, fever, and multiple organ failure. We found that addition of increasing concentrations of TSST-1-VP1035-16A, but not TSST-PEC, TSST-SEC, staphylococcal enterotoxin A or B, progressively decreased human neutrophil bactericidal activity against S. aureus, 502A in vitro. TSST-1-VP1035-16A, but not the other toxins, also decreased superoxide anion and hydrogen peroxide concentrations in mixtures containing neutrophils and phorbol myristate acetate (PMA) in vitro. The results indicate that various preparations of TSST have different effects on neutrophil function in vitro and, accordingly, may have different effects in other in vitro and in vivo models of TSS.

Cefodizime (HR 221) potentiation of human neutrophil oxygen-independent bactericidal activity.

The enhanced bactericidal activity of human neutrophils induced by cefotaxime and cefodizime, two methoxy-imino-amino- 2-thiazolyl cephalosporins, is linked to the cell stimulation of oxygen-dependent and oxygen-independent killing systems, respectively. Cefotaxime enhances both the killing and the oxidative response of neutrophils to opsonized particulate stimuli (bacteria for both activities and opsonized zymosan for the oxidative burst). These effects were not observed with non-opsonized particles (bacteria or zymosan) or soluble stimuli. On the contrary, cefodizime enhances killing of opsonized and non-opsonized bacteria by neutrophils regardless of treatment with phenylbutazone which blocks neutrophil oxidative metabolism. Cefodizime does not universally alter the oxidative burst induced by various stimuli, but has been shown to enhance the bactericidal activity of crude extracts of neutrophil granules. The data suggest that cefodizime and non O2-dependent killing systems of neutrophils cooperate in killing bacteria.

Enhancement of Human Neutrophil Bactericidal Activity by Chemotactic Factors

Neutrophils are important effector cells in the defense against microorganisms. They migrate into infected sites and then phagocytose and kill bacteria. Chemotactic factors may be important for initiating neutrophil migration. We investigated whether chemotactic factors might also influence an event subsequent to chemotaxis, namely bacterial killing. It was found that preincubation (20 min at 37 degrees C) of human leukocytes with chemotactic substances such as zymosan-activated serum, a C5a-containing fraction of zymosan-activated serum, N-formyl methionyl phenylalanine or N-formyl methionyl-leucine-phenylalanine, enhanced leukocyte killing of Staphylococcus aureus, Escherichia coli, and Streptococcus faecalis in a dose-dependent fashion. The concentration of chemotactic factor required to enhance killing was similar to that required to induce neutrophil chemotaxis. In addition, zymosan-activated serum, C5a fraction, and the two N-formyl methionyl peptides increased the hexose monophosphate shunt activity of resting and phagocytosing neutrophils by two- to threefold. In contrast, bacterial killing by sodium azide-treated neutrophils and neutrophils from a patient with chronic granulomatous disease was not increased by any chemotactic factor. These findings suggest that chemotactic factors stimulate neutrophil oxygen-dependent microbicidal pathways. These observations illustrate another important contribution of biologically active molecules to effector cell function and host defense.