Intracellular Killing Of Bacteria Research Articles

Iron Administration in the Critically III

Anemia is a common pathology in intensive care unit (ICU) patients. The pathophysiology of anemia includes altered iron metabolism with decreased erythropoiesis. Under normal conditions, there is a balance between iron transport by transferrin, making iron available for incorporation in hemoglobin, and iron storage as ferritin. In inflammatory processes, this balance is disturbed and plays a central role in the development of anemia. Typically, the inflammatory process is associated with a low concentration of serum iron, high ferritin and low transferrin. Effective erythropoiesis requires both erythropoietin (EPO) and iron. Critically ill patients have inappropriately low EPO levels, and several studies have been conducted to assess the potential benefits of exogenous EPO supplementation. EPO treatment plus iron administration reduced the number of red blood cell (RBC) transfusions needed but had no effects on outcome in terms of ICU infection rates or mortality. Iron can have adverse effects, including inhibition of phagocytosis, inhibition of intracellular killing of bacteria, and altered polymorphonuclear cell function. Iron overload has also been shown to cause increased apoptosis in patients with hemochromatosis. Further study is needed to accurately define the precise role of iron in the development of anemia in critically ill patients, and to determine the potential benefits and risks of iron supplementation.

Factors influencing the intracellular activity of fluoroquinolones: a study using levofloxacin in a Staphylococcus aureus THP-1 monocyte model

Recent studies have raised the question of whether the intracellular activity of quinolones is optimal with respect to their cellular accumulation. The aim of this study was to compare the intracellular and extracellular activities of a commonly used quinolone, levofloxacin, and to examine the causes of the possible inconsistency between intracellular and extracellular effects. The bactericidal activity of levofloxacin at therapeutic levels, alone or in combination with various efflux-pump inhibitors or alkalinizing agents, was studied against Staphylococcus aureus ATCC 25923 in Mueller-Hinton (MH) broth and in a THP-1 monocytic cell model, using intracellular salt medium (ISM) mimicking the phagolysosomal environment, and in cell lysate. Levofloxacin accumulation was 2-fold higher in uninfected than in infected cells. Intracellular activity was significantly lower than extracellular activity (decrease in the inoculum of < or = 1 log10 cfu/mL at 4 or 8 mg/L versus > or = 2 log10 units at > or = 1 mg/L in MH broth over 5 h). Persisters remained fully susceptible to the drug. The efflux pump inhibitors verapamil and gemfibrozil did not affect killing of intracellular bacteria, although gemfibrozil increased cellular accumulation of levofloxacin 1.7-fold. The lysosomotropic alkalinizing agents chloroquine and ammonium chloride significantly enhanced intracellular killing by levofloxacin. The bactericidal activity of levofloxacin, abolished in ISM, was partially restored when the pH was neutralized from 5.0 to 7.4. Binding to intracellular components (20%) substantially decreased the efficiency of levofloxacin. Levofloxacin exhibited substantially lower intracellular activity than extracellular activity. Cellular compartmentalization of the drug, phagolysosomal environment and antibiotic binding to cellular components most likely contribute to this failure.

Mycobacteriophage and their application to disease control

The resurgence of tuberculosis and emergence of drug resistant disease has stimulated fresh research into mycobacteriophage. Studies are currently underway to develop phage-based tools for therapeutic and diagnostic use. Previous attempts at mycobacteriophage therapy in experimentally infected animals were not successful and alternative strategies of phage delivery that enable killing of intracellular bacteria are required. Replication of mycobacteriophage provides a simple means of detecting viable bacteria and good progress has been made towards the development of new phage-based diagnostic tools. When screening isolates for resistance to the major antituberculosis drug rifampicin phage-based tests have been shown to have high sensitivity. For the diagnosis of pulmonary tuberculosis evaluation studies indicate that current phage tests are not as sensitive as traditional culture methods. Further trials are needed to determine whether they might have a role in the detection of smear negative tuberculosis. A second generation of phage tests are under development following the construction of luciferase reporter phage. Preliminary data suggests they may offer rapid detection of mycobacteria and simple screening for drug resistance. The potential of mycobacteriophage to detect and treat other mycobacterial diseases remains largely unexplored. 2. INTRODUCTION

Interferon‐γ Mediates Neuronal Killing of Intracellular Bacteria

Neurons can be targets for microbes, which could kill the neurons. Just in reverse, we, in this study, report that bacteria can be killed when entering a neuron. Primary cultures of foetal mouse hippocampal neurons and a neuronal cell line derived from mouse hypothalamus were infected by Listeria monocytogenes. Treatment with interferon-gamma (IFN-gamma) did not affect bacterial uptake, but resulted in increased killing of intracellular bacteria, whereas the neuronal cell remained intact. The IFN-gamma-mediated bacterial killing was mapped to the neuronal cytosol, before listerial actin tail formation. Treatment with IFN-gamma induced phosphorylation of the transcription factor STAT-1 in neurons and IFN-gamma-mediated listerial killing was not observed in STAT-1(-/-) neurons or neurons treated with IFN regulatory factor-1 antisense oligonucleotides. IFN-gamma-treated neuronal cells showed increased levels of inducible nitric oxide synthase (iNOS) mRNA, and antisense iNOS oligonucleotides hampered the bacterial killing by neurons upon IFN-gamma treatment. This novel neuronal function - i.e., that of a microbe killer - could play a crucial role in the control of infections in the immuno-privileged nervous system.

Alveolar bone response in an experimental model of renal failure and periodontal disease: a histomorphometric and histochemical study.

Chronic destructive periodontal disease is characterized by gingival inflammation, periodontal pocket formation, and bacterial plaque that lead to alveolar bone destruction. Polymorphonuclear neutrophil leukocytes (PMNs) are the first line of defense against infection caused by dental plaque bacteria. Renal patients present functional abnormalities of PMN, including impaired chemotaxis, phagocytosis, and intracellular killing of bacteria. In view of the above, the aim of this work was to evaluate the effect of renal failure on bone damaged by periodontal disease using histomorphometric and histochemical parameters. Twenty male Wistar rats weighing 250 g were assigned to one of the following four groups: 1) control (no treatment); 2) renal failure (RF); 3) periodontal disease (PD); and 4) renal failure plus periodontal disease (RF+PD). All the animals were sacrificed 31 days after the onset of the experiment. Mesio-distally oriented sections of the first lower molar were obtained for histomorphometric and histochemical evaluation. Total erosion, active erosion, and total number of tartrate-resistant acid phosphatase-positive (TRAP+) osteoclasts were found to be increased in the RF+PD group compared with the PD group. Our results demonstrate increased bone resorption in animals with untreated renal failure and periodontal disease, and thus indicate that the release of different factors by inflammatory cells is magnified, accelerating the progression of the disease in this animal model.

Hypertonic saline enhances host response to bacterial challenge by augmenting receptor-independent neutrophil intracellular superoxide formation.

This study sought to determine whether hypertonic saline (HTS) infusion modulates the host response to bacterial challenge. Sepsis was induced in 30 Balb-C mice by intraperitoneal injection of Escherichia coli (5 x 107 organisms per animal). In 10 mice, resuscitation was performed at 0 and 24 hours with a 4 mL/kg bolus of HTS (7.5% NaCl), 10 animals received 4 mL/kg of normal saline (0.9% NaCl), and the remaining animals received 30 mL/kg of normal saline. Samples of blood, spleen, and lung were cultured at 8 and 36 hours. Polymorphonucleocytes were incubated in isotonic or hypertonic medium before culture with E. coli. Phagocytosis was assessed by flow cytometry, whereas intracellular bacterial killing was measured after inhibition of phagocytosis with cytochalasin B. Intracellular formation of free radicals was assessed by the molecular probe CM-H(2)DCFDA. Mitogen-activated protein (MAP) kinase p38 and ERK-1 phosphorylation, and nuclear factor kappa B (NFkappaB) activation were determined. Data are represented as means (SEM), and an analysis of variance test was performed to gauge statistical significance. Significantly reduced bacterial culture was observed in the animals resuscitated with HTS when compared with their NS counterparts, in blood (51.8 +/- 4.3 vs. 82.0 +/- 3.3 and 78.4 +/- 4.8, P = 0.005), lung (40.0 +/- 4.1 vs. 93.2 +/- 2.1 and 80.9 +/- 4.7, P = 0.002), and spleen (56.4 +/- 3.8 vs. 85.4 +/- 4.2 and 90.1 +/- 5.9, P = 0.05). Intracellular killing of bacteria increased markedly (P = 0.026) and superoxide generation was enhanced upon exposure to HTS (775.78 +/- 23.6 vs. 696.57 +/- 42.2, P = 0.017) despite inhibition of MAP kinase and NFkappaB activation. HTS significantly enhances intracellular killing of bacteria while attenuating receptor-mediated activation of proinflammatory cascades.

Defect in neutrophil killing and increased susceptibility to infection with nonpathogenic gram-positive bacteria in peptidoglycan recognition protein-S (PGRP-S)–deficient mice

AbstractInsect peptidoglycan recognition protein-S (PGRP-S), a member of a family of innate immunity pattern recognition molecules conserved from insects to mammals, recognizes bacterial cell wall peptidoglycan and activates 2 antimicrobial defense systems, prophenoloxidase cascade and antimicrobial peptides through Toll receptor. We show that mouse PGRP-S is present in neutrophil tertiary granules and that PGRP-S–deficient (PGRP-S-/-) mice have increased susceptibility to intraperitoneal infection with gram-positive bacteria of low pathogenicity but not with more pathogenic gram-positive or gram-negative bacteria. PGRP-S-/- mice have normal inflammatory responses and production of tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6). Neutrophils from PGRP-S-/- mice have normal phagocytic uptake of bacteria but are defective in intracellular killing and digestion of relatively nonpathogenic gram-positive bacteria. Therefore, mammalian PGRP-S functions in intracellular killing of bacteria. Thus, only bacterial recognition by PGRP-S, but not its effector function, is conserved from insects to mammals.

MODULATORY EFFECTS OF SALMONELLA LAP-LPS ON MURINE MACROPHAGES

Purpose: To study the modulatory effects of Salmonella lipid associated protein - lipopolysaccharides (LAP-LPS) on murine macrophages as the intracellular survival within the host macrophages is an important feature for a number of gram-negative pathogens like S.typhi. Methods: Macrophage functions were studied in two groups of mice immunized with either LPS or LAP-LPS. Results: Comparison of protective efficacy of mice preimmunized with LPS based preparations, against challenge infectious doses, showed higher protection in LAP-LPS complex immunized mice group as compared to the mice group immunized with LPS alone. Aggregation of S.typhi cells was lesser with intestinal mucus extracted from LAP-LPS immunized mice as compared to LPS immunized challenged group. A significant increase in the number of macrophages in LAP-LPS immunized mice was also observed in comparison to control and LPS immunized mice groups. Nitric oxide (NO) and superoxide dismutase (SOD) production were also more in macrophages derived from LAP-LPS immunized mice group. Phagocytic uptake studies showed that there was enhanced uptake of bacteria in the LAP-LPS immunized animals in comparison to LPS immunized and controls. Similar trend was observed in intracellular killing of bacteria by the macrophages. Conclusions: The results indicated the involvement of protein moiety in LAP on modulation of effects of LPS on macrophages.

Hypertonic saline enhances host response to bacterial challenge by augmenting neutrophil intracellular Superoxide formation

HTS significantly enhances neutrophil-mediated intracellular killing of bacteria. This provides further evidence of the beneficial effects of hypertonic resuscitation in the critically ill patient.

Cooperation between reactive oxygen and nitrogen intermediates in killing of Rhodococcus equi by activated macrophages.

Rhodococcus equi is a facultative intracellular bacterium of macrophages which can infect immunocompromised humans and young horses. In the present study, we examine the mechanism of host defense against R. equi by using a murine model. We show that bacterial killing is dependent upon the presence of gamma interferon (IFN-gamma), which activates macrophages to produce reactive nitrogen and oxygen intermediates. These two radicals combine to form peroxynitrite (ONOO(-)), which kills R. equi. Mice deficient in the production of either the high-output nitric oxide pathway (iNOS(-/-)) or the oxidative burst (gp91(phox-/-)) are more susceptible to lethal R. equi infection and display higher bacterial burdens in their livers, spleens, and lungs than wild-type mice. These in vivo observations, which implicate both nitric oxide (NO) and superoxide (O(2)(-)) in bacterial killing, were reexamined in cell-free radical-generating assays. In these assays, R. equi remains fully viable following prolonged exposure to high concentrations of either nitric oxide or superoxide, indicating that neither compound is sufficient to mediate bacterial killing. In contrast, brief exposure of bacteria to ONOO(-) efficiently kills virulent R. equi. The intracellular killing of bacteria in vitro by activated macrophages correlated with the production of ONOO(-) in situ. Inhibition of nitric oxide production by activated macrophages by using N(G)-monomethyl-L-arginine blocks their production of ONOO(-) and weakens their ability to control rhodococcal replication. These studies indicate that peroxynitrite mediates the intracellular killing of R. equi by IFN-gamma-activated macrophages.

Influence of Microbial Flora on Macrophages

The purpose of this article was to collect the existing data on the influence of bacterial flora on the properties of macrophages which represent one of the first lines of non specific defence against microbial invasion and tumours. To the effects of indigenous microflora were added those of lactic acid bacteria potentially usable as dietary supplements which have been widely investigated. The results of these studies indicate that functions important in host defence, such as chemotaxis, phagocytosis, cytotoxicity, intracellular killing of bacteria as well as cytokine production, appear to be, at least in part, under the control of the indigenous microflora. Though lactic acid bacteria exert stimulating effects on macrophage functions, their impact is less clearly established and seems to be strain- and test system-dependent. Results showing macrophage activation in humans by orally administered lactic bacteria are very limited and more studies are needed to correlate the stimulation of macrophage functions and positive health effects of these probiotic products. Keywords: macrophage, immunity, microbial flora, lactic acid bacteria, probiotics.

Bactericidal activity of a monocytic cell line (THP-1) against common respiratory tract bacterial pathogens is depressed after infection with respiratory syncytial virus.

Non-typable Haemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis and respiratory syncytial virus (RSV) are commonly isolated from patients during the course of chronic obstructive pulmonary disease (COPD). Earlier studies found that virus infection enhanced binding of bacterial respiratory pathogens to epithelial cells in vitro. The objective of the present study was to assess the effect of RSV infection of a human monocytic cell line on bactericidal activity and cytokine production in response to these bacterial respiratory pathogens. The effect of RSV infection on binding, uptake and intracellular killing of bacteria by a human monocytic leukaemia cell line, THP-1, was assessed. Cell culture supernates were examined with a mouse fibroblast cell assay for tumour necrosis factor-alpha (TNF-alpha) bioactivity. Expression of CD14, CD11a, CD18, CD15 and CD29 on uninfected and RSV-infected THP-1 cells was assessed by flow cytometry in relation to differences in bacterial binding. RSV infection of THP-1 cells significantly decreased their ability to bind and kill bacteria. Compared with uninfected cells, fewer bacteria bound to RSV-infected THP-1 cells and the surface antigens that have been reported to bind bacteria were expressed at lower levels on RSV-infected cells. RSV-infected cells incubated with bacteria exhibited less TNF-alpha bioactivity than uninfected cell incubated with bacteria. The results elucidate some of the mechanisms involved in the increased susceptibility of virus-infected patients to secondary bacterial infection. Reduced bacterial killing by virus-infected monocytes might contribute to reduced clearance of bacteria from the respiratory tract and damage elicited by the bacteria or cytokine response in COPD patients.

Influence of Microbial Flora on Macrophages

The purpose of this article was to collect the existing data on the influence of bacterial flora on the properties of macrophages which represent one of the first lines of non specific defence against microbial invasion and tumours. To the effects of indigenous microflora were added those of lactic acid bacteria potentially usable as dietary supplements which have been widely investigated. The results of these studies indicate that functions important in host defence, such as chemotaxis, phagocytosis, cytotoxicity, intracellular killing of bacteria as well as cytokine production, appear to be, at least in part, under the control of the indigenous microflora. Though lactic acid bacteria exert stimulating effects on macrophage functions, their impact is less clearly established and seems to be strain- and test system-dependent. Results showing macrophage activation in humans by orally administered lactic bacteria are very limited and more studies are needed to correlate the stimulation of macrophage functions and positive health effects of these probiotic products.

Tumor necrosis factor receptor p55 controls the severity of arthritis in experimental Yersinia enterocolitica infection.

To dissect the host defense mechanisms in relation to the development of Yersinia-associated arthritis by evaluating the impact of tumor necrosis factor receptor p55 (TNFRp55) deficiency on Yersinia enterocolitica infection. TNFRp55-/- and C57BL/6 mice were inoculated intravenously with arthritogenic strain 8081 of Yenterocolitica serotype 0:8. Mice were observed daily for generating survival curves and monitoring arthritis. In subsequent sets of experiments, mice were sacrificed at day 14 after infection for examination of histopathology of joints, bacterial clearance, macrophage microbicidal activity, nitric oxide (NO) production, oxidative burst generation, and cytokine production. There was an 80% mortality rate in TNFRp55-/- mice compared with 25% in the controls at 8 weeks after inoculation with 70 colony-forming units of Y. enterocolitica 0:8. Histologic examination of joint tissues revealed that TNFRp55-/- mice developed more severe arthritis, including cartilage degradation and bony destruction, than controls at day 14 after infection. The more extensive joint pathology in TNFRp55-/- mice was correlated with the higher bacterial load in liver, spleen, and lungs, and with the increased levels of interleukin-10. TNFRp55-/- mice displayed impaired intracellular killing of bacteria by macrophages. This was associated with decreased NO production and impaired oxidative burst activity. This study demonstrates that TNF signaling through TNFRp55 controls the severity of Yersinia-induced arthritis and implicates TNF-mediated macrophage microbicidal activity as a central event in this process.

β2-agonist-induced inhibition of neutrophil chemotaxis is not associated with modification of LFA-1 and Mac-1 expression or with impairment of polymorphonuclear leukocyte antibacterial activity

Patients with chronic obstructive lung disorders often show increased susceptibility to airway infections. As β2-adrenoceptor agonists, in addition to reversing the contractile response of bronchial smooth muscles, may inhibit a variety of inflammatory and immuno-effector cell functions, it is possible that these drugs interfere with host defence mechanisms.The present study was designed to test in vitro whether fenoterol, a short-acting β2-adrenoceptor agonist, could modify human blood neutrophil recruitment and antimicrobial activity.Pre-exposure to fenoterol significantly reduced neutrophil migration towards the complement component C5a, at concentrations ranging from 10−7m to 10−5m , or towards lipopolysaccharide, at a concentration of 10−5m (P<0·05, each comparison). In contrast, the drug (10−8–10−5m) did not significantly modify the increased expression of lymphocyte function-associated antigen (LFA-1, i.e. CD11a/CD18) the macrophage antigen-1 (Mac-1, i.e. CD11b/CD18) induced by N -formylmethionylleucylphenylalanine (fMLP) (P>0·05, each comparison). Finally, incubation of neutrophils with fenoterol (10−8–10−5m) did not significantly influence phagocytosis or intracellular killing of bacteria (Staphylococcus aureus) or H2O2release induced by tetradecanoyl-phorbol-acetate (P>0·1 for each comparison).These results suggest that short-acting β2-adrenoceptor agonists, such as fenoterol, are able partially to reduce neutrophil recruitment in the airways without interfering with the processes involved in phagocytic activity against bacteria.

Cell-mediated immunity: Dealing a direct blow to pathogens

Cytotoxic T lymphocytes are essential for defence against viral infections. Recent data demonstrating direct killing of intracellular bacteria by granulysin, a protein released from the granules of cytotoxic T lymphocytes, emphasize the contribution of these lymphocytes to the control of tuberculosis.

Killing kinetics of intracellular Afipia felis treated with amikacin.

Afipia felis is a facultative intracellular bacterium which multiplies in macrophages following inhibition of phagosome-lysosome (P-L) fusion. When A. felis-infected cells are incubated for 72 h with various antibiotics, only aminoglycosides are found to be bactericidal. We therefore studied the killing of intracellular A. felis by amikacin, and its relationship with the restoration of P-L fusion. Amikacin reduced the number of A. felis from 8.5 x 10(5) to 3.5 x 102 cfu/mL within 94 h. P-L fusion was restored after 30-40 h of incubation with amikacin. Both mechanisms may participate in the intracellular killing of bacteria.

Monocytes incubated with surfactant: a model for human alveolar macrophages?

Monocytes migrate to the lungs and enter the alveoli where they come into contact with surfactant and differentiate into alveolar macrophages. This study focused on the question of the extent to which monocytes and monocyte-derived macrophages (MDM) incubated with surfactant resemble alveolar macrophages. Surfactant-incubated monocytes shared with alveolar macrophages the intracellular presence of surfactant, efficient phagocytosis of opsonized Staphylococcus aureus, and poor intracellular killing of ingested bacteria. The suppressive effect of surfactant on bactericidal activities of monocytes could not be attributed to either the surfactant lipid fraction or surfactant protein A. Monocytes incubated with surfactant differed from alveolar macrophages with respect to expression of various Fc and complement receptors involved in intracellular killing of bacteria. Surfactant-incubated monocytes produced significantly more H2O2 upon stimulation with phorbol ester than alveolar macrophages, but significantly less than control monocytes. Together, monocytes and MDM incubated with surfactant, although similar to alveolar macrophages in some aspects, are not an adequate model for alveolar macrophages. Most likely, factors other than surfactant in the microenvironment of the alveoli, such as oxygen tension, play a role in the differentiation of monocytes to alveolar macrophages as well.

Increased intracellular survival of Mycobacterium smegmatis containing the Mycobacterium leprae thioredoxin-thioredoxin reductase gene.

The thioredoxin (Trx) system of Mycobacterium leprae is expressed as a single hybrid protein containing thioredoxin reductase (TR) at its N terminus and Trx at its C terminus. This hybrid Trx system is unique to M. leprae, since in all other organisms studied to date, including other mycobacteria, both TR and Trx are expressed as two separate proteins. Because Trx has been shown to scavenge reactive oxygen species, we have investigated whether the TR-Trx gene product can inhibit oxygen-dependent killing of mycobacteria by human mononuclear phagocytes and as such could contribute to mycobacterial virulence. The gene encoding M. leprae TR-Trx was cloned into the apathogenic, fast-growing bacterium Mycobacterium smegmatis. Recombinant M. smegmatis containing the gene encoding TR-Trx was killed to a significantly lesser extent than M. smegmatis containing the identical vector with either no insert or a control M. leprae construct unrelated to TR-Trx. Upon phagocytosis, M. smegmatis was shown to be killed predominantly by oxygen-dependent macrophage-killing mechanisms. Coinfection of M. smegmatis expressing the gene encoding TR-Trx together with Staphylococcus aureus, which is known to be killed via oxygen-dependent microbicidal mechanisms, revealed that the TR-Trx gene product interferes with the intracellular killing of this bacterium. A similar coinfection with Streptococcus pyogenes, known to be killed by oxygen-independent mechanisms, showed that the TR-Trx gene product did not influence the oxygen-independent killing pathway. The data obtained in this study suggest that the Trx system of M. leprae can inhibit oxygen-dependent killing of intracellular bacteria and thus may represent one of the mechanisms by which M. leprae can deal with oxidative stress within human mononuclear phagocytes.

Regulation of ciprofloxacin uptake in human promyelocytic leukemia cells and polymorphonuclear leukocytes.

Polymorphonuclear leukocytes (PMNs) actively internalize ciprofloxacin, a capability that can enhance killing of intracellular bacteria and facilitate delivery of the antimicrobial agent to infection sites by migrating PMNs. In this study we investigated mechanisms for up-regulation of this process. Activation with N-formyl-methionyl-leucyl-phenylalanine (fMLP; 100 nM) enhanced PMN ciprofloxacin uptake by 50% (P < 0.05). Phorbol myristate acetate (PMA; > or = 10 nM) enhanced uptake by at least 36-fold, mainly by stimulating an increase in the Vmax of the ciprofloxacin transporter. This effect of PMA was inhibited by antagonists of protein kinase C (H7 and chelerythrine) and the mitogen-activated protein kinase cascade downstream (PD 098059). Under resting and PMA-activated conditions, ciprofloxacin uptake by immature human promyelocytic leukemia (HL-60) cells was much lower than in PMNs. However, when HL-60 cells were induced to mature into PMN-like cells, their ciprofloxacin uptake activity increased markedly. These findings implicate a role for protein kinase C in up-regulation of the ciprofloxacin transporter and suggest that myeloid cells acquire an enhanced ability to take up ciprofloxacin as they mature to end-stage PMNs.