Impairment of Growth of<i>Listeria monocytogenes</i>in THP‐1 Macrophages by Granulocyte Macrophage Colony‐Stimulating Factor: Release of Tumor Necrosis Factor–α and Nitric Oxide

Oritavancin, a lipoglycopeptide antibiotic in development, accumulates to high levels in the lysosomes of eukaryotic cells. We examined specific functions of macrophages (phagocytic capacity, lysosomal integrity, metabolic activity, and production of reactive oxygen species [ROS]) in correlation with the cellular accumulation of the drug, using J774 mouse macrophages and THP-1 human monocytes differentiated into macrophages using phorbol 12-myristate 13-acetate. Oritavancin did not affect Pseudomonas aeruginosa phagocytosis, lysosomal integrity, or metabolic activity in cells incubated for 3 h with extracellular concentrations ranging from 5 to 50 g/ml. At extracellular concentrations of >25 g/ml, oritavancin reduced latex bead phagocytosis by approximately 50% and doubled ROS production in J774 macrophages only. This may result from the fact that the cellular accumulation of oritavancin was 15 times higher in J774 cells than in activated THP-1 cells at 3 h. Human pharmacokinetic studies estimate that the concentration of oritavancin in alveolar macrophages could reach approximately 560 g/ml after administration of a cumulative dose of 4 g, which is below the cellular concentration needed in the present study to impair latex bead phagocytosis (1,180 g/ml) or to stimulate ROS production (15,000 g/ml) by J774 cells. The data, therefore, suggest that, in spite of its substantial cellular accumulation, oritavancin is unlikely to markedly affect macrophage functions under the conditions of use investigated in current phase III trials (a single dose of 1,200 mg).

Section: Methodsmentioning

confidence: 99%

Study of Macrophage Functions in Murine J774 Cells and Human Activated THP-1 Cells Exposed to Oritavancin, a Lipoglycopeptide with High Cellular Accumulation

Lemaire

Mingeot‐Leclercq

Tulkens

et al. 2014

“…Fractionation studies were performed with J774 murine macrophages. Both cell lines were maintained in our laboratory as previously described, using RPMI medium supplemented with 10% fetal calf serum (15,29).…”

Section: Methodsmentioning

confidence: 99%

Contrasting Effects of Acidic pH on the Extracellular and Intracellular Activities of the Anti-Gram-Positive Fluoroquinolones Moxifloxacin and Delafloxacin againstStaphylococcus aureus

Lemaire

Tulkens

Bambeke

2011

169

140

In contrast to currently marketed fluoroquinolones, which are zwitterionic, delafloxacin is an investigational fluoroquinolone with an anionic character that is highly active against Gram-positive bacteria. We have examined the effect of acidic pH on its accumulation in Staphylococcus aureus and in human THP-1 cells, in parallel with its activity against extracellular and intracellular S. aureus. Moxifloxacin was used as a comparator. Delafloxacin showed MICs 3 to 5 log 2 dilutions lower than those of moxifloxacin for a collection of 35 strains with relevant resistance mechanisms and also proved to be 10-fold more potent against intracellular S. aureus ATCC 25923. In medium at pH 5.5, this difference was further enhanced, with the MIC decreasing by 5 log 2 dilutions. In infected cells incubated in acidic medium, the relative potency was 10-fold higher than that at neutral pH and the maximal relative efficacy reached a bactericidal effect at 24 h. These results can be explained by a 10-fold increase in delafloxacin accumulation in both bacteria and cells at acidic pH, making delafloxacin one of the most efficient drugs tested in this model. Opposite effects were seen for moxifloxacin with respect to both activity and accumulation. As reported for zwitterionic fluoroquinolones, delafloxacin was found associated with the soluble fraction in homogenates of eukaryotic cells. Taken together, these properties may confer to delafloxacin an advantage for the eradication of S. aureus in acidic environments, including intracellular infections.Fluoroquinolones are one of the first families of fully synthetic antibiotics with large clinical use, and they represent a wide range of molecules, which permits the establishment of in-depth structure-activity relationships (16,17). Over the last 20 years, most fluoroquinolone research efforts have been focused on new molecules with improved activity toward Grampositive cocci because of the increasing spread of multiresistant staphylococci and streptococci. The 8-methoxy fluoroquinolone moxifloxacin is the first example of this new generation to reach the commercial market. It combines many desirable microbiological and pharmacokinetic properties (rapid bactericidal activity, a spectrum covering pertinent pathogens, excellent oral bioavailability, a large tissue distribution, and a propensity to accumulate within eukaryotic cells [see reference 45 for a review]). Moxifloxacin shows extensive activity toward bacteria thriving in the cytosol (Listeria monocytogenes [14]), phagosomes (Legionella pneumophila, Mycobacterium avium, and Mycobacterium tuberculosis [5,9,46]), and phagolysosomes (Staphylococcus aureus [6]), suggesting that it easily diffuses between different subcellular compartments. Yet moxifloxacin activity is partially defeated by the acidic pH prevailing in vacuolar subcellular compartments (6).Delafloxacin [RX-3341; 1-(6-amino-3,5-difluoropyridin-2-yl)-8-chloro-6-fluoro-7-(3-hydroxyazetidin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid] is an investigational fluoro...

“…All conditions for measurement of the MICs (at pH 7.3 and 5.0) and the minimal bactericidal concentrations (MBCs) were exactly the same as those described earlier (57). Dose-kill curve studies were performed as described previously (57), with the following modifications: (i) RPMI 1640 medium supplemented with 10% fetal calf serum rather than broth was systematically used to measure the extracellular activities to better mimic a true extracellular environment, and (ii) enumeration of colonies (for determination of CFU) was performed with an automated detector (14). All samples (diluted as needed) were prepared in a final volume of 1 ml, of which 50 l was used to seed 8.2-cm-diameter petri dishes containing 12.5 ml of nutrient agar.…”

Section: Methodsmentioning

confidence: 99%

“…For each of these antibiotics, the assay method was checked for linearity (telithromycin, 0.03 to 4 mg/liter; azithromycin, 0.05 to 4 mg/liter; ampicillin, 0.21 to 10 mg/liter, nafcillin, 1.1 to 120 mg/liter; oxacillin, 1.6 to 30 mg/liter; penicillin V, 0.04 to 10 mg/liter; rifampin, 0.2 to 15 mg/liter; linezolid, 12 to 190 mg/liter, vancomycin, 2.7 to 150 mg/liter; teicoplanin, 2.9 to 250 mg/liter; gentamicin, 0.6 to 240 mg/liter) and for reproducibility (coefficient of variation, Ͻ10%). Ciprofloxacin, moxifloxacin, and levofloxacin concentrations were measured by fluorimetry (12,56), and garenoxacin and oritavancin concentrations were measured by radiometry by using 14 C-labeled drugs (41,64). In pilot studies we checked that these assays detected genuine, bioactive drug.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Pharmacodynamic Evaluation of the Intracellular Activities of Antibiotics against Staphylococcus aureus in a Model of THP-1 Macrophages

Barcia-Macay

Seral

Mingeot‐Leclercq

et al. 2006

232

341

Staphylococcus aureus, which often causes chronic or relapsing diseases (68), is reported to persist as an opportunistic intracellular organism both in vitro and in vivo (8,10,18,30,31,34,39). Antibiotic treatments should therefore be optimized not only toward the extracellular forms of S. aureus but also toward the intracellular forms of S. aureus to avoid creating a niche where bacteria may persist, cause cell alterations, and possibly, be selected for resistance if they are exposed to subtherapeutic concentrations (1). A large body of literature on the activities of antibiotics against intracellular S. aureus in various cellular models is available (see references 54, 66, 67, and 70 for reviews). Yet, many of these studies yield contradictory results, and we still lack a clear understanding of which parameters are truly critical for the expression of antibiotic activity in the intracellular milieu (11). In a previous study, we measured the activities of selected antibiotics characterized by a fair to high level of cellular accumulation against intracellular S. aureus in a model of unstimulated murine J774 macrophages (57). We observed that cellular accumulation was only partially and nonconsistently predictive of activity. In a subsequent pilot study, performed with human THP-1 macrophages, we also noted that ␤-lactams, which notoriously do not accumulate in cells, actually showed significant activity against intracellular S. aureus when their extracellular concentration was brought to a sufficiently high but still clinically meaningful level (36). This triggered us to broaden and systematize our approach. For this purpose, we selected typical representatives of seven classes of antibiotics with known activities against S. aureus and included in commonly used guidelines for the handling of staphylococcal infections. We concentrated our effort on THP-1 macrophages because these cells present many of the characteristics of human monocytes while forming a homogeneous and reproducible population (6). THP-1 macrophages have been successfully used in various studies aimed at characterizing the interactions between S. aureus and macrophages in a clinical context (19,28,49) and to analyze the potential relationship between the accumulation of antibiotics in cells and intracellular activity (48). In contrast to many other models, however, we explored a large array of extracellular concentrations (including the range observed in the serum of patients receiving conventional doses) and used incubation times up to 24 h. Our purpose, indeed, was to analyze the pharmacodynamic parameters governing the activities of these antibiotics against intra-* Corresponding author. Mailing address: