Differential effects of antibiotics on neutrophils exposed to lipoteichoic acid derived from Staphylococcus aureus

Abstract

Background: Persistent bacteremia occurs in at least 30% of patients with Staphylococcus aureus bloodstream infection (SAB) and may be attributable to a dysregulated host immune response. Neutrophils interact with a variety of S. aureus microbial factors, including lipoteichoic acid (LTA), to activate phagocytic function in a concentration-dependent manner. Antibiotics have been shown to exert both direct antimicrobial action as well as immunomodulatory effects. In this study, we compared the effects of different anti-staphylococcal antibiotics on LTA-mediated immune activation of neutrophils.

Methods: Neutrophils obtained from healthy volunteers were exposed to two levels of LTA (1 and 10 μg/ml) with or without addition of antibiotics from different pharmacologic classes (vancomycin, daptomycin, ceftaroline). Neutrophil function was assessed by examining phagocytic response, activation (CD11b, CD62L expression), Toll-like receptor-2 expression, cell survival and apoptosis, and CXCL8 release.

Results: Differential LTA-mediated antibiotic effects on neutrophil function were observed primarily at the high LTA exposure level. Ceftaroline in the presence of 10 μg/ml LTA had the most prominent effects on phagocytosis and CD11b and CD62L expression, with trends towards increased neutrophil survival and preservation of CXCL8 release when compared to daptomycin and vancomycin with the latter significantly dampening PMN CXCL8 release.

Conclusions: Select antimicrobial agents, such as ceftaroline, exert immunostimulatory effects on neutrophils exposed to S. aureus LTA, which when confirmed in vivo, could be leveraged for its dual immunomodulatory and antibacterial actions for the treatment of persistent SAB mediated by a dysregulated host response.

Keywords: Antibiotics; Ceftaroline; Immunomodulation; Lipoteichoic acid; Staphylococcus aureus bacteremia.

Fig. 1

Effects of high (10 μg) and low (1 μg) level of LTA and antibiotics on CD11b (a) and CD62L expression (b) in human neutrophils isolated from healthy volunteers (n = 5). CD11b and CD62L expression were determined at 4 h following stimulation using flow cytometry. CD62L and CD11b are inversely correlated markers of PMN activation, with CD62L shedding and CD11b upregulation indicative of activation. Representative scatter plots of unstimulated cells and cells exposed to high LTA with and without antibiotics are shown in (c). Statistical significance was determined using Two-way ANOVA * p < 0.05; **p < 0.01. VAN, vancomycin; CFT, ceftaroline; DAP, daptomycin

Fig. 2

Effects of high (10 μg) and low (1 μg) levels of LTA and antibiotics on phagocytosis in human neutrophils isolated from healthy volunteers (n = 5). Phagocytosis was assessed at 4 h after stimulation using pHrodo S. aureus bioparticles and a fluorescent plate reader. Percent effect, based on the unstimulated control is shown in (a). Mean fluorescence values, as determined via fluorescence spectrophotometry, are depicted in (b). Statistical significance was determined using Two-way ANOVA * p < 0.05. VAN, vancomycin; CFT, ceftaroline; DAP, daptomycin

Fig. 3

Effects of high (10 μg) and low (1 μg) levels of LTA and antibiotics on TLR-2 expression in human neutrophils isolated from healthy volunteer donors (n = 5). TLR-2 expression was determined at 4 h following stimulation using flow cytometry. Percentage of PE + cells is shown in (a). Representative histograms of PE fluorescence intensity in unstimulated cells and cells exposed to high LTA with and without antibiotics are shown in (b). Statistical significance was determined using Two-way ANOVA * p < 0.05. VAN, vancomycin; CFT, ceftaroline; DAP, daptomycin

Fig. 4

Correlation analysis of select variables: a TLR2 expression and phagocytosis; b TLR2 expression and CXCL8 release; c PMN survival and CXCL-8 release; and d CD62L expression and CXCL8 release. Correlation was determined using Pearson’s r value, as shown on each graph

Fig. 5

Effects of high (10 μg) and low (1 μg) levels of LTA and antibiotics on PMN lifespan (a) and survival (b) in human neutrophils isolated from healthy volunteers (n = 5). Effects on PMN lifespan and survival were determined at 16 h following stimulation using flow cytometry with Annexin V and propidium iodide (PI). Live cells are unstained, whereas Annexin V stains apoptotic cells and PI permeates dead cells. Double-stained cells are considered necrotic. Representative scatter plots of unstimulated cells and cells exposed to high LTA with and without antibiotics are shown in c. Statistical significance was determined using two way ANOVA * p < 0.05, **p < 0.01. VAN, vancomycin; CFT, ceftaroline; DAP, daptomycin

Fig. 6

Effects of high (10 μg) and low (1 μg) concentrations of LTA and antibiotics on production of CXCL8 in human neutrophils isolated from healthy volunteers (n = 5). CXCL8 release was determined at 16 h following stimulation using ELISA. Statistical significance was determined using Two-way ANOVA. * p < 0.05, **p < 0.01, ****, p < 0.0001. VAN, vancomycin; CFT, ceftaroline; DAP, daptomycin