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Azithromycin suppresses CD4(+) T-cell activation by direct modulation of mTOR activity.

Ratzinger F, Haslacher H, Poeppl W, Hoermann G, Kovarik JJ, Jutz S, Steinberger P, Burgmann H, Pickl WF, Schmetterer KG - Sci Rep (2014)

Bottom Line: Analysis of molecular signaling pathways revealed that exposure to AZM reduced the phosphorylation of the S6 ribosomal protein, a downstream target of mTOR.This effect was also observed at 40 mg/L CLM.Our results might have implications for the clinical use of macrolides.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, Medical University of Vienna, Austria.

ABSTRACT
Advanced macrolides, such as azithromycin (AZM) or clarithromycin (CLM), are antibiotics with immunomodulatory properties. Here we have sought to evaluate their in vitro influence on the activation of CD4(+) T-cells. Isolated CD4(+) T-cells were stimulated with agonistic anti-CD3/anti-CD28 monoclonal antibodies in the presence of 0.6 mg/L, 2.5 mg/L, 10 mg/L or 40 mg/L AZM or CLM. Cell proliferation, cytokine level in supernatants and cell viability was assessed. Intracellular signaling pathways were evaluated using reporter cell lines, FACS analysis, immunoblotting and in vitro kinase assays. AZM inhibited cell proliferation rate and cytokine secretion of CD4(+) T-cells in a dose-dependent manner. Similarly, high concentrations of CLM (40 mg/L) also suppressed these T-cell functions. Analysis of molecular signaling pathways revealed that exposure to AZM reduced the phosphorylation of the S6 ribosomal protein, a downstream target of mTOR. This effect was also observed at 40 mg/L CLM. In vitro kinase studies using recombinant mTOR showed that AZM inhibited mTOR activity. In contrast to rapamycin, this inhibition was independent of FKBP12. We show for the first time that AZM and to a lesser extent CLM act as immunosuppressive agents on CD4(+) T-cells by inhibiting mTOR activity. Our results might have implications for the clinical use of macrolides.

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Suppression of CD4+ T-cells proliferation following activation.(A) Median proliferation values (with quartile 1 and quartile 3) normalized to the respective antibiotic-free medium control from thymidine incorporation assays from CD4+ T-cells from ten apparently healthy volunteers; if not otherwise indicated, all statistical comparisons relate to the antibiotic-free medium control; n.s. not significant. (B) Proliferation of eFluor670 CPD labeled CD4+ T-cells 96 hours after stimulation in the presence of antibiotic-free medium or the indicated concentrations of AZM and CLM; numbers indicate division index; one representative experiment (n = 4) is depicted.
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f1: Suppression of CD4+ T-cells proliferation following activation.(A) Median proliferation values (with quartile 1 and quartile 3) normalized to the respective antibiotic-free medium control from thymidine incorporation assays from CD4+ T-cells from ten apparently healthy volunteers; if not otherwise indicated, all statistical comparisons relate to the antibiotic-free medium control; n.s. not significant. (B) Proliferation of eFluor670 CPD labeled CD4+ T-cells 96 hours after stimulation in the presence of antibiotic-free medium or the indicated concentrations of AZM and CLM; numbers indicate division index; one representative experiment (n = 4) is depicted.

Mentions: Highly purified CD4+ T-cells were activated in the presence of titrated levels of AZM (Zithromax®), CLM (Klacid®), or antibiotic-free medium. In thymidine incorporation assays, cell proliferation levels of CD4+ T-cells were significantly reduced in cells exposed to indicated macrolides (p < 0.001, see: Figure 1A; for absolute values from one representative donor see Supplementary Figure 1). A slight reduction of cellular proliferation was found already at 0.6 mg/L AZM, which did not reach statistical significance (p = 0.21). At a fourfold higher concentration (2.5 mg/L) a significant reduction of cellular proliferation was observed in T-cells exposed to AZM (p = 0.001), but not following exposure to CLM (p > 0.999). A concentration of 10 mg/L AZM reduced T-cell proliferation to a median proliferation rate of 0.45 (p < 0.001). In contrast to AZM, the presence of CLM at this concentration had no significant influence on the proliferation rate (median: 0.72, p = 0.066). At 40 mg/L AZM, T-cell proliferation was strongly reduced (median: 0.10, p < 0.001), whereas exposure to 40 mg/L CLM suppressed the median proliferation to 0.54 (p = 0.002). Generally, the magnitude of the suppressive effect of AZM was significantly higher than the effect of CLM (0.6 mg/L: p = 0.023, 2.5 mg/L: p = 0.008, 10 mg/L p = 0.008, 40 mg/L: p = 0.014). Results did not depend on the used drug formulation (i.v. formulation versus highly purified substance, data not shown). Each increase of AZM dose led to a significant reduction of the T-cell proliferation (p values: ≤0.001), indicating a dose-dependency in this suppression. For confirmation of these effects of AZM and CLM, a second proliferation test was performed, using T-cells labeled with a fluorescent tracer. Figure 1B presents the graphical results, demonstrating a similar suppressive effect for AZM at a concentration of 10 mg/L, as for 40 mg/L CLM (p = 0.475).


Azithromycin suppresses CD4(+) T-cell activation by direct modulation of mTOR activity.

Ratzinger F, Haslacher H, Poeppl W, Hoermann G, Kovarik JJ, Jutz S, Steinberger P, Burgmann H, Pickl WF, Schmetterer KG - Sci Rep (2014)

Suppression of CD4+ T-cells proliferation following activation.(A) Median proliferation values (with quartile 1 and quartile 3) normalized to the respective antibiotic-free medium control from thymidine incorporation assays from CD4+ T-cells from ten apparently healthy volunteers; if not otherwise indicated, all statistical comparisons relate to the antibiotic-free medium control; n.s. not significant. (B) Proliferation of eFluor670 CPD labeled CD4+ T-cells 96 hours after stimulation in the presence of antibiotic-free medium or the indicated concentrations of AZM and CLM; numbers indicate division index; one representative experiment (n = 4) is depicted.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4262884&req=5

f1: Suppression of CD4+ T-cells proliferation following activation.(A) Median proliferation values (with quartile 1 and quartile 3) normalized to the respective antibiotic-free medium control from thymidine incorporation assays from CD4+ T-cells from ten apparently healthy volunteers; if not otherwise indicated, all statistical comparisons relate to the antibiotic-free medium control; n.s. not significant. (B) Proliferation of eFluor670 CPD labeled CD4+ T-cells 96 hours after stimulation in the presence of antibiotic-free medium or the indicated concentrations of AZM and CLM; numbers indicate division index; one representative experiment (n = 4) is depicted.
Mentions: Highly purified CD4+ T-cells were activated in the presence of titrated levels of AZM (Zithromax®), CLM (Klacid®), or antibiotic-free medium. In thymidine incorporation assays, cell proliferation levels of CD4+ T-cells were significantly reduced in cells exposed to indicated macrolides (p < 0.001, see: Figure 1A; for absolute values from one representative donor see Supplementary Figure 1). A slight reduction of cellular proliferation was found already at 0.6 mg/L AZM, which did not reach statistical significance (p = 0.21). At a fourfold higher concentration (2.5 mg/L) a significant reduction of cellular proliferation was observed in T-cells exposed to AZM (p = 0.001), but not following exposure to CLM (p > 0.999). A concentration of 10 mg/L AZM reduced T-cell proliferation to a median proliferation rate of 0.45 (p < 0.001). In contrast to AZM, the presence of CLM at this concentration had no significant influence on the proliferation rate (median: 0.72, p = 0.066). At 40 mg/L AZM, T-cell proliferation was strongly reduced (median: 0.10, p < 0.001), whereas exposure to 40 mg/L CLM suppressed the median proliferation to 0.54 (p = 0.002). Generally, the magnitude of the suppressive effect of AZM was significantly higher than the effect of CLM (0.6 mg/L: p = 0.023, 2.5 mg/L: p = 0.008, 10 mg/L p = 0.008, 40 mg/L: p = 0.014). Results did not depend on the used drug formulation (i.v. formulation versus highly purified substance, data not shown). Each increase of AZM dose led to a significant reduction of the T-cell proliferation (p values: ≤0.001), indicating a dose-dependency in this suppression. For confirmation of these effects of AZM and CLM, a second proliferation test was performed, using T-cells labeled with a fluorescent tracer. Figure 1B presents the graphical results, demonstrating a similar suppressive effect for AZM at a concentration of 10 mg/L, as for 40 mg/L CLM (p = 0.475).

Bottom Line: Analysis of molecular signaling pathways revealed that exposure to AZM reduced the phosphorylation of the S6 ribosomal protein, a downstream target of mTOR.This effect was also observed at 40 mg/L CLM.Our results might have implications for the clinical use of macrolides.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, Medical University of Vienna, Austria.

ABSTRACT
Advanced macrolides, such as azithromycin (AZM) or clarithromycin (CLM), are antibiotics with immunomodulatory properties. Here we have sought to evaluate their in vitro influence on the activation of CD4(+) T-cells. Isolated CD4(+) T-cells were stimulated with agonistic anti-CD3/anti-CD28 monoclonal antibodies in the presence of 0.6 mg/L, 2.5 mg/L, 10 mg/L or 40 mg/L AZM or CLM. Cell proliferation, cytokine level in supernatants and cell viability was assessed. Intracellular signaling pathways were evaluated using reporter cell lines, FACS analysis, immunoblotting and in vitro kinase assays. AZM inhibited cell proliferation rate and cytokine secretion of CD4(+) T-cells in a dose-dependent manner. Similarly, high concentrations of CLM (40 mg/L) also suppressed these T-cell functions. Analysis of molecular signaling pathways revealed that exposure to AZM reduced the phosphorylation of the S6 ribosomal protein, a downstream target of mTOR. This effect was also observed at 40 mg/L CLM. In vitro kinase studies using recombinant mTOR showed that AZM inhibited mTOR activity. In contrast to rapamycin, this inhibition was independent of FKBP12. We show for the first time that AZM and to a lesser extent CLM act as immunosuppressive agents on CD4(+) T-cells by inhibiting mTOR activity. Our results might have implications for the clinical use of macrolides.

Show MeSH
Related in: MedlinePlus