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

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.

Engagement of distinct epitopes on CD43 induces different co‐stimulatory pathways in human T cells

“…To analyse the activation of downstream signalling pathways, a multi‐channel reporter cell line Jurkat E6 expressing reporter gene under the control of NF‐ κ B, NFAT and AP‐1 promoter element was used as described previously . Briefly, a reporter cell line was generated by introducing constructs encoding NF‐ κ B‐CFP, NFAT‐eGFP and AP‐1‐mCherry into Jurkat E6 cells.…”

“…To analyse the activation of downstream signalling pathways, a multi-channel reporter cell line Jurkat E6 expressing reporter gene under the control of NF-jB, NFAT and AP-1 promoter element was used as described previously. 27 Briefly, a reporter cell line was generated by introducing constructs encoding NF-jB-CFP, NFAT-eGFP and AP-1-mCherry into Jurkat E6 cells. A cell clone that was negative for fluorescent proteins in an unstimulated state and strongly up-regulated CFP, eGFP and mCherry expression upon PMA/ionomycin treatment was selected for further use.…”

Is clarithromycin a potential treatment for cachexia in people with lung cancer? A feasibility study

“…For example, although azithromycin has no 'track record' in cachexia, it also has immunomodulatory properties [17,18], is used long-term [19,20], and has a lower risk of a drug-drug interaction, such that only 10 of our patients would have been excluded on this basis. A formal feasibility study is required to confirm improved recruitment and to explore other aspects of the study still untested in our patient group.…”

Changes in circulating lymphocyte subpopulations in pigs receiving therapeutic doses of ceftiofur and tulathromycin

“…Counter to this suggestion, however, is the observation concerning treatment of CD4+ T-cells obtained from healthy humans with azithromycin or clarithromycin macrolides as used in human medicine. This results in inhibition of the CD4+ T-cells proliferation rate and cytokine secretion, which are essential in evoking an effective immune response to pathogens (24).…”