Macrolide derivatives reduce proinflammatory macrophage activation and macrophage-mediated neurotoxicity

Abstract

Introduction: Azithromycin (AZM) and other macrolide antibiotics are applied as immunomodulatory treatments for CNS disorders. The immunomodulatory and antibiotic properties of AZM are purportedly independent.

Aims: To improve the efficacy and reduce antibiotic resistance risk of AZM-based therapies, we evaluated the immunomodulatory and neuroprotective properties of novel AZM derivatives. We semisynthetically prepared derivatives by altering sugar moieties established as important for inhibiting bacterial protein synthesis. Bone marrow-derived macrophages (BMDMs) were stimulated in vitro with proinflammatory, M1, stimuli (LPS + INF-gamma) with and without derivative costimulation. Pro- and anti-inflammatory cytokine production, IL-12 and IL-10, respectively, was quantified using ELISA. Neuron culture treatment with BMDM supernatant was used to assess derivative neuroprotective potential.

Results: Azithromycin and some derivatives increased IL-10 and reduced IL-12 production of M1 macrophages. IL-10/IL-12 cytokine shifts closely correlated with the ability of AZM and derivatives to mitigate macrophage neurotoxicity.

Conclusions: Sugar moieties that bind bacterial ribosomal complexes can be modified in a manner that retains AZM immunomodulation and neuroprotection. Since the effects of BMDMs in vitro are predictive of CNS macrophage responses, our results open new therapeutic avenues for managing maladaptive CNS inflammation and support utilization of IL-10/12 cytokine profiles as indicators of macrophage polarization and neurotoxicity.

Keywords: M2; brain; erythromycin; microglia; spinal cord injury; stroke.

Figure 1

Altering the antibiotic properties of azithromycin does not decrease macrophage viability. Bone marrow‐derived macrophages (BMDMs) were isolated from adult mice and were treated with AZM, AZM1, AZM4, AZM5, and AZM7 at concentrations of 1, 5, 25, and 125 μmol/L for 24 h Cell viability was measured by using MTT assay. AZM or AZM derivatives exhibited no cytotoxicity at any tested concentration as compared to unstimulated, nontreated BMDM control (dotted line). Moreover, AZM and AZM derivatives at 25 and/or 125 μmol/L significantly increased proliferation of BMDMs as compared to unstimulated controls at *P < 0.05, **P < 0.01, ***P < 0.001. Data are mean ±SD and representative of three independent biological replicate experiments

Figure 2

Nonantibiotic macrolides polarize proinflammatory macrophages to an anti‐inflammatory phenotype. BMDMs were polarized to be M1 macrophages by stimulating with LPS +INF‐gamma. AZM, AZM1, AZM4, AZM5, and AZM7 were coapplied to M1 cells at concentrations of 1, 5, 25, and 125 μmol/L for 6 h Protein levels of IL‐12 (A) and IL‐10 (B) in cell culture medium were analyzed by ELISA and expressed as fold change over M1 of mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 vs M1. Data are representative of three independent biological replicate experiments. Each experiment was performed in triplicates per treatment group. (A) AZM and AZM 5 at the concentrations of 25 and 125 μmol/L significantly decreased proinflammatory cytokine IL‐12, while AZM 4 and AZM 7 significantly reduced IL‐12 level only at the concentration of 125 μmol/L. AZM 1 showed no effect in changing IL‐12 secretion. (B) The anti‐inflammatory cytokine IL‐10 level was significantly increased in M1 macrophages coincubated with AZM and AZM 5 at the concentrations of 25 and 125 μmol/L; while AZM 1, AZM 4, and AZM 7 significantly increased IL‐10 expression only at the highest tested concentration of 125 μmol/L (B)

Figure 3

Nonantibiotic macrolides decrease the neurotoxic potential of proinflammatory macrophages. Neuron 2A (N2A) cells were incubated with MCM for 24 h MCM from M1 macrophages reduced neuron cell viability significantly as compared to control (N2a cells treated with MCM from unstimulated, nontreated BMDMs, dotted line), M1 vs Control at $$$$ P < 0.0001). MCM from M1 macrophages coincubated with AZM and/or AZM 4, AZM 5, and AZM 7 at 25 and/or 125 μmol/L restored neuron cell viability as compared to M1 MCM (*P < 0.05, P < 0.01, P < 0.0001 vs M1). Derivative AZM 1 exhibited no effect in modulating M1 MCM‐induced neurotoxicity. Data are mean ± SD and representative of three independent biological replicate experiments