Mycolactone displays anti-inflammatory effects on the nervous system

Abstract

Background: Mycolactone is a macrolide produced by the skin pathogen Mycobacterium ulcerans, with cytotoxic, analgesic and immunomodulatory properties. The latter were recently shown to result from mycolactone blocking the Sec61-dependent production of pro-inflammatory mediators by immune cells. Here we investigated whether mycolactone similarly affects the inflammatory responses of the nervous cell subsets involved in pain perception, transmission and maintenance. We also investigated the effects of mycolactone on the neuroinflammation that is associated with chronic pain in vivo.

Methodology/ principle findings: Sensory neurons, Schwann cells and microglia were isolated from mice for ex vivo assessment of mycolactone cytotoxicity and immunomodulatory activity by measuring the production of proalgesic cytokines and chemokines. In all cell types studied, prolonged (>48h) exposure to mycolactone induced significant cell death at concentrations >10 ng/ml. Within the first 24h treatment, nanomolar concentrations of mycolactone efficiently suppressed the cell production of pro-inflammatory mediators, without affecting their viability. Notably, mycolactone also prevented the pro-inflammatory polarization of cortical microglia. Since these cells critically contribute to neuroinflammation, we next tested if mycolactone impacts this pathogenic process in vivo. We used a rat model of neuropathic pain induced by chronic constriction of the sciatic nerve. Here, mycolactone was injected daily for 3 days in the spinal canal, to ensure its proper delivery to spinal cord. While this treatment failed to prevent injury-induced neuroinflammation, it decreased significantly the local production of inflammatory cytokines without inducing detectable cytotoxicity.

Conclusion/ significance: The present study provides in vitro and in vivo evidence that mycolactone suppresses the inflammatory responses of sensory neurons, Schwann cells and microglia, without affecting the cell viability. Together with previous studies using peripheral blood leukocytes, our work implies that mycolactone-mediated analgesia may, at least partially, be explained by its anti-inflammatory properties.

Fig 1. Cytotoxicity of mycolactone on PNS and CNS cell subsets.

(A-B) Cytotoxicity of mycolactone (ML) on dorsal root ganglion (DRG) neurons, identified by β-III tubulin staining. (A) Proportion of TUNEL⁺ DRG neurons, relative to the total number of DRG neurons, after 24 h of exposure to increasing doses of ML. Red lines indicate mean percentages. Data are from two independent experiments, with at least 10 acquisition fields per dose, and 200 cells per dose. Statistics: Mann whitney, * p<0.05, ** p<0.01, *** p<0.001. (B) Viability of DRG neurons after 24 or 48 h of exposure to ML expressed as percentages relative to vehicle-treated controls. Data are from two independent experiments, with at least 10 acquisition fields per dose, and 200 cells per dose (C-F) Cell viability, as assessed by MTT reduction, of primary mouse Schwann cells incubated with ML or vehicle for 48 h (C), primary cortical astrocytes for 72 h (D), cortical neurons for 72 h (E) and microglia for 48 h (F). IC₅₀ indicates the concentration of ML leading to 50% cell death, compared to vehicle-treated controls. Data are mean percentages ± SD of triplicates, and are representative of three independent experiments.

Fig 2. Anti-inflammatory effects of mycolactone on primary DRG and Schwann cells.

Production of CCL-2 (A), IL-6 (B) and TNF-α (C) by mouse dorsal root ganglion (DRG) cells exposed to subtoxic doses of mycolactone (ML) or equivalent volume of vehicle (DMSO) for 30 min prior to 16 h of stimulation with 1 μg/ml LPS. Controls (NA) are vehicle-treated, non-activated cells. (D) IL-6 production by Schwann cells (SCs) exposed to ML or vehicle (DMSO) for 30 min prior to stimulation with 1 μg/m LPS + 20 ng/ml IFN-γ or not (NA). (E) Flow cytometry analysis of TLR4 surface expression by SCs exposed to increasing doses of ML for 16 h. Data are mean values of OD or MFI ± SEM of triplicates, and are representative of two independent experiments.

Fig 3. Mycolactone suppresses the production of pro-inflammatory mediators by activated microglia.

IL-6 (A) and TNF-α (B) production by primary mouse cortical microglia exposed to mycolactone (ML) or vehicle for 16 h, prior to a 8 h activation with 100 ng/ml LPS. (C) Flow cytometry analysis of intracellular NOS-2 in primary cortical microglia pre-treated with ML for 30 min, prior to 16 h activation with 100 ng/ml LPS + 20 ng/ml IFN-γ, in presence of ML. (D) Flow cytometry analysis of surface expression of TLR4 (black) and IFN-γ receptor (CD119, gray) in microglia exposed to ML for 16 h. Data are means IL-6 or TNF-α levels ± SEM (A-B), mean fluorescence intensity ± SEM (C) and mean percentage of suppression compared to vehicle (D) of duplicates, and are representative of two independent experiments.

Fig 4. Intrathecal injection of mycolactone triggers a decrease of pro-inflammatory cytokines in spinal cord of Sham rats.

(A) Experimental procedure: chronic constriction injury (CCI) was induced in rats by partial ligation of the sciatic nerve. At day 2 (D2) post-operation, rats were treated daily by intrathecal injections of mycolactone (ML) or vehicle (Veh) during 3 days and were sacrificed at D5. Sham-operated rats were submitted to the same procedure without CCI. Ipsilateral DRGs from lumbar regions 4 to 6 (L4-L6) and ipsilateral dorsal horn of the spinal cord were collected. (B-G) Expression levels of the pro-inflammatory mediators Il-1β, TIMP-1, IL-6, IFN-γ and IL-2 in the spinal cord (SpC) or in the dorsal root ganglion (DRG), 5 days post CCI or Sham treatment, in Vehicle or ML injected rats. Mean fold changes ± SEM compared to sham treated rats injected with vehicle (n = 6–9). Statistics: Mann whitney, * p<0.05, ** p<0.01, *** p<0.001. (H) Colocalization of Dapi and TUNEL stainings in the ipsilateral region of spinal cord slices from rats injected with DMSO vehicle (left) or ML (middle) daily during three days via intrathecal route. TUNEL positive controls are spinal cord slices from rats injected with vehicle, treated with DNase before staining. Scale bar = 50μm.