The effect of IL-1β inhibitor canakinumab (Ilaris®) on IL-6 production in human skeletal muscle cells

Abstract

Muscle inflammation is one of the hallmarks of Duchenne muscular dystrophy (DMD). Dystrophin-deficient skeletal muscle cells produce higher levels of pro-inflammatory cytokines such as interleukin 1β (IL-1β) in response to toll-like receptor stimulation compared to normal muscle skeletal cells. IL- 1β induces the human skeletal muscle secretion of the myokine Interleukin-6 (IL-6). Here, we evaluated the effect of a human IgG1κ monoclonal antibody (canakinumab (Ilaris®)) that specifically blocks the IL-1β effect on IL-6 secretion by human skeletal muscle cells. Canakinumab is an excellent candidate for therapeutic repositioning to treat DMD because it is an FDA-approved drug to treat periodic fever syndromes and systemic juvenile idiopathic arthritis. Unlike previous generations of IL-1 inhibitors, canakinumab is highly specific for the IL-1β ligand, has a longer half-life, and does not interfere with other IL-1-activated inflammatory pathways. Following cell culture optimization and viability assays to assess toxicity, skeletal muscle cells were stimulated with IL-1β (10 ng/mL) for 48 hours in the presence of nine concentrations of canakinumab ranging from 0.001 nM to 1000 nM, and IL-6 production was measured with an enzyme-linked immunosorbent assay. Pre-incubation of myoblasts with canakinumab before IL-1β-stimulation, significantly reduced IL-6 production at concentrations of 1, 10, 100, 250, and 1000 nM relative to controls, yielding an IC50 of 0.264 nM. On the other hand, co-incubation of canakinumab with IL-1β before addition to myoblasts resulted in a significant inhibition with the IC50 reducing to 0.126 nM, less than half of the previous method. Canakinumab also did not affect myotube viability at 10 nM and was also able to significantly reduce the production of IL-6, when the cells were stimulated with IL-1β (10 ng/ml). Taken together, our results show that canakinumab is a potent inhibitor of IL-1β signaling in muscle cells. These results align with previously published pre-clinical work with other IL-1 inhibitors in the mdx mouse model and support further investigation into the clinical utility of repositioning canakinumab to treat DMD.

Fig 1. Desmin staining of primary human myoblasts (20x magnification).

Most of the cells stained positive for desmin, suggesting that these are myogenic cells. Bar =  100 µm.

Fig 2. Cell viability (MTT) assay for four conditions - media control and three concentrations of IL-1

β (1 ng/ml, 10 ng/ml, 100 ng/ml) after 72 hrs of treatment (n = 3 biological replicates, n = 8 technical replicates, media vs treatment groups is not significant, p = ns). Data shown as mean ±  SEM.

Fig 3. Treatment with IL-1

β triggers IL-6 production in SkMC myoblasts. Treatment of SkMCs myoblasts with IL-1β increases IL-6 compared to media alone. We observed a significant increase of IL-6 after 48h and with both 10 and 100 ng/ml of IL-1β (p < 0.05) (n = 2 biological replicates, n = 8 technical replicates). Individual graphs for each timepoint and dose are provided in the supplementary data (Fig S2 and Tables S15-22; and Fig S3 and Tables S23-30, respectively).

Fig 4. Cell viability analysis after canakinumab treatment for 72 hr.

MTT assay for ten conditions- media control, six concentrations of canakinumab (0.001 nm, 0.01 nm, 0.1 nm, 1 nm, 10 nm, 100 nm) and three concentrations of 1 hr heat-shocked canakinumab (1 nm, 10 nm, and 100 nm) after 72 hrs of treatment (n = 2 biological replicates, n = 8 technical replicates, p = ns).

Fig 5. IL-6 ELISA measurements for 12 treatment conditions (media controls, canakinumab (0.001 nm, 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 250 nM, 500 nM, and 1000 nM), and 1-hr 100 nM heat-shocked canakinumab stimulated with IL-1

β using two treatment schedules for 48 h. The left graphs show 30 min pre-treatment with canakinumab before administering 10 ng/ml of IL-1β for 48 hrs. The right graphs indicate treatment with a 30 min co-incubation of canakinumab with 10 ng/ml of IL-1β before administration of solution to myoblasts for 48 hrs. A) Measured IL-6 (ng/ml) per treatment condition. B) IC₅₀ calculation using 8 different concentrations of canakinumab treatment. For all assays shown above, n = 2 biological replicates, n = 4 technical replicates.

Fig 6. Cell viability (MTT) assay for four conditions - media control, IL-1

β 10 ng/ml, Canakinumab 10 nM, and the co-incubation of IL-1β 10 ng/ml and canakinumab 10 nM after 72 hrs of treatment (n = 2 biological replicates, n = 3 technical replicates). Data shown as mean ±  SEM.

Fig 7. Evaluation of the effects of canakinumab in reducing IL-6 production in myoblasts (A) and myotubes, the terminally differentiated and functional muscle cells

(B). The ELISA assay showed significant reduced levels of IL-6 after 30 min IL-1β (10 ng/ml) coincubation with canakinumab (10 nM) in both cell types (p < 0.0001).