CB2 Receptor as Emerging Anti-Inflammatory Target in Duchenne Muscular Dystrophy

Abstract

Duchenne Muscular Dystrophy (DMD) is a very severe X-linked dystrophinopathy. It is due to a mutation in the DMD gene and causes muscular degeneration in conjunction with several secondary co-morbidities, such cardiomyopathy and respiratory failure. DMD is characterized by a chronic inflammatory state, and corticosteroids represent the main therapy for these patients. To contradict drug-related side effects, there is need for novel and more safe therapeutic strategies. Macrophages are immune cells stringently involved in both physiological and pathological inflammatory processes. They express the CB2 receptor, one of the main elements of the endocannabinoid system, and have been proposed as an anti-inflammatory target in several inflammatory and immune diseases. We observed a lower expression of the CB2 receptor in DMD-associated macrophages, hypothesizing its involvement in the pathogenesis of this pathology. Therefore, we analyzed the effect of JWH-133, a CB2 receptor selective agonist, on DMD-associated primary macrophages. Our study describes the beneficial effect of JWH-133 in counteracting inflammation by inhibiting pro-inflammatory cytokines release and by directing macrophages' phenotype toward the M2 anti-inflammatory one.

Keywords: CB2 receptor; Duchenne muscular dystrophy; inflammation; macrophage phenotype.

Figure 1

Levels of IFN-γ (A), IL-6 (B), IL-4 (C), and IL-10 (D) from control macrophages (CTR) and Duchenne muscular dystrophy macrophages (DMD) investigated through an enzyme-linked immunosorbent assay (ELISA). The graphs show interleukins levels [pg/mL] as the mean ± standard deviation (SD). (B) Fe³⁺ intracellular concentrations (nmol/µL) in CTR and DMD-associated macrophages, determined by iron assay. Histogram shows Fe³⁺ concentration as the mean ± SD. Students’ t-test has been used for statistical analysis. *, p ≤ 0.05 compared to CTR (p-values: (A) 0.0457; (B) 0.0066; (D) 0.0417; (E) 0.0098).

Figure 2

pSTAT6 (A), CD206 (B), CCR7 (C), and CD86 (D) protein expression levels in CTR macrophages and DMD-associated macrophages evaluated by Western blot technique, starting from 15 μg of total lysates. The most representative images are displayed. The protein bands were detected through Image Lab. Ink software “BIORAD”, and the intensity ratios of immunoblots compared to CTR, taken as 1, were quantified after normalizing with respective controls. The relative quantification for these proteins, normalized for the housekeeping protein β-actin, is represented in the histogram as the mean ± SD. Students’ t-test has been used for statistical analysis. *, p ≤ 0.05 compared to NT (p-values: (A) 0.0003; (B) 0.0012; (C) 0.0069; (D) 0.0521).

Figure 3

CB2 receptor protein expression levels in CTR and DMD-associated macrophages, determined by Western blot, starting from 15 μg of total lysates. The most representative images are displayed. The protein bands were detected through Image Lab. Ink software “BIORAD”, and the intensity ratios of immunoblots compared to CTR, taken as 1, were quantified after normalizing with respective controls. The relative quantification for CB2 receptor expression, normalized for the housekeeping protein β-actin, is represented in the histogram as the mean ± SD. Students’ t-test has been used for statistical analysis. *, p ≤ 0.05 compared to CTR (p-value: 0.0249).

Figure 4

Levels of IL-6 (A) and IL-10 (B) from DMD-associated macrophages after 24-h treatment with JWH-133 and AM630, investigated through enzyme-linked immunosorbent assay (ELISA). The graphs show interleukins levels [pg/mL] as the mean ± standard deviation (SD). Students’ t-test has been used for statistical analysis. *, p ≤ 0.05 compared to NT (p-value: (A) 0.0350).

Figure 5

(A) Fe³⁺ intracellular concentrations (nmol/µL) in DMD-associated macrophages after 24-h treatment with JWH-133 and AM630, determined by iron assay. Histogram shows Fe³⁺ concentration as the mean ± SD. TfR1 (B) and FPN-1 (C) protein expression levels in DMD-associated macrophages after 24-h treatment with JWH-133 and AM630, determined by Western blotting, starting from 15 μg of total lysates. The most representative images are displayed. The protein bands were detected through Image Lab. Ink software “BIORAD”, and the intensity ratios of immunoblots compared to non-treated (NT), taken as 1, were quantified after normalizing with respective controls. The relative quantification for TfR1 and FPN-1 expression, normalized for the housekeeping protein β-actin, is represented in the histogram as the mean ± SD. Students’ t-test has been used for statistical analysis. *, p ≤ 0.05 compared to NT (p-values: (A) 0.0169; (B) 0.0509).

Figure 6

pSTAT6 (A), CD206 (B), CCR7 (C), and CD86 (D) protein expression levels in DMD-associated macrophages after 24-h treatment with JWH-133 and AM630, determined by Western blotting, starting from 15 μg of total lysates. The most representative images are displayed. The protein bands were detected through Image Lab. Ink software “BIORAD”, and the intensity ratios of immunoblots compared to non-treated (NT), taken as 1, were quantified after normalizing with respective controls. The relative quantification for these proteins’ expression, normalized for the housekeeping protein β-actin, is represented in the histogram as the mean ± SD. Students’ t-test has been used for statistical analysis. *, p ≤ 0.05 compared to NT (p-values: (A) 0.0541; (B) 0.0101; (C) 0.0066; (D) 0.0146).