Serum inflammatory cytokines as disease biomarkers in the DE50-MD dog model of Duchenne muscular dystrophy

Abstract

Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disease, caused by mutations in the dystrophin gene, characterised by cycles of muscle degeneration, inflammation and regeneration. Recently, there has been renewed interest specifically in drugs that ameliorate muscle inflammation in DMD patients. The DE50-MD dog is a model of DMD that closely mimics the human DMD phenotype. We quantified inflammatory proteins in serum from wild-type (WT) and DE50-MD dogs aged between 3 and 18 months, to identify biomarkers for future pre-clinical trials. Significantly higher concentrations of C-C motif chemokine ligand 2 (CCL2), granulocyte-macrophage colony-stimulating factor, keratinocyte chemotactic-like, TNFα, and interleukins (IL)-2, IL6, IL7, IL8, IL10, IL15 and IL18, were detected in DE50-MD compared to WT serum. Of these, CCL2 best differentiated the 2 genotypes. Relative quantity of CCL2 mRNA was greater in DE50-MD vastus lateralis muscle than WT dogs, and CCL2 was expressed both within and at the periphery of damaged myofibres. Serum CCL2 concentration was significantly associated with acid phosphatase staining in vastus lateralis biopsy samples in DE50-MD dogs. In conclusion, the serum cytokine profile suggests that inflammation is a feature of the DE50-MD phenotype. Quantification of serum CCL2 in particular is a useful non-invasive biomarker of the DE50-MD phenotype.

Longitudinal assessment of skeletal muscle functional mechanics in the DE50-MD dog model of Duchenne muscular dystrophy

“…To characterise the phenotype of the DE50-MD model, we conducted an extensive natural history study, monitoring disease progression longitudinally over 18 months via multiple complementary approaches (including the muscle physiology measurements described here). This work recently concluded, and results of other investigations suggest that the model exhibits disease progression comparable to that seen in young human patients with DMD: covering the period from first symptomatic manifestation (at approximately 3 years of age) to before loss of ambulation (typically between 10 and 12 years of age) ( Hildyard et al, 2018a ; Riddell et al, 2021 , 2022 ; Hornby et al, 2021 ). Although magnetic resonance imaging (MRI) measurements of DE50-MD dogs muscle volumes did not differ from WT dogs at 3 months of age, the volumes were significantly smaller in DE50-MD dogs at 6 months of age and onwards (except for the cranial sartorius, as discussed above; Hornby et al, 2021 ).…”

“…Conducting pre-clinical trials in large animal models has many practical and financial challenges. Findings from this paper and others ( Hornby et al, 2021 ; Riddell et al, 2021 , 2022 ; Hildyard et al, 2022 ) characterising the DE50-MD model have indicated that an n of six dogs per genotype would be optimal for a select panel of biomarkers with high statistical power to detect a phenotypic improvement in response to treatment. ECD would thus be an excellent addition to this panel of biomarkers for future pre-clinical trials in the DE50-MD model.…”

“…The work presented in this paper forms part of an extensive natural history study for work on this dog model, which followed a large number of dystrophic animals alongside their WT littermates for 18 months. This huge undertaking produced a wealth of information regarding the progression of disease in this model, which includes already published findings on blood biomarkers ( Riddell et al, 2021 , 2022 ), skeletal muscle histological phenotype ( Hildyard et al, 2022 ) and skeletal muscle MRI ( Hornby et al, 2021 ), all of which support the similarity of the model to the human phenotype. The results of this study confirm that muscle function is impaired in DE50-MD dogs.…”

“…RNA was isolated and RT-qPCR conducted as previously described ( Riddell et al, 2022 ), from the same samples used for histological analysis (as described above): following sectioning and mounting for histology, additional unmounted serial cryosections (20-40, approximately 100 mg of tissue) were collected directly into a pre-chilled 1.5 ml Eppendorf tube. RNA was isolated from the frozen tissue using TRIzol (Invitrogen, 15596026) according to the manufacturer's instructions, with the addition of a 1:1 chloroform extraction step and using a modified precipitation protocol [isopropanol (one volume), 3 M sodium acetate pH 5.5 (0.1 volume) and 10 µg/ml glycogen].…”

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

“…We firstly described the inflammatory state of these patients, focusing on prevailing macrophages phenotype and on their secretory activity by studying the released cytokine profile. In literature, it has been already reported that inflammation is a typical feature of DMD; in particular, high serum levels of TNF-α, IL-6, IL-7, and other pro-inflammatory molecules have been described [ 53 ]. In accordance, we observed an elevated release of pro-inflammatory cytokines IL-6 and IFN-γ by DMD-associated macrophages.…”