The dual CCR2/CCR5 chemokine receptor antagonist Cenicriviroc reduces macrophage infiltration and disease severity in Duchenne muscular dystrophy (Dmdmdx-4Cv) mice

Abstract

Duchenne muscular dystrophy (DMD) is characterized by progressive muscle weakness which is ultimately fatal, most often due to involvement of the diaphragm. Macrophage infiltration of dystrophic muscles has been strongly linked to muscle damage and fibrosis in DMD. We hypothesized that cenicriviroc (CVC), a dual chemokine receptor (CCR2/CCR5) antagonist currently under clinical evaluation for other diseases, could prevent macrophage accumulation and blunt disease progression in the diaphragms of mdx mice (genetic homologue of DMD). Treatment with CVC (20 mg/kg/day intraperitoneally) or vehicle was initiated in mdx mice at 2 weeks of age (prior to the onset of muscle necrosis) and continued for 4 weeks. Flow cytometry to assess inflammatory cell subsets as well as histological and force generation parameters were determined in mdx diaphragms at the conclusion of the treatment. CVC therapy induced a major (3.9-fold) reduction in total infiltrating macrophages, whereas total numbers of neutrophils and T lymphocytes (CD4+ and CD8+) were unaffected. No changes in macrophage polarization status (inflammatory versus anti-inflammatory skewing based on iNOS and CD206 expression) were observed. Muscle fiber size and fibrosis were not altered by CVC, whereas a significant reduction in centrally nucleated fibers was found suggesting a decrease in prior necrosis-regeneration cycles. In addition, maximal isometric force production by the diaphragm was increased by CVC therapy. These results suggest that CVC or other chemokine receptor antagonists which reduce pathological macrophage infiltration may have the potential to slow disease progression in DMD.

Fig 1. CVC effects on macrophage accumulation.

(A) Representative flow cytometry plots of CD45+ leukocytes from diaphragms of vehicle- and CVC-treated mdx mice, demonstrating a marked reduction in the percentage of macrophages (CD11b+ F4/80+) present in the CVC group. (B) Group mean data (± SE) for macrophage proportion among CD45+ leukocytes in the two groups. (C) Total numbers of macrophages (normalized to muscle weight) present in mdx diaphragms after vehicle or CVC treatment. * P <0.05 for vehicle (n = 8) vs. CVC (n = 7).

Fig 2. CVC effects on the macrophage polarization markers iNOS and CD206.

(A) Representative flow cytometry plots of iNOS and CD206 expression on CD11b+ F4/80+ macrophages in diaphragms of vehicle- and CVC-treated mdx mice. (B) Group mean data (± SE) indicating the proportion of diaphragm macrophages showing different patterns of iNOS and CD206 expression. (C) Mean fluorescence intensity (MFI) for iNOS and CD206 expression by diaphragm macrophages in the two groups of mice. There were no statistically significant differences between the vehicle and CVC groups.

Fig 3. CVC effects on neutrophil and lymphocyte accumulation.

Representative flow cytometry plots of (A) neutrophils (Ly6G+) and (B) lymphocytes (CD3+) in diaphragms of vehicle- and CVC-treated mdx mice. (C) Group mean data (± SE) indicating the proportion of CD45+ cells expressing characteristic neutrophil and lymphocyte markers. (D) Total numbers of neutrophils and lymphocytes (normalized to muscle weight) present in mdx diaphragms after vehicle or CVC treatment. There were no statistically significant differences between the vehicle and CVC groups.

Fig 4. CVC effects on antecedent necrosis-regeneration cycles.

Representative mdx diaphragm histological images stained with (A) Haematoxylin and eosin and (B) Agglutinin and Hoechst (blue nuclei) to reveal the location (either central or peripheral) of muscle fiber nuclei. (C) Quantification of the percentage of fibers containing centrally located nuclei, which was significantly reduced in the CVC-treated group. Values are group mean data (± SE). * P <0.05 for vehicle vs. CVC (n = 8 per group).

Fig 5. CVC effects on muscle fiber size and fibrosis.

The cross-sectional area (CSA) and minimal Feret's diameter of fibers with (A, B) central and (C, D) peripheral nuclei, as well as (E) the degree of muscle fibrosis as determined by extracellular agglutinin staining, were unchanged (n = 8 mice per group for vehicle- and CVC-treated). In addition, the (F) hydroxyproline content (n = 4 mice per group) did not differ between groups. Values are group mean data (± SE).

Fig 6. CVC effects on force generation.

Ex vivo force generating capacity of the mdx diaphragm was tested (A) at different electrical stimulation frequencies, revealing (B) increased maximal isometric force generation in CVC-treated mice. Values are group mean data (± SE). * P <0.05 for vehicle vs. CVC (n = 7 per group).