Splenic Ly6Chi monocytes are critical players in dystrophic muscle injury and repair
Giuseppe Rizzo, Rosanna Di Maggio, Anna Benedetti, Jacopo Morroni, Marina Bouche, Biliana Lozanoska-Ochser
Giuseppe Rizzo, Rosanna Di Maggio, Anna Benedetti, Jacopo Morroni, Marina Bouche, Biliana Lozanoska-Ochser
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Research Article Inflammation Muscle biology

Splenic Ly6Chi monocytes are critical players in dystrophic muscle injury and repair

Abstract

Dystrophic muscle is characterized by chronic injury and a steady recruitment of inflammatory Ly6Chi monocytes. Recent studies have identified the spleen as the dominant reservoir of these cells during chronic inflammation. Here, we investigated the contribution of splenic Ly6Chi monocytes to dystrophic muscle pathology. Using the mdx mouse model of muscular dystrophy, we show that Ly6Chi monocytes accumulate in great numbers in the spleen over the course of the disease. The chemokine receptor CCR2 was upregulated on Ly6Chi monocytes in mdx spleen before disease onset, thereby enabling their recruitment to dystrophic muscle. Splenectomy performed before disease onset significantly reduced the number of Ly6Chi monocytes infiltrating dystrophic limb muscle. Moreover, in the absence of splenic Ly6Chi monocytes there was a significant reduction in dystrophic muscle inflammation and necrosis, along with improved regeneration during early disease. However, during late disease, a lack of splenic Ly6Chi monocytes adversely affected muscle fiber repair, due to a delay in the phenotypic shift of proinflammatory F4/80+Ly6ChiCD206lo to antiinflammatory F4/80+Ly6CloCD206+ macrophages. Overall, we show that the spleen is an indispensable source of Ly6Chi monocytes in muscular dystrophy and that splenic monocytes are critical players in both muscle fiber injury and repair.

Authors

Giuseppe Rizzo, Rosanna Di Maggio, Anna Benedetti, Jacopo Morroni, Marina Bouche, Biliana Lozanoska-Ochser

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Figure 4

Splenectomy reduces dystrophic muscle inflammation and early necrosis.

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Splenectomy reduces dystrophic muscle inflammation and early necrosis. (...
(A) Representative H&E staining of tibialis anterior (TA) muscle sections showing reduced areas of infiltration (arrows) in splenectomized (– sp) mdx mice compared with control (+ sp) at 4 weeks of age. Scale bars: 100 μm. (B) A summary graph of histological analysis showing the percentage of infiltration area (n = 7, mdx control; n = 8, mdx splenectomized). Following splenectomy at 2 weeks of age the inflammatory cell infiltrate in hind limb muscle was analyzed by flow cytometry at 4, 8, and 12 weeks of age (C–J). (C) Total number of CD45+ cells normalized to muscle mass in control and splenectomized mdx mice at 4 weeks (n = 6, WT; n = 12, mdx control; n = 14, mdx splenectomized), (D) 8 weeks (n = 5 for all groups), and (E) 12 weeks of age (n = 5 for all groups). (F) Summary graph showing the kinetics of CD45+ cells in muscle of control and splenectomized mdx mice at 4, 8, and 12 weeks of age. (G) Total number of Ly6g+ neutrophils in muscle of control and splenectomized mdx mice at 4 and (H) 8 weeks of age. (I) Total number of CD3+ T cells in muscle of control and splenectomized mdx mice at 4 and (J) 8 weeks of age. (K) Representative TA muscle sections stained with IgG (red) to reveal necrotic muscle fibers showing reduced necrosis in muscle of splenectomized mdx mice compared with control. Scale bars: 100 μm. (L) Quantification of necrosis (n = 7, control; n = 9, splenectomized mdx mice) at 4 weeks. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01 by Student’s t test (B, D, G, H, and L) and 1-way ANOVA (C and F).