Depletion of Mast Cells and Macrophages Impairs Heterotopic Ossification in an Acvr1R206H Mouse Model of Fibrodysplasia Ossificans Progressiva

Abstract

Heterotopic ossification (HO) is a clinical condition that often reduces mobility and diminishes quality of life for affected individuals. The most severe form of progressive HO occurs in those with fibrodysplasia ossificans progressiva (FOP; OMIM #135100), a genetic disorder caused by a recurrent heterozygous gain-of-function mutation (R206H) in the bone morphogenetic protein (BMP) type I receptor ACVR1/ALK2. In individuals with FOP, episodes of HO frequently follow injury. The first sign of active disease is commonly an inflammatory "flare-up" that precedes connective tissue degradation, progenitor cell recruitment, and endochondral HO. We used a conditional-on global knock-in mouse model expressing Acvr1^R206H (referred to as Acvr1^cR206H/+ ) to investigate the cellular and molecular inflammatory response in FOP lesions following injury. We found that the Acvr1 R206H mutation caused increased BMP signaling in posttraumatic FOP lesions and early divergence from the normal skeletal muscle repair program with elevated and prolonged immune cell infiltration. The proinflammatory cytokine response of TNFα, IL-1β, and IL-6 was elevated and prolonged in Acvr1^cR206H/+ lesions and in Acvr1^cR206H/+ mast cells. Importantly, depletion of mast cells and macrophages significantly impaired injury-induced HO in Acvr1^cR206H/+ mice, reducing injury-induced HO volume by ∼50% with depletion of each cell population independently, and ∼75% with combined depletion of both cell populations. Together, our data show that the immune system contributes to the initiation and development of HO in FOP. Further, the expression of Acvr1^R206H in immune cells alters cytokine expression and cellular response to injury and unveils novel therapeutic targets for treatment of FOP and nongenetic forms of HO. © 2017 American Society for Bone and Mineral Research.

Keywords: ACVR1; BMP; BONE MORPHOGENETIC PROTEIN SIGNALING; CHRONIC INFLAMMATION; FIBRODYSPLASIA OSSIFICANS PROGRESSIVA; FOP; HETEROTOPIC OSSIFICATION; TISSUE INJURY.

Fig. 1.

Progression of heterotopic ossification formation following injury in Acvr1^cR206H/+ mice. Skeletal muscle tissue sections from Acvr1^cR206H/+ and Acvr1^+/+ mice from indicated days following cardiotoxin injury were detected for cartilage (Alcian Blue), bone (Orange G), cell nuclei/immune cells (Hematoxylin), and skeletal muscle (Eosin). (A) Skeletal muscle histology is similar in mutant and control mice before injury (day 0). (B, C) Skeletal muscle degradation and increasingly abundant immune cells are present by days 1 to 2 in both mutant and control mice. (D) At day 3, immune cells increased further, but to a greater extent in Acvr1^cR206H/+ tissue. (E–G) In control tissue, muscle repair progresses through the fibroproliferative stage and clearance of immune cells (days 4 to 6); however, GAG proteins are detected (Alcian Blue) in Acvr1^cR206H/+ tissue. (H–J) In control tissue, skeletal muscle repair continues and is completed over days 7 to 14. In Acvr1^cR206H/+ lesions, endochondral ossification proceeds with maturation to hypertrophic chondrocytes and mineralized bone. Ectopic bone with adjacent regions of mature cartilage and remaining fibroproliferation at day 14 is shown. Representative images from n = 4 mice per day, per genotype are shown. Scale bar = 100 μm for all images. Dotted lines indicate tissue areas as noted. M = skeletal muscle; I = immune infiltration; FP = fibroproliferation; C = chondrocytes; B = bone.

Fig. 2.

Immune cell numbers are elevated and prolonged in Acvr1^cR206H/+ lesions. Specific immune cell populations were detected and quantified in injured Acvr1^cR206H/+ and Acvr1^+/+ skeletal muscle over time; early-stage (day 2), intermediate-stage (day 6), and late-stage (day 14) postinjury are shown. (A) Neutrophils were detected by myeloperoxidase IHC. (B) Monocytes/macrophages were detected with F4/80 IHC. (C) Mast cells were detected by C.E. M. stain kit, and indicated by red arrows. (D) T cells were detected with CD3 IHC. Cells were quantified from three fields of view per independent sample; n = 3 for neutrophils, monocytes/macrophages, T cells; n = 4 for mast cells. Representative images are shown. Scale bar = 50 μm. Data shown are mean values ± SE; two-way ANOVA with Sidak’s multiple comparisons test comparing Acvr1^cR206H/+ versus Acvr1^+/+ per day was performed; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. IHC = immunohistochemistry.

Fig. 3.

Proinflammatory cytokine expression is elevated and prolonged in Acvr1^cR206H/+ lesions and primary Acvr1^cR206H/+ mast cells. Specific proinflammatory cytokines were detected by immunostaining of injured tissues from early-stage (day 2), intermediate-stage (day 6), and late-stage (day 14) Acvr1^cR206H/+ and Acvr1^+/+ lesions. (A) TNFα, (B) IL-6, and (C) IL-1β. n = 3 to 4 per day, per genotype; representative images are shown. Scale bar = 100 μm for all images. (D, E) mRNA expression of specific proinflammatory cytokines in mast cells and macrophages was detected by qRT-PCR following mast cell treatment with 100 μM Substance P or macrophage treatment with 1 ng/mL lipopolysaccharides, then 15 ng/mL BMP4 for 1 hour. Data were normalized to Gapdh and are shown as mean values ± SE; Student’s t test compared expression in Acvr1^cR206H/+ versus Acvr1^+/+; *p < 0.05. ns = not significant.

Fig. 4.

Variable expression of MCP-1, IL-13, and Activin A in Acvr1^cR206H/+ and Acvr1^+/+ lesions. Sections from early-stage (day 2), intermediate-stage (day 6), and late-stage (day 14) Acvr1^cR206H/+ and Acvr1^+/+ lesions were immunostained for expression of multiple proinflammatory cytokines. Immunohistochemistry was performed to detect (A) MCP-1, (B) IL-13, and (C) Activin A expression. (D) Higher magnification insets demonstrating fibroblast and chondrocyte Activin A expression in Acvr1^cR206H/+ lesions are shown, indicated by dotted rectangles in C. n = 3 to 4; representative images per day, per genotype, are shown. Scale bar = 100 μm for all images in A–C; scale bar = 50 μm for all images in D.

Fig. 5.

Mast cell and macrophage depletion impairs formation of heterotopic ossification in Acvr1^cR206H/+ mice. Cardiotoxin-injured skeletal muscles of Acvr1^cR206H/+ mice with intact or depleted mast cells and/or macrophages were examined by μCT to detect heterotopic ossification after 17 days. Mast cells were genetically ablated in Acvr1^cR206H/+;c-Kit^W-sh/W-sh mice or partially ablated in Acvr1^cR206H/+;c-Kit^W-sh/+ mice. Macrophages were chemically depleted in Clo-treated mice. Data are compared to Acvr1^cR206H/+ (positive HO control) and Acvr1^+/+ (negative control). (A) Representative μCT 3D volume renderings showing mean HO volume per hind limb are shown. (B) Quantification of HO for each cohort (n = 5 to 20); HO per hind limb was added for total HO per mouse. Data shown are mean values ± SE; one-way ANOVA with Tukey’s multiple comparisons test compared Acvr1^cR206H/+ versus other cohorts; *p < 0.05, **p < 0.01. ns = not significant; Clo = Clodronate-liposomes.