Aberrant muscle antigen exposure in mice is sufficient to cause myositis in a Treg cell-deficient milieu

Abstract

Objective: Myositis is associated with muscle-targeted inflammation and is observed in some Treg cell-deficient mouse models. Because an autoimmune pathogenesis has been strongly implicated, the aim of this study was to investigate the hypothesis that abnormal exposure to muscle antigens, as observed in muscle injury, can induce autoimmune-mediated myositis in susceptible hosts.

Methods: FoxP3 mutant (scurfy) mice were mated to synaptotagmin VII (Syt VII) mutant mice, which resulted in a new mouse strain that combines impaired membrane resealing with Treg cell deficiency. Lymphocyte preparations from double-mutant mice were adoptively transferred intraperitoneally, with or without purified Treg cells, into recombination-activating gene 1 (RAG-1)-null recipients. Lymph node cells from mice with the FoxP3 mutation were transferred into RAG-1-null mice either 1) intraperitoneally in conjunction with muscle homogenate or purified myosin protein or 2) intramuscularly with or without cotransfer of purified Treg cells.

Results: FoxP3-deficient mouse lymph node cells transferred in conjunction with myosin protein or muscle homogenate induced robust skeletal muscle inflammation. The infiltrates consisted predominantly of CD4+ and CD8+ T cells, a limited number of macrophages, and no B cells. Significant inflammation was also seen in similar experiments using lymph node cells from FoxP3/Syt VII double-mutant mice but was absent in experiments using adoptive transfer of FoxP3 mutant mouse cells alone. The cotransfer of Treg cells completely suppressed myositis.

Conclusion: These data, derived from a new, reproducible model, demonstrate the critical roles of Treg cell deficiency and aberrant muscle antigen exposure in the priming of autoreactive cells to induce myositis. This mouse system has multifaceted potential for examining the interplay in vivo between tissue injury and autoimmunity.

Figure 1

Muscle tissue inflammation in mice with the synaptotagmin VII (Syt VII) mutation is marked at 4 weeks but subsides over time. A, Muscle tissue inflammation in heterozygous (Syt VII^+/−) and mutant (Syt VII^−/−) mice. Mice were killed at the indicated times, and paraffin-embedded skeletal muscle tissue sections were stained with hematoxylin and eosin. All images were cropped for enhanced resolution and are representative of trends observed in at least 3 mice at each time point in independent experiments. Original magnification × 200. B, Extent of myositis in Syt VII^−/− mice at the 4-week time point, as determined by blinded histopathologic scoring. Values are the mean ± SD (n = 3 mice of each genotype). ∗ = P ≤ 0.05 versus Syt VII^+/−.

Figure 2

FoxP3 and synaptotagmin VII (Syt VII) double-mutant mice develop significant myositis. Male Syt VII^+/− mice were bred with female FoxP3^+/− mice to produce male wild-type, Syt VII mutant (Syt VII^−/−), FoxP3 mutant (FoxP3^−/Y), and double-mutant (Foxp3^−/Y/Syt VII^−/−) mice. A, Hematoxylin and eosin–stained muscle sections obtained from mice killed at 2 weeks or 4 weeks (±2 days). Original magnification × 200. B, Extent of myositis in Foxp3^−/Y/Syt VII^+/− and Foxp3^−/Y/Syt VII^−/− mice at the 4-week time point. Each symbol represents an individual mouse. Bars show the mean ± SD. C, Immunohistochemical staining for CD3 (T cell marker), F4/80 (macrophage cell marker), and B220 (B cell marker) in muscle sections obtained from FoxP3/Syt VII double-mutant mice at the 2-week and 4-week time points. Original magnification × 400. Results are representative of observations made in at least 3 mice at each time point and were independently verified in subsequent experiments. ∗ = P ≤ 0.005 versus Syt VII^+/−.

Figure 3

Intraperitoneal (IP) adoptive transfer of FoxP3/synaptotagmin VII (Syt VII) double-mutant mouse lymph node cells induces robust myositis in recombination-activating gene 1 (RAG-1)–null recipients. FoxP3-deficient (FoxP3^−/Y) lymph node preparations from mice with either a heterozygous or mutant Syt VII genotype were made at the time the mice were killed, and transferred by intraperitoneal (IP) injection into RAG-1–null mice. A, Tissue inflammation in muscle sections obtained 4 weeks after adoptive transfer of FoxP3^−/Y/Syt VII^+/− mouse cells or FoxP3^−/Y/Syt VII^−/− mouse cells. Hematoxylin and eosin (H&E) stained; original magnification × 80 (left) and × 200 (right). B, Histopathologic scores of the H&E-stained sections, as determined by blinded analysis. Each symbol represents an individual mouse. Bars show the mean ± SD. C, Immunohistochemical staining for CD4, CD8, B220, and F4/80 in muscle sections obtained from FoxP3^−/Y/Syt VII^−/− mice 4 weeks after adoptive transfer. All images are representative of trends observed in 6 mice (n = 2 individual experiments). Original magnification × 200. D, Results of digital image analysis showing the pixel intensity values for CD4 T cells, CD8 T cells, F4/80 macrophages, and B220 B cells. Values are the mean ± SD. ∗ = P ≤ 0.001 versus Syt VII^+/− (B) and versus all other cell subtype markers (D).

Figure 4

Myositis is induced in RAG-1–null mouse recipients of FoxP3-deficient mouse lymph node cells exposed to muscle antigen. Lymph nodes from FoxP3-deficient mice were prepared and transferred by intraperitoneal injection, intramuscular (IM) injection, intraperitoneal coinjection with muscle tissue homogenate, or intraperitoneal coinjection with purified myosin protein. A, H&E-stained muscle tissue sections obtained from mice in the 4 treatment groups 4 weeks after adoptive transfer. Original magnification × 200. B, Results of digital image analysis showing the pixel intensity values for the inflammatory response induced by intraperitoneal and intramuscular injections. Values are the mean ± SD. C, Histopathologic scores of the H&E-stained muscle tissue sections, as determined by blinded analysis. Each symbol represents an individual mouse. Bars show the mean ± SD. Results are representative of trends observed in at least 3 mice (n = 2 individual experiments). ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.005 versus intraperitoneal injection. See Figure 3 for other definitions.

Figure 5

Inflammatory responses in myositis induced by exposure to muscle protein are predominated by CD4+ cells. Preparations of lymph node cells from FoxP3-deficient mice were adoptively transferred into recombination-activating gene 1–null recipients by intramuscular (IM) or intraperitoneal (IP) injection, with or without coadministration of muscle homogenate or purified myosin protein. A, Expression of CD4, CD8, F4/80, and B220 within inflammatory infiltrates, as determined by immunohistochemistry. Original magnification × 200. B, Results of digital image analysis showing the pixel intensity values in areas of infiltration for each leukocyte marker. Results are representative of similar trends observed in at least 5 mice (n = 4 independent experiments). Values are the mean ± SD. ∗ = P < 0.05; ∗∗ = P < 0.0001 versus all other markers in the same experimental group.

Figure 6

Treg cells suppress immune-mediated muscle tissue inflammation. CD4+CD25+ Treg cells from wild-type C57BL/6 mice were isolated by immunomagnetic sorting, and the purity was confirmed by flow cytometry to be >80%. A and B, Representative hematoxylin and eosin–stained paraffin-embedded muscle tissue sections from recombination-activating gene 1–null mice that received intramuscular injections of lymph node cells from Foxp3-deficient mice (A) or intraperitoneal injections of lymph node cells from Foxp3/Syt VII double-mutant mice (B), either with or without CD4+CD25+ Treg cells. Original magnification × 200. C, Extent of inflammation induced by adoptive transfer, with and without Treg cell supplementation, as assessed quantitatively by digital image analysis and semiquantitatively by histopathologic scoring. Bars show the mean ± SD; values above the bars are the mean ± SD histopathologic scores (n = 3 independent experiments). ∗ = P < 0.001 versus no Treg cell supplementation.