Scurfy Mice Develop Features of Connective Tissue Disease Overlap Syndrome and Mixed Connective Tissue Disease in the Absence of Regulatory T Cells

Due to a missense mutation in the Foxp3 gene, scurfy mice are deficient in functional regulatory T cells (Treg). The consequent loss of peripheral tolerance manifests itself by fatal autoimmune mediated multi-organ disease. Previous studies have outlined the systemic inflammatory disease and demonstrated production of anti-nuclear antibodies (ANA) in scurfy mice. However, specific autoantibody targets remained to be defined. ANA are immunological markers for several connective tissue diseases (CTD) and target a large number of intracellular molecules. Therefore, we examined scurfy sera for the presence of different ANA specificities and further assessed the organ involvement in these animals. Indirect immunofluorescence was used as a screen for ANA in the sera of scurfy mice and dilutions of 1/100 were considered positive. Addressable laser bead immunoassays (ALBIA) were used to detect specific autoantibody targets. Subsequent histological tissue evaluation was verified by hematoxylin and eosin (H&E) staining. In our study, we observed that nearly all scurfy mice produced ANA. The most prevalent pattern in scurfy sera was nuclear coarse speckled, also known as the AC-5 pattern according to the International Consensus on ANA Patterns. U1-ribonucleoprotein (U1RNP) was found to be the most common target antigen recognized by autoantibodies in scurfy mice. Additionally, scurfy mice exhibited a mild myositis with histological characteristics similar to polymyositis/dermatomyositis. Myopathy-specific autoantibody profile revealed significantly increased levels of anti-SMN (survival of motor neuron) as well as anti-Gemin3 antibodies in scurfy sera. Overall, we demonstrate that the impaired peripheral tolerance in the absence of regulatory T cells in scurfy mice is associated with features of mixed connective tissue disease (MCTD). This includes, along with our previous findings, very high titers of anti-U1RNP antibodies and an inflammatory myopathy

Regulatory T cell-deficient scurfy mice develop systemic autoimmune features resembling lupus-like disease

Introduction: Scurfy mice are deficient in regulatory T cells (Tregs), develop a severe, generalized autoimmune disorder that can affect almost every organ and die at an early age. Some of these manifestations resemble those found in systemic lupus erythematosus (SLE). In addition, active SLE is associated with low Treg numbers and reduced Treg function, but direct evidence for a central role of Treg malfunction in the pathophysiology of lupus-like manifestations is still missing. In the present study, we characterize the multiorgan pathology, autoantibody profile and blood count abnormalities in scurfy mice and show their close resemblances to lupus-like disease. Methods: Scurfy mice have dysfunctional Tregs due to a genetic defect in the transcription factor Forkhead box protein 3 (Foxp3). We analyzed skin, joints, lung and kidneys of scurfy mice and wild-type (WT) controls by conventional histology and immunofluorescence (IF) performed hematological workups and tested for autoantibodies by IF, immunoblotting and enzyme-linked immunosorbent assay. We also analyzed the intestines, liver, spleen and heart, but did not analyze all organs known to be affected in scurfy mice (such as the testicle, the accessory reproductive structures, the pancreas or the eyes). We transferred CD4+ T cells of scurfy or WT mice into T cell-deficient B6/nude mice. Results: We confirm previous reports that scurfy mice spontaneously develop severe pneumonitis and hematological abnormalities similar to those in SLE. We show that scurfy mice (but not controls) exhibited additional features of SLE: severe interface dermatitis, arthritis, mesangioproliferative glomerulonephritis and high titers of anti-nuclear antibodies, anti-double-stranded DNA antibodies, anti-histone antibodies and anti-Smith antibodies. Transfer of scurfy CD4+ T cells (but not of WT cells) induced autoantibodies and inflammation of lung, skin and kidneys in T cell-deficient B6/nude mice. Conclusion: Our observations support the hypothesis that lupus-like autoimmune features develop in the absence of functional Tregs

Regulatory T-cell deficiency leads to features of autoimmune liver disease overlap syndrome in scurfy mice

Scurfy mice have a complete deficiency of functional regulatory T cells (Treg) due to a frameshift mutation in the Foxp3 gene. The impaired immune homeostasis results in a lethal lymphoproliferative disorder affecting multiple organs, including the liver. The autoimmune pathology in scurfy mice is in part accompanied by autoantibodies such as antinuclear antibodies (ANA). ANA are serological hallmarks of several autoimmune disorders including autoimmune liver diseases (AILD). However, the underlying pathogenesis and the role of Treg in AILD remain to be elucidated. The present study therefore aimed to characterize the liver disease in scurfy mice

Regulatory T-cell deficiency leads to features of autoimmune liver disease overlap syndrome in scurfy mice

IntroductionScurfy mice have a complete deficiency of functional regulatory T cells (Treg) due to a frameshift mutation in the Foxp3 gene. The impaired immune homeostasis results in a lethal lymphoproliferative disorder affecting multiple organs, including the liver. The autoimmune pathology in scurfy mice is in part accompanied by autoantibodies such as antinuclear antibodies (ANA). ANA are serological hallmarks of several autoimmune disorders including autoimmune liver diseases (AILD). However, the underlying pathogenesis and the role of Treg in AILD remain to be elucidated. The present study therefore aimed to characterize the liver disease in scurfy mice.MethodsSera from scurfy mice were screened for ANA by indirect immunofluorescence assay (IFA) and tested for a wide range of AILD-associated autoantibodies by enzyme-linked immunosorbent assay, line immunoassay, and addressable laser bead immunoassay. CD4+ T cells of scurfy mice were transferred into T cell-deficient B6/nude mice. Monoclonal autoantibodies from scurfy mice and recipient B6/nude mice were tested for ANA by IFA. Liver tissue of scurfy mice was analyzed by conventional histology. Collagen deposition in scurfy liver was quantified via hepatic hydroxyproline content. Real-time quantitative PCR was used to determine fibrosis-related hepatic gene expression. Hepatic immune cells were differentiated by flow cytometry.ResultsAll scurfy mice produced ANA. AILD-associated autoantibodies, predominantly antimitochondrial antibodies, were detected at significantly higher levels in scurfy sera. CD4+ T cells from scurfy mice were sufficient to induce anti-dsDNA autoantibodies and ANA with an AILD-related nuclear envelope staining pattern. Liver histology revealed portal inflammation with bile duct damage and proliferation, as in primary biliary cholangitis (PBC), and interface hepatitis with portal-parenchymal necroinflammation, as found in autoimmune hepatitis (AIH). In scurfy liver, TNFα and fibrosis-related transcripts including Col1a1, Timp1, Acta2, Mmp2, and Mmp9 were upregulated. The level of proinflammatory monocytic macrophages (Ly-6Chi) was increased, while M2-type macrophages (CD206+) were downregulated compared to wildtype controls. Despite severe hepatic inflammation, fibrosis did not develop within 25 days, which is close to the lifespan of scurfy mice.DiscussionOur findings suggest that Treg-deficient scurfy mice spontaneously develop clinical, serological, and immunopathological characteristics of AILD with overlapping features of PBC and AIH

DataSheet_1_Regulatory T-cell deficiency leads to features of autoimmune liver disease overlap syndrome in scurfy mice.docx

IntroductionScurfy mice have a complete deficiency of functional regulatory T cells (Treg) due to a frameshift mutation in the Foxp3 gene. The impaired immune homeostasis results in a lethal lymphoproliferative disorder affecting multiple organs, including the liver. The autoimmune pathology in scurfy mice is in part accompanied by autoantibodies such as antinuclear antibodies (ANA). ANA are serological hallmarks of several autoimmune disorders including autoimmune liver diseases (AILD). However, the underlying pathogenesis and the role of Treg in AILD remain to be elucidated. The present study therefore aimed to characterize the liver disease in scurfy mice.MethodsSera from scurfy mice were screened for ANA by indirect immunofluorescence assay (IFA) and tested for a wide range of AILD-associated autoantibodies by enzyme-linked immunosorbent assay, line immunoassay, and addressable laser bead immunoassay. CD4+ T cells of scurfy mice were transferred into T cell-deficient B6/nude mice. Monoclonal autoantibodies from scurfy mice and recipient B6/nude mice were tested for ANA by IFA. Liver tissue of scurfy mice was analyzed by conventional histology. Collagen deposition in scurfy liver was quantified via hepatic hydroxyproline content. Real-time quantitative PCR was used to determine fibrosis-related hepatic gene expression. Hepatic immune cells were differentiated by flow cytometry.ResultsAll scurfy mice produced ANA. AILD-associated autoantibodies, predominantly antimitochondrial antibodies, were detected at significantly higher levels in scurfy sera. CD4+ T cells from scurfy mice were sufficient to induce anti-dsDNA autoantibodies and ANA with an AILD-related nuclear envelope staining pattern. Liver histology revealed portal inflammation with bile duct damage and proliferation, as in primary biliary cholangitis (PBC), and interface hepatitis with portal-parenchymal necroinflammation, as found in autoimmune hepatitis (AIH). In scurfy liver, TNFα and fibrosis-related transcripts including Col1a1, Timp1, Acta2, Mmp2, and Mmp9 were upregulated. The level of proinflammatory monocytic macrophages (Ly-6Chi) was increased, while M2-type macrophages (CD206+) were downregulated compared to wildtype controls. Despite severe hepatic inflammation, fibrosis did not develop within 25 days, which is close to the lifespan of scurfy mice.DiscussionOur findings suggest that Treg-deficient scurfy mice spontaneously develop clinical, serological, and immunopathological characteristics of AILD with overlapping features of PBC and AIH.</p