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Loss of the lupus autoantigen Ro52/Trim21 induces tissue inflammation and systemic autoimmunity by disregulating the IL-23-Th17 pathway.

Espinosa A, Dardalhon V, Brauner S, Ambrosi A, Higgs R, Quintana FJ, Sjöstrand M, Eloranta ML, Ní Gabhann J, Winqvist O, Sundelin B, Jefferies CA, Rozell B, Kuchroo VK, Wahren-Herlenius M - J. Exp. Med. (2009)

Bottom Line: Ro52/Trim21 is targeted as an autoantigen in systemic lupus erythematosus and Sjögren's syndrome.Ro52, which was recently identified as an E3 ligase, mediates ubiquitination of several members of the interferon regulatory factor (IRF) family, and the Ro52-deficient mice have an enhanced production of proinflammatory cytokines that are regulated by the IRF transcription factors, including cytokines involved in the Th17 pathway (interleukin [IL] 6, IL-12/IL-23p40, and IL-17).These data reveal that the lupus-associated Ro52 protein is an important negative regulator of proinflammatory cytokine production, and they provide a mechanism by which a defective Ro52 function can lead to tissue inflammation and systemic autoimmunity through the IL-23-Th17 pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Karolinska Institute, Stockholm SE-171 77, Sweden.

ABSTRACT
Ro52/Trim21 is targeted as an autoantigen in systemic lupus erythematosus and Sjögren's syndrome. Polymorphisms in the Ro52 gene have been linked to these autoimmune conditions, but the molecular mechanism by which Ro52 may promote development of systemic autoimmune diseases has not been explored. To address this issue, we generated Ro52- mice (Ro52(-/-)), which appear phenotypically normal if left unmanipulated. However, Ro52(-/-) mice develop severe dermatitis extending from the site of tissue injury induced by ear tags. The affected mice further develop several signs of systemic lupus with hypergammaglobulinemia, autoantibodies to DNA, proteinuria, and kidney pathology. Ro52, which was recently identified as an E3 ligase, mediates ubiquitination of several members of the interferon regulatory factor (IRF) family, and the Ro52-deficient mice have an enhanced production of proinflammatory cytokines that are regulated by the IRF transcription factors, including cytokines involved in the Th17 pathway (interleukin [IL] 6, IL-12/IL-23p40, and IL-17). Loss of IL-23/IL-17 by genetic deletion of IL-23/p19 in the Ro52(-/-) mice conferred protection from skin disease and systemic autoimmunity. These data reveal that the lupus-associated Ro52 protein is an important negative regulator of proinflammatory cytokine production, and they provide a mechanism by which a defective Ro52 function can lead to tissue inflammation and systemic autoimmunity through the IL-23-Th17 pathway.

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Ro52-deficient mice develop tissue inflammation and systemic autoimmunity. (A) Photographs of the progressive dermatitis developing spontaneously from tissue injury mediated by ear tags in Ro52-deficient mice. Metal tag length, 1 cm. (B) Histology with Toluidine blue staining of ears of tagged Ro52+/+ and Ro52−/− mice. Bars, 150 µm. A and B are representative of mice in C. (C) Incidence of dermatitis. 13 independent Ro52−/− mice born during a period of 1.5 yr and their Ro52+/+ littermates (n = 11) were followed for dermatitis development. Dermatitis developed at different ages but, eventually, in all tagged Ro52−/− mice, whereas none of their littermate controls developed the disease. (D) Serum immunoglobulin levels determined by ELISA in triplicates in Ro52−/− mice with dermatitis (n = 6) and Ro52+/+ mice (n = 12; mean± SEM; Mann-Whitney U test). Data represent one of two experimental repeats. (E) Ro52−/− mice with dermatitis develop ANA. Presence of ANA was tested by immunofluorescence of Hep-2 cells and summarized as the percent positive in each group, and anti-DNA antibody levels were measured by ELISA (triplicates) in ANA-positive mice with dermatitis Ro52−/− (n = 5) and Ro52+/+ (n = 16; Mean± SEM; Mann-Whitney U test). Data represent one of two experimental repeats. Bar, 75 µm. (F, top) Electron microscopy photographs of renal glomeruli with increased mesangium and immunoglobulin deposition in Ro52−/− mice indicated by arrows. Bar, 2 µm. (F, bottom) Immunofluorescence IgG staining of renal glomeruli visualizing deposited IgG (red) and proteinuria (g/liter) in mice with dermatitis Ro52−/− (n = 7) and age-matched Ro52+/+ mice (n = 8; mean± SEM; Mann-Whitney U test). Bar, 10 µm. (G) Increased cytokine expression in draining lymph node cells ex vivo from Ro52−/− mice with dermatitis compared with Ro52+/+ littermates. Cytokine expression was investigated by quantitative RT-PCR in triplicates and calculated relative to HPRT. Data are representative for five Ro52−/− and five Ro52+/+ mice analyzed in five independent experiments and each assay was performed at least twice. (H) Cytokine production in triplicate supernatants was measured by ELISA after culture of cells from draining lymph nodes with 1 µg/ml anti-CD3 (IFN-γ, IL-4, and IL-17) or 1 µg/ml LPS (IL-12/IL-23p40) for 48 h. Data are representative of one Ro52−/− mouse with dermatitis compared with one Ro52+/+ mouse in eight independent experiments. Assays were performed at least twice. (I) Cells producing IL-17 were determined by intracellular cytokine staining and flow cytometry of cells from draining lymph nodes after in vitro culture with anti-CD3 for 48 h followed by restimulation with PMA/ionomycin for 4 h. One out of three independent experiments is shown. Numbers in quadrants represent the percentage of cells in each.
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fig2: Ro52-deficient mice develop tissue inflammation and systemic autoimmunity. (A) Photographs of the progressive dermatitis developing spontaneously from tissue injury mediated by ear tags in Ro52-deficient mice. Metal tag length, 1 cm. (B) Histology with Toluidine blue staining of ears of tagged Ro52+/+ and Ro52−/− mice. Bars, 150 µm. A and B are representative of mice in C. (C) Incidence of dermatitis. 13 independent Ro52−/− mice born during a period of 1.5 yr and their Ro52+/+ littermates (n = 11) were followed for dermatitis development. Dermatitis developed at different ages but, eventually, in all tagged Ro52−/− mice, whereas none of their littermate controls developed the disease. (D) Serum immunoglobulin levels determined by ELISA in triplicates in Ro52−/− mice with dermatitis (n = 6) and Ro52+/+ mice (n = 12; mean± SEM; Mann-Whitney U test). Data represent one of two experimental repeats. (E) Ro52−/− mice with dermatitis develop ANA. Presence of ANA was tested by immunofluorescence of Hep-2 cells and summarized as the percent positive in each group, and anti-DNA antibody levels were measured by ELISA (triplicates) in ANA-positive mice with dermatitis Ro52−/− (n = 5) and Ro52+/+ (n = 16; Mean± SEM; Mann-Whitney U test). Data represent one of two experimental repeats. Bar, 75 µm. (F, top) Electron microscopy photographs of renal glomeruli with increased mesangium and immunoglobulin deposition in Ro52−/− mice indicated by arrows. Bar, 2 µm. (F, bottom) Immunofluorescence IgG staining of renal glomeruli visualizing deposited IgG (red) and proteinuria (g/liter) in mice with dermatitis Ro52−/− (n = 7) and age-matched Ro52+/+ mice (n = 8; mean± SEM; Mann-Whitney U test). Bar, 10 µm. (G) Increased cytokine expression in draining lymph node cells ex vivo from Ro52−/− mice with dermatitis compared with Ro52+/+ littermates. Cytokine expression was investigated by quantitative RT-PCR in triplicates and calculated relative to HPRT. Data are representative for five Ro52−/− and five Ro52+/+ mice analyzed in five independent experiments and each assay was performed at least twice. (H) Cytokine production in triplicate supernatants was measured by ELISA after culture of cells from draining lymph nodes with 1 µg/ml anti-CD3 (IFN-γ, IL-4, and IL-17) or 1 µg/ml LPS (IL-12/IL-23p40) for 48 h. Data are representative of one Ro52−/− mouse with dermatitis compared with one Ro52+/+ mouse in eight independent experiments. Assays were performed at least twice. (I) Cells producing IL-17 were determined by intracellular cytokine staining and flow cytometry of cells from draining lymph nodes after in vitro culture with anti-CD3 for 48 h followed by restimulation with PMA/ionomycin for 4 h. One out of three independent experiments is shown. Numbers in quadrants represent the percentage of cells in each.

Mentions: Individual mice are routinely identified either by ear punching or by metallic ear tags. After ear tagging with metal clips, no visible tissue reaction, other than transient erythema and swelling, is normally observed. However, 25 wk after application of metal ear tags, >90% of Ro52−/− mice had developed a very severe and progressive dermatitis emanating from the tagged ear, which did not occur in Ro52+/+ WT littermates (Fig. 2, A-C). Dermatitis also developed in Ro52+/− mice but was not present in Ro52-deficient mice that had lost their ear tag. Histological analysis of the skin lesions showed epidermal hyperplasia with ulcerations and inflammatory infiltrates consisting primarily of neutrophils (Fig. 2 B). Serum immunoglobulin analysis revealed increased IgG levels in Ro52−/− mice with dermatitis (Fig. 2 D), and the affected mice also developed antinuclear antibodies (ANAs), with specificity for DNA, and autoantibodies to several lupus-associated autoantigens and other autoantigens (Fig. 2 E and Fig. S4). Furthermore, affected mice developed kidney pathology with substantiated mesangium and intraglomerular immunoglobulin depositions, as well as proteinuria, showing impaired renal function (Fig. 2 F). The systemic nature of the inflammation was further supported by the development of splenomegaly and lymphadenopathy in affected mice (Fig. S4 and not depicted). Ro52−/− mice that were not ear tagged did not develop either dermatitis or systemic autoimmunity when observed up to 40 wk of age, suggesting that initial tissue injury induced by physical ear tagging results in uncontrolled tissue inflammation in Ro52−/− mice and, subsequently, leads to development of systemic autoimmunity.


Loss of the lupus autoantigen Ro52/Trim21 induces tissue inflammation and systemic autoimmunity by disregulating the IL-23-Th17 pathway.

Espinosa A, Dardalhon V, Brauner S, Ambrosi A, Higgs R, Quintana FJ, Sjöstrand M, Eloranta ML, Ní Gabhann J, Winqvist O, Sundelin B, Jefferies CA, Rozell B, Kuchroo VK, Wahren-Herlenius M - J. Exp. Med. (2009)

Ro52-deficient mice develop tissue inflammation and systemic autoimmunity. (A) Photographs of the progressive dermatitis developing spontaneously from tissue injury mediated by ear tags in Ro52-deficient mice. Metal tag length, 1 cm. (B) Histology with Toluidine blue staining of ears of tagged Ro52+/+ and Ro52−/− mice. Bars, 150 µm. A and B are representative of mice in C. (C) Incidence of dermatitis. 13 independent Ro52−/− mice born during a period of 1.5 yr and their Ro52+/+ littermates (n = 11) were followed for dermatitis development. Dermatitis developed at different ages but, eventually, in all tagged Ro52−/− mice, whereas none of their littermate controls developed the disease. (D) Serum immunoglobulin levels determined by ELISA in triplicates in Ro52−/− mice with dermatitis (n = 6) and Ro52+/+ mice (n = 12; mean± SEM; Mann-Whitney U test). Data represent one of two experimental repeats. (E) Ro52−/− mice with dermatitis develop ANA. Presence of ANA was tested by immunofluorescence of Hep-2 cells and summarized as the percent positive in each group, and anti-DNA antibody levels were measured by ELISA (triplicates) in ANA-positive mice with dermatitis Ro52−/− (n = 5) and Ro52+/+ (n = 16; Mean± SEM; Mann-Whitney U test). Data represent one of two experimental repeats. Bar, 75 µm. (F, top) Electron microscopy photographs of renal glomeruli with increased mesangium and immunoglobulin deposition in Ro52−/− mice indicated by arrows. Bar, 2 µm. (F, bottom) Immunofluorescence IgG staining of renal glomeruli visualizing deposited IgG (red) and proteinuria (g/liter) in mice with dermatitis Ro52−/− (n = 7) and age-matched Ro52+/+ mice (n = 8; mean± SEM; Mann-Whitney U test). Bar, 10 µm. (G) Increased cytokine expression in draining lymph node cells ex vivo from Ro52−/− mice with dermatitis compared with Ro52+/+ littermates. Cytokine expression was investigated by quantitative RT-PCR in triplicates and calculated relative to HPRT. Data are representative for five Ro52−/− and five Ro52+/+ mice analyzed in five independent experiments and each assay was performed at least twice. (H) Cytokine production in triplicate supernatants was measured by ELISA after culture of cells from draining lymph nodes with 1 µg/ml anti-CD3 (IFN-γ, IL-4, and IL-17) or 1 µg/ml LPS (IL-12/IL-23p40) for 48 h. Data are representative of one Ro52−/− mouse with dermatitis compared with one Ro52+/+ mouse in eight independent experiments. Assays were performed at least twice. (I) Cells producing IL-17 were determined by intracellular cytokine staining and flow cytometry of cells from draining lymph nodes after in vitro culture with anti-CD3 for 48 h followed by restimulation with PMA/ionomycin for 4 h. One out of three independent experiments is shown. Numbers in quadrants represent the percentage of cells in each.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2722164&req=5

fig2: Ro52-deficient mice develop tissue inflammation and systemic autoimmunity. (A) Photographs of the progressive dermatitis developing spontaneously from tissue injury mediated by ear tags in Ro52-deficient mice. Metal tag length, 1 cm. (B) Histology with Toluidine blue staining of ears of tagged Ro52+/+ and Ro52−/− mice. Bars, 150 µm. A and B are representative of mice in C. (C) Incidence of dermatitis. 13 independent Ro52−/− mice born during a period of 1.5 yr and their Ro52+/+ littermates (n = 11) were followed for dermatitis development. Dermatitis developed at different ages but, eventually, in all tagged Ro52−/− mice, whereas none of their littermate controls developed the disease. (D) Serum immunoglobulin levels determined by ELISA in triplicates in Ro52−/− mice with dermatitis (n = 6) and Ro52+/+ mice (n = 12; mean± SEM; Mann-Whitney U test). Data represent one of two experimental repeats. (E) Ro52−/− mice with dermatitis develop ANA. Presence of ANA was tested by immunofluorescence of Hep-2 cells and summarized as the percent positive in each group, and anti-DNA antibody levels were measured by ELISA (triplicates) in ANA-positive mice with dermatitis Ro52−/− (n = 5) and Ro52+/+ (n = 16; Mean± SEM; Mann-Whitney U test). Data represent one of two experimental repeats. Bar, 75 µm. (F, top) Electron microscopy photographs of renal glomeruli with increased mesangium and immunoglobulin deposition in Ro52−/− mice indicated by arrows. Bar, 2 µm. (F, bottom) Immunofluorescence IgG staining of renal glomeruli visualizing deposited IgG (red) and proteinuria (g/liter) in mice with dermatitis Ro52−/− (n = 7) and age-matched Ro52+/+ mice (n = 8; mean± SEM; Mann-Whitney U test). Bar, 10 µm. (G) Increased cytokine expression in draining lymph node cells ex vivo from Ro52−/− mice with dermatitis compared with Ro52+/+ littermates. Cytokine expression was investigated by quantitative RT-PCR in triplicates and calculated relative to HPRT. Data are representative for five Ro52−/− and five Ro52+/+ mice analyzed in five independent experiments and each assay was performed at least twice. (H) Cytokine production in triplicate supernatants was measured by ELISA after culture of cells from draining lymph nodes with 1 µg/ml anti-CD3 (IFN-γ, IL-4, and IL-17) or 1 µg/ml LPS (IL-12/IL-23p40) for 48 h. Data are representative of one Ro52−/− mouse with dermatitis compared with one Ro52+/+ mouse in eight independent experiments. Assays were performed at least twice. (I) Cells producing IL-17 were determined by intracellular cytokine staining and flow cytometry of cells from draining lymph nodes after in vitro culture with anti-CD3 for 48 h followed by restimulation with PMA/ionomycin for 4 h. One out of three independent experiments is shown. Numbers in quadrants represent the percentage of cells in each.
Mentions: Individual mice are routinely identified either by ear punching or by metallic ear tags. After ear tagging with metal clips, no visible tissue reaction, other than transient erythema and swelling, is normally observed. However, 25 wk after application of metal ear tags, >90% of Ro52−/− mice had developed a very severe and progressive dermatitis emanating from the tagged ear, which did not occur in Ro52+/+ WT littermates (Fig. 2, A-C). Dermatitis also developed in Ro52+/− mice but was not present in Ro52-deficient mice that had lost their ear tag. Histological analysis of the skin lesions showed epidermal hyperplasia with ulcerations and inflammatory infiltrates consisting primarily of neutrophils (Fig. 2 B). Serum immunoglobulin analysis revealed increased IgG levels in Ro52−/− mice with dermatitis (Fig. 2 D), and the affected mice also developed antinuclear antibodies (ANAs), with specificity for DNA, and autoantibodies to several lupus-associated autoantigens and other autoantigens (Fig. 2 E and Fig. S4). Furthermore, affected mice developed kidney pathology with substantiated mesangium and intraglomerular immunoglobulin depositions, as well as proteinuria, showing impaired renal function (Fig. 2 F). The systemic nature of the inflammation was further supported by the development of splenomegaly and lymphadenopathy in affected mice (Fig. S4 and not depicted). Ro52−/− mice that were not ear tagged did not develop either dermatitis or systemic autoimmunity when observed up to 40 wk of age, suggesting that initial tissue injury induced by physical ear tagging results in uncontrolled tissue inflammation in Ro52−/− mice and, subsequently, leads to development of systemic autoimmunity.

Bottom Line: Ro52/Trim21 is targeted as an autoantigen in systemic lupus erythematosus and Sjögren's syndrome.Ro52, which was recently identified as an E3 ligase, mediates ubiquitination of several members of the interferon regulatory factor (IRF) family, and the Ro52-deficient mice have an enhanced production of proinflammatory cytokines that are regulated by the IRF transcription factors, including cytokines involved in the Th17 pathway (interleukin [IL] 6, IL-12/IL-23p40, and IL-17).These data reveal that the lupus-associated Ro52 protein is an important negative regulator of proinflammatory cytokine production, and they provide a mechanism by which a defective Ro52 function can lead to tissue inflammation and systemic autoimmunity through the IL-23-Th17 pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Karolinska Institute, Stockholm SE-171 77, Sweden.

ABSTRACT
Ro52/Trim21 is targeted as an autoantigen in systemic lupus erythematosus and Sjögren's syndrome. Polymorphisms in the Ro52 gene have been linked to these autoimmune conditions, but the molecular mechanism by which Ro52 may promote development of systemic autoimmune diseases has not been explored. To address this issue, we generated Ro52- mice (Ro52(-/-)), which appear phenotypically normal if left unmanipulated. However, Ro52(-/-) mice develop severe dermatitis extending from the site of tissue injury induced by ear tags. The affected mice further develop several signs of systemic lupus with hypergammaglobulinemia, autoantibodies to DNA, proteinuria, and kidney pathology. Ro52, which was recently identified as an E3 ligase, mediates ubiquitination of several members of the interferon regulatory factor (IRF) family, and the Ro52-deficient mice have an enhanced production of proinflammatory cytokines that are regulated by the IRF transcription factors, including cytokines involved in the Th17 pathway (interleukin [IL] 6, IL-12/IL-23p40, and IL-17). Loss of IL-23/IL-17 by genetic deletion of IL-23/p19 in the Ro52(-/-) mice conferred protection from skin disease and systemic autoimmunity. These data reveal that the lupus-associated Ro52 protein is an important negative regulator of proinflammatory cytokine production, and they provide a mechanism by which a defective Ro52 function can lead to tissue inflammation and systemic autoimmunity through the IL-23-Th17 pathway.

Show MeSH
Related in: MedlinePlus