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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−/− related tissue inflammation is dependent on the IL-23–IL-17 pathway. (A) Ear thickness after Oxazolone challenge. Ro52−/−p19+/+, n = 5; Ro52−/−p19−/−, n = 5; p19−/−Ro52+/+, n = 3; Ro52+/+p19+/+, n = 3 (ANOVA mixed model). (B) Microphotographs of Oxazolone-treated ears. Ears of all mice in A were analyzed. Original magnification, 10×. Bars, 100 µm. (C) Flow cytometry analysis of draining lymph node cells of Oxazolone-treated mice (Ro52−/−p19+/+, n = 5; Ro52−/−p19−/−, n = 5). (D) Cytokine expression in cells from draining lymph nodes ex vivo measured by RT-PCR with expression relative to HPRT (Ro52−/−p19+/+, n = 5; Ro52−/−p19−/−, n = 5; Ro52+/+p19+/+, n = 3). Data in A–D depicts one out of two representative experiments. Error bars represent SEM. (E) Mice without dermatitis in Ro52−/−p19+/+ compared with Ro52−/−p19−/− mice. Number of mice observed at different ages (Ro52−/−p19+/+; Ro52−/−p19−/−): 5 wk (11; 21), 10 wk (11; 18), 15 wk (11; 14), 20 wk (11; 6), 25 wk (11; 5), and 30 wk (11; 4; Fischer's exact test). (F) Ear with tag in Ro52−/−p19−/− mouse showing no inflammation or dermatitis. Data are representative of mice in E. Metal tag length, 1 cm. (G) ANA analysis tested by immunofluorescence of Hep-2 cells and summarized as the percent positive in each group, and anti-DNA antibody levels measured by ELISA (triplicates) in Ro52−/− p19+/+ (n = 7), Ro52−/−p19−/− (n = 10), and Ro52+/+p19+/+ (n = 7; mean ± SEM; Mann-Whitney U test). Bar, 50 µm. (H) Electron microscopy image of kidneys depicting reversal of pathological changes in Ro52−/−p19−/− mice (arrows). All mice in A were analyzed. Bar, 2 µm.
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fig5: Ro52−/− related tissue inflammation is dependent on the IL-23–IL-17 pathway. (A) Ear thickness after Oxazolone challenge. Ro52−/−p19+/+, n = 5; Ro52−/−p19−/−, n = 5; p19−/−Ro52+/+, n = 3; Ro52+/+p19+/+, n = 3 (ANOVA mixed model). (B) Microphotographs of Oxazolone-treated ears. Ears of all mice in A were analyzed. Original magnification, 10×. Bars, 100 µm. (C) Flow cytometry analysis of draining lymph node cells of Oxazolone-treated mice (Ro52−/−p19+/+, n = 5; Ro52−/−p19−/−, n = 5). (D) Cytokine expression in cells from draining lymph nodes ex vivo measured by RT-PCR with expression relative to HPRT (Ro52−/−p19+/+, n = 5; Ro52−/−p19−/−, n = 5; Ro52+/+p19+/+, n = 3). Data in A–D depicts one out of two representative experiments. Error bars represent SEM. (E) Mice without dermatitis in Ro52−/−p19+/+ compared with Ro52−/−p19−/− mice. Number of mice observed at different ages (Ro52−/−p19+/+; Ro52−/−p19−/−): 5 wk (11; 21), 10 wk (11; 18), 15 wk (11; 14), 20 wk (11; 6), 25 wk (11; 5), and 30 wk (11; 4; Fischer's exact test). (F) Ear with tag in Ro52−/−p19−/− mouse showing no inflammation or dermatitis. Data are representative of mice in E. Metal tag length, 1 cm. (G) ANA analysis tested by immunofluorescence of Hep-2 cells and summarized as the percent positive in each group, and anti-DNA antibody levels measured by ELISA (triplicates) in Ro52−/− p19+/+ (n = 7), Ro52−/−p19−/− (n = 10), and Ro52+/+p19+/+ (n = 7; mean ± SEM; Mann-Whitney U test). Bar, 50 µm. (H) Electron microscopy image of kidneys depicting reversal of pathological changes in Ro52−/−p19−/− mice (arrows). All mice in A were analyzed. Bar, 2 µm.

Mentions: Ro52−/− mice developing dermatitis and systemic autoimmunity produced IL-6 and IL-12/IL-23p40 and had increased levels of Th17 cells in the draining lymph nodes. IL-6 and IL-23 are needed for differentiation and maturation of Th17 cells (Cua et al., 2003; Bettelli et al., 2006), and the IL-23–IL-17 axis appears to be hyperactive in lupus and Sjögren's syndrome (Crispin et al., 2008; Nguyen et al., 2008). We therefore hypothesized that genetic disruption of the IL-23–Th17 pathway rather than the IL-12–IFN-γ pathway would result in inhibition of the tissue inflammation and systemic autoimmunity in Ro52−/− mice. To address this hypothesis, we crossed Ro52−/− mice with IL-23p19−/− mice to disrupt the IL-23–Th17 pathway. The increased hypersensitivity with dermatitis and inflammatory infiltrates observed in Ro52−/− mice after Oxazolone application was completely reversed in the Ro52−/− mice lacking IL-23p19 chains (Fig. 5, A and B). The enhanced T cell activation observed in Ro52−/− mice was also reversed in the double knockout mice (Fig. 5 C). In addition, the increased expression of IL-23R, IL-12/IL-23p40, IL-17, and RoRγt in the draining lymph nodes of Ro52−/− mice was reversed to baseline levels in the Ro52−/−p19−/− mice (Fig. 5 D). Furthermore, the skin inflammation after ear tagging in the Ro52−/− mice did not develop in ear-tagged Ro52−/−p19−/− in up to 6 mo (30 wk) of observation (Fig. 5, E and F). The Ro52−/−p19−/− mice did not develop ANA or anti-DNA antibodies and did not develop kidney pathology as did the ear-tagged Ro52−/− mice (Fig. 5, G and H). Together, these data demonstrate that abrogating the IL-23–Th17 pathway abolishes tissue inflammation and systemic autoimmunity observed in the Ro52-deficient mice after ear tagging and Oxazolone treatment.


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−/− related tissue inflammation is dependent on the IL-23–IL-17 pathway. (A) Ear thickness after Oxazolone challenge. Ro52−/−p19+/+, n = 5; Ro52−/−p19−/−, n = 5; p19−/−Ro52+/+, n = 3; Ro52+/+p19+/+, n = 3 (ANOVA mixed model). (B) Microphotographs of Oxazolone-treated ears. Ears of all mice in A were analyzed. Original magnification, 10×. Bars, 100 µm. (C) Flow cytometry analysis of draining lymph node cells of Oxazolone-treated mice (Ro52−/−p19+/+, n = 5; Ro52−/−p19−/−, n = 5). (D) Cytokine expression in cells from draining lymph nodes ex vivo measured by RT-PCR with expression relative to HPRT (Ro52−/−p19+/+, n = 5; Ro52−/−p19−/−, n = 5; Ro52+/+p19+/+, n = 3). Data in A–D depicts one out of two representative experiments. Error bars represent SEM. (E) Mice without dermatitis in Ro52−/−p19+/+ compared with Ro52−/−p19−/− mice. Number of mice observed at different ages (Ro52−/−p19+/+; Ro52−/−p19−/−): 5 wk (11; 21), 10 wk (11; 18), 15 wk (11; 14), 20 wk (11; 6), 25 wk (11; 5), and 30 wk (11; 4; Fischer's exact test). (F) Ear with tag in Ro52−/−p19−/− mouse showing no inflammation or dermatitis. Data are representative of mice in E. Metal tag length, 1 cm. (G) ANA analysis tested by immunofluorescence of Hep-2 cells and summarized as the percent positive in each group, and anti-DNA antibody levels measured by ELISA (triplicates) in Ro52−/− p19+/+ (n = 7), Ro52−/−p19−/− (n = 10), and Ro52+/+p19+/+ (n = 7; mean ± SEM; Mann-Whitney U test). Bar, 50 µm. (H) Electron microscopy image of kidneys depicting reversal of pathological changes in Ro52−/−p19−/− mice (arrows). All mice in A were analyzed. Bar, 2 µm.
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fig5: Ro52−/− related tissue inflammation is dependent on the IL-23–IL-17 pathway. (A) Ear thickness after Oxazolone challenge. Ro52−/−p19+/+, n = 5; Ro52−/−p19−/−, n = 5; p19−/−Ro52+/+, n = 3; Ro52+/+p19+/+, n = 3 (ANOVA mixed model). (B) Microphotographs of Oxazolone-treated ears. Ears of all mice in A were analyzed. Original magnification, 10×. Bars, 100 µm. (C) Flow cytometry analysis of draining lymph node cells of Oxazolone-treated mice (Ro52−/−p19+/+, n = 5; Ro52−/−p19−/−, n = 5). (D) Cytokine expression in cells from draining lymph nodes ex vivo measured by RT-PCR with expression relative to HPRT (Ro52−/−p19+/+, n = 5; Ro52−/−p19−/−, n = 5; Ro52+/+p19+/+, n = 3). Data in A–D depicts one out of two representative experiments. Error bars represent SEM. (E) Mice without dermatitis in Ro52−/−p19+/+ compared with Ro52−/−p19−/− mice. Number of mice observed at different ages (Ro52−/−p19+/+; Ro52−/−p19−/−): 5 wk (11; 21), 10 wk (11; 18), 15 wk (11; 14), 20 wk (11; 6), 25 wk (11; 5), and 30 wk (11; 4; Fischer's exact test). (F) Ear with tag in Ro52−/−p19−/− mouse showing no inflammation or dermatitis. Data are representative of mice in E. Metal tag length, 1 cm. (G) ANA analysis tested by immunofluorescence of Hep-2 cells and summarized as the percent positive in each group, and anti-DNA antibody levels measured by ELISA (triplicates) in Ro52−/− p19+/+ (n = 7), Ro52−/−p19−/− (n = 10), and Ro52+/+p19+/+ (n = 7; mean ± SEM; Mann-Whitney U test). Bar, 50 µm. (H) Electron microscopy image of kidneys depicting reversal of pathological changes in Ro52−/−p19−/− mice (arrows). All mice in A were analyzed. Bar, 2 µm.
Mentions: Ro52−/− mice developing dermatitis and systemic autoimmunity produced IL-6 and IL-12/IL-23p40 and had increased levels of Th17 cells in the draining lymph nodes. IL-6 and IL-23 are needed for differentiation and maturation of Th17 cells (Cua et al., 2003; Bettelli et al., 2006), and the IL-23–IL-17 axis appears to be hyperactive in lupus and Sjögren's syndrome (Crispin et al., 2008; Nguyen et al., 2008). We therefore hypothesized that genetic disruption of the IL-23–Th17 pathway rather than the IL-12–IFN-γ pathway would result in inhibition of the tissue inflammation and systemic autoimmunity in Ro52−/− mice. To address this hypothesis, we crossed Ro52−/− mice with IL-23p19−/− mice to disrupt the IL-23–Th17 pathway. The increased hypersensitivity with dermatitis and inflammatory infiltrates observed in Ro52−/− mice after Oxazolone application was completely reversed in the Ro52−/− mice lacking IL-23p19 chains (Fig. 5, A and B). The enhanced T cell activation observed in Ro52−/− mice was also reversed in the double knockout mice (Fig. 5 C). In addition, the increased expression of IL-23R, IL-12/IL-23p40, IL-17, and RoRγt in the draining lymph nodes of Ro52−/− mice was reversed to baseline levels in the Ro52−/−p19−/− mice (Fig. 5 D). Furthermore, the skin inflammation after ear tagging in the Ro52−/− mice did not develop in ear-tagged Ro52−/−p19−/− in up to 6 mo (30 wk) of observation (Fig. 5, E and F). The Ro52−/−p19−/− mice did not develop ANA or anti-DNA antibodies and did not develop kidney pathology as did the ear-tagged Ro52−/− mice (Fig. 5, G and H). Together, these data demonstrate that abrogating the IL-23–Th17 pathway abolishes tissue inflammation and systemic autoimmunity observed in the Ro52-deficient mice after ear tagging and Oxazolone treatment.

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