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Neutrophil-specific deletion of the CARD9 gene expression regulator suppresses autoantibody-induced inflammation in vivo.

Németh T, Futosi K, Sitaru C, Ruland J, Mócsai A - Nat Commun (2016)

Bottom Line: Neutrophil-specific deletion of CARD9 is sufficient to induce that phenotype.In vivo deletion of CARD9 reduces tissue levels of pro-inflammatory chemokines and cytokines but not LTB4.The CARD9-mediated signalling pathway involves Src-family kinases, Syk, PLCγ2, Bcl10/Malt1 and NFκB.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Semmelweis University School of Medicine, 1094 Budapest, Hungary.

ABSTRACT
Neutrophils are terminally differentiated cells with limited transcriptional activity. The biological function of their gene expression changes is poorly understood. CARD9 regulates transcription during antifungal immunity but its role in sterile inflammation is unclear. Here we show that neutrophil CARD9 mediates pro-inflammatory chemokine/cytokine but not lipid mediator release during non-infectious inflammation. Genetic deficiency of CARD9 suppresses autoantibody-induced arthritis and dermatitis in mice. Neutrophil-specific deletion of CARD9 is sufficient to induce that phenotype. Card9(-/-) neutrophils show defective immune complex-induced gene expression changes and pro-inflammatory chemokine/cytokine release but normal LTB4 production and other short-term responses. In vivo deletion of CARD9 reduces tissue levels of pro-inflammatory chemokines and cytokines but not LTB4. The CARD9-mediated signalling pathway involves Src-family kinases, Syk, PLCγ2, Bcl10/Malt1 and NFκB. Collectively, CARD9-mediated gene expression changes within neutrophils play important roles during non-infectious inflammation in vivo and CARD9 acts as a divergence point between chemokine/cytokine and lipid mediator release.

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CARD9 is important for the development of autoantibody-induced arthritis.Intact wild type (WT), Card9−/− (a–d) or Card9−/− (EUCOMM) (e,f) mice were injected with B × N (control) or K/B × N (arthritic) serum i.p. on day 0. Arthritis development was followed by photographing on day 7 (a), clinical scoring of the hind limbs (b,e), ankle-thickness measurement (c,f) and an articular function test (hanging on a wire grid; (d)). Images are representative of, and quantitative data show mean and s.e.m. from 8 to 9 control and 12 to 13 arthritic serum-treated individual mice per group from four independent experiments (a–d) or 5 to 6 control and 8 to 9 arthritic serum-treated mice per group from three independent experiments (e,f). (d) Results from functional test performed 12 times on each mouse between days 7–10. *P<0.05 (two-way ANOVA); see the text for actual P values.
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f1: CARD9 is important for the development of autoantibody-induced arthritis.Intact wild type (WT), Card9−/− (a–d) or Card9−/− (EUCOMM) (e,f) mice were injected with B × N (control) or K/B × N (arthritic) serum i.p. on day 0. Arthritis development was followed by photographing on day 7 (a), clinical scoring of the hind limbs (b,e), ankle-thickness measurement (c,f) and an articular function test (hanging on a wire grid; (d)). Images are representative of, and quantitative data show mean and s.e.m. from 8 to 9 control and 12 to 13 arthritic serum-treated individual mice per group from four independent experiments (a–d) or 5 to 6 control and 8 to 9 arthritic serum-treated mice per group from three independent experiments (e,f). (d) Results from functional test performed 12 times on each mouse between days 7–10. *P<0.05 (two-way ANOVA); see the text for actual P values.

Mentions: To test the role of CARD9 in non-infectious inflammation, we tested the effect of CARD9 deficiency on autoantibody-induced arthritis development in the K/B × N serum-transfer model30. We have used two independent CARD9-deficient mouse strains (Supplementary Fig. 1a) that were homozygous either for the conventional Card9tm1Jrld knockout mutation (referred to as Card9−/− mice) or for the so-called knockout-first33Card9tm1a(EUCOMM)Hmgu mutation (referred to as Card9−/− (EUCOMM) mice). CARD9 was absent from bone marrow (Supplementary Fig. 1b) and spleen (Supplementary Fig. 1c) cell lysates of both mutant strains. As indicated in Fig. 1a, Card9−/− mice showed substantially reduced signs of arthritis development. Quantification of the visible clinical signs (Fig. 1b; P=0.0028; two-way analysis of variance (ANOVA)), ankle thickening (Fig. 1c; P=0.0047; two-way ANOVA) and the ability of the mice to hold on to the bottom of a wire grid (Fig. 1d; P=2 × 10−4; two-way ANOVA) all confirmed highly significant protection from disease development. Similarly, Card9−/− (EUCOMM) mice were also strongly protected from visible signs of arthritis (Fig. 1e; P=6.6 × 10−4; two-way ANOVA) and ankle thickening (Fig. 1f; P=0.0038; two-way ANOVA). Therefore, CARD9 plays a major role in the development of autoantibody-induced arthritis.


Neutrophil-specific deletion of the CARD9 gene expression regulator suppresses autoantibody-induced inflammation in vivo.

Németh T, Futosi K, Sitaru C, Ruland J, Mócsai A - Nat Commun (2016)

CARD9 is important for the development of autoantibody-induced arthritis.Intact wild type (WT), Card9−/− (a–d) or Card9−/− (EUCOMM) (e,f) mice were injected with B × N (control) or K/B × N (arthritic) serum i.p. on day 0. Arthritis development was followed by photographing on day 7 (a), clinical scoring of the hind limbs (b,e), ankle-thickness measurement (c,f) and an articular function test (hanging on a wire grid; (d)). Images are representative of, and quantitative data show mean and s.e.m. from 8 to 9 control and 12 to 13 arthritic serum-treated individual mice per group from four independent experiments (a–d) or 5 to 6 control and 8 to 9 arthritic serum-treated mice per group from three independent experiments (e,f). (d) Results from functional test performed 12 times on each mouse between days 7–10. *P<0.05 (two-way ANOVA); see the text for actual P values.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4821996&req=5

f1: CARD9 is important for the development of autoantibody-induced arthritis.Intact wild type (WT), Card9−/− (a–d) or Card9−/− (EUCOMM) (e,f) mice were injected with B × N (control) or K/B × N (arthritic) serum i.p. on day 0. Arthritis development was followed by photographing on day 7 (a), clinical scoring of the hind limbs (b,e), ankle-thickness measurement (c,f) and an articular function test (hanging on a wire grid; (d)). Images are representative of, and quantitative data show mean and s.e.m. from 8 to 9 control and 12 to 13 arthritic serum-treated individual mice per group from four independent experiments (a–d) or 5 to 6 control and 8 to 9 arthritic serum-treated mice per group from three independent experiments (e,f). (d) Results from functional test performed 12 times on each mouse between days 7–10. *P<0.05 (two-way ANOVA); see the text for actual P values.
Mentions: To test the role of CARD9 in non-infectious inflammation, we tested the effect of CARD9 deficiency on autoantibody-induced arthritis development in the K/B × N serum-transfer model30. We have used two independent CARD9-deficient mouse strains (Supplementary Fig. 1a) that were homozygous either for the conventional Card9tm1Jrld knockout mutation (referred to as Card9−/− mice) or for the so-called knockout-first33Card9tm1a(EUCOMM)Hmgu mutation (referred to as Card9−/− (EUCOMM) mice). CARD9 was absent from bone marrow (Supplementary Fig. 1b) and spleen (Supplementary Fig. 1c) cell lysates of both mutant strains. As indicated in Fig. 1a, Card9−/− mice showed substantially reduced signs of arthritis development. Quantification of the visible clinical signs (Fig. 1b; P=0.0028; two-way analysis of variance (ANOVA)), ankle thickening (Fig. 1c; P=0.0047; two-way ANOVA) and the ability of the mice to hold on to the bottom of a wire grid (Fig. 1d; P=2 × 10−4; two-way ANOVA) all confirmed highly significant protection from disease development. Similarly, Card9−/− (EUCOMM) mice were also strongly protected from visible signs of arthritis (Fig. 1e; P=6.6 × 10−4; two-way ANOVA) and ankle thickening (Fig. 1f; P=0.0038; two-way ANOVA). Therefore, CARD9 plays a major role in the development of autoantibody-induced arthritis.

Bottom Line: Neutrophil-specific deletion of CARD9 is sufficient to induce that phenotype.In vivo deletion of CARD9 reduces tissue levels of pro-inflammatory chemokines and cytokines but not LTB4.The CARD9-mediated signalling pathway involves Src-family kinases, Syk, PLCγ2, Bcl10/Malt1 and NFκB.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Semmelweis University School of Medicine, 1094 Budapest, Hungary.

ABSTRACT
Neutrophils are terminally differentiated cells with limited transcriptional activity. The biological function of their gene expression changes is poorly understood. CARD9 regulates transcription during antifungal immunity but its role in sterile inflammation is unclear. Here we show that neutrophil CARD9 mediates pro-inflammatory chemokine/cytokine but not lipid mediator release during non-infectious inflammation. Genetic deficiency of CARD9 suppresses autoantibody-induced arthritis and dermatitis in mice. Neutrophil-specific deletion of CARD9 is sufficient to induce that phenotype. Card9(-/-) neutrophils show defective immune complex-induced gene expression changes and pro-inflammatory chemokine/cytokine release but normal LTB4 production and other short-term responses. In vivo deletion of CARD9 reduces tissue levels of pro-inflammatory chemokines and cytokines but not LTB4. The CARD9-mediated signalling pathway involves Src-family kinases, Syk, PLCγ2, Bcl10/Malt1 and NFκB. Collectively, CARD9-mediated gene expression changes within neutrophils play important roles during non-infectious inflammation in vivo and CARD9 acts as a divergence point between chemokine/cytokine and lipid mediator release.

Show MeSH
Related in: MedlinePlus