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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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Upstream and downstream components of the CARD9 signalling pathway.(a–c) Wild type (WT), Hck−/−Fgr−/−Lyn−/− (3 × SFK KO), Syk−/−, Plcg2−/− or Card9−/− neutrophils were placed on immobilized immune complex (IC) surfaces. Their superoxide release (a), MIP-2 levels (b) and LTB4 (c) release were determined. (d) WT and Card9−/− neutrophils were plated on IC surfaces for 10 min, the phosphorylation and the total amount of Syk in cell lysates was determined by western blot after immunoprecipitation. (e–h) Bcl10−/− and Malt1−/− neutrophils showed similar functional phenotypes on IC surfaces like Card9−/− neutrophils: they had intact superoxide release (e,g) and defective chemokine production (f,h). (i) WT and Card9−/− neutrophils were stimulated for 20 min; the phosphorylation and degradation of IκBα was followed by western blotting from whole cell lysates. (j) IC-activated WT and Card9−/− neutrophils were lysed after 20 min; the activation and nuclear translocation of NF-κB was determined by infrared dye-labelled NFκB consensus binding site probes. Kinetic curves in a,e and g show mean and s.e.m. of three to four independent experiments. Control data points were subtracted. Graphs in b,c,f and h represent data from two to four independent experiments. (d,i,j) Representative of two to three independent experiments. *P<0.05; NS, statistically not significant (two-way ANOVA); see the text for actual P values.
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f7: Upstream and downstream components of the CARD9 signalling pathway.(a–c) Wild type (WT), Hck−/−Fgr−/−Lyn−/− (3 × SFK KO), Syk−/−, Plcg2−/− or Card9−/− neutrophils were placed on immobilized immune complex (IC) surfaces. Their superoxide release (a), MIP-2 levels (b) and LTB4 (c) release were determined. (d) WT and Card9−/− neutrophils were plated on IC surfaces for 10 min, the phosphorylation and the total amount of Syk in cell lysates was determined by western blot after immunoprecipitation. (e–h) Bcl10−/− and Malt1−/− neutrophils showed similar functional phenotypes on IC surfaces like Card9−/− neutrophils: they had intact superoxide release (e,g) and defective chemokine production (f,h). (i) WT and Card9−/− neutrophils were stimulated for 20 min; the phosphorylation and degradation of IκBα was followed by western blotting from whole cell lysates. (j) IC-activated WT and Card9−/− neutrophils were lysed after 20 min; the activation and nuclear translocation of NF-κB was determined by infrared dye-labelled NFκB consensus binding site probes. Kinetic curves in a,e and g show mean and s.e.m. of three to four independent experiments. Control data points were subtracted. Graphs in b,c,f and h represent data from two to four independent experiments. (d,i,j) Representative of two to three independent experiments. *P<0.05; NS, statistically not significant (two-way ANOVA); see the text for actual P values.

Mentions: Having established a critical role for CARD9-mediated neutrophil gene expression changes and chemokine/cytokine release, we finally aimed to characterize the signalling components upstream and downstream of CARD9 in neutrophils. We and others have previously shown that myeloid Src-family kinases (Hck, Fgr and Lyn)32, Syk3137 and PLCγ2 (refs 29, 30) are required for immune complex-induced neutrophil functions and the K/B × N serum-transfer arthritis, supposedly by forming a receptor-proximal signalling cluster. We therefore compared immune complex-induced functional responses of neutrophils lacking those molecules with responses of Card9−/− cells. As shown in Fig. 7a, Hck−/−Fgr−/−Lyn−/− (3 × SFK KO), Syk−/− and Plcg2−/− neutrophils showed complete defect in immune complex-induced superoxide production (P values between 7.9 × 10−5 and 1.6 × 10−4; two-way ANOVA), whereas Card9−/− cell showed a normal response (P=0.73; two-way ANOVA). Similarly, as shown in Fig. 7b,c, while Hck−/−Fgr−/−Lyn−/−, Syk−/− and Plcg2−/− neutrophils showed complete defect in immune complex-induced MIP-2 and LTB4 release (P values between 2.9 × 10−4 and 9.4 × 10−3; two-way ANOVA), Card9−/− neutrophils displayed a strong but incomplete reduction of MIP-2 release (P=0.027; two-way ANOVA) and no significant reduction of LTB4 release (P=0.38; two-way ANOVA). The Card9−/− neutrophil phenotype was not due to defective receptor-proximal signalling since Card9−/− neutrophils showed normal Syk phosphorylation on activation by immobilized immune complexes (Fig. 7d). Those results suggest that CARD9 functions downstream of the receptor-proximal Src-family/Syk/PLCγ2 complex during immune complex-induced neutrophil activation.


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)

Upstream and downstream components of the CARD9 signalling pathway.(a–c) Wild type (WT), Hck−/−Fgr−/−Lyn−/− (3 × SFK KO), Syk−/−, Plcg2−/− or Card9−/− neutrophils were placed on immobilized immune complex (IC) surfaces. Their superoxide release (a), MIP-2 levels (b) and LTB4 (c) release were determined. (d) WT and Card9−/− neutrophils were plated on IC surfaces for 10 min, the phosphorylation and the total amount of Syk in cell lysates was determined by western blot after immunoprecipitation. (e–h) Bcl10−/− and Malt1−/− neutrophils showed similar functional phenotypes on IC surfaces like Card9−/− neutrophils: they had intact superoxide release (e,g) and defective chemokine production (f,h). (i) WT and Card9−/− neutrophils were stimulated for 20 min; the phosphorylation and degradation of IκBα was followed by western blotting from whole cell lysates. (j) IC-activated WT and Card9−/− neutrophils were lysed after 20 min; the activation and nuclear translocation of NF-κB was determined by infrared dye-labelled NFκB consensus binding site probes. Kinetic curves in a,e and g show mean and s.e.m. of three to four independent experiments. Control data points were subtracted. Graphs in b,c,f and h represent data from two to four independent experiments. (d,i,j) Representative of two to three independent experiments. *P<0.05; NS, statistically not significant (two-way ANOVA); see the text for actual P values.
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Related In: Results  -  Collection

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f7: Upstream and downstream components of the CARD9 signalling pathway.(a–c) Wild type (WT), Hck−/−Fgr−/−Lyn−/− (3 × SFK KO), Syk−/−, Plcg2−/− or Card9−/− neutrophils were placed on immobilized immune complex (IC) surfaces. Their superoxide release (a), MIP-2 levels (b) and LTB4 (c) release were determined. (d) WT and Card9−/− neutrophils were plated on IC surfaces for 10 min, the phosphorylation and the total amount of Syk in cell lysates was determined by western blot after immunoprecipitation. (e–h) Bcl10−/− and Malt1−/− neutrophils showed similar functional phenotypes on IC surfaces like Card9−/− neutrophils: they had intact superoxide release (e,g) and defective chemokine production (f,h). (i) WT and Card9−/− neutrophils were stimulated for 20 min; the phosphorylation and degradation of IκBα was followed by western blotting from whole cell lysates. (j) IC-activated WT and Card9−/− neutrophils were lysed after 20 min; the activation and nuclear translocation of NF-κB was determined by infrared dye-labelled NFκB consensus binding site probes. Kinetic curves in a,e and g show mean and s.e.m. of three to four independent experiments. Control data points were subtracted. Graphs in b,c,f and h represent data from two to four independent experiments. (d,i,j) Representative of two to three independent experiments. *P<0.05; NS, statistically not significant (two-way ANOVA); see the text for actual P values.
Mentions: Having established a critical role for CARD9-mediated neutrophil gene expression changes and chemokine/cytokine release, we finally aimed to characterize the signalling components upstream and downstream of CARD9 in neutrophils. We and others have previously shown that myeloid Src-family kinases (Hck, Fgr and Lyn)32, Syk3137 and PLCγ2 (refs 29, 30) are required for immune complex-induced neutrophil functions and the K/B × N serum-transfer arthritis, supposedly by forming a receptor-proximal signalling cluster. We therefore compared immune complex-induced functional responses of neutrophils lacking those molecules with responses of Card9−/− cells. As shown in Fig. 7a, Hck−/−Fgr−/−Lyn−/− (3 × SFK KO), Syk−/− and Plcg2−/− neutrophils showed complete defect in immune complex-induced superoxide production (P values between 7.9 × 10−5 and 1.6 × 10−4; two-way ANOVA), whereas Card9−/− cell showed a normal response (P=0.73; two-way ANOVA). Similarly, as shown in Fig. 7b,c, while Hck−/−Fgr−/−Lyn−/−, Syk−/− and Plcg2−/− neutrophils showed complete defect in immune complex-induced MIP-2 and LTB4 release (P values between 2.9 × 10−4 and 9.4 × 10−3; two-way ANOVA), Card9−/− neutrophils displayed a strong but incomplete reduction of MIP-2 release (P=0.027; two-way ANOVA) and no significant reduction of LTB4 release (P=0.38; two-way ANOVA). The Card9−/− neutrophil phenotype was not due to defective receptor-proximal signalling since Card9−/− neutrophils showed normal Syk phosphorylation on activation by immobilized immune complexes (Fig. 7d). Those results suggest that CARD9 functions downstream of the receptor-proximal Src-family/Syk/PLCγ2 complex during immune complex-induced neutrophil activation.

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