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Susceptibility to tuberculosis is associated with variants in the ASAP1 gene encoding a regulator of dendritic cell migration.

Curtis J, Luo Y, Zenner HL, Cuchet-Lourenço D, Wu C, Lo K, Maes M, Alisaac A, Stebbings E, Liu JZ, Kopanitsa L, Ignatyeva O, Balabanova Y, Nikolayevskyy V, Baessmann I, Thye T, Meyer CG, Nürnberg P, Horstmann RD, Drobniewski F, Plagnol V, Barrett JC, Nejentsev S - Nat. Genet. (2015)

Bottom Line: In the combined analysis of these subjects and the follow-up cohort (15,087 TB patients and controls altogether), we found an association between TB and variants located in introns of the ASAP1 gene on chromosome 8q24 (P = 2.6 × 10(-11) for rs4733781; P = 1.0 × 10(-10) for rs10956514).The ASAP1 protein is involved in actin and membrane remodeling and has been associated with podosomes.Therefore, genetically determined excessive reduction of ASAP1 expression in M. tuberculosis-infected DCs may lead to their impaired migration, suggesting a potential mechanism of predisposition to TB.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Cambridge, Cambridge, UK.

ABSTRACT
Human genetic factors predispose to tuberculosis (TB). We studied 7.6 million genetic variants in 5,530 people with pulmonary TB and in 5,607 healthy controls. In the combined analysis of these subjects and the follow-up cohort (15,087 TB patients and controls altogether), we found an association between TB and variants located in introns of the ASAP1 gene on chromosome 8q24 (P = 2.6 × 10(-11) for rs4733781; P = 1.0 × 10(-10) for rs10956514). Dendritic cells (DCs) showed high ASAP1 expression that was reduced after Mycobacterium tuberculosis infection, and rs10956514 was associated with the level of reduction of ASAP1 expression. The ASAP1 protein is involved in actin and membrane remodeling and has been associated with podosomes. The ASAP1-depleted DCs showed impaired matrix degradation and migration. Therefore, genetically determined excessive reduction of ASAP1 expression in M. tuberculosis-infected DCs may lead to their impaired migration, suggesting a potential mechanism of predisposition to TB.

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In DCs, the ASAP1 protein is partly associated with podosomes and its depletion leads to impaired matrix degradation and migration(a) Confocal microscopy showing ASAP1 (green), actin (red), vinculin (blue) and nuclei (white) in DCs derived from monocytes of a healthy volunteer. Podosomes are structures characterized by the actin core (visible as red dots) surrounded by other proteins. Boxed area is shown in grayscale enlarged on the right. Scale bar represents 7.5μm.(b) Representative confocal microscopy images (left panel) showing monocyte-derived DCs treated with either ASAP1 or control siRNA and seeded on FITC-conjugated gelatine (green) for 4 hours. The cells were stained to visualize actin (phalloidin-red) and nuclei (DAPI-blue). White arrows show examples of the DAPI-stained nuclei of cells that do not degrade matrix. Scale bar represents 50μm.In the same experiment cell lysates were collected for western blotting and probed with anti-ASAP1 and anti-actin antibodies (right panel) to assess knockdown efficiency.Area of degradation of the FITC-conjugated gelatine matrix by DCs (lower panel). In total 394 and 317 DCs treated with either ASAP1 or control siRNA, respectively, were counted in 4 independent experiments.(c) Migration velocity of DCs in a Dunn chamber. In total 162 and 186 DCs treated with either ASAP1 or control siRNA, respectively, were tracked in 5 independent experiments.Graphs show mean values ± SEM. P-values were calculated using two-tailed Student T test. Unadjusted P-values are shown.
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Figure 3: In DCs, the ASAP1 protein is partly associated with podosomes and its depletion leads to impaired matrix degradation and migration(a) Confocal microscopy showing ASAP1 (green), actin (red), vinculin (blue) and nuclei (white) in DCs derived from monocytes of a healthy volunteer. Podosomes are structures characterized by the actin core (visible as red dots) surrounded by other proteins. Boxed area is shown in grayscale enlarged on the right. Scale bar represents 7.5μm.(b) Representative confocal microscopy images (left panel) showing monocyte-derived DCs treated with either ASAP1 or control siRNA and seeded on FITC-conjugated gelatine (green) for 4 hours. The cells were stained to visualize actin (phalloidin-red) and nuclei (DAPI-blue). White arrows show examples of the DAPI-stained nuclei of cells that do not degrade matrix. Scale bar represents 50μm.In the same experiment cell lysates were collected for western blotting and probed with anti-ASAP1 and anti-actin antibodies (right panel) to assess knockdown efficiency.Area of degradation of the FITC-conjugated gelatine matrix by DCs (lower panel). In total 394 and 317 DCs treated with either ASAP1 or control siRNA, respectively, were counted in 4 independent experiments.(c) Migration velocity of DCs in a Dunn chamber. In total 162 and 186 DCs treated with either ASAP1 or control siRNA, respectively, were tracked in 5 independent experiments.Graphs show mean values ± SEM. P-values were calculated using two-tailed Student T test. Unadjusted P-values are shown.

Mentions: To investigate the role of the ASAP1 protein in DCs, we first studied its cellular localization. Confocal microscopy showed that the ASAP1 protein was localized in the cytoplasm and partly was associated with podosomes (Fig. 3a). Therefore, we hypothesized that reduced ASAP1 expression may affect matrix degradation by DCs and their migration. To test this, we isolated primary monocytes from healthy volunteers, derived DCs in vitro and transfected them with siRNA to reduce ASAP1 expression. We found that degradation of a gelatine matrix by the ASAP1-depleted DCs was impaired (Fig. 3b) and such cells migrated significantly slower than control cells (Fig. 3c).


Susceptibility to tuberculosis is associated with variants in the ASAP1 gene encoding a regulator of dendritic cell migration.

Curtis J, Luo Y, Zenner HL, Cuchet-Lourenço D, Wu C, Lo K, Maes M, Alisaac A, Stebbings E, Liu JZ, Kopanitsa L, Ignatyeva O, Balabanova Y, Nikolayevskyy V, Baessmann I, Thye T, Meyer CG, Nürnberg P, Horstmann RD, Drobniewski F, Plagnol V, Barrett JC, Nejentsev S - Nat. Genet. (2015)

In DCs, the ASAP1 protein is partly associated with podosomes and its depletion leads to impaired matrix degradation and migration(a) Confocal microscopy showing ASAP1 (green), actin (red), vinculin (blue) and nuclei (white) in DCs derived from monocytes of a healthy volunteer. Podosomes are structures characterized by the actin core (visible as red dots) surrounded by other proteins. Boxed area is shown in grayscale enlarged on the right. Scale bar represents 7.5μm.(b) Representative confocal microscopy images (left panel) showing monocyte-derived DCs treated with either ASAP1 or control siRNA and seeded on FITC-conjugated gelatine (green) for 4 hours. The cells were stained to visualize actin (phalloidin-red) and nuclei (DAPI-blue). White arrows show examples of the DAPI-stained nuclei of cells that do not degrade matrix. Scale bar represents 50μm.In the same experiment cell lysates were collected for western blotting and probed with anti-ASAP1 and anti-actin antibodies (right panel) to assess knockdown efficiency.Area of degradation of the FITC-conjugated gelatine matrix by DCs (lower panel). In total 394 and 317 DCs treated with either ASAP1 or control siRNA, respectively, were counted in 4 independent experiments.(c) Migration velocity of DCs in a Dunn chamber. In total 162 and 186 DCs treated with either ASAP1 or control siRNA, respectively, were tracked in 5 independent experiments.Graphs show mean values ± SEM. P-values were calculated using two-tailed Student T test. Unadjusted P-values are shown.
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Figure 3: In DCs, the ASAP1 protein is partly associated with podosomes and its depletion leads to impaired matrix degradation and migration(a) Confocal microscopy showing ASAP1 (green), actin (red), vinculin (blue) and nuclei (white) in DCs derived from monocytes of a healthy volunteer. Podosomes are structures characterized by the actin core (visible as red dots) surrounded by other proteins. Boxed area is shown in grayscale enlarged on the right. Scale bar represents 7.5μm.(b) Representative confocal microscopy images (left panel) showing monocyte-derived DCs treated with either ASAP1 or control siRNA and seeded on FITC-conjugated gelatine (green) for 4 hours. The cells were stained to visualize actin (phalloidin-red) and nuclei (DAPI-blue). White arrows show examples of the DAPI-stained nuclei of cells that do not degrade matrix. Scale bar represents 50μm.In the same experiment cell lysates were collected for western blotting and probed with anti-ASAP1 and anti-actin antibodies (right panel) to assess knockdown efficiency.Area of degradation of the FITC-conjugated gelatine matrix by DCs (lower panel). In total 394 and 317 DCs treated with either ASAP1 or control siRNA, respectively, were counted in 4 independent experiments.(c) Migration velocity of DCs in a Dunn chamber. In total 162 and 186 DCs treated with either ASAP1 or control siRNA, respectively, were tracked in 5 independent experiments.Graphs show mean values ± SEM. P-values were calculated using two-tailed Student T test. Unadjusted P-values are shown.
Mentions: To investigate the role of the ASAP1 protein in DCs, we first studied its cellular localization. Confocal microscopy showed that the ASAP1 protein was localized in the cytoplasm and partly was associated with podosomes (Fig. 3a). Therefore, we hypothesized that reduced ASAP1 expression may affect matrix degradation by DCs and their migration. To test this, we isolated primary monocytes from healthy volunteers, derived DCs in vitro and transfected them with siRNA to reduce ASAP1 expression. We found that degradation of a gelatine matrix by the ASAP1-depleted DCs was impaired (Fig. 3b) and such cells migrated significantly slower than control cells (Fig. 3c).

Bottom Line: In the combined analysis of these subjects and the follow-up cohort (15,087 TB patients and controls altogether), we found an association between TB and variants located in introns of the ASAP1 gene on chromosome 8q24 (P = 2.6 × 10(-11) for rs4733781; P = 1.0 × 10(-10) for rs10956514).The ASAP1 protein is involved in actin and membrane remodeling and has been associated with podosomes.Therefore, genetically determined excessive reduction of ASAP1 expression in M. tuberculosis-infected DCs may lead to their impaired migration, suggesting a potential mechanism of predisposition to TB.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Cambridge, Cambridge, UK.

ABSTRACT
Human genetic factors predispose to tuberculosis (TB). We studied 7.6 million genetic variants in 5,530 people with pulmonary TB and in 5,607 healthy controls. In the combined analysis of these subjects and the follow-up cohort (15,087 TB patients and controls altogether), we found an association between TB and variants located in introns of the ASAP1 gene on chromosome 8q24 (P = 2.6 × 10(-11) for rs4733781; P = 1.0 × 10(-10) for rs10956514). Dendritic cells (DCs) showed high ASAP1 expression that was reduced after Mycobacterium tuberculosis infection, and rs10956514 was associated with the level of reduction of ASAP1 expression. The ASAP1 protein is involved in actin and membrane remodeling and has been associated with podosomes. The ASAP1-depleted DCs showed impaired matrix degradation and migration. Therefore, genetically determined excessive reduction of ASAP1 expression in M. tuberculosis-infected DCs may lead to their impaired migration, suggesting a potential mechanism of predisposition to TB.

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Related in: MedlinePlus