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RGS1 regulates myeloid cell accumulation in atherosclerosis and aortic aneurysm rupture through altered chemokine signalling.

Patel J, McNeill E, Douglas G, Hale AB, de Bono J, Lee R, Iqbal AJ, Regan-Komito D, Stylianou E, Greaves DR, Channon KM - Nat Commun (2015)

Bottom Line: Regulator of G-Protein Signalling-1 (RGS1) deactivates G-protein signalling, reducing the response to sustained chemokine stimulation.Rgs1 reduces macrophage chemotaxis and desensitizes chemokine receptor signalling.Collectively, these data reveal a role for Rgs1 in leukocyte trafficking and vascular inflammation and identify Rgs1, and inhibition of chemokine receptor signalling as potential therapeutic targets in vascular disease.

View Article: PubMed Central - PubMed

Affiliation: 1] Division of Cardiovascular Medicine, British Heart Foundation Centre of Research Excellence, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, UK [2] Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK.

ABSTRACT
Chemokine signalling drives monocyte recruitment in atherosclerosis and aortic aneurysms. The mechanisms that lead to retention and accumulation of macrophages in the vascular wall remain unclear. Regulator of G-Protein Signalling-1 (RGS1) deactivates G-protein signalling, reducing the response to sustained chemokine stimulation. Here we show that Rgs1 is upregulated in atherosclerotic plaque and aortic aneurysms. Rgs1 reduces macrophage chemotaxis and desensitizes chemokine receptor signalling. In early atherosclerotic lesions, Rgs1 regulates macrophage accumulation and is required for the formation and rupture of Angiotensin II-induced aortic aneurysms, through effects on leukocyte retention. Collectively, these data reveal a role for Rgs1 in leukocyte trafficking and vascular inflammation and identify Rgs1, and inhibition of chemokine receptor signalling as potential therapeutic targets in vascular disease.

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Rgs1 deletion enhances monocyte–macrophage chemotaxis and impairs homologous desensitization.Migration of peritoneal macrophages from ApoE−/− and Rgs1−/−ApoE−/− mice through an 8-μm filter towards increasing concentrations of recombinant murine (a) CCL5, (b) CCL3 and (c) CCL2 placed in the lower chamber of a Boyden chamber. (d) Migration of peritoneal macrophages pretreated with 0, 0.1, 1 and 10 nM CCL5 and exposed to 1 nM CCL5. Quantification of migration is presented relative to results of untreated cells, set as 1. RPMI media was used as a negative control. Graphs indicate migration index±s.e.m. for each treatment group (triplicates; n=5–6 per group). In vivo chemotaxis was assessed by i.p. injection of 100 μg zymosan and recruited, peritoneal 7/4hiLy6G− monocytes quantified by flow cytometry at (e) 4 h and (f) 16 h after injection (n=2–4 for saline and n=6–11 for zymosan) (g) The expression of CCR5 on the surface of circulating monocytes after zymosan (n=6–7). Mean fluorescence intensity (MFI) is shown for CCR5 on monocytes at 4 h after zymosan above isotype control (grey). P<0.01 in a,b; P<0.05 in c,d calculated by two-way analysis of variance with significance at individual doses indicated by stars calculated by Bonferroni post-tests. P<0.05 in e–g calculated by Student’s t-test.
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f2: Rgs1 deletion enhances monocyte–macrophage chemotaxis and impairs homologous desensitization.Migration of peritoneal macrophages from ApoE−/− and Rgs1−/−ApoE−/− mice through an 8-μm filter towards increasing concentrations of recombinant murine (a) CCL5, (b) CCL3 and (c) CCL2 placed in the lower chamber of a Boyden chamber. (d) Migration of peritoneal macrophages pretreated with 0, 0.1, 1 and 10 nM CCL5 and exposed to 1 nM CCL5. Quantification of migration is presented relative to results of untreated cells, set as 1. RPMI media was used as a negative control. Graphs indicate migration index±s.e.m. for each treatment group (triplicates; n=5–6 per group). In vivo chemotaxis was assessed by i.p. injection of 100 μg zymosan and recruited, peritoneal 7/4hiLy6G− monocytes quantified by flow cytometry at (e) 4 h and (f) 16 h after injection (n=2–4 for saline and n=6–11 for zymosan) (g) The expression of CCR5 on the surface of circulating monocytes after zymosan (n=6–7). Mean fluorescence intensity (MFI) is shown for CCR5 on monocytes at 4 h after zymosan above isotype control (grey). P<0.01 in a,b; P<0.05 in c,d calculated by two-way analysis of variance with significance at individual doses indicated by stars calculated by Bonferroni post-tests. P<0.05 in e–g calculated by Student’s t-test.

Mentions: Since Rgs1 expression in macrophages is high and upregulated with activation, we reasoned that RGS1 would inhibit the migration of macrophages to atherogenic chemokines. We compared the chemotactic responses between ApoE−/− and Rgs1−/−ApoE−/− peritoneal macrophages in vitro. Rgs1−/−ApoE−/− macrophage chemotaxis was significantly increased in response to CCL2, CCL3 and CCL5 (Fig. 2a–c) suggesting a broad specificity for RGS1 to Gαi-coupled chemokine receptors. We also confirmed the role of RGS1 in lymphocyte chemotaxis, by showing increased migration of Rgs1−/−ApoE−/− splenocytes to the homeostatic chemokine CXCL12 (Supplementary Fig. 2) at a similar magnitude to published studies10. Because RGS1 promotes the formation of the inactive G-protein heterotrimer and accelerates the termination of chemokine signalling, we tested the effect of RGS1 on chemokine receptor desensitization in macrophages. We observed that RGS1 reduced the migration to sustained CCL5 stimulation of macrophages. Pretreatment of peritoneal macrophages with increasing doses of CCL5 before chemotaxis to 1 nM CCL5 markedly impaired chemotaxis in ApoE−/− macrophages, whereas Rgs1-deficient macrophages continued to migrate, regardless of previous exposure to chemokine (Fig. 2d). To further address the role of RGS1 in myeloid cell chemotaxis, we used a chemokine-dependent model of sterile inflammation—zymosan-induced peritonitis16 to assess cellular recruitment in vivo. At an early time point after zymosan administration, coinciding with the phase of cellular recruitment to the peritoneum, we observed that the number of monocytes in the peritoneum was significantly increased in Rgs1−/−ApoE−/− mice (Fig. 2e). However, at 16 h after zymosan administration, when cellular recruitment has plateaued and the resolution phase is beginning, we observed a significant decrease in the numbers of monocytes in Rgs1−/−ApoE−/− mice (Fig. 2f) suggesting that an early increase in cell number in Rgs1−/−ApoE−/− mice is then followed by reduced accumulation. To address if there were any alterations in chemokine receptor signalling that may be crucial for trafficking, we assessed CCR5 and CCR2 surface expression on circulating monocytes in mice treated with zymosan. At 4 h, coinciding with the increase in monocytes in the peritoneum of Rgs1−/−ApoE−/− mice, there was an increase in CCR5 on the circulating monocytes compared with monocytes in ApoE−/− mice. In contrast, at 16 h, there was no difference in the cell surface level of CCR5 between Rgs1−/−ApoE−/− and ApoE−/− monocytes (Fig. 2g). However, CCR2 surface expression was not detectable on circulating monocytes from either Rgs1−/−ApoE−/− and ApoE−/− mice after 4 h of zymosan.


RGS1 regulates myeloid cell accumulation in atherosclerosis and aortic aneurysm rupture through altered chemokine signalling.

Patel J, McNeill E, Douglas G, Hale AB, de Bono J, Lee R, Iqbal AJ, Regan-Komito D, Stylianou E, Greaves DR, Channon KM - Nat Commun (2015)

Rgs1 deletion enhances monocyte–macrophage chemotaxis and impairs homologous desensitization.Migration of peritoneal macrophages from ApoE−/− and Rgs1−/−ApoE−/− mice through an 8-μm filter towards increasing concentrations of recombinant murine (a) CCL5, (b) CCL3 and (c) CCL2 placed in the lower chamber of a Boyden chamber. (d) Migration of peritoneal macrophages pretreated with 0, 0.1, 1 and 10 nM CCL5 and exposed to 1 nM CCL5. Quantification of migration is presented relative to results of untreated cells, set as 1. RPMI media was used as a negative control. Graphs indicate migration index±s.e.m. for each treatment group (triplicates; n=5–6 per group). In vivo chemotaxis was assessed by i.p. injection of 100 μg zymosan and recruited, peritoneal 7/4hiLy6G− monocytes quantified by flow cytometry at (e) 4 h and (f) 16 h after injection (n=2–4 for saline and n=6–11 for zymosan) (g) The expression of CCR5 on the surface of circulating monocytes after zymosan (n=6–7). Mean fluorescence intensity (MFI) is shown for CCR5 on monocytes at 4 h after zymosan above isotype control (grey). P<0.01 in a,b; P<0.05 in c,d calculated by two-way analysis of variance with significance at individual doses indicated by stars calculated by Bonferroni post-tests. P<0.05 in e–g calculated by Student’s t-test.
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f2: Rgs1 deletion enhances monocyte–macrophage chemotaxis and impairs homologous desensitization.Migration of peritoneal macrophages from ApoE−/− and Rgs1−/−ApoE−/− mice through an 8-μm filter towards increasing concentrations of recombinant murine (a) CCL5, (b) CCL3 and (c) CCL2 placed in the lower chamber of a Boyden chamber. (d) Migration of peritoneal macrophages pretreated with 0, 0.1, 1 and 10 nM CCL5 and exposed to 1 nM CCL5. Quantification of migration is presented relative to results of untreated cells, set as 1. RPMI media was used as a negative control. Graphs indicate migration index±s.e.m. for each treatment group (triplicates; n=5–6 per group). In vivo chemotaxis was assessed by i.p. injection of 100 μg zymosan and recruited, peritoneal 7/4hiLy6G− monocytes quantified by flow cytometry at (e) 4 h and (f) 16 h after injection (n=2–4 for saline and n=6–11 for zymosan) (g) The expression of CCR5 on the surface of circulating monocytes after zymosan (n=6–7). Mean fluorescence intensity (MFI) is shown for CCR5 on monocytes at 4 h after zymosan above isotype control (grey). P<0.01 in a,b; P<0.05 in c,d calculated by two-way analysis of variance with significance at individual doses indicated by stars calculated by Bonferroni post-tests. P<0.05 in e–g calculated by Student’s t-test.
Mentions: Since Rgs1 expression in macrophages is high and upregulated with activation, we reasoned that RGS1 would inhibit the migration of macrophages to atherogenic chemokines. We compared the chemotactic responses between ApoE−/− and Rgs1−/−ApoE−/− peritoneal macrophages in vitro. Rgs1−/−ApoE−/− macrophage chemotaxis was significantly increased in response to CCL2, CCL3 and CCL5 (Fig. 2a–c) suggesting a broad specificity for RGS1 to Gαi-coupled chemokine receptors. We also confirmed the role of RGS1 in lymphocyte chemotaxis, by showing increased migration of Rgs1−/−ApoE−/− splenocytes to the homeostatic chemokine CXCL12 (Supplementary Fig. 2) at a similar magnitude to published studies10. Because RGS1 promotes the formation of the inactive G-protein heterotrimer and accelerates the termination of chemokine signalling, we tested the effect of RGS1 on chemokine receptor desensitization in macrophages. We observed that RGS1 reduced the migration to sustained CCL5 stimulation of macrophages. Pretreatment of peritoneal macrophages with increasing doses of CCL5 before chemotaxis to 1 nM CCL5 markedly impaired chemotaxis in ApoE−/− macrophages, whereas Rgs1-deficient macrophages continued to migrate, regardless of previous exposure to chemokine (Fig. 2d). To further address the role of RGS1 in myeloid cell chemotaxis, we used a chemokine-dependent model of sterile inflammation—zymosan-induced peritonitis16 to assess cellular recruitment in vivo. At an early time point after zymosan administration, coinciding with the phase of cellular recruitment to the peritoneum, we observed that the number of monocytes in the peritoneum was significantly increased in Rgs1−/−ApoE−/− mice (Fig. 2e). However, at 16 h after zymosan administration, when cellular recruitment has plateaued and the resolution phase is beginning, we observed a significant decrease in the numbers of monocytes in Rgs1−/−ApoE−/− mice (Fig. 2f) suggesting that an early increase in cell number in Rgs1−/−ApoE−/− mice is then followed by reduced accumulation. To address if there were any alterations in chemokine receptor signalling that may be crucial for trafficking, we assessed CCR5 and CCR2 surface expression on circulating monocytes in mice treated with zymosan. At 4 h, coinciding with the increase in monocytes in the peritoneum of Rgs1−/−ApoE−/− mice, there was an increase in CCR5 on the circulating monocytes compared with monocytes in ApoE−/− mice. In contrast, at 16 h, there was no difference in the cell surface level of CCR5 between Rgs1−/−ApoE−/− and ApoE−/− monocytes (Fig. 2g). However, CCR2 surface expression was not detectable on circulating monocytes from either Rgs1−/−ApoE−/− and ApoE−/− mice after 4 h of zymosan.

Bottom Line: Regulator of G-Protein Signalling-1 (RGS1) deactivates G-protein signalling, reducing the response to sustained chemokine stimulation.Rgs1 reduces macrophage chemotaxis and desensitizes chemokine receptor signalling.Collectively, these data reveal a role for Rgs1 in leukocyte trafficking and vascular inflammation and identify Rgs1, and inhibition of chemokine receptor signalling as potential therapeutic targets in vascular disease.

View Article: PubMed Central - PubMed

Affiliation: 1] Division of Cardiovascular Medicine, British Heart Foundation Centre of Research Excellence, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, UK [2] Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK.

ABSTRACT
Chemokine signalling drives monocyte recruitment in atherosclerosis and aortic aneurysms. The mechanisms that lead to retention and accumulation of macrophages in the vascular wall remain unclear. Regulator of G-Protein Signalling-1 (RGS1) deactivates G-protein signalling, reducing the response to sustained chemokine stimulation. Here we show that Rgs1 is upregulated in atherosclerotic plaque and aortic aneurysms. Rgs1 reduces macrophage chemotaxis and desensitizes chemokine receptor signalling. In early atherosclerotic lesions, Rgs1 regulates macrophage accumulation and is required for the formation and rupture of Angiotensin II-induced aortic aneurysms, through effects on leukocyte retention. Collectively, these data reveal a role for Rgs1 in leukocyte trafficking and vascular inflammation and identify Rgs1, and inhibition of chemokine receptor signalling as potential therapeutic targets in vascular disease.

Show MeSH
Related in: MedlinePlus