Defective cholesterol metabolism in haematopoietic stem cells promotes monocyte-driven atherosclerosis in rheumatoid arthritis

doi:10.1093/eurheartj/ehy119

. 2018 Jun 14;39(23):2158-2167.

doi: 10.1093/eurheartj/ehy119.

Defective cholesterol metabolism in haematopoietic stem cells promotes monocyte-driven atherosclerosis in rheumatoid arthritis

Dragana Dragoljevic^{1

2}, Michael J Kraakman^{1

3}, Prabhakara R Nagareddy⁴, Devi Ngo⁵, Waled Shihata^{1

6}, Helene L Kammoun^{1

2}, Alexandra Whillas¹, Man Kit Sam Lee^{1

2}, Annas Al-Sharea^{1

2}, Gerard Pernes^{1

2}, Michelle C Flynn^{1

2}, Graeme I Lancaster^{1

2}, Mark A Febbraio⁷, Jaye Chin-Dusting⁶, Beatriz Y Hanaoka⁸, Ian P Wicks^{5

9}, Andrew J Murphy^{1

2}

Affiliations

¹ Haematopoiesis and Leukocyte Biology Laboratory, Division of Immunometabolism, Baker Heart and Diabetes Institute, 75 Commercial Rd, 3004 Melbourne, Victoria, Australia.
² Department of Immunology, Monash University, 89 Commercial Road, 3004 Melbourne, Victoria, Australia.
³ Human Nutrition, Naomi Berrie Diabetes Centre, Columbia University, New York, 1150 St Nicholas Ave, 10032 NY, USA.
⁴ Department of Nutrition Sciences, University of Alabama at Birmingham, Birmingham, 1720 2nd Ave South, 35294 AL, USA.
⁵ Inflammation Division, Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, 3052 Melbourne, Victoria, Australia.
⁶ Department of Pharmacology, Monash University, Wellington Road, 3800 Clayton, Victoria, Australia.
⁷ Cellular and Molecular Metabolism, Division of Diabetes & Metabolism, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, 2010 Sydney, New South Wales, Australia.
⁸ Department of Medicine, Division of Clinical Immunology and Rheumatology, University of Alabama at Birmingham, Birmingham, 1720 2nd Ave South, 35294 AL, USA.
⁹ Rheumatology Unit, Royal Melbourne Hospital, 300 Grattan St, 3050 Melbourne, Victoria, Australia.

PMID: 29905812
PMCID: PMC6001889
DOI: 10.1093/eurheartj/ehy119

Defective cholesterol metabolism in haematopoietic stem cells promotes monocyte-driven atherosclerosis in rheumatoid arthritis

Dragana Dragoljevic et al. Eur Heart J. 2018.

. 2018 Jun 14;39(23):2158-2167.

doi: 10.1093/eurheartj/ehy119.

Authors

Affiliations

¹ Haematopoiesis and Leukocyte Biology Laboratory, Division of Immunometabolism, Baker Heart and Diabetes Institute, 75 Commercial Rd, 3004 Melbourne, Victoria, Australia.
² Department of Immunology, Monash University, 89 Commercial Road, 3004 Melbourne, Victoria, Australia.
³ Human Nutrition, Naomi Berrie Diabetes Centre, Columbia University, New York, 1150 St Nicholas Ave, 10032 NY, USA.
⁴ Department of Nutrition Sciences, University of Alabama at Birmingham, Birmingham, 1720 2nd Ave South, 35294 AL, USA.
⁵ Inflammation Division, Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, 3052 Melbourne, Victoria, Australia.
⁶ Department of Pharmacology, Monash University, Wellington Road, 3800 Clayton, Victoria, Australia.
⁷ Cellular and Molecular Metabolism, Division of Diabetes & Metabolism, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, 2010 Sydney, New South Wales, Australia.
⁸ Department of Medicine, Division of Clinical Immunology and Rheumatology, University of Alabama at Birmingham, Birmingham, 1720 2nd Ave South, 35294 AL, USA.
⁹ Rheumatology Unit, Royal Melbourne Hospital, 300 Grattan St, 3050 Melbourne, Victoria, Australia.

PMID: 29905812
PMCID: PMC6001889
DOI: 10.1093/eurheartj/ehy119

Abstract

Aim: Rheumatoid arthritis (RA) is associated with an approximately two-fold elevated risk of cardiovascular (CV)-related mortality. Patients with RA present with systemic inflammation including raised circulating myeloid cells, but fail to display traditional CV risk-factors, particularly dyslipidaemia. We aimed to explore if increased circulating myeloid cells is associated with impaired atherosclerotic lesion regression or altered progression in RA.

Methods and results: Using flow cytometry, we noted prominent monocytosis, neutrophilia, and thrombocytosis in two mouse models of RA. This was due to enhanced proliferation of the haematopoietic stem and progenitor cells (HSPCs) in the bone marrow and the spleen. HSPCs expansion was associated with an increase in the cholesterol content, due to a down-regulation of cholesterol efflux genes, Apoe, Abca1, and Abcg1. The HSPCs also had enhanced expression of key myeloid promoting growth factor receptors. Systemic inflammation was found to cause defective cellular cholesterol metabolism. Increased myeloid cells in mice with RA were associated with a significant impairment in lesion regression, even though cholesterol levels were equivalent to non-arthritic mice. Lesions from arthritic mice exhibited a less stable phenotype as demonstrated by increased immune cell infiltration, lipid accumulation, and decreased collagen formation. In a progression model, we noted monocytosis, enhanced monocytes recruitment to lesions, and increased plaque macrophages. This was reversed with administration of reconstituted high-density lipoprotein (rHDL). Furthermore, RA patients have expanded CD16+ monocyte subsets and a down-regulation of ABCA1 and ABCG1.

Conclusion: Rheumatoid arthritis impairs atherosclerotic regression and alters progression, which is associated with an expansion of myeloid cells and disturbed cellular cholesterol handling, independent of plasma cholesterol levels. Infusion of rHDL prevented enhanced myelopoiesis and monocyte entry into lesions. Targeting cellular cholesterol defects in people with RA, even if plasma cholesterol is within the normal range, may limit vascular disease.

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Figures

**Figure 1**
Murine models of inflammatory arthritis display leucocytosis and thrombocytosis. Experimental arthritis was induced by: (A) the collagen-induced arthritis (CIA) model or (E) the K/BxN serum transfer model. (*B, F*) Blood leucocytes were quantified by flow cytometry, (C, G) Circulating platelets counts, and (*D, H*) reticulated thrombocytes as determined by flow cytometry and normalized to platelet counts. n = 3–5. All data are mean ± standard deviation.

**Figure 2**
Inflammatory arthritis promotes extramedullary haematopoiesis in the spleen. Blood, spleen and bone marrow cell populations were assessed using flow cytometry in the collagen-induced arthritis (blue bars) and K/BxN (red bars) models of inflammatory arthritis. (*A, B, H, I*) Populations and (C,J) proliferation of bone marrow haematopoietic stem and progenitor cells, common myeloid progenitors, granulocyte-macrophage progenitors, and megakaryocyte-erythroid progenitors. (*D, K*) Blood stem and progenitor cells. (*E, L*) Spleen weights. (*F, M*) Haematopoietic stem and progenitor cell and progenitor cell abundance in the spleen. (*G, N*) Splenic leucocyte populations. n = 3–5. All data are mean ± standard deviation.

**Figure 3**
Systemic inflammation causes enhanced myelopoiesis in inflammatory arthritis, and is associated with disturbed cellular cholesterol handling. (A) Membrane lipid rafts staining of bone marrow haematopoietic stem and progenitor cells from control and collagen-induced arthritis visualized by confocal microscopy (CT-xB staining; red, 4′,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) counterstained; blue, 40× magnification). (B) Haematopoietic stem and progenitor cell representative plot and quantified membrane cholesterol on bone marrow haematopoietic stem and progenitor cells, common myeloid progenitors, and granulocyte-macrophage progenitors using boron-dipyrromethene (BODIPY)-cholesterol mean fluorescence intensity (MFI) via flow cytometry. (C) Cholesterol efflux gene expression from sorted bone marrow haematopoietic stem and progenitor cells. Ly6-C^hi (D) BODIPY-cholesterol content, and (E) gene expression. *n =* 4–11. (F) Experimental overview. (G) Blood leucocyte abundance. (H) Platelet levels. (I) Representative flow plots, and the ratio of CD45.2 to CD45.1 cells. (*J, K*) Bone marrow haematopoietic stem and progenitor cell abundance and haematopoietic stem and progenitor cell CD45.2/CD45.1 cell ratio. (L) Haematopoietic stem and progenitor cell proliferation and the ratio of CD45.2/CD45.1 proliferation indicated in brackets. (M) Gene expression from isolated haematopoietic stem and progenitor cells. n = 3–8. All data are mean ± standard deviation.

**Figure 4**
Inflammatory arthritis impairs atherosclerotic regression independent of circulating cholesterol. (A) *Ldlr^−/−* mice were fed a western type diet for 14 weeks to induce atherogenesis [(i) baseline]. The remaining mice were switched to a chow diet to induce atherosclerotic lesion regression [(ii) control regression group] and a (iii) arthritic group (K/BxN). (B) Arthritic clinical scores. (C) Plasma cholesterol. The aortic sinus was characterized for (D) lesion size, (E) lipid abundance (Oil Red O; ORO staining) and (F) macrophage content (CD68⁺). (G) Peripheral blood cell abundance, (H) spleen weight, (I) total and reticulated platelet counts, and (J) leucocyte–platelet interaction. *n =* 8–11. All data are mean ± standard deviation.

**Figure 5**
Reconstitute high-density lipoprotein prevents the rheumatoid arthritis-driven Ly6-C^hi monocyte entry into atherosclerotic lesions and reduces macrophage abundance. (A) 15-week-old *Apoe^−/−* mice, on a chow diet, were either (i) left as controls, (ii) made arthritic, or (iii) made arthritic and treated with reconstitute high-density lipoprotein. Mice were injected with fluorescent tracking beads on Day 4 (−72 h before sacrifice) and the following day with EdU. (B) Clinical arthritic scores. (C) Representative flow cytometry gating and quantified abundance of blood leucocytes. (D) Monocyte (Ly6-C) fluorescent staining (Ly6-C—green; nuclei—red) and (E) EdU⁺ cells in atherosclerotic lesions (EdU—green; nuclei—blue). Indicated with white arrows. (F) Lesional macrophage (CD68) content. n = 5–11. All data are mean ± standard deviation.

**Figure 6**
Patients with rheumatoid arthritis display peripheral blood monocytosis. (A) Monocyte subsets were identified using flow cytometry, from healthy controls and patients with rheumatoid arthritis. (B) Total monocytes, (C) classical CD14⁺CD16^dim monocytes, (D) intermediate CD14⁺CD16⁺ monocytes, and (E) non-classical CD14^dimCD16⁺⁺ monocyte levels. (F) Gene expression in peripheral blood mononuclear cells. n = 3–12. All data are mean ± standard deviation.

**Take home figure**
Rheumatoid arthritis impairs atherosclerotic regression, independent of circulating cholesterol levels. Systemic inflammation in rheumatoid arthritis causes an accumulation of cellular cholesterol in bone marrow haematopoietic stem and progenitor cells that contributes to enhanced proliferation and myeloid skewing towards myeloid progenitors. Enhanced myelopoiesis causes increased levels of circulating monocytes (monocytosis), neutrophils (neutrophilia), and platelets (thrombocytosis). Rheumatoid arthritis promotes increased Ly6-C^hi monocyte entry into lesions, and consequently increases macrophage burden. Rheumatoid arthritis impairs the regression of established atherosclerotic lesions, which contained more macrophages and lipid, even when cholesterol levels were controlled. Therefore, rheumatoid arthritis appears to impair atherosclerotic lesion regression, associated with an expansion of myeloid cells and disturbed cellular cholesterol handling, independent of plasma cholesterol levels. Promoting cholesterol efflux with reconstitute high-density lipoprotein could reverse the abundance and entry of monocytes into the lesion, and consequently reduce plaque macrophage burden.

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Comment in

Is defective cholesterol efflux an integral inflammatory component in myelopoiesis-driven cardiovascular diseases?
Yvan-Charvet L, Swirski FK. Yvan-Charvet L, et al. Eur Heart J. 2018 Jun 14;39(23):2168-2171. doi: 10.1093/eurheartj/ehy269. Eur Heart J. 2018. PMID: 29771312 No abstract available.

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References

1. Gonzalez-Juanatey C, Llorca J, Testa A, Revuelta J, Garcia-Porrua C, Gonzalez-Gay MA.. Increased prevalence of severe subclinical atherosclerotic findings in long-term treated rheumatoid arthritis patients without clinically evident atherosclerotic disease. Medicine 2003;82:407–413. - PubMed
1. McInnes IB, Schett G.. The pathogenesis of rheumatoid arthritis. N Engl J Med 2011;365:2205–2219. - PubMed
1. Meune C, Touze E, Trinquart L, Allanore Y.. High risk of clinical cardiovascular events in rheumatoid arthritis: levels of associations of myocardial infarction and stroke through a systematic review and meta-analysis. Arch Cardiovasc Dis 2010;103:253–261. - PubMed
1. Crowson CS, Matteson EL, Roger VL, Therneau TM, Gabriel SE.. Usefulness of risk scores to estimate the risk of cardiovascular disease in patients with rheumatoid arthritis. Am J Cardiol 2012;110:420–424. - PMC - PubMed
1. Agca R, Heslinga SC, Rollefstad S, Heslinga M, McInnes IB, Peters MJL, Kvien TK, Dougados M, Radner H, Atzeni F, Primdahl J, Södergren A, Wallberg Jonsson S, van Rompay J, Zabalan C, Pedersen TR, Jacobsson L, de Vlam K, Gonzalez-Gay MA, Semb AG, Kitas GD, Smulders YM, Szekanecz Z, Sattar N, Symmons DPM, Nurmohamed MT.. EULAR recommendations for cardiovascular disease risk management in patients with rheumatoid arthritis and other forms of inflammatory joint disorders: 2015/2016 update. Ann Rheum Dis 2017;76:17–28. - PubMed

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[1] Gonzalez-Juanatey C, Llorca J, Testa A, Revuelta J, Garcia-Porrua C, Gonzalez-Gay MA.. Increased prevalence of severe subclinical atherosclerotic findings in long-term treated rheumatoid arthritis patients without clinically evident atherosclerotic disease. Medicine 2003;82:407–413. - PubMed

[2] Gonzalez-Juanatey C, Llorca J, Testa A, Revuelta J, Garcia-Porrua C, Gonzalez-Gay MA.. Increased prevalence of severe subclinical atherosclerotic findings in long-term treated rheumatoid arthritis patients without clinically evident atherosclerotic disease. Medicine 2003;82:407–413. - PubMed

[3] McInnes IB, Schett G.. The pathogenesis of rheumatoid arthritis. N Engl J Med 2011;365:2205–2219. - PubMed

[4] McInnes IB, Schett G.. The pathogenesis of rheumatoid arthritis. N Engl J Med 2011;365:2205–2219. - PubMed

[5] Meune C, Touze E, Trinquart L, Allanore Y.. High risk of clinical cardiovascular events in rheumatoid arthritis: levels of associations of myocardial infarction and stroke through a systematic review and meta-analysis. Arch Cardiovasc Dis 2010;103:253–261. - PubMed

[6] Meune C, Touze E, Trinquart L, Allanore Y.. High risk of clinical cardiovascular events in rheumatoid arthritis: levels of associations of myocardial infarction and stroke through a systematic review and meta-analysis. Arch Cardiovasc Dis 2010;103:253–261. - PubMed

[7] Crowson CS, Matteson EL, Roger VL, Therneau TM, Gabriel SE.. Usefulness of risk scores to estimate the risk of cardiovascular disease in patients with rheumatoid arthritis. Am J Cardiol 2012;110:420–424. - PMC - PubMed

[8] Crowson CS, Matteson EL, Roger VL, Therneau TM, Gabriel SE.. Usefulness of risk scores to estimate the risk of cardiovascular disease in patients with rheumatoid arthritis. Am J Cardiol 2012;110:420–424. - PMC - PubMed

[9] Agca R, Heslinga SC, Rollefstad S, Heslinga M, McInnes IB, Peters MJL, Kvien TK, Dougados M, Radner H, Atzeni F, Primdahl J, Södergren A, Wallberg Jonsson S, van Rompay J, Zabalan C, Pedersen TR, Jacobsson L, de Vlam K, Gonzalez-Gay MA, Semb AG, Kitas GD, Smulders YM, Szekanecz Z, Sattar N, Symmons DPM, Nurmohamed MT.. EULAR recommendations for cardiovascular disease risk management in patients with rheumatoid arthritis and other forms of inflammatory joint disorders: 2015/2016 update. Ann Rheum Dis 2017;76:17–28. - PubMed

[10] Agca R, Heslinga SC, Rollefstad S, Heslinga M, McInnes IB, Peters MJL, Kvien TK, Dougados M, Radner H, Atzeni F, Primdahl J, Södergren A, Wallberg Jonsson S, van Rompay J, Zabalan C, Pedersen TR, Jacobsson L, de Vlam K, Gonzalez-Gay MA, Semb AG, Kitas GD, Smulders YM, Szekanecz Z, Sattar N, Symmons DPM, Nurmohamed MT.. EULAR recommendations for cardiovascular disease risk management in patients with rheumatoid arthritis and other forms of inflammatory joint disorders: 2015/2016 update. Ann Rheum Dis 2017;76:17–28. - PubMed

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Defective cholesterol metabolism in haematopoietic stem cells promotes monocyte-driven atherosclerosis in rheumatoid arthritis

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Defective cholesterol metabolism in haematopoietic stem cells promotes monocyte-driven atherosclerosis in rheumatoid arthritis

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