The Open Rheumatology Journal




ISSN: 1874-3129 ― Volume 13, 2019
REVIEW ARTICLE

Vitamin D and Anti-Phospholipid Antibody Syndrome: A Comprehensive Review



Roberta Gualtierotti1, 2, *, Alessio Di Giacomo3, Elena Raschi3, Maria Orietta Borghi2, 3, Pier Luigi Meroni3
1 Lupus Clinic, Department of Rheumatology and Clinical Sciences, ASST Pini-CTO, Milan, Italy
2 Department of Clinical Sciences and Community Health, University of Milan, P.za Cardinal Ferrari, 1 20122, Milan, Italy
3 Immunorheumatology Research Laboratory, Instituto Auxologico Italiano, IRCCS, Milan, Italy

Abstract

Vitamin D is a steroid hormone that exerts a variety of biological effects that range from the well-known regulation of bone metabolism to the modulation of cellular growth, of apoptosis, and the regulation of both innate and adaptive immunity. Evidence supports a correlation between low vitamin D levels and a high risk to develop chronic inflammatory diseases including autoimmune diseases.

Anti-phospholipid antibody (aPL) syndrome (APS) is an autoimmune chronic condition characterized by recurrent arterial and/or venous thrombosis and/or obstetric complications associated with persistent aPL positivity. Secondary prevention of thrombosis is widely accepted in these patients and relies on life-long anticoagulant drugs. On the contrary, primary prevention in isolated aPL positivity in healthy carriers and treatment of obstetric manifestations in APS are still debated.

Epidemiological data have shown that vitamin D deficiency (serum levels <30 ng/ml) is frequent in APS patients and that it may be associated with an increased risk of thrombosis in these patients. Experimental data show that vitamin D is able to reduce the expression of adhesion molecules, of toll-like receptors and the secretion of proinflammatory chemokines, thus playing a protective role on endothelial activation and the subsequent development of thrombosis in APS.

Although these observations need to be confirmed in prospective studies and randomized clinical trials, it is tempting to speculate that vitamin D supplementation could be very useful for the prevention of clinical manifestations in APS patients, in particular as a primary prevention countermeasure in aPL carriers.

Keywords: Anti-phospholipid syndrome, Vitamin D, Endothelial cells, Inflammation, Toll-like receptor, TLR, Adhesion molecules, IFN.


Article Information


Identifiers and Pagination:

Year: 2018
Volume: 12
Issue: Suppl-1, M5
First Page: 248
Last Page: 260
Publisher Id: TORJ-12-248
DOI: 10.2174/1874312901812010248

Article History:

Received Date: 4/2/2017
Revision Received Date: 28/3/2018
Acceptance Date: 22/4/2018
Electronic publication date: 27/12/2018
Collection year: 2018

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© 2018 Gualtierotti et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: (https://creativecommons.org/licenses/by/4.0/legalcode). This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


* Address correspondence to this author at the Department of Clinical Sciences and Community Health, University of Milan, P.za Cardinal Ferrari, 1 20122, Milano, Italy; Tel: 0258296272; E-mail: roberta.gualtierotti@unimi.it




1. INTRODUCTION

Vitamin D is a steroid hormone generated by cholesterol, exerting a wide variety of biological effects that go beyond the well-known role in the regulation of bone metabolism and calcium homeostasis [1Anderson PH, Vitamin D. Vitamin D activity and metabolism in bone. Curr Osteoporos Rep 2017; 15(5): 443-9.[http://dx.doi.org/10.1007/s11914-017-0394-8] [PMID: 28808890] , 2Cutolo M, Pizzorni C, Sulli A. Vitamin D endocrine system involvement in autoimmune rheumatic diseases. Autoimmun Rev 2011; 11(2): 84-7.[http://dx.doi.org/10.1016/j.autrev.2011.08.003] [PMID: 21864722] ]. Vitamin D has been later recognized as responsible for many non-skeletal effects, mainly based on observational studies conducted in large cohorts showing a significant association between low levels of vitamin D (i.e., <20 ng/mL of 25-hydroxy-vitamin D) and the risk of developing cardiovascular (CV), metabolic, neoplastic and autoimmune diseases [3Dobnig H, Pilz S, Scharnagl H, et al. Independent association of low serum 25-hydroxyvitamin d and 1,25-dihydroxyvitamin d levels with all-cause and cardiovascular mortality. Arch Intern Med 2008; 168(12): 1340-9.[http://dx.doi.org/10.1001/archinte.168.12.1340] [PMID: 18574092] -6Hewison M. Vitamin D and innate immunity. Curr Opin Investig Drugs 2008; 9(5): 485-90.[PMID: 18465658] ].

The biological activity of the active form of vitamin D is triggered by its interaction with the high-affinity Vitamin D Receptor (VDR), which belongs to the superfamily of nuclear hormone receptors [7Zehnder D, Bland R, Williams MC, et al. Extrarenal expression of 25-hydroxyvitamin d(3)-1 alpha-hydroxylase. J Clin Endocrinol Metab 2001; 86(2): 888-94.[PMID: 11158062] , 8Adams JS, Hewison M. Extrarenal expression of the 25-hydroxyvitamin D-1-hydroxylase. Arch Biochem Biophys 2012; 523(1): 95-102.[http://dx.doi.org/10.1016/j.abb.2012.02.016] [PMID: 22446158] ]. After interaction with its ligand, VDR behaves as a transcription factor binding another receptor, Retinoid X Receptor (RXR), and thus generating a heterodimeric complex that recognizes specific DNA sequences. The VDR is also found in caveoles, invaginations of the plasma membrane with high concentrations of sphingolipids and steroids. Vitamin D binds its specific receptor also at this level, but the transduction pathway of this signal is currently unknown [9Huhtakangas JA, Olivera CJ, Bishop JE, Zanello LP, Norman AW. The vitamin D receptor is present in caveolae-enriched plasma membranes and binds 1 alpha,25(OH)2-vitamin D3 in vivo and in vitro. Mol Endocrinol 2004; 18(11): 2660-71.[http://dx.doi.org/10.1210/me.2004-0116] [PMID: 15272054] ]. Activation of VDR induces not only osteometabolic responses, but also a wide variety of biological effects, such as modulation of cellular growth, proliferation, apoptosis, and immune cell activation [10Peelen E, Knippenberg S, Muris AH, et al. Effects of vitamin D on the peripheral adaptive immune system: A review. Autoimmun Rev 2011; 10(12): 733-43.[http://dx.doi.org/10.1016/j.autrev.2011.05.002] [PMID: 21621002] ]. Vitamin D regulates both innate and adaptive immunity by means of VDR, which is present in almost all immune cells [2Cutolo M, Pizzorni C, Sulli A. Vitamin D endocrine system involvement in autoimmune rheumatic diseases. Autoimmun Rev 2011; 11(2): 84-7.[http://dx.doi.org/10.1016/j.autrev.2011.08.003] [PMID: 21864722] , 11Penna G, Amuchastegui S, Giarratana N, et al. 1,25-Dihydroxyvitamin D3 selectively modulates tolerogenic properties in myeloid but not plasmacytoid dendritic cells. J Immunol 2007; 178(1): 145-53.[http://dx.doi.org/10.4049/jimmunol.178.1.145] [PMID: 17182549] -13Almerighi C, Sinistro A, Cavazza A, Ciaprini C, Rocchi G, Bergamini A. 1Alpha,25-dihydroxyvitamin D3 inhibits CD40L-induced pro-inflammatory and immunomodulatory activity in human monocytes. Cytokine 2009; 45(3): 190-7.[http://dx.doi.org/10.1016/j.cyto.2008.12.009] [PMID: 19186073] ]. Furthermore, the presence of VDR allelic polymorphisms has been associated with a major susceptibility to develop an autoimmune disease such as Systemic Lupus Erythematosus (SLE) and Rheumatoid Arthritis (RA) [14Xiong J, He Z, Zeng X, Zhang Y, Hu Z. Association of vitamin D receptor gene polymorphisms with systemic lupus erythematosus: A meta-analysis. Clin Exp Rheumatol 2014; 32(2): 174-81.[PMID: 24321519] , 15Mosaad YM, Hammad EM, Fawzy Z, et al. Vitamin D receptor gene polymorphism as possible risk factor in rheumatoid arthritis and rheumatoid related osteoporosis. Hum Immunol 2014; 75(5): 452-61.[http://dx.doi.org/10.1016/j.humimm.2014.02.009] [PMID: 24530824] ].

1.1. Immunomodulatory Effects of Vitamin D

Evidence speaks in favor of a regulatory role of vitamin D both for innate and adaptive immunity [16Yang CY, Leung PS, Adamopoulos IE, Gershwin ME. The implication of vitamin D and autoimmunity: A comprehensive review. Clin Rev Allergy Immunol 2013; 45(2): 217-26.[http://dx.doi.org/10.1007/s12016-013-8361-3] [PMID: 23359064] , 17Adorini L, Penna G. Control of autoimmune diseases by the vitamin D endocrine system. Nat Clin Pract Rheumatol 2008; 4(8): 404-12.[http://dx.doi.org/10.1038/ncprheum0855] [PMID: 18594491] ]. VDR is constitutively expressed in Antigen Presenting Cells (APC), such as macrophages and Dendritic Cells (DC), and is inducible in activated T lymphocytes [11Penna G, Amuchastegui S, Giarratana N, et al. 1,25-Dihydroxyvitamin D3 selectively modulates tolerogenic properties in myeloid but not plasmacytoid dendritic cells. J Immunol 2007; 178(1): 145-53.[http://dx.doi.org/10.4049/jimmunol.178.1.145] [PMID: 17182549] , 18Veldman CM, Cantorna MT, DeLuca HF. Expression of 1,25-dihydroxyvitamin D(3) receptor in the immune system. Arch Biochem Biophys 2000; 374(2): 334-8.[http://dx.doi.org/10.1006/abbi.1999.1605] [PMID: 10666315] ]. Vitamin D promotes a balance of innate and adaptive immunity [19Wang TT, Nestel FP, Bourdeau V, et al. Cutting edge: 1,25-dihydroxyvitamin D3 is a direct inducer of antimicrobial peptide gene expression. J Immunol 2004; 173(5): 2909-12.[http://dx.doi.org/10.4049/jimmunol.173.5.2909] [PMID: 15322146] ] by regulating molecules related to immune activation such as MHC class II and CD40 among others and the production of Interleukin (IL)-1β, IL-6, IL-12, Tumor Necrosis Factor (TNF)-α, and Macrophage-Colony Stimulating Factor (M-CSF) [20Boltz-Nitulescu G, Willheim M, Spittler A, Leutmezer F, Tempfer C, Winkler S. Modulation of IgA, IgE, and IgG Fc receptor expression on human mononuclear phagocytes by 1 alpha,25-dihydroxyvitamin D3 and cytokines. J Leukoc Biol 1995; 58(2): 256-62.[http://dx.doi.org/10.1002/jlb.58.2.256] [PMID: 7643018] -22Sochorová K, Budinský V, Rozková D, et al. Paricalcitol (19-nor-1,25-dihydroxyvitamin D2) and calcitriol (1,25-dihydroxyvitamin D3) exert potent immunomodulatory effects on dendritic cells and inhibit induction of antigen-specific T cells. Clin Immunol 2009; 133(1): 69-77.[http://dx.doi.org/10.1016/j.clim.2009.06.011] [PMID: 19660988] ]. In addition, vitamin D receptor agonists favor DC proliferation and tolerance [16Yang CY, Leung PS, Adamopoulos IE, Gershwin ME. The implication of vitamin D and autoimmunity: A comprehensive review. Clin Rev Allergy Immunol 2013; 45(2): 217-26.[http://dx.doi.org/10.1007/s12016-013-8361-3] [PMID: 23359064] ].

Furthermore, vitamin D can modulate Toll Like Receptor (TLR) response to bacterial infections, with an immunosuppressive effect due to the reduction of the expression of TLR2 and TLR4 on monocytes/macrophages [23Gambhir V, Kim J, Siddiqui S, et al. Influence of 1,25-dihydroxy vitamin D3 on TLR4-induced activation of antigen presenting cells is dependent on the order of receptor engagement. Immunobiology 2011; 216(9): 988-96.[http://dx.doi.org/10.1016/j.imbio.2011.03.011] [PMID: 21529994] -25Sadeghi K, Wessner B, Laggner U, et al. Vitamin D3 down-regulates monocyte TLR expression and triggers hyporesponsiveness to pathogen-associated molecular patterns. Eur J Immunol 2006; 36(2): 361-70.[http://dx.doi.org/10.1002/eji.200425995] [PMID: 16402404] ]. Moreover, vitamin D inhibits lipopolysaccharide (LPS)-induced TNF-α production in a concentration-dependent manner [26Cohen ML, Douvdevani A, Chaimovitz C, Shany S. Regulation of TNF-alpha by 1alpha,25-dihydroxyvitamin D3 in human macrophages from CAPD patients. Kidney Int 2001; 59(1): 69-75.[http://dx.doi.org/10.1046/j.1523-1755.2001.00467.x] [PMID: 11135059] -28Takahashi K, Horiuchi H, Ohta T, Komoriya K, Ohmori H, Kamimura T. 1 alpha,25-dihydroxyvitamin D3 suppresses interleukin-1beta-induced interleukin-8 production in human whole blood: An involvement of erythrocytes in the inhibition. Immunopharmacol Immunotoxicol 2002; 24(1): 1-15.[http://dx.doi.org/10.1081/IPH-120003399] [PMID: 12022438] ]. Vitamin D is capable of inhibiting LPS-mediated production of various inflammatory chemokines and cytokines and TLR4 and 5 signaling in myometrial smooth muscle cells; in contrast, IL-10 and TLR10 expression increases in response to treatment with vitamin D, suggesting an inhibitory role in infection-related inflammation [29Thota C, Farmer T, Garfield RE, Menon R, Al-Hendy A. Vitamin D elicits anti-inflammatory response, inhibits contractile-associated proteins, and modulates Toll-like receptors in human myometrial cells. Reprod Sci 2013; 20(4): 463-75.[http://dx.doi.org/10.1177/1933719112459225] [PMID: 23012315] ]. In addition, VDR is expressed also on trophoblast cells, which are influenced by the locally synthesized 1,25(OH)2D, either in an autocrine or paracrine way [30Evans KN, Bulmer JN, Kilby MD, Hewison M. Vitamin D and placental-decidual function. J Soc Gynecol Investig 2004; 11(5): 263-71.[http://dx.doi.org/10.1016/j.jsgi.2004.02.002] [PMID: 15219879] -32Pospechova K, Rozehnal V, Stejskalova L, et al. Expression and activity of vitamin D receptor in the human placenta and in choriocarcinoma BeWo and JEG-3 cell lines. Mol Cell Endocrinol 2009; 299(2): 178-87.[http://dx.doi.org/10.1016/j.mce.2008.12.003] [PMID: 19133314] ]. The local synthesis of active vitamin D seems to play a key role in placental innate immunity regulation [30Evans KN, Bulmer JN, Kilby MD, Hewison M. Vitamin D and placental-decidual function. J Soc Gynecol Investig 2004; 11(5): 263-71.[http://dx.doi.org/10.1016/j.jsgi.2004.02.002] [PMID: 15219879] ]. In addition, there is evidence that vitamin D exerts an anti-inflammatory role on trophoblast cells isolated both from normal and preeclampsia subjects [33Díaz L, Noyola-Martínez N, Barrera D, et al. Calcitriol inhibits TNF-alpha-induced inflammatory cytokines in human trophoblasts. J Reprod Immunol 2009; 81(1): 17-24.[http://dx.doi.org/10.1016/j.jri.2009.02.005] [PMID: 19501915] , 34Noyola-Martínez N, Díaz L, Avila E, Halhali A, Larrea F, Barrera D. Calcitriol downregulates TNF-α and IL-6 expression in cultured placental cells from preeclamptic women. Cytokine 2013; 61(1): 245-50.[http://dx.doi.org/10.1016/j.cyto.2012.10.001] [PMID: 23103122] ].

1.2. Anti-Phospholipid Antibody Syndrome (APS)

APS is characterized by arterial or venous thrombosis and/or pregnancy morbidity with persistent positivity of aPL detected by means of three formal diagnostic assays: anti-cardiolipin (aCL), anti-β2glycoprotein I (anti-β2GPI) and Lupus Anticoagulant (LA) [35Miyakis S, Lockshin MD, Atsumi T, et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Haemost 2006; 4(2): 295-306.[http://dx.doi.org/10.1111/j.1538-7836.2006.01753.x] [PMID: 16420554] -37Giannakopoulos B, Krilis SA. The pathogenesis of the antiphospholipid syndrome. N Engl J Med 2013; 368(11): 1033-44.[http://dx.doi.org/10.1056/NEJMra1112830] [PMID: 23484830] ]. The reactivity with the LA assay is mainly mediated by antibodies directed against prothrombin and β2GPI, whereas aCL positivity is mainly caused by β2GPI-dependent aPL. Despite persistent positivity of aPL antibodies, thrombotic events in patients with APS occur occasionally. Based on this clinical observation, a “two hit” hypothesis has been proposed in which the antibody (representing the first hit) induces a thrombophilic state, and the presence of another thrombophilic condition provides the second hit, triggering clotting [36Meroni PL, Borghi MO, Raschi E, Tedesco F. Pathogenesis of antiphospholipid syndrome: Understanding the antibodies. Nat Rev Rheumatol 2011; 7(6): 330-9.[http://dx.doi.org/10.1038/nrrheum.2011.52] [PMID: 21556027] ].

Immunoglobulin G (IgG) from APS patients are able to induce endothelial dysfunction, monocyte and platelet activation, as well as overexpression of Tissue Factor (TF), adhesion molecules, and proinflammatory cytokines through activation of TLR4 [38Lambrianides A, Carroll CJ, Pierangeli SS, et al. Effects of polyclonal IgG derived from patients with different clinical types of the antiphospholipid syndrome on monocyte signaling pathways. J Immunol 2010; 184(12): 6622-8.[http://dx.doi.org/10.4049/jimmunol.0902765] [PMID: 20483743] , 39Kornberg A, Blank M, Kaufman S, Shoenfeld Y. Induction of tissue factor-like activity in monocytes by anti-cardiolipin antibodies. J Immunol 1994; 153(3): 1328-32.[PMID: 8027560] ]. There is evidence that aPL may also activate endothelial cells via TLR2- and TLR4-mediated signaling [40Alard JE, Gaillard F, Daridon C, Shoenfeld Y, Jamin C, Youinou P. TLR2 is one of the endothelial receptors for beta 2-glycoprotein I. J Immunol 2010; 185(3): 1550-7.[http://dx.doi.org/10.4049/jimmunol.1000526] [PMID: 20601596] , 41Borghi MO, Raschi E, Grossi C, Chighizola CB, Meroni PL. Toll-like receptor 4 and β2 glycoprotein I interaction on endothelial cells. Lupus 2014; 23(12): 1302-4.[http://dx.doi.org/10.1177/0961203314536479] [PMID: 25228733] ]. LPS is the natural ligand of TLR4 and experimental data demonstrate that LPS increases the expression of β2GPI in vascular tissues and triggers aPL-mediated thrombosis [42Raschi E, Chighizola CB, Grossi C, et al. β2-glycoprotein I, lipopolysaccharide and endothelial TLR4: Three players in the two hit theory for anti-phospholipid-mediated thrombosis. J Autoimmun 2014; 55: 42-50.[http://dx.doi.org/10.1016/j.jaut.2014.03.001] [PMID: 24685231] ]. It is well known that the second hit can be provided by several triggers, such as surgery, inflammatory and infectious processes [36Meroni PL, Borghi MO, Raschi E, Tedesco F. Pathogenesis of antiphospholipid syndrome: Understanding the antibodies. Nat Rev Rheumatol 2011; 7(6): 330-9.[http://dx.doi.org/10.1038/nrrheum.2011.52] [PMID: 21556027] , 43Shoenfeld Y, Blank M, Cervera R, Font J, Raschi E, Meroni PL. Infectious origin of the antiphospholipid syndrome. Ann Rheum Dis 2006; 65(1): 2-6.[http://dx.doi.org/10.1136/ard.2005.045443] [PMID: 16344491] ]. In particular, the combination of a second hit plus the perturbation of endothelial cells mediated by aPL may overcome the threshold for triggering thrombosis [38Lambrianides A, Carroll CJ, Pierangeli SS, et al. Effects of polyclonal IgG derived from patients with different clinical types of the antiphospholipid syndrome on monocyte signaling pathways. J Immunol 2010; 184(12): 6622-8.[http://dx.doi.org/10.4049/jimmunol.0902765] [PMID: 20483743] , 41Borghi MO, Raschi E, Grossi C, Chighizola CB, Meroni PL. Toll-like receptor 4 and β2 glycoprotein I interaction on endothelial cells. Lupus 2014; 23(12): 1302-4.[http://dx.doi.org/10.1177/0961203314536479] [PMID: 25228733] ].

It is widely recognized that pathogenic mechanisms in obstetric APS are different from those involved in thrombotic manifestations. Intraplacental thrombosis, with subsequent impairment of maternal-fetal blood exchange, was initially suggested as the main pathogenic mechanism of aPL-induced fetal loss and heparin was introduced in the treatment of pregnant APS patients based on this assumption. Indeed, in vitro studies have shown that aPL may induce a procoagulant state at the placental level through several mechanisms [44Peaceman AM, Rehnberg KA. The effect of immunoglobulin G fractions from patients with lupus anticoagulant on placental prostacyclin and thromboxane production. Am J Obstet Gynecol 1993; 169(6): 1403-6.[http://dx.doi.org/10.1016/0002-9378(93)90408-B] [PMID: 8267036] , 45Nayar R, Lage JM. Placental changes in a first trimester missed abortion in maternal systemic lupus erythematosus with antiphospholipid syndrome; A case report and review of the literature. Hum Pathol 1996; 27(2): 201-6.[http://dx.doi.org/10.1016/S0046-8177(96)90377-9] [PMID: 8617465] ]. However, it has become clear that thrombotic mechanisms do not fully explain recurrent fetal loss in these patients, and proinflammatory rather than prothrombotic mechanisms have been advocated, such as complement activation, inhibition of trophoblast proliferation, differentiation and migration, defective angiogenesis, embryonal toxicity on preimplantation embryo, decreased production of human Chorionic Gonadotropin (hCG) and human placental lactogen, decreased expression of annexin V as well as acute placental inflammation [36Meroni PL, Borghi MO, Raschi E, Tedesco F. Pathogenesis of antiphospholipid syndrome: Understanding the antibodies. Nat Rev Rheumatol 2011; 7(6): 330-9.[http://dx.doi.org/10.1038/nrrheum.2011.52] [PMID: 21556027] , 46Girardi G, Berman J, Redecha P, et al. Complement C5a receptors and neutrophils mediate fetal injury in the antiphospholipid syndrome. J Clin Invest 2003; 112(11): 1644-54.[http://dx.doi.org/10.1172/JCI200318817] [PMID: 14660741] -55Pantham P, Abrahams VM, Chamley LW. The role of anti-phospholipid antibodies in autoimmune reproductive failure. Reproduction 2016; 151(5): R79-90.[http://dx.doi.org/10.1530/REP-15-0545] [PMID: 26884418] ]. Furthermore, aPL have been shown to specifically destroy trophoblast, inhibit syncytium formation, halt hCG production, and limit trophoblast invasion [56Rote NS, Vogt E, DeVere G, Obringer AR, Ng AK. The role of placental trophoblast in the pathophysiology of the antiphospholipid antibody syndrome. Am J Reprod Immunol 1998; 39(2): 125-36.[http://dx.doi.org/10.1111/j.1600-0897.1998.tb00344.x] [PMID: 9506210] , 57McIntyre JA. Antiphospholipid antibodies in implantation failures. Am J Reprod Immunol 2003; 49(4): 221-9.[http://dx.doi.org/10.1034/j.1600-0897.2003.01197.x] [PMID: 12852496] ]. The effect of heparin in the management of patients with obstetric APS seems to be based on mechanisms of action other than anticoagulation [58Han CS, Mulla MJ, Brosens JJ, et al. Aspirin and heparin effect on basal and antiphospholipid antibody modulation of trophoblast function. Obstet Gynecol 2011; 118(5): 1021-8.[http://dx.doi.org/10.1097/AOG.0b013e31823234ad] [PMID: 22015869] , 59Girardi G, Redecha P, Salmon JE. Heparin prevents antiphospholipid antibody-induced fetal loss by inhibiting complement activation. Nat Med 2004; 10(11): 1222-6.[http://dx.doi.org/10.1038/nm1121] [PMID: 15489858] ].

The clinical spectrum of APS actually comprises non-criteria manifestations such as APS nephropathy and Central Nervous System (CNS) symptoms (epilepsy and cognitive abnormalities). Also in this case, clinical manifestations are not fully explained by ischemic mechanisms. A direct effect of aPL on glomerular microcirculation and on neuronal cells has been proposed [60Shoenfeld Y, Meroni PL, Toubi E. Antiphospholipid syndrome and systemic lupus erythematosus: Are they separate entities or just clinical presentations on the same scale? Curr Opin Rheumatol 2009; 21(5): 495-500.[http://dx.doi.org/10.1097/BOR.0b013e32832effdd] [PMID: 19593144] , 61Katzav A, Shoenfeld Y, Chapman J. The pathogenesis of neural injury in animal models of the antiphospholipid syndrome. Clin Rev Allergy Immunol 2010; 38(2-3): 196-200.[http://dx.doi.org/10.1007/s12016-009-8154-x] [PMID: 19557316] ].

1.3. Management of APS and Primary Prevention in aPL Carriers

Due to the risk of relapse after the first episode of thrombosis, the management of patients with APS envisages long-term anticoagulation [62Erkan D, Aguiar CL, Andrade D, et al. 14th International Congress on Antiphospholipid Antibodies: Task force report on antiphospholipid syndrome treatment trends. Autoimmun Rev 2014; 13(6): 685-96.[http://dx.doi.org/10.1016/j.autrev.2014.01.053] [PMID: 24468415] ]. Bleeding complications may occur as a result of anticoagulation therapy, thrombocytopenia, use of NSAIDs and congenital or acquired hemorrhagic syndromes such as the rare but possible Lupus anticoagulant-hypoprothrombinemia syndrome [63Erkan D, Bateman H, Lockshin MD. Lupus anticoagulant-hypoprothrombinemia syndrome associated with systemic lupus erythematosus: Report of 2 cases and review of literature. Lupus 1999; 8(7): 560-4.[http://dx.doi.org/10.1191/096120399678840846] [PMID: 10483036] , 64Cugno M, Gualtierotti R, Tedeschi A, Meroni PL. Autoantibodies to coagulation factors: From pathophysiology to diagnosis and therapy. Autoimmun Rev 2014; 13(1): 40-8.[http://dx.doi.org/10.1016/j.autrev.2013.08.001] [PMID: 23954454] ]. Non-pharmacological management of APS is aimed at managing other CV risk factors such as arterial hypertension, obesity (body mass index >30 kg/m2), diabetes mellitus, smoking, active or treated cancer, use of oral contraceptives, underlying systemic autoimmune diseases and genetic hypercoagulable states [65Raso S, Sciascia S, Kuzenko A, Castagno I, Marozio L, Bertero MT. Bridging therapy in antiphospholipid syndrome and antiphospholipid antibodies carriers: Case series and review of the literature. Autoimmun Rev 2015; 14(1): 36-42.[http://dx.doi.org/10.1016/j.autrev.2014.09.002] [PMID: 25242343] ], or venous prothrombotic factors [66Legault KJ, Ugarte A, Crowther MA, Ruiz-Irastorza G. Prevention of recurrent thrombosis in antiphospholipid syndrome: Different from the general population? Curr Rheumatol Rep 2016; 18(5): 26.[http://dx.doi.org/10.1007/s11926-016-0573-0] [PMID: 27032789] ].

Isolated positivity of aPL in healthy carriers is often a conundrum for clinicians. Primary prophylaxis in aPL carriers with Low Dose Aspirine (LDA) remains controversial due to the lack of relevant evidence-based data. In particular, it is unclear whether the benefits of LDA in a low-risk population may outweigh the increased risk of bleeding [67Arnaud L, Conti F, Massaro L, Denas G, Chasset F, Pengo V. Primary thromboprophylaxis with low-dose aspirin and antiphospholipid antibodies: Pro’s and Con’s. Autoimmun Rev 2017; 16(11): 1103-8.[http://dx.doi.org/10.1016/j.autrev.2017.09.003] [PMID: 28911988] ]. Based on aPL specificity and isotype, it is possible to stratify patients, with LA and triple positivity bearing the highest risk for a first thrombotic event [68Galli M, Luciani D, Bertolini G, Barbui T. Anti-beta 2-glycoprotein I, antiprothrombin antibodies, and the risk of thrombosis in the antiphospholipid syndrome. Blood 2003; 102(8): 2717-23.[http://dx.doi.org/10.1182/blood-2002-11-3334] [PMID: 12816875] ]. In particular, LA raises the risk of thrombosis by approximately 4-fold [69de Groot PG, Lutters B, Derksen RH, Lisman T, Meijers JC, Rosendaal FR. Lupus anticoagulants and the risk of a first episode of deep venous thrombosis. J Thromb Haemost 2005; 3(9): 1993-7.[http://dx.doi.org/10.1111/j.1538-7836.2005.01485.x] [PMID: 16102105] ] and aPL IgG isotype is considered clinically more meaningful compared to IgM isotype [70Ruffatti A, Del Ross T, Ciprian M, et al. Risk factors for a first thrombotic event in antiphospholipid antibody carriers. A multicentre, retrospective follow-up study. Ann Rheum Dis 2009; 68(3): 397-9.[http://dx.doi.org/10.1136/ard.2008.096669] [PMID: 18812393] ]. Although hydroxychloroquine has been demonstrated to reduce the risk for venous thromboembolism in SLE [71Ruiz-Irastorza G, Egurbide MV, Pijoan JI, et al. Effect of antimalarials on thrombosis and survival in patients with systemic lupus erythematosus. Lupus 2006; 15(9): 577-83.[http://dx.doi.org/10.1177/0961203306071872] [PMID: 17080912] -73Becker-Merok A, Nossent J. Prevalence, predictors and outcome of vascular damage in systemic lupus erythematosus. Lupus 2009; 18(6): 508-15.[http://dx.doi.org/10.1177/0961203308099233] [PMID: 19395452] ], and to have a CV protective effect [74Fasano S, Pierro L, Pantano I, Iudici M, Valentini G. Longterm hydroxychloroquine therapy and low-dose aspirin may have an additive effectiveness in the primary prevention of cardiovascular events in patients with systemic lupus erythematosus. J Rheumatol 2017; 44(7): 1032-8.[http://dx.doi.org/10.3899/jrheum.161351] [PMID: 28507183] ], evidence is controversial for primary prevention of thrombotic events [75Petri M. Use of hydroxychloroquine to prevent thrombosis in systemic lupus erythematosus and in antiphospholipid antibody-positive patients. Curr Rheumatol Rep 2011; 13(1): 77-80.[http://dx.doi.org/10.1007/s11926-010-0141-y] [PMID: 20978875] ]. Also statins have been advocated as effective drugs for primary and secondary prevention of thrombotic events in APS, not only due to their lipid-lowering effect, but also because of their pleiotropic immunomodulatory, anti-inflammatory, and antithrombotic properties [76Danesh FR, Anel RL, Zeng L, Lomasney J, Sahai A, Kanwar YS. Immunomodulatory effects of HMG-CoA reductase inhibitors. Arch Immunol Ther Exp (Warsz) 2003; 51(3): 139-48.[PMID: 12894868] ]. However, in the absence of sound evidence, statins cannot be recommended in APS patients in the absence of hyperlipidemia [58Han CS, Mulla MJ, Brosens JJ, et al. Aspirin and heparin effect on basal and antiphospholipid antibody modulation of trophoblast function. Obstet Gynecol 2011; 118(5): 1021-8.[http://dx.doi.org/10.1097/AOG.0b013e31823234ad] [PMID: 22015869] ]. Therefore, primary prevention in aPL carriers is still an unmet need.

1.4. Endothelial Perturbation in Anti-Phospholipid Antibody Syndrome

It is widely accepted that anti-β2GPI antibodies, in the presence of β2GPI, can induce endothelial cell perturbation [77Allen KL, Fonseca FV, Betapudi V, Willard B, Zhang J, McCrae KR. A novel pathway for human endothelial cell activation by antiphospholipid/anti-β2 glycoprotein I antibodies. Blood 2012; 119(3): 884-93.[http://dx.doi.org/10.1182/blood-2011-03-344671] [PMID: 22106343] , 78Cugno M, Borghi MO, Lonati LM, et al. Patients with antiphospholipid syndrome display endothelial perturbation. J Autoimmun 2010; 34(2): 105-10.[http://dx.doi.org/10.1016/j.jaut.2009.07.004] [PMID: 19656656] ]. In the past, our group has demonstrated that anti-β2GPI is able to induce a proinflammatory phenotype with up-regulation of Endothelial-Leukocyte Adhesion Molecule 1 (ELAM-1) and Intercellular Adhesion Molecule 1 (ICAM-1) [42Raschi E, Chighizola CB, Grossi C, et al. β2-glycoprotein I, lipopolysaccharide and endothelial TLR4: Three players in the two hit theory for anti-phospholipid-mediated thrombosis. J Autoimmun 2014; 55: 42-50.[http://dx.doi.org/10.1016/j.jaut.2014.03.001] [PMID: 24685231] ]. Although the precise mechanism of aPL-induced activation of endothelial cells remains to be determined, TLR4 has been demonstrated to play a central role [36Meroni PL, Borghi MO, Raschi E, Tedesco F. Pathogenesis of antiphospholipid syndrome: Understanding the antibodies. Nat Rev Rheumatol 2011; 7(6): 330-9.[http://dx.doi.org/10.1038/nrrheum.2011.52] [PMID: 21556027] , 79Laplante P, Fuentes R, Salem D, et al. Antiphospholipid antibody-mediated effects in an arterial model of thrombosis are dependent on Toll-like receptor 4. Lupus 2016; 25(2): 162-76.[http://dx.doi.org/10.1177/0961203315603146] [PMID: 26391610] ]. More recently, other TLRs have been investigated as potentially involved in APS pathogenesis. TLR3 activation via polyinosinic:polycytidylic acid (Poly I:C) seems to enhance LPS/TLR4-induced production of Interferon (IFN) [80Koch SR, Lamb FS, Hellman J, Sherwood ER, Stark RJ. Potentiation and tolerance of toll-like receptor priming in human endothelial cells. Transl Res 2017; 180(53): 53-67. e4]. TLR9 recognizes viral and bacterial DNA, which contain unmethylated CpG dinucleotides. CpG DNA was found to exert potent proinflammatory actions, such as the expression of adhesion molecules, IL-8, and monocyte chemoattractant protein-1 in Human Endothelial Cells (HUVECs), thus facilitating leukocyte trafficking [81El Kebir D, József L, Pan W, Wang L, Filep JG. Bacterial DNA activates endothelial cells and promotes neutrophil adherence through TLR9 signaling. J Immunol 2009; 182(7): 4386-94.[http://dx.doi.org/10.4049/jimmunol.0803044] [PMID: 19299739] , 82József L, Khreiss T, El Kebir D, Filep JG. Activation of TLR-9 induces IL-8 secretion through peroxynitrite signaling in human neutrophils. J Immunol 2006; 176(2): 1195-202.[http://dx.doi.org/10.4049/jimmunol.176.2.1195] [PMID: 16394009] ]. Evidence suggests that aPL can also directly activate neutrophils, as a consequence of enhanced granule release, oxidative burst, and increase IL-8 production [83Arvieux J, Jacob MC, Roussel B, Bensa JC, Colomb MG. Neutrophil activation by anti-beta 2 glycoprotein I monoclonal antibodies via Fc gamma receptor II. J Leukoc Biol 1995; 57(3): 387-94.[http://dx.doi.org/10.1002/jlb.57.3.387] [PMID: 7884309] , 84Gladigau G, Haselmayer P, Scharrer I, et al. A role for Toll-like receptor mediated signals in neutrophils in the pathogenesis of the anti-phospholipid syndrome. PLoS One 2012; 7(7): e42176.[http://dx.doi.org/10.1371/journal.pone.0042176] [PMID: 22860075] ]. After activation, adhesion to endothelial cells and transmigration, neutrophils further amplify the inflammatory process. Furthermore, after stimulation within endosomes TLRs activate transcription factors Interferon Regulatory Factor (IRF)3 or IRF7 and induce type I IFNs [85Lee BL, Barton GM. Trafficking of endosomal Toll-like receptors. Trends Cell Biol 2014; 24(6): 360-9.[http://dx.doi.org/10.1016/j.tcb.2013.12.002] [PMID: 24439965] ].

1.5. Vitamin D and the Anti-Phospholipid Antibody Syndrome

The role of vitamin D in immune system regulation could contribute to APS pathogenesis. To date, no data are available regarding VDR polymorphisms and APS. Vitamin D deficiency (<10–20 ng/ml) and insufficiency (<30 ng/mL) are relatively common in autoimmune diseases [86Agmon-Levin N, Theodor E, Segal RM, Shoenfeld Y. Vitamin D in systemic and organ-specific autoimmune diseases. Clin Rev Allergy Immunol 2013; 45(2): 256-66.[http://dx.doi.org/10.1007/s12016-012-8342-y] [PMID: 23238772] ], including APS [87Orbach H, Zandman-Goddard G, Amital H, et al. Novel biomarkers in autoimmune diseases: Prolactin, ferritin, vitamin D, and TPA levels in autoimmune diseases. Ann N Y Acad Sci 2007; 1109: 385-400.[http://dx.doi.org/10.1196/annals.1398.044] [PMID: 17785327] ]. Vitamin D deficiency has been reported to be significantly correlated with arterial and venous thrombosis as well as with non-criteria APS manifestations such as neurological and ophthalmic manifestations, pulmonary hypertension, livedo reticularis and skin ulcerations in APS patients [88Agmon-Levin N, Blank M, Zandman-Goddard G, et al. Vitamin D: an instrumental factor in the anti-phospholipid syndrome by inhibition of tissue factor expression. Ann Rheum Dis 2011; 70(1): 145-50.[http://dx.doi.org/10.1136/ard.2010.134817] [PMID: 20980705] -90Piantoni S, Andreoli L, Allegri F, Meroni PL, Tincani A. Low levels of vitamin D are common in primary antiphospholipid syndrome with thrombotic disease. Reumatismo 2012; 64(5): 307-13.[http://dx.doi.org/10.4081/reumatismo.2012.307] [PMID: 23256106] ]. Similarly, in a large Swedish study an increase of 50% in the risk of venous and arterial thrombosis was observed during winter compared with other seasons, whereas a significantly lower risk of thrombosis was reported in women who were more sun-exposed [91Lindqvist PG, Epstein E, Olsson H. Does an active sun exposure habit lower the risk of venous thrombotic events? A D-lightful hypothesis. J Thromb Haemost 2009; 7(4): 605-10.[http://dx.doi.org/10.1111/j.1538-7836.2009.03312.x] [PMID: 19335448] ].

Vitamin D is able to suppress the expression of TLRs such as TLR4, which is responsible for the activation of nuclear factor κB and the signaling cascade that ultimately induces a prothrombotic state in endothelial cells by aPL. Reduced expression of TLRs is accompanied by impaired nuclear factor κB translocation to the nucleus and by reduced TLR-dependent signal transduction [63Erkan D, Bateman H, Lockshin MD. Lupus anticoagulant-hypoprothrombinemia syndrome associated with systemic lupus erythematosus: Report of 2 cases and review of literature. Lupus 1999; 8(7): 560-4.[http://dx.doi.org/10.1191/096120399678840846] [PMID: 10483036] ]. Vitamin D can reduce TF expression induced by proinflammatory stimuli such as TNF-α or LPS on monocytes [92Chung J, Koyama T, Ohsawa M, Shibamiya A, Hoshi A, Hirosawa S. 1,25(OH)(2)D(3) blocks TNF-induced monocytic tissue factor expression by inhibition of transcription factors AP-1 and NF-kappaB. Lab Invest 2007; 87(6): 540-7.[http://dx.doi.org/10.1038/labinvest.3700550] [PMID: 17401435] , 93Ohsawa M, Koyama T, Yamamoto K, Hirosawa S, Kamei S, Kamiyama R. 1alpha,25-dihydroxyvitamin D(3) and its potent synthetic analogs downregulate tissue factor and upregulate thrombomodulin expression in monocytic cells, counteracting the effects of tumor necrosis factor and oxidized LDL. Circulation 2000; 102(23): 2867-72.[http://dx.doi.org/10.1161/01.CIR.102.23.2867] [PMID: 11104746] ].

Vitamin D deficiency has been linked to a three-fold increase in preeclampsia risk [94Wei SQ, Audibert F, Hidiroglou N, et al. Longitudinal vitamin D status in pregnancy and the risk of pre-eclampsia. BJOG 2012; 119(7): 832-9.[http://dx.doi.org/10.1111/j.1471-0528.2012.03307.x] [PMID: 22462640] , 95Robinson CJ, Alanis MC, Wagner CL, Hollis BW, Johnson DD. Plasma 25-hydroxyvitamin D levels in early-onset severe preeclampsia. Am J Obstet Gynecol 2010; 203(4) :366 e1-6]. In a cross-sectional study conducted in women with recurrent pregnancy loss, an association between low vitamin D levels and positivity for aPL was found [96Ota K, Dambaeva S, Han AR, Beaman K, Gilman-Sachs A, Kwak-Kim J. Vitamin D deficiency may be a risk factor for recurrent pregnancy losses by increasing cellular immunity and autoimmunity. Hum Reprod 2014; 29(2): 208-19.[http://dx.doi.org/10.1093/humrep/det424] [PMID: 24277747] ]. In particular, during weeks 24-26 of pregnancy, women with low serum levels of vitamin D have significantly increased risk [97Baker AM, Haeri S, Camargo CA Jr, Espinola JA, Stuebe AM. A nested case-control study of midgestation vitamin D deficiency and risk of severe preeclampsia. J Clin Endocrinol Metab 2010; 95(11): 5105-9.[http://dx.doi.org/10.1210/jc.2010-0996] [PMID: 20719829] ].

Vitamin D can inhibit TLR4 signaling in peripheral blood monocytes of pregnant women at risk for preeclampsia, thereby down-regulating inflammatory pathways and reducing the risk of endothelial cell damage [98Qian L, Wang H, Wu F, Li M, Chen W, Lv L. Vitamin D3 alters Toll-like receptor 4 signaling in monocytes of pregnant women at risk for preeclampsia. Int J Clin Exp Med 2015; 8(10): 18041-9.[PMID: 26770399] ]. Notably, Gysler et al. found that vitamin D, either alone or in combination with low molecular weight heparin, is able to attenuate the inflammatory response in trophoblast cells after exposure to a monoclonal murine anti-human β2GPI [99Gysler SM, Mulla MJ, Stuhlman M, et al. Vitamin D reverses aPL-induced inflammation and LMWH-induced sFlt-1 release by human trophoblast. Am J Reprod Immunol 2015; 73(3): 242-50.[http://dx.doi.org/10.1111/aji.12301] [PMID: 25070806] ].

1.6. Vitamin D and Endothelial Perturbation in Anti-Phospholipid Syndrome

There is evidence that vitamin D is able to directly modulate endothelial perturbation due to its anti-inflammatory effect. Vitamin D was shown to reduce the expression of adhesion molecules such as ELAM-1 and ICAM-1 on endothelial cells [100Chen SF. 1 alpha, 25-Dihydroxyvitamin D3 decreased ICAM-1 and ELAM-1 expressions on pulmonary microvascular endothelial cells and neutrophil motivation. J Steroid Biochem Mol Biol 1995; 52(1): 67-70.[http://dx.doi.org/10.1016/0960-0760(94)00153-D] [PMID: 7532002] ]. Equils et al. found that pretreatment of human microvessel endothelial cells with vitamin D inhibited LPS-induced activation of transcription factor NF-κB and secretion of IL-8 [101Equils O, Naiki Y, Shapiro AM, et al. 1,25-Dihydroxyvitamin D inhibits lipopolysaccharide-induced immune activation in human endothelial cells. Clin Exp Immunol 2006; 143(1): 58-64.[http://dx.doi.org/10.1111/j.1365-2249.2005.02961.x] [PMID: 16367934] ]. Furthermore, a reduction of IL-8 secretion was shown in coronary artery endothelial cells [102Kudo K, Hasegawa S, Suzuki Y, et al. 1α,25-Dihydroxyvitamin D(3) inhibits vascular cellular adhesion molecule-1 expression and interleukin-8 production in human coronary arterial endothelial cells. J Steroid Biochem Mol Biol 2012; 132(3-5): 290-4.[http://dx.doi.org/10.1016/j.jsbmb.2012.07.003] [PMID: 22841897] ]. Despite the well-known role of endothelial cell perturbation in APS, evidence of a direct role of vitamin D on aPL-mediated endothelial cell perturbation is still scarce. Indeed, Agmon-Levin et al reported that vitamin D is a potent inhibitor of the in vitro expression of TF in endothelial cells stimulated by anti-β2GPI antibodies derived from APS patients [88Agmon-Levin N, Blank M, Zandman-Goddard G, et al. Vitamin D: an instrumental factor in the anti-phospholipid syndrome by inhibition of tissue factor expression. Ann Rheum Dis 2011; 70(1): 145-50.[http://dx.doi.org/10.1136/ard.2010.134817] [PMID: 20980705] ].

With this as background, we investigated the expression of ELAM-1 and ICAM-1, of TLR3 and 9, the secretion of the proinflammatory chemokine IL-8 and the expression of type I IFNs (IFN-α, IFN-β) in cultures of HUVEC pretreated with vitamin D and incubated with inflammatory stimuli such as LPS, TNF-α or anti-β2GPI IgG isolated by APS subjects. After informed consent, we recruited two subjects with high titer of anti-β2GPI and aCL IgG and IgM and with LA activity fulfilling APS 2006 criteria [35Miyakis S, Lockshin MD, Atsumi T, et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Haemost 2006; 4(2): 295-306.[http://dx.doi.org/10.1111/j.1538-7836.2006.01753.x] [PMID: 16420554] ]. Polyclonal IgG from five age- and sex-matched healthy subjects were included as controls (HD). Serum IgG were isolated as as previously described [103Tincani A, Spatola L, Prati E, et al. The anti-beta2-glycoprotein I activity in human anti-phospholipid syndrome sera is due to monoreactive low-affinity autoantibodies directed to epitopes located on native beta2-glycoprotein I and preserved during species’ evolution. J Immunol 1996; 157(12): 5732-8.[PMID: 8955227] ].

ELAM-1 is one of the main endothelial activation markers whose expression is up-regulated in response to inflammatory stimuli (e.g. LPS and TNF-α) and to anti-β2GPI antibodies [104Betapudi V, Lominadze G, Hsi L, Willard B, Wu M, McCrae KR. Anti-β2GPI antibodies stimulate endothelial cell microparticle release via a nonmuscle myosin II motor protein-dependent pathway. Blood 2013; 122(23): 3808-17.[http://dx.doi.org/10.1182/blood-2013-03-490318] [PMID: 23954892] ]. Overnight pre-treatment with vitamin D significantly reduced ELAM-1 expression induced by both inflammatory stimuli (LPS and TNF-α) and anti-β2GPI IgG (p<0.05) in our in vitro model. We observed no changes in ELAM-1 expression in all other experimental conditions (Fig. 1). ICAM-1 is an adhesion molecule whose expression is both constitutive and up-regulated in response to inflammatory stimuli (e.g. LPS and TNF-α) or antibodies to anti-β2GPI [104Betapudi V, Lominadze G, Hsi L, Willard B, Wu M, McCrae KR. Anti-β2GPI antibodies stimulate endothelial cell microparticle release via a nonmuscle myosin II motor protein-dependent pathway. Blood 2013; 122(23): 3808-17.[http://dx.doi.org/10.1182/blood-2013-03-490318] [PMID: 23954892] ]. Overnight pre-treatment with vitamin D significantly reduced ICAM-1 transcription induced by inflammatory stimuli (LPS and TNF-α) and anti-β2GPI polyclonal IgG (p<0.05) (data not shown).

IL-8 is a proinflammatory chemokine whose secretion is up-regulated in response to inflammatory stimuli. It is one of the key regulators of leukocyte trafficking/activation in inflammation and it is also involved in tissue injury, fibrosis and angiogenesis [105Russo RC, Garcia CC, Teixeira MM, Amaral FA. The CXCL8/IL-8 chemokine family and its receptors in inflammatory diseases. Expert Rev Clin Immunol 2014; 10(5): 593-619.[http://dx.doi.org/10.1586/1744666X.2014.894886] [PMID: 24678812] ]. IL-8 secretion increases in response to LPS and aPL [84Gladigau G, Haselmayer P, Scharrer I, et al. A role for Toll-like receptor mediated signals in neutrophils in the pathogenesis of the anti-phospholipid syndrome. PLoS One 2012; 7(7): e42176.[http://dx.doi.org/10.1371/journal.pone.0042176] [PMID: 22860075] ]. In agreement with previous studies, we found that overnight pre-treatment with vitamin D resulted in a significant reduction of IL-8 concentration in supernatants from HUVEC cultures stimulated with LPS, Poly I:C and aPL IgG (p<0.05) (Fig. 2).

Fig. (1)
ELAM-1 expression in endothelial cells treated with APS IgG and vitamin D. Overnight pretreatment with vitamin D (1α,25-dihydroxy-vitamin D3, 1,25-OH-D3; SIGMA-Aldrich - Saint Louis, MI, USA) 80 nM significantly reduced the expression of Endothelial-Leukocyte Adhesion Molecule 1 (ELAM-1) induced by inflammatory stimuli (LPS and TNF-α) and anti-β2GPI IgG. No changes were detected in all other experimental conditions. Human umbilical vein endothelial cells (HUVECs) isolated from the umbilical cord vein were cultured in E199 (Gibco-Life Technologies - Groningen, The Netherlands), supplemented with 20% heat-injected Fetal Bovine Serum (FBS, PAA-GE Healthcare - Buckinghamshire, United Kingdom) in the presence of LPS (100 ng/mL, SIGMA-Aldrich - Saint Louis, MI, USA); TNF-α (40 ng/mL, R&D Systems – Minneapolis, MN, USA); or polyclonal IgG directed against β2GPI isolated from APS patient sera and IgG (200 μg/mL) from a pool of Healthy Donor (HD) sera respectively. IgG were isolated on a protein-G-Sefarose column (HiTrap Protein G, GE Healthcare Bio-Science AB, Uppsala, Sweden) as previously described by Tincani et al (97). A cyto-ELISA monoclonal IgG murine antibody was used to detect human ELAM-1 (R&D systems - Minneapolis, MN, USA). The choice of the doses and regimens was based on previous unpublished experimental data from our laboratory.
anti-b2GPI: Anti-b2glycoprotein I; HD: Healthy Donor; LPS: lipopolysaccharide; NT: No Treatment; TNF: Tumor Necrosis Factor-alpha. (*p <0.05)


Fig. (2)
IL-8 secretion in endothelial cells treated with APS IgG and vitamin D Overnight pretreatment with vitamin D (1α,25-dihydroxy-vitamin D3, 1,25-OH-D3; SIGMA-Aldrich - Saint Louis, MI, USA) 80 nM resulted in a significant reduction of upregulated levels of interleukin (IL)-8 (R&D Systems – Minneapolis, MN, USA). No significant difference was observed after treatment with Healthy Donor (HD) IgG and culture medium alone. Human Umbilical Vein Endothelial Cells (HUVECs) cultured in E199 (Gibco-Life Technologies - Groningen, The Netherlands), supplemented with 20% heat-injected Fetal Bovine Serum (FBS, PAA-GE Healthcare - Buckinghamshire, United Kingdom) in the presence of LPS (100 ng/mL, SIGMA-Aldrich - Saint Louis, MI, USA); Poly (I:C) (20 μg/mL, InvivoGen - San Diego, CA, USA); polyclonal IgG directed against β2GPI isolated from APS patient sera and HD IgG (200 μg/mL) from a pool of HD sera respectively. IgG were isolated on a protein-G-Sefarose column (HiTrap Protein G, GE Healthcare Bio-Science AB, Uppsala, Sweden) as previously described by Tincani et al., (97). The choice of the doses and regimens was based on previous unpublished experimental data from our laboratory
anti-b2GPI: anti-b2glycoprotein I, HD: Healthy Donor, IL-8: Interleukin-8, LPS: Lipopolysaccharide, NT: No Treatment, Poly (I:C): Polyinosinic:polycytidylic acid. (*p <0.05).


Both TLR3 and TLR9 are expressed on the endosomal membranes [85Lee BL, Barton GM. Trafficking of endosomal Toll-like receptors. Trends Cell Biol 2014; 24(6): 360-9.[http://dx.doi.org/10.1016/j.tcb.2013.12.002] [PMID: 24439965] ]. Therefore, we studied gene expression of these two receptors. The expression of TLR3 was significantly increased by Poly I:C and LPS, but not by anti-β2GPI, and was reduced by overnight pretreatment with vitamin D (p<0.05). TLR9 was up-regulated by LPS and IgG of patients with APS, but not in the presence of culture medium alone. A slight increase was found in the presence of HD IgG compared to the only culture medium. Overnight pretreatment with vitamin D resulted in increased TLR9 transcription in basal conditions, but significantly reduced receptor expression levels in the presence of LPS, anti-β2GPI IgG (p <0.01) and HD IgG (p <0.05) (data not shown).

Type I IFNs (IFN-α, IFN-β) are important mediators of the immune response that modulate the differentiation and proliferation of immune system cells and contribute to the pathogenesis of several autoimmune diseases [106Di Domizio J, Cao W. Fueling autoimmunity: Type I interferon in autoimmune diseases. Expert Rev Clin Immunol 2013; 9(3): 201-10.[http://dx.doi.org/10.1586/eci.12.106] [PMID: 23445195] ]. For example, patients with SLE display high and constant levels of IFN-α, responsible for disease activity and organ damage [107Rönnblom LE, Alm GV, Oberg KE. Autoimmunity after alpha-interferon therapy for malignant carcinoid tumors. Ann Intern Med 1991; 115(3): 178-83.[http://dx.doi.org/10.7326/0003-4819-115-3-178] [PMID: 2058872] , 108Eloranta ML, Lövgren T, Finke D, et al. Regulation of the interferon-alpha production induced by RNA-containing immune complexes in plasmacytoid dendritic cells. Arthritis Rheum 2009; 60(8): 2418-27.[http://dx.doi.org/10.1002/art.24686] [PMID: 19644885] ]. The analysis of IFN-α and IFN-β mRNA expression showed that pathological Poly I:C, LPS and IgG from APS patients resulted in a significant increase in transcription of both cytokines and a slight increase was also observed in the presence of HD IgG compared to the culture medium alone. The expression of type I IFNs was reduced after overnight pretreatment with vitamin D in all experimental conditions (p<0.01 for LPS and anti-β2GPI IgG; p<0.05 for Poly I:C and HD IgG) (data not shown).

4. DISCUSSION

The therapeutic role of vitamin D supplementation, including the dosage and definition of treatment targets (among which the standardization of the definition of “vitamin D deficiency”) in aPL-positive patients is still to be determined in prospective studies and randomized clinical trials.

Based on the available literature, the administration of vitamin D with a target serum level of vitamin D >30 ng/ml may be beneficial in APS patients. Vitamin D may act as a further countermeasure for secondary prevention in full-blown APS because of its ability to interfere with the prothrombotic mechanisms mediated by aPL. Inflammatory conditions, such as infectious events, may favor the occurrence of the first thrombotic event in asymptomatic aPL carriers [66Legault KJ, Ugarte A, Crowther MA, Ruiz-Irastorza G. Prevention of recurrent thrombosis in antiphospholipid syndrome: Different from the general population? Curr Rheumatol Rep 2016; 18(5): 26.[http://dx.doi.org/10.1007/s11926-016-0573-0] [PMID: 27032789] , 70Ruffatti A, Del Ross T, Ciprian M, et al. Risk factors for a first thrombotic event in antiphospholipid antibody carriers. A multicentre, retrospective follow-up study. Ann Rheum Dis 2009; 68(3): 397-9.[http://dx.doi.org/10.1136/ard.2008.096669] [PMID: 18812393] ]. In these patients, vitamin D could provide a potential new pharmacological approach for primary prevention. Finally, its ability to interfere with the mechanisms leading to miscarriages may be useful in preventing clinical manifestations also in obstetric patients. Indeed, vitamin D supplementation should be considered in all APS patients showing vitamin D deficiency and insufficiency [62Erkan D, Aguiar CL, Andrade D, et al. 14th International Congress on Antiphospholipid Antibodies: Task force report on antiphospholipid syndrome treatment trends. Autoimmun Rev 2014; 13(6): 685-96.[http://dx.doi.org/10.1016/j.autrev.2014.01.053] [PMID: 24468415] ].

Our experimental data, although preliminary, are in agreement with previous reports. We used an in vitro model of endothelial cell activation with HUVEC stimulated with proinflammatory stimuli - TNF-α and TLR specific agonists such as LPS and Poly I:C - or with anti-β2GPI IgG from APS patients compared with medium culture and HD IgG. We showed that vitamin D can reduce the expression of adhesion molecules such as ELAM-1 and ICAM-1, both expressed as a consequence of an inflammatory stimulus or incubation with aPL IgG [42Raschi E, Chighizola CB, Grossi C, et al. β2-glycoprotein I, lipopolysaccharide and endothelial TLR4: Three players in the two hit theory for anti-phospholipid-mediated thrombosis. J Autoimmun 2014; 55: 42-50.[http://dx.doi.org/10.1016/j.jaut.2014.03.001] [PMID: 24685231] , 100Chen SF. 1 alpha, 25-Dihydroxyvitamin D3 decreased ICAM-1 and ELAM-1 expressions on pulmonary microvascular endothelial cells and neutrophil motivation. J Steroid Biochem Mol Biol 1995; 52(1): 67-70.[http://dx.doi.org/10.1016/0960-0760(94)00153-D] [PMID: 7532002] ]. Furthermore, the secretion of IL-8, a proinflammatory chemokine which increases in response to aPL and LPS and is involved in leukocyte recruitment was significantly reduced [84Gladigau G, Haselmayer P, Scharrer I, et al. A role for Toll-like receptor mediated signals in neutrophils in the pathogenesis of the anti-phospholipid syndrome. PLoS One 2012; 7(7): e42176.[http://dx.doi.org/10.1371/journal.pone.0042176] [PMID: 22860075] ]. TLR9 has been involved in the pathogenesis of APS, whereas TLR3 role has never been investigated in APS pathogenesis [42Raschi E, Chighizola CB, Grossi C, et al. β2-glycoprotein I, lipopolysaccharide and endothelial TLR4: Three players in the two hit theory for anti-phospholipid-mediated thrombosis. J Autoimmun 2014; 55: 42-50.[http://dx.doi.org/10.1016/j.jaut.2014.03.001] [PMID: 24685231] , 109Aguilar-Valenzuela R, Nickerson K, Romay-Penabad Z. Involvement of TLR7 and TLR9 in the production of antiphospholipid antibodies. Arthritis Rheum 2011; 63(10): s281.]. TLR9 was significantly increased both in the presence of inflammatory stimuli and IgG isolated from APS patients and was completely inhibited by pretreatment with vitamin D. Conversely, the expression of TLR3, although reduced by overnight pretreatment with vitamin D, was significantly increased by Poly I:C and LPS, but not by anti-β2GPI. Based on this observation, we assume that TLR9 may be involved in the pathogenesis of APS and could provide a possible target of vitamin D whereas TLR3 may not be directly involved in APS pathogenesis.

TLRs are able to up-regulate expression of type I IFNs; in particular IFN-α transcription is dependent on TLR9 while IFN-β depends on TLR3 and TLR4 activation [110Simons KH, Peters HAB, Jukema JW, de Vries MR, Quax PHA. A protective role of IRF3 and IRF7 signalling downstream TLRs in the development of vein graft disease via type I interferons. J Intern Med 2017; 282(6): 522-36.[http://dx.doi.org/10.1111/joim.12679] [PMID: 28857295] -112Lövgren T, Eloranta ML, Kastner B, Wahren-Herlenius M, Alm GV, Rönnblom L. Induction of interferon-alpha by immune complexes or liposomes containing systemic lupus erythematosus autoantigen- and Sjögren’s syndrome autoantigen-associated RNA. Arthritis Rheum 2006; 54(6): 1917-27.[http://dx.doi.org/10.1002/art.21893] [PMID: 16729300] ]. We observed an increased gene expression of both IFN-α and IFN-β in the presence of both TLR specific agonists and IgG isolated from APS patients and a significant reduction with pretreatment with vitamin D, further confirming its anti-inflammatory effect.

Although our results are preliminary and should be validated with larger experiments and cohort studies, they further support the hypothesis that vitamin D may display a protective effect on endothelial cell perturbation in APS.

CONCLUSION

The anti-inflammatory and antithrombotic role of vitamin D is supported by epidemiological data on vitamin D deficiency in APS subjects, in particular in those with thrombotic manifestations.

It is tempting to speculate that aPL carriers, who do not have an indication for antiplatelet and anticoagulation, may benefit from vitamin D supplementation. Vitamin D may be considered in addition to standard care for obstetric and thrombotic APS patients.

Future mechanistic research should be aimed at investigating the potential effects of vitamin D in APS.

CONSENT FOR PUBLICATION

Not applicable.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENTS

Gruppo LES Italiano provided a grant for RG to run the Lupus Clinic

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