Pathogenesis of Antiphospholipid Syndrome

Transcription

1 La Sapienza Università di Roma Maggio 2016 Pathogenesis of Antiphospholipid Syndrome Guido Valesini

2 - The twin paradox - The Amalia paradox

3 HUGHES SYNDROME THROMBOSIS, ABORTION, CEREBRAL DISEASE, AND THE LUPUS ANTICOAGULANT G.R.V. HUGHES Br. Med. J. 1983; 287:

4 Clinical criteria 1. Vascular thrombosis 2. Pregnancy morbidity Laboratory criteria (present on two or more occasions, at least 12 weeks apart) Lupus anticoagulant Anticardiolipin (acl) antibody Anti-β2 glycoprotein-i antibody

5 Pathogenetic mechanisms mediated by apl THROMBOSIS FETAL LOSS..& more Several umet needs!

6 Pathogenetic mechanisms mediated by apl THROMBOSIS Most of the pathogenic mechanisms have been demonstrated using in vitro models In vivo models of thrombosis induced in mice have confirmed that apl are able to increase thrombus formation in the venous and arterial trees. Passive infusion of human apl IgG together with a small amount of LPS was shown to trigger clotting in the rat mesenteric microcirculation. - Pierangeli S. S. et al, Circulation Jankowski M. et al, Blood Fischetti F. et al, Blood 2005 FETAL LOSS association is supported by several epidemiological studies and by experimental models showing that passive transfer of apl IgG induces fetal loss and growth retardation in pregnant naive mice. - Meroni PL et al, Lupus 2010

7 THROMBOSIS Clotting formation mediated by apl apl interact with endothelial cells, primarily through binding of β2gpi on cell surfacei Meroni PL et al, Nat Rev Rheumatol 2011

8 Endothelial cells activation by anti-β2gpi antibodies β2gpi adheres to endothelial cell membranes: electrostatic interaction between the cationic phospholipid-binding site (domain V) and anionic structures, such as heparan sulfate, on the cell membrane; as a ligand for annexin A2; cell signaling that results in activation of NFκB or p38 MAPK or both; interaction of the β2gpi clusters with TLR2/TLR4 might be responsible for MyD88 and TRAF6-dependent signaling Meroni PL et al, Nat Rev Rheumatol 2011

9 Clotting formation mediated by apl apl upregulate tissue factor expression on endothelial cells and blood monocytes endothelial leukocyte adhesion, cytokine secretion and PGE2 synthesis

10 β2gpi mrna expression by human peripheral blood monocytes Monocytes synthesize b2-gpi β2gpi expression on cell surface of monocytes flow cytometric analysis β2gpi expression on monocytes is significantly increased in patients APS and correlates with tissue factor expression on monocytes

11 - monocytes are able to synthesize β2-gpi - patients with APS may have increased β2-gpi exposure on cell surface, which leads to persistently high monocyte tissue factor expression and consequently to - prothrombotic diathesis.

12 Clotting formation mediated by apl apl recognize phospholipid-binding proteins expressed on platelets apl binding potentiates platelet aggregation Meroni PL et al, Nat Rev Rheumatol 2011

13 Clotting formation mediated by apl apl interfere with plasma components of the coagulation cascade: - inhibiting anticoagulant activity, - affecting fibrinolysis, - displacing the binding of the natural anticoagulant annexin A5 to anionic structures

14 Peptide of β2gpi-domain I shares 88% identity with an epitope within the extracellular domain of human TLR4 Autoantibodies specific to the peptide in a high percentage of sera from patients with SLE and APS, but not in sera from healthy donors The Abs, trigger a TLR4 signaling cascade with consequent IRAK phosphorylation and NF-B translocation, promote VCAM expression on endothelial cells and TNF- release by monocytes, thus leading to a proinflammatory microenvironment.

15 FETAL LOSS In vitro studies showed that apl might induce a procoagulant state (several mechanisms) ability of the apl (anti- β2gpi) antibodies to disrupt the anticoagulant annexin A5 shield on trophoblast and endothelial cell monolayers Rand J e al, Lupus 2010 Proinflammatory mediators (complement, TNF, chemokines) have been shown to have a role in animal models of apl-induced fetal loss Meroni PL et al, Nat Rev Rheumatol 2011 Use of glucocorticoids?? No evidence to support the routine use of glucocorticoids Ruiz-Irastorza G et al, Lancet 2010

16 FETAL LOSS Another mechanism DEFECTIVE PLACENTATION β2gpi-dependent antibodies bind to human trophoblasts and affect several cell functions in vitro: cell injury apoptosis inhibition of proliferation and syncitia formation decreased production of human chorionic gonadotrophin defective secretion of growth factors Expression of β2gpi on trophoblast cell membranes placental tropism of anti-β2gpi antibodies β2gpi binds to exposed phosphatidylserine on the external cell membranes of trophoblasts undergoing syncitium formation Meroni PL et al, Nat Rev Rheumatol 2011

17 Pathogenetic mechanisms mediated by apl THROMBOSIS FETAL LOSS..& more Several umet needs!

18 TWO-HIT HYPOTHESIS A two hit hypothesis has been suggested to explain the clinical observation that thrombotic events occur only occasionally in spite of the persistent presence of apl.

19 Clotting formation mediated by apl Procoagulant and proinflammatory state (apl-mediated) is necessary but not sufficient for clotting TWO-HIT HYPOTHESIS Supporting this mechanism the administration of a small amount of LPS was required for human β2gpi-specific apl IgG to produce a thrombogenic effect in rat mesenteric microcirculation Fischetti F, et al. Blood 2005 FIRST HIT (apl) SECOND HIT: - Infections and recent surgery (stress) - Oxidative stress such as smoking - Complement activation seems to be necessary to clot formation in vivo Giannakopoulos B et al, NEJM 2013

20 FETAL LOSS The main pathogenetic mechanism intra-placental thrombosis impairment of maternal fetal blood exchange TWO-HIT HYPOTHESIS (with the single exception of the increased risk of venous thromboembolism during pregnancy) Passive infusion of IgG fractions with apl activity induces fetal loss in naive pregnant mice without necessarily requiring a second hit Meroni PL et al, Nat Rev Rheumatol 2011

21 INFECTIONS and APS Infectious triggers induction of transient antiphospholipid antibodies If microbes permanently colonize us role in the pathogenesis of APS? MICROBIOTA the collection of all microorganisms colonizing humans, necessary for normal host physiology One potential piece in the APS puzzle could be the contribution of commensal bacteria to the development and maintenance of autoreactive CD4+ T cells and autoantibodies In genetically predisposed individuals, the gut microbiota may drive the induction of anti-β2gpi antibodies by several mechanisms Ruff WE et al, Curr Rheumatol Rep 2015

22 GUT MICROBIOTA and APS 1. Gut commensal antigens recognized by mucosal DCs (phagocytosis after barrier disruption or apoptosis of intestinal epithelial cells) 2. Commensal-derived LPS, phospholipids and oxidative stress conformational change of β2gpi that exposes cryptic epitopes in domains I of β2gpi DCs take up unfolded β2gpi bounds to LPS via receptors (e.g., TLRs) 3. DCs present cross-reactive commensal and cryptic β2gpi antigens to CD4+ helper T cells that assist antigen-specific B cells via CD40 ligand (co-stimulatory receptors). B cells then secrete IgA/IgG in the mucosal lumen and in the systemic circulation. Ruff WE et al, Curr Rheumatol Rep 2015

23 The clinical spectrum of the antiphospholipid syndrome (APS) is not limited to vascular thrombosis or miscarriages but includes additional manifestations that cannot be explained solely by a thrombophilic state.

24 APS, NOT JUST THROMBOSIS & MISCARRIAGE G.R.V. Hughes, The Rheumatologist. Volume 10. N.2, 2016

25 «CRITERIA NON CRITERIA» APS, NOT JUST THROMBOSIS & MISCARRIAGE BRAIN BONE & JOINTS Migraine Stroke Bone fractures Seizures Avascular necrosis (AVN) Memory Loss Multiple Sclerosis SKIN Autonomic disturbance (Postural Orthostatic Tachycardia Syndrome-POTS) Livaedo reticularis Psychiatric disorders GI TRACT & LIVER KIDNEY Renal artery stenosis Microangiopathy Abdominal angina Budd-Chiari syndrome HELLP syndrome (in pregnancy women) G.R.V. Hughes, The Rheumatologist. Volume 10. N.2, 2016

26 WHY THE BRAIN? APS, NOT JUST THROMBOSIS & MISCARRIAGE Platelet activation Tissue factor induction Disruption of the annexin A5 anticoagulant shield Endothelial activation Alteration of the permeability of the blood-brain-barrier Direct binding to neuronal cell surface Possible cross-reactivity between anti-lymphocyte antibodies and brain G.R.V. Hughes, The Rheumatologist. Volume 10. N.2, 2016

27 APS, NOT JUST THROMBOSIS & MISCARRIAGE Anti-β2GPI might contribute to CNS pathology by either a direct interaction with astrocytes and neurons or an interaction with cerebral vascular endothelial cells Reverse transcriptase-polymerase chain reaction followed by restriction enzyme digestion of the product obtained demonstrated the presence of beta2-gpi mrna in endothelial cells, astrocytes and neurones

28 ANTIPHOSPHOLIPID ANTIBODIES Heterogeneous group of autoantibodies; Bind different anionic phospholipids, proteins or phospholipid-protein complex; a-cl a-factor-xii a-pth LAC??? a-lbpa a-β 2 -GPI a-pe a-sulfatides a-ps a-annexin-2 Valesini & Alessandri - Ann N Y Acad Sci, 2005

29 ANTIPHOSPHOLIPID ANTIBODIES Heterogeneous group of autoantibodies; Bind different anionic phospholipids, proteins or phospholipid-protein complex; a-cl a-factor-xii a-pth LAC??? a-lbpa a-β 2 -GPI a-pe a-sulfatides a-ps a-annexin-2 Valesini & Alessandri - Ann N Y Acad Sci, 2005

30 APL spectrum albpa aps ape LAC acl apth aβ2gpi af XII aanx A5 aanx 2..?

31 β2gpi: the playmaker in APS β2gpi circular form, with domain I interacting with domain V CRIPTIC EPITOPE hidden to the immune system Binding to an anionic phospholipid surface through domain V, the circular form of β2gpi opens up to a fishhook configuration exposing the domain I epitope Chighizola CB, Curr Rheumatol Rep 2014

32 Anti-β2GPI-domain I antibodies Anti-β2GPI-domain I antibodies the main apl pathogenic subset Patients with triple apl-positivity higher anti-b2gpi domain I antibody titres compared with double/single apl-positive patients The most recent studies have further confirmed the role of anti-domain I antibody positivity as a strong predictor of thrombotic events and pregnancy losses - Pengo V, et al. J Thromb Haemost Andreoli L, et al. Arthritis Rheumatol 2015

33 Time to challenge the dogma.. dominant thought

34 Many researchers propose that the only relevant antigens in the APS are the cofactors β2gpi the most important Antibodies binding phospholipids in a cofactor independent manner are considered insignificant for the pathogenesis of the APS REVIEW THIS EVIDENCE They don t negate a role for anti-β2gpi, but challenge the view that cofactor independent apl are irrelevant for the pathogenesis of the APS

35 Patients with the APS have quite heterogeneous antibody profiles. Contrary to common belief, there is ample evidence in the literature that a significant proportion if not the majority of APS patients harbor cofactor independent antibodies against cardiolipin.

36 Time to Challenge the Dogma Review of clinical studies: (1) LA is the apl test most consistently and strongly associated with thrombotic events (2) Anticardiolipin is still the immunoassay with the strongest and best documented association with thromboembolic events (3) None of the clinical studies was designed to differentiate between cofactor dependent and independent apl clinical data cannot support a pathophysiologic role of one or the other acl are pathogenic and this is independent of cofactor requirement the differentiation of pathogenic and non-pathogenic apl must be based on other criteria, not yet defined Lackner & Muller-Calleja, Journal of Thromb & Haemost 2016

37 .an «experimental model» in humans the so-called SERONEGATIVE APS

38 APS Clinical Spectrum PAPS, primary Microangiophatic APS SAPS, secondary Seronegative APS CAPS, cathastrophic GV, 2007

39 SERONEGATIVE APS Patients with clinical manifestations indicative of APS but persistently negative for the commonly used assays to detect apl Other autoantibodies? Other laboratory methods to detect apl? A new diagnostic tool: immunostaining on thin layer chromatography has been proposed to evaluate apl reactivity

40 SERONEGATIVE APS TLC immunostaining SN-APS pts=36 acl + in 47.2% of SN-APS albpa + in 41.7% of SN-APS ape + in 30.5% of SN-APS TLC immunostaining was performed with a second sample obtained at least 12 weeks later the same result was confirmed except in 5 sera: - 3 positive pts were negative; - 2 negative pts were positive. a representative TLC immunostaining with two positive and one negative samples Conti F, et al Clin Exp Immunol 2011

41 SERONEGATIVE APS Other results: IgG from SN-APS induce IRAK phosphorylation and NF-kB activation (a) IgG from SN-APS induce VCAM-1 surface expression and tissue factor release by endothelial cells (b) Conti F, et al Clin Exp Immunol 2011

42 We tested sera from patients with SN-APS by a proteomic approach, analysing endothelial cellsurface membrane proteins. Sera from SN-APS patients revealed two reactive spots, corresponding to vimentin, a protein which is shown to bind cardiolipin in vitro. TLC immunostaining SN-APS pts = 29 IgG anti-vimentin-cardiolipin in 55.2% IgM anti-vimentin-cardiolipin in 37.9% Confirmed in a second sample (after 12 weeks)

43 Affinity-purified antivimentin/cardiolipin antibodies from SN-APS induce: A) IRAK1 phosphorylation B) NF-kB activation in endothelial cells

44 SERONEGATIVE APS - small epidemiological weight - great conceptual value

45 Akwnoledgement DMISM Lupus Clinic Fabrizio Conti Cristiano Alessandri Francesca Spinelli Fulvia Ceccarelli Tania Colasanti Carlo Perricone Simona Truglia Francesca Miranda Laura Massaro Dipartimento Medicina Sperimentale Maurizio Sorice Roberta Misasi Antonella Capozzo Tina Garofalo Istituto Superiore Sanità Elena Ortona Paola Margutti

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48 NEUTROPHILS and APS Like monocytes, circulating neutrophils from patients with APS are in an activated state their increased production of reactive ROS Effector function of neutrophils capacity to form extracellular traps (NETs) NETosis = a cell death process Figure by Valesini G et al, Autoimmun Rev 2015 NETosis is known to be impaired in SLE patients Recent studies indicate that NET formation and degradation are equally affected patients with PAPS Van den Hoogen, Autoimmun Rev 2015

49 NEUTROPHILS and PAPS Incubation of neutrophils from healthy controls with apl stimulates NET formation and also, that neutrophils from PAPS patients are more prone to undergo NETosis compared with neutrophils from healthy controls

50 NEUTROPHILS and SAPS/SLE Serum from both SLE and primary and secondary APS patients have a reduced capacity to degrade NETs which implies that these patients are strongly exposed to NETs Inhibition of NETosis targeting the inhibitory receptor SIRL-1 was recently proposed for SLE

51 COMPLEMENT ACTIVATION Case reports document the use of the C5 inhibitor eculizumab to prevent APS associated thrombotic microangiopathy that complicates renal transplantation to treat patients with acute CAPS Shapira I et al, Arthritis Rheum 2012 In vivo murine studies implicating the activation of the classical complement pathway in thrombosis associated with APS - Pierangeli SS et al, Arthr Rheum Fischetti F et al, Blood 2005 Activation of complement by apl generates C5a binds and activates neutrophils tissue factor expression Ritis K et al, J Immunol 2006

52 INNATE IMMUNE RESPONSE apl from patients with APS up-regulate the expression of TLR7 and TLR8 in plasmacytoid dendritic cells and in monocyte (respectively) and promote their translocation from the endoplasmic reticulum to the endosome Prinz N et al, Blood 2011 Inhibition of TLR7 and TLR8 may be an attractive therapeutic target in patients with SLE and apl antibodies HYDROXYCHLOROQUINE inhibits TLR7 Kuznik A et al, J Immunol 2011 B-cell activating factor (BAFF) is a cytokine crucial for B-cell survival BAFF inhibition prevented thrombosis in NZW x BXSB F1 male mice a role in the prevention of thrombosis associated with the APS in high-risk patients with SLE Kahn P et al, Arthritis Rheum 2008

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55 Antiphospholpid antibodies in asymptomatic carriers

56 APS Classification Primary APS (53%) Secondary APS (47%) Secondary to Autoimmune Diseases

57 Citrullinazione e Artrite Reumatoide G. Valesini Felix qui potuit rerum cognoscere causas

58 LAC methods Step 1 activated Partial Thromboplastin Time (aptt); Dilute Russel viper venom time (drvvt); Kaolin clotting time (KCT); (LAC positive if 1 or more test prolungation) Step 2 Mixing test: mix (1:1) normal human plasma with Pts plasma (LAC positive if persist prolungation) Step 3 Neutralization test: add phospholipids or platelets to Pts plasma (LAC positive if the tests become normal)

59 - Classification of APS should be avoided if less than 12 weeks or more than 5 years separate the positive apl test and the clinical manifestation. - Coexisting inherited or acquired factors for thrombosis are not reasons for excluding patients from APS trials. - Superficial venous thrombosis is not included in the clinical criteria. 64

60 Peptide of β2gpi-domain I shares 88% identity with an epitope within the extracellular domain of human TLR4 Complete sequence omology between β2gpi and P.Gingivalis

61 β2gpi-domain I: immunodominant epitope Pathogenic effect of anti-domain I antibodies using anti-β2gpi minibody (MBB2) a human monoclonal IgG antibody -- targeting b2gpi domain I. Its infusion induced fetal losses in pregnant mice and blood clots in rat mesenteric microcirculation after LPS priming. MBB2DCH2 displays the same antigen specificity of MBB2, but lacks the CH2 domain, being unable to activate complement. Therapeutic implications: MBB2DCH2 has been shown to prevent apl effects in vivo by competing with autoantibodies for binding to b2gpi Agostinis C et al, Blood 2014

62 Oxidative stress and APS Paraoxonase activity (antioxidant properties) significantly decreased in APS pts Plasma levels of β2gpi-oxidized-ldl complexes are elevated in APS patients as compared with healthy controls Oxidative stress plays a direct role in the structure and function of β2gpi In healthy subjects, the free thiol form of β2gpi predominates in the plasma characterized by a broken disulfide bridge at Cys32 and Cys60 and at Cys288 andcys326 Under conditions of oxidative stress, disulfide bonds form at these sites unmask criptic epitope Giannakopoulos B et al, NEJM 2013

63 β2gpi: a bridge between circulating apl and target cells Second hit hypothesis VCAM-I, ICAM-I and E-selectine Del Papa N, 1997 Ab-specific peptide β2gpi cross-react with TLR4 Colasanti T, 2012 TF expression Conti F, 2003 β2gpi- Receptor (Annexin II) and Sorice via M, TLR Sherer Y, 2007 Platelets aggregatio n Asherson RA, 2007 Protein C Annexin V activation Meroni PL, 2004 Sherer Y, 2007 Modified from Meroni PL, Art Res & Therapy 2014

64 The phosphatidylinositol 3-kinase (PI3K) AKT pathway recently identified as an additional signalling cascade engaged by apls This pathway culminates in the recruitment of mammalian target of rapamycin (mtor), a kinase modulating cellular growth, proliferation and apoptosis.

65 Confirmed at immunostaining: the phosphorylation rate of both S6RP and AKT was markedly increased in vascular endothelial cells in renal specimens from APS patients with apl nephropathy and in the carotid and left anterior descending arteries of catastrophic APS mtor pathway might mediate the intimal hyperplasia leading to chronic vasculopathy mtor inhibitor SIROLIMUS was prescribed to patients with APS nephropathy undergoing kidney transplantation who developed no vascular lesion recurrence Canaud G. et al, NEJM 2014

66 A schematic overview of the most recent insights into the intracellular pathways engaged by anti-b2gp Chighizola CB et al, Curr Opin Rheumatol. 2015

67 FROM PATHOGENESIS TO TREATMENT Giannakopoulos B et al, NEJM 2013

68 Activation of NFκB or p38 MAPK The cellular steps leading to NF-kB translocation in monocytes have been recently clarified: apls engaged NF-kB via a clathrin-dependent endocytic pathway requiring CD14 (TLR4 co-receptor) and AnnexinA2 Brandt KJ et al, J.Thromb.Haemost 2014 NF-kB potential future pharmacological target: in BALB/c mice, treatment with a specific NF-kB inhibitor (Dehydroxymethylepoxyquinomicin), prevented apl-mediated thrombus formation Nishimura M et al, J Nephropathol 2013

69 FETAL LOSS Immunohistological analysis of abortive material or full-term placentae from APS women has not provided conclusive information about the pathogenic contributions of acute local inflammatory events and complement deposition lack of histological evidence of inflammation in the human placentae - Park, A. SpringerVerlag Gerosa, M. et al Ann Rheum Dis 2009 apl abnormalities at the maternal side of the placenta In endometrial biopsies impaired endometrial differentiation and reduced expression of complement decay-accelerating factor (CD55) were found These alterations before conception might compromise implantation and predispose to complement-mediated pregnancy failure Francis J et al, Mol Hum Reprod 2006

70 Non Criteria APS clinical manifetations Thrombocytopenia Heart valve disease Skin ( livedo, ulcers, superficial thrombophlebitis) Kidney ( renal Arterial stenosis, APS nephropathy) CNS Migraine/headache, Epilepsy, MS like disese, Cognitive impairment, Dementia, Transverse myelopathy, Retinal Thtombosis, Amaurosis fugax, Pulmonary alveolar hemorrhage

71 Criteria for pathogenicity of apl 1. Presence in many or most APS patients 2. Induction of humoral or cellular responses compatible with the clinical manifestation of the APS 3. Induction of clinical manifestations of the APS in vivo 4. Association with clinical manifestations of the APS in humans Lackner & Muller-Calleja, Journal of Thromb & Haemost 2016

72 McNeil HP, Simpson RJ, Chesterman CN, Krilis SA. Anti-phospholipid antibodies are directed against a complex antigen that includes a lipid-binding inhibitor of coagulation: beta 2- glycoprotein I (apolipoprotein H). Proc Natl Acad Sci U S A 1990; 87: Galli M, Comfurius P, Maassen C, Hemker HC, de Baets MH, van Breda- Vriesman PJ, Barbui T, Zwaal RF, Bevers EM. Anticardiolipin antibodies (ACA) directed not to cardiolipin but to a plasma protein cofactor. Lancet 1990; 335: Matsuura E, Igarashi Y, Fujimoto M, Ichikawa K, Suzuki T, Sumida T, Yasuda T, Koike T. Heterogeneity of anticardiolipin antibodies defined by the anticardiolipin cofactor. J Immunol 1992; 148: