IS PRE-CLINICAL AUTOIMMUNITY BENIGN?

IS PRE-CLINICAL AUTOIMMUNITY BENIGN? Darcy S. Majka MD, MS Disclosures I have no significant financial or other relationship with the manufacturers of any product or services. Dr. Teodorescu (coauthor) is the owner of TheraTest Laboratories where the autoantibody testing for my research was completed. Northwestern University Feinberg School of Medicine, Chicago, IL Key References Maradit-Kremers H, Crowson CS, Nicola PJ, Ballman KV, Roger VL, Jacobsen SJ, Gabriel SE et al. Increased unrecognized coronary heart disease and sudden deaths in rheumatoid arthritis: A population-based cohort study. Arthritis Rheum 2005;52(2):402-411. Pre-clinical in the Paradigm of RA Development Tomasson G, Aspelund T, Jonsson T, Valdimarsson H, Felson DT, Gudnason V. Effect of rheumatoid factor on mortality and coronary heart disease. Ann Rheum Dis 2010;69:1649-54. Kramer HR, Giles JT. Cardiovascular disease risk in rheumatoid arthritis: Progress, debate and opportunity. Arthritis Care and Research 2011;63(4):484-99. Bykerk, VP, et al. Curr Opin Rheumatol 2011;23:179 184. Phases in the Development of Pathogenic Autoimmunity in Lupus Genetic influence Are autoantibodies in unaffected individuals benign? Normal Immunity Benign Autoimmunity Environmental factors Pathogenic Autoimmunity Clinical Illness in normals without any clinical sequelae: o Increase in the number and titer of autoantibodies with age related to loss of tolerance. o Healthy siblings of patients with autoimmune disorders. Arbuckle MR et al. N Engl J Med 2003;349:1526-1533. 1

are markers for autoreactive B cell activation Autoreactive B cells can drive disease pathogenesis through a variety of mechanisms: o Generation of autoantibody secreting plasma cells o Formation of immune complexes o Presentation of autoantigens to T cells and costimulation o Production of proinflammatory cytokines, chemokines and lymphangiogenic growth factors o Formation of ectopic tertiary lymphoid structures 1. Townsend MJ et al. Immunological Reviews 2010; 237:264-83. 2. Anolik JH. Bull NYU Hosp Jt Dis 2007;65(3):182-6. Transition from Benign to Pathogenic Autoimmunity? Autoantigen activates self-reactive B cells causing formation of short lived plasma cells which secrete autoantibodies. Self-reactive B cells can enter germinal centers: o Somatic hypermutation and affinity maturation of BCR s. o Immunoglobulin class switching o Generation of long lived self-reactive B cells. o Differentiation into long lived plasma cells secreting high affinity Fc receptor binding autoantibodies. CTD-related Conceptual Model Possible Progression Inflammation Autoimmune CTD Coronary Atherosclerosis (Coronary Artery Calcium [CAC] is surrogate measure) Cardiovascular Events and CHD There are preliminary data showing that circulating autoantibodies are associated with coronary heart disease. These observations have led to hypotheses that coronary heart disease has autoimmune features. 1. Grainger DJ, et al. Ann Rheum Dis 2002, 61(2):110-114. 2. Sherer Y, et al. Cardiology 2001, 95(1):20-24. 3. Soltesz P, et al. Autoimmun Rev 2007, 6(6):379-386. 4. Liang KP, et al. J Rheumatol 2009, 36(11):2462-2469. 5. Shoenfeld Y, et al. Ann Med 2000, 32 Suppl 1:37-40. APA and atherosclerosis Antiphospholipid antibodies (APA) have been found in the sera and plaques of individuals with atherosclerosis and clinical cardiovascular events. APA and atherosclerosis It has been hypothesized that APA contribute to atherosclerosis through both thrombotic and inflammatory pathways. If APA are associated with subclinical atherosclerosis in humans prior to the onset of clinical thrombotic events, then it is possible that their association with atherosclerosis is due to non-thrombotic pathways. 1. Sherer Y, et al. Cardiology 2001, 95(1):20-24. 2. Soltesz P, et al. Autoimmun Rev 2007, 6(6):379-386. 3. Shoenfeld Y, et al. Ann Med 2000, 32 Suppl 1:37-40. 4. Brey RL, et al. Stroke 2001, 32(8):1701-1706. 1. Soltesz P, et al. Autoimmun Rev 2007, 6(6):379-386. 2. Kobayashi K, et al. Ann N Y Acad Sci 2007, 1108:489-496. 3. Hasunuma Y, et al. Clin Exp Immunol 1997, 107(3):569-573. 4. George J, et al. Circulation 2000, 102(15):1822-1827. 5. George J, et al. Circulation 1998, 98(11):1108-1115. 6. Matsuura E, et al. Clin Dev Immunol 2004, 11(2):103-111. 2

APA and Subclinical Atherosclerosis-IMT A few studies demonstrated a correlation between APA, or antiphospholipid syndrome, and intima media thickness. However, the results were inconsistent and most associations were found in individuals with active autoimmune CTDs or primary antiphospholipid syndrome. APA and subclinical atherosclerosiscoronary artery calcification (CAC) In a small group of lupus patients (N = 60), Plazak et al recently demonstrated an increased risk of CAC in patients with acl IgG and anti-β2gpi IgG levels (RR 4.1). We also tested the hypothesis that circulating APA are associated with subsequent sub-clinical atherosclerosis measured as CAC in a cohort of community based young adults. 1. Ames PR, et al. Lupus 2002, 11(4):208-214. 2. Dropinski J, et al. Med Sci Monit 2003, 9(4):BR105-109. 3. Margarita A, et al. Ann N Y Acad Sci 2007, 1108:475-480. Plazak W, et al. Inflamm Res 2011, 60(10):973-980. Methods Multicenter, longitudinal study to examine physiological, psychological and lifestyle factors affecting CVD development. In 1985, ~5000 participants aged 18-30. Gender and Race distribution 25% in each group. o Black women White women o Black men White men Participants are examined at 2-5 year intervals. 1985 1992 2000 2005 CARDIA Baseline: 5115 participants aged 18-30 enrolled CARDIA Year 7: Serum Collected. later measured in stored serum of 2284 participants CARDIA Year 15: CAC measured CARDIA Year 20: CAC measured EBCT Coronary Calcium Screen Selection of Study Participants No Coronary Artery Calcium Extensive Coronary Artery Calcium CARDIA: N = 5115 enrolled in 1985 N = 2819 with serum Yr 7 for autoantibody testing Excluded 536 without any CAC data at Yr 15 or Yr 20 70 without covariate data 10 pregnant N = 1838 with data for CAC Yr 15 N=1515 with data for both CAC Yr 15 and Yr 20 N = 1880 with data for CAC Yr 20 Majka DS, et al. Inflammation Research 2013, 10.1007/s00011-013-0652-x. 3

Odds Ratios (95%CI) for Year 15 CAC>0 by Autoantibody Positivity acl IgM 0.8 (0.2-3.6) acl IgG 5.1 (1.4-18.6) Beta-2-GPI IgA 5.6 (2.3-13.2) Beta-2-GPI IgM 1.7 (1.0-3.1) Beta-2-GPI IgG 6.4 (2.4-16.8) All ppts* Women Men Black White 1.3 (0.2-10.6) 1.8 (0.2-15.6) 10.3 (2.8-37.2) 3.3 (1.4-7.5) 7.4 (1.8-30.6) 0.5 (0.1-4.2) 10.4 (1.6-68.0) 4.1 (1.2-13.7) 1.0 (0.4-2.3) 5.5 (1.4-22.3) NA 1.0 (0.2-5.1) 5.0 (1.1-23.4) 7.4 (2.0-27.1) 1.8 (0.7-5.2) 5.0 (1.1-23.8) * Models adjusted for year 7 race, gender, age, LDL-C, BMI, BP, smoking status, and CRP Models adjusted for year 7 race, age, LDL-C, BMI, BP, smoking status, and CRP Models adjusted for year 7 gender, age, LDL-C, BMI, BP, smoking status, and CRP 3.9 (0.3-47.3) 4.2 (1.3-13.9) 1.7 (0.8-3.4) 7.2 (2.1-25.5) Majka DS, et al. Inflammation Research 2013, 10.1007/s00011-013-0652-x. Odds Ratios (95%CI) for Year 20 CAC>0 by Anti-Beta-2-GPI Positivity Beta-2-GPI IgA 1.7 (0.7-4.2) Beta-2-GPI IgM 0.9 (0.5-1.5) Beta-2-GPI IgG 3.1 (1.2-7.7) All ppts* Women Men Black White 2.3 (0.6-8.3) 1.3 (0.6-2.7) 5.7 (1.8-18.2) 1.4 (0.4-4.9) 0.7 (0.4-1.5) 1.6 (0.4-5.8) 2.6 (0.8-8.5) 1.1 (0.5-2.6) 4.2 (1.1-16.5) 0.8 (0.2-3.7) 0.8 (0.4-1.5) 2.4 (0.7-8.3) * Models adjusted for year 7 race, gender, age, LDL-C, BMI, BP, smoking status, and CRP Models adjusted for year 7 race, age, LDL-C, BMI, BP, smoking status, and CRP Models adjusted for year 7 gender, age, LDL-C, BMI, BP, smoking status, and CRP Majka DS, et al. Inflammation Research 2013, 10.1007/s00011-013-0652-x. Odds Ratios (95%CI) for CAC>0 by Number of Anti-Beta-2-GPI Isotypes All Ppts Women Men Black White CAC>0 Year 15 as Outcome 0 isotypes 1 1 1 1 1 1 pos isotype 1.4 (0.8-2.6) >2 pos isotypes 8.6 (3.3-22.9) 2.6 (1.1-6.4) 12.6 (3.1-50.7) 1.0 (0.5-2.3) 7.3 (1.7-31.8) 1.0 (0.3-3.1) 17.7 (3.4-92.4) 1.6 (0.8-3.3) 6.4 (1.9-21.9) These findings suggest APA may promote other autoimmune, non-thrombotic pathways in CHD β2-gpi and anti-β2-gpi derived from an APA mouse model enhance oxidized LDL/β2-GPI complex binding to macrophages. CAC>0 Year 20 as Outcome 0 isotypes 1 1 1 1 1 1 pos isotype 1.0 (0.6-1.7) 1.6 (0.8-3.3) 0.8 (0.4-1.5) 1.3 (0.6-2.8) 0.8 (0.4-1.6) >2 pos isotypes 2.0 (0.7-5.4) 2.5 (0.6-9.6) 1.5 (0.3-6.6) 3.2 (0.7-14.6) 1.3 (0.3-5.2) Majka DS, et al. 2013, 10.1007/s00011-013-0652-x. 1. Kobayashi K, et al. Ann N Y Acad Sci 2007, 1108:489-496. 2. Hasunuma Y, et al. Clin Exp Immunol 1997, 107(3):569-573. 3. Matsuura E, et al. Clin Dev Immunol 2004, 11(2):103-11. Possible mechanisms for associations between APA and CHD APA increased atherosclerotic lesions in a mouse model of atherosclerosis. o LDL-receptor knockout mouse (LDL-RKO) Mouse acl lead to increased fatty streak formation. George J, et al. Circulation 2000, 102(15):1822-27. George J, et al. Circulation 1998, 98(11):1108-15. Matsuura E, et al. Clin Dev Immunol 2004, 11(2):103-11. Titers of ANA autoantibodies among subjects with triple vessel disease compared with controls. Group Titre 1/40 1/80 1/160 1/320 1/640 1/1280 1/2560 Total No CHD 0 2 1 0 1 1 0 5 (17%) Triple Vessel CHD 1 4 6 7 8 1 1 28 (70%) Participants with triple vessel CHD had higher odds of positive ANA compared to controls with positive stress tests/negative angiograms (OR 11.7 for ANA > 1:40). Grainger DJ et al. Ann Rheum Dis 2002;61:110 114 4

CHD risk in pre-clinical RA Retrospective longitudinal cohort study using data from the Rochester Epidemiology Project. RA incidence cohort compared with controls. In the two year period prior to RA fulfillment of ACR criteria, RA patients had: o Increased odds acute MI hospitalization: OR 3.17 (1.16, 8.68) o Increased odds sudden death: HR 1.94 (1.06, 3.55) o Increased odds unrecognized MI OR 5.86 (1.29, 26.64) o Reduced odds of angina: OR 0.58 (0.34, 0.99) Maradit-Kremers H, et al. Arthritis Rheum. 2005;52(2):402-11 RA-related autoantibodies and atherosclerosis in the pre-clinical period Liang et al used RF, anti-ccp and ANA results previously measured in participants from the Rochester Epidemiology Project and ICD-9 codes for cardiovascular outcomes. Subjects found to have autoantibody testing: o 6783 subjects with RF tested (10.4% positive) o 7852 with ANA (23.9% positive) o 299 with CCP (14.7% positive) Liang KP, et al. J Rheumatol 2009;36(11):2462-9. Risk of Autoantibody Positivity on Cardiovascular Outcomes and Death Outcome Events Hazard ratio (95% CI) Unadjusted for presence of rheumatic disease * Adjusted for presence of rheumatic disease * RF MI 365 1.36 (1.03, 1.79) 1.22 (0.91, 1.64) MI, HF or PVD 871 1.32 (1.10, 1.59) 1.24 (1.01, 1.51) Death 998 1.55 (1.33, 1.80) 1.43 (1.21, 1.68) ANA MI 371 1.32 (1.06, 1.65) 1.29 (1.03, 1.61) MI, HF or PVD 890 1.28 (1.10, 1.48) 1.26 (1.09, 1.46) RF and ischemic heart disease (IHD) in a general population cohort 567 men and 589 women recruited from the Hertfordshire Cohort Study. The HCS population is representative of the rest of the population of England. Prevalent IHD was identified: o Typical angina (Rose chest pain questionnaire) o Self report of previous CABG or angioplasty o Pathological Q waves on ECG. Death 1142 1.19 (1.05, 1.35) 1.18 (1.04, 1.34) Liang KP, et al. J Rheumatol 2009; 36(11):2462-9. Edwards CJ, et al. Heart 2007;93:1263 67. RF and IHD Autoantibody positivity: o RF: 16% men, 12% women o ANA: 11% men, 12% women o ACA: 24% men, 20% women RF was associated with an increased likelihood of IHD in men after adjustment for traditional risk factors o Men, OR = 2.9 (95% CI 1.6 to 5.3) o Women, OR = 1.3 (95% CI 0.6 to 2.8) No significant association between ANA or ACA with IHD in men or women. Edwards CJ, et al. Heart 2007;93:1263 67. RF and CVD in a population based study Participants drawn from the Reykjavik study. Followed prospectively for o All cause mortality o Incident CHD o CV mortality 2010 by BMJ Publishing Group Ltd and European League Against Rheumatism 5

Kaplan Meier curves according to RF status Model 1: Model 2: Further Model 3: Further Model 4: Further Adjustment for adjustment for adjustment for CV adjustment for age and sex smoking risk factors ESR Effects of RF (140 subjects with positive RF) All-cause mortality 1.47 (1.19, 1.80) 1.38 (1.12, 1.70) 1.40 (1.14, 1.72) 1.31 (1.06, 1.61) CV mortality 1.57 (1.15, 2.13) 1.47 (1.08, 2.00) 1.57 (1.15, 2.14) 1.45 (1.07, 1.98) Non-CV mortality 1.40 (1.06, 1.85) 1.32 (1.00, 1.74) 1.29 (0.97, 1.70) 1.20 (0.91, 1.59) Incident CHD 1.32 (0.96, 1.81) 1.25 (0.91, 1.72) 1.33 (0.97, 1.83) 1.24 (0.90, 1.70) Effect of positive RF and no joint symptoms (82 subjects) All-cause mortality 1.29 (0.98, 1.70) 1.24 (0.95, 1.63) 1.30 (0.99, 1.71) 1.33 (1.01, 1.74) CV mortality 1.45 (0.98, 2.15) 1.40 (0.94, 2.07) 1.57 (1.05, 2.32) 1.60 (1.08, 2.37) Non-CV mortality 1.17 (0.80, 1.72) 1.13 (0.77, 1.65) 1.12 (0.77, 1.64) 1.14 (0.78, 1.67) Incident CHD 1.20 (0.80, 1.81) 1.16 (0.77, 1.75) 1.28 (0.85, 1.93) 1.30 (0.86, 1.97) 2010 by BMJ Publishing Group Ltd and European League Against Rheumatism Effect of positive RF and inflammatory joint symptoms (29 subjects) All-cause mortality 2.25 (1.45, 3.50) 2.04 (1.31, 3.17) 2.24 (1.44, 3.48) 1.56 (1.00, 2.43) CV mortality 1.41 (0.58, 3.38) 1.26 (0.52, 3.03) 1.55 (0.64, 3.72) 1.06 (0.44, 2.56) Non-CV mortality 2.84 (1.71, 4.71) 2.59 (1.56, 4.30) 2.63 (1.58, 4.36) 1.87 (1.11, 3.14) Incident CHD 2.34 (1.22, 4.51) 2.14 (1.11, 4.12) 2.54 (1.32, 4.88) 1.76 (0.91, 3.42) Model 1: Model 2: Further Model 3: Further Model 4: Further Adjustment for adjustment for adjustment for CV adjustment for age and sex smoking risk factors ESR Effects of RF (140 subjects with positive RF) All-cause mortality 1.47 (1.19, 1.80) 1.38 (1.12, 1.70) 1.40 (1.14, 1.72) 1.31 (1.06, 1.61) CV mortality 1.57 (1.15, 2.13) 1.47 (1.08, 2.00) 1.57 (1.15, 2.14) 1.45 (1.07, 1.98) Non-CV mortality 1.40 (1.06, 1.85) 1.32 (1.00, 1.74) 1.29 (0.97, 1.70) 1.20 (0.91, 1.59) Incident CHD 1.32 (0.96, 1.81) 1.25 (0.91, 1.72) 1.33 (0.97, 1.83) 1.24 (0.90, 1.70) Effect of positive RF and no joint symptoms (82 subjects) All-cause mortality 1.29 (0.98, 1.70) 1.24 (0.95, 1.63) 1.30 (0.99, 1.71) 1.33 (1.01, 1.74) CV mortality 1.45 (0.98, 2.15) 1.40 (0.94, 2.07) 1.57 (1.05, 2.32) 1.60 (1.08, 2.37) Non-CV mortality 1.17 (0.80, 1.72) 1.13 (0.77, 1.65) 1.12 (0.77, 1.64) 1.14 (0.78, 1.67) Incident CHD 1.20 (0.80, 1.81) 1.16 (0.77, 1.75) 1.28 (0.85, 1.93) 1.30 (0.86, 1.97) Effect of positive RF and inflammatory joint symptoms (29 subjects) All-cause mortality 2.25 (1.45, 3.50) 2.04 (1.31, 3.17) 2.24 (1.44, 3.48) 1.56 (1.00, 2.43) CV mortality 1.41 (0.58, 3.38) 1.26 (0.52, 3.03) 1.55 (0.64, 3.72) 1.06 (0.44, 2.56) Non-CV mortality 2.84 (1.71, 4.71) 2.59 (1.56, 4.30) 2.63 (1.58, 4.36) 1.87 (1.11, 3.14) Incident CHD 2.34 (1.22, 4.51) 2.14 (1.11, 4.12) 2.54 (1.32, 4.88) 1.76 (0.91, 3.42) Model 1: Model 2: Further Model 3: Further Model 4: Further Adjustment for adjustment for adjustment for CV adjustment for age and sex smoking risk factors ESR Effects of RF (140 subjects with positive RF) All-cause mortality 1.47 (1.19, 1.80) 1.38 (1.12, 1.70) 1.40 (1.14, 1.72) 1.31 (1.06, 1.61) CV mortality 1.57 (1.15, 2.13) 1.47 (1.08, 2.00) 1.57 (1.15, 2.14) 1.45 (1.07, 1.98) Non-CV mortality 1.40 (1.06, 1.85) 1.32 (1.00, 1.74) 1.29 (0.97, 1.70) 1.20 (0.91, 1.59) Incident CHD 1.32 (0.96, 1.81) 1.25 (0.91, 1.72) 1.33 (0.97, 1.83) 1.24 (0.90, 1.70) Effect of positive RF and no joint symptoms (82 subjects) All-cause mortality 1.29 (0.98, 1.70) 1.24 (0.95, 1.63) 1.30 (0.99, 1.71) 1.33 (1.01, 1.74) CV mortality 1.45 (0.98, 2.15) 1.40 (0.94, 2.07) 1.57 (1.05, 2.32) 1.60 (1.08, 2.37) Non-CV mortality 1.17 (0.80, 1.72) 1.13 (0.77, 1.65) 1.12 (0.77, 1.64) 1.14 (0.78, 1.67) Incident CHD 1.20 (0.80, 1.81) 1.16 (0.77, 1.75) 1.28 (0.85, 1.93) 1.30 (0.86, 1.97) Effect of positive RF and inflammatory joint symptoms (29 subjects) All-cause mortality 2.25 (1.45, 3.50) 2.04 (1.31, 3.17) 2.24 (1.44, 3.48) 1.56 (1.00, 2.43) CV mortality 1.41 (0.58, 3.38) 1.26 (0.52, 3.03) 1.55 (0.64, 3.72) 1.06 (0.44, 2.56) Non-CV mortality 2.84 (1.71, 4.71) 2.59 (1.56, 4.30) 2.63 (1.58, 4.36) 1.87 (1.11, 3.14) Incident CHD 2.34 (1.22, 4.51) 2.14 (1.11, 4.12) 2.54 (1.32, 4.88) 1.76 (0.91, 3.42) Model 1: Model 2: Further Model 3: Further Model 4: Further Adjustment for adjustment for adjustment for CV adjustment for age and sex smoking risk factors ESR Effects of RF (140 subjects with positive RF) All-cause mortality 1.47 (1.19, 1.80) 1.38 (1.12, 1.70) 1.40 (1.14, 1.72) 1.31 (1.06, 1.61) CV mortality 1.57 (1.15, 2.13) 1.47 (1.08, 2.00) 1.57 (1.15, 2.14) 1.45 (1.07, 1.98) Non-CV mortality 1.40 (1.06, 1.85) 1.32 (1.00, 1.74) 1.29 (0.97, 1.70) 1.20 (0.91, 1.59) Incident CHD 1.32 (0.96, 1.81) 1.25 (0.91, 1.72) 1.33 (0.97, 1.83) 1.24 (0.90, 1.70) Effect of positive RF and no joint symptoms (82 subjects) All-cause mortality 1.29 (0.98, 1.70) 1.24 (0.95, 1.63) 1.30 (0.99, 1.71) 1.33 (1.01, 1.74) CV mortality 1.45 (0.98, 2.15) 1.40 (0.94, 2.07) 1.57 (1.05, 2.32) 1.60 (1.08, 2.37) Non-CV mortality 1.17 (0.80, 1.72) 1.13 (0.77, 1.65) 1.12 (0.77, 1.64) 1.14 (0.78, 1.67) Incident CHD 1.20 (0.80, 1.81) 1.16 (0.77, 1.75) 1.28 (0.85, 1.93) 1.30 (0.86, 1.97) Effect of positive RF and inflammatory joint symptoms (29 subjects) All-cause mortality 2.25 (1.45, 3.50) 2.04 (1.31, 3.17) 2.24 (1.44, 3.48) 1.56 (1.00, 2.43) CV mortality 1.41 (0.58, 3.38) 1.26 (0.52, 3.03) 1.55 (0.64, 3.72) 1.06 (0.44, 2.56) Non-CV mortality 2.84 (1.71, 4.71) 2.59 (1.56, 4.30) 2.63 (1.58, 4.36) 1.87 (1.11, 3.14) Incident CHD 2.34 (1.22, 4.51) 2.14 (1.11, 4.12) 2.54 (1.32, 4.88) 1.76 (0.91, 3.42) Are RA-related autoantibodies associated with sub-clinical CHD? RF, acl, anti-β 2 -GPI and ANA were elevated in patients with symptomatic ischemic heart disease. APA have been associated with subclinical atherosclerosis. It remains unknown whether RA-related autoantibodies are associated with sub-clinical atherosclerosis in individuals without RA. 6

Objectives Multi-Ethnic Study of Autoimmunity and Atherosclerosis To investigate the cross-sectional association between RA-related autoantibodies measured from serum collected at MESA baseline examination and subclinical atherosclerosis defined as CAC in a population based cohort. To examine the prospective association between RArelated autoantibodies and subsequent clinical CHD and CVD events after 7.1 years of follow-up. The Multi-Ethnic Study of Atherosclerosis Community-based sample of 6,814 men and women. Age 45 to 84 at enumeration Recruited from 7/2000 8/2002 No physician-diagnosed clinical CVD at baseline - MI/angina - Chronic Atrial Fib - TIA/stroke - CVD procedures - CHF Collaborating Centers in MESA Univ of Washington Univ of MN U of Vermont MESA Ancillary Study: Multi-Ethnic Study of Autoimmunity and CVD CVD Endpoint Data Collected continually at Follow Ups conducted every 9 months. Columbia Exam 1 Exam 2 Exam 3 Exam 4 Exam 5 UCLA Northwestern Univ Johns Hopkins Wake Forest 2000-2002 MESA baseline Exam 1: -CAC measured -Serum samples stored (and later tested for RF/anti-CCP) Average of 7.1 years follow-up data available 2010-2012 MESA Exam 5 completed. Caucasian 38%, African-American 28%, Hispanic 22%, Chinese 12% 53% Women 6,557 MESA participants included in the analysis sample Adjusted Odds Ratios (95% CI) of Having Degrees of CAC by Autoantibody Positivity RA-related 0<CAC<99 CAC Levels 99<CAC<300 Caucasian Men (N=1205) CAC>300 RF IgM 1.1 (0.7-1.7) 1.0 (0.6-1.8) 0.8 (0.5-1.4) RF IgA 0.4 (0.2-0.9) 0.8 (0.4-1.7) 0.8 (0.4-1.5) Either RF isotype 0.9 (0.6-1.3) 1.0 (0.6-1.7) 0.9 (0.5-1.4) Both RF isotypes 0.4 (0.1-1.2) 0.5 (0.1-1.7) 0.4 (0.1-1.3) CCP 0.7 (0.2-2.1) 0.5 (0.1-2.1) 0.5 (0.1-1.7) Caucasian Women (N=1323) RF IgM 2.2 (1.5-3.3) 1.4 (0.8-2.7) 1.5 (0.8-2.8) RF IgA 1.7 (0.9-3.1) 0.9 (0.3-2.5) 2.3 (1.01-5.5) Either RF isotypes 2.0 (1.4-3.0) 1.2 (0.7-2.2) 1.7 (0.95-3.1) Both RF isotypes 2.2 (0.9-5.0) 1.5 (0.5-4.9) 2.1 (0.7-6.7) CCP 0.9 (0.3-2.7) 1.5 (0.5-5.3) 0.9 (0.2-4.4) * Adjusted for age, smoking status, BP, BMI, HDL-c, LDL-c, DM, aspirin use, and cholesterol and BP medication use Adjusted Odds Ratios (95% CI) of Having Degrees of CAC by Autoantibody Positivity RA-related 0<CAC<99 CAC Levels 99<CAC<300 AA Men (N=794) CAC>300 RF IgM 1.1 (0.7-1.7) 0.6 (0.3-1.3) 1.0 (0.5-1.9) RF IgA 1.1 (0.6-1.9) 0.9 (0.4-2.0) 1.2 (0.6-2.4) Either RF isotype 1.1 (0.7-1.6) 0.7 (0.4-1.4) 1.1 (0.6-1.9) Both RF isotypes 1.2 (0.6-2.6) 0.5 (0.1-2.0) 1.1 (0.4-3.1) CCP 0.9 (0.4-2.4) 0.3 (0.03-2.4) NA** AA Women (N=1000) RF IgM 1.2 (0.8-1.8) 1.4 (0.8-2.5) 1.5 (0.8-2.8) RF IgA 1.0 (0.6-1.8) 2.2 (1.1-4.2) 2.4 (1.2-5.0) Either RF isotype 1.2 (0.8-1.7) 1.6 (0.9-2.7) 1.4 (0.8-2.6) Both RF isotypes 1.1 (0.5-2.3) 2.7 (1.1-6.3) 4.0 (1.6-9.6) CCP 2.0 (0.9-4.8) 1.1 (0.2-5.1) 4.0 (1.3-12.5) * Adjusted for age, smoking status, BP, BMI, HDL-c, LDL-c, DM, aspirin use, and cholesterol and BP medication use 7

Adjusted Hazard Ratios (95% CI) for the Incidence of Cardiovascular Clinical Events CTD-related CVD Hard CHD Hard CVD All CHD All Caucasian Men (N=1203) RF IgM 1.4 (0.8-2.5) 1.5 (0.8-2.9) 1.2 (0.7-1.9) 1.1 (0.6-1.9) RF IgA 0.8 (0.3-1.9) 0.9 (0.4-2.4) 0.6 (0.3-1.4) 0.6 (0.3-1.5) Either RF isotype 1.0 (0.6-1.8) 1.2 (0.6-2.2) 0.9 (0.6-1.5) 0.9 (0.5-1.5) Both RF isotypes 1.8 (0.6-4.9) 1.7 (0.5-5.6) 1.0 (0.4-2.8) 0.9 (0.3-2.8) CCP 1.2 (0.3-4.9) 0.7 (0.1-5.3) 1.0 (0.3-3.3) 0.8 (0.2-3.2) Caucasian Women (N=1323) RF IgM 1.8 (0.9-3.4) 1.2 (0.4-3.0) 1.5 (0.8-2.6) 1.0 (0.4-2.3) RF IgA 0.5 (0.1-2.2) 1.1 (0.3-4.5) 0.8 (0.3-2.3) 1.3 (0.5-3.8) Either RF isotype 1.6 (0.8-3.0) 1.2 (0.5-3.0) 1.3 (0.7-2.3) 1.0 (0.4-2.1) Both RF isotypes 0.4 (0.1-3.1) 0.8 (0.1-6.1) 1.1 (0.3-3.5) 1.8 (0.6-6.0) CCP NA** NA NA NA * Adjusted for age, smoking status, BP, BMI, HDL-c, LDL-c, diabetes, aspirin use, and cholesterol and BP medication use. ** NA: Not applicable Adjusted Hazard Ratios (95% CI) for the Incidence of Cardiovascular Clinical Events RA-related CVD Hard CHD Hard CVD All CHD All AA Men (N=794) RF IgM 1.0 (0.5-2.0) 0.8 (0.3-2.0) 0.8 (0.4-1.5) 0.6 (0.3-1.4) RF IgA 1.3 (0.6-2.9) 1.1 (0.4-3.1) 1.1 (0.5-2.1) 0.7 (0.2-1.8) Either RF isotypes 0.9 (0.5-1.8) 0.8 (0.3-1.8) 0.8 (0.4-1.4) 0.6 (0.3-1.2) Both RF isotypes 2.0 (0.7-5.6) 1.6 (0.4-6.8) 1.5 (0.6-3.8) 0.9 (0.2-3.6) CCP NA** NA 0.4 (0.1-3.0) NA AA Women (N=999) RF IgM 2.1 (1.1-4.0) 1.7 (0.7-4.5) 1.7 (0.9-3.0) 1.4 (0.6-3.1) RF IgA 3.4 (1.7-6.9) 5.0 (1.9-12.7) 2.8 (1.5-5.1) 3.5 (1.6-7.6) Either RF isotypes 2.7 (1.4-5.1) 2.5 (1.0-6.4) 2.0 (1.2-3.5) 1.9 (0.9-4.0) Both RF isotypes 3.3 (1.5-7.3) 4.5 (1.6-13.0) 2.7 (1.3-5.7) 3.3 (1.3-8.2) CCP 2.3 (0.7-7.7) 1.1 (0.1-8.3) 1.6 (0.5-5.1) 0.8 (0.1-5.9) * Adjusted for age, smoking status, BP, BMI, HDL-c, LDL-c, diabetes, aspirin use, and cholesterol and BP medication use. ** NA: Not applicable Conclusion RA-related autoantibodies were associated with both subclinical and clinical atherosclerosis in a community based cohort. These findings taken together with previous work indicate that autoimmune factors may play a role in the pathogenesis of atherosclerosis, even in individuals without RA. Areas for further investigation Risk for CHD cannot be explained completely by conventional cardiac risk factors. Consequently, new approaches to cardiovascular risk assessment and identification of novel biomarkers for early CHD are being sought in order to identify more patients who are asymptomatic, but at sufficiently high risk to justify risk factor modification. Hlatky MA, et al. Circulation 2009;119(17):2408-16. Greenland P, et al. Circulation 2001;104(15):1863-7. Areas for further investigation Could autoantibodies be utilized as novel risk markers for CVD? Large scale long term prospective studies needed: o To confirm our findings and establish causality o To better characterize the degree of risk conferred by RA-related and other autoantibodies. o To gather serial serum specimens so that we can determine whether epitope spreading and increased specificities are relevant to CVD risk. Kiang Liu 1, PhD Rowland W. Chang 1, MD, MPH Richard M. Pope 1, MD Thanh-Huyen T. Vu 1, MD, PhD Joseph Kang 1, PhD Acknowledgements Elizabeth Karlson 3, MD Marius Teodorescu 2, PhD Mary Cushman 4, MD, MSc 1 Northwestern University Feinberg School of Medicine, Chicago, IL 2 TheraTest Laboratories, University of Illinois College of Medicine, Chicago, IL 3 Brigham and Women's Hospital, Boston, MA 4 The University of Vermont, Colchester, VT NIH Arthritis Foundation R01 HL104047 (NHLBI) Arthritis Investigator Award K23AI064397-01A2 (NIAID/NIAMS) N01-HC-48047-50, N01-HC-95095 (NHLBI) P60 AR048098 (NIAMS) Multidisciplinary Clinical Research Center in Rheumatology TheraTest Laboratories Marius Teodorescu, MD, PhD 8

Table 2. Percent Positive RA-related in Study Samples by Race and Gender RA-related Auto-antibodies (%) Caucasian Chinese African American Hispanic P Value, Men Women Men Women Men Women Men Women N. 1205 1323 380 406 794 1000 696 753 2-sided RF IgM 12.6 12.8 12.2 13.8 18. 9 22.9 15.2 17.4 <.001 RF IgA 7.0 5.0 9.5 5.9 11.5 12.3 10.5 9.4 <.001 Either RF isotype 17.4 14. 9 18.2 16.8 25.1 28.9 20.8 22.7 <.001 Both RF isotypes 2.2 2.9 3.4 3.0 5.3 6.3 4.9 4.1 <.001 Anti-CCP 1.7 1.8 0.8 1.0 2.8 3.4 1.2 1.6.003 P value for overall group comparison based on χ2. Table 2. Percent Positive CTD-related (Ppts with Data for CAC Year 15 and Year 7) CTD-related Black Women Black Men White Women White Men N 489 331 495 523 P value* acl IgM 14.3 10.9 17.4 11.9 0.024 acl IgG 6.3 4.2 4.0 3.4 0.139 ANA 18.8 12.4 15.0 10.1 <0.001 Beta-2-GPI IgM 6.8 4.5 9.9 6.5 0.023 Beta-2-GPI IgG 1.4 1.2 1.2 1.3 0.989 Beta-2-GPI IgA 1.6 2.4 1.4 1.9 0.743 RF IgM 24.1 22.7 13.9 18.9 <0.001 RF IgA 11.7 11.2 6.3 5.7 <0.001 CCP 0.6 0 1.0 0.8 0.353 *P value for overall group comparison based on χ2 9