The Pathogenesis of Bone Erosions in RA FULL VERSION

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1 The Pathogenesis of Bone Erosions in RA FULL VERSION 1

2 Key Learning Objectives Understand the role of osteoclasts in normal bone remodeling Comprehend the key processes in pathologic osteoclast functions Discuss the pathogenesis of bone erosion in RA before and after clinical onset Evaluate the mechanism of ACPA-induced osteoclastogenesis in RA ACPA = anti-citrullinated protein antibody; RA = rheumatoid arthritis. 2

3 OSTEOCLAST BIOLOGY IN HEALTH AND DISEASE 3

4 Bone Formation and Resorption Are Closely Coupled Processes Bone formation Osteoblasts make bone by producing a matrix that then becomes mineralized Bone mass is maintained by a balance between the activity of osteoblasts and osteoclasts Bone resorption Osteoclasts break down bone and are responsible for bone resorption Osteoblasts Osteoclasts New bone Bone Adapted from Logothetis CJ, Lin SH. Nat Rev Cancer. 2005;5:

5 Osteoclast Formation Is Triggered by CSF-1, RANKL, and OPG Monocyte Macrophage CSF-1R CSF-1 Osteoclast precursor RANKL RANK TRAP Cathepsin K MMP-9 OPG Osteoblast Osteoclast Sealing Zone Ruffled border The RANKL-RANK-OPG axis is crucial to physiologic osteoclast differentiation Osteoclast expression of bone-degrading enzymes, TRAP, cathepsin K and MMP-9, regulates the bone resorption process CSF-1 = macrophage colony-stimulating factor 1; CSF-1R = CSF-1 receptor; MMP-9 = matrix metalloproteinase 9; OPG = osteoprotegerin; RANK = receptor activator of nuclear factor κb; RANKL = RANK ligand; TRAP = tartrate-resistant acid phosphatase. Adapted from Adamopoulos IE, Mellins ED. Nat Rev Rheumatol. 2015;11:

6 Pathological Osteoclastogenesis in RA Involves Many Components, Including ACPA Blood vessel membrane Monocytes CSF-1, GM-CSF T H 1 cell IFN-γ TNF M1 Macrophage M2 IL-4 IL-10 IL-13 T H 2 cell Tissue IL-10 Citrullinated Proteins (e.g. cit-vimentin) Bone Immature Dendritic cell IL-23 TNF IL-6 IL-1 IL-8 Osteoclast precursor RANKL Osteoclast ACPA B cell Adapted from Adamopoulos IE & Mellins ED. Nat Rev Rheumatol.2015;11:

In RA, Osteoclasts Are Found in the Junction Zone Between the Synovium, Cartilage, and Bone Synovial Tissue Higher Magnification At The Cartilage and Bone Interface Interface of

7 In RA, Osteoclasts Are Found in the Junction Zone Between the Synovium, Cartilage, and Bone Synovial Tissue Higher Magnification At The Cartilage and Bone Interface Interface of unmineralized and mineralized cartilage Osteoclast interface of mineralized cartilage and cortical bone Reprinted from Schett G, et al. Nat Clin Pract Rheumatol. 2005;1:

8 Our Understanding of RA Pathogenesis Has Evolved Preclinical Overt Clinical Disease Risk of developing RA ACPA-associated disease ACPA+ RA Genetic susceptibility and environmental challenges Loss of immune tolerance Pain Bone loss Sick leave Pain Inflammation Erosions Antibody production Osteoclast activation PAD activation IL-8 production Epitope spreading Increasing antibody titers Second hit IL = interleukin; PAD = peptidylarginine deiminase. Reprinted from Catrina AI, et al. Nat Rev Rheumatol. 2017;13:

Osteoclastogenesis May Occur Before Clinical Disease Onset Plasma cell ACPA Osteoclast precursor B cell T cell

osteoclast differentiation and lead to initial bone loss in the bone marrow adjacent to the joint Clinical

osteoclastogenesis via RANKL, and enhancing bone erosion Established Disease Large bone erosions filled with

9 Osteoclastogenesis May Occur Before Clinical Disease Onset Plasma cell ACPA Osteoclast precursor B cell T cell Osteoclast Mesenchymal cell Cytokines Pannus and erosion Preclinical Phase ACPA expression can stimulate osteoclast differentiation and lead to initial bone loss in the bone marrow adjacent to the joint Clinical Disease Onset Synovitis at the onset of clinical disease leads to production of cytokines, stimulating osteoclastogenesis via RANKL, and enhancing bone erosion Established Disease Large bone erosions filled with synovially-derived pannus tissue are present in established RA Adapted from Schett G, Gravallese E. Nat Rev Rheumatol. 2012;8:

10 THE EFFECTS OF ACPA ON BONE EROSION IN RA BEFORE AND AT CLINICAL ONSET 10

Citrullination Is a Post-translational Modification of a Protein Enzymatic conversion of arginine to citrulline, a non-classical amino acid H O I N NH +H 2 0 H 2 N+ NH 2 Peptidylarginine (+) PAD Ca

11 Citrullination Is a Post-translational Modification of a Protein Enzymatic conversion of arginine to citrulline, a non-classical amino acid H O I N NH +H 2 0 H 2 N+ NH 2 Peptidylarginine (+) PAD Ca 2+ H O I N + NH 3 + H NH O NH 2 Peptidylcitrulline (neutral) Post-translational citrullination of self-proteins is an essential step for the induction of ACPA autoantibodies PAD citrullination PAD = peptidylarginine deiminase. Top figure reprinted from Vossenaar ER, et al. Bioessays. 2003;25(11): Bottom image based on Willemze A, et al. Nat Rev Rheumatol. 2012;8(3):

ACPAs Are a Collection of Antibodies That Recognize

that can become citrullinated 1-3 Vimentin Fibrinogen

ACPAs recognize citrullinated protein, and some are

Arthritis Res Ther. 2014;16:R167. 2. Ioan-Facsinay A, et al.

12 ACPAs Are a Collection of Antibodies That Recognize Citrullinated Proteins Amongst the many self-proteins that can become citrullinated 1-3 Vimentin Fibrinogen Type II collagen α-enolase Myelin basic protein Histones ACPAs recognize citrullinated protein, and some are cross-reactive 1,2 IgG IgG IgG = immunoglobulin G. 1. Ossipova E, et al. Arthritis Res Ther. 2014;16:R Ioan-Facsinay A, et al. Ann Rheum Dis. 2011;70(1): Pratesi F, et al. Ann Rheum Dis. 2014;73: Image based on Willemze A, et al. Nat Rev Rheumatol. 2012;8(3):

13 Autoantibodies May Arise Years Before the Clinical Onset of RA 60 Analysis of Blood Samples From 79 RA Patients Prior to the Onset of Disease Symptoms Positive Patients, % IgM-RF or ACPA ACPA IgM-RF 49.4% 40.5% 27.8% Years Before the Start of Symptoms IgM = immunoglobulin M. Reprinted from Nielen MM, et al. Arthritis Rheum. 2004;50(2):

Bone Loss Can Be Detected Before the Clinical Onset of RA in ACPA+ Subjects A study of 15 ACPA+ subjects evaluated the effect of ACPA serostatus on bone preceding the onset of clinical disease ACPA+

14 Bone Loss Can Be Detected Before the Clinical Onset of RA in ACPA+ Subjects A study of 15 ACPA+ subjects evaluated the effect of ACPA serostatus on bone preceding the onset of clinical disease ACPA+ subjects had no concomitant symptoms or signs of RA Mean (±SD) age of ACPA+ subjects was 52.1±11.3 years Micro-CT results were compared to an ACPA cohort of patients (n=11) Healthy ACPA Subjects Healthy ACPA+ Subjects Arrows show corroded cortical bone in threedimensional reconstructions of the metacarpophalengeal joint Reprinted from Kleyer A, et al. Ann Rheum Dis. 2014;73(5):

15 Bone Density Is Reduced Before Clinical Onset In ACPA+ Subjects ACPA Healthy Subjects ACPA+ Healthy Subjects Arrows show cortical thinning, cortical fenestration as well as small bone erosions Reprinted from Kleyer A, et al. Ann Rheum Dis. 2014;73(5):

16 ACPA at Baseline Are Strong Predictors of Radiographic Progression An observational study of 125 RA patients evaluated the prognostic capacity of anti-cyclic citrullinated peptide (anti-ccp) Mean disease duration (SD) was 2.2 (1.2) years 75.2% were female 59.2% and 61.5 % were Anti-CCP and Anti-MCV positive at baseline, respectively Analyses compared the effect of a positive anti-ccp test on various measurements of radiographic progression Progression Measurement Total SHS Univariate Analyses OR (95% CI) 5.7 ( ) a Erosion Score 4.6 ( ) a JSN Score 2.6 ( ) a OR a P<0.01 CI = confidence interval; JSN = joint space narrowing; OR = odds ratio; SHS = Sharp van der Heijde Score. Syversen SW, et al. Ann Rheum Dis. 2010;69:

Seropositive RA Patients (n=38) Reprinted from Kocijan R, et

17 After RA Diagnosis, More Severe Disease Is Observed in Seropositive RA Patients Seronegative RA Patients (n=22) Seropositive RA Patients (n=38) Reprinted from Kocijan R, et al. Ann Rheum Dis. 2014;73: % were ACPA+ 87% were RF+ 17

18 ACPA+, But Not RF+ Is Significantly Associated With Erosion RF+ (vs RF ) ACPA+ (vs ACPA ) Age per 5 years Female (vs male) White (vs others) BMI per 5 kg/m 2 RA duration per 5 years Number of comorbidities Any biologic DMARD MHAQ Odds ratio and 95% CI for erosion Does Not Favor Erosion Favors Erosion anti-ccp = anti-cyclic-citrullinated peptide; BMI = body mass index; DMARD = disease-modifying antirheumatic drug; MHAQ = Modified Health Assessment Questionnaire; RF = rheumatoid factor. Alemao E, et al. Ann Rheum Dis. 2016;75(suppl 2):211. Abstract THU

19 Patients With RA Who Were ACPA+ Had Higher Rates of Prevalent Erosions Than Those Who Were ACPA A study of 9759 RA patients identified from the Corrona registry evaluated the proportion of patients with erosive disease stratified by disease duration and ACPA status Prevalence of Erosions According to Disease Duration and Serological Status Disease Duration, years >10 Serological status Anti-CCP, % 16.1% 22.7% 21.8% 28.1% (n/n) (359/2226) (169/744) (128/588) (206/733) Anti-CCP+, % 22.1% 32.8% 41.8% 59.4% (n/n) (546/2473) (306/934) (341/816) (740/1245) Harrold LR, et al. Arthritis Rheumatol. 2017;69(suppl 10): Abstract

20 A Broader Isotype Profile Is Associated With Greater Radiographic Progression EAC Netherlands EURIDISS Norway 40 a 80 a a 30 a 60 Median SHS 20 a a Median SHS 40 a Baseline Years of follow-up >5 ACPA isotypes 4 ACPA isotypes ACPA 0 Baseline 5 10 Years of follow-up a Comparison 4 isotypes vs 5 isotypes: P<0.05 ACPA IgG1, IgG2, IgG3, IgG4, IgA, and IgM were determined using a sandwich ELISA technique Note: EAC inception cohort, recent-onset RA, n=171; EURIDISS cohort, disease duration 4 years, n=238. Association between ACPA isotypes and radiographic progression: Mann-Whitney U tests and logistical regression analysis. EAC = Early Arthritis Clinic; EURIDISS = European Research on Incapacitating Diseases and Social Support; SHS = Sharp van der Heijde Score. van der Woude D, et al. Ann Rheum Dis. 2010;69(6):

21 THE MECHANISM OF ACPA-INDUCED OSTEOCLASTOGENESIS 21

ACPA From RA Patients Can Induce Osteoclast Differentiation and Enhance Bone Erosion ACPA 0 ng/ml ACPA 1 ng/ml ACPA 10 ng/ml ACPA 100 ng/ml ACPA 0 ng/ml ACPA 1 ng/ml ACPA 10

22 ACPA From RA Patients Can Induce Osteoclast Differentiation and Enhance Bone Erosion ACPA 0 ng/ml ACPA 1 ng/ml ACPA 10 ng/ml ACPA 100 ng/ml ACPA 0 ng/ml ACPA 1 ng/ml ACPA 10 ng/ml ACPA 100 ng/ml *P<0.05 Note: Osteoclasts were stained for TRAP (tartrate-resistant acid phosphatase)..reprinted from Harre U, et al. J Clin Invest. 2012;122(5):

citrullinated vimentin during osteoclast differentiation 0.0006 PAD2 Relative expression 0.0004 0.0002 0.

23 Anti-Citrullinated Vimentin, an ACPA, Is Expressed During Osteoclast Differentiation In the same study, osteoclast cultures demonstrated the increased activity of PAD enzymes and surface expression of citrullinated vimentin during osteoclast differentiation PAD2 Relative expression Days in culture Reprinted from Harre U, et al. J Clin Invest. 2012;122(5):

24 ACPA-Treated Mice Experienced Enhanced Expression of Osteoclast Precursors, TNF-α, and RANK Lymphocyte-deficient Rag1 / mice were challenged with mutated-citrullinated vimentin (MCV)-specific ACPA or control IgG pg/ml TNF-α * MFI RANK * Positive cells, % Osteoclast Precursor * Untreated IgG MCV-ACPA Untreated IgG MCV-ACPA Untreated IgG MCV-ACPA *P<0.05. MCV = mutated citrullinated vimentin; MFI = multiplex fluorescent immunoassay. Reprinted from Harre U, et al. J Clin Invest. 2012;122(5):

25 ACPA-Treated Mice Show Greater Bone Loss Compared to Controls Control IgG-Treated ACPA-Treated Note: Osteoclasts were stained for TRAP. Reprinted from Harre U, et al. J Clin Invest. 2012;122(5):

ACPAs Can Induce Osteoclastogenesis in Macrophages From Healthy Patients Total IgGs were isolated from the synovial fluid (SF, n=25) and peripheral blood (PB, n=35) of patients with RA, then ACPA

26 ACPAs Can Induce Osteoclastogenesis in Macrophages From Healthy Patients Total IgGs were isolated from the synovial fluid (SF, n=25) and peripheral blood (PB, n=35) of patients with RA, then ACPA affinity purified Purified ACPA IgG pools were effective in inducing osteoclastogenesis from PB-derived macrophages of healthy individuals OC numbers (fold) IgG ACPA * * Tartrate-resistant acid phosphatase (TRAP) staining of mature OC 0 SF PB *P<0.05 OC = osteoclast; PB = peripheral blood; SF = synovial blood. Reprinted from Krishnamurthy A, et al. Ann Rheum Dis. 2016;75:

27 Osteoclastogenesis Is Induced by Specific ACPA Epitopes OC numbers TRAP staining of mature osteoclasts and microscopic visualization of calcium phosphate resorption areas in the presence of 4 monoclonal ACPAs B02 Anti cit-peptides Abs B02, D10, B09 and C07 D10 B09 C07 OC numbers (fold) * E02 * B02 D10 B09 C07 Bone loss B02 D10 B09 D1 C07 0 Resorption area (fold) * * E02 B02 D10 B09 C07 *P<0.05 Reprinted from Krishnamurthy A, et al. Ann Rheum Dis. 2016;75:

28 Early Inhibition of PAD Enzymes Abrogates ACPA-Induced Osteoclastogenesis Early PAD inhibition (Day 0) Late PAD inhibition (Day 6) * * OC numbers (fold) * OC numbers (fold) # PADi (mm) PADi (mm) Control IgG-treated 1 µg/ml ACPA-treated 1 µg/ml Control IgG-treated 1 µg/ml ACPA-treated 1 µg/ml *P<0.05 PADi = peptidylarginine deiminase 2/4 inhibitor. Reprinted from Krishnamurthy A, et al. Ann Rheum Dis. 2016;75:

29 ACPA Binds to and Activates Human Osteoclasts to Produce IL-8 In Vitro IL-8 concentrations (pg/ml) Control * IgG 1 µg/ml * ACPA 1 µg/ml IL-8 concentrations (pg/ml) Control lgg 1µg/mL ACPA 1µg/mL * Day 4 Day 12 * *P<0.05 Reprinted from Krishnamurthy A, et al. Ann Rheum Dis. 2016;75:

30 IL-8 Plays a Vital Role in Perpetuating ACPA-Induced Osteoclastogenesis IL-8 increases osteoclastogenesis in a dose-dependent manner in culture Anti-IL-8 neutralizing antibodies inhibited macrophage-derived osteoclasts maturation dose dependently OC numbers OC numbers (fold) IL8 (µg/ml) Anti-IL8 (µg/ml) OC numbers (fold) Anti-IL-8 neutralizing antibodies completely abolished the effect of ACPAs No Anti-IL-8 * IgG Anti-IL-8 1 mg/ml No Anti-IL-8 * ACPA Anti-IL-8 1 mg/ml *P<0.05 Note: Reparixin is a CXCR1/2 antagonist blocking the murine IL-8 homologues. Reprinted from Krishnamurthy A, et al. Ann Rheum Dis. 2016;75:

$number/mm 1.5 1.0 0.5 Trabecular number * * BV/TV (%) 8 6 4 2 Trabecular bone volume fraction * * 0.$ 0 Saline ACPA ACPA + IL-8 inhibitor 0 Saline ACPA ACPA + IL-8 inhibitor. *P<0.

0 Saline ACPA ACPA + IL-8 inhibitor 0 Saline ACPA ACPA + IL-8 inhibitor. *P<0.

31 IL-8 Blocking Prevents ACPA-induced Bone Loss In Vivo Saline ACPA ACPA + IL-8 inhibitor Trabecular number/mm Trabecular number * * BV/TV (%) Trabecular bone volume fraction * * 0.0 Saline ACPA ACPA + IL-8 inhibitor 0 Saline ACPA ACPA + IL-8 inhibitor. *P<0.05 BMD = bone mineral density; RV/TV = bone volume/total volume. Reprinted from Krishnamurthy A, et al. Ann Rheum Dis. 2016;75:

Summary Citrullinated protein Osteoclast precursor PAD Precursor fusion ACPA ACPA bound to RF Bone loss in RA is a result of osteoclastogenesis, which may precede the onset of overt disease

32 Summary Citrullinated protein Osteoclast precursor PAD Precursor fusion ACPA ACPA bound to RF Bone loss in RA is a result of osteoclastogenesis, which may precede the onset of overt disease Studies suggest pathologic osteoclastogenesis may be induced via an ACPA-dependent mechanism PAD FCR Osteoclast IL-8 Bone loss Adapted from Catrina A, et al. Nat Rev Rheumatol. 2017;13:

33 Appendix 33

34 Abbreviations ACPA = anti-citrullinated protein antibody Anti-CCP = anti-cyclic citrullinated peptide Anti-MCV = anti-mutated citrullinated vimentin BMD = bone mineral density BMI = body mass index CI = confidence interval CSF-1 = macrophage colony-stimulating factor 1 CSF-1R = CSF-1 receptor CXCR1/2 = C-X-C motif chemokine receptor 1/2 DMARD= disease modifying antirheumatic drug EAC = Early Arthritis Clinic ELISA = enzyme-linked immunosorbent assay EURIDISS = European Research on Incapacitating Diseases and Social Support IgG = Immunoglobulin G IgM = Immunoglobulin M IL-8 = interleukin-8 JSN = joint space narrowing MCV = mutated citrullinated vimentin MFI = multiplexfluorescent immunoassay MHAQ = modified health assessment questionnaire MMP-9 = matrix metalloproteinase 9 OC = osteoclast OPG = osteoprotegerin OR = odds ratio 34

35 Abbreviations (cont d) PAD = peptidylarginine deiminase PADi = peptidylarginine deiminase 2/4 inhibitor PB = peripheral blood RA = rheumatoid arthritis RAG1 = recombination-activating gene 1 RANK = receptor activator of nuclear factor RANKL = RANK ligand RF = rheumatoid factor RV/TV = bone volume/total volume SD = standard deviation SF = synovial blood SHS = Sharp van der Heijde Score TNF = tumor necrosis factor TRAP = tartrate-resistant acid phosphatase 35

36 References Adamopoulos IE, Mellins ED. Alternative pathways of osteoclastogenesis in inflammatory arthritis. Nat Rev Rheumatol. 2015;11: Alemao E, et al. Association of the rheumatoid arthritis prognostic factors anti-citrullinated peptide antibodies, rheumatoid factor and erosions with disease activity. EULAR Poster THU0090. Catrina AI, et al. Mechanisms leading from systemic autoimmunity to joint-specific disease in rheumatoid arthritis. Nat Rev Rheumatol. 2017;13: Harre U, et al. Induction of osteoclastogenesis and bone loss by human autoantibodies against citrullinated vimentin. J Clin Invest. 2012;122(5): Harrold LR, et al. The impact of anti-cyclic citrullinated peptide seropositivity on erosion prevalence among patients with RA of varying disease duration.arthritis Rheumatol. 2017;69(suppl 10). Abstract Ioan-Facsinay A, et al. Anti-cyclic citrullinated peptide antibodies are a collection of anti-citrullinated protein antibodies and contain overlapping and non-overlapping reactivities. Ann Rheum Dis. 2011;70(1): Klareskog L, et al. Immunity to citrullinated proteins in rheumatoid arthritis. Annu Rev Immunol. 2008;26: Kleyer A, et al. Bone loss before the clinical onset of rheumatoid arthritis in subjects with anticitrullinated protein antibodies. Ann Rheum Dis. 2014;73(5): Kocijan R, et al. Differences in bone structure between rheumatoid arthritis and psoriatic arthritis patients relative to autoantibody positivity. Ann Rheum Dis. 2014;73: Krishnamurthy A, et al. Identification of a novel chemokine-dependent molecular mechanism underlying rheumatoid arthritis-associated autoantibody-mediated bone loss. Ann Rheum Dis. 2016;75:

37 References Logothetis CJ, Lin S-H. Osteoblasts in prostate cancer metastasis to bone. Nat Rev Cancer. 2005;5: Nielen MM, et al. Specific autoantibodies precede the symptoms of rheumatoid arthritis: a study of serial measurements in blood donors. Arthritis Rheum. 2004;50(2): Ossipova E, et al. Affinity purified anti-citrullinated protein/peptide antibodies target antigens expressed in the rheumatoid joint. Arthritis Res Ther. 2014;16:R167. Pratesi F, et al. Antibodies from patients with rheumatoid arthritis target citrullinated histone 4 contained in neutrophils extracellular traps. Ann Rheum Dis. 2014;73: Schett G, et al. Mechanisms of disease: the link between RANKL and arthritic bone disease. Nat Clin Pract Rheumatol. 2005;1: Schett G, Gravallese E. Bone erosion in rheumatoid arthritis: mechanisms, diagnosis and treatment. Nat Rev Rheumatol. 2012;8: Syversen SW, et al. Prediction of radiographic progression in rheumatoid arthritis and the role of antibodies against mutated citrullinated vimentin: results from a 10-year prospective study. Ann Rheum Dis. 2010;69: van der Woude D, et al. The ACPA isotype profile reflects long-term radiographic progression in rheumatoid arthritis. Ann Rheum Dis. 2010;69(6): Vossenaar ER, et al. PAD, a growing family of citrullinating enzymes: genes, features and involvement in disease. Bioessays. 2003;25(11): Willemze A, et al. The influence of ACPA status and characteristics on the course of RA. Nat Rev Rheumatol. 2012;8(3): IMMUS Jan/18 37

PATHOGENESIS OF RHEUMATOID ARTHRITIS

PATHOGENESIS OF RHEUMATOID ARTHRITIS Division of Rheumatology Department of Internal Medicine College of Medicine Seoul National University Seoul National University Bundang Hospital Yun Jong Lee Rheumatoid