1. Professor of Rheumatology and Chief ImmunoRheumatology Clinical Unit, University Hospital University of L’Aquila, L’aquila ITALY IMMOTA MANET Prof. Roberto Giacomelli MD, PhD Alexandria March 2016 Bone–Cartilage cross-talk in inflammatory arthritis patients

2. Introduction The ostheocondral junction Encompasses the region between the deeper layers of articular cartilage and the underlying subchondral bone. In the normal joint it comprises the deeper non-calcified cartilage, the tidemark, calcified cartilage, the cement line, and subchondral bone plate. S. Suri, D.A. Walsh / Bone 51 (2012) 204–211

3. Cross-talk between subchondral bone and articular cartilage Subchondral bone influences the overlying articular cartilage not only through its biomechanical properties, but also biochemically. Osteoblasts, osteoclasts, osteocytes and bone-lining cells may each engage in molecular interactions with the articular cartilage. The close physical relationship between subchondral bone and the cartilage has introduced the concept of biochemical and molecular cross-talk across this region. S. Suri, D.A. Walsh / Bone 51 (2012) 204–211

4. Cross-talk between subchondral bone and articular cartilage II S. Suri, D.A. Walsh / Bone 51 (2012) 204–211

5. Normal articular cartilage is hostile to vascular invasion, in part due to its matrix composition. In normal adult cartilage, chondrocytes synthesize matrix components very slowly. Important products of their activity to mantein the homeostasis are anti-angiogenic factors such as the regulatory peptides troponin I and thrombospondin-1, and inhibitors of matrix degrading enzymes, including tissue inhibitors of metalloproteinases (TIMPs), secretory leukocyte proteinase inhibitor and plasminogen activator inhibitor-1. S. Suri, D.A. Walsh / Bone 51 (2012) 204–211

6. An important pathway of signalling across the osteochondral junction may be illustrated by hepatocyte growth factor (HGF) and its receptor c-Met C-Met is expressed by articular chondrocytes, in which HGF can promote type II collagen and proteoglycan synthesis C-met signaling pathway D. Pfander et al.: Hepatocyte growth factor in osteoarthritis

7. C-met signaling pathway II In normal human cartilage HGF proteins were found in the territorial matrix surrounding chondrocytes of the lower deep zone and within calcified cartilage. Only weak or absent HGF RNA transcripts were identified in these locations by in-situ hybridization. The HGF receptor protein was detected in normal cartilage in a cellular or pericellular distribution. mainly in the chondrocytes of the deep zone and within calcified cartilage. D. Pfander et al.:Osteoarthritis and Cartilage (1999) 7, 548–559

8. The chondrocytes reaction pattern after an injury is at first glance rather heterogeneous and they can basically be summarized in different categories: Chondrocyte response to injury Sandell LJ, Arthritis Res 2001, 3:107–113 proteoglycan Phenotypical modulation

9. Human chondrocytes were isolated from OA cartilage and cultured in alginate beads for 4 or 10 days in the absence or in the presence of osteoblasts in monolayer. The osteoblasts were either isolated from non- sclerotic (N) or sclerotic (SC) zones of human subchondral bone. Before co-culture, osteoblasts were incubated for 72 h with IL-1b, IL-6 or oncostatin M SOX9, COL1, COL2, COL10, osteoblasts-stimulating factor, ALP, PTHrP and its receptor PTH-R mRNA levels in chondrocytes are incresed OsteoArthritis and Cartilage (2005) 13, 988e997

11. SC osteoblasts induced a phenotypic shift in OA chondrocytes that could lead to hypertrophic phenotype and subsequently to matrix mineralization. IL-1b, IL-6 or OSM-treated osteoblasts isolated from N subchondral bone fail to reproduce the SC osteoblasts effects, suggesting that the factor(s) involved in this process is (are) not produced or produced in an inactive form by IL-1b, IL-6 or OSM stimulated normal osteoblasts.

12. Crosstalk Between Chondrocytes and Osteoblasts in OA Chondrocytes in response to changes in their chemical and mechanical environment are activate and after they produce several inflammatory response proteins, such as cytokines, including IL-1β, IL-6, and TNF, and matrix-degrading enzymes including the metalloproteinases and a disintegrin and metalloproteinase with thrombospondin-like motifs. The innate immune system is activated in osteoarthritis. Chondrocytes express many toll-like receptors, which are activated by damage-associated molecular patterns. In osteoarthritis, these patterns consist of extracellular matrix molecules that include the glycosaminoglycan hyaluronan. Calcium pyrophosphate and sodium urate crystals also bind chondrocyte toll-like receptors and might therefore play a part in the aetiology of osteoarthritis. Lancet 2015; 386: 376–87

13. Pan J, Bone 51 (2012) 212–217

14. Morphology and vascularity at the osteochondral junction Smooth muscle a-actin positive cells Vascular channels stained for CD34-positive ECs Walsh D, Rheumatology 2010;49:1852–1861 Vascular densities for OA and RA were higher in the non-calcified cartilage, and lower in the subchondral bone.. RA cases were more likely to display proliferating ECs in vascular channels than were PM cases.

15. Vascular growth factor expression in articular cartilage and bone Walsh D, Rheumatology 2010;49:1852–1861 VEGF-positive cells PDGF-positive cells Chondrocytes displaying VEGF immunoreactivity in deep (RA) and superficial (OA) articular cartilage VEGF-positive Chondrocytes were predominantly localized to the superficial zone of articular cartilage of OA cases. PDGF-positive cells in vascular channels were most abundant in RA

16. Cartilage damage and chondrocyte response secondary to bone/cartilage cross-talk is a key feature in inflammatory joint diseases, such as rheumatoid arthritis and in degenerative joint disorders- osteoarthritis. Joint damage in OA and RA proceeds via different pathways that nonetheless share certain mechanistic similarities. Comparison of OA and RA Nature Reviews Rheumatology 11, 606–615 (2015)

17. In OA, cartilage loss occurs as part of a complex programme that resembles aspects of embryonic bone formation through endochondral ossification. Interaction of cartilage matrix and chondrocytes in OA Nature Reviews Rheumatology 11, 606–615 (2015)

18. Inflammatory cells in subchondral bone spaces are associated vascular channels Walsh D, Rheumatology 2010;49:1852–1861 CD3-positive lymphocytes within fibrovascular tissue occupying subchondral bone spaces in RA and OA Mononuclear CD68-positive cells

19. In RA, early cartilage damage is a key trigger of cellular reactions in the synovium. In a proposed model of RA as a site-specific manifestation of a systemic autoimmune disorder, early cartilage damage in the context of immune activation leads to a specific cellular response within articular joints that could explain not only the organ specificity of RA, but also the chronic nature and perpetuation of the disease Cartilage damage in RA triggers the tissue response and FLS activation Nature Reviews Rheumatology 11, 606–615 (2015)

21. Lymphoid aggregates with lymphoid neogenetic features are detectable on the subchondral side of the joint in established RA. The local inflammation/aggregation process appears to be related to osteoclast differentiation on the marrow side of subchondral bone, supporting a functional role of the bone compartment in local carthilage damage.

22. Cross-talk between synovial fibroblasts and immune cells is suggested to play a crucial role in inflammation and chronification of RA. In B cells-SF cocultures the concentrations of IL-6 and IL-8 increased manifold compared with single cultures and remained stable for several days after B-cell removal. Intracellular staining confirmed SF as key producers of IL-6 and IL-8, and B cells as main producers of TNFα and IL-1ß. Blocking experiments with a combination of anti-TNFα-antibodies and rIL- 1RA significantly reduced SF cytokine production by up to 90%, suggesting that B-cell-derived TNFα and IL-1ß were crucial mediators of SF activation. Besides cytokines, B-cell-activated SF also upregulated secretion of matrix metalloproteases such as MMP-3, thereby acquiring potential tissue destructive properties. Storch H, Ann Rheum Dis. 2015 May 18

24. Histologic features of femoral heads obtained from patients with ankylosing spondylitis In areas without cartilage the density of mononuclear cell infiltrates in the subchondral bone marrow was clearly lower or absent Dense rims of osteoblasts along the bone trabeculae were present in areas without cartilage

25. CD3 T cell aggregates The number of CD3 T cell follicles in AS patients was significantly higher than in OA patients, but was similar to that in RA patients. In AS patients, the number of lymphocytic infiltrates was significantly reduced in areas of complete cartilage destruction as compared with areas with remaining. This clearly differed from the findings in RA patients, where T cell follicles persisted even in the absence of surface cartilage. In RA patients, the number of CD3 T cell follicles was slightly increased in areas without cartilage as compared with areas with remaining cartilage

26. Interstitial T cells positive for CD4 and CD8 In femoral heads with cartilage on the surface, CD4 T cells and CD8 T cells were significantly increased in AS patients as compared with OA patients. In RA patients, in contrast, the number of CD4 T cells at sites with cartilage on the surface was similar to that in AS patients. The number of CD8 T cells in AS patients was significantly higher than that in RA patients.

27. CD68 osteoclastic foci at the bone–cartilage interface

28. These findings suggest that the subchondral bone marrow and bone–cartilage interface are primary sites of inflammation in AS and that cartilage might be necessary for the induction of inflammation, and cross-talk between cartilage and subchondral bone may be different from RA and OA

29. The crosstalk at the bone/cartilage interface may be elevated in inflammatory arthritis patients in vivo and in vitro. Increased vascularisation and formation of microcracks associated with abnormal bone remodelling in joints facilitate molecular transport from cartilage to bone and vice versa. Recent reports suggest that several critical signalling pathways and biological factors are key regulators and activate cellular and molecular processes in crosstalk among joint compartments. Therapeutic interventions including angiogenesis inhibitors, agonists/antagonists of molecules and drugs targeting bone remodelling are potential candidates for this interaction. A better understanding of crosstalk in bone/cartilage interface may lead to development of more effective strategies for treating these patients. Conclusions