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Projekt HRZZ: COPERA

Projekt Hrvatske zaklade za znanost br. 5699

Characterization of osteoclast progenitor responses to arthritis

ACRONIM: COPERA

Principal Investigator (PI) – prof.dr.sc. Danka Grčević

Project duration in months: 36 (1.9.2014-1.9.2017)

Project funding: 750.000 HRK (250.000 HRK / year)

Project synopsis:

Chronic joint diseases affect more than a third of the world’s population and represent a major health problem because they cause disability, have progressive morbidity, and therapy is only partially successful. Several forms are classified in clinical practice, depending on the pathogenic mechanism and primary target tissue: osteoarthritis, rheumatoid arthritis (RA) and spondyloarthritis (including ankylosing spondylitis, psoriatic arthritis, and several other forms). Mouse models of arthritis, with collagen-induced arthritis being the most widely used as a model of rheumatoid arthritis, still represent the necessary tool to study the disease pathogenesis in light of newly discovered regulatory molecules and novel therapeutic strategies.

Through the production of osteoresorptive mediators by inflammatory and immune cells, chronic inflammation creates microenvironment that promotes osteoresorption by osteoclasts. Osteoclasts are large multinucleated bone-resorbing cells arising from monocyte/macrophage hematopoietic lineage. A number of systemic and local stimuli, such as hormones and cytokines/chemokines, promote osteoclastogenesis by enhancing osteoclast differentiation, activation, lifespan and function, but the exact mechanism of how the cytokine/chemokine network works in vivo in the context of arthritis is largely unclear. Particularly, the human osteoclast precursor subpopulations are not precisely defined and it is, as yet, unknown which critical mediators have the potential to activate osteoclast precursors and attract them to joint lesions.

Using the mouse model of collagen-induced arthritis and samples from arthritic patients, we aim to understand the differentiation sequence of early progenitors to mature osteoclasts, shifts in osteoclast progenitor populations and their migration during arthritis. We plan to identify distinct osteoclast precursor subpopulations at joint lesions and in periphery, their migratory and homing patterns, and their functional link to bone loss in arthritis. In addition, we expect to define the properties of inflammatory microenvironment and the role of specific mediators in the regulation of osteoclast trafficking, differentiation and activity in arthritic conditions. The mouse model of arthritis will enable us to monitor the disease progression and manipulate the immune response in vivo whereas the analysis of human samples will allow for the translation to the clinical setting.

A better modulation of the immune response could offer the potential to design therapeutic approaches to not only control inflammation but also limit the bone destruction in the context of chronic inflammatory joint diseases.

DETALJNIJE INFORMACIJE O PROJEKTU

Extended Synopsis of the project

Chronic joint diseases affect more than a third of the world’s population and represent a major health problem because they cause disability, have progressive morbidity, and therapy is only partially successful (Romas E. Rheum Dis Clin North Am 2006; Sipos W. Curr Med Chem 2008; Suematsu A. Mod Rheumatol 2007; Smolen JS. Ann Rheum Dis 2010). Several forms of chronic joint diseases are classified in clinical practice, depending on their pathogenic mechanisms and primary target tissue: osteoarthritis, rheumatoid arthritis (RA) and spondyloarthritis (including ankylosing spondylitis, psoriatic arthritis, and several other forms) (Lories RJU. Cyt Grow Fac Rev 2005; Sangha O. Rheumatology (Oxford) 2000). Inflammatory arthritides comprise a heterogeneous group of joint disorders that are characterized by chronic inflammatory response as well as periarticular and generalized bone loss due to deregulation of bone metabolism – bone resorption by osteoclasts and bone formation by osteoblasts (Takayanagi H. Nat Rev Rheumatol 2009; Goldring SR. Ann Rheum Dis 2013). Through the production of osteoresorptive mediators (interleukin (IL)-1, IL-6, IL-11, IL-17, tumor necrosis factor (TNF)-alpha and receptor-activator of nuclear factor-kappaB ligand (RANKL), CCL2, CXCL12, etc) by inflammatory/immune cells, inflammation creates conditions that promote differentiation of bone-resorbing osteoclasts. Thus, osteoclasts represent a link between joint inflammation and structural damage in arthritis (Takayanagi H. Arthritis Rheum 2000; Choi Y. Nat Rev Rheumatol 2009).

Animal models of autoimmune arthritis still represent an idispensible tool to study the disease pathogenic mechanisms especially in light of newly discovered regulatory molecules and novel therapeutic strategies (De Benedetti F. Arthritis Rheum 2006; Kwak HB. Arthritis Rheum 2008; Chao CC. Autoimmunity 2010). Different animal models are exploited to better understand arthritis pathogenesis, with collagen-induced arthritis (CIA) being the most widely used as an experimental model of RA (Campbell IK. Eur J Immunol 2000)

Erosions of the periarticular bone are the central feature of RA and also occur in SpA and the osteoresortive form of OA (Deal C. Curr Rheumatol Rep 2012; van der Heijde D. Ann Rheum Dis 2013). Development of bone erosions is critically dependent on osteoclasts, highly specialized bone cells capable of resorbing the mineralized matrix (Le Goff B. Arthritis Rheum 2013), arising from myeloid hematopoietic progenitor cells (Boyce BF. Arch Bichem Biophys 2008). Physiologically, osteoclast progenitors (OCPs) reside within spleen, bone marrow and peripheral blood mononuclear cells. Committed OCPs express the receptor activator of nuclear factor κΒ (RANK) and can be matured in vitro into fully functional osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF) and RANKL (Boyle WJ. Nature 2003). RANKL, expressed on osteoblasts and hypertrophying chondrocytes, as well as on activated T lymphocytes, is up-regulated by pro-resorptive hormones, cytokines and inflammatory mediators, such as vitamin D3, parathyroid hormone, prostaglandin E2, IL-1, IL-7, IL-11, IL- 17, and IL-23. Although it is known that a number of systemic and local stimuli promote osteoclastogenesis by enhancing osteoclast differentiation, activation, lifespan and function, dissection of OCP populations is necessary to more clearly elucidate those effects.

Studies on human samples have revealed that OCPs could be found in the peripheral blood and synovial tissue of patients with RA and SpA, mediating bone loss locally, in the form of bone erosions and joint osteolysis, and systemically, with loss of skeletal bone density (Schett G. Ann Rheum Dis 2010; Kikuta J. Rheumatology (Oxford) 2013). Under pathological conditions associated to arthritis, the process of osteoclastogenesis is markedly enhanced by various proinflammatory factors (Ritchlin CT. J Clin Invest 2003; Dalbeth N. Arthritis Res Ther 2010). A wide range of proinflammatory cytokines (IL-1, IL-6, IL-17, IL-18, tumor necrosis factor α (TNF-α)), chemokines (such as CC-chemokine ligand 2 (CCL2), CCL3, CCL4), growth factors (vascular endothelial growth factor (VEGF), hypoxia-inducible factors), and apoptotic mediators (TNF-related apoptosis-inducing ligand, herpesvirus entry mediator ligand) are disturbed in arthritis, which directly or indirectly, through RANK/RANKL/osteoprotegerin (OPG) system, promote osteoclastogenesis (Grčević D. J Rheumatol 2010; Dalbeth N. Arthritis Res Ther 2010; Bugatti S. Curr Rheumatol Rep 2011). It has been shown that even a small rise in the level of systemic inflammation can precipitate osteodestruction, leading to fractures and disabilities related to arthritis (Kim MS. J Cell Biochem 2006). A number of treatment modalities are currently under investigation or in use, including biological therapeutics aimed to block proinflammatory cytokines (ie IL-1, TNF-α, IL-6, IL-12/IL-23), but without satisfactory effects in reducing bone resorption, indicating the need for further investigations.

Objectives

– Using the mouse model of CIA and samples from arthritic patients with RA, we propose to elucidate the differentiation sequence of early progenitors to mature osteoclasts, shifts in the discrete OCP subpopulations as well as their migration pattern during arthritis.

– We plan to identify discrete OCP subpopulations induced by arthritis at joint lesions, juxta-articular bone marrow and in periphery, their migratory and homing patterns, and their functional link to bone loss in arthritis.

– In addition, we expect to define the properties of the inflammatory microenvironment and the role of specific mediators in the regulation of OCPs trafficking, homing, differentiation and activity in conditions related to arthritis.

– The mouse CIA model will enable us to monitor the disease progression in the context of increased osteoclastogenesis associated to inflammatory arthritis in vivo whereas human samples will allow for the characterization of OCPs in RA and translation to the clinical setting.

– A better characterization of the arthritis-induced OCP subpopulations induced by arthritis could offer the potential to design therapeutic approaches to control their migration and curtail their osteoresorptive potential thus limiting bone destruction in the context of arthritis.

Hypothesis

We hypothesize that systemic immune-mediated chronic inflammatory response in RA, as a prototype of osteoresorptive arthritis, promotes bone loss by altering the discrete subpopulation(s) of OCPs in the bone marrow, circulation and synovial compartment. The responses of the induced OCP subpopulation(s) associated to arthritis include activation, recruitment and homing of OCPs to subchondral region and joint lesions, becoming mature bone-resorbing osteoclasts. Thus, discrete osteoclast subpopulation that is the most sensitive to inflammation associated to RA could be identify and represents the crucial source of effectors of bone destruction. Activation and migration of arthritis-sensitive OCPs are orchestrated by the specific attraction signals of complex inflammatory environment not only in synovial compartment, but also within bone marrow compartment and in circulation.

PRELIMINIRANE STUDIJE I OČEKIVANI REZULTATI

Preliminary studies

Mouse model of CIA was induced according to the modified protocol by Cambell et al (Eur J Immunol 2000), in C57BL/6 mice. We had been optimized clinical scoring and anti-CII antibody detection, and found that in the follow-up period (up to 100 days) mice developed moderate to severe arthritis, with significant production of anti-CII antibodies in serum. Peripheral (metatarsophalangeal) joints were affected, mostly in the form of articular space narrowing and mild synovial thickening and infiltration. Changes in the subchondral bone marrow compartment were consistently observed, with fatty marrow replacement by diffuse infiltration of inflammatory cells. Moreover, subchondral bone volume was reduced, with increase in the number of active osteoclasts presented at the subchondral bone surfaces. Number of TRAP+ osteoclasts differentiated in vitro from bone marrow cells of mice with CIA were higher compared to non-arthritic controls at all analyzed time-points. In addition to bone marrow changes, we confirmed that peripheral blood of mice with CIA contained approximately 2-times more OCPs (defined as B220-CD3-N.K1.1-CD11b+CD115+ cells) paralleled with increased number of TRAP+ osteoclasts differentiated in vitro. Our results on human samples indicated that OCPs, capable for differentiation into mature osteoclasts, are contained among both peripheral blood and synovial fluid-derived cells but not with the same frequency in different forms of arthritis. Patients with RA have greater osteoclastogenic potential of peripheral blood OCPs compared with PsA.

Expected impact

By accomplishing project aims, we expect the following outcomes and impact beneficiary for the host institution and Croatian scientific community in general:

  1. Establishment of internationally competitive research groups. Proposed project will help Dr. Grcevic to further establish herself as an independent senior researcher and to stimulate association of complementary research groups (participating in the project) into the interdisciplinary field of osteoimmunology/rheumatology as a center of excellence. Project achievements and publications in leading scientific journal would add to the international recognition of the supporting institution, PI (Dr. Grcevic) and her team members as competitive research environment.
  2. Development of Croatian scientific and research space. Scientific achievements will be disseminating at national and international scientific meetings, symposia and university (web page, lectures, workshops) and provide a basis for future applications for other sources of funding. D. Grcevic most closely collaborates with several research groups in USA and Europe and she will actively work to attract international funding to UZSM.
  3. Training of young scientists. Transfer of knowledge will be stimulated by the recruitment of young scientists to the novel and competitive scientific approaches at UZSM and during the research visits to the collaborative institution (Prof. H.L. Aguila and Prof. I. Kalajzic at the University of Connecticut Health Center, CT, USA). Visiting researchers will transfer acquired knowledge and techniques and implement them at UZSM to help create a state-of-the art laboratory for the field of osteoimmunology/rheumatology.
  4. Strengthening of mentoring capacities in institution. The experience in research and organizational activity related to the proposed project will help Dr. Grcevic to further establish herself and research team as experts in the field of osteoimmunology/rheumatology, and to increase the research competence and mentoring capacities for training and education of young scientists at UZSM.
  5. Strengthening of Croatian economy and the welfare of society. Our study may have clinical relevanc by offering the potential for designing novel therapeutic approaches in which osteoclast differentiation and activity could be modulated by controlling immune system pathways. The idea is to locally deliver siRNA that would block targeted cytokine/chemokine or corresponding receptor expression, and consequently suppress osteoclast differentiation.

METODOLOGIJA I FINANCIJSKI PLAN

Methodology

For the project, which will utilize the mouse model of CIA and samples of patients with RA, the number of state-of the art and novel procedures are planed to be used for several applications. In vivo CIA model in C57BL/6 mice as well as osteoclastogenic in vitro assays, hematopoietic cell phenotyping, cell sorting and human sample analysis are already ongoing in the involved laboratories, therefore we do not expect major technical difficulties to introduce those procedures for the proposed applications. Preliminary studies on mouse CIA model and human samples from patients with arthritis confirmed proposed methodology as suitable to test the research hypothesis. The most innovative steps, which would be introduced by the research team, are separate dissection of OCP subpopulations at one side and immune-cell subpopulations on the other side, in vivo tracking system for OCP migration pattern, high-power preclinical MRI imaging and studies for efficient delivery of siRNA in vivo.

Financial plan

Total project budget: 750000.00 HRK (250000.00/year)

SUDIONICI PROJEKTA I OPREMA

Scientific project leader

DANKA GRČEVIĆ, M.D., Ph.D., associate professor

Department of Physiology and Immunology, University of Zagreb School of Medicine, Croatia

Croatian Institute for Brain Research; Laboratory for Molecular Immunology, Zagreb, Croatia

Project team members

ZRINKA JAJIC, M.D., Ph.D., associate professor

University Department of rheumatology, physical medicine and rehabilitation Clinical hospital center “Sestre milosrdnice”, University of Zagreb

ASJA STIPIC MARKOVIC, M.D., Ph.D., associate professor

Head of Department of Clinical Immunology, Pulmology and Rheumatology, University Hospital “Sveti Duh”, Zagreb, Croatia

IVO KALAJZIC, M.D., Ph.D., associate professor

Center of Regenerative Medicine and Skeletal Development, University of Connecticut Health Center, Farmington, Connecticut, USA

HECTOR LEONARDO AGUILA, M.D., Ph.D., associate professor

Center for Immunotherapy of Cancer and Infectious Diseases, Department of Immunology, University of Connecticut Health Center, Farmington, Connecticut, USA

PETER IAN CROUCHER, M.D., Ph.D., associate professor

Chair, Musculoskeletal Medicine Division, Garvan Institute of Medical Research, Conjoint Professor, Faculty of Medicine, University of New South Wales, Australia.

VEDRAN KATAVIC, M.D., Ph.D., associate professor

Department of Anatomy, University of Zagreb School of Medicine, Croatia

NATASA KOVACIC, M.D., Ph.D., assistant professor

Department of Anatomy, University of Zagreb School of Medicine, Croatia.

MARINA IKIC MATIJASEVIC, M.D., PhD, clinical immunologist

Department of Internal Medicine, University Hospital “Sveti Duh”, Zagreb, Croatia

ELVIRA LAZIC MOSLER, M.D., Ph.D., dermatovenerologist

University Department for Dermatovenereology, University Hospital Centre Zagreb, Croatia

FRANE PAIC, molecular biologist, PhD, senior assistant

Department of Medical Biology, University of Zagreb School of Medicine, Zagreb, Croatia.

ALAN SUCUR, M.D., assistant, PhD student

Department of Physiology and Immunology, University of Zagreb School of Medicine, Croatia

DARJA FLEGAR, M.D., HRZZ project associate, PhD student

Department of Physiology and Immunology, University of Zagreb School of Medicine, Croatia

Used facilities at UZSM

– Laboratory for Immunology and Cell culture located at the Department of Physiology and Immunology, UZSM consists of two laboratory rooms: first laboratory (two cooling centrifuges, standard PCR unit (2700, Applied Biosystems by LT), devices for the electrophoresis of DNA and proteins, UV/VIS spectrophotometer (Eppendorf BioSpectrometer), autoclave, sonicator (Qsonica Sonicator Q500, Fisher Scientific)), second laboratory (equipped with two CO2 incubators (Heracell 150, Haereus), laminar flow chamber (Aura-V, Ehret), invert microscope (Axiovert, CarlZeiss), liquid nitrogen container, centrifuge (5810/5810, Eppendorf)).

– Laboratory for Molecular Immunology located in the building of the Croatian Institute for Brain Research, within ZUSM as a part of the research team facilities, consists of two laboratory rooms (first laboratory – equipped with cooling centrifuge, standard PCR unit (2700, Applied Biosystems), devices for the electrophoresis of DNA and proteins, equipments for routine histology and immunohistochemistry, fume hood; second laboratory – equipped with CO2 incubator (Heracell 150, Haereus), laminar flow chamber (Aura-V, Ehret), spectrophotometer (NanoDrop 1000, Thermo Scientific), cooling centrifuges (Hettich Universal 32R and Hettich Micro 120), vacuum centrifuge (Concentrator 5301, Eppendorf) and a real-time PCR system (ABI7000 and ABI7500 SDS, Applied Biosystems by LT)); and a cabinet (used for microscopic analysis and histomorphometry. It is equipped with a fluorescent invert microscope (Axiovert, Carl Zeiss), with camera and software (AxioVision, Carl Zeiss)).

– Croatian Institute for Brain Research core facilities and capital equipment (including IVIS Spectrum Pre-clinical In Vivo Imaging System, PerkinElmer), Animal facility, Flow-cytometry facility (Attune, Applied Biosystems by LT, FACSAria I, BDB).

– Laboratory for Microscopy and Morphometry at the Department of Anatomy equipped with: fluorescent microscope (AxioImager, Carl Zeiss) with camera and software for image analysis and reconstruction (Osteomeasure, Ostoemtrix). Preclinical magnetic resonance technology (Preclinical MRI BioSpec 7T, Bruker; still under installation, will be fully functional by June 2014) and Micro Computed-tomography instrument (SkyScan1076, SkyScan).

REZULTATI PROJEKTA

1. GODINA PROJEKTA

DARJA FLEGAR, ALAN ŠUĆUR, ANTONIO MARKOTIĆ, NATAŠA KOVAČIĆ, DANKA GRČEVIĆ; Osteoporosis in the view of osteoimmunology: common feature underlined by different pathogenic mechanisms
Alan Šućur, Vedran Katavić, Tomislav Kelava, Zrinka Jajić, Natasa Kovačić & Danka Grčević. Induction of osteoclast progenitors in inflammatory conditions: key to bone destruction in arthritis
Dan doktorata, Alan Šućur
12th International Medical Postgraduate Conference in Hradec Kralove, Alan Šućur

2. GODINA PROJEKTA

Ikić Matijašević M, Flegar D, Kovačić N, Katavić V, Kelava T, Šućur A, Ivčević S, Cvija H, Lazić Mosler E, kALAJZIĆ I, Marušić A, Grčević D. Increased chemotaxis and activity of circulatory myeloid progenitor cells may contribute to enhanced osteoclastogenesis and bone loss in the C57BL/6 mouse model of collagen-induced arthritis. Clin Exp Immunol 2016, Sep 10. doi: 10.1111/cei.12862

D. Flegar, A. Sucur, A. Markotic, N. Kovacic, T. Kelava, V. Katavic, S. Ivcevic, K. Zrinski Petrovic, D. Grcevic. Osteoclast progenitors are attracted by CCL2/CCR2 and ccl5/ccr5 chemotactic signals to the sites of osteitis associated with collagen induced arthritis. Ann Rheum Dis 2016;75(Suppl2): 925

A. Sucur, Z. Jajic, M. Artukovic, D. Flegar, D. Grcevic. CD32+ B lymphocytes and IL21R+ T lymphocytes are associated with disease activity and increased levels of proinflammatory cytokines in patients with rheumatoid and psoriatic arthritis. Ann Rheum Dis 2016;75(Suppl2): 181

Z. Jajic, A. Sucur, T. Kelava, M. Artukovic, A. Stipic-Markovic, S. Ivcevic, F. Grubišic, D. Flegar, N. Kovacic, V. Katavic, D. Grcevic. Expression of chemokines and chemokine receptors on peripheral blood mononuclear cells of patients with rheumatoid arthritis. Ann Rheum Dis 2016;74(Suppl2): 912

Sucur, Z. Jajic, M. Artukovic, A. Stipic Markovic, S. Ivcevic, K. Zrinski Petrovic, D. Flegar, T. Kelava, V. Katavic, N. Kovacic, D. Grcevic. Chemokine receptor profile of osteoclast progenitor cells in patients with rheumatoid arthritis. ECTS-IBMS Abstracts (2015) P10

Hrvatsko imunološko društvo predavanje na temu "CHARACTERIZATION OF OSTEOCLAST PROGENITORS IN PATIENTS WITH RHEUMATOID ARTHRITIS", 10.07.2016.,HIIM, ZAGREB
3rd Meeting of Middle European Societies of immunology and Alergology predavanje na temu "Lymphocytic subpopulations associated with disease activity and levels of proinflammatory cytokines in rheumatoid and psoriatic arthritis", Alan Šućur

3. GODINA PROJEKTA

Sucur A, Jajic z, Artukovic M, Ikic Matijasevic M, Anic B, Flegar D, i sur. Chemokine signals are crucial for enhanced homing and differentiation of circulating osteoclast progenitor cells. Arthritis Res The. 2017;19(1):142.

Darja Flegar, Alan Sucur, Antonio Markotic, Natasa Kovacic, Tomislav Kelava, Vedran Katavic, Sanja Ivcevic and Danka Grcevic. Immunophenotyping of chemokine receptor profile on osteoclast progenitors may help reveal mechanisms of their increased migration in collagen-induced arthritis. J Immunol May 1, 2017, 198 (1 Supplement) 143.15;

A Sucur, Z Jajic, M Artukovic, M Ikic Matijasevic, F Grubisic, B Anic, S Ivcevic, D Flegar, D Grcevic. Chemokine signals are critical for homing and enhanced differentiation of circulating osteoclast progenitor cells. Ann Rheum Dis 2017;76(Suppl2)

Doktorska disertacija "Fenotipska i funkcijska karakterizacija osteoklastnih progenitora u bolesnika s reumatoidnim artritisom", Alan Šućur