Short summary

Fibrocytes in bone marrow, peripheral blood and synovial tissue. Association with clinical presentation, disease course and joint magnetic resonance imaging.

Rationale

The intimal lining of a synovial joint consists of unique cells called fibroblast-like synoviocytes (FLS cells) with interspersed macrophage-like synoviocytes. In the healthy joint, macrophage-like synoviocytes are implicated in the innate immune defense and support adaptive immunity, while FLS cells function to regulate the release of nutrients and molecules, into the joint cavity.

Rheumatoid arthritis (RA) is a chronic systemic disease with autoimmune traits, which primarily affects synovial joints. The intimal lining of the synovium expands and exhibits characteristics of unregulated growth and loss of contact inhibition. The expanding synovium degrades cartilage and erodes into the bone at the cartilage-bone interface, creating the joint erosion.

The RA FLS cell, has a markedly different phenotype than the healthy cell, and plays a key role in the destructive process by the release of proinflammatory cytokines and matrix destructive enzymes. FLS cells expand during the joint destructive process, however, there is minimal mitotic activity. The origin of the expanding FLS population is therefore uncertain. Possible explanations to this phenomenon could be decreased senescence, migration of mesenchymal stem cells from the circulation, epithelial to mesenchymal cell-transition or expansion of a stem cell pool in the synovium.

A possible FLS cell precursor has been identified in peripheral blood and called fibrocytes. Fibrocytes are cells that express surface receptors of both stromal and hematopoietic cells. Circulating fibrocytes are rare comprising appr. 0.5% of leucocytes.

Increased numbers of circulating fibrocytes have been found in murine RA models. In a small sample of RA patients (six patients, ten controls), peripheral blood fibrocytes had elevated phosphorylation activation compared to controls, and cells were demonstrated in the synovium. Here fibrocyte activation was correlated with arthritis disease activity (DAS28). In a murine model, activated circulating fibrocytes where found to migrate to arthritis joints in the preclinical phase of the disease, and to accumulate in the FLS cell layer of the synovium.  Adoptive transfer of circulating fibrocytes in this murine model, led to worsening of joint disease indicating that circulating fibrocytes are involved in RA joint pathology, possibly as a FLS-cell precursor.

The bone marrow is a possible central compartment in the RA pathogenesis. In RA patients bone marrow abnormalities have been reported both in the subchondral bone marrow adjacent to the join and in the iliac bone marrow. The number of mononuclear cells in the bone marrow of RA patients with longstanding disease has increased compared to non-RA controls. Fibrocytes have been found using flow cytometry in the bone marrow in a murine model, but to date not in humans.

Another part of the project is a minor study of fibrocyte levels in patients with RA, Systemic Scleroderma, Idiopathic pulmonary fibrosis and severe treatment refractory asthma.

The presented study focuses on the fibrocytes role in RA and lung disease pathology. This project systematically searches for fibrocytes in the bone marrow, blood and synovial compartment in RA patients and controls and compares this with clinical and radiographic disease activity. The study could enforce that fibrocytes as a future target for RA treatment, possibly already in the bone marrow compartment.

This project will further introduce a new safe method of research and diagnostics in Denmark; ultrasound-guided synovial biopsies to be performed in the rheumatologically ambulatory setting. The method is introduced in collaboration with the leading center in Londonand can be applied to a broad range of other diseases affecting the synovium.

Description of the cohort

RA synovial biopsy study:

• 20 RA patients from Department of Rheumatology or Department of Medicine OUH, with newly diagnosed RA (according to ACR/EULAR 2010 classification criteria) who have been diagnosed under 6 months ago, and who have at least one swollen joint in hands/fingers.
• 20 RA patients with longstanding RA (over 5 years duration).
• Synovial control group: 20 Non-RA patients who are referred to arthroscopy in a joint of the hand at OUH Department of Orthopedics, Section of hand surgery.
• Bone marrow control group: 20 Non-RA patients to undergo bone marrow aspiration as part of their diagnostic workup at Diagnostic Centre OUH.

Exclusion criteria:

• Under 18 years of age.
• Rheumatic autoimmune disease other than RA, including systemic lupus erythematosus (SLE), mixed connective tissue disease (MCTD), scleroderma, polymyositis or primary vasculitis.
• History of or current inflammatory joint disease other than RA (e.g. gout, reactive arthritis, psoriatic arthirits, spondyloarthropathy, lyme disease).
• Allergy to local anesthetics.
• Hereditary or acquired (e.g. Anticoagulant Treatment, apart from low dose aspirin) coagulation abnormality.
• Prednisolone above 7.5 mg/day
• Skin pathology at site of joint biopsy.
• Obesity, BMI over 30.
• Functional class IV as defined by the ACR criteria for classification of functional status in RA.
• Heart failure (NYHA class III/IV), immunodeficiency, current or past malignant disease (except for non-melanoma skin cancer), recurrent or chronic infections (viral, fungal or bacterial), inflammatory bowel disease, myeloproliferative disorder or other bone marrow disease.
• Severe nervous system, liver, pulmonary, renal or endocrine disease (including Type 1 diabetes, stable type 2 diabetes is allowable).
• Major surgery within 8 weeks.
• Persons unable to give informed consent.

Laboratory exclusion Criteria

Platelet count under 100 x 10⁹ /L or Hemoglobin under 5 mmol/L or White blood cells under 3 x 10⁹/L or estimated Glomerular Filtration Rate (eGFR) under 60.

The RA patients are followed for 6 months with three visits. Control patients only one visit.

Fibrocytes and Lung disease

• 10 RA Patients from Department of Rheumatology or Department of Medicine OUH, with RA (according to ACR/EULAR 2010 classification criteria) who have at least one swollen joint in hands/fingers.
• 10 Patients from Department of Lung medicine with severe asthma.
• 10 Patients from Department of Lung medicine with Idiopathic Pulmonary Fibrosis.
• 10 Patients from Department of Rheumatology with Systemic Sclerodermia.
• 10 Blood Donors

Exclusion criteria

• Over 18 years of age.
• Heart failure (NYHA class III/IV), immunodeficiency, current or past malignant disease (except for non-melanoma skin cancer), recurrent or chronic infections (viral, fungal or bacterial), inflammatory bowel disease, myeloproliferative disorder or other bone marrow disease.
• Major surgery within 8 weeks.
• Platelet count under 100 x 10⁹ /L or Hemoglobin under 5 mmol/L or White blood cells under 3 x 10⁹/L or estimated Glomerular Filtration Rate (eGFR) under 60.
• Persons unable to give informed consent.

Data and biological material

RA synovial biopsy study:

Visit 1 (2eek 0)

• Medical history
• Physical examination
• Biochemistry (peripheral blood)
• IgM-RF/anti-CCP (peripheral blood)
• DANBIO registration
• X-ray hands/wrist, upper feet
• Informed consent
• Biobank (plasma, serum and buffy coat)
• Flow cytometry (peripheral blood)
• MRI of hand
• Joint tissue biopsy
• Iliac Bone marrow biopsy

• Biochemistry (peripheral blood)
• DANBIO registration
• Biobank (plasma, serum and buffy coat) 
•  Flow cytometry (peripheral blood)

• Biochemistry (peripheral blood)
• DANBIO registration
• Biobank (plasma, serum and buffy coat)
• Flow cytometry (peripheral blood)
• MRI of hand (Only early RA)
• Joint tissue biopsy

Fibrocytes and Lung disease:

One peripheral blood sample of 20 ml, for fibrocyte level determination, further:

• Results of the newest blood sample taken as part of disease management in the ambulatory where the patient was recruited.
• Disease activity as evaluated by the ambulatory where the patient is followed. (Most current Lung function test, radiographic results, disease activity score for RA patients). 

Collaborating researchers and departments

Department of Rheumatology, Odense University Hospital

• Consultant Hanne Lindegaard, MD, PhD

OPEN Odense Patient data Explorative Network, Odense University Hospital

• Professor Torben Barington, MD, DMSc

Department of Clinical Immunology, Odense University Hospital

• Christian Nielsen, Cand.scient, PhD

Department of Clinical Pathology, Odense University Hospital

• Professor Henrik Daa Schrøder, MD, DMSc

Department of Clinical Pathology, Odense University Hospital

• Eva Killdal Hejbøll, Cand.scient

Department of Molecular Medicine, Odense University Hospital

• Associate Professor Søren Hansen, Cand.scient, PhD

Department of Orthopedics, Section of hand surgery, Odense University Hospital

• Hans Tromborg, MD, PhD

Department of Radiology, Odense University Hospital

• Trine Torfing, MD, PhD

Department of Medicine, Odense University Hospital, Svendborg

• Inger Marie Jensen Hansen, MD, DMSc, PhD