What are learning objectives?
- describe the different forms of bone loss in RA
- how does RA affect osteoclast differentiation and function?
- how does RA affect osteoblast differentiation and function?
- how is RANKL and OPG expression regulated in RA?
- how is Wnt signalling regulated in RA?
- how do IL-1 and TNF affect bone cells?
- why is it important for inflammation to be controlled in RA for bone health? how would you detect if inflammation is completely resolved within in a joint?
What do factors produced by inflammatory cells cause?
- destruction of cartilage and bone
- proinflammatory cytokines e.g.: TNFa, IL-1, IL-6, IL-17, RANKL
What are patterns of bone loss in RA?
Juxta-articular/peri-articular osteopenia
- occurs early in disease within the trabecular/cancellous bone near the affected joint
focal bone erosion
- occurs within cortical bone within the arthritic joint, initially at junction of cartilage and bone
systemic osteoporosis
- present in many of the patients: thinning of trabecular/cancellous bone and cortical bone at sites remote from affected joints e.g. hip, vertebrae
What is osteopenia?
- reduced bone mass
- not as severe as osteoporosis
- loss of opacity in radiograph
When were osteoclasts identified as the cell responisble for bone erosion?
1998
- done by staining RA tissue sections for markers identifying osteoclasts
- RNA probe to a particular gene
- in situ hybridisation
- tartrate resistant acid phosphatase mRNA
- cathepsin K mRNA
- key osteoclast differentiation factor: calcitonin receptor mRNA (only seen in osteoclasts on the bone)
What are the multiple cellular sources of RANKL in RA synovium?
- osteoblast-lineage cells
- synovial fibroblasts
- T cells
- (B cells… not as prolific)
What do we see in immunohistochemistry of RA patients?
- TRAP: multinucleated cells on bone surface
- RANK: multinucleated cells expressing RANK, cells in infiltrative tissue that are rank+
- RANKL: focal in expression pattern, at the sites where osteoclasts are resorbing bone
OPG: completely absent at bone erosion sites
RANKL expression “outweighs” OPG expression at the pannus-bone interface in RA
net bone loss
What is the bone phenotype of mice that have no functional RANKL?
Osteopetrotic
The RANKL ko-mice have?
No functional osteoclasts and thick dense bone
Compared to their wild type littermates what happens to bone when RA is induced in RANKL KO?
RANKL KO mice are protected from bone loss
So what is seen in mice that lack RANKL?
- resistant to arthritis induced focal-erosion but have similar severity of synovial inflammation compared to Wild type (WT) mice
What was seen in OPG-Fc treatment?
- reduces osteoclasat nmbers and focal bone erosion and systemic bone loss in animal models of RA
- done in both hTNF.Tg mice and CIA rats
- decreased number of osteoclasts within the inflamed joints
- leads to decreased bone erosion and decreased systemic bone loss
- no effect on inflammation
What is osteoclast differentiation and function in RA?
- osteoclasts are the only cell responsible for bone erosion in RA
- at pannus-bone interface there is increased expression of RANKL relative to OPG, promoting osteoclast differentiation and function at this site
- in RA, synovial fibroblasts and T cells as well as osteoblast-lineage cells are ADDITIONAL sources of RANKL
- inhibition of RANKL (either by gene deletion or blockade with OPG.Fc) protects against focal bone erosion in mouse arthritis models but has NO effect on synovial inflammation
Osteoblast differentiation and function in RA?
- net bone loss: either osteoblasts can’t keep up or they are not functioning properly
- effective therapy –> attenuated focal bone erosion
- but erosive lesions often persist
- suggests there is something wrong with the osteoblasts at that site
How is osteoblast maturation/bone formation impaired in RA?
- at the interface of inflammation and bone
mouse models:
- injected fluorochromes incorporated into newly formed bone shows lack of bone formation at bone surfaces adjacent inflammation
- lack of mature osteoblast-lineage cells at bone surfaces adjacent inflammation: prevalence of Runx2+ osteoblast-lineage cells but these lack the more mature osteoblast markers osteocalcin and alkaline phosphatase expression
What is increased in inflammatory arthritis?
- expression of multiple antagonists of Wnt signalling
- inhibits osteoblast maturation and function
- in synovial tissue isolated from mouse arthritic joints:
- Increased DKK1/2/3
- increased sFRP1/2/4
RA patients –> increased serum DKK1
TNF –> increased DKK1 in synovial fibroblasts
What happens to Wnt signalling in RA?
inflammation –>
increased Wnt antagonists e.g. DKK1, sFRP1
decreased activation of Wnt signalling
inhibitory to bone formation:
promotes bone resorption (increased RANKL:OPG)
net bone loss
What happens to osteoblast differentation and function in RA?
- bone formation is impaired at bone surfaces adjacent inflammation (pannus) in RA
- osteoblasts fail to mature properly leading to failure to form proper mineralised bone efficiently
- expression of Wnt signalling antagonists (including DKK proteins and sFRP proteins) is increased in inflamed synovial tissue (including pannus)
- -> inhibition of Wnt signalling at these sites
- -> inhibition of osteoblast maturation and bone formation at these sites
- DKK1 expression is increased by TNF in synovial fibroblasts
What was seen in inhibition of DKK1 in hTNF.Tg mouse model of RA?
- protection from bone loss due to active bone formation and reduced osteoclasts
- protection from bone erosion (increase in OPG, inhibition of osteoclasts), increase in bone formation and osteophyte formation
How do cytokines/factors produced by inflammatory infiltrat act in RA?
- augment osteoclastogenesis
direct effect on osteoclasts
- RANKL –> OCL differentiation
- IL-1
- TNF
indirect effect on osteoclasts
- TNF
- IL-1
- IL-6
- IL-17
- PGE2
- IL-11
- oncostatin M
- IL-7
–> osteoblast-lineage cells/synovial fibroblasts and T cells –> increase RANKL:OPG –> OCL
What is the effect of TNF and IL-1 on osteoclast differentiation?
- directly augment
- increase RANK expression on osteoclast progenitors, increase number of osteoclast progenitors with increasing TNF
- TNF acts more on osteoclast precursors
- IL-1 acts more at later stages
- promotes cell fusion and survival
What is the direct effect of TNF on osteoblasts in vitro?
- decreased Wnt signalling in the osteoblast-lineage cell
- decreased RUNX2 protein levels (TNF signalling targets RUNX2 for degradation) (this prevents further differentiation)
- decreased alkaline phosphatase (needed for proper mineralisation of bone matrix) and osteocalcin (helps maintain Ca++ within bone matrix) gene expression
- increased RANKL expression (promoting osteoclastogenesis)
- decreased capacity of osteoblast-lineage cells to form properly mineralised bone
- increased apoptosis
role of IL-1 in bone formation in the context of RA has not been thoroughly investigated
What is critical for repair of focal bone erosion in RA?
- strict control of inflammation and synovitis is critical for repair of focal bone erosion in RA
in human studies of RA:
- evidence for repair of focal bone erosions limited to ~10% erosions
- -> most in patients who are in remission, or who have had low diease activity
- smoldering synovitis, detected by MRI in clinically “normal” joints, is still an issue
- -> may compromise bone repair, even when patients appear to be in remission
- “healing of erosions” more common in patients treated with TNF inhibitor
in mouse model of arthritis:
- focal bone erosions do repair
- BUT ONLY when synovitis and local inflammation resolves
Could bone repair in RA be improved by targeting bone in addition to inflammation control?
targeting inflammation
- methotrexate (and other DMARDS)
- anti-TNF (e.g. infliximab, etanercept, adalimumab)
- anti-IL-6R (toculizumab)
- CTLA4-UG (abatacept, T cells)
- Rituximab (anti-CD20, B cells)
targeting bone
- bisphosphinates
- anti-RANKL Ab (denosumab, approved for post-menopausal osteoporosis)
- rhPTH 1-34 (teriparatide)
- anti-sclerostin (not yet)
- anti-DKK1 (pre-clinical for osteoporosis)
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