Fibroblasts in RA Flashcards
why have RA outcomes improved?
Outcomes have been improved in last 20 years due to drugs targeting the immune system
- Drugs are DMARDs and biologics that target immune system
- Broad: methotrexate
- Highly selective: anti-TNF, anti-CD20, anti-IL-6R
- All DMARDs target immune system to antagonise joint inflammation - pain, swelling, stiffness, loss of function
- 50% patients achieve sustained disease-free on-drug remission, where they must keep taking drugs
- Other 50% can still have disease control
what happens when drugs are removed?
If drugs are removed, 50% relapse between first 12 months of treatment
- Not cure, just suppression
- active therapeutic suppression of disease can antagonise inflammation, but doesn’t reverse disease process in target tissue effector phase, hence relapse– how can we cause true remission with drug removal
- Drugs can have long-term toxicity – immunosuppression (increased risk of infection), liver function issues, bone marrow issues
What occurs in tissue itself and what disease processes should we reverse to enable full remission?
how can we find what can reverse disease processes?
access to target tissue is needed
- Synovial tissue biopsy - minimally invasive, ultra-sound guided
- Guide wire into synovial lining
- See bone, fluid, thickened lining
- Needle takes small sample of tissue from lining
- Can do this longitudinally before and after treatment, and in early stage
- means we don’t have to wait to get tissue samples when someone is having surgery, and can instead do it in early stages
how can the synovial tissue sample be studied?
Tissue staining of synovial lining
- Blue = lining layer
- Red = blood vessels = angiogenesis
- Areas of inflammation – immune cell aggregates – T cells, B cells, macrophages - infiltration of inflammatory cells
what is the normal synovial joint like?
Thin lining:
- Some fibroblasts – mesenchymal structural cells that are tissue-resident
- TRMs form protective barrier around synovium – phagocytose cartilage debris in synovial fluid from joint during locomotion
Sublining tissue – mostly made of fatty cells, scattered fibroblasts, blood vessels
- in health, its mainly the lining layer present
what is the RA synovium like?
Inflammation
- Lining layer barrier is disrupted – macrophages become active – more clearance of debris, change morphology
- Expansion and activation of lining layer fibroblasts – release factors to drive damage
- Pathogenic remodelling of sublining tissue: more blood vessels, fibroblast expansion, infiltration of inflammatory cells from bloodstream, T B cells monocytes which differentiate to macrophages, neutrophil
how have fibroblasts been shown to play a pathogenic role in RA?
Take human RA fibroblasts, grow in culture for months:
- Implant fibroblasts in a human cartilage implant into SCID mouse – immunodeficient for T and B cells, depleted adaptive system, so no GVHD
- When human cartilage implant is removed – human fibroblasts invade mouse cartilage and degrade it
These fibroblasts have an abnormal pathogenic phenotype maintained out of joint - actively contribute to damage
- in inflammation, there’s more fibroblasts with changed phenotype
- they aren’t bystanders, they contribute to disease
what is fibroblast inflammatory priming?
One inflammatory challenge = fibroblast responds with cytokines e.g. IL-6
Once challenge settles and hit again with inflammation = fibroblast has amplified response to second challenge – becomes more inflammatory and abnormal - primed
This is due to rewiring of fibroblast metabolism to be proinflammatory
- Gene pathway for metabolism primed to respond to second challenge
What cells are in joint in health vs disease?
Biopsy of synovium tissue and matched blood and synovial fluid samples
- Looked at cells present within using scRNA sequencing – profile of RNA from cells of disaggregated from tissue, blood and fluid
- Gene expression gives identify of cells which cluster with shared identity of gene expression
- Within tissue – blood vessels, pericytes, immune cells, fibroblasts
- Fibroblasts not detectable in blood and synovial fluid as they’re tissue resident
- Fibroblasts drive inflammation within joint
what does spatial transcriptomics show?
Spatial transcriptomics – take the gene expression data and project onto tissue to show where the different cell types are in the tissue
- H&E imaging – lining layer becomes densely infiltrated with inflammatory cells and expansion of tissue resident cells
Single-cell transcriptomics projection onto image:
- See lining layer fibroblasts, macrophages
- T cells populations beneath, along with blood vessels and perivascular fibroblasts surrounding them
Disaggregation of tissue loses spatial localisation – this method allows us to see organisation
- Can see how cells communicate within tissue space
is cell type composition in the joint the same across patients?
Different cells present in joint known
- Different cells map to different patient groups – joint composition is different across patients
- Heterogeneity of inflammation within tissue and within patients
- Population of RA patients have fibroblast-rich synovial pathotype
Heterogeneity explains why patients respond differently to drugs
- Helps determine what drugs to use
what different roles can fibroblasts have in disease?
Resolving inflammation - good
Drive inflammation/damage - bad
what drives the phenotype of fibroblasts depends on their tissue location
how do fibroblast phenotypes depend on their location in the tissue?
Where cells were located and gene expression data combined
- Synovial tissue stained for blood vessels, perivascular fibroblasts, and lining layer fibroblasts and macrophages
- Near endothelium = fibroblasts are more inflammatory and produce chemokines/cytokines to recruit and retain inflammatory cells from blood to joint - signal to endothelium to drive cells into tissue
- Interstitial fibroblasts - form niches to support survival of inflammatory infiltrating cells
how should fibroblasts be targeted in RA?
Want to intervene with these 2 populations that recruit and maintain inflammatory cells from the blood in the joint
- need to inhibit specific chronic, aberrant inflammation that drives disease without affecting normal inflammation e.g. important for healing
- identify mechanisms in tissue that promote survival of inflammatory cells and intervene long term to return tissue to homeostatic state = remission
what phenotype to fibroblasts have in the lining layer?
Fibroblasts in lining layer of RA = gene expression profile of damage
- Loss of normal function - no longer lubricate joint
- with inflammation, they drive bone damage and osteoclastogenesis
how was it shown that different fibroblast subsets drive different aspects of disease?
Mouse model:
- Isolated fibroblasts depending on location in tissue and gave back to mouse
- Perivascular inflammatory fibroblasts injected into joint of RA model causes increased inflammation that persists
- Lining layer fibroblasts injected into joint have no effect on inflammation severity but drive bone damage
There are different subsets of fibroblasts, similar to T cell effector populations
- Fibroblasts are not just bystanders, they actively drive damage and inflammation and resolution
- Enables selective targeting
how do fibroblasts differentiate to an inflammatory phenotype?
Perivascular fibroblasts around blood vessels
- In inflammation, arterial endothelium is activated and signals via DLL4 to notch 3 expressed on fibroblasts nearby
- Causes fibroblast to differentiate to proinflammatory sublining fibroblast which drives inflammation
can inhibit this differentiation process
what happens to fibroblasts with notch 3 is K/O or blocked?
Mouse with notch 3 K/O = inflammation is less severe as fibroblasts can’t differentiate
- mAb to notch 3 has same effect
how do some fibroblasts for an immune exclusion zone? is this good?
Aggregations of T and B cells around blood vessels
- Stain for matrix proteins e.g. collagen released by fibroblasts support tissue
- Where there are aggregations of cells, fibroblasts secrete MMPs to break down the matrix and make space to support lymphocyte infiltration
- During repair and resolution of inflammation, there is a physical barrier of collagen around blood vessels to prevent migration of cells into tissue – this is formed by good fibroblasts – helping resolution and repair by forming areas of scarring to physically prevent infiltration
what are the two key pathogenic populations of fibroblasts that have been identified?
Profiled fibroblasts using sc-seq in RA patients
2 key pathogenic populations that drive immune cells:
- Some stimulate inflammatory cell recruitment via T cell crosstalk
- Perivascular – recruit T cells - notch 3 response
how can fibroblasts be targeted?
- Block differentiation of pathogenic fibroblasts
- Deplete established pathogenic fibroblasts in fibroblast-rich patients (these patients have disease driven by fibroblasts, not immune cells, so anti-TNF doesn’t work)
what is fibroblast activated protein?
FAP: serine protease on surface of fibroblast which helps attach the fibroblast to cartilage and supports their migration in the joint
In joint FAP is not usually present, it is upregulated in inflammation by fibroblasts
- FAP doesn’t drive inflammation itself, it just is upregulated in pathogenic fibroblasts
what happens when FAP is K/O?
Mouse K/O of FAP-expressing cells
- Suppression of inflammatory arthritis and reduced damage to bone
- Before depletion = high chemokine and cytokine signals at RNA and protein levels to recruit immune cells
- After depletion – these signals are reduced
Main producer of inflammatory signals is pathogenic FAP+ fibroblasts - by depleting these, global inflammation in the joint is reduced
how is FAP expressed in RA?
Patients with early joint inflammation that went on to get RA, there was more FAP in joint compared to resolving or control
- with high levels FAP in the joint in early inflammation, these patients are more likely to be diagnosed with RA
- FAP may be biomarker for population of pathogenic fibroblasts
- To deplete these cells, they need to only be present in diseased tissue, and in healthy tissue
