L6 - cytokine regulation of Rheumatoid Arthritis Flashcards
- Describe the clinical features of rheumatoid arthritis (RA) and the key players (e.g., cells, autoantibodies and cytokines) involved in joint inflammation and damage - Discuss risk factors and early immunological events associated with the evolution of RA in patients - Compare how different inflammatory cytokines direct joint inflammation and erosive tissue damage in rheumatoid arthritis - Appraise regulatory cytokines as potential new therapies for the treatment of rheumatoid arthritis -
What is rheumatoid arthritis (RA) and what tissue damage does it cause?
RA is a chronic inflammatory disease affecting joints, causing damage to cartilage and bone. The inflammation-driven erosion of these tissues leads to irreversible joint damage.
What experimental evidence demonstrates cartilage erosion in RA?
In a lab model of inflammatory arthritis, red staining highlights proteoglycans in cartilage. In arthritic joints, reduced red staining shows cartilage erosion due to inflammation
What historical anecdote is associated with early RA treatment in Australia?
In the late 1800s, RA patients were advised to sit inside rotting whale carcasses, supposedly inspired by a man who claimed being cured of rheumatism after drunkenly falling into a whale.
How have RA treatments evolved from historical approaches?
Modern RA treatments involve multimillion-pound blockbuster drugs targeting cytokine-driven mechanisms, providing effective management of the disease.
What are the key clinical features of RA?
Swollen, painful, stiff joints
Symmetrical arthritis (affecting both sides equally)
Leads to irreversible disability due to bone and cartilage damage
How prevalent is RA, and who does it affect most?
RA affects 1% of people worldwide (400,000 in the UK) and is more common in females than males. Onset typically occurs at ages 40-60, slightly later in men.
What is the female to male ratio of rheumatoid arthritis
3:1
What factors may explain why RA affects females more than males?
Possible factors include hormonal influences, particularly the anti-inflammatory effects of oestrogen, though this remains controversial.
Why is early treatment crucial in RA?
Early intervention prevents inflammation and irreversible damage to bone and cartilage, which cannot be reversed once it occurs.
What comorbidities are associated with RA?
Most common: Cardiovascular disease
Others: Mental health issues - fatigue and depression
Severe cases can lead to hand and joint deformations, including joint dislocations
What does an X-ray of a joint affected by RA show compared to a healthy joint?
Healthy joint: Clear joint space and defined bone structure
RA-affected joint: Loss of joint space due to cartilage erosion, painful bone-on-bone movement, and bone erosion
How does RA have both systemic and local characteristics?
Systemic: Autoimmunity (breach of tolerance leading to presence of auto antibodies) and inflammation markers (CRP, ESR)
Local: Immune cell infiltration of the synovium, synovial thickening, and hyperactivated tissue cells
What percentage of RA patients are autoantibody positive, and what autoantibodies are involved?
Around 70% of patients are autoantibody positive. Key autoantibodies include:
Rheumatoid factor (RF): Targets the Fc portion of IgG
Anti-citrullinated protein antibodies (ACPAs): Target post-translationally modified self-proteins
What inflammatory markers are elevated in RA patients?
C-reactive protein (CRP): Acute phase protein indicating systemic inflammation
Erythrocyte sedimentation rate (ESR): Clinical marker for inflammation
What is the role of the synovium in a healthy joint, and how is it affected in RA?
Healthy synovium: Thin lining that produces synovial fluid for joint lubrication
RA synovium: Thickened lining with immune cell infiltration, leading to swelling and tissue damage
How do synovial fibroblasts and osteoclasts contribute to joint damage in RA?
Synovial fibroblasts: Hyperactivated, contributing to inflammation
Osteoclasts: Hyperactivated, leading to bone resorption and erosion
What changes occur in the synovium during RA?
Thickening and swelling of the synovial lining
Immune cell infiltration
Hyperactivation of tissue cells such as fibroblasts and osteoclasts
What role do single nucleotide polymorphisms (SNPs) play in rheumatoid arthritis (RA)?
Over 100 SNIPs have been associated with the development of rheumatoid arthritis, and while a single SNP may only minimally increase the risk, combinations of SNPs can raise the likelihood of developing the disease by up to 40-fold.
How were the single nucleotide polymorphisms (SNPs) associated with rheumatoid arthritis identified
through genome wide association studies (GWAS) and meta analyses
What is the most significant SNP in terms of genetic risk factors for developing rheumatoid arthritis
shared epitope encoded by HLA-DRB1
What is the shared epitope in HLA-DRB1, and why is it significant?
The shared epitope is a five-amino acid sequence in the HLA-DRB1 chain, which is associated with more severe disease, greater damage to cartilage and bone, and higher autoantibody titres. Its discovery led to the shared epitope hypothesis, which suggests that individuals with this epitope are more efficient at presenting autoantigens to T cells.
What non-HLA SNPs are associated with RA, and what processes do they regulate?
Non-HLA SNPs involved in rheumatoid arthritis regulate key processes such as T cell activity, cytokine and chemokine signalling, and immune regulation. FoxO3 is associated with a milder course of RA, while PTPN2 negatively regulates signalling downstream of cytokine and T cell receptors.
What have bioinformaticians looked and compared
GWAS and SNPs to understand their impact on gene expression in patients with RA. The studies found that the genes associated with these SNPs are involved in various processes, including the activation of the immune system, regulation of cytokines and cytokine receptors, and T cell activation.
What is PTPN2
a protein tyrosin phophatase (PTP) that negatively regulates TCR and cytokine signalling (effectively shuts off downstream signalling)
How does haploinsufficiency of PTPN2 exacerbate arthritis in mice?
Haploinsufficiency of PTPN2 in mice leads to increased production of inflammatory cytokines IL-6 and IL-17 and the formation of ectopic lymphoid structures within joints, while regulatory T cell levels are reduced. Conditional knockout of PTPN2 in T cells alone is sufficient to exacerbate the disease.
What is FOXO3
A transcription factor that suppresses inflammatory cytokine production by immune cells ( a more recently identified SNP)
What is the significance of FOXO3 in RA development?
FOXO3 is notable because it is associated with a milder course of rheumatoid arthritis, with people carrying this SNP typically experiencing lower disease severity.
What do twin studies reveal about the genetic and environmental contributions to RA?
Twin studies reveal that genetics play an important role, as monozygotic twins show a disease concordance rate of about 12-15%, compared to 2-5% for dizygotic twins and 1% in the general population, although the differences also indicate significant environmental contributions.
what are monozygotic vs dizygotic twins
Monozygotic (MZ) twins are identical twins, while dizygotic (DZ) twins are fraternal twins
What environmental factors have been linked to RA development?
Environmental factors linked to the development of rheumatoid arthritis include
- microbiota and inflammation of mucosal sites ( e.g. Gingiva AKA gums)
- exposure to silica dust, as seen in increased autoimmunity among firefighters involved in the 9/11 Trade Tower collapse,
- western diet, obesity,
- long term alcohol consumption (decreased risk),
- low socioeconomic status,
- low education levels
- vitamin D deficiency ( sunshine).
What environmental factor is linked to increased rates of rheumatoid arthritis (RA) in factory workers?
Exposure to silica dust in low- to middle-income countries has been associated with higher rates of rheumatoid arthritis and autoimmunity.
How does periodontitis relate to rheumatoid arthritis?
There is a strong link between periodontitis, which is inflammation of the gums, and rheumatoid arthritis, with periodontitis often being a comorbidity in RA patients.
How can smoking contribute to the development of RA
Smoking induces inflammation and stress at mucosal sites, leading to post-translational modifications of proteins, such as citrination, which may trigger autoimmune responses.
What is the two-hit hypothesis in RA?
The two-hit hypothesis proposes that RA development involves an initial immunological hit that breaches tolerance, followed by a second hit that triggers clinical symptoms.
what causes the clinical symtoms
immune cells infiltrating the joint causing pain
What occurs during the first hit of the two-hit hypothesis in RA?
The first hit involves a breach of tolerance, where autoantibodies such as anti-citrullinated protein antibodies (ACPAs) develop in a seemingly healthy individual.
What role does citrination play in RA development?
Citrination, the conversion of arginine to citrulline in proteins, creates neoepitopes that may trigger autoantibody production in RA.
Which proteins are commonly citrullinated in RA?
Histone and things that are part of the external matrix e.g. fibronectin, collagen, fibrinogen, and vimentin are commonly citrullinated in rheumatoid arthritis.
How does periodontitis promote protein citrination? (1st hit)
The bacteria Porphyromonas gingivalis, which causes periodontitis, produces enzymes (peptidyl deaminase ) that drive the citrination of proteins.
What immune process follows the presentation of neoepitopes during the first hit of RA?
APC presentation to T cells results in activation, leading to the production of autoantibodies against citrullinated proteins.
What are other autoantibodies in RA other than rheumatoid factor (RF) and anti citrullinated protein antibodies (ACPA)
- anti-carbamylated protein antibodies (anti-CarP)
- anti-peptidylarginine deiminase 4 (anti-PAD4)
- anti-BRAF antibodies
What are PAD4 antibodies, and what role do they play in RA?
PAD4 antibodies are specialized antibodies that interact with the PAD4 enzyme, a member of the protein arginine deiminase family. These antibodies can bind to PAD4, leading to a process called auto-citrullination, where the enzyme modifies its own structure. This modification enhances the visibility of PAD4 to the immune system, increasing its recognition by human autoantibodies. As a result, these antibodies not only target PAD4 but also activate it, boosting its catalytic efficiency. This heightened activity promotes inflammation by facilitating the conversion of arginine residues in proteins to citrulline, a process known as citrination. Consequently, elevated levels of PAD4 antibodies can play a significant role in autoimmunity / RA.
what is BRAF
a serine-threonine protein kinase that is part of the RAF proteins group and regulates the mitogen-activated protein kinase (MAPK) signalling involved in the production of pro-inflammatory cytokines
What are anti-BRAF antibodies, and how do they affect RA?
Anti-BRAF antibodies activate intracellular signalling pathways that lead to the production of pro-inflammatory cytokines in RA.
What triggers joint inflammation in experimental models of arthritis in mice?
Joint inflammation is induced initial collagen exposure from a different species e.g. chicks (which causes them to develop autoantibodies against their own collogen) followed by a second hit which comes by a booster injection or an inflammatory stimulus like LPS
What are potential second hits in the two-hit hypothesis for RA?
Increased vascular permeability from systemic inflammation may allow cells to infiltrate the synovial tissue establishing inflammation in the joint and microtrauma, and transient infections can also lead to the release of endogenous antigens that contribute to autoimmunity through mechanisms like epitope spreading, molecular mimicry and bystander activation
What immune cells are involved in driving joint inflammation in RA?
T cells, B cells, macrophages, and other immune cells release cytokines and chemokines to promote inflammation in joints e.g. fibroblasts and osteoclasts
Which innate immune cells release cytokines and chemokines to sustain inflammation
Macrophages, Monocytes, Neutrophils and Dendritic cells
which adaptive immune cells release cytokines and chemokines to sustain inflammation
T cells, B cells and plasma cells
what are example chemokines and cytokines that sustain inflammation
TNF, IL-6, IL-17, IL-2, RANKL, GM-CSF
What are fibroblast-like synoviocytes, and what role do they play in RA?
Fibroblast-like synoviocytes release cytokines, chemokines, and matrix metalloproteinases (MMPs 1,3,9 and 13) that contribute to inflammation and cartilage erosion.
How do osteoclasts contribute to joint damage in RA?
Osteoclasts resorb bone by releasing acids, matric metalloproteinases (MMP9, MMP13) and enzymes like cathepsin K, contributing to cartilage and bone erosion.
how do osteoclasts make acid
through the action of carbonic anhydrase within the cell they convert water and carbon dioxide into acid (which gets released into erosive pits)
What is the role of MMPs in rheumatoid arthritis?
Matrix metalloproteinases (MMPs) like MMP-1, MMP-3, and MMP-9 break down cartilage, contributing to joint damage in RA.
How do cytokines contribute to RA pathology?
Cytokines drive inflammation by recruiting immune cells and promoting the release of additional inflammatory molecules.
At what distance do cytokines exert their effects?
Usually work locally over short distances ( released by one cell and act on another through binding to receptors typically present on the surface of the cell)
What happens when cytokines bind to receptors on a reciprocating cell
a change in behavior e.g.
- proliferation
- trafficking/recruitment (to a site of inflammation)
- differentiation
- effector function
- death / survival
what do cytokines shape
the inflammatory landscape with what it looks like and how much tissue damage occurs. e.g.
- pro-inflammatory cytokines which drive activation of immune cells e.g. T cells, B cells and fibroblasts
- regulatory cytokines which promote the development of immunosuppressive and regulatory populations of cells e.g. T regs and B regs cells
- some cytokines antagonise the action of other cytokines e.g. IL-27 inhibits IL-6
- other cytokines in combination act in synergy
- drive cascade effects making it a complex environment ( hard to know which cytokine to target in an inflamed tissue)
What is molecular mimicry, and how might it contribute to RA?
Molecular mimicry occurs when the immune system mistakes self-antigens for foreign antigens, leading to autoimmune responses in RA.
What are the effects of increased vascular permeability in RA?
Increased vascular permeability allows immune cells and antigens to infiltrate the synovial tissue, establishing inflammation within joints.
What are cytokines?
Cytokines are communication molecules that act over short distances, released by one cell to promote changes in another cell through receptor binding.
What changes in cell behaviour can cytokines drive?
Proliferation, trafficking to inflammation sites, differentiation, and signalling for survival or apoptosis.
What are pro-inflammatory cytokines?
Cytokines that drive the activation of many immune cells and contribute to inflammation.
What are regulatory or anti-inflammatory cytokines?
Cytokines that promote the development of immunosuppressive cell populations, such as Tregs and Bregs, which inhibit pro-inflammatory responses.
How can cytokines interact with one another?
Some cytokines antagonise others (e.g., IL-27 inhibits IL-6), while others synergise, producing effects greater than the sum of their individual actions.
Why are cytokines good therapeutic targets?
TThey are highly potent and produced in small amounts, which means that only a low quantity of inhibitors is needed to neutralize them. These substances are accessible to antibodies in extracellular environments and can influence downstream cytokine cascades which makes them very effective. Many of them are upregulated at sites of inflammation, and they can target either the cytokine itself or its receptor.
What are some cons to using cytokines as therapeutic targets
they have potential or real redundancy and they aren’t easily blocked by small molecule inhibitors
What is cytokine redundancy?
When another cytokine substitutes for the function of a targeted cytokine, reducing therapeutic effectiveness.
how do we determine which of the multiple cytokines produced in inflamed tissues contribute to pathogenesis are are potential therapeutic targets
by correlating their levels with disease severity, conducting in vitro and animal studies, using blocking experiments, and validating findings through clinical trials? ( “try to think about which is the best cytokine to target and build a case against it)
What is a keystone cytokine?
A cytokine that holds together inflammatory processes, similar to the keystone in an arch. Targeting it can disrupt multiple pathways.
What discovery did studies on TNF in rheumatoid arthritis reveal ( when synovial fluid was taken from patients, they were treated with collagenase and DNAse to make a single cell suspension and cultured them)
TNF was found to be hierarchically dominant, driving the production of inflammatory cytokines such as IL-1, IL-6, and GM-CSF and matrix metalloproteins.
What did in vitro experiments with TNF antibodies show?
Anti-TNF treatment dramatically reduced the production of inflammatory cytokines and matrix metalloproteinases in synovial tissue cultures.
How was TNF inhibition tested in vivo?
Mice with collagen-induced arthritis were either treated with a control or various doses of anti-TNF antibodies. and they found that anti TNF reduced joint swelling and histopathological signs of inflammation.
how were the collagen-induced arthritis mice immunised and how long did it take for them to develop arthritis
with collagen type II from chicks which induced arthritis within ~21 days
What are TNF antagonists in clinical use today
Anti TNF monoclonal antibodies (e.g. Adalimumab and Infliximab) which prevent its interaction with surface receptors on cells.and soluble TNF receptors (e.g. Etanercept) which also inhibit TNF by binding to it and blocking receptor engagement.
Why isn’t targeting TNF effective for all diseases?
Different diseases have different key cytokines. For example, IL-6 is more effective for RA, while IL-17 is crucial for psoriasis.
How do soluble TNF receptors increase drug stability in the body?
Soluble TNF receptors are fused to the Fc portion of IgG, which increases their circulation time by enhancing stability and reducing degradation.
What did the discovery of IL-6 lead to in drug development?
The discovery of IL-6 and its receptor allowed the development of drugs targeting IL-6, like anti-IL-6 receptor antibodies.
What role does IL-6 play in rheumatoid arthritis, and what are some treatments targeting it?
IL-6 contributes to inflammation in RA. Treatments like tocilizumab (anti-IL-6 receptor antibody) block IL-6 signalling, reducing disease symptoms.
What are the two modes of IL-6 signalling, and how do they differ?
- Classical signalling: IL-6 binds to a membrane-bound receptor, which requires the GP130 co-receptor for signalling.
- Trans-signalling: A soluble IL-6 receptor binds IL-6 and associates with GP130 on any cell, enabling broader activation.
What is soluble GP130, and what is its role in IL-6 trans-signalling?
Soluble GP130 binds to the IL-6/soluble receptor complex, preventing its interaction with cell-surface GP130, thereby inhibiting IL-6 trans-signalling and potentially reducing inflammation.
What is activated when IL-6 binds to the IL-6R
it associates with the gp130 homodimer and activates JAK-STAT signalling (STAT1 STAT2) / downstream signalling pathways
Which cells express IL-6R
leukocytes (immune cells) and hepatocytes (liver cells)
What is methotrexate used for in RA treatment?
Methotrexate is an immunosuppressant often used alongside TNF-targeting drugs to treat RA.
When was anti-IL-6 receptor antibody Tosylsum approved?
It was approved in 2010 for the treatment of RA (in the USA)
What have been in development from 2015
alternative IL-6 blockers (looking for possible treatments that maybe give us an advantage over some of the drug that are currently available)
What is IL-6 trans-signalling?
A form of IL-6 signalling where soluble IL-6 receptors bind IL-6 and activate GP130 on nearly all cells in the body.
How is soluble IL-6 receptor generated?
It is generated by alternative splicing of mRNA or proteolytic cleavage from the surface of cells by enzymes like Adam10 and Sentine.
Why is IL-6 trans-signalling significant?
It is an agonist which can associate with gp130 and activate nearly every cell in the body, potentially leading to widespread inflammation, like cytokine storms (when not tightly regulated properly)
which cells have gp130
almost every cell in the body
How is IL-6 trans signalling regulated?
It is regulated by soluble GP130 (typically found circulating in the blood) , which prevents the soluble IL-6 receptor from activating GP130 on cells.
What therapeutic potential does soluble GP130 have?
It may block IL-6 trans-signalling and reduce inflammation in diseases like RA
What experiments have been done to investigate the therapeutic potential of soluble GP130
investigating whether IL-6 trans signalling can drive the development of pathogenic T cell subsets (e.g. Th17 cells) and seeing whether it has any therapeutic potential by blocking its effects in vivo
Which two cytokines are required for Th17 differentiation and why is this important to know experimentally
TGF-β (transforming growth factor beta) and IL-6 (interleukin-6. by knowing this you can specifically culture Th17 cells
How does soluble GP130 affect TH17 cell development?
Soluble GP130 can block the expansion of TH17 cells, which are involved in inflammation.
What effect did soluble GP130 FC have in experimental arthritis?
Soluble GP130 FC improved the clinical score of collagen-induced arthritis (CIA), reducing inflammation.
Give examples of emerging IL-6 therapies that are approved, in clinical trial and recently licensed for the treatment of RA and their different modes of action
Anti IL-6 receptor blockers (approved) e.g. Tocilizumab and Sarilumab
Anti IL-6 blockers (clinicla trial) e.g. Olokizumab
IL-6/sIL-6R blockers ( recently licensed)
What is the drug Olokizimab and where is it licensed?
Olokizimab targets IL-6 and is licensed in Russia for treating RA.
What is Olamkst, and where is it in clinical trials?
Olamkst targets soluble GP130 FC and is in clinical trials for treating inflammatory bowel disease (IBD).
What are possible ways that drugs can target cytokine signalling
by targeting the receptor, the cytokine itself or blocking the signal …. more recently we have a the new class of drug called JAK inhibitors
What are Jak inhibitors?
Jak inhibitors block downstream signalling of cytokine receptors, targeting proteins like JAK1 and JAK3.
What does JAK signalling involve
- When a cytokine binds to its receptor, the receptor dimerizes, bringing associated JAKs into proximity.
- JAKs phosphorylate each other, activating their kinase activity.
- Activated JAKs then phosphorylate key tyrosine residues on the C terminal domain of cytokine receptors / STAT proteins which act as binding sites for transcription factors
- Phosphorylated STATs translocate to the nucleus to activate gene expression
What is the advantage of Jack inhibitors?
They target multiple cytokine receptors, making them effective in treating various diseases, especially in patients who don’t respond to single-target therapies.
e.g. what does JAK3 inhibition inhibit downstream signalling of
cytokines of the gamma chain :
* IL-2
* IL-4
* IL-7
* IL-9
* IL-15
* IL-21
What side effects are associated with Jack inhibitors?
Jack inhibitors may increase the risk of infections and some cancers.
What is the role of anti-inflammatory cytokines in treatment?
Anti-inflammatory cytokines like IL-10 and IL-27 are being researched as potential treatments for RA due to their ability to reduce inflammation.
What are pro-inflammatory cytokines and examples mentioned in the lecture?
Pro-inflammatory cytokines are signalling proteins that promote inflammation. Examples include TNF and IL-6.
What are anti-inflammatory cytokines and regulatory cytokines?
What are anti-inflammatory cytokines and regulatory cytokines?
Back: Anti-inflammatory cytokines reduce inflammation and regulate immune responses. Examples include IL-10 and IL-27.
Why is IL-27 of particular interest in rheumatoid arthritis research?
IL-27’s receptor is highly expressed on T cells in the joints of patients with rheumatoid arthritis, indicating its potential role in modulating inflammation.
What is the structure of IL-27?
IL-27 is a heterodimeric cytokine composed of two subunits: p28 and EBI3.
What happens in IL-27 receptor knockout mice when antigen-induced arthritis is introduced?
IL-27 receptor knockout mice develop exacerbated arthritis with more joint inflammation, cell infiltration, and bone/cartilage erosion compared to wild-type mice.
What role do Th17 cells play in the arthritis model discussed?
Th17 cells are critical drivers of pathology in the antigen-induced arthritis model.
How does IL-27 affect Th17 cell development?
IL-27 inhibits the development of Th17 cells and promotes the expansion of Th1 cells, which do not drive arthritis in this model.
Why did IL-10 fail in clinical trials for rheumatoid arthritis treatment?
IL-10 likely failed because it was not effectively delivered to the joints.
What is Decavil, and how does it improve cytokine delivery?
Decavil is a modified form of IL-10 fused to the F8 antibody, which enhances its delivery to inflamed tissues, including joints.
