Inflammasomes and autoimmune diseasesfmf.igh.cnrs.fr/ISSAID/doc/FMF SAID...

Leeds Institute of Molecular MedicineLeeds Institute of Molecular Medicine

Michael McDermott([email protected])

Section of Musculoskeletal Disease,Leeds Institute of Molecular Medicine,

University of Leeds, UK

Inflammasomes andautoimmune diseases


Definition of autoimmunity

• Breakdown of mechanisms for self-tolerance - inductionof an immune response against components of self

• Aberrant B and T cell responses lead to breaking of tolerance - immune reactivity towards native antigens

• Adaptive immune system plays predominant role in the eventual clinical expression of autoimmune disease

• Organ-specific autoantibodies may predate clinical disease expression by years (e.g CCP, dsDNA, ICA)


Antigen presentation, T cell function,including B cell help

MHC associationsMost autoimmunediseases

Regulation of T lymphocyte activation

Regulation of T lymphocyte activation

CTLA-4/CTLA-4

PTPN22/PTPN22

SLE, T1D, AITD

RA, SLE

Polygenic Autoimmune Diseases

Thymic epithelium/neg T cell selectionRegulatory T cells/Immunomodulation

Widespread/lymphocyte apoptosis

AIRE/AIREFOXP3/FOXP3

FAS/FAS

APS-1/APECEDIPEX

ALPS

Monogenic Autoimmune Diseases

Cellular Distribution/DefectGene/ ProteinInflammatoryDisorder

Genetic and Cellular basis forAutoimmunity


Problems encountered with the autoimmunityparadigm in certain diseases

• Some diseases lack specific autoantibodies –ankylosing spondylitis

• Difficult to prove role of cell-mediated immunity -Behçet’s Syndrome

• Autoantibodies may be secondary e.g. apoptosis• Diseases lacking MHC associations – Crohn’s

Disease• Lack of efficacy of therapies e.g multiple sclerosis• Heterogeneous clinical disease expression – sero-

negative forms of rheumatoid arthritis (RA)


Rare Monogenic Autoinflammatory DiseasesFMF, TRAPS, HIDS, PAPABlau Syndrome

Polygenic Autoinflammatory DiseasesCrohn’s Disease, Ulcerative ColitisDegenerative diseases e.g. osteoarthritisGout/Pseudogout/other crystal arthropathiesSome categories of reactive arthritis and psoriasis (no MHC associations)Self-limiting inflammatory arthritis including diseases clinically presenting as RAStorage Diseases/Congenital Diseases with associated tissue inflammationNon-antibody associated vasculitis including Giant Cell ArteritisIdiopathic Uveitis, Acne and Acneform associated diseasesErythema Nodosum associated disease, including Sarcoidosis

Mixed Pattern Diseases with MHC associations and autoinflammatory componentsAnkylosing SpondylitisReactive ArthritisPsoriasis/Psoriatic ArthritisBehçet’s SyndromeUveitis (HLA-B27 associated)

Classic Polygenic Autoimmune Diseases (Organ Specific and Non Specific)Rheumatoid ArthritisCoeliac Disease, Primary Biliary CirrhosisAutoimmune gastritis/Pernicious anaemia, Autoimmune Thyroid DiseaseAddison’s Disease, Pemphigus, Pemphigoid, Myasthenia GravisDermatomyositis/polymyositis/sclerodermaVitiligo, ANCA-associated VasculitisType 1 diabete, Systemic Lupus Erythematosus

Rare Monogenic Autoimmune DiseasesALPS, IPEX, APS-1/APECED

Innate ImmunityAdaptive Immunity

Anti-B cell andAnti-T cell Therapy


NALPs in vitiligo and hypertension

Jin Y et al. NALP1 in vitiligo-associated multipleautoimmune disease. N Engl J Med 2007 356:1216-22

• NALP1 SNPs associated with several autoimmune and autoinflammatorydiseases, implicating innate immunity in the pathogenesis of these disorders• these diseases include autoimmune thyroid disease (Graves' disease andautoimmune hypothyroidism), Addison's disease, rheumatoid arthritis,psoriasis, pernicious anaemia, and SLE among patients with generalised vitiligo• NALP1 expressed in T-lymphocytes, granulocytes and monocytes

T. Omi et al. An intronic VNTRs of the cold-inducedautoinflammatory syndrome 1 (CIAS1) gene modifies geneexpression and is associated with essential hypertension.Eur J Hum Genet. 2006 14:1295–1305

• inflammation oxidative stress-related genes in development of hypertension



PYDCCSPRY bZIPPyrin

PYD NACHT LRR

NALP1

CARD Caspase-1

CARD

Caspase-1

FIIND CARD

MDP

PYD

CARDASC

Pro-caspase-1

NALP1 Inflammasome earlier

IL1βPro- IL1βIL-1β activation


NACHT LRR

Kinase

CARD

NOD2 signalasome

CARD

CARD

NF-κB

MDP/DAP?

mutations in the LRRs leads to

CROHNʼs DISEASE

mutations in the NACHT leads to

BLAU SYNDROME


NALP3 Inflammasome

PYDCCSPRY bZIPPyrin

PYD NACHT LRRNALP3

CARDCaspase-1

CARD

Caspase-1

FIIND

CARDCardinal

MDPUric AcidATPCytosolic DNA

PYD

CARDASC

Pro-caspase-1

IL1βPro- IL1β


NALP3 is upregulated in synovium of RApatients compared to OA

Rosengren et al. (2005), Ann Rheum Dis. 64: 708-714.

NALP3 (cryopyrin), ASC, andpyrin mRNA expression wassignificantly raised in RAcompared to OA

Detection of NALP3 mRNA in synovialtissue by in situ hybridisation

A. NALP3 expressed in both lining andsublining in RA synovium

B. Negative control using NALP3 senseprobe

A B


DAS28 response criteria and methods

Bingham et al (2004), Rheumatology 43: 364-368

Wk 0 Wk 2 Wk 6 Wk 14

PBMC

mRNA

cDNA

qRT- PCR

ASC NALP3

Infliximab regime / sample collection


Pre-therapy baseline levels of NALP3were predictive of response to treatment

Lower baseline NALP3mRNA levels in RApatients subsequentlyresponding to infliximabtreatment compared tonon-responders

Mann-Whitney U test.

Responders Non-Responders 0

10

20

30

(n=12) (n=11)

p=0.04

NA

LP

3 e

xp

res

sio

n


NALP3 mRNA levels were furtherdecreased from baseline in response to

infliximab treatment

Week 0 Week 2 Week 140

5

10

15 p=0.007n=12

Time

NA

LP

3 e

xp

ressio

n

Wilcoxon signed-rank test

Week 0 Week 2 Week 140

10

20

30 n= 11

TimeN

AL

P3

ex

pre

ss

ion


Preliminary data suggest that infliximab alsoreduces NALP3 expression in the synovium

NALP3 mRNA fromsynovial tissuebiopsies pre- andpost- infliximabtreatment

Mann-Whitney U test.

Pre Post0

10

20

30

Non Responders (n=2)

Responders (n=3)

NA

LP

3 e

xp

res

sio

n


Infliximab Responders

Wk0 Wk2 Wk140

20

40

60

80

100

n=9p=0.036

AS

C e

xp

ressio

n

Infliximab non-responders

Wk0 Wk2 Wk140

50

100

150

n=10

AS

C e

xp

ressio

n

ASC mRNA increased during infliximabtreatment in responders

ASC

ActinWk0

Wk2

Wk14

Protein expression inone representativeRA patient whoresponded to therapy


Summary

• baseline NALP3 a potential predictor of response toinfliximab in RA patients - levels further reduced inthose who respond to therapy

• ASC mRNA levels are increased in patientsresponding to infliximab after week 14 of treatment

• Future work• examine ASC and NALP3 mRNA in synovial tissue

• determine the changes in different leukocyte populations


Etanercept Infliximab

VariableNo changeProtein

Variable+mRNA

No change++Protein

No change++mRNA

ASC

Pyrin

Different effects of infliximab and etanercept onASC levels in PBMCs of RA patients

Pronounced changes inASC mRNA and proteinexpression wereobserved in RA patientsreceiving infliximab, butnot etanercept

(EWRR 2007)



Mutations in genes encoding the RNASEH2complex and TREX1 cause AGS

TREX1

Ch. 3 AGS1

RNASEH2B/FLJ11712

Ch. 13 AGS2

RNASEH2A

Ch. 19 AGS4

RNASEH2C/AYP1

Ch. 11 AGS3


Aicardi-Goutieres syndrome (AGS) is a genetic encephalopathywhose clinical features mimic acquired in utero viral infection.

• early-onset disease with calcification of basal-ganglia, white-matterabnormalities, and a chronic cerebrospinal fluid (CSF) lymphocytosis

• raised levels of antiviral cytokine interferon alpha (IFN-α) in the CSF

• nucleases defective in AGS (TREX1 and RNASEH2A, 2B and 2C)remove endogenous nucleic acids during normal cellular processes

• possibly act on a common substrate, and failure of nuclease activityresults in an (inappropriate) activation of the innate immune system

• heterozygous missense changes and frameshift in 6/218 SLE patients(UK), 6/199 (Germany) compared with 0/200 and 2/1,512 in controls

• dual role of TREX1 as DNA-degrading enzyme in granzymeA–mediated apoptosis and cytosolic DNA sensor induce autoimmunitywhen this nuclease is mutated

Aicardi-Goutieres syndrome (AGS) and SLE


Virus

ss/dsDNA ssRNA dsRNA

Endosomal nucleic acid sensing TLRs

TLR9 TLR7(8) TLR3RIG-1

‘Self’ nucleic acids

Type I IFN induced antiviral state

Virus

MDA5DAI

Inflamma-some


Receptors of the innateimmune system

TLR

2

TLR

4

TLR

5

TLR

3

TLR

7

TLR

8

TLR

9

Endosome

Caspase-1/ -5

Pro-IL-1βPro-IL-18

IL-1βIL-18

MyD88 dependent/independent pathways

Pathogen clearance

IKK

NF-κB

Proinflammatory cytokines

Toll-like receptors (TLRs)

NA

IP5

IPA

F

Nod

1/2

NA

LPs

Intracellular pathogensendogenous signals Danger

Nod-like receptors (NLRs)

Leeds Institute of Molecular MedicineLeeds Institute of Molecular MedicineIFN-α

PlasmacytoidDendritic cell

MyD88

TLR-9

Nucleosomes (Host derived)DNA Protein

Role of TLRs in Lupus

TLR-7ssRNA


TLR-9

MyD88

YY

Y Y Y Y

Nucleosomes (Host-derived)

AutoreactiveB cell

Autoantibodies

DNA

Self IgG2aRF

Protein

IgG2a = immunoglobulin G2a; RF = rheumatoid factor.

TLRs as Therapeutic Targets:TLR-9 and Autoantibodies


INDUCTION OF CO-STIMULATORY MOLECULES CD80/86INDUCTION OF TNF

Courtesy of Steffen Gay and colleagues

sense control probeanti-sense probe

Expression of TLR2 mRNA in RA synovialtissue


Toll-like Receptors

10 TLRs

MyD88 Mal Trif Tram

Products ofinflamed tissues

Modulation of immune and inflammatory genes

Immune and inflammatory effector mechanisms

Microbialproducts

NLRs


Cytokines and chemokinesAnti-viral proteinsproIL-1 and proIL-18

Mature IL-1 and IL18

Anti-viral proteins

MyD88MalTrifTram

MICROBES

TLRs

NLRs

RLRs

NF-κBIRFsMAPKs

Caspase-1

IRFs

MyD88

MAVS/IPS-1/VISA/Cardiff

SENSOR SIGNAL RESPONSE ADAPTER

The Innate World as we Know it:Host Defence



Why is Autoimmunity paradigmso strong?

• Approx 1,000,000, 000 T cell receptors in man• T cell clones expand x 64,000 in 4 days• Each activated Th1 cell attracts up to 1,000

macrophages• T cells stimulate macrophages to make IFN etc

• Repertoire- an almost unlimited number of epitopes

• Memory with secondary responses: occur sooner,steeper, faster, higher and with a lower lowerthreshold


Aicardi-Goutières syndrome (AGS)


Adaptive vs innate immunity in rheumatoidarthritis

• Rheumatoid arthritis (RA) has traditionally been describedas an adaptive immune response, but, more recently, therehas been a gradual appreciation of the role of the innateimmune system

• The innate immune system recognises-microbial pathogen associated molecular patterns (PAMPs)-endogenous signals; danger associated molecular patterns (DAMPs)

• The detection of DAMPs and PAMPs is through Toll-likereceptors (TLRs) and Nod-like receptors (NLRs)

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