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Investigation of Systemic Juvenile
Idiopathic Arthritis (SJIA): a disease of
dysregulated innate inflammation
Betsy Mellins, MD
Divisions of Human Gene Therapy and Pediatric
Rheumatology
Interdisciplinary Program in Immunology
[email protected]
Autoimmune versus Autoinflammatory disease
Autoimmune (adaptive)
• Autoinflammatory (innate)
• Pathogenic cells
– T cells, B cells
• Mechanism
– Failure of peripheral or
central tolerance to selfantigens
• Features
– Autoreactive T cells
– Autoantibodies
– HLA class II associations
• Pathogenic cells
– Monocytes, macroΦ, polys, NK
• Mechanism
– Excessive sensor activation or
failure of inhibitory or
resolution mechanisms
• Features
– No autoreactive T cells or
autoantibodies
– No HLA class II associations
– Pro-inflammatory cytokines
• Examples
– FMF, NOMID, MWS, FCU, TRAPS
– SJIA
• Examples
– RA, T1D (IDDM), MS,
– Graves Disease
Juvenile Idiopathic Arthritis (JIA)
• A group of conditions
– 7 subtypes
• Characterized by:
– Arthritis (joint inflammation) for > 6 weeks
– Onset age < 16 years
• Prevalence: 8-150 per 100,000 children
Weiss, Ped Clin N Am 2005
• Unknown etiology
Systemic JIA (SJIA)
• Subtype of JIA =10-20% of all JIA
Schneider et al, Baillieres Clin Rheum 1998
• Onset throughout childhood; Adult Stills
• No diagnostic test; clinical diagnosis
• Features: spiking fevers, rash,
systemic inflammation (pericarditis,
pleuritis) and arthritis;  ESR, CRP
• Unknown etiology
SJIA
• Course: monocyclic, polycyclic,
persistent
• Up to ½ of SJIA patients have
chronic destructive joint disease
• SJIA: 2/3 of mortality in JIA
Wallace CA et al, Rheum Dis Clin N Am 1991
• Complication: macrophage activation syndrome,
amyloidosis (less in recent decade)
• Rx: Challenging to treat (steroids, NSAIDS); significant
proportion has relatively poor response to current drugs
that show benefit in rheumatoid arthritis (e.g.,MTX, antiTNFα).
The SJIA project
• Comprehensive immunological phenotyping
of patients in association with clinical data
– To identify correlations with clinical status =
“biomarker discovery”
• Example: Find high levels of a set of acute phase
proteins (and derivatives) in association with disease
flare
– Diagnostic : distinguish SJIA from other causes of fever
– Prognostic: identify those SJIA patients with impending
disease flare
– To identify correlations that suggest disease
mechanism
• Example: Find evidence for IL-1 driven phenotypes in
association with disease flare
Plasma Proteins
Tirumalai, R. S. (2003)
Mol. Cell. Proteomics 2: 1096-1103
Schematic of 2D Gel System
2D Gel Sample Image
Green – flare
Red – quiescent
Yellow – no change
Cell distribution by FACS
Microarray analysis of PBMC
PBMC microarray
CD14: canonical marker of CD14+ monocytes
CXCL16: associated with CD16+ monocytes;
induced by IFNγ and TNF in monocytes
SOD2: associated with M1 monocyte/macrophages
TREM1: associated with CD14+; induced by TNF in monocytes
BCL2A1: associated with M1 and CD16+
ARG1: associated with M2
SLP1: associated with M1
MMP9: associated with CD16+; induced by TNF in monocytes
• Candidate Gene Expression
• Tested 81 genes of interest by
kinetic PCR (kPCR)
• Found 11 genes whose expression
pattern was statistically
significantly different between SJIA
flare & quiescent clinical states, but
• Flare signature found was related
to IL-1
SJIA plasma induces APC activation
IL-1
• Molecularly characterized in the 1980’s
• The term “IL-1” refers to 2 distinct proteins: IL-1a
and IL-1b, that signal through the same receptor
complex and have identical biological activities in
solution
• multiple and varied biological functions: fever
induction, hepatic acute-phase proteins
stimulation, lymphocyte responses increase,
induction of degenerative changes in joints and
increase of the number of bone marrow cells
IL-1 family
• Several other members of the IL-1 family have
been identified
• Currently there are 11 members:
– IL-1a, IL-1b, IL-1 receptor antagonist (IL1-Ra), IL18, IL-33 and IL-1F5 to IL-1F10
• Probably arose from duplication of a common
ancestral gene
• Except for IL-18 and IL-33, all the IL-1 family
genes are in chromosome 2
Structure
• All the cytokines in the IL-1 family are extracellular
• But only IL1RN (the gene that encodes IL-1Ra) encodes a classical signal
peptide that enables secretion of the cytokine by the endoplasmic
reticulum and Golgi apparatus
• IL-1b and IL-18 have pro-domains at their amino termini that require
cleavage by a protein assembly known as the inflammasome to generate
the biologically active forms and to be secreted
• IL-1α also has a pro-domain, which can be cleaved by the cysteine protease
calpain, but this is not required for its biological activity
• IL-1F5, IL-1F6, IL-1F8, IL-1F9 and IL-33 all have biological activity as fulllength molecules, although they are less potent than forms lacking the
complete N termini. They are not processed by the inflammasome
Receptors
• IL-1 family members signal through a group of closely
related receptors
• Many of the genes are also encoded in chromosome 2
• The receptors contain extracellular immunoglobulin
domains and a cytoplasmic Toll/IL-1 receptor (TIR)
domain portion
• The response is initiated when the ligand binds to its
primary receptor subunit; in the case of IL-1, IL-1
receptor type I (IL-1R1)
• Binding of the ligand allows the recruitment of a
second receptor subunit; for IL-1, the IL-1R accessory
protein (IL-1RAP)
Signaling
• Formation of the receptor heterodimer induces
signaling:
– the juxtaposition of the two TIR domains enables the
recruitment of myeloid differentiation primary
response protein 88 (MYD88), IL-1R-associated kinase
4 (IRAK4), TNFR-associated factor 6 (TRAF6) and other
signaling intermediates
• The ensuing biological response typically involves
the activation of the nuclear factor-κB (NF-κB)
and mitogen-activated protein kinase (MAPK)
pathways
Expression
• IL-1β is mainly produced by monocytes and
macrophages
• IL-1α expression is more widespread; for
example, it is highly expressed by
keratinocytes and endothelial cells
• IL-1β is secreted and circulates systemically
• IL-1α is generally associated with the plasma
membrane of the producing cell and so acts
locally
Regulation
• Because of their potency and extensive functions,
the biological activity of IL-1α and IL-1β is tightly
regulated
• IL-1α and IL-1β are expressed at low levels under
normal conditions and require induction at both
the transcriptional and translational levels
• Their processing and secretion are also regulated
processes, and loss of this regulation step results
in syndromes characterized by fever, rash and
arthritis
Regulation
• 2 physiological mechanisms can block the
action of active cytokines released by cells:
– Binding of IL-1Ra to IL-1R1, thus blocking binding
of IL-1α and IL-1β (this also inhibits recruitment of
IL-1RAP)
– another IL-1-binding protein, IL-1R type II (IL-1R2),
acts as a decoy receptor: it has an extracellular
region that is similar to IL-1R1 but has a short
cytoplasmic domain that cannot signal
Cellular sources of IL-1 family members
and their effects on innate immune cells
The effects of IL-1 family members on CD4+ T cells
The IL-1 family and immune-mediated
diseases
• Arthritis: IL-1b (JIA)
• Skin diseases:
– Psoriasis (IL-1 driving Th17, IL18) and atopic dermatitis
(IL-1, IL-18, IL-33)
• Multiple sclerosis:
– IL-1 induces Th17 in animal models
• Systemic Lupus Erythemathosus: IL-18
• Asthma: IL-1b, IL-18, IL-33
• Crohn’s disease and ulcerative colitis: IL-1a, IL-1b,
IL-18
• Reference:
• The IL-1 family: regulators of immunity. Sims
JE, Smith DE. Nat Rev Immunol. 2010
Feb;10(2):89-102. Epub 2010 Jan 18.
 IL-6 was first discovered in 1986, in a search for factors that promote plasma
cell differentiation and antibody production of B cells.
 Cytokines of the IL-6 family include IL-6, IL-11, oncostatin M (OSM),
cardiotrophin-1 (CT-1), ciliary neurotrophic factor (CNTF), cardiotophin-like
cytokine (CLC), leukemia inhibitory factor (LIF), and the recently identified IL27p28.
 As a pleiotropic cytokine, IL-6 is widely implicated in multiple processes
including immune response, hematopoiesis, neurogenesis, embryogenesis, and
oncogenesis.
 IL-6 is considered as an important proinflammatory cytokine that regulates
inflammatory response and immune reaction.
 Overproduction of IL-6 is observed in inflammatory autoimmune diseases such
as multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus.
IL-6 signaling pathway
David E. Levy & J. E. Darnell, Jr Nature Reviews Molecular Cell Biology 3, 651-662, 2002
IL-6 signaling pathway
• IL-6 stimulation also activates the transcription factor C/EBPβ through the
ras-Erk MAPK cascade and further upregulates the expression of C/EBPβ.
Lastly, phosphatidyl-inositol (PI)3-kinase has been described as a signal
transducer of IL-6 triggering the activation of Akt and subsequently
promoting survival in many cell types.
• In addition to membrane-bound IL-6R, a soluble form of IL-6R (sIL-6Ra),
which has been found in various human fluids, significantly enhances IL-6
tissue response by a process termed “trans-signaling”. The sIL-6Ra is
produced by two mechanisms: translation from an alternative spliced mRNA
transcript or metalloprotease-dependent proteolytic cleavage of a
membrane-anchored protein at a site close to the cell surface. The soluble IL6-IL-6Ra complex can initiate IL-6 signaling on any cell type that only express
gp130. While gp130 is ubiquitously expressed, IL-6R is present mostly on
leukocytes and hepatocytes. Therefore, IL-6 trans-signaling significantly
expands the repertoire of IL-6 responsive cells.
Negative regulation of IL-6 signaling
• Ligand-induced internalization and degradation of IL-6Rα and gp130 has
been identified as a proximal mechanism for negating signaling.
• STAT3-dependent recruitment of suppressor of cytokine signaling 3
(SOCS3) to the gp130 Tyr 759 residue and inhibits JAK1 activity.
• SOCS proteins also act as adaptor molecules for an E3 ubiquitin ligase
complex that target activated cell signaling proteins to the protein
degradation pathway.
• IL-6-gp130 signaling is also attenuated by a phosphorylation-dependent
induction of SHP-2 tyrosine phosphatase activity which dephosphorylate
gp130 and JAKs.
• PIAS1 and PIAS3 are E3 SUMO-protein ligase. They specifically interact
with STAT1 and STAT3 respectively and to block their DNA binding activity
as well as STAT mediated gene activation.
Negative regulatory pathways of gp130 signaling.
• SHP2 dephosphorylates JAK2
inactivating it.
• SOCS1 and SOCS3 interact
with JAK2 and inhibit its
activity.
• PIAS1 and 3 act at a different
level interacting with STATs
and blocking their binding to
DNA.
• Question marks indicate that
the roles of
ubiquitination/proteosome
mediated degradation of
SOCS and sumorylation of
STATs by PIAS proteins are not
clear.
Alberto Carbia-Nagashima and Eduardo ArztIUBMB Life, 2004, 56(2): 83–88.
Molecular mechanism of IL-6 induced IL-4 production.
Oliver Dienz and Mercedes Rincon. Clinical Immunology 2009, 130(1): 27-33
IL-6 exerts its effects on cytokine production through a
diverse set of key molecules.
Oliver Dienz and Mercedes Rincon. Clinical Immunology 2009, 130(1): 27-33
Contribution of IL-6 to T helper cell differentiation and
subsequent cytokine production by various T cell subsets.
Oliver Dienz and Mercedes Rincon. Clinical Immunology 2009, 130(1): 27-33