Investigation of Systemic Juvenile Idiopathic Arthritis (SJIA): a disease of

dysregulated innate inflammation

Betsy Mellins, MDDivisions of Human Gene Therapy and Pediatric

RheumatologyInterdisciplinary Program in Immunology

mellins@stanford.edu

Autoimmune versus Autoinflammatory diseaseAutoimmune (adaptive)• Pathogenic cells

– T cells, B cells• Mechanism

– Failure of peripheral or central tolerance to self-antigens

• Features– Autoreactive T cells– Autoantibodies– HLA class II associations

• Examples– RA, T1D (IDDM), MS, – Graves Disease

• Autoinflammatory (innate)• 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

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 childrenWeiss, Ped Clin N Am 2005

• Unknown etiology

Systemic JIA (SJIA)

• Subtype of JIA =10-20% of all JIASchneider 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, anti-TNFα).

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 ImageGreen – flare

Red – quiescentYellow – no change

Cell distribution by FACS

Microarray analysis of PBMC

CD14: canonical marker of CD14+ monocytes

BCL2A1: associated with M1 and CD16+

CXCL16: associated with CD16+ monocytes; induced by IFNγ and TNF in monocytes

ARG1: associated with M2

MMP9: associated with CD16+; induced by TNF in monocytes SLP1: associated with M1

SOD2: associated with M1 monocyte/macrophages

TREM1: associated with CD14+; induced by TNF in monocytes

PBMC microarray

• 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-1

and IL-1that 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-1, IL-1, IL-1 receptor antagonist (IL1-Ra), IL-

18, 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-1 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 full-length 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-1 (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-1IL-18, IL-33• Crohn’s disease and ulcerative colitis: IL-1, IL-

1, 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 IL-27p28. 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 IL-6-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.

• 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.

Negative regulatory pathways of gp130 signaling.

Alberto Carbia-Nagashima and Eduardo ArztIUBMB Life, 2004, 56(2): 83–88.

Oliver Dienz and Mercedes Rincon. Clinical Immunology 2009, 130(1): 27-33

Molecular mechanism of IL-6 induced IL-4 production.

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