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

Autoimmune versus Autoinflammatory disease Autoimmune (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: 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, 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:

Schematic of 2D Gel System

2D Gel Sample Image Green – flare Red – quiescent Yellow – 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-1  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-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-1  IL-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 Feb;10(2): 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, , 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): 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