Doctoral Seminar

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Doctoral Seminar

Title: The Interleukin 12 Family of Cytokines

Presented by: M. Sh. Mojadadi

Advisor: Dr. M. Ebtekar

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Outline

Overview of the Interleukin 12 family of cytokines

Biology and functions of Interleukin 12




Summary and future perspectives

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Overview

The interleukin-12 (IL-12) family is composed of IL-12, IL-23, IL-27, and the newly identified IL-35.

Members of the IL-12 cytokine family differ from other members of the type I cytokine superfamily in that they are heterodimeric complexes.

The structure of IL-12 cytokine family:

α chain: p19, p28 and p35

β chain: p40 and Ebi3

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Overview

1. IL-12: p35 and p40

2. IL-23: p19 and p40

3. IL-27: p28 and Ebi3 (Ebstein-barr virus-induced gene 3)

4. IL-35: p35 and Ebi3

Like IL-12 family cytokines, their receptors also are heterodimeric.

1. IL-12 receptor: IL-12Rβ1 and IL-12Rβ2

2. IL-23 receptor: IL-12Rβ1 and IL-23R

3. IL27 receptor: gp130 and WSX-1

4. IL-35 receptor: ?

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Overview

Like type I and type II cytokine receptors, the IL-12 family of cytokines also engage Jak-STAT signaling pathways.

DCs, macrophages and B cells (APCs) produce IL-12, IL-23 and IL-27 when stimulated by PAMPs through TLRs.

In contrast, IL-35 is produced by Foxp3+ Treg cells.

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Overview

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Introduction

IL-12 was independently discovered by Trinchieri et al. (in 1989) and by Gately et al. (in 1990) as ‘‘natural killer–stimulating factor’’ and as ‘‘cytotoxic lymphocyte maturation factor’’, respectively.

It was present in the supernatant of phorbol-ester–induced EBV-transformed B cell lines.

IL-12 is a heterodimer cytokine formed by a 35-kDa light chain (known as p35 or IL-12α) and a 40-kDa heavy chain (known as p40 or IL-12β).


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Introduction

p35 has homology to other single-chain cytokines (eg. IL-6).

p40 is homologous to the extracellular domain of members of the hematopoietic cytokine-receptor family (eg. IL-6Rα).

The IL-12 receptor is composed of two chains: IL-12Rβ1 and IL-12Rβ2.

IL-12R is very similar to gp130 (the β chain of IL-6R).

Hence it is hypothesized that IL-12 might have evolved from a primordial cytokine of the IL-6 family and one of its receptors.


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Structure and putative evolution of IL-12 and its receptor


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Regulation of IL-12 production


IL-12 is now known to be mainly a product of activated inflammatory cells (monocytes, macrophages, neutrophils, microglia and DCs).

The production of biologically active IL-12 heterodimer (IL-12p70) by phagocytic cells require:

1: Priming signals (from bacterial product through TLRs)

2: Amplification signals (cytokines and cell-cell interactions)

It has been shown that in vivo, CD8α+ DCs, and not macrophages, are the first cells to synthesize IL-12 in the spleens of mice exposed to a soluble extract of T. gondii or to LPS (independent of Amplification signals)

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Positive regulation of IL-12 production


1. Products from microorganisms including bacteria, intracellular parasites, fungi, double-stranded RNA, bacterial DNA and CpG-containing oligonucleotides are strong inducers of IL-12 production by macrophages, monocytes, neutrophils and DCs.

Particularly for phagocytes, TLR ligands alone are not sufficient to induce production of IL-12 heterodimer.

2. Cytokines such as IFN-γ and surprisingly, the two TH2 cytokines IL-4 and IL-13 are also potent enhancers of IL-12 production.

IFN-γ enhances transcription of the genes encoding both p40 and p35, but it has a particularly marked effect on production of the heterodimer.

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Positive regulation of IL-12 production


The effects of IL-4 and IL-13 on expression of the gene encoding p40 are bimodal:

At early times during treatment (<24 hours), they inhibit p40 production, whereas

At later times, they strongly enhance it.

These cytokines augment production of the heterodimer (IL-12p70) even more efficiently than does IFN-γ.

3. T cells also enhance the production of IL-12 through direct cell–cell interactions mostly through interaction of CD40L on activated T cells with CD40 on DCs or macrophages.

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Negative regulation of IL-12 production


1. IL-10 and TGF-β are potent inhibitor of IL-12 production by blocking transcription of both of its encoding genes through induction of the synthesis of an as-yet-unidentified protein.

TGF-β also reduces the stability of IL-12 p40 mRNA.

2. IFN-α, IFN-β and TNF are also potent inhibitor of IL-12 production .

3. Production of IL-12 is inhibited markedly by ligand binding to Gαs-linked GPCRs, mainly through their induction of cyclic AMP.

The efficacy of Gαs-mediated inhibition of IL-12 production was supported by the ability of cholera toxin, which is an activator of Gαs, to inhibit the production of IL-12 and TH1 responses both in vivo and in vitro.

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IL-12 receptor and signal transduction


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Biological functions of IL-12


Initial characterization of IL-12 biological activities revealed that it, when added to human peripheral blood lymphocytes, induced IFN-γ production, increased NK cell cytotoxicity as well as T cell proliferation.

Subsequent studies indicated that IL-12 could boost the generation of CTLs by promoting the transcription of genes encoding cytolytic factors including perforin and granzymes.

In 1993, Hsieh et al.discovered that IL-12, produced by macrophages in response to microbial pathogens, was a key cytokine in TH1 T cell differentiation.

This findings established the central role of IL-12 in a pathway in which innate immune cells drove the adaptive immune response.

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1- Induction of Th1 responsesIL-12 might be more important for the clonal expansion of, as well as amplifying and fixing the phenotype of, already committed TH1 cells than for priming naive CD4+ T cells for TH1-cell differentiation directly.

2- Resistance to infectionOverall, IL-12, by inducing an efficient TH1 immune response, seems to be important for resistance to most bacteria, intracellular protozoa and fungal pathogens.

3- Anti-tumour activity

4- Anti-angiogenesis activityThrough induction of anti-angiogenic facors: IP10 (IFN-inducible protein10) and MIG (Monokine-induced by IFN-γ)

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Anti-tumour activity of IL-12


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5- Induction of IgG2a production from B cells

6- Role of IL-12 in autoimmunityExperimental colitis, Collagen-induced arthritis (CIA), IDDM

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Introduction

Oppmann et al. in 2000 discovered IL-23.

IL-23 is comprised of the IL-12p40 subunit and a novel p19 subunit.

There is 70% homology between the mouse and human forms of IL-23p19.

IL-23 is secreted by activated DCs, Macrophages and monocytes.

It induces proliferation of memory T cells and productio of IFN-γ from activated T cells.

Furthermore it promotes the expansion and survival of a distinct lineage of T cells, Th17.


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IL-23R is expressed on:T cellsNK cellsNKT CellsMonocytesmacrophages DCs

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Expression of the p19 and p40 chains is usually induced by:

1- TLR pathways: the same TLR stimuli (TLR 2,3 and 8)

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2- Non-TLR pathways:Signalin through C-type lectins: the β-glucan curdlan, an agonist of the C-type lectin dectin-1, induces IL-23 synthesis by DCs without inducing IL-12p70 production.

3- Cyclic AMP (cAMP) pathways:Triggering of the E prostanoid 2 (EP2) and EP4 by PGE2 and the purinergic P2y receptors by extracellular nucleotides such as ATP inhibits TLR-mediated IL-12p35 expression while enhancing IL-23 expression.

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1- Expansion and survival of Th17 cells

Naïve CD4 T cell Th17 cell (IL23R) Expansion and survival

2- Enhances the proliferation of memory T cells.

3- Enhances the production of IFN-γ, IL-12 and TNF-α from activated T cells.

4- Effect on APCsInduces production of IFN-γ and IL-12 from DCs.IL-23 promotes differentiation of macrophages to the M1 type.

M1 type: secretion of low levels of TGF-β1, high levels of NO production

TGF-β+IL-6 IL-23

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5- Anti-tumor activity

IL-23-transduced tumors:

IL-23 has potent anti-tumour and anti-metastatic effects in murine CT26 and B16F1 melanoma models.

70% animals completely rejected IL-23-transduced tumors

Free from tumor for the entire 120-day observation period

These effects mediated by CD8 T cells and memory CTLs

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6- Roles of IL-23 in autoimmunity

IL-23 mediated survival and expansion of Th17 cells

Th17 cells are causative agent of more autoimmune diseases such as:

EAE

CIA

IBD

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So it is noteworthy that many of therapeutic targets for the treatment of autoimmune inflammation are associated with the IL-23/Th17 axis.

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Introduction


IL-27 is composed of p28 (a p35-related polypeptide) and Epstein–Barr virus-induced gene 3 (EBI3), a p40-related protein.

IL-27 receptor is also heterodimer composed WSX1 and gp130.

In 1996 Devergne et al. identified EBI3.

In 1998 Sprecher et al., discovered WSX1.

In 2002 Kastelein et al. discovered the p28 subunit of IL-27 (IL-27p28).

After that Pflanz et al. recognize that IL-27p28 partners EBI3 to form IL-27 and that WSX1 is required for its signalling.

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Introduction


IL-27 is a pleiotropic cytokine with contrasting effects (pro and anti-inflammatory effects).

IL-27 is produced by APCs and it signals through a heterodimeric receptor complex consisting of IL-27R also called WSX-1, and gp130.

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Transcriptional regulation of IL‑27


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1-Pro-inflammatory effects (through induction of Th1 responses)

WSX1 deficient mice produce low levels of IFN-γ

WSX1 deficient mice are more susceptible to L. major infection

IL-27 can promote CD8+ T-cell responses

Transgenic overexpression of IL-27 during viral hepatitis or by mouse carcinomas leads to increased CD8+ T-cell, IFN-γ production, cytotoxicity and tumour clearance.

severity of adjuvant-induced arthritis in rats and EAE in mice can be ameliorated by IL-27-specific antibodies.

IL-27 can directly induce mast cells and monocytes to produce IL-1 and TNF.

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2-Anti-inflammatory effects

WSX1 deficient mice infected with T. gondii develop normal CD4 and CD8 responses but fail to downregulate the adaptive immune response and develop a lethal CD4+ T-cell-dependent inflammatory disease.

WSX1-deficient mice show increased sensitivity to conA-induced hepatitis that correlates with increased production of IL-4 and IFN-γ by NKT cells.

WSX1 is expressed more on surface of antigen-experienced T cells than naïve T cell counterparts.

So IL-27 can influence the function of multiple T-cell subsets, including naive, effector, regulatory and memory T cells.

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Interleukin-27 regulates the intensity and duration of T-helper-1 cell andT-helper-2 cell responses.

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3- IL-27 has a regulatory role in immune privileged organs

p28 is produced by activated astrocytes, and both p28 and EBI3 are detected in microglia.

4- IL-27 can inhibit Th17 cell expansion (through STAT1 pathway)

5- IL-27 inhibits iTreg development

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IL-27 orchestrates effective immunity by coordinating Th1, Th17, and Tregs cytokine production.

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6- IL-27 augment cytotoxic activity of TCD8 cells and NK cells.

7- IL-27 induces IgG2a isotype switching in B cells (through STAT1 and T-bet).

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Therapeutic implications of IL-27


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Introduction


IL-35

Devergne et al. in 1997 reported that EBI3 in association with the p35 form a heterodimeric hematopoietin in vivo.

Wanda et al. in 2007 named this heterodimer (p35-Ebi3) IL-35.

Bioactive IL-35 is secreted only in forkhead box protein 3 (Foxp3)Treg.

Ebi3 and p35 are co-expressed in peripheral γδ T cells, CD8 T cells, and placental trophoblasts.

α chain: p35

β chain: Ebi3

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IL-35 receptor and signaling pathway are still unknown.

But it is reasonable to hypothesize that IL-35, like its siblings, will signal through the pairing of known receptor chains utilized by the IL-12 cytokine family.

In addition, it is interesting to speculate, based on the known signaling pathways involved in IL-12 and IL-27 activity, that STAT4 and perhaps STAT1 and STAT3 might be involved in the IL-35 signaling pathway.

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1- Induction of Th1 responses (through T-bet expression)

2- Proliferation of CD4CD25 Treg and production of IL-10

3- Inhibition of Th17 responses

4- Suppression of Tef proliferation

5- Immunomodulator at the feto-maternal border

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Summary


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Future perspectives


There is still much that needs to be determined about IL-35 biology and the role of the IL-12 family cytokines in general.

1- Can IL-35 inhibit other T-cell (Th1, Th2, Th17, CD8 T cell, etc.) and non-T-cell (DCs, macrophages, NK cells, neutrophils, etc.)?

2- The composition of the IL-35 receptor and the signaling cascade it initiates and the expression pattern of this receptor is still unclear.

3- Do the other cell types produce biologically active IL-35?

4-Many of the current knockout mice in fact lack production of two cytokines, new mutant mouse models are clearly required.

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Thank You For Attention

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Doctoral Seminar