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Doctoral Seminar

Doctoral Seminar. Title: The Interleukin 12 Family of Cytokines Presented by: M. Sh. Mojadadi Advisor: Dr. M. Ebtekar. Outline. Overview of the Interleukin 12 family of cytokines Biology and functions of Interleukin 12 Biology and functions of Interleukin 23

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Doctoral Seminar

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  1. Doctoral Seminar Title: The Interleukin 12 Family of Cytokines Presented by: M. Sh. Mojadadi Advisor: Dr. M. Ebtekar

  2. Outline • Overview of the Interleukin 12 family of cytokines • Biology and functions of Interleukin 12 • Biology and functions of Interleukin 23 • Biology and functions of Interleukin 27 • Biology and functions of Interleukin 35 • Summary and future perspectives

  3. Overview Overview of the Interleukin 12 family of cytokines • 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 βchain: p40 and Ebi3

  4. Overview Overview of the Interleukin 12 family of cytokines • IL-12: p35 and p40 • IL-23: p19 and p40 • IL-27: p28 and Ebi3 (Ebstein-barr virus-induced gene 3) • IL-35: p35 and Ebi3 • Like IL-12 family cytokines, their receptors also are heterodimeric. • IL-12 receptor: IL-12Rβ1 and IL-12Rβ2 • IL-23 receptor: IL-12Rβ1 and IL-23R • IL27 receptor: gp130 and WSX-1 • IL-35 receptor: ?

  5. Overview Overview of the Interleukin 12 family of cytokines • 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.

  6. Overview Overview of the Interleukin 12 family of cytokines

  7. Introduction Biology and functions of Interleukin 12 • 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).

  8. Introduction Biology and functions of Interleukin 12 • 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.

  9. Structure and putative evolution of IL-12 and its receptor Biology and functions of Interleukin 12

  10. Regulation of IL-12 production Biology and functions of Interleukin 12 • 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)

  11. Positive regulation of IL-12 production Biology and functions of Interleukin 12 • 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. • 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.

  12. Positive regulation of IL-12 production Biology and functions of Interleukin 12 • 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. • 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.

  13. Negative regulation of IL-12 production Biology and functions of Interleukin 12 • 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. • IFN-α, IFN-β and TNF are also potent inhibitor of IL-12 production . • 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.

  14. IL-12 receptor and signal transduction Biology and functions of Interleukin 12

  15. Biological functions of IL-12 Biology and functions of Interleukin 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.

  16. Biological functions of IL-12 Biology and functions of Interleukin 12 • 1- Induction of Th1 responses • IL-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 infection • Overall, 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 activity • Through induction of anti-angiogenic facors: IP10 (IFN-inducible protein10) and MIG (Monokine-induced by IFN-γ)

  17. Anti-tumour activity of IL-12 Biology and functions of Interleukin 12

  18. Biological functions of IL-12 Biology and functions of Interleukin 12 5- Induction of IgG2a production from B cells 6- Role of IL-12 in autoimmunity Experimental colitis, Collagen-induced arthritis (CIA), IDDM

  19. Introduction Biology and functions of Interleukin 23 • 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.

  20. IL-23 receptor and signal transduction Biology and functions of Interleukin 23 • IL-23R is expressed on: • T cells • NK cells • NKT Cells • Monocytes • macrophages • DCs

  21. Regulation of IL-23 production Biology and functions of Interleukin 23 • Expression of the p19 and p40 chains is usually induced by: • 1-TLR pathways: the same TLR stimuli (TLR 2,3 and 8)

  22. Regulation of IL-23 production Biology and functions of Interleukin 23 • 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.

  23. Biological functions of IL-23 Biology and functions of Interleukin 23 • 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 APCs • Induces 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

  24. Biological functions of IL-23 Biology and functions of Interleukin 23 • 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

  25. Biological functions of IL-23 Biology and functions of Interleukin 23 • 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

  26. Biological functions of IL-23 Biology and functions of Interleukin 23 • So it is noteworthy that many of therapeutic targets for the treatment of autoimmune inflammation are associated with the IL-23/Th17 axis.

  27. Introduction Biology and functions of Interleukin 27 • 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.

  28. Introduction Biology and functions of Interleukin 27 • 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.

  29. Transcriptional regulation of IL‑27 Biology and functions of Interleukin 27

  30. IL-27 receptor and signal transduction Biology and functions of Interleukin 27

  31. Biological functions of IL-27 Biology and functions of Interleukin 27 • 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.

  32. Biological functions of IL-27 Biology and functions of Interleukin 27 • 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.

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

  34. Biological functions of IL-27 Biology and functions of Interleukin 27 • 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

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

  36. Biological functions of IL-27 Biology and functions of Interleukin 27 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).

  37. Therapeutic implications of IL-27 Biology and functions of Interleukin 27

  38. Introduction Biology and functions of Interleukin 35 α chain: p35 β chain: Ebi3 • 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.

  39. IL-35 receptor and signal transduction Biology and functions of Interleukin 35 • 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.

  40. Biological functions of IL-35 Biology and functions of Interleukin 35 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

  41. Summary Summary and future perspectives

  42. Future perspectives Summary and 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.

  43. Thank You For Attention

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