Regulation of the kinetics of Interleukin-12 (IL12) and Interleukin-10 (IL10) in a dendritic cell

Natalie KnappCCRI2011

Today‘s topics

• Kinetics of IL12/IL10 production• Detailed information about IL12/IL10• Regulation of the kinetics

- impact of IL10 on IL12 expression- regulation of time-dependent secretion of IL10 and IL12

Kinetic differentiation model in a human DC

Interleukin-12• Structure

- heterodimer (p70):35-kDa light chain (p35) and 40-kDa heavy chain (p40) - both SU encoded by 2 different genes on 2 distinct chromosomes:p40 (5q31-33)p35 (3p12-q13.2) [1/7]

p40 SU - large excess p35 SU –constitutively expressed [8]

Main producers: phagocytes, DCs [7]

• IL-12 Receptor (IL12R)

- composed of 2 chains:IL12Rβ1 and IL12Rβ2 [1]

present on activated T-cells,NK cells, but also B-cells and DCs [7]

TLR4 signaling pathway MyD88-dependent/independent pathway

MyD88 - Myeloid differentiation primary response gene 88IRAK - IL-1R associated kinase TRAF6 - TNF receptor associated factor 6MKK – MAPK kinasesIKK - heterotrimer kinase complexJnk - c-Jun N-terminal kinases[9]

[9]

IL12 receptor and the JAK-STAT pathway

JAK – Janus kinaseSTAT - Signal Transducers and Activators of TranscriptionTYK2/JAK2 – Janus kinases [7]

Interleukin 10Structure:• HomodimerIL-10 Receptor (IL10R)• Type II cytokine receptor• Jak1 and Tyk2 Janus kinases [1]

Producers:Th1,Th2 and Th17 subsets, Tregs, CD8+ T-cells and B cells but also by DCs, macrophages, mast cells, NK (natural killer) cells, eosinophils and neutrophils. [11]

Regulation of IL10 productionTLR-dependent expression TLR-independent expression

TPL2 (tumour progression locus 2; MEK, MAPK/ERK1 kinase; MSK (mitogen- and stress-activated protein kinase); TRIF (TIR-domain containing adaptor protein inducing IFNβ);TRAF3 (TNFR-associated factor 3)[11]

DC-SIGN (DC-specific ICAM3-grabbing non integrin)RAF1 (proto-oncogene serine/threonine-protein kinase)SYK (spleen tyrosine kinase)[11]

Regulation of gene-encoding IL12p40 [7]

Regulation of IL10 expression by ERK strength [11]

Positive/Negative regulation of IL12/IL10 production

Positive regulation: ERK pathwayp38TPL2 (tumor progression locus 2)SYK (spleen tyrosine kinase)RAF1 kinaseNFkB (Nuclear factor kB)FOS (depends on ERK activation)SP1/3(specific protein 1/3),C/EBPbeta (CCAAT/enhancer binding protein-β)IRF1 (IFN-regulatory factor 1)STAT3

[11]

Negative regulation:IFNgGSK3 (glycogen synthase kinase 3)DUSP1 (dual specificity protein phosphatase 1)‑IL27 (monocytes)

CIITA (MHCII transactivator-mouse DCs)

Positive regulation: IFNg, IL4/IL13, CD40-CD40L interaction TFs: C/EBP(CCAAT/enhancer-binding protein), NFkB (nuclear factor-kb), ETS2 /PU.1 (E-twenty six-family of TFs),IRF1/ IRF8 (Interferon regulatory factor)

Negative regulation:IL10!!! IFNα/β, TNFβ (Transforming growth factor-β), TNF (Tumor necrosis factor), CCL2/8/7/13, complement C5aformyl peptide fMLP1, 25-Dihydroxyvitamin D3 Fc -receptor ligation GAP12 repressor (GA12-binding protein) [7]

IL12 IL10

Regulation of the kinetics

TLR signaling leads to the production of bothcytokines IL10 and IL12. So how should a distinctsecretion in a time-dependent manner be

possible? only core-signaling pathway the same to all TLRs different adaptor proteins lead to differential gene expression [10]

Integration of activation and instruction signals in vivo

[10]

Why is IL12 just shortly produced?

IL10 inhibits IL12 expression [10, 15, 16, 17, 14, 8, 11, 18, 6]

HOW???

battle between TRANSCRIPTIONAL versus POST-TRANSCRIPTIONAL theories

Theory of post-transcriptional regulation (1)

In monocyte-derived dendritic cells:-Downregulation of MyD88, c-Rel, Rel-B and IRF -3/8 (no blockade of

MAPK kinase pathway –p38 unaffected)-Downregulation for TLR2/3/4, IRAK1, STAT3, TRAF6 and

PU-1 could be noted (not affected on mRNA transcriptional level)-Suppression of IKK activity and NFkB activation-Upregulation of DC-SIGN

[15]

Possible ExplanationsDownregulation of TLR2/3/4, MyD88, IRAK1 and

TRAF6 -> core signaling pathwayDownregulation of STAT3 -> negative fb loop NO effect on MAPK kinases (rather downstream)Decreased level of c-Rel, Rel-B and IKK activityNFkB is a family of TFs including Rel A (p65),

NFkB1 (p50 and p105), NFkB2 (p52 and p100), c-Rel and Rel B

Previous observations: only NFkB1(p50) is involved in IL10 induction, other members of NFkB family are important for IL12 induction

Lack of kB binding sites in the human IL10 promoter

Upregulation of DC-SIGN -> TLR-independent pathways

Theory of transcriptional regulation (2)

-Inhibition of transcriptional rate of IL12p40 (not p35), TNFα (BMDMs and Monocytes) [8]-Use of protein-synthesis inhibitor (CHX) shows superinduction of the expression of IL12p40

gene (BMDMs and Monocytes) [8]-Blocking in the recruitment of RNA Pol II by treatment with IL10(Macrophages) [6]-But no effect on nucleosome remodeling at IL12p40 promoter

(restriction enzyme assays –SpeI) (Macrophages) [6]- No inhibition of p50/c-Rel complex, AP-1, C/EBPβ binding [6]

CONCLUSION

IL12 and IL10 are CONTRADICTORY REGULATED!!!

IMMATURE STATE: low level of IL10 (20-50pg/ml/10^6c) [14]-> tolerogenic state

Upon LPS STIMULATION: TLR SIGNALING -> IL12 production but with time IFNg unresponsivness [19]

IMMUNE-SUPRESSIVE state: IL10 increases and inhibits IL12

PARALYZED state of a DC: after 48 hours both cytokines diminish

REFERENCE1• [1] Abbas A. K. et al., Cellular and Molecular Immunology. 6th edition

[2] Banchereau J. et al., Immunobiology of dendritic cells. Annu. Re. Immunol. 2000.18:767-811[3] Mahnke, K., Immature, but not inactive: the tolerogenic function of immature dendritic cells. Immunology and Cell Biology, 2002. 80: p.477-483[4] Steinman, R.M and J. Banchereau, Dendritic cells and the control of immunity. Nature, 1998.p. 245-252[5] Langenkamp A. et al., Kinetics of dendritic cell activation: impact on priming of Th1, Th2 and nonpolarized T cells. Nature Immunology, 2000. p.311-116[6] Zhou L., et al., Interleukin-10 inhibits interleukin-12p40 gene transcription by targeting a late event in the activation pathway. Molecular and cellular biology, 2004.p.2385-2396[7] Trinchieri Giorgio, Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nature reviews, 2003 p.133-146[8] Aste-Amezaga M. et al., Molecular mechanisms of the induction of IL-12 and its inhibition by IL-10. J Immunol, 1998; 160; p.5936-5944[9] Akira S. et al., Toll-like receptors: critical proteins linking innate and acquired immunity. Nature Immunology, 2001; p.675-680[10] Mazzoni A. and Segal D.M., Controlling the Toll road to dendritic cell polarization. Journal of Leukocyte Biology, 2004; 75; p. 1-10[11] Saraiva M. and O’Garra A. et al., The regulation of IL-10 production by immune cells. Nature reviews, 2010. p. 170-181[12] Liu Yong-Jun et al., Dendritic cell lineage, plasticity and cross-regulation. Nature Immunology, 2001.p. 585- 589[13] Qi H. et al., Differential induction of Interleukin-10 and Interleukin-12 in dendritic cells by microbial Toll-like receptor activators and skewing of T-cell cytokine profiles. Infection and Immunity, 2003, p.3337-3342[14] Corinti S. et al., Regulatory activity of Autocrine IL-10 on dendritic cell functions. J Immunol, 2001; 166, p. 4312-4318

• [15] Knödler A. et al., Post-transcriptional regulation of adapter molecules by IL-10 inhibits TLR-mediated activation of antigen-presenting cells. Leukemia, 2009; 23, p. 535-544

REFERENCE2• [16] Cao S. et al., NFkB (p50) homodimers differentially regulate pro- and anti-inflammatory cytokines in macrophages.

Journal of Biological chemistry, 2006; p. 26041-26050[17] Rahim S.S. et al., Interleukin-10 (IL-10) mediated suppression of IL-12 production in RAW 264.7 cells also involves c-rel transcription factor. Immunology, 114, p.313-321[18] Murray P.J., The primary mechanism of the IL-10-regulated anti-inflammatory response is to selectively inhibit transcription. PNAS, 2005, p.8686-8691[19] Polumuri S.K. et al., Role of phosphatidylinositol-3 kinase in transcriptional regulation of TLR-induced IL-12 and IL-10 by Fc γ receptor ligation in murine macrophages. J Immunology, 2007; 179 p. 236-246[20] Bondeson J. et al., Selective Regulation of Cytokine Induction by Adenoviral Gene Transfer of IkBa into Human Macrophages: Lipopolysaccharide-Induced, But Not Zymosan-Induced, Proinflammatory Cytokines Are Inhibited, But IL-10 Is Nuclear Factor-kB Independent. J Immunology, 1999; 162: p. 2939-2945