Regulation of the kinetics of Interleukin-12 (IL12) and
Interleukin-10 (IL10) in a dendritic cell
Natalie Knapp
CCRI
2011
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 - Myeloid differentiation primary response gene 88
IRAK - IL-1R associated kinase
TRAF6 - TNF receptor associated factor 6
MKK – MAPK kinases
IKK - heterotrimer kinase complex
Jnk - c-Jun N-terminal kinases
[9]
MyD88-dependent/independent
pathway
[9]
IL12 receptor and the JAK-STAT pathway
JAK – Janus kinase
STAT - Signal Transducers and Activators of Transcription
TYK2/JAK2 – Janus kinases
[7]
Interleukin 10
Structure:
• Homodimer
IL-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 production
TLR-dependent 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]
TLR-independent expression
DC-SIGN (DC-specific ICAM3-grabbing
non integrin)
RAF1 (proto-oncogene
serine/threonine-protein kinase)
SYK (spleen tyrosine kinase)
[11]
Regulation of
IL10
expression by
ERK strength
[11]
Regulation of geneencoding IL12p40 [7]
Positive/Negative regulation of
IL12/IL10 production
IL12
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 C5a
formyl peptide fMLP
1, 25-Dihydroxyvitamin D3
Fc -receptor ligation
GAP12 repressor (GA12-binding protein) [7]
IL10
Positive regulation:
ERK pathway
p38
TPL2 (tumor progression locus 2)
SYK (spleen tyrosine kinase)
RAF1 kinase
NFkB (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:
IFNg
GSK3 (glycogen synthase kinase 3)
DUSP1 (dual-specificity protein phosphatase 1)
IL27 (monocytes)
CIITA (MHCII transactivator-mouse DCs)
Regulation of the kinetics
TLR signaling leads to the production of both
cytokines IL10 and IL12. So how should a distinct
secretion 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 Explanations
Downregulation of TLR2/3/4, MyD88, IRAK1
and TRAF6 -> core signaling pathway
Downregulation of STAT3 -> negative fb loop
NO effect on MAPK kinases (rather
downstream)
Decreased level of c-Rel, Rel-B and IKK
activity
NFkB 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
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Nature Immunology, 2000. p.311-116
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[12] Liu Yong-Jun et al., Dendritic cell lineage, plasticity and cross-regulation. Nature Immunology, 2001.p. 585- 589
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[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
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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
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Macrophages: Lipopolysaccharide-Induced, But Not Zymosan-Induced, Proinflammatory Cytokines Are Inhibited, But IL10 Is Nuclear Factor-kB Independent.
J Immunology, 1999; 162: p. 2939-2945