1. Regulation of the kinetics of Interleukin-12 (IL12) and Interleukin-10 (IL10) in a dendritic cell
Natalie Knapp CCRI 2011

2. 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

3. Kinetic differentiation model in a human DC
Our hypothesis is that upon PAMP recognition, as LPS, a DC starts to undergo a differentiation program. First, a rapid induction of proinflammatory cytokines, upregulation of co-stimulatory molecules and major histocompatibility complex takes place., also referred as the immune-stimulatory window. One of theses important cytokines is IL12. After about one day to keep a balance in the IS the so called immune-suppressive window follows and anti-inflammatory cytokines, such as IL10 are produced.

4. 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]
IL12 is a heterodimer, composed of a 35kDa light chain and 40kDa heavy chain. Both subunits are encoded by two distinct genes on different chromosomes. What explains why they are independently regulated. P35 is constitutively expressed in low amounts in most cells, whereas p40 is produced in large excess over the IL12 heterodimer and restricted to those cells which are able to express the heterodimer. Expression of p35 can be seen as the rate-limiting facotr of heterodimer formation. Only the binding of both SU with high affinity to the IL12 receptor can induce signaling.

5. TLR4 signaling pathway MyD88-dependent/independent pathway
Different TLRs recognize a variety of ligands, such as LPS, Lipoproteins, glycolipids, flagellin, bacterial DNA. LPS from Gram-negative bacteria is responsible for the TLR4 signaling. MD2 is associated with the extracellular domain of TLR4 and as a complex is needed for LPS signalling. Activated TLR4 receptors leads to the recruitment of a cytoplasmic adaptor protein MyD88. IRAK, a kinase binds to the TLR/MyD88 complex by ist TIR domain. Phosphorylated IRAK gets bound by TRAF6, which activates MAP kinases or heterotrimer IKK complex. Phosphorylation and nuclear translocation of IRF3 can occur in a MyD88 independent manner and is involved in IFN-inducible
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]
[9]

6. IL12 receptor and the JAK-STAT pathway
Now to the JAK STAT pathway, IL12R/IL10R upon binding their ligand (in this case IL12), JAK kinases Tyk2/Jak2 come together upon dimerization of the chains, and transphorphorylate each other and then phosphorylate tyrosin residues of cytoplasmic IL12R domains. SH2 domain -> Stat proteins bind to the receptors get phosphorylated by JAKs, detach and form dimers, translocate to the nucleus and regulate IL12 gene expression. Different translocated STAT dimers regulate specifically different cytokine genes.
JAK – Janus kinase STAT - Signal Transducers and Activators of Transcription TYK2/JAK2 – Janus kinases [7]

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]
IL10, the immunosuppressive cytokine, is a homodimer composed of 2 domains intercalating non-covalently with each other.

8. Regulation of IL10 production
TLR-dependent expression
TLR-independent expression
As well as for IL12 production TLR4 signalling is necessary for IL10 production. As mentioned before upon TLR4 activation, adaptor proteins as MyD88 or TRIF, bind activate various kinases. Important is that there are also TLR-independent pathways for IL10 production. Dectin1 ligation leads to the recruitment of SYK and activates the ERK signaling pathway. And DC-SIGN ligation…
DC-SIGN (DC-specific ICAM3-grabbing non integrin) RAF1 (proto-oncogene serine/threonine-protein kinase) SYK (spleen tyrosine kinase) [11]
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]

9. Regulation of IL10 expression by ERK strength [11]
Regulation of gene-encoding IL12p40 [7]
POS/NEGATIVE Regulation of each cytokines. ERK activation is part of many signaling pathways and it depends on the strength of ist activation which results in distinct secretion profiles in various cells as macrophages, myeloid and plasmacytoid DCs. That‘s a picture of the mechanisms how the IL12p40 locus might be regulated on transcriptional level. Specific DNA-sequences for different DNA binding proteins as TF, this region here nucleosome1, which must be loosened for the accessibility of AP1 and CEBP. A NFkB element was found to be important for promotor activation downstream. ETS consensus element might bind 2 members of the ETS family of transcription factors ETS2 and PU.1, they form a large supramolecular complex, with c-REL and other IRF members. IRF1/8 pos. regulators. IRF2 negative regulators. Binding of the transcription complex to the ETS site important for enhanced transcription that is induced by IFNg and LPS. Also some inhibitors a….you see GATA sequence, where GAP12 repressor binds..enhanced by treatment with IL4 and Prostaglandin….most potent inhibitor is IL10…

10. Positive/Negative regulation of IL12/IL10 production
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)
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]
Upon inhibition of p38 and ERK kinases, they observed an almost entire suppression of IL10 IL10 also acts by a positive feedback loop on its own regulation by upregulating TPL2.

11. 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]
How we have seen how each cytokines can be regulated on molecular level. We have seen that TLR signaling is important for both expressions. The core-signaling pathway is common to the production of all cytokines. It starts with binding of MyD88 to the TLR, the recruitment of IRAK and TRAF6

12. Integration of activation and instruction signals in vivo
On this picture I wanted to show you that the signals which drive a DC to cytokine production are due to environmental(instruction signals) and activation signals. Activation signals, are signals which initiate the signal transduction pathways of cytokine production…as LPS whereas instruction signals rather modify the activating signals…as IFNg is e.g important for boosting IL12 production…IL10 inhibits it… On the picture depending on the interplay of the (instruction/activation signals) a DC might maturate into distinct phenotypes by either secreting IL10 and therefore priming Tregs or secreting IL12 to prime TH1 cells but contrary to that our theory is that a DC can go through all these phenotypes in a time-dependent manner.
[10]

13. 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
IL12 is only transiently produced. IL10 most potent inhibitor of IL12. thousands of publications supporting this. HOW? I thought i would find a simple answer to this question but unfortunately i found out that it‘s a very controversial issue.

14. 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]
In comparison with other studies it‘s rather recently published and the experiments were done in monocyte-derived DCs. It was found out that IL10 downregulates MyD88, c-Rel, Rel-B and IRF-3/8, but this wasn‘t due to a blockade of the MAP kinase pathways because the activation of p38 was unaffected Additionally a downregulation for TLR2/3/4 IRAK1, STAT3, TRAF6, PU.1 could be noted . Astonishing that on mRNA level none of these genes were affected

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

16. 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]
Some studies confirmed that IL10 suppressed the expression of IL12p40 at transcriptional level without affecting ist mRNA stability. Also made use of cylcoheximide, so that the inhibitory effect of IL10 was abrogated…superinduction due to the stabilization of the mRNA, because mRNA degradation depends upon protein synthesis Blocking recruitment of POlII to the p40 promoter but no neg. effect on the nucleosome remodeling of p40 locus or inhibition of TF on binding of DNA

17. IL12 and IL10 are CONTRADICTORY REGULATED!!!
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
So considering all my results and those published data I have read I state that a very low level of IL10 exists in Immature DCs to avoid spontanous maturation -> so a tolerogenic state… Upon LPS stimulation, the activating pathways enhance IL12 production by TLR signaling. And because a DC undergoing final maturation become unresponsive to stimulation, which is associated with reduced expression of IFNgR. IL10 production arises and starts to inhibit IL12 expression….

18. 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 : [3] Mahnke, K., Immature, but not inactive: the tolerogenic function of immature dendritic cells. Immunology and Cell Biology, : p [4] Steinman, R.M and J. Banchereau, Dendritic cells and the control of immunity. Nature, 1998.p [5] Langenkamp A. et al., Kinetics of dendritic cell activation: impact on priming of Th1, Th2 and nonpolarized T cells. Nature Immunology, p [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 [7] Trinchieri Giorgio, Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nature reviews, 2003 p [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 [9] Akira S. et al., Toll-like receptors: critical proteins linking innate and acquired immunity. Nature Immunology, 2001; p [10] Mazzoni A. and Segal D.M., Controlling the Toll road to dendritic cell polarization. Journal of Leukocyte Biology, 2004; 75; p [11] Saraiva M. and O’Garra A. et al., The regulation of IL-10 production by immune cells. Nature reviews, p [12] Liu Yong-Jun et al., Dendritic cell lineage, plasticity and cross-regulation. Nature Immunology, 2001.p [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 [14] Corinti S. et al., Regulatory activity of Autocrine IL-10 on dendritic cell functions. J Immunol, 2001; 166, p
[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

19. REFERENCE2
[16] Cao S. et al., NFkB (p50) homodimers differentially regulate pro- and anti-inflammatory cytokines in macrophages. Journal of Biological chemistry, 2006; p [17] Rahim S.S. et al., Interleukin-10 (IL-10) mediated suppression of IL-12 production in RAW cells also involves c-rel transcription factor. Immunology, 114, p [18] Murray P.J., The primary mechanism of the IL-10-regulated anti-inflammatory response is to selectively inhibit transcription. PNAS, 2005, p [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 [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