REGULATION OF INTERLEUKIN-12 AND INTERLEUKIN-23 PRODUCTION BY TRISTETRAPROLIN (TTP) LOW PEY YNG BSc (Honours), NUS A THESIS SUBMITED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2012 ACKNOWLEDGEMENTS All these would not have been possible without the help from my supervisor, Prof David M Kemeny, my dear lab mates for the past years as well as my family and friends. First, I would like to thank Prof Kemeny for his advice and support for the past years. Thank you for always being supportive and believing in my work even during the most difficult period. I have learnt a lot over the four years of research and also through the overseas conference opportunities that you have given us. I would also like to thank our collaborators, Dr Perry J Blackshear (Research Triangle Park, NC, US) for providing us with the TTP-/- mice and their invaluable advice. The years of PhD study would not have been so ‘bearable’ without the nice companions of DMK lab. Thank you all for not only the fun and laughter but also for the encouragements whenever the experiments fail to work. To the Yafang and Shuzhen, it has been great knowing you girls and I will never forget the time we had during our overseas trip. To Nayana, Zhou Qian, Isaac, Adrian, Kenneth and Kok loon, thank you all for the advice and help in one way or another. Hope we will always stay in contact and all the best for your future endeavours. To Christopher Yang, thank you for all the help with the experimental techniques. To Benson Chua, thank you for helping us take good care of the mice and also with their genotyping. Our experiments would not have been progressing so smoothly without your help. I would also like to thank members of the flow cytometry facility, Paul Hutchinson, Fei Chuin and Guo Hui for helping with the cell sorting and advice regarding flow cytometry. Of course I would also like to thank my most beloved family for their unwavering support and faith in me for the past years. Especially my parents, aunt and grandma who have been taking good care of me all these years. In addition, I would like to thank my husband for being very encouraging and understanding for the past few years. Thank you for helping me with the housework whenever I am busy with my thesis or lab work and thank you for being accommodating to my schedule especially during this period of time. ABSTRACT IL-12 and IL-23 form a crucial link to adaptive immunity through their ability to influence the development of TH1 and TH17 cells respectively. Despite the importance of the TH1/IL-12 and TH17/IL-23 axis in protective immunity, excessive production of IL-12 and IL-23 can be a nuisance as it leads to immunopathology. This highlights the importance of regulating IL-12 and IL-23 production and we sought to investigate the ability of the mRNA-destabilizing protein, Tristetraprolin (TTP) in regulating IL-12 and IL-23 production by bone-marrow derived dendritic cells (BMDCs). TTP involvement in the regulation of IL-12 and IL-23 production was suggested by the rapid kinetics of IL-23p19 mRNA induction and the sensitivity of IL-12p40, IL-12p35 and IL-23p19 mRNA stability to p38 MAPK inhibitor (SB202190). Using TTP-/- BMDCs, there was enhanced production of IL-23 as compared to WT BMDCs. This enhancement was due to enhanced mRNA stability of IL-23p19 as the half-life of IL-23p19 mRNA was increased. The role of TTP in regulation of IL-23p19 was further confirmed with the overexpression of TTP in HEK293/Tet-off cells as a reduction of IL-23p19 mRNA half-life was observed. Besides IL-23, TTP also negatively regulates the production of IL-12p70 and IL-6. Coculture of naïve CD4 T cells with WT and TTP-/- BMDCs revealed a role of TTP in negatively regulating TH1 responses as the proportion of IFN- producing cells was enhanced in cocultures with TTP-/- BMDCs. This enhancement of TH1 responses results from increased IL-12p70 production by TTP-/- BMDCs. Hence, our study has revealed the importance of TTP as a negative regulator of inflammatory dendritic cell function through the suppression of excessive IL-12, IL-23, TNF- and IL-6 production and the inhibition of their TH1 polarizing potential. Table of contents Chapter 1: Introduction . 17 1.1 Recognition of pathogen by innate immunity . 17 1.1.1 Toll-like receptors (TLRs) . 17 1.1.2 Nod-like receptors (NLR) 19 1.1.3 Retinoic acid inducible gene-I (RIG-I)-like receptors (RLRs) 19 1.1.4 C-type lectin receptors (CLRs) 20 1.2 Subsets of dendritic cells and function . 21 1.2.1 Migratory dendritic cells and their origin 22 1.2.2 Lymphoid tissue resident dendritic cells and their function 24 1.2.3 Inflammatory dendritic cells, the protector and the destroyer . 28 1.3 Interleukin-12 and interleukin-23: Linking innate and adaptive immunity 30 1.3.1 Interleukin-12 and interleukin-23 and their receptors 31 1.3.2 Interleukin-12 and the TH1 lineage . 33 1.3.3 Role of interleukin-23 in the TH17 lineage 35 1.3.4 IL-12 and IL-23 in resistance to infection 39 1.3.5 IL-12 and IL-23 in autoimmunity inflammation 41 1.3.6 Roles of IL-12 and IL-23 in innate immunity 44 1.4 Transcriptional regulation of IL-12 and IL-23 production . 48 1.4.1 Regulation of IL-12p40 promoter 48 1.4.2 Regulation of IL-12p35 promoter 50 1.4.3 Regulation of IL-23p19 promoter 50 1.5 Post-transcriptional control of cytokine production . 52 1.5.1 Post-transcriptional control of cytokines via the AU-rich elements 52 1.5.2 ARE-binding proteins 53 1.5.3 The central role of ARE-binding proteins in ARE-mRNA degradation 54 1.5.4 Tristetraprolin and its regulation through covalent modifications . 57 1.6 Aims of this thesis . 59 1.7 Specific aims . 60 1.8 Hypothesis 60 Chapter 2: Material and Methods 61 2.1 Preparation of buffers and culture media 61 2.1.1 Phosphate-buffered saline (PBS) . 61 2.1.2 MACS/FACS buffer . 61 2.1.3 Complete medium for cell culture 62 2.1.4 Optiprep density centrifugation media for splenic DC isolation . 62 2.1.5 Digestion buffer for splenic dendritic cells isolation . 63 2.1.6 Buffers for ELISA 63 2.1.7 Buffers for SDS-PAGE and Western Blot . 63 2.2 Cell isolation . 63 2.2.1 Generation of GM-CSF-derived bone marrow dendritic cell (BMDCs) . 63 2.2.2 Isolation of splenic dendritic cells 64 2.2.3 Isolation of splenic CD4 or CD8 T cells 66 2.2.4 Stimulation of dendritic cells with microbial components and cytokines . 67 2.2.5 Coculture of dendritic cells and T cells 68 2.3 Fluorescent activated cells sorting (FACS) analysis 68 2.3.1 Surface staining of cells . 68 2.3.2 Intracellular cytokine staining of cells . 69 2.3.3 Preparation of cells for sorting . 71 2.3.4 List of antibodies used for FACS analysis and cell sorting . 71 2.4 ELISA for detection of cytokines . 72 2.5 SDS-PAGE and Western Blot Analysis . 73 2.5.1 Reagents . 73 2.5.2 SDS-PAGE and Western Blot 74 2.6 Qualitative and quantitative analysis of nucleic acid 74 2.6.1 Quantification of mRNA and DNA levels . 74 2.6.2 Extraction of mRNA from cells . 74 2.6.3 Reverse Transcription 75 2.6.4 Real-time PCR . 76 2.6.5 Isolation of genomic DNA from mouse tail . 77 2.6.6 Polymerase chain reaction (PCR) for the genotyping of TTP-/- mice 78 2.7 Molecular cloning and transfection in human embryonic kidney cells (HEK293) 79 2.7.1 Reagents . 79 2.7.2 Preparation of LB broth and LB agar . 79 2.7.3 Purification of DNA from gel 80 2.7.4 Plasmid DNA purification using the QIAprep Spin Miniprep Kit (Qiagen)81 2.7.5 Plasmid DNA purification using Qiagen HiSpeed Plasmid Maxi Kit . 81 2.7.6 Polyfect of HEK293 cell line . 83 2.7.7 Creation of a stable Tet-off Advanced HEK293 cell line 84 2.7.8 pcDNA 3.1/V5-His TOPO® TA Expression of V5-His-Tristetraprolin . 84 2.7.9 Topo TA cloning of IL-23p19, IL-23p19Δ763, IL-23p19Δ1219 & IL23p19Δ1284 86 2.7.10 Ligation of IL-23p19, IL-23p19Δ763, IL-23p19Δ1219 and IL23p19Δ1284 DNA fragment into pTre-tight vector. . 87 2.8 Statistics 89 Chapter 3: Interleukin-23 production by murine GM-CSF-derived bone marrow dendritic cells upon microbial stimuli 90 3.1 Introduction . 90 3.2 Results . 92 3.2.1 Generation of GM-CSF-derived BMDCs 92 3.2.2 Purification of splenic dendritic cells . 92 3.2.3 Differential ability of microbial stimuli to induce IL-12 and IL-23 production . 96 3.2.4 Production of IL-12 and IL-23 are enhanced by CD4 T cells and are dependent on CD40-CD40L interaction . 101 3.2.5 Differential ability of BMDCs and splenic DC to produce IL-12 and IL-23 . 105 3.3 Discussion . 107 Chapter 4: IL-23 production is dependent on Tristetraprolin (TTP) mediated mRNA decay . 112 4.1 Introduction . 112 4.2 Results . 114 4.2.1 IL-23p19 production follows a rapid kinetics of mRNA accumulation 114 4.2.2 mRNA degradation of IL-23p19, IL-12p40, IL-12p35 and TNF- are dependent on p38 MAPK 117 4.2.3 LPS induces the mRNA and protein expression of Tristetraprolin (TTP) . 119 4.2.4 Genotyping of TTP deficient mice . 121 4.2.5 Characterization of TTP-/- bone marrow derived dendritic cells 123 4.2.6 Tristetraprolin negatively regulates the expression of IL-23 by enhancing mRNA degradation of IL-23p19 . 125 4.3 Discussion . 130 Chapter 5: Overexpression of Tristetraprolin in HEK293 cell line enhances the breakdown of IL-23p19 mRNA . 134 5.1 Introduction . 134 5.2 Results . 136 5.2.1 Creating a HEK293 cell line stably expressing tetR-VP-16 fusion protein (HEK293/Tet off) and cloning of IL-23p19, IL-23p19∆1219, IL-23p19∆1284 into pTRETIGHT vector 136 5.2.2 Cloning of V5/His-tagged Tristetraprolin 143 5.2.3 Transfection of pTRE-IL-23p19 into HEK293 stably expressing Tet-off Advanced . 146 5.3 Discussion . 151 Chapter 6: Tristetraprolin negatively regulates production of IL-12p70 by BMDCs and suppresses TH1 development 153 6.1 Introduction . 153 6.2 Results . 156 6.2.1 Tristetraprolin negatively regulates the expression of IL-12p70 through enhancing mRNA degradation of IL-12p35 . 156 6.2.2 Tristetraprolin negatively regulates both TH1 and TH17 promoting cytokines . 159 6.2.3 Tristetraprolin KO BMDCs demonstrated enhanced polarization of naïve CD4 T cells to IFN- producing TH1 cells while inhibiting TH17 polarization 160 6.2.4 Enhanced production of IL-12p70 from Tristetraprolin deficient BMDCs resulted in enhanced TH1 polarization . 165 6.3 Discussion . 170 Chapter 7: Final Discussion 173 7.1 Summary of findings 173 7.2 Limitations of current studies . 175 7.2.1 GM-CSF-derived BMDCs as an in vitro equivalent of inflammatory dendritic cells and their role in the polarization of naïve CD4 T cells . 175 7.2.2 Usage of HEK293 cell line 176 7.3 Targeting IL-12 and IL-23 in chronic diseases . 177 7.3.1 Targeting IL-12 and IL-23 in inflammatory and autoimmune diseases 177 7.3.2 Targeting IL-12 and IL-23 in asthma? . 178 7.4 Tristetraprolin as a possible target for immunotherapy 179 7.4.1 Tristetraprolin as a global regulator of cytokine production 179 7.4.2 Tristetraprolin as a negative regulator of TH1 development . 180 7.4.3 Targeting tristetraprolin for immunotherapy . 181 7.5 Future studies 182 7.5.1 Effect of TTP deficiency on asthma . 182 7.5.2 Effect of TTP deficiency on protection against influenza . 182 REFERENCES . 184 List of Figures and Tables Figure 1.1 Model of 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IL-23p19 3’UTR deletion contructs 139 Figure 5.3 Schematic diagram showing TOPO TA cloning of IL-23p19, IL23p19 121 9 and IL-23p19 128 4 140 Figure 5.4 Schematic diagram showing EcoRI restriction digest and ligation of IL23p19, IL-23p19 121 9 and IL-23p19 128 4 into pTRETIGHT vector 141 Figure 5.5 Identification of bacterial clones expressing pTRE-IL-23p19, pTRE-IL23p19 128 4, pTRE-IL23p19 121 9... enhanced production of IL-12p70 but not IL -23, IL-1 or IL-6 166 Figure 6.9 Enhanced development of IFN-producing CD4 T cells by TTP-/- BMDCs results from enhanced production of IL-12p70 but not IL -23, IL-1 or IL-6 167 Figure 6.10 Supplementation of IL -12 but not IL -23 reversed -IL-12p40 suppression of TH1 development 168 Figure 6.11 Supplementation of IL -12 but not IL -23 reversed... role of IL -23 but not IL -12 in the stimulation of memory CD4 T cells to produce IL-17A and IL-17F This was the pivotal finding that first associated IL -23 with the production of IL-17 Subsequently, IL -23 was identified as a crucial factor required for the maintenance of TH17 lineage 1.3.1 Interleukin- 12 and interleukin- 23 and their receptors Despite their divergent roles in adaptive immunity, IL -12 and. .. costimulation with CD28 and by IL -12 itself (Rogge and Sinigaglia, 1997; Szabo et al., 1997) Besides sharing the same IL-12p40 subunit, similarity between IL -12 and IL -23 is extended to their receptors as IL -23 receptor (IL-23R) comprises of the IL-23R subunit as well as the IL-12R1 subunit that is shared with IL-12R (Oppmann et al., 2000; Parham et al., 2002) Similar to IL-12R, expression of IL-23R was absent... domains The similarity of IL-6 and IL -12 extended to their receptors as IL12 receptor components (IL-12R1 and IL-12R2) and gp130 component of the IL-6 receptor both belong to the gp130 (glycoprotein 130) family of receptors On the other hand, IL-12p40 is structurally related to the soluble IL-6 receptor (IL-6R) (Hunter, 2005) IL-12R is mainly expressed by activated T cells and NK cells (Presky et... 1993; Trinchieri, 2003) The role of IL -12 in the induction of TH1 responses has been demonstrated by the addition of recombinant IL -12 in both in vitro and in vivo models Furthermore, treatment of animals with neutralizing antibodies specific for IL -12 or using animals genetically deficient for IL-12p40, IL-12p35, IL-12R1, IL-12R2 and STAT4 resulted in a suppression of TH1 responses (Trinchieri, 2003)... IL -23 secretion post-LPS stimulation 127 Figure 4.8 TTP-/- BMDCs exhibit enhanced levels of IL-23p19 and TNF- mRNA expression 128 Figure 4.9 TTP-/- BMDCs exhibit prolonged expression of IL-23p19 mRNA through enhanced mRNA stability 129 Figure 5.1 Schematic of gene regulation in Tet-Off gene expression system 137 Figure 5.2 Diagram depicting a series of. .. expression of IL-12R has also been reported on other cell types such as DCs and B-cell lines (Airoldi et al., 2002; Grohmann et al., 1998) IL-12R is undetectable on most resting T cells and is expressed at low level by resting NK cells Upon 31 activation through TCR, the transcription and expression of both chains of IL-12R is upregulated, and this upregulation is further enhanced in the presence of IFN-,... infection by the balance of IL -12 and IL-4, which favours TH1 and TH2 development respectively Indeed, IL -12 when present early during clonal expansion, primes both CD4 and CD8 T cells for the production of high levels of IFN- upon restimulation (Manetti et al., 1994; Seder et al., 1993) However, in experiments involving single cell cloning, IL -12 has minimal ability to reduce T-cell production of IL-4,... first evidence of IL -23 in the regulation of T cell effector function comes from the finding that IL -23, but not IL -12, induced the production of IL-17 by activated and memory T cells (Aggarwal et al., 2003) Subsequently, with an experimental autoimmune encephalomyelitis (EAE) model, IL -23 was unable to induce the development of IFN- producing TH1 cells but instead, promoted development of a T cell subset . protector and the destroyer 28 1.3 Interleukin-12 and interleukin-23: Linking innate and adaptive immunity 30 1.3.1 Interleukin-12 and interleukin-23 and their receptors 31 1.3.2 Interleukin-12 and. 1 REGULATION OF INTERLEUKIN-12 AND INTERLEUKIN-23 PRODUCTION BY TRISTETRAPROLIN (TTP) LOW PEY YNG BSc (Honours), NUS A THESIS SUBMITED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY. 44 1.4 Transcriptional regulation of IL-12 and IL-23 production 48 1.4.1 Regulation of IL-12p40 promoter 48 1.4.2 Regulation of IL-12p35 promoter 50 1.4.3 Regulation of IL-23p19 promoter 50