DISCOVERY OF NOVEL REGULATORS OF TYPE i INTERFERON RESPONSE

DISCOVERY OF NOVEL REGULATORS OF TYPE-I INTERFERON RESPONSE LAM, IRIS WING TO (B.Sc (Biochemistry), The Hong Kong University of Science and Technology) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE IN INFECTIOUS DISEASES, VACCINOLOGY AND DRUG DISCOVERY NATIONAL UNIVERSITY OF SINGAPORE & UNIVERSITY OF BASEL 2015 Declaration I hereby declare that this thesis is my original work and it has been written by me in its entirety I have duly acknowledged all the sources of information which have been used in the thesis This thesis has also not been submitted for any degree in any university previously LAM, Iris Wing To 20th December, 2014 Table of contents Acknowledgement P.2 Abstract P.3 Introduction P.4 Materials and Methods P.22 Results P.30 Discussion P.54 Bibliography P.59 Acknowledgement My deepest gratitude goes to my supervisor, Dr Manoj Krishnan, for his tremendous support and guidance throughout the year He is an inspiring mentor who always keeps his door open and enlightens me with insightful ideas about science and life in general Without his guidance, this thesis would not have been made possible I really appreciate his support My gratitude also goes to all members in Dr Krishnan’s laboratory: Niyas, Shailendra, Pradeep, Jitesh and Sajith They have bestowed me with handson knowledge and techniques in the lab and have given me generous support and assistance that contributed a great deal to my thesis Also, I am genuinely grateful to my parents and my brother, who have formed the best support network throughout all these years I thank them for the unlimited love and care which gave me the emotional support I needed to survive through this year of intensive learning and work Last but not least, I want to express my heartfelt gratefulness to an important person in my life, Simon Lau, for standing by me all the time Thank you! Abstract Innate-immune pattern-recognition-receptor TLR3 is critical for sensing RNAviruses, and triggering transcription of potent antiviral type-I interferons While ubiquitination is well known to regulate interferon response, a genome-wide understanding of the role of ubiquitin ligases in TLR3 signaling is yet to be generated To fill this knowledge-gap, the effect of ectopic expression of the human ubiquitin ligases and their adaptors (UBL/A) encoded in human genome on TLR3 mediated type-I interferon b (IFNb) response was assessed In the first part of the study, HEK293T cells over-expressing each one of the UBL/A genes were generated, stimulated with TLR3 ligand Poly (I:C), and the IFNb gene transcription was determined using IFNb gene promoter driven GFP-reporter Microscopy based data collection and analysis identified novel positive or negative regulator UBL/A genes of IFNb production An overexpression screen was then carried out to identify the genes that regulate the IFN production by modulating the cellular level of TLR3 The capacity of the identified gene to regulate IFNb response was subsequently validated with a gene knock-down approach Finally, the mechanism by which one selected hit gene (TRAF4) regulates the IFN response was investigated It was determined that TRAF4 interacted with TLR3, and promoted degradation of the TLR3 Furthermore, Sendai virus infection resulted in transcriptional upregulation of TRAF4 In summary, this thesis identified TRAF4 as a negative regulator of TLR3 mediated interefron response, throught the ergulation of TLR3 protein turnover Thus, this study generated both a novel global as well as specific mechanistic understanding of the role of UBL/A genes in TLR3 signalling Introduction Innate immunity- An overview The innate immune system constitutes the first line of defence against invading pathogens In principle, it consists of the physical and chemical defence and the cellular defence The physical defence is basically a physical barrier to block the entry pathogens, thereby prevents pathogens from crossing epithelia and colonizing in tissues, while the chemical defence clears the pathogens by degrading them with chemical substances The cellular component of the innate immune system is more complex, and consists of various types of myeloid cells, which are professional immune cells that function to engulf and clear pathogens from the host (Medzhitov & Janeway, 2000) These cells can perform their functions on their own, such as in the case of neutrophils which are sufficient on their own to destroy bacteria (Mayadas, Cullere, & Lowell, 2014) On the other hand, these cells can also act in conjunction with one another or with the components of the adaptive immune system For instance, antibodies opsonize pathogens and mark them for ingestion by myeloid cells (Gasteiger & Rudensky, 2014) Myeloid cells mentioned above can be broadly classified into two classes: mononuclear phagocytes and polymorphonuclear phagocytes (Yan & Hansson, 2007) Mononuclear phagocytes include macrophages and dendritic cells, which originate from monocytes (Gordon & Mantovani, 2011) Macrophages constitute a functionally important population of phagocytes that are capable of phagocytosis, a process through which pathogens are internalized and destroyed More precisely, after the internalization of the pathogen, phagosome containing various reactive oxygen species and hydrolytic enzymes are formed to destroy the engulfed pathogens (Vernon & Tang, 2013) Also, macrophages can secrete different chemotactic cytokines to recruit other kinds of phagocytes to the site of infection to coordinate the overall immune response (Poon, Ho, Chiu, & Chang, 2013) Dendritic cells, as one of the members of the professional antigen-presenting cells, act in a similar fashion to gather the other classes of phagocytes to initiate an immune response The so by taking up and destroying the pathogens by phagocytosis The pathogens are then digested into small peptides, further processed and displaying the antigens on class II MHC proteins to present them to CD4+ T cells On the other hand, dendritic cells also present antigens to CD8+ T cells via class I MHC or cross presentation (Mildner & Jung, 2014) The polymorphonuclear phagocytes, on the other hand, work in a more independent manner The short-lived yet highly motile neutrophils, are responsible for destroying pathogens by phagocytosis and degranulation, a process through which the granules with anti-microbial properties are released to combat pathogens (Wang & Arase, 2014) Eosinophils and basophils are found more rarely than neutrophils, and are mainly responsible for releasing inflammatory mediators to regulate inflammation (Geering, Stoeckle, Conus, & Simon, 2013) To distinguish foreign from self-molecules, the innate immune system employs a strategy to recognize features associated with a broad class of pathogens that are absent in host molecules with a limited number of molecular sensors (Tang, Kang, Coyne, Zeh, & Lotze, 2012) These features, also known as pathogen-associated molecular patterns (PAMPs), are essential for the survival and/or pathogenicity of the pathogens, and so are evolutionarily conserved and not easily altered by mutation or selection (Paul & Grossman, 2014) Primarily speaking, the detection of PAMPs is carried out by germline-encoded pattern-recognition receptors (PRRs) After identifying the invading pathogens by the PRRs, the innate immune system responds to remove the infectious pathogens by employing complement activation and initiating phagocytosis and autophagy by the various immune cells mentioned above (Kumar, Kawai, & Akira, 2011) Pattern recognition receptors - how viruses are recognized as foreign by the immune system Pattern recognition receptors (PRRs) can be broadly classified into two types based on their cellular localization: Membrane-bound receptors including tolllike receptors (TLRs) and C-type lectin receptors (CLRs) recognize PAMPs that are generally associated with extracellular pathogens such as lipoproteins, lipopolysaccharide (LPS), flagellin and peptidoglycan present in bacteria (Tang et al., 2012) Cytoplasmic receptors such as NOD-like receptors (NLRs), pyrin and HIN domain-containing (PYHIN) family members, RIG-I-like receptors (RLRs) and other cytoplasmic nucleic acid sensors are more steered towards recognizing features commonly associated with intracellular pathogens such as viral nucleic acids (Suresh & Mosser, 2013) Toll-like receptors are one of the best characterized families of PRRs They are evolutionarily conserved and can be classified into three main categories, namely Toll-like receptors (TLRs), RIG-I-like receptors (RLRs) and Nod-like receptors (NLRs) Up till now, 13 TLRs have been identified (Kawai & Akira, 2011) All these TLRs consist of an extracellular domain which is made up of leucine-rich repeats that are responsible for PAMP recognition, a transmembrane domain and a conserved cytoplasmic toll/IL-1 receptor domain (TIR), which mediates the interaction with downstream signalling molecules (Uematsu & Akira, 2006) TLRs are located at different cellular compartments for sensing pathogens from different sources: in humans, TLR3, TLR7, TLR8 and TLR9 are found on intracellular compartments such as the endoplasmic reticulum and endosome, and are responsible for recognizing viral nucleic acids; while the other TLRs, like TLR1, TLR2, TLR4, TLR5 and TLR6 are localized on the cell surface to recognize mainly bacterial cell wall components (Uematsu & Akira, 2008) Figure summarizes the location and ligands of TLR 1-9 (adopted from Borden et al., 2007) Figure 1: TLR 1-9 and their respective ligands Those that are located n cell membrane recognized extracellular pathogen-derived molecules, while those that are located in endosomal membrane recognize intracellular pathogenderived molecules Virus receptors Among all the identified TLRs, a subset is important for detecting PAMPs found in viruses and plays a critical role in anti-viral defense, and it includes TLR3, TLR7/8, and TLR9 Of these, TLR3 is specifically responsible for recognizing double-stranded RNA structures which are found commonly in RNA viruses, especially in retroviruses (Perales-Linares & Navas-Martin, 2013) It is expressed in immune cells including macrophages and conventional dendritic cells, as well as in non-immune cells such as epithelial could mean that it plays a regulatory role in the overall immune response elicited to eliminate the virus As the over-production of IFN can overwhelm the local environment of immune cells and cause deleterious consequences such as septic shock, (Trinchieri 2010) TRAF4 may act as a negative regulator of the IFN production to maintain a relatively stable environment of immune cells at the site of infection to prevent excessive or unwanted responses while the cells try to secrete an enormous amount of IFN to clear the virus After the role of TRAF4 as a negative regulator of the TLR3-mediated IFN production was confirmed, it was speculated that it interacts with TLR3 and directly degrades it by facilitating the process of proteolysis To confirm this, a co-immunoprecipitation experiment was performed to check whether TRAF4 interacts with TLR3 A band in the lane where both TRAF4 and TLR3 were overexpressed indicates a interaction between the two proteins However, the intensity of the band is quite low, which may be due to the handling of samples during experimentation that led to the degradation of the two proteins Nonetheless, the interaction between the two proteins was confirmed, but further experiments such as ubiquitination assay are required to prove that TRAF4 degrades TLR3, possibly through tagging it for proteolysis Also, as TRAF4 is a RING domain-containing E3 ligase, it is highly possible that it degrades TLR3 with the action of its RING domain, and ubiquitination It would be interesting to test this hypothesis using TRAF4 mutants that lack the functional RING domain 57 The association of TRAF4 with the TLR3-mediated anti-viral response was previously known, as it has already been reported earlier to be a negative regulator of the TLR3-mediated signalling cascade acting through the degradation of TRIF, the adaptor of TLR3 The content of this thesis expanded our understanding of the role of TRAF4 in TLR3 signalling, by identifying that it regulates the cellular level of TLR3 directly In the same report, it was also reported that TRAF4 interacts with p47phox, IRAK1, TRAF6, and TRIF, which are all key players downstream of TLR3 in the signalling cascade leading to IFN production (Takeshita, Ishii et al 2005) This further supports the idea that TRAF4 plays a role in regulating innate response against viral infection However, further experiments such as in vivo experiments using a TRAF4-deficient mouse model are required to fully characterize the functions of TRAF4 in the context 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