gC1qR was originally discovered as a C1q receptor specific to the globular head domain of C1q, the first subcomponent of the classical pathway of complement activation. During the same period, calreticulin (CRT), formerly called as cC1qR, was described as a receptor for the collagen region of C1q and collectins. Although much work has been carried out with relation to CRT-CD91 complex, the biological implications and structure-function studies of C1q-gC1qR interaction has not been further explored. With passage of time since 1994, it has become evident that gC1qR is also a multi-functional pattern recognition receptor that can recognise pathogens in addition to acting as a modulator of inflammation at the site of injury or infection. In this thesis, a recombinant form of gC1qR using a T7 promotor expression system was expressed and examined for its interaction with individual globular head modules of C1q A, B and C chains (ghA, ghB and ghC, respectively). A number of single residue substitution mutants of ghA, ghB and ghC modules were also analysed for their interaction with gC1qR in order to map complementary binding sites. Concomitant expression of gC1qR and C1q in the adherent monocytes with, and without proinflammatory stimuli was analysed by qPCR in order to establish autocrine/paracrine basis of C1q-gC1qR interaction. In addition, experiments were carried out to examine if C1q-mediated anti-lymphoproliferative effect can be altered by gC1qR. Subsequently, using the wild type and mutants of ghA, ghB and ghC modules, the interaction of DC-SIGN and SIGN-R, a newly discovered partner of C1q and gC1qR on the dendritic cell surface, was examined. Experiments are underway to understand how a trimolecular complex involving C1q, gC1qR and DC-SIGN participate during HIV-1 infection. Structure-function studies involving gC1qR and HCV core protein and HIV-1 gp41 have also been carried out to localise domains of gC1qR responsible for viral pathogenesis. The last chapter dwells on a newly discovered ability of gC1qR to upregulate bradykinin 1 receptor on the endothelial cell surface, thus its role in altering vascular permeability and the contact system. The thesis describes (1) localisation of interacting sites between C1q and gC1qR and their togetherness in co-expression under pro-inflammatory conditions and possibly suppression of immune cell proliferative response; (2) localisation of complementary binding sites between DC-SIGN, gC1qR and C1q and its possible implications in HIV-1 infection and antigen presenting cells such as dendritic cells; (3) localisation of interacting sites between gC1qR and HCV core protein as well as HIV-1 gp41 peptides with potential to propose a therapeutic peptide; and (4) ability of gC1qR to upregulate bradykinin 1 and 2 receptors on endothelial cells and its newly identified function as a modifier of inflammation.

Endogenous retroviruses (ERVs) are fossils of ancient retroviral infection in the germline. In primates they represent around 5% of the genome sequence. During time spent in the genome, being transmitted in a Mendelian fashion, copies of ERVs have accumulated mutations, which rendered them inactive. However, some of them (the most recently integrated ones) are still able to transcribe and produce viral proteins, although few are capable of re-infection. In the past often considered as unharmful 'junk DNA', recent evidence link ERVs with cancer and several inflammatory diseases. For example, a few reports demonstrate that ERVs are involved in tumour development using shRNA knock-down and over-expression systems, and their overexpression tends to correlate with inflammation status, generating the hypothesis that they can act as pathogen-associated molecular patterns (PAMPs) and bind to innate sensors. Focusing on the Human (Homo sapiens) and the rhesus macaque (Macaca mulatta), the main aims of this thesis are to look for further evidence linking ERVs to tumour development, with possible implications for therapies, and test the hypothesis that ERVs are PAMPs by seeing if individuals with higher levels of ERV expression exhibit a higher innate immune response. The work on ERVs in cancer involved the human ERV type-K HML2 lineage (HERV-K (HML2)), an ERV lineage found in humans, in Merlin-deficient tumours. These are schwannomas that arise from Schwann cells and for which effective drug therapy is urgently needed. The work on ERVs in inflammation involved the Papio cynocephalus ERV (PcEV), in rhesus macaques infected with simian immunodeficiency virus (SIV) infection. The main outcomes are as follows: regarding HERV-K (HML2) in human schwannomas, (i) HERV-K (HML2) proteins are overexpressed in schwannoma compared to Schwann cells; (ii) these proteins are released from the tumour; (iii) regulation of HERV-K (HML2) expression in the tumour appears to involve the transcription factor TEAD; (iv) schwannomas are potentially treatable using anti-HERV-K (HML2) monoclonal antibodies and antiretroviral drugs since both decreased proliferation in vitro. Regarding PcEV in SIV-infected macaques: (i) PcEV is transcriptionally active; (ii) PcEV can be retrieved at low levels in the blood of some macaque animals; (iii) the levels of PcEV in cells correlates strongly with the strength of the innate response as measured by cellular levels of STAT1 transcripts - an interferon-stimulated gene (ISG). Other recent research has shown that human ERV lineages, namely HERV-W and HERV-H, have been co-opted and are involved in placentation and pluripotency during development, respectively. The present work suggests that ERVs are involved in a wide range of biological process and supports the need for further research into the biological significance of ERVs for their hosts.

<p>The larva of the fruit fly Drosophila melanogaster is an excellent model to study the molecular control of innate cellular immune responses. Cellular responses take place, and can be studied, following infestation of the wasp Leptopilina boulardi. This response includes proliferation and activation (differentiation) of the blood cells (hemocytes). In a successful anti-parasitic response, an immune-induced lineage of hemocytes, the lamellocytes, forms a cellular capsule covering and killing the foreign intruder. I will in this thesis present data about the finding and characterization of a novel marker that is expressed specifically in the hemocytes, the Hemese gene. I furthermore describe the construction of a useful tool, the transgenic Hemese-Gal4 fly, which enables blood cell specific expression of any gene of interest. By using the Hemese-Gal4 fly in a directed screen, I have found that a surprisingly large number of genes, that in turn are members of seemingly diverse signaling pathways, are able to induce a cellular response. In many cases their expression is also associated with a blood cell tumor phenotype. Overexpression of certain genes, such as hopscotch (a Drosophila Jak homologue) and hemipterous (a c-jun kinase kinase) lead to the formation of lamellocytes. Other genes may control the cell number, such as Egfr and Ras, as their expression produced a massive in increase the numbers of hemocytes. A third group of genes, including, e.g. Alk, Rac1 and Pvr give a mixed response, promoting both hemocyte proliferation and activation. Surprisingly, the suppression of WNT signaling in hemocytes lead to hemocyte activation. In one case, with a UAS-Pvr dominant negative construct, we observe a reduction of the circulating blood cells in uninfested larva. The expression of DN-Pvr additionally contributes to reduce encapsulation rates in larvae subjected to Leptopilina infestation. In conclusion: the control of blood cells in larval hematopoiesis, and during parasitic wasp attacks, is complex and may involve multiple pathways. In a broader sense, the gene functions found in the directed screen may have implications also for understanding the molecular control of mammalian myeloid lineage blood cells.</p>

The innate immune response is a primary transcriptional defence network activated by interferons (IFNs) α/ β in response to viral infection. A cell must have the capability to detect the virus, activate signalling cascades, and engage transcriptional anti-viral networks. IFNs trigger the Signal Transducer and Activator of Transcription (STAT) family, which in turn induce anti-viral gene expression. Recruitment of STATs to IFN stimulated gene (ISG) promoters and the ensuing gene induction requires Brahma Related Gene 1 (BRG1), the catalytic component of the SWI/ SNF chromatin remodelling (or BAF) complex. Cell lines with high BRG1 expression are hyper-responsive to IFN induced transcription, conversely BRG1 low cells exhibit impaired induction. However, BRG1 high cells that are resistant to Encephalomyocarditis virus infection did not require signalling through the IFN receptor complex for anti-viral immunity. This suggested 2F-BRG1 cells must rely on BRG1 dependent non-ISGs or an as yet uncharacterized subset of basally expressed BRG1-dependent ISGs that do not require IFN enhanced expression for anti-viral activity. Utilizing genome wide microarrays we identified five genes with potent anti-viral activity. These genes may restrict viral infection through alterations in integrin signalling, endosomal trafficking, and activation of host transcriptional responses. We also investigated the role of Special AT-Rich Binding Protein (SATB1) in regulation of IFN responsive genes. The loss of this chromatin binding protein is associated with transcriptional changes in the MHC locus that mimic IFNγ induced expression. Through microarray analysis we discovered a remarkable 47% of IFNα regulated genes were co-regulated by SATB1; 42% of IFNα induced genes were induced by SATB1 knock down, while 63% of IFNα repressed genes were SATB1 dependent. Functionally, knock down of SATB1 protected cells from EMCV induced cell death at low multiplicity of infection (MOI), and increased the cytoprotective effect of IFNα against EMCV at higher MOIs. Analysis of IFNα, SATB1 and BRG1 regulated genes revealed a subset of core genes regulated by all three factors that may be critical to robust anti-viral immunity. The potent immunosuppressive properties of SATB1 suggest this protein may be involved in complex immunopathologies. The immuno-modulatory properties of SATB1 and BRG1 established in this thesis provide substantive evidence for the development of pharmaceutical therapies targeting these proteins.

The innate immune response is a primary transcriptional defence network activated by interferons (IFNs) α/ β in response to viral infection. A cell must have the capability to detect the virus, activate signalling cascades, and engage transcriptional anti-viral networks. IFNs trigger the Signal Transducer and Activator of Transcription (STAT) family, which in turn induce anti-viral gene expression. Recruitment of STATs to IFN stimulated gene (ISG) promoters and the ensuing gene induction requires Brahma Related Gene 1 (BRG1), the catalytic component of the SWI/ SNF chromatin remodelling (or BAF) complex. Cell lines with high BRG1 expression are hyper-responsive to IFN induced transcription, conversely BRG1 low cells exhibit impaired induction. However, BRG1 high cells that are resistant to Encephalomyocarditis virus infection did not require signalling through the IFN receptor complex for anti-viral immunity. This suggested 2F-BRG1 cells must rely on BRG1 dependent non-ISGs or an as yet uncharacterized subset of basally expressed BRG1-dependent ISGs that do not require IFN enhanced expression for anti-viral activity. Utilizing genome wide microarrays we identified five genes with potent anti-viral activity. These genes may restrict viral infection through alterations in integrin signalling, endosomal trafficking, and activation of host transcriptional responses. We also investigated the role of Special AT-Rich Binding Protein (SATB1) in regulation of IFN responsive genes. The loss of this chromatin binding protein is associated with transcriptional changes in the MHC locus that mimic IFNγ induced expression. Through microarray analysis we discovered a remarkable 47% of IFNα regulated genes were co-regulated by SATB1; 42% of IFNα induced genes were induced by SATB1 knock down, while 63% of IFNα repressed genes were SATB1 dependent. Functionally, knock down of SATB1 protected cells from EMCV induced cell death at low multiplicity of infection (MOI), and increased the cytoprotective effect of IFNα against EMCV at higher MOIs. Analysis of IFNα, SATB1 and BRG1 regulated genes revealed a subset of core genes regulated by all three factors that may be critical to robust anti-viral immunity. The potent immunosuppressive properties of SATB1 suggest this protein may be involved in complex immunopathologies. The immuno-modulatory properties of SATB1 and BRG1 established in this thesis provide substantive evidence for the development of pharmaceutical therapies targeting these proteins.

The mammalian immune system has evolved a complex and diverse set of mechanisms to detect and respond to pathogens by recognizing conserved molecular structures and inducing protective immune responses. While many of these mechanisms are capable of sensing diverse molecular structures, a large fraction of pathogen sensors recognize nucleic acids. Pathogen-derived nucleic acids trigger nucleic acid sensors that typically induce anti-viral or anti-microbial immunity, however host-derived nucleic acids may also activate these sensors and lead to increased risk of inflammatory or autoimmune disease. Animal models and humans lacking key DNA nucleases, such as Trex1/Dnase3, accumulate intracellular DNA and develop progressive autoimmunity marked by increased Type-I Interferon (IFN) expression and inflammatory signatures. Double-stranded DNA (dsDNA) is a potent inducer of the Type-I IFN response. Many of the sensors and signaling components that drive the IFN signature following simulation with transfected dsDNA (also called 'Interferon Stimulatory DNA' or 'ISD') remain unknown. We set out to identify novel components of the ISD pathway by developing a large-scale loss-of-function genetic perturbation screen of 1003 candidate genes. We interrogated multiple human and murine primary and immortalized cells, tested several Type-I IFN reporters, and considered multiple loss-of-function strategies before proceeding with an RNAi screen whereby mouse embryonic fibroblasts were stimulated with ISD and Type-IFN pathway activation was assessed by measuring Cxcl10 protein by ELISA. Candidate genes for testing in the RNAi screen were curated from quantitative proteomic screens, IFN-beta and ISD stimulated mRNA expression profiles, and a selection of domain-based proteins including helicases, cytoplasmically located DNA- binding proteins and a set of potential negative regulators including phosphatases, deubiquitinases and known signaling proteins. We identified a number of novel ISD pathway components including Abcf1, Ptpn1 and Hells. We validated hits through siRNA-resistant cDNA rescue, chemical inhibition or targeted knockout. Additionally, we evaluated protein-protein interactions of our strongest validated hits to develop a network model of the ISD pathway. In addition to the identification of novel ISD pathway components, our enriched screening data set may provide a useful resource of candidate genes involved in the response to cytosolic DNA.

The larva of the fruit fly Drosophila melanogaster is an excellent model to study the molecular control of innate cellular immune responses. Cellular responses take place, and can be studied, following infestation of the wasp Leptopilina boulardi. This response includes proliferation and activation (differentiation) of the blood cells (hemocytes). In a successful anti-parasitic response, an immune-induced lineage of hemocytes, the lamellocytes, forms a cellular capsule covering and killing the foreign intruder. I will in this thesis present data about the finding and characterization of a novel marker that is expressed specifically in the hemocytes, the Hemese gene. I furthermore describe the construction of a useful tool, the transgenic Hemese-Gal4 fly, which enables blood cell specific expression of any gene of interest. By using the Hemese-Gal4 fly in a directed screen, I have found that a surprisingly large number of genes, that in turn are members of seemingly diverse signaling pathways, are able to induce a cellular response. In many cases their expression is also associated with a blood cell tumor phenotype. Overexpression of certain genes, such as hopscotch (a Drosophila Jak homologue) and hemipterous (a c-jun kinase kinase) lead to the formation of lamellocytes. Other genes may control the cell number, such as Egfr and Ras, as their expression produced a massive in increase the numbers of hemocytes. A third group of genes, including, e.g. Alk, Rac1 and Pvr give a mixed response, promoting both hemocyte proliferation and activation. Surprisingly, the suppression of WNT signaling in hemocytes lead to hemocyte activation. In one case, with a UAS-Pvr dominant negative construct, we observe a reduction of the circulating blood cells in uninfested larva. The expression of DN-Pvr additionally contributes to reduce encapsulation rates in larvae subjected to Leptopilina infestation. In conclusion: the control of blood cells in larval hematopoiesis, and during parasitic wasp attacks, is complex and may involve multiple pathways. In a broader sense, the gene functions found in the directed screen may have implications also for understanding the molecular control of mammalian myeloid lineage blood cells.

The last decade has been witness to a number of seminal discoveries in the field of innate immunity. The discovery that microbial molecules and endogenous danger signals can be detected by germ-line encoded receptors has changed the way we study the immune system. Indeed, the characterization of Toll in Drosophila as a sensor of microbial products in 1997 then led to the discovery of a family of Toll Like Receptors (TLRs) in mammals. TLRs are critical for the induction of inflammatory responses and the generation of a successful adaptive immune response. The array of ligands that these transmembrane proteins recognized mediates defense against bacteria, viruses, fungus and parasites, as well as, possibly, cancerous cells. In addition to this membrane-bound family of recognition proteins, two families of pattern recognition receptors have been recently shown to respond to microbial and chemical ligands within the cytosol. These represent the Nod Like Receptors (NLRs) and RIGI-like helicase receptor (RLH) families. Nod1 and Nod2 are members of the NLR family of proteins, which are responsible for the recognition of components derived from the bacterial cell wall, more precisely, moieties of peptidoglycan. As such, Nod1 and Nod2 are implicated in the recognition and the defense against bacterial pathogens. Importantly, the genes encoding these two proteins have also been linked to the etiology of several inflammatory disorders such as Crohn’s disease and asthma. In this thesis, we show that recognition of Nod1 and Nod2 ligands generates a rapid and transient inflammatory response in vivo. When co-injected with a model protein, Nod1 and Nod2 ligands exhibit adjuvant properties that lead to the generation of an antigen-specific Th2 type adaptive immune response. Surprisingly, recognition of the Nod1 ligand in non-hematopoietic cells is critical for the generation of this immune response. In contrast, TLRs classically tip the balance towards a Th1 response and interestingly, co-injection of TLR and Nod ligands synergize to generate a more potent immune response characterized by the generation of Th1, Th2 and Th17 T cell respones. To study the role of Nod1 and Nod2 in the context of a bacterial infection in vivo, we used an intestinal mouse pathogen, Salmonella enterica serovar Typhimurium. We were able to show that Nod1-deficient mice, but not Nod2-deficient mice, are more susceptible to the strain of this bacterium, which enters the host through the active pickup in the intestinal lumen by underlying myeloid cells. This sampling mechanism is mediated by a subset of dendritic cells that populate the intestinal lamina propria. Accordingly, the defect seen in Nod1-deficient mice localizes to the mucosal barrier where these dendritic cells appear to have an impaired response towards the bacteria. Taken together, these results increase our knowledge on the general role of Nod1 and Nod2 in immunity and might generate new avenues of research and potential therapeutic targets.

No description available.

Prion diseases are curious neurodegenerative diseases characterized by the conversion of a cellular protein, PrPC, into an infectious isoform, PrPSc. One of the earliest hallmarks of disease and concurrent with prion deposition, is the activation of the brain’s principal immune effector cells, microglia. In prion disease, activated microglia synthesize fairly low levels of pro-inflammatory cytokines, presumably to ameliorate the severe pathology that can arise in host tissue as a result of an acute inflammatory response. The specific stimuli and signaling pathways that lead to this modulation of function are as yet unknown. However, the involvement of miRNAs, a recently identified class of regulatory molecules, is likely. Recently, miR-146a was found to be upregulated in the brains of prion infected mice. In addition, its expression was found to be enriched in cells of microglial origin. It was hypothesized that, given the immunomodulatory role ascribed to miR-146a in macrophages, upregulation of miR-146a may function to attenuate the microglial immune response to prion infection. The first objective was to identify inflammatory related miRNAs associated with prion disease in microglia. Using Taqman Low Density Arrays, allowing for the detection of hundreds of miRNAs at once, the miRNAs of microglia treated with inflammatory agonists were profiled. The miRNA profile of activated microglia was found to be similar to that of macrophages. Furthermore, among the miRNAs profiled, miR-146a and miR-155 were the most highly induced and persistently expressed over 24 hours. The second objective was to investigate miR-146a induction. Therefore, microglia were treated with various agonists and miR-146a expression was determined using Taqman miR-146a assays. Although treatment with a PrP-mimic did not induce miR-146a expression, stimulation of TLRs 1, 2, 4, and 5, resulted in significant over-expression similar to what has been described previously. Moreover, in contrast to the rapid and transient induction of inflammatory mediators, miR-146a follows alternate kinetics functioning to prolong the dampening of the innate immune response following activation via TLR4 and TLR2. By employing a functional proteomic strategy, the third objective was to identify miR-146a regulated proteins. First, miR-146a expression was manipulated using miR-146a mimics and miR-146a inhibitors. Secondly, the functional model was validated by confirming decreased expression of IL6 by ELISA in miR-146a over-expressing microglia cells. Lastly, using Tandem Mass Tag labels to discriminate between treatment group (miR-146a mimic and TLR2 agonist) and control group (scrambled-miR and TLR2 agonist), the effect of miR-146a on the proteome was determined. In total, 172 proteins were identified as being miR-146a regulated and gene ontology assignment resulted in an over-representation of proteins involved in cellular dynamics capable of altering the activation state of microglia. After filtering for bioinformatically predicted targets and those implicated in a similar genomic study, it was decided to further investigate proteins ARF6, RhoA and NOS2 based on their role in modulating the phagocytic potential of microglia. The final objective was to validate miR-146a putative direct targets identified from the proteomics analysis. Luciferase expression of the 3’UTR of targets upon transfection with miR-146a were determined. Based on luciferase analysis, NOS2 appears to be directly targeted by miR-146a and this was also confirmed by western blot. While production of NOS2 by microglia under an acute activation state serves to support and protect CNS homeostasis, chronic expression of this factor can lead to neurotoxicity. Therefore, miR-146a appears to have an overarching role in altering microglial activation during prion disease thus protecting neurons from bystander damage. Taken together, these results suggest that miR-146a could play an important role in the prion disease process by specifically attenuating the microglial induced immune response. Therefore, manipulation of miR-146a may represent a novel therapeutic strategy. Furthermore, given that miR-146a de-regulation has been observed in other neurodegenerative diseases, these results may well extend beyond the realm of prion disease. Lastly, although practical limitations relating to the sensitivity of the comparative proteomics methodology meant that it alone were not sufficient to identify miRNA targets, an integrated approach that takes into consideration genomic and bioinformatic strategies is most promising.