published_or_final_version / Paediatrics / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Surgery / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Medicine / Doctoral / Doctor of Philosophy

The innate immune system is an important mechanism that protects the host from infection. Viral and bacterial infection triggers activation of the transcription factors interferon response factor (IRF) 3 and nuclear factor (NF)-kB. These transcription factors collaborate to induce transcription of type I interferons (IFNs) cytokines and the interleukin (IL)-12 family of cytokines. Type I IFN and the IL-12 family of cytokines play a critical role in establishing innate immune responses as well as initiating and directing adaptive responses. Our study focused on the role of protein kinase C (PKC) isoforms in Toll-like (TLR)-dependent and –independent activation of IRF-3 and NF-kB and their subsequent regulation of IFN-beta and the IL-12 family of cytokines. TLR3, TLR4 and retinoic acid-inducible gene 1 (RIG-1)/melanoma differentiation associated gene 5 (MDA-5) activation by double stranded (ds) RNA mimic polyinosine-polycytidylic acid (poly(I:C)), lipopolysaccharide (LPS) and synthetic ds-B-DNA respectively, mediated IFN-beta as well as TNF-alpha and IL-8 synthesis in monocyte-derived DCs. Using the pharmacological inhibitor of conventional PKCs (cPKCs), Gö6976, we demonstrated that this family of kinases was involved in TLR3, TLR4 and RIG-1/ MDA-5 signaling pathways leading to the production of IFN-beta but not of TNF-alpha and IL-8. Further analysis with the use of specific kinase inactive cPKC isoforms and siRNA targeted to PKCalpha, we established that PKCalpha was the isoform involved in the TLR3 signaling pathway. In the case of TLR3, we show that PKCalphaexerts its effect downstream of TRIF and TBK1. Moreover, we show that inactivation of PKCalpha specifically inhibits the activation of IRF-3 and not that of NF-kB. Through biochemical analysis, we assessed the contribution of PKCalpha in the critical events of IRF-3 activation: a) phosphorylation b) homodimerization c) nuclear translocation d) DNA-binding and e) recruitment of creb-binding protein (CBP). We conclude that inhibition of cPKCs severely hinders the association of IRF-3 with CBP. Overall, these data revealed the critical role of cPKCs in TLR-dependent and -independent pathways leading to IFN-beta synthesis. The selective targeting of IRF-3 by cPKCs prompted us to study the possible implications of cPKCs in the transcriptional control of IL-12 family members, some of which are regulated by IRF3. Indeed, recent studies have emerged demonstrating the essential role of IRF-3 in IL-12p35 and IL-27p28 gene expression (1;2). Likewise, we investigated the role of cPKCs in the regulation of LPS- and poly(I:C)-induced expression of IL-12(p40/p35), IL-23(p40/p19) and IL-27(p28/EBI3) in monocyte-derived DCs. Treatment of monocyte-derived DCs with Gö6976 down-regulated LPS- and poly(I:C)-induced IL-12 and IL-27 synthesis while it did not alter IL-23 production. Next, we showed that impaired IL-12 and IL-27 synthesis was due to repressed IL-12p35 and IL-27p28 gene expression downstream of TLR3 and TLR4 whereas IL-23p19 and IL-27EBI3 gene expression were not modified. Reporter gene assays demonstrated that cPKCs are involved in LPS- and poly(I:C)-induced IL-12p35 and IL-27p28 promoter activity. Finally, experiments in bone marrow-derived DCs from IRF-3-/- and wild type mice showed that IL-23 synthesis does not require IRF-3 activation. We conclude that cPKCs through the control of IRF-3 activity are critically involved in the regulation of IL-12 and IL-27 synthesis downstream of TLR3 and TLR4 while they do not participate in IRF-3-independent IL-23 synthesis. On whole, we demonstrated a novel function for cPKCs in the regulation of IRF-3 and IRF-3 dependent gene expression, specifically IFN-beta, IL-12 and IL-27. In light of the important and divergent roles of IFN-beta and IL-12 family of cytokines on the development of T helper (Th) Th1, Th2, Th17-mediated immune responses, cPKCs represent a potential target for therapeutic immunomodulation. This modulation needs to be carefully administered due to the complex interplay of the IL-12 family members in immunity.

The leukocyte immunoglobulin like receptors (LILRs) are a group of receptors with immunomodulatory effects. Group 1 LILRs comprise of LILRB1, among others, and bind to class 1 MHC molecules and transmits inhibitory signals. Studies have shown that LILRB1 ligation during the monocyte differentiation process into dendritic cells (DCs) results in the generation of a population of cells that are tolerogenic. Here we hypothesize that this tolerogenic nature of the resultant cells is due to the high expression of nuclear factor kappa – light chain enhancer of activated B cells (NF-κB) inhibitor – A20 binding inhibitor of NF-κB signalling 1 (ABIN1). In this study we analyzed the effect that ABIN1 exerts on the maturation of DCs and CD14+HLA-DRlow/- monocytes - a population of cells that have been recently been identified as myeloid derived suppressor cells (MDSCs) in humans. LILRB1 ligated DCs and CD14+HLA-DRlow/- monocytes, when treated with ABIN1 siRNA, displayed an increase in the expression of antigen presentation and co-stimulatory molecules such as CD80, CD86, HLA-DR and HLA-ABC and displayed a greater capacity to produce cytokines like IL-12 and IFN-α. Additionally, they displayed a greater capacity to stimulate the adaptive component of the immune system in terms of IFN-γ production, cell proliferation and adapter molecule and mitogen activated protein kinase (MAPK) activation in T cells. Based on the results we obtained, it can be concluded that ABIN1 plays a significant role in maintaining the immature and suppressive phenotype of immature myeloid cells (IMCs) by dampening NF-κB signalling, while also exerting a negative effect on antiviral signalling.

Dendritic cells play an essential role in activating immune responses upon recognition of pathogens. This results in maturation and migration to the lymph nodes, where T cells are stimulated by upregulated antigen presentation, co-stimulation and cytokine secretion. DCs are also considered important in inhibiting inappropriate immune responses against self-peptides which could lead to the development of autoimmunity. This has been attributed to DCs that demonstrate inhibited co-stimulation and cytokine secretion. It has been previously shown that the continuous ligation of an immunomodulatory receptor, LILRB1, during DC differentiation results in such a DC population that demonstrates an immature phenotype even after exposure to bacterial components and resulted in inhibiting primary T cell responses. The mechanisms by which LILRB1-DCs promote tolerance are, therefore, here investigated. Previous studies revealed significantly altered expression for a large number of gene targets which varied from immune to cytoskeletal and bone-related functions. One of these includes DcR3, a soluble protein with a poorly defined role in immune regulation. It is here demonstrated that DcR3 has a positive role in the induction of IL-17, a cytokine implicated in autoimmunity. However, DcR3 was not secreted by LILRB1-DCs, possibly accounting for some of their tolerogenic functions. In addition, the expression of several cytoskeletal proteins was significantly changed in response to LILRB1 ligation and was associated with decreased ability for phagocytosis and migration. Lastly, it has been recently identified that DCs are able to trans-differentiate into osteoclasts, the main cell type linked with inflammatory bone disorders, such as rheumatoid arthritis. It is here shown for the first time that ligation of LILRB1 inhibits this process and results in decreased bone resorption. Overall, these data provides evidence that ligation of LILRB1 on DCs affects normal inflammatory functions and suggests its potential for the development of new treatments against several autoimmune diseases.

No description available.

The alternative activation of macrophage populations by Interleukin-4 (IL-4) is well characterised. Alternatively activated macrophages (AAM) express high levels of the arginine converting enzyme arginase-1, and express a plethora of IL-4 driven molecules including the resistin like molecule alpha (RELMα) and the chitinase like molecule Ym1/2. Dendritic cells (DCs) are the professional antigen presenting cells (APC) of the immune system, responsible for the detection of invading pathogens, secretion of cytokines and the subsequent activation of T-cells. This thesis addresses whether IL-4 is able to ‘alternatively activate’ DCs both in vitro and in vivo, in a manner similar to that of AAM. The impact of IL-4 on DC and macrophage activation was compared and contrasted, and it was confirmed for the first time that IL-4 can alternatively activate DCs, inducing high level expression of a range of alternative activation associated markers including RELMα, Ym1/2, CCL24 and dectin-1, with the exception of arginase. DCs were significantly more capable at the in vivo priming of T-cell responses in the context of both Th1 and Th2 polarising antigens than similarly exposed macrophages, confirming their superior capacity as APC. The requirements for DC IL-4Rα expression were assessed, and IL-4 responsiveness was found to be required for the optimal induction of Th1 responses. Conversely, selective loss of only one facet of the IL-4 response, namely RELMα expression, limited the ability of IL-4 exposed DCs to induce the regulatory cytokine IL-10 both in vitro and in vivo. Furthermore, alternatively activated DCs (AADCs) were found in the spleen following 8 weeks of infection with the parasitic trematode Schistosoma mansoni, highlighting a role for DC alternative activation in a disease setting. IL-4 was shown to induce expression of the vitamin A converting enzyme aldehyde dehydrogenase, and the product of such activity, retinoic acid (RA), was found to promote the expression of RELMα in IL-4 exposed DCs. Aldehyde dehydrogenase activity was found to inversely correlate with DC expression of Ym1/2 and inhibition of RA signalling limited IL-4 driven RELMα and promoted Ym1/2.

Dendritic cells (DCs) play an essential role in bridging the innate and adaptive immune system, activating T cell responses against invading pathogens. It has been shown definitively that DCs fulfil the vital role of activating Th2 responses in the liver and spleen during infection with the parasitic helminth Schistosoma mansoni. However, DCs are an incredibly heterogeneous cell type, with diverse subsets displaying different phenotypes and functions in specific tissues in the body. Moreover, relatively little is known about how DCs become activated and stimulate T cells in response to Th2-associated parasitic helminths. This thesis addresses how distinct DC subsets function in response to schistosomes, both in vitro and in vivo. The primary DC differentiation factor, Flt3-L, generates DC subsets in vitro that are analogous to the subsets resident in the lymphoid organs in the steady-state: CD24+ conventional DCs (cDCs, CD8α+ equivalents), CD11b+ cDCs and plasmacytoid DCs (pDCs). These different DC subsets displayed distinct responses to the strongly Th2- polarising soluble egg antigens (SEA) from S. mansoni. pDCs are unlikely to play a role in priming the Th2 response against SEA, although pDCs upregulated surface expression of MHC II and co-stimulatory molecules, these markers were expressed only at very low-levels, and pDCs failed to migrate to the draining lymph node (dLN) following adoptive transfer. In contrast, cDCs migrated efficiently to the T cell zone of the dLN. CD11b+ and CD24+ cDCs also significantly upregulated expression of the surface markers associated with T cell priming in response to SEA, however, this was a muted surface phenotype when compared to the classical activation elicited by a bacterial stimulus. The DC subsets produced very little cytokine in response to SEA stimulation, with the exception of Type I Interferons (IFN-I), which were uniquely secreted by CD24+ cDCs. The Toll-like receptor (TLR) adaptor proteins, TRIF and MyD88, were revealed to have contrasting roles in the control of SEA-specific IFN-I production. TRIF was essential for this response, whilst MyD88 acted as a negative regulator. TLRs are not the only receptors involved in this response however, as the C-type lectin CD205 was also required for optimal IFN-I production by SEA-stimulated cells. IFN-I proved critical to the ability of Flt3L-generated cDCs (FL-cDCs) to polarise responses following adoptive transfer, as IFN-I receptor-deficient (Ifnar1-/-) cells failed to prime an SEA-specific Th2 response in the dLN. Ifnar1-/- cells were almost completely unresponsive to SEA stimulation, failing to upregulate co-stimulatory molecules on their surface or to produce IFN-I. However Ifnar1-/- FL-cDCs displayed no deficiency in their ability to initiate T cell proliferation or IL-4-dependent Th2 polarisation in vitro. As T cell priming was abrogated in vivo only, this suggests that optimal cDC migration may be abrogated in the absence of the IFN-I receptor, although this is yet to be demonstrated definitively. The importance of IFN-I responsiveness for optimal Th2 induction during helminth infection was also assessed. Th2 responses were normal in the liver of S. mansoni Ifnar1-/- mice; however, IL-4 and IL-13 levels in the mesenteric LN (MLN) were drastically reduced. It was found that Th2 induction in the MLN was also ablated in mice infected with the gastrointestinal helminth Heligomosoides polygyrus. This suggests that there is a selective dependency on IFN-I for the activation of Th2 responses in lymphoid organs. The small intestine and the MLN provided an ideal site for further investigation of the development of the schistosome-specific immune response in peipheral tissues versus the draining lymph nodes, as this site is directly affected by parasite egg traffic during S. mansoni infection. The intestine is a unique immune environment – with a propensity towards regulation and tolerance, and a large population of innate effectors. Intestinal DCs depend on Flt3-L for their generation; however, the importance of DCs resident in the intestinal lamina propria (LP) for the initiation of Th2 inflammation in response to helminths is not yet known. Characterisation of LP DCs indicated that the activation of these cells is modulated during acute S. mansoni infection, whilst a novel model of schistosome egg deposition in the intestinal tissue illustrated that CD11c+ cells are essential for induction of the egg-specific Th2 response in both the LP and MLN following egg challenge. These data demonstrate the importance of IFN-I signalling for the development of helminth-specific immune responses, highlighting for the first time a role for this pluripotent innate effector in Th2 induction. Development of an egg challenge model in the intestine also provides an ideal setting with which to further explore the importance of IFN-I for Th2 polarisation in peripheral tissues and lymphoid organs.

Dendritic cells (DCs) constitute a large family of immune cells with a dendritic morphology and a critical role in all aspects of an immune response and immune regulation, from immunogenicity to tolerance. One of the important characteristic of DCs is maturation, during which DCs undergo significant changes in their phenotypic and functional properties and change from phagocytic cells to highly efficient antigen presenting cells (APCs). Dendritic cells have recently been at the centre of attention as a promising tool in treatment or control of cancer and infectious diseases. Accordingly, DCs have been generated, matured, and loaded with tumor-associated or microbial antigens ex vivo, to be subsequently used as therapeutic tools or vaccine carriers.<p> Hepatitis C virus (HCV) is a hepatotropic virus, which infects the liver in humans and results in a chronic infection in most cases. The persistent infection of the liver eventually results in cirrhosis and/or hepatocellular carcinoma in 15-20 years. Chronic hepatitis C (CHC) has recently become a serious health concern and the leading cause of liver transplantation. The mechanism of persistence of the virus is not clear yet, but as a Th1-type immune response is strongly correlated with elimination of HCV in vivo, it is evident that insufficient cellular immunity is a contributing factor. Non-cytopathic viruses such as HCV may infect immune cells to modify and evade a protective immune response. Dendritic cells, which are the most potent APCs, and uniquely capable of initiating a primary immune response, have been considered as a target for HCV. Inhibition of DC maturation by HCV has been suggested as a potential contributing factor in immune evasion; however, this issue remains controversial as many contradictory results have been reported.<p> To investigate this contention, we initially planned to evaluate the effects of HCV on DCs of CHC patients; however, due to limited access to patients blood, we instead elected to examine the effects of HCV genes products on in vitro generated DCs from healthy volunteers. Specific attention was paid to the generation, maturation, and transfection of DCs in vitro, as variability in procedures might have been responsible for the controversial reports. Viral vectors have generally been used to transfect DCs; however, a vector and HCV genes might have synergistic effects on DC maturation. Thus, our first objective was to develop an efficient non-viral transfection method while retaining high viability of the DCs, as previous efforts in this regard resulted either in low efficiency or in low viability of DCs after transfection. In order to improve the viability of DCs after transfection, we established a new method for fast generation of monocyte-derived DCs (Mo-DCs) in two to three days. By performing a comprehensive study on transfection reagents, electroporation, and nucleofection with DNA or in vitro transcribed (IVT) RNA, we successfully established a new, highly efficient non-viral method for transfection of DCs with long-term viability. This method is based on the use of the X1 program of a nucleofection device with IVT RNA and results in high transfection efficiency of 93%, with 75% viability of DCs 72 h after transfection.<p> Subsequently, we performed a comprehensive study on the effects of different maturation methods on the phenotype, function and gene expression profile of DCs. Three commonly used treatments, TNF-á, LPS and a maturation cocktail (MC) consisting of IL-1â, IL-6, TNF-á, and prostaglandin E2 (PGE2) were compared. Our results showed that there is a significant difference in the level of maturity between these treatments, and MC generated more functionally competent mDCs than TNF-á or LPS. In addition, MC induced Th1-promoting changes in the transcriptional profile of mDCs. This observation was important, as the presence of PGE2 in MC was previously challenged based on the potential induction of Th2-biased immune responses. However, our results suggest retaining PGE2 in the cocktail because of the fact that MC generated highly competent and functional mDCs with a Th1-promoting transcriptional profile. Finally, Mo-DCs were transfected with IVT HCV RNAs, individually or in combination. While HCV genes had no inhibitory effect on DC maturation, transfection of DCs with IVT core RNA appeared to result in changes compatible with maturation. To investigate this in more detail, the transcriptional profiles of DCs transfected with IVT core, NS3 or green fluorescent protein (GFP) RNA were examined using a DC-specific membrane array. Of the 288 genes on the array, 46 genes were distinctively up- or down-regulated by transfection with IVT core RNA in comparison to NS3 or GFP RNA treatments, 42 of which are involved in DC maturation. The effects of core on maturation of DCs were further confirmed by a significant increase in surface expression of CD83 and HLA-DR, a reduction of phagocytosis, as well as an increase in proliferation and IFN-ã secretion by T cells in a mixed lymphocyte reaction assay. These results show that HCV core does not have an inhibitory effect on human DC maturation, but could be a target for the immune system.<p> The use of a non-viral method of transfection combined with confirmed transcriptional profiles of DCs in this study may make these results conclusive for in vitro generated DCs from healthy volunteers. However, further investigations are required to confirm the effects on DCs from CHC patients.