Toll-like receptor 9 (TLR9) activates the innate immune system in response to microbial DNA or mimicking oligodeoxynucleotides. While the discrimination of host and microbial DNA is presumed to reflect TLR9-mediated recognition of CpG motifs, little information is available to verify this hypothesis. Cell stimulation experiments demonstrate preferential activation of TLR9 by CpG-containing nucleic acids, however direct binding investigations have reached contradictory conclusions with respect to the ability of TLR9 to bind nucleic acids in a sequence-specific fashion. Here we report expression of the soluble, ectodomain of human TLR9 with characterization of its ligand-binding properties. TLR9 has a high degree of ligand specificity in being able to discriminate not only CpG dinucleotides, but also higher order six nucleotide motifs that mediate species-specific activation. However, TLR9 ligand binding is also functionally influenced by nucleic acids in a sequence-independent manner both in vitro and in cell proliferation experiments. A model is proposed in which TLR9 activation is mediated specifically by CpG-containing ligands while sensitivity is mediated specifically by the absolute concentration of nucleic acids in a sequence-independent manner.<p>The bovine hsp70A promoter was used to direct the heat-regulated synthesis of the ectodomain of human TLR9 in transfected cultured bovine cells. The protein was efficiently secreted from transfected cells in a temperature-dependent manner and the recombinant receptor produced was found to be relatively pure. A stably transfected cell line with regulated expression of the protein was obtained and repeated thermal cycling of the cultures enabled high-yield production of the receptor in an active ligand-binding form. Using this recombinant receptor to study the ligand binding properties of TLR9, a model of positive cooperativity is proposed in which the sensitivity of TLR9 ligand binding is modulated by the absolute concentration of nucleic acids in a sequence-independent fashion, while activation of TLR9 is highly dependent on DNA sequence. That is to say that TLR9 is primed for activation by interaction with non-activating sequences but activation itself occurs in a sequence-specific fashion.

Toll-like receptor 9 (TLR9) activates the innate immune system in response to microbial DNA or mimicking oligodeoxynucleotides. While the discrimination of host and microbial DNA is presumed to reflect TLR9-mediated recognition of CpG motifs, little information is available to verify this hypothesis. Cell stimulation experiments demonstrate preferential activation of TLR9 by CpG-containing nucleic acids, however direct binding investigations have reached contradictory conclusions with respect to the ability of TLR9 to bind nucleic acids in a sequence-specific fashion. Here we report expression of the soluble, ectodomain of human TLR9 with characterization of its ligand-binding properties. TLR9 has a high degree of ligand specificity in being able to discriminate not only CpG dinucleotides, but also higher order six nucleotide motifs that mediate species-specific activation. However, TLR9 ligand binding is also functionally influenced by nucleic acids in a sequence-independent manner both in vitro and in cell proliferation experiments. A model is proposed in which TLR9 activation is mediated specifically by CpG-containing ligands while sensitivity is mediated specifically by the absolute concentration of nucleic acids in a sequence-independent manner.<p>The bovine hsp70A promoter was used to direct the heat-regulated synthesis of the ectodomain of human TLR9 in transfected cultured bovine cells. The protein was efficiently secreted from transfected cells in a temperature-dependent manner and the recombinant receptor produced was found to be relatively pure. A stably transfected cell line with regulated expression of the protein was obtained and repeated thermal cycling of the cultures enabled high-yield production of the receptor in an active ligand-binding form. Using this recombinant receptor to study the ligand binding properties of TLR9, a model of positive cooperativity is proposed in which the sensitivity of TLR9 ligand binding is modulated by the absolute concentration of nucleic acids in a sequence-independent fashion, while activation of TLR9 is highly dependent on DNA sequence. That is to say that TLR9 is primed for activation by interaction with non-activating sequences but activation itself occurs in a sequence-specific fashion.

Drosophila TGFbeta-Activating-Kinase 1 (dTAK1) is an essential component of both the Immune Deficiency (IMD) innate immune and TNF/Eiger apoptotic cascades. The IMD and JNK pathways bifurcate at the level of dTAK1. Hence, elucidating the regulatory mechanism of dTAK1 is pertinent to understanding the regulation of both innate immunity and apoptosis. In this study, Trabid was identified as a novel negative regulator of the Drosophila IMD pathway. Trabid interacted with dTAK1 and decreased K63-linked ubiquitination, thereby reducing immune signalling. Three tandem Npl4 Zinc Fingers (NZF) and C518 were required for Trabid activity. Lysines 142 &amp; 156 were identified as the K63 Ub acceptor sites of dTAK1, required for K63-linked ubiquitination and signalling. Also, results show Lys 156 functioned as the K48 Ub acceptor site. Further, the ZF domain of TAK1-associated Binding Protein 2 (dTAB2) was important in modulating dTAK1 K63-linked ubiquitination and thereby the immune signal. These results indicate an elaborate and multi-tiered mechanism for regulating dTAK1 activity and modulating the immune signal. Further, Ariadne-2 (Ari-2) was identified as a novel component of the Drosophila TNF/Eiger pathway which functioned at the level of dTAK1. Results indicate that Ari-2 is essential for normal development and longevity. It enhances the apoptotic signal when concomitantly over-expressed with Eiger. Further, Ari-2 interacts with dTAK1, dTAB2 and dTRAF2 which are all implicated in TNF/Eiger signalling. Thus, evidence supports the hypothesis that Ari-2 functions as an adaptor, involved in assembling a distinct signalling complex which transduces the apoptotic signal without activating immunity.

The neonatal period represents a critical time period in the development of the immune system. Adaptive human immune responses are generally viewed as immature at birth. However little is known about innate immune capacity at birth. The TLR system plays an integral role as pattern recognition receptors in the innate immune response. It is also critical in initiating and regulating the adaptive immune response. Preterm birth is associated with increased risk of developing infections in early life and has been associated with increased risk of development of other chronic disorders in later life; however the underlying mechanisms are not at all well understood. Recently, late preterm neonates (34-36 weeks gestation vs full term, 37+ weeks) have been identified as having significantly greater risks of morbidity and mortality in the perinatal period than their full term counterparts. Hence, we focus on examination of TLR responses in late preterm and full term neonates to better understand immune potential and function in these populations. We examined cord blood cytokine and chemokine responses following stimulation with a broad range of TLR agonists. Our results show for the first time that late preterm neonates have reduced capacity to produce both pro- and anti-inflammatory cytokines following stimulation with a panel of TLR agonists. This reduced responsiveness was not due to a reduction in the number of responding cells, but instead appears to be mediated by a reduction in the intrinsic levels of expression of TLRs and associated adaptor proteins. Because little is known about how the innate immune system develops throughout life, we next compared TLR responses in full term neonates to 7 children, adolescents and adults. We found that neonates had selective impairments in TLR responses, most notably in anti-inflammatory cytokine production and anti-viral immune responses compared to the other age groups. Epigenetic modifications, such as the addition or removal of acetyl groups to histone proteins by histone acetyl transferase (HAT) and histone deactylase (HDAC) respectively, are able to modify the expression of genes. Hence, environmental stimuli have been shown to influence gene expression in part by modifying the level or activity of these epigenetic regulators. Currently there are no studies which have examined how epigenetic modifications may influence neonatal innate immune responses. Hence, we sought to determine how modulation of endogenous HDAC activity would affect neonatal innate immune responses. We found that inhibition of HDAC had both inhibitory and enhancing effects on cytokine expression depending on the TLR pathway activated, indicating that the endogenous HDAC expression does not have a global inhibitory impact on all TLR-dependent responses. In summary, this body of work demonstrates that neonatal innate immune responses vary depending on gestational age, indicating that the final few weeks of gestation are crucial for maturation of responses to both bacteria and viruses. Neonates respond differently to TLR stimuli than do older individuals, further highlighting a maturation process of the innate immune system which continues throughout life. Finally, we have shown that environmental exposures may have powerful effects on immune responses in early life.

The neonatal period represents a critical time period in the development of the immune system. Adaptive human immune responses are generally viewed as immature at birth. However little is known about innate immune capacity at birth. The TLR system plays an integral role as pattern recognition receptors in the innate immune response. It is also critical in initiating and regulating the adaptive immune response. Preterm birth is associated with increased risk of developing infections in early life and has been associated with increased risk of development of other chronic disorders in later life; however the underlying mechanisms are not at all well understood. Recently, late preterm neonates (34-36 weeks gestation vs full term, 37+ weeks) have been identified as having significantly greater risks of morbidity and mortality in the perinatal period than their full term counterparts. Hence, we focus on examination of TLR responses in late preterm and full term neonates to better understand immune potential and function in these populations. We examined cord blood cytokine and chemokine responses following stimulation with a broad range of TLR agonists. Our results show for the first time that late preterm neonates have reduced capacity to produce both pro- and anti-inflammatory cytokines following stimulation with a panel of TLR agonists. This reduced responsiveness was not due to a reduction in the number of responding cells, but instead appears to be mediated by a reduction in the intrinsic levels of expression of TLRs and associated adaptor proteins. Because little is known about how the innate immune system develops throughout life, we next compared TLR responses in full term neonates to 7 children, adolescents and adults. We found that neonates had selective impairments in TLR responses, most notably in anti-inflammatory cytokine production and anti-viral immune responses compared to the other age groups. Epigenetic modifications, such as the addition or removal of acetyl groups to histone proteins by histone acetyl transferase (HAT) and histone deactylase (HDAC) respectively, are able to modify the expression of genes. Hence, environmental stimuli have been shown to influence gene expression in part by modifying the level or activity of these epigenetic regulators. Currently there are no studies which have examined how epigenetic modifications may influence neonatal innate immune responses. Hence, we sought to determine how modulation of endogenous HDAC activity would affect neonatal innate immune responses. We found that inhibition of HDAC had both inhibitory and enhancing effects on cytokine expression depending on the TLR pathway activated, indicating that the endogenous HDAC expression does not have a global inhibitory impact on all TLR-dependent responses. In summary, this body of work demonstrates that neonatal innate immune responses vary depending on gestational age, indicating that the final few weeks of gestation are crucial for maturation of responses to both bacteria and viruses. Neonates respond differently to TLR stimuli than do older individuals, further highlighting a maturation process of the innate immune system which continues throughout life. Finally, we have shown that environmental exposures may have powerful effects on immune responses in early life.

Mutations in the Leucine-rich repeat kinase 2 (Lrrk2) gene are associated with familial and sporadic cases of Parkinson’s disease but are also found in inflammatory-related disorders such as Crohn’s disease, systemic lupus erythematosus, tuberculosis and leprosy. There is also evidence that LRRK2 is highly expressed in immune cells, particularly in macrophages, and has been functionally linked to pathways pertinent to immune cell function such as modulating the course of infections, cytokine release, autophagy and phagocytosis. Indeed, G2019S mutation in Lrrk2 is the most common mutation in Parkinson’s disease. Accordingly, we hypothesized that G2019S mutation in Lrrk2 might enhance the activation of the innate immune system. We tested our hypothesis by performing challenge experiments in a mouse model of Listeria monocytogenes, and by measuring the activation of bone marrow derived macrophages (BMDMs) following in vitro infection with the bacterium. We found that Lrrk2G2019S mutant mice controlled L. monocytogenes better than WT mice. The mechanism behind the better control of L. monocytogenes by the G2019S mutation of Lrrk2 was investigated in BMDMs following in vitro infection with L. monocytogenes. Interestingly, we found that Lrrk2G2019S mutation enhances the production of TNF-α, IL-1β and IL-10 by infected BMDMs. The impact on TNF-α and IL-1β was specifically due to the G2019S mutation of Lrrk2 since there was no impact on the expression of these cytokines in Lrrk2 knockout macrophages. Western blotting experiments revealed that the G2019S mutation of Lrrk2 enhances MAPK signaling (TAK1, p38 and ERK). Modulation of the expression of the pro-inflammatory cytokines, TNF-α and IL-1β by G2019S mutation of Lrrk2 occurred via p38 MAPK activation. The impact on IL-10 expression occurred through increased ERK activation by the G2019S mutation of Lrrk2. We did not observe any impact of G2019S mutation of Lrrk2 on the activation of NF-κB and JNK MAPK pathways. Increased expression of IL-1β by G2019S mutation of Lrrk2 revealed increased inflammasome signaling. Inflammasome signaling in response to L. monocytogenes was mainly mediated by the AIM2- and partly by NLRP3- inflammasome and was dependent on activation of caspase-1. We found that Lrrk2G2019S mutation enhanced the expression of NLRP3 and caspase-1. Finally, we found that the expression of reactive oxygen species (ROS) following infection with L. monocytogenes was augmented by G2019S mutation of Lrrk2, and this can be an important mechanism that promotes the enhanced clearance of the bacterium in vivo. Overall, these results present new insights into the signaling mechanisms through which the G2019S mutation of Lrrk2 augments innate immune response which leads to better control of infection.

The phenomenon known as immunothrombosis has garnered increased attention over the last few years. Much work has been done to characterize the cross talk between hemostasis and the innate immune system. This thesis outlines the role of the vascular endothelial cells during immunothrombotic events as regulators of coagulation, platelet-, and leukocyte recruitment. A newly developed method for investigating the interaction between endothelial cells and the blood compartment illustrated the procoagulant and proinflammatory effects elicited by tumor necrosis factor α activated endothelial cells upon exposure to whole blood. The method was utilized in evaluating treatment of endothelial dysfunction and disruption with a heparin conjugate. Damaged or hypoxic endothelial cells, in addition to basement membrane collagen, that were pretreated with the heparin conjugate prior to contact with blood were found to have reduced activation of coagulation, platelet-, and leukocyte recruitment; in contrast to unfractionated heparin, which had no effect on the aforementioned parameters. The treatment was then investigated in the setting of ischemia reperfusion injury during kidney transplantation and the heparin conjugate was found to bind cultured endothelial cells with high avidity under cold storage conditions. Furthermore, it was found to bind to the renal vasculature during static cold storage and was subsequently found to be beneficial with regard to early graft function in an experimental mouse model of syngeneic kidney transplantation. Recipients of kidneys treated with the heparin conjugate had reduced serum creatinine compared to controls 24 hours after transplantation. Lastly, the anticoagulant properties of the heparin conjugate were investigated in comparison to unfractionated heparin. While the conjugate exerted reduced capacity with regard to thrombin inhibition, it rapidly inhibited the binding of platelets to exposed collagen. The conjugate was furthermore found to preferentially locate to sites of endothelial cell activation at early stage during endotoxic shock in mice. In conclusion, this thesis demonstrates that disrupted functioning of the vascular endothelial cells actively contributes to immunothrombosis, and that it is possible to model endothelial cell function using whole blood assays. Furthermore, this thesis presents a treatment that enhances the hemocompatibility of damaged endothelial cells and subsequently improves the early renal function after kidney transplantation.

Several epidemiological and clinical studies have indicated a possible role of Enterovirus (EV) infection in type 1 diabetes (T1D) development. However, the exact casual mechanism of these viruses in T1D development is not known. The aim of this thesis is to study various EVs that have been shown to differ in their immune phenotype, lytic ability, association with induction of islet autoantibodies, ability to replicate, cause islet disintegration and induce innate antiviral pathways in infected pancreatic cells in vitro. Furthermore, EV presence and pathogenic process in pancreatic tissue and isolated islets of T1D patients was also studied. Studies in this thesis for first time show the detection of EV RNA and protein in recent onset live T1D patients supporting the EV hypothesis in T1D development. Further all EV serotypes studied were able to replicate in islets, causing variable amount of islet disintegration ranging from extensive islet disintegration to not affecting islet morphology at all. However, one of the EV serotype replicated in only two out of seven donors infected, highlighting the importance of individual variation between donors. Further, this serotype impaired the insulin response to glucose stimulation without causing any visible islet disintegration, suggesting that this serotype might impaired the insulin response by inducing a functional block. Infection of human islets with the EV serotypes that are differentially associated with the development of islet autoantibodies showed the islet cell disintegration that is comparable with their degree of islet autoantibody seroconversion. Suggesting that the extent of the epidemic-associated islet autoantibody induction may depend on the ability of the viral serotypes to damage islet cells. Furthermore, one of the EV strains showed unique ability to infect and replicate both in endo and exocrine cells of the pancreas. EV replication in both endo and exocrine cells affected the genes involved in innate and antiviral pathways and induction of certain genes with important antiviral activity significantly varied between different donors. Suggesting that the same EV infection could result in different outcome in different individuals. Finally, we compared the results obtained by lytic and non lytic EV strains in vitro with the findings reported in fulminant and slowly progressing autoimmune T1D and found some similarities. In conclusion the results presented in this thesis further support the role of EV in T1D development and provide more insights regarding viral and host variation.  This will improve our understanding of the possible causative mechanism by EV in T1D development.

Η βρουκέλλα, ένα προαιρετικώς ενδοκυττάριο βακτήριο που προκαλεί μελιταίο πυρετό, ενδοκαρδίτιδα, αρθρίτιδα και οστεομυελίτιδα στους ανθρώπους, εγκαθιστά χρόνιες λοιμώξεις μολύνοντας, επιβιώνοντας και πολλαπλασιαζόμενη σε διαφόρους τύπους κυττάρων, συμπεριλαμβανομένων των μονοκυττάρων και των δενδριτικών κυττάρων. Οι B. abortus, B. melitensis και B. suis είναι τα κύρια είδη που προκαλούν βρουκέλλωση στους ανθρώπους και η B. melitensis προκαλεί την πλειονότητα των περιστατικών και την πιο βαριά συμπτωματολογία. Η ικανότητα της βρουκέλλας να παραμένει στα μολυσμένα κύτταρα εξαρτάται από την ιδιότητά της να αποφεύγει ή να αλληλεπιδρά με στοιχεία των απαντήσεων της φυσικής και επίκτητης ανοσίας. Η αρχική άμυνα του ξενιστή έναντι βακτηριακών λοιμώξεων διεγείρεται από PAMP, που αναγνωρίζονται από τον ξενιστή. Πληθώρα αποδείξεων εμπλέκουν διαφορετικά μέλη της οικογένειας των TLR στην αναγνώριση της βρουκέλλας και/ή στην εκκαθάριση της λοιμώξεως. Ένας απαραίτητος κλάδος των σηματοδοτικών οδών που ξεκινά από τους TLR είναι η οικογένεια των MAP κινασών. Οι MAP κινάσες διαμεσολαβούν κυτταρικές απαντήσεις σε ποικιλία εξωτερικών διεγέρσεων, όπως το φυσικό στρες, οι φλεγμονώδεις κυτταροκίνες, οι αυξητικοί παράγοντες και συστατικά των βακτηρίων. Αντικείμενο της μελέτης ήταν η διερεύνηση της ενορχήστρωσης των απαντήσεων της φυσικής ανοσίας έναντι ζωντανών παθογόνων κλινικών στελεχών B. melitensis σε ανθρώπινα μονοκύτταρα από τις MAP κινάσες. Αρχικά απεδείχθη ότι η βρουκέλλα προκάλεσε ισχυρή προφλεγμονώδη απάντηση με αποτέλεσμα την απελευθέρωση υψηλών επιπέδων IL-1β, IL-6 και TNF-α και ταυτοχρόνως πυροδότησε έντονη, μικρής διαρκείας αντιφλεγμονώδη απάντηση. Δηλαδή, η ζωντανή βρουκέλλα δεν αποφεύγει την αρχική αναγνώριση και κινητοποιεί ισχυρή προφλεγμονώδη απάντηση από τη φυσική ανοσία. Επιπλέον, η παραγωγή TNF-α, IL-6 και IL-10 που προκλήθηκε από τη βρουκέλλα ανεστάλη παρουσία αντι-TLR2, ενώ παρέμεινε ανεπηρέαστη παρουσία αντι-TLR4 αντισώματος. Η IL-1β δεν επηρεάστηκε από την εξουδετέρωση είτε του TLR2 ή του TLR4. Η διακοπή της σηματοδότησης διαμέσου του TLR2, αλλά όχι του TLR4, μείωσε σημαντικά την ενεργοποίηση αμφοτέρων των p38 και ERK ως απάντηση στη μόλυνση με βρουκέλλα. Επιπρόσθετα, η παραγωγή IL-1β από μονοκύτταρα μολυσμένα με βρουκέλλα παρέμεινε ανεπηρέαστη από την προσθήκη αναστολέων των MAP κινασών. Αναστολή της p38 ελάττωσε σημαντικά την παραγωγή IL-6 και εμπόδισε σχεδόν πλήρως την απελευθέρωση TNF-α, ενώ αναστολή της ERK1/2 μείωσε αξιοσημείωτα και τις δύο. Αναστολή της JNK δεν επηρέασε την παραγωγή TNF-α και IL-6. Η παραγωγή IL-10 μειώθηκε σημαντικά από αναστολή των p38 ή JNK, αλλά όχι από αυτήν της MAP2K. Τα συγκεκριμένα αποτελέσματα υποδηλώνουν ότι η ενεργοποίηση των MAP κινασών είναι σημαντικό ενδοκυττάριο στάδιο στην παραγωγή κυτταροκινών στην πορεία της βρουκελλώσεως. Τέλος, η ενεργοποίηση των MAP κινασών επηρεάζει την επιβίωση της βρουκέλλας εντός των ανθρωπίνων μονοκυττάρων. Η αναστολή της p38 ή της JNK κατέστειλε σχεδόν πλήρως την αύξηση της βρουκέλλας, ενώ αναστολή της ERK δεν μείωσε τον πολλαπλασιασμό της. Συμπερασματικά, καταδεικνύεται ότι η λοίμωξη με B. melitensis προκαλεί όψιμη ενεργοποίηση των ERK και p38 η οποία επηρεάζει την απελευθέρωση κυτταροκινών διαμέσου του TLR2. Ακόμη, η δράση των MAP κινασών είναι επωφελής για τον πολλαπλασιασμό της βρουκέλλας εντός των ανθρωπίνων μονοκυττάρων. Οι MAP κινάσες ενδέχεται να επηρεάζουν διαφορετικούς μηχανισμούς που εμπλέκονται στην ενδοκυττάρια επιβίωση της B. melitensis. Επί παραδείγματι, εμπλέκονται στη μεταφορά στο πρώιμο ενδόσωμα, την πρώιμη οξίνιση του φαγοσώματος, τη σηματοδότηση της επαγωγής του εκκριτικού συστήματος τύπου IV VirB ή τη ρύθμιση της σηματοδότησης της αυτοφαγίας. Τρέχουσες έρευνες του εργαστηρίου εξετάζουν τα συγκεκριμένα ενδεχόμενα. / Brucella, a facultative intracellular bacterium that causes undulant fever, endocarditis, arthritis and osteomyelitis in humans, establishes chronic infections by infecting, surviving and replicating in different cell types, including macrophages and dendritic cells. B. abortus, B. melitensis and B. suis are the main species that cause human brucellosis, with B. melitensis causing the majority of cases and the most severe symptoms. The capacity of Brucella to persist in infected cells depends on its stealthy strategy to avoid or interfere with components of the host innate and acquired immune responses. Initial host defenses against bacterial infection are stimulated by PAMPs, which are recognized by the host. Ample evidence implicates the different members of TLR family in recognition of Brucella and/or clearance of infection. One essential branch of signaling cascades initiated by TLR is the ubiquitously expressed family of MAPKs. MAPKs mediate cellular responses to a variety of extracellular stimuli, such as physical stress, inflammatory cytokines, growth factors and bacterial components. Object of the study was the investigation of MAPK orchestration of the innate immune response against pathogenic live clinical strains of B. melitensis in fresh human monocytes. Initially it has been shown that Brucella induced a strong pro-inflammatory response resulting in the release of high levels of IL-1β, IL-6 and TNF-α and simultaneously triggered a strong anti-inflammatory response that lasted for a short time. Namely, live Brucellae do not avoid the initial recognition and trigger a strong inflammatory response. Moreover, TNF-α, IL-6 and IL-10 production induced by Brucella was strongly inhibited in the presence of anti-TLR2, whereas it remained unaffected by the presence of anti-TLR4. IL-1β was not influenced either by TLR2 or TLR4 neutralization. Blocking by anti-TLR2, but not anti-TLR4, markedly reduced both p38 and ERK activation to basal levels in response to Brucella infection. Additionally, IL-1β production by Brucella-infected monocytes remained unaffected by the addition of MAPK inhibitors. Inhibition of p38 significantly diminished IL-6 production and almost completely prevented TNF-α release, whereas inhibition of ERK1/2 significantly reduced both. JNK inhibition had no effect on TNF-α and IL-6 production. IL-10 production was markedly reduced by p38 or JNK inhibition, but not MAP2K. These results suggest that MAPK activation is an important intracellular event leading to cytokine production in the course of Brucella infection. Finally, MAPK activation affects the survival of Brucella in human monocytes. Inhibition of p38 or JNK almost completely repressed Brucella growth, whereas inhibition of ERK did not reduce the multiplication rate of Brucella. In conclusion, it has been demonstrated that infection with B. melitensis induces a late activation of ERK and p38 that affects cytokine release in a TLR2-dependent manner. Moreover, MAPK activity is beneficial for replication of Brucella inside human monocytes. MAPK activation could affect a number of mechanisms involved in the intracellular survival of B. melitensis. MAPK activity is involved in the transport to the early endosome, early acidification of the phagosome, signaling for induction of the VirB Type IV secretion system, or regulation of the autophagic pathway. Current studies in our laboratory investigate these possibilities.

Virus infection of mammalian cells induces several stress mechanisms, including autophagy and type-I interferon (IFN). Autophagy, a cellular homeostatic mechanism in which intracellular materials are sequestered into double-membrane vesicles and targeted to lysosomes for degradation, is also activated in response to virus infection. Most positive single-stranded RNA viruses studied to date utilise autophagy to increase virus replication. IFN is a potent anti-viral mechanism, which can be divided into two parts: (i) induction and secretion of IFN and (ii) IFN signalling and priming of uninfected cells for a rapid response upon infection and induction of an anti-viral state in infected cells. Alphaviruses are medically important RNA viruses. Semliki Forest virus (SFV) provides a well-characterised model for studying alphavirus infection. A number of strains have been identified, which differ in virulence in adult mice. In this thesis three hypotheses were investigated: (i) that SFV infection induces autophagy in cell culture and utilises this response to enhance virus replication, (ii) that the quality, quantity and/or protective efficacy of the IFN response differ between virus strains and between human and murine cells and (iii) that non-structural protein (nsP)-2 and/or nsP3 antagonise the IFN response. SFV4, SFV L10 and SFV A7(74) infection induced autophagy in Huh7 cells as early as one hour post-infection. Pharmacological induction or inhibition of autophagy had no affect on SFV4 replication, except at a very low multiplicity of infection. NsP3, capsid and dsRNA rarely colocalised with the autophagosome marker LC3. Taken together these results indicate that SFV does not use autophagosomes for replication and autophagy is not important in controlling SFV4 infection at a high MOI, at least in Huh7 cells. However, autophagy may be important in controlling SFV4 spread at a low MOI. An IFN bioassay was established. In fibroblasts, SFV4, SFV L10 and SFV A7(74) induced relatively little IFN in comparison to that induced by Sendai virus. In human fibroblasts, similar levels of IFN were induced by all three virus strains. In mouse fibroblasts, SFV4 induced more IFN than SFV L10. Treatment of fibroblasts with IFN prior to infection greatly reduced, but did not abolish, the replication and spread of all three strains. Therefore, SFV is sensitive to IFN. Analysis of IFN signalling demonstrated that all three strains of SFV inhibited STAT1 phosphorylation during infection of fibroblasts. The growth and viability of SFV infected cells varied between human and mouse cells. The complete genetic sequences of SFV L10 and SFV A7(74) were determined using Solexa (Illumina) sequencing and compared to the sequence of SFV4. The sequences of SFV L10 and SFV4 were extremely similar; only seven differences were identified. Multiple amino acid substitutions were identified in SFV A7(74) compared to SFV4, these mostly mapped to nsP3. To investigate the hypothesis that nsP2 and or nsP3 antagonise the IFN response, two virus mutants were studied: SFV4nsP2RDR and SFV4nsP3Δ50. SFV4nsP2RDR encodes a point mutation in the nuclear localisation signal of nsP2, which largely restricts nsP2 to the cell cytoplasm. SFV4nsP3Δ50 contains a deletion of 50 amino acids in the C-terminus hyperphosphorylated region of nsP3. Neither mutant inhibited STAT1 phosphorylation as efficiently as WT SFV4; SFV4nsP2RDR was particularly poor at inhibiting STAT1 phosphorylation. Both mutants induced more IFN in fibroblasts than SFV4. In summary, autophagy had a limited affect on SFV replication. In contrast, strains of SFV were highly sensitive to IFN, but antagonised this response through the nsP2 protein inhibiting STAT1 phosphorylation.