Innate sensing of nucleic acids lies at the heart of antiviral host defense. However, aberrant activation of innate sensors by host nucleic acids can also lead to the development of autoimmune diseases. Such host nucleic acids can also be released from stressed, damaged or dying cells into the tissue microenvironment. It however remains unclear how the extracellular nucleic acids impacts the quality of the host immune responses against viral infections. Using a mouse model of influenza A virus (IAV) infection, we uncovered an important immune-regulatory pathway that tempers the intensity of the host-response to infection. We found that host-derived DNA from necrotic cells accumulates in the lung microenvironment during IAV infection, and is sensed by the DNA receptor Absent in Melanoma 2 (AIM2). AIM2-deficiency resulted in severe immune pathology highlighted by enhanced recruitments of immune cells, and excessive systemic inflammation after IAV challenge, which led to increased morbidity and lethality in IAV-infected mice. Interestingly, these effects of AIM2 were largely independent of its ability to mediate IL-1β maturation through inflammasome complexes. Finally, ablation of accumulated DNA in the lung by transgenic expression of DNaseI in vivo had similar effects. Collectively, our results identify a novel mechanism of cross talk between PRR pathways, where sensing of hostderived nucleic acids limits immune mediated damage to virus infected tissues.

Despite Neospora caninum being recognised as a major cause of bovine abortion, its pathogenesis is only partially understood. Evidence of immune mediated placental pathology has been reported as being responsible for compromising pregnancy probably due to an exacerbated Th1 immune response at the maternal-foetal interface. Different clinical outcomes are known to follow experimental infections at different stages of gestation, with foetal death being the most common finding during early gestation infections, and the birth of live congenitally infected calves following infection in mid or late gestation. The aim of the current study was to characterise the placental cellular immune responses and cytokine expression following experimental Neospora infection during pregnancy. Placentomes were collected from cattle experimentally inoculated with the tachyzoites of the Nc-1 strain during early, mid and late gestation. Inflammation in early gestation was generally moderate to severe. Differently in mid gestation, inflammation was mild to moderate and minimal to mild in late gestation. Generally cellular infiltrates were mainly characterised by the presence of CD3+, CD4+ and γδ T-cells; whereas CD8+ and NK cells were less numerous. Macrophages were detected in larger numbers during later time-points after infection. A moderate to severe infiltration of IL-12, IFN-γ and TNF-α expressing cells was observed in the placentas collected in early gestation. This infiltration was more pronounced in the samples of placentome collected from dams carrying a dead foetus or in those that had aborted, compared with mothers carrying live foetuses at the time of sampling. The distribution of the cellular subsets observed in the three studies was similar. However, cellular infiltrates were more severe following infection during the first trimester in comparison to the second and third trimester. Similarly, the infiltration of Th1 cytokine expressing-cells was more severe in early gestation compared with the milder and more minimal infiltrations observed following N. caninum infection in mid and late gestation, respectively. These results may explain the milder clinical outcome observed when animals are infected in later stages of pregnancy.

The studies submitted herein have contributed to our understanding of ruminant immunology, host-parasite interactions during ruminant infection with nematode parasites, and potential vaccine strategies to combat parasitic gastroenteritis (PGE). PGE of sheep and cattle, caused by T. circumcincta and O. ostertagia respectively, is a major problem for the global farming industry both in terms of productivity and animal welfare. To date control of these parasites has relied on the use of anthelmintic drugs however the emergence of widespread anthelmintic resistance is driving the search for alternative methods of control. As ruminants do acquire immunity in the field, vaccination is one such alternative under investigation. The first three papers contributing to this thesis used modern immunological tools alongside a locally developed surgical technique to revisit a model of nematode infection in sheep, investigating the composition and kinetics of the ovine local immune response to infection with Teladorsagia circumcincta via cannulation of the efferent gastric lymph duct. A protective local secondary immune response was observed in sheep which had previously experienced infection with T. circumcincta, but was absent from naive sheep. This immune response consisted initially of a rise in TE and BE cell activity peaking at 3 and 5 days post challenge respectively, followed by a secondary parasiteEspecific IgA response from 5 days post challenge which correlated with stunting of parasite growth. Significant parasite loss occurred by 2 days post challenge, prior to detection of the secondary immune response, suggesting critical early events in the host-parasite interaction and the potential importance of larval antigens in these interactions. No difference was observed in either the manifestations of immunity, or the magnitude and quality of the immune response, between adult sheep and lambs. The fourth and fifth papers describe vaccine trials carried out in bovine and ovine hosts using detergent soluble proteins derived from 4th larval stage Ostertagia ostertagi and Teladorsagia circumcincta respectively as antigens. Substantial reduction in total faecal egg output of up to 85% was observed in the calf trials, but not in the sheep trials which attained a maximum reduction of 29% in total faecal egg output. The sixth paper is a transcriptomic study carried out using the Roche 454 sequencing platform to investigate the immediate responses of Teladorsagia circumcincta upon encountering ovine host tissue of either immune or naive status. Following larval exsheathing and 4 hours of exposure to either immune or naive abomasal environments the transcript level of several genes was observed to differ. Genes which were most upregulated in response to encountering the immune environment included a peptidyl-glycine alpha-amidating mono-oxygenase homologue and a small heat shock protein. The studies described herein represent a body of work carried out using up-to-date tools and technologies. The first three papers confirmed the existence of critical early events in the host-parasite interaction, pointing to the potential use of larval antigens as vaccine candidates described in the trials in papers 4 and 5, and leading to the in-depth transcriptomic analysis described in paper 6. Papers 4 and 5 demonstrated that while Teladorsagia circumcincta and Ostertagia ostertagi have similar life cycles and host-site predilection, and both the ovine and bovine host can develop immunity to incoming parasitic larvae in the field, important differences may exist in either the proteome of the fourth stage larvae and/or the nature of the host response. Paper 6 revealed that changes in T. circumcincta transcript levels in response to ovine-host immune status can be detected early in the host-parasite interaction.

Single Nucleotide Polymorphisms (SNPs) in the intracellular pattern recognition receptor gene NLRP3 are associated with susceptibility to Crohn’s disease, a form of inflammatory bowel disease (IBD). Following cell damage or infection, NLRP3 triggers the formation of inflammasomes, a multimolecular protein complex containing NLRP3, ASC and caspase-1, which mediate secretion of IL-1β and IL-18. NLRP3 inflammasome activation in macrophages has been implicated in protection against several pathogens, but whether NLRP3 activation in tissue cells contributes to protective immunity against bacterial pathogens is unknown. We show that upon infection with the attaching/effacing (A/E) intestinal pathogen Citrobacter rodentium, Nlrp3-/- and Asc-/- mice displayed higher bacterial colonization, more weight loss and exacerbated intestinal inflammation. We further show that Nlrp3 inflammasome activation in intestinal epithelial cells (IECs) acts rapidly after infection to limit bacterial replication and penetration, and inhibits the development of inflammatory pathology in the gut. We also show that epithelial Nlrp3-mediated protection is independent of the classical inflammasome cytokines IL-1β and IL-18. Thus an Nlrp3-Asc circuit in IECs regulates early defense against a mucosal pathogen and limits inflammation in the intestine. Nlrp3 inflammasome activation has also been implicated in protection in acute models of experimental colitis, but its role in chronic models of colitis is unknown. We found that Asc signaling is necessary for the development of innate chronic intestinal inflammation driven by Helicobacter hepaticus. Thus while deficient inflammasome signaling in tissue cells increases susceptibility towards enteric pathogens, excessive inflammasome activation can drive chronic intestinal inflammation.

No description available.

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.

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: The immune system matures progressively from infancy to adulthood, thus children may differ from adults in their immune function. The immature immune system demonstrates a higher naive to memory T cell ratio, defective macrophage function and antigen presentation which, cumulatively, results in diminished production of cytokines such as IFN-y. This cytokine has been shown to play a pivotal role in protection against Mycobacterium tuberculosis (M. tuberculosis) disease. Other cytokines, such as IL-12 and TNF-a, are also involved in the defence against M. tuberculosis. Epidemiological evidence suggests an agerelated incidence of tuberculosis (TB) irrespective of prevalence in a given region. Reports in the literature also demonstrate depressed immune responses in TB patients, at diagnosis, (before TB therapy) with subsequent improvement after TB therapy. The aims of this study were to optimise a whole blood assay in order to characterise immune responses, as measured by proliferation and cytokine production, in TB patients (after TB therapy) and healthy controls of different ages. Immune responses of TB patients would also be compared, before, and after TB therapy. A total of 68 subjects were included in this study. These comprised 27 TB patients and 41 healthy Mantoux positive controls. All subjects were stratified into two age groups: <12 years and >12 years. Diluted whole blood was cultured and stimulated with the mitogen, phytohaemagglutinin (PHA) and the specific mycobacterial antigen, purified protein derivative (PPD) to measure proliferation and IFN-y, IL-2, TNF-a and IL-10 production in the supernatant of cultures. Age was a significant variable for the following PHA-stimulated cytokines: IFN-y, TNF-a and IL-10. Proliferation and IL-2 production after PHA stimulation did not demonstrate any relationship with age. None of the PPD-stimulated proliferative or cytokine responses demonstrated any correlation with age. Concentrations of PHA- and PPD-induced IFN-y for all subjects (patients and controls) were increased “after therapy”, compared to “before therapy”. This phenomenon could possibly be due to maturation in the capacity of the immune system to produce this cytokine. Patients >12yrs demonstrated improvement in all proliferative and cytokine responses (except for PPD-induced IL-2 and TNF-a) “after therapy”, compared to “before therapy”. This is probably a valid finding and is thus in accordance with the literature. The whole blood assay is a simple, non-laborious assay that, according to the literature, produces results that seem to correlate well with that of conventionally used PBMCs. Age appears to be an important variable in the quantitative assessment of cellular immune responses (when the mitogen, PHA is used as a stimulant) and immune responses of older TB patients appear to improve after TB therapy, compared to before TB therapy. / AFRIKAANSE OPSOMMING: Die immuunsisteem matureer stelselmatig van kind na volwassene. Dus sal kinders se immuniteit verskil van volwassenes s’n. Die immature immuunsisteem het ‘n hoer nai'witeit vir geheue T-sel verhouding, defektiewe makrofaag funksie en antigeen presentering wat gesamentlik lei tot verminderde produksie van sitokiene soos byvoorbeeld IFN-y. Daar is bewys dat hierdie sitokien ‘n deurslaggewende rol speel in die beskerming teen Mycobacterium tuberculosis (M. tuberculosis). Ander sitokiene, soos IL-12 en TNF-a speel ook ‘n rol in die beskerming teen M. tuberculosis. Epidemiologiese data dui aan dat daar ‘n ouderdomverwante insidensie van tuberkulose (TB) is sonder dat dit beinvloed word deur die voorkoms van TB in ‘n sekere area. Verslae in die literatuur wys ook op onderdrukte immuniteitrespons in TB-pasiente by diagnose (voor TB-behandeling) met uiteindelike verbetering na TB-behandeling. Die doel van hierdie studie was om ’n volbloed metode te optimaliseer in ’n poging om die immuunrespons te karakteriseer soos gemeet met behulp van proliferasie en sitokien produksie by TB-pasiente (na TB-behandeling) en gesonde kontrole persone van verskillende ouderdomme. Die immuunrespons van TB-pasiente word ook vergelyk voor en na TBbehandeling. ‘n Totaal van 68 gevalle is vir die studie gebruik. Dit sluit in 27 TB-pasiente en 41 gesonde Mantoux positiewe kontroles. A1 die gevalle is in twee ouderdomsgroepe verdeel: <12 jaar en >12 jaar. Kulture is gemaak van verdunde volbloed en gestimuleer met phytohaemaglutinin (PHA) en gesuiwerde proteien derivaat (purified protein derivative-PPD) om proliferasie en IFN-y, IL- 2, TNF-a en IL-10- produksie in die supernatant van die kulture te meet. Ouderdom was ‘n beduidende veranderlike vir die volgende PHA-gestimuleerde sitokiene: IFN-y, TNF- a en IL-10. Daar was geen korrelasie tussen proliferasie en IL-2-produksie na PHA-stimulasie aan die een kant en ouderdom aan die ander kant nie. Geen van die PPDgestimuleerde proliferasie response of sitokien response het enige korrelasie met ouderdom getoon nie. Konsentrasies van PHA- en PPD-geinduseerde IFN-y vir alle gevalle (pasiente en kontrole) was verhoog “na behandeling”, vergeleke met “voor behandeling”. Hierdie fenomeen kan moontlik toegeskryf word aan maturasie in die vermoe van die immuunsisteem om sitokiene te vervaardig. Pasiente >12 jaar het bewyse getoon van verbetering in alle proliferasie en sitokien response (behalwe vir PPD-gei'nduseerde IL-2 en TNF-a) “na behandeling”, vergeleke met “voor behandeling”. Dit is waarskynlik ‘n geldige bevinding en is dus in ooreenstemming met verslae in die literatuur. Die volbloed metode is ‘n eenvoudige metode wat nie baie arbeidsintensief is nie, wat volgens die literatuur, resultate lewer wat goed korreleer met die konvensionele gebruik van perifere bloed mononukliere selle (PBMC’s). Dit wil voorkom asof ouderdom ‘n belangrike veranderlike is in die kwantitatiewe beoordeling van sellulere immuunrespons (wanneer PHA gebruik word as ‘n stimulant), en of die immuunrespons van ouer TB-pasiente verbeter na TB-behandeling in vergeleke met die respons voor TB-behandeling.

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.

Lipid rafts are operationally defined as cholesterol-rich membrane microdomains resistant to solubilization in nonionic detergents at low temperatures. Lipid rafts, which are quite different in lipid composition from the surrounding membranes, are of great importance to signal transduction, protein sorting and membrane transport. They have been implicated in a range of biosynthetic and endocytic processes and systems-signaling, molecular trafficking, diseases as well as being involved in the immune, vascular, digestive and reproductive systems. Dietary nutrients like fatty acids and vitamins of different types also play a critical and decisive role in the regulation of lipid rafts. / text