Injuries to connective tissues such as ligaments and tendons are common, and rather than healing, repair typically results in fibrosis, or the formation of mechanically inferior and disorganized scar tissue. This fibrotic repair response is due in part to inflammation, during which the injury site is invaded by a number of cell types, including macrophages, neighboring fibroblasts, and homed stem cells or progenitor cells. Activation of macrophages is believed to be modulated by communications with fibroblasts and stem cells, prompting either a pro-fibrotic or a pro-regenerative response. Beyond changes to the cellular microenvironment, fibrosis also results in changes to the organization and mechanical properties of the matrix microenvironment. For healthy fibrous connective tissues, the matrix is comprised of aligned collagen fibers, while scar tissue is disorganized and exhibits weaker mechanical properties than healthy tissue. To date, the nature of the cell-cell and cell-matrix interactions and their relevance in tissue healing or repair remain understudied. To better understand the cellular and matrix-based cues that direct scar formation versus tissue regeneration, and using anterior cruciate ligament (ACL) injuries as a model, Aim 1 of this thesis tests the hypothesis that in vitro models of cellular communications between fibroblasts, macrophages, and mesenchymal stem cells (MSC) can be used to determine the effects of cellular interactions on macrophage activation and fibrosis. In Aim 2, the contribution of matrix-based cues (alignment and mechanical properties) to the inflammatory and fibrotic response, as well as their modulation of cellular interactions, were examined. Findings from these two aims reveal that 1) communications between native tissue fibroblasts and macrophages drive inflammation and fibrosis, while stem cells modulate the repair process through a combination of trophic signaling and immunomodulatory roles, and 2) matrix alignment and mechanical properties exert combined regulation on cell response during inflammation. From a clinical application perspective, stem cells delivered in conjunction with an engineered matrix that provides the critical cues for driving stem cell immunomodulation and trophic signaling will be essential for promoting tissue regeneration and minimizing fibrosis. In particular, an aligned matrix with an elastic modulus similar to that of developing connective tissue may serve to further minimize inflammation and scar formation, and activate stem cell-guided regeneration of mechanically functional connective tissue.

Thesis (MScMedSc (Biomedical Sciences))--University of Stellenbosch, 2010. / Bibliography / ENGLISH ABSTRACT: Mycothiol (MSH) is unique to mycobacteria as the major low molecular weight cellular thiol responsible for protection of bacteria against oxidative stress. The design of drugs and inhibitors against enzymes of the mycothiol pathway was based on the premise that mycothiol is unique to mycobacteria, and is thus important for its survival. A total of 80 inhibitors designed against enzymes of the mycothiol pathway were screened for inhibition of growth on in vitro growing M. tuberculosis using the BACTEC 460TM assay. The most active compounds were further tested for inhibitory potential of M. tuberculosis within macrophages. Initial screening in the macrophage system was done using the human-like THP1 cell line and then mouse bone marrow-derived macrophages. In this investigation we established that phenothiazine can be exploited as an inhibitor of enzymes of the mycothiol pathway. Although tunicamycin significantly inhibited the growth of M. tuberculosis both in vitro and ex vivo; it was found to be cytotoxic to host macrophages. To this end we provide proof-of-concept that compounds which can inhibit the expression of mycothiol enzymes have potential as anti-tubercular drugs. The response of M. tuberculosis to stress conditions was assessed via LC-MS in which maximal levels of mycothiol were produced during the early time points of exposure to isoniazid. We used mycothiol-deficient (mshA) M. tuberculosis to investigate the role of mycothiol for survival as well as the resultant phenotype when such mutants are exposed to stress conditions. The mshA deletion mutants in M. tuberculosis were resistant to INH at concentrations which inhibited growth in the wild-type strains. We postulated that katG and inhA, the genes involved in INH metabolism, required mycothiol for their activation. Morphological alterations of M. tuberculosis within macrophages were assessed using electron microscopy approaches. In this way we attempted to follow the fate of M. tuberculosis within the phagosomes, and how mycobacteria is processed in phagosomes in terms of replication, survival and degradation. The establishment of a successful infection by M. tuberculosis depends on the initial encounter with host macrophages, which represent the first line of cellular defense against microbial invasion. At the interface between mycobacteria and macrophages, the complex outermost layer of the mycobacterial cell wall probably plays a role in facilitating host cell entry. Under normal conditions (i.e. ingestion of non pathogenic microorganisms), newly formed phagosomes intermingle contents and membrane with the successive compartments of the endocytic pathway (early endosomes, late endosomes, lysosomes) through a complex series of fusion and fission. As they are processed into phagolysosomes, they undergo gradual modifications by specific addition and removal of membrane constituents. In addition, they become acidified due to the vacuolar proton pump ATPase located in the membrane and acquire toxic constituents, including hydrolases that will ultimately destroy bacteria. / AFRIKAANSE OPSOMMING: Mycothiol (MSH) is uniek aan mycobacteria as die belangrikste lae molekulêre gewig sellulêre thiol verantwoordelik vir die beskerming van bakterieë teen oksidatiewe stres. Die ontwerp van dwelms en inhibeerders teen ensieme van die mycothiol pad is gebaseer op die veronderstelling dat mycothiol uniek is aan mycobacteria, en is dus belangrik vir sy oorlewing. 'n Totaal van 80 inhibeerders ontwerp teen ensieme van die mycothiol pad is gekeur vir die inhibisie van groei op in vitro groeiende M. tuberculosis met behulp van die BACTEC 460TM toets. Die mees aktiewe verbindings is verder getoets vir inhiberende potensiaal van M. tuberculosis binne makrofage. Aanvanklike sifting in die makrofage stelsel is gedoen met behulp van die mens-soos THP1 sel lyn dan makrofage afkomstig van muis beenmurg. In hierdie ondersoek het ons vasgestel dat fenotiasien kan gebruik word as 'n inhibitor van ensieme van die mycothiol pad. Alhoewel tunicamycin aansienlik die groei van M. tuberculosis beide in vitro en ex vivo inhibeer, was dit gevind word sitotoksies is vir makrofage. Om hierdie rede het ons bewys-van-konsep wat verbindings dat die uitdrukking van mycothiol ensieme inhibeer, die potensiaal het as anti-tuberkulose dwelms. Die reaksie van M. tuberkulose op stress is geëvalueer deur LC-MS waarin maksimum vlakke van mycothiol gedurende die vroeë tyd punte van blootstelling aan isoniasied geproduseer is. Ons gebruik mycothiol-deficient (mshA) M. tuberculosis om die rol van mycothiol vir oorlewing sowel as die gevolglike fenotipe te ondersoek wanneer sodanige mutanten blootgestel word aan stres kondisies. Die mshA-weglating mutanten van M. tuberculosis was bestand teen INH konsentrasies wat groei geïnhibeer in die wilde-tipe-stamme. Ons veronderstel dat katG en inhA, die gene wat betrokke is in INH metabolisme, mycothiol vereis vir hulle aktivering. Morfologiese veranderinge van M. tuberculosis binne makrofage is beoordeel met behulp van elektronmikroskopie. In hierdie manier waarop ons probeer om die lot van M. tuberkulose te volg binne die phagosomes, en hoe mycobacteria verwerk word in phagosomes in terme van replikasie, oorlewing en agteruitgang. Die vestiging van „n suksesvolle infeksie deur M. tuberculosis hang af van die aanvanklike ontmoeting met host makrofage, wat die eerste lyn 'n sellulêre verdediging teen mikrobiese inval verteenwoordig. Op die grens tussen mycobacteria en makrofage, speel die komplekse buitenste laag van die mikobakteriese selwand waarskynlik 'n rol in die intog van die gasheersel. Onder normale omstandighede (dws inname van non patogene mikroörganismes), nuutgevormde phagosomes meng inhoud en membraan met die opeenvolgende kompartemente van die endositiese roete (vroeë endosomes, laat endosomes, lisosome) deur 'n komplekse reeks van samesmelting en fisie. Soos hulle verwerk word tot phagolysosomes, ondergaan hulle geleidelike veranderinge deur spesifieke optel en verwydering van membraan komponente. Benewens, raak hulle versuur as gevolg van die vacuolair proton pomp ATPase geleë in die membraan en verkry giftige bestanddele, insluitend hydrolase wat uiteindelik bakterieë vernietig.

Les Toll-Like Receptors (TLRs) jouent un rôle majeur dans la réponse immunitaire innée, en reconnaissant des motifs moléculaires précis retrouvés exclusivement chez différents pathogènes (lipopolysaccharides (LPS), peptidoglycane, flagelline, ...). Le LPS est reconnu pour favoriser la production de cytokines pro-inflammatoires (ex. IL-1, IL-8, et TNF-ot) par les monocytes/macrophages via l'induction du facteur de transcription NK-KB par le TLR4. D'autre part, le virus d'immunodéficience humaine de type I (VIH-1) possède deux sites de liaison pour NF-KB dans sa région promotrice. L'objectif de cette étude était de vérifier l'effet de la stimulation au LPS sur la réplication et la production du VIH-1 dans une lignée cellulaire macrophagique ainsi que dans les macrophages primaires humains. L'ensemble des résultats obtenus montre que le LPS augmente l'activation de la transcription dans la lignée THP89GFP, alors que le contraire est observé dans les macrophages primaires humains et ce même si l'activation de NF-KB et la production de TNF-a sont observées dans ces cellules suite à un traitement au LPS. Cette étude révèle que les conclusions faites à partir d'une lignée cellulaire bien établie ne peuvent directement être appliquées à une un modèle plus physiologique. Ainsi, la réponse aux ligands des TLR devrait être étudiée dans un système de culture cellulaire beaucoup plus proche de la situation in vivo. / Toll-Like Receptors (TLRs) play a major role in the innate immune response, by recognizing precise molecular patterns found exclusively on different pathogens (lipopolysaccharides/LPS, peptidoglycans, flagellin, etc.). LPS is known to induce the production of pro-inflammatory cytokines (e.g. IL-1, IL-8, and TNF-a) by monocytes/macrophages through TLR4-mediated induction of the transcription factor NFKB. Interestingly, HIV-1 carries two consensus NF-KB binding sequences within its promoter region. The main goal of this study is a comparison of the modulatory effect of LPS treatment on HFV-1 transcription and virus production in a promonocytic cell line and primary monocyte-derived macrophages (MDM). We found that LPS increases virus gene expression in THP89GFP cells by activation of NF-KB, which is in sharp contrast with what is seen in primary MDM even though NF-KB and TNF-a were both stimulated upon treatment of the latter cell type by LPS. More importantly, our findings reveals that conclusions made with established human-derived cell lines cannot be directly applied to the situation prevailing in a more natural cellular setting. Thus, the response to TLR ligands should be studied in an appropriate cell culture System to more closely parallel the in vivo situations.

Le CD154 joue un rôle crucial dans l’activation des cellules présentatrices d’antigènes. Il a été démontré que le CD154 est incorporé à la surface du VIH-1 et que de tels virus activent des lymphocytes B. Nous avons vérifié s’ils ont également la capacité d’activer des macrophages humains. Nous avons observé une augmentation de la production d’IL-8 par les macrophages dérivés de monocytes (MDM), mais celle-ci varie grandement en fonction des différentes productions virales et des donneurs utilisés. L’induction de la sécrétion d’IL-8 est spécifique au CD154 puisqu’une forme mutée ne l’induit pas et est indépendante de la gp120. L’expression des MMP-1 et -2 est influencée par le CD154 sur les cellules 293T, mais l’effet spécifique de CD154 est perdu lorsque des virus sont utilisés. L’ensemble de ces résultats suggère que l’incorporation de CD154 par le virus pourrait être un moyen additionnel d’attirer des cellules cibles au site d’infection et ainsi favoriser la réplication virale. / CD154 interacts with CD40 found on antigen-presenting cells such as macrophages and plays a crucial role in activating these cells and initiating immune responses. We have previously reported that CD154 is incorporated within HIV-1 envelope and is effective at activating primary B lymphocytes. In this study, we tested if such CD154-bearing virions are also effective in activating human monocyte-derived-macrophages (MDM). We observed an increase in IL-8 secretion, but it is highly variable depending on viral productions and donors used. The induction of IL-8 production is specific to CD154 since a mutant form does not induce it and is independent of gp120. The production of MMP-1 and -2 is influenced by CD154 on 293T cells but the specificity is lost when viral particles are used. These results suggest a possible way used by the virus to attract target cells to the site of infection and thereby favour its own replication.

Submitted by Haroudo Xavier Filho (haroudo.xavierfo@ufpe.br) on 2016-04-05T15:42:35Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese Luís final (2).pdf: 2354969 bytes, checksum: 99edddc3a89f16f62d7e6fc6517c4a93 (MD5) / Made available in DSpace on 2016-04-05T15:42:35Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese Luís final (2).pdf: 2354969 bytes, checksum: 99edddc3a89f16f62d7e6fc6517c4a93 (MD5) Previous issue date: 2013-09-09 / FACEPE / As lectinas são proteínas conhecidas por sua capacidade de ligar específica e reversivelmente a carboidratos resultando em uma variedade de propriedades biológicas. Este trabalho tem por objetivo avaliar os efeitos imunomodulador e citoprotetor das lectinas Cramoll 1,4 (pCramoll) e rCramoll 1 (rCramoll). Na determinação da ação imunomoduladora macrófagos peritoneais de camundongos Balb/c foram tratados com diferentes concentrações das lectinas (0,625-10 μM) e foram analisados os efeitos na produção óxido nítrico (NO), viabilidade celular, produção de ânion superóxido, alterações no potencial de membrana mitocondrial (ΔΨm) e na fagocitose de Staphylococcus aureus. A produção de citocinas pró-inflamatorias (IL-1β, IL-6, INF-γ e TNF-α) foi avaliada em macrófagos infectados e não infectados com S. aureus. Ambas lectinas induziram significantemente a produção de NO e de citocinas. As proteínas foram consideradas não-citotóxicas pelo ensaio com MTT, entretanto a análise por Citometria de Fluxo revelaram um aumento de células mortas. A produção de superóxido foi estimulada pelas lectinas o que foi confirmado pelas mudanças induzidas no ΔΨm. A ação fagocítica dos macrófagos foi aumentada em 27,1% e 22,47% após o tratamento destes com pCramoll e rCramoll . Por fim, as lectinas inibiram a expressão de TNF-α e IL-6 e estimularam a produção de IL-1β e INF-γ por macrófagos infectados por S. aureus. Paralelamente, o potencial protetor das lectinas contra a morte celular induzida pelo estresse oxidativo foi avaliada. Para tanto, células Vero (fibroblastos renais de macaco) foram pré-tratadas com crescentes concentrações das lectinas (0,625-10 μM) por 30 minutos e em seguida foram expostas ao H2O2 (1 mM). Após 24 horas, o efeito citoprotetor foi avaliado. Os mecanismos celulares envolvidos foram determinados por citometria de fluxo envolvendo: proteção contra morte celular, danos nos lisossomos e DNA, produção de ânion superóxido (MitoSOX), alterações no potencial de membrana mitocondrial (ΔΨm) e proliferação celular. pCramoll e rCramoll atenuaram a citotoxicidade induzida por H2O2 de forma dose-dependente, os efeitos máximos foram 96.85 ± 15.59% (rCramoll) e 59.48 ± 23.44% (pCramoll). A análise com Live/Dead mostrou redução da morte celular de 65.04 ± 3.29% (H2O2) para 39.77 ± 2.93% (pCramoll) e 13.90 ± 9.01% (rCramoll). Os efeitos deletérios de H2O2 na proliferação celular foram reduzidos em 10.83% (pCramoll) e 24.17% (rCramoll). As lectinas atenuaram a produção excessiva de superóxido, o collapso do ΔΨm e os danos lisossomais e ao DNA das células Vero expostas à H2O2. Em conclusão nossos estudos demonstram que pCramoll e rCramoll possuem elevado potencial imunomodulador e citoprotetor. / This study aims to evaluate the immunomodulatory effects on macrophages and cytoprotector action against oxidative stress of Cramoll 1.4 (pCramoll) and rCramoll 1 (rCramoll). In the determination of the immunomodulory action, peritoneal macrophages from Balb/c mice were treated with different concentrations of lectins (0.625 to 10 mM) and we analyzed the effect on NO production (Griess Reagent), cell viability (MTT Reagent), induction of apoptosis (Kit Live/Dead and Acridine orange), superoxide anion production (MitoSOX), changes in mitochondrial membrane potential (ΔΨm) and phagocytosis of Staphylococcus aureus. The production of proinflammatory cytokines (IL-1β, IL-6, INF-γ e TNF-α) was analyzed in S. aureus infected and non-infected macrophages. Both lectins significantly enhanced Macrophages NO production and cytokines. The lectins were not cytotoxic as observed by MTT assay. However Live/Dead analysis revealed an increase of apoptosis in treated cells, the reduction of lysosomal activity was also reduced. The superoxide production was stimulated by both lectins, which was confirmed with the reduction on ΔΨm. S. aureus phagocytic activity of macrophages were enhanced in 27.1% and 22.47% by pCramoll and rCramoll, respectively. Finally, pCramoll and rCramoll downregulated the production of IL-6 and TNF-α and upregulated the expression of IL-1β, INF-γ during S. aureus infection of macrophages. In addtion, the protective effects of lectins against cell death induced by oxidative stress were evaluated. For this purpose, Vero cells (monkey kidney fibroblasts) were pretreated with increasing concentrations of lectins (0.625 to 10 μM) for 30 minutes and then were exposed to H2O2 (1mM). After 24 hours, the cytoprotective effect was evaluated and the cellular mechanisms involved were determined by flow cytometry involving: protection against cell death (Live/Dead kit), damage to lysosomes (Acridine Orange) and DNA (TUNEL), superoxide anion production (MitoSOX), changes in mitochondrial membrane potential (ΔΨm) (Rhodamine 123) and cell proliferation. pCramoll and rCramoll significantly attenuated the H2O2-induced cytotoxicity in a dose-dependent way, the maximum protective effects were 96.85 ± 15.59% (rCramoll) and 59.48 ± 23.44% (pCramoll). The Live/Dead analysis showed a reduction in apoptotic cells from 65.04 ± 3.29% to 39.77 ± 2.93% (pCramoll) and 13.90 ± 9.01% (rCramoll). The deleterious effects of H2O2 on cell proliferation were reduced 10.83% (pCramoll) and 24.17% (rCramoll). The lectins attenuated the excessive superoxide production, the collapse of ΔΨm, lysosomal and DNA damage that occurred in Vero cells exposed to H2O2. In conclusion, our results show that pCramoll and rCramoll have high immunomodulatory potential on macrophages and cytoprotective effects against H2O2.

Lors d'une infection, la principale voie d'activation du macrophage passe par l'IFNY . Par conséquent, la connaissance des événements qui surviennent préalablement à l'activation du macrophage revêt un intérêt certain. Cette thèse s'est donc intéressée à l'effet de l'infection par le parasite protozoaire intracellulaire Trypanosoma cruzi sur deux fonctions IFN-Y -dépendantes du macrophage murin: la production d'oxyde nitrique et la synthèse de la machinerie de présentation antigénique restreinte au CMH de classe I. Les résultats obtenus démontrent que l'infection préalable du parasite pré-active le macrophage à produire de l'oxyde nitrique en réponse à l'IFN-y à des niveaux nettement supérieurs de ceux atteints par le macrophage uniquement stimulé à l'IFN-Y • Cette pré-activation résulte de l'engagement par le parasite des voies NF-KB, Erkl/Erk2 et SAPK/JNK. L'activation subséquente de la voie Jak/STAT par l'IFN-y induit la translocation au noyau de STATla et à la transcription d'un ARNm stable d'iNOS. Une dégradation ralentie de l'ARNm d'iNOS mène à des niveaux élevés de protéine iNOS et d'oxyde nitrique. La stabilité de l'ARNm d'iNOS résulte de l'activation des voies Erkl/Erk2 et SAPK/JNK par le parasite, alors que le facteur de transcription NF-KB favorise probablement une expression maximale d'iNOS. Les résultats présentés dans cette thèse montrent également que l'infection préalable du macrophage par T. cruzi réduit l'expression IFN-Y -dépendante du CMH de classe I et des immuno-sous-unités du protéasome, que cette diminution est due à l'activation de c-Jun suite à l'inhibition des phosphatases murines qui, en retour, est une conséquence des radicaux libres d'oxygène générés par le macrophage en réponse au contact de T. cruzi. Bien que ces résultats soient intéressant en soi, l'ensemble des travaux présentés dans cette thèse vont quelque peu au-delà des objectifs initiaux, en faisant ressortir le rôle essentiel que semble jouer la voie de signalisation SAPK/JNK dans la modulation des fonctions IFN-y -dépendantes du macrophage.

Le but de ce travail était de mieux comprendre les mécanismes de régulation influençant le recrutement des macrophages cérébraux et leur activation dans différentes affections du système nerveux central. À cette fin, trois objectifs ont été définis : (1) Mettre en évidence le rôle du TNF dans l'infiltration et l'activation des macrophages dans un modèle de démyélinisation induit par la cuprizone; (2) Identifier les précurseurs sanguins des macrophages périvasculaires du cerveau et le rôle de l'angiopoïétine-2 dans leur recrutement suite à une endotoxinémie; (3) Définir le rôle du récepteur GPR84 lors de la réponse microgliale dans un modèle murin de la maladie d'Alzheimer. L'identification des différents facteurs pouvant influencer le phénomène de démyélinisation est un enjeu majeur de la recherche sur la sclérose de plaques. Dans cette maladie et dans les modèles animaux associés, l'expression de TNF par les macrophages cérébraux suggérait que la cytokine pro-inflammatoire jouerait un rôle dans le recrutement des macrophages dérivés du sang et de la microglie. Afin de déterminer quelles fonctions jouaient le TNF et ses récepteurs TNF-R1 et TNF-R2 sur les macrophages cérébraux, nous avons utilisé des souris chimériques dont le système hématopoïétique exprimait la GFP, et nous les avons traitées avec la toxine cuprizone pour reproduire un modèle de démyélinisation. Nous avons montré que les macrophages étaient très fortement recrutés depuis la périphérie et activés dans le SNC, mais selon un mécanisme indépendant du TNF. De plus, chez des souris déficientes en TNF traitées avec la cuprizone, l'étendue de la démyélinisation n'était pas diminuée par rapport aux souris contrôles. Finalement, par la technique d'hybridation in situ, il a été prouvé que le TNF n'était pas exprimé dans le cerveau dont la démyélinisation était provoquée par la cuprizone. Le système nerveux est infiltré de façon continue par les macrophages périvasculaires dérivés du système sanguin. Les précurseurs immédiats de ces cellules n'ont pas encore été identifiés et les mécanismes gouvernant leur recrutement sont encore largement méconnus. Dans cette étude, nous apportons la preuve que les monocytes CD68+GR1" pouvaient se différencier en macrophages périvasculaires chez des souris souffrant d'endotoxinémie. Après leur adhésion à l'endothélium, ces monocytes rampaient, adoptaient une morphologie dite en bâtonnet lors de leur passage dans les capillaires, pouvaient proliférer et former de longues protubérances cytoplasmiques. Ils étaient recrutés en grand nombre lors d'une endotoxinémie grâce à la combinaison de molécules vasorégulatrices, incluant le TNF, l'interleukine-ip et l'angiopoïétine-2. Après quelques heures, quelques unes de ces cellules traversaient l'endothélium afin d'accroître la population de macrophages périvasculaires du SNC. L'élimination des monocytes adhérents et des macrophages périvasculaires nouvellement recrutés pouvait être provoquée par l'injection d'une protéine de fusion couplant un peptide-Fc et un anti-angiopoïétine-2. Cette seconde étude permet ainsi une meilleure compréhension du comportement monocytaire à l'interface sang-cerveau et fournit une nouvelle façon de contrer leur infiltration dans le tissu nerveux en conditions inflammatoires. Le GPR84 est un récepteur transmembranaire couplé aux protéines G récemment identifié comme étant exprimé de façon sélective par la microglie chez des souris souffrant d'endotoxinémie ou d'encéphalomyélite autoimmune expérimentale, mais sa fonction demeure inconnue. Nos travaux rapportent que le GPR84 est également exprimé par les cellules microgliales entourant les plaques amyloïdes chez les souris transgéniques APP/PS1, un modèle de la maladie d'Alzheimer. Les symptômes d'une endotoxinémie ou de l'EAE ne différaient pas chez des souris déficientes en GPR84, mais un déclin cognitif plus précoce et plus sévère était observé chez les souris APP/PS1 déficientes en GPR84. Dans le cerveau de ces dernières, le nombre de microglies global ainsi que par plaque était diminué; toutefois, aucune différence de dépôt d'amyloïde n'était observée. Nous en concluons dans cette troisième étude que le GPR84 joue un rôle dans le recrutement des cellules microgliales lors de certaines conditions inflammatoires et que son absence réduit la capacité de la microglie à protéger partiellement le cerveau contre la P-amyloïde toxique sans pouvoir l'éliminer. L'ensemble de ces

D. Tech. (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal University of Technology. / Salmonella enterica serovar Typhimurium and Campylobacter jejuni are the leading causes of Salmonellosis and Campylobacteriosis that is characterised by gastroenteritis. These waterborne diseases can be easily prevented by home water treatment methods such as solar disinfection (SODIS). The SODIS process involves placing microbiologically unsafe water in clear plastic or glass bottles and exposing them to direct sunlight for approximately six to eight hours. SODIS kills microbes through a combination of DNA-damaging effects of ultraviolet (UV) radiation and thermal inactivation from solar heating. The result is microbiologically safe water. Continuous drinking of SODIS treated water may confer some immunological effects on the consumer. These immunological effects have not been thoroughly explored. Therefore, the objectives of this study were to firstly, characterise the effects of solar irradiation on the viability of S. Typhimurium and C. jejuni; secondly, to determine the cytotoxicity and modulation of cell death of solar irradiated S. Typhimurium and C. jejuni on macrophages. Thirdly, to analyse the chemokine and cytokine profiles of macrophages infected with solar irradiated S. Typhimurium and C. jejuni. Lastly, to analyse the host-cell interactions of macrophages infected with solar-irradiated and non-solar irradiated S. Typhimurium and C. jejuni using a proteomic approach. In all the experiments, S. Typhimurium and C. jejuni were (i) heat/chemically treated, (ii) solar and non-solar irradiated for 4 and 8 hours. A murine macrophage cell line RAW264.7 was co-cultured with the differentially treated bacteria species for 3 and 24 hours. Appropriate controls were included. The impact of solar irradiated S. Typhimurium and C. jejuni on intracellular growth, proliferation, cytotoxicity, and apoptosis on macrophages was assessed. Intracellular growth of the both bacterial species was assessed with the gentamicin protection assay, and cytotoxicity was determined by Lactate Dehydrogenase Assay (LDH). The macrophages treated with solar irradiated S. Typhimurium and C. jejuni showed no intracellular growth after 48 hours post-infection. However, the non-irradiated S. Typhimurium survived within the macrophages and were highly toxic to the macrophages (average cytotoxicity of 91%±32). The non-solar irradiated C. jejuni were metabolically active but non-culturable, whereas the solar-irradiated C. jejuni was metabolically inactive. Thus, solar irradiated C. jejuni showed a lower percentage cytotoxicity (2.57% ± 0.32%) in comparison to non-solar irradiated C. jejuni at 24 hours post-infection (p.i.) (30.28% ± 0.05%). Flow cytometric analysis showed that the non-irradiated S. Typhimurium brought about a statistically significant increase in the percentage of necrotic cells (48% ± 2.99%), whereas bacteria irradiated for 8 hours produced a lower percentage of necrotic cells (25% ± 5.87%). The heat/chemical attenuated samples had the lowest percentage of necrotic cells (21.15% ± 5.36%) at 24 h p.i. Macrophages treated with solar irradiated and non-solar irradiated C. jejuni did not induce necrosis, but apoptotic cell death. At 24 h p.i., the highest proportion of apoptotic cell death was observed in macrophages treated with non-solar irradiated C. jejuni whereas the solar irradiated C. jejuni showed a lower percentage of apoptotic cell death. Therefore, there is great possibility that S. Typhimurium and C. jejuni could become avirulent after SODIS treatment and this could prevent gastroenteritis in consumers of SODIS-treated water. The activation of macrophages infected with solar irradiated S. Typhimurium and C. jejuni was also assessed in this study. The production of nitric oxide (NO) was determined using the Greiss Reagent Assay, whereas the production of chemokines, cytokines, and growth stimulating factors by the RAW264.7 cells in vitro was measured using the Luminex 200. The results showed that both solar and non-solar irradiated S. Typhimurium inhibited the production of nitric oxide in the RAW264.7 cells. The heat/chemically attenuated S. Typhimurium induced a significant increase (p<0.0.5) in the production of NO2− in the macrophages when compared to the unstimulated RAW264.7. The chemokine and cytokine levels produced by the macrophages were similar in the solar inactivated S. Typhimurium and the live untreated S. Typhimurium. However, macrophages treated with heat/chemically attenuated S. Typhimurium showed an anti-inflammatory response by inhibiting the production of pro-inflammatory cytokines such as IL-1, IL-1, IL-2, IL-6, and IL-17 in macrophages. The macrophages treated with solar and non-solar irradiated C. jejuni possibly produced an anti-inflammatory effect since the amount of pro-inflammatory cytokines in the samples was significantly reduced during the late infection period (24 h p.i.). This study also analysed the proteomic profiles of macrophages treated with LPS, non-solar irradiated, solar irradiated, heat/ chemical inactivated S. Typhimurium, and C. jejuni. This was carried out using SWATH-mass spectrophotometry-based proteomics. Proteins were extracted from infected macrophages after 24 hours p.i. HILIC-based sample clean-up and digestion, DDA LCMS-MS (spectral library), SWATH LCMS-MS, and data processing were carried out. A total of 15,077 peptides matching to 2,778 proteins were identified at 1% FDR with numerous differentially expressed proteins (DEPs) detected in macrophages treated with lipopolysaccharide (LPS), non-solar irradiated C. jejuni (NS), heat-attenuated C. jejuni (HA) and 4h-solar irradiated (SI4) and 8h-solar irradiated (SI8) C. jejuni, respectively. Pathway analysis revealed that most of the upregulated proteins in macrophages treated with solar irradiated C. jejuni were involved in oxidation-reduction processes, endoplasmic reticulum stress, transport, antigen processing and presentation of exogenous peptide antigens via MHC class I (TAP-dependant) and ATP-biosynthetic processes. The KEGG-pathways also revealed the roles of some upregulated proteins in lysosomal and phagosome pathways. In conclusion, our results revealed that there is coordinated up-regulation of MHC-I processing pathways occurred at 24 h p.i. It is likely that proteins from solar irradiated C. jejuni may undergo proteasomal degradation, and the peptides are transported to the endoplasmic reticulum (ER) and loaded onto MHC-I molecules. Peptide loading results in class I complexes consolidation and transit to the cell surface where antigens can be presented to circulating CD8 + T cells. Additionally, solar irradiated C. jejuni also undergoes degradation in the phagosome. The phagosome has the potential to create antigens that can be expressed on the cell surface of macrophages to stimulate different lymphocytes and induce appropriate immune responses, thus, connecting the innate to adaptive immunity, and this could also have health benefits via the consumption of SODIS treated water. However, proteomic analysis of S. Typhimurium showed no significant differentially expressed proteins in macrophages treated with LPS, non-solar irradiated, and solar irradiated S. Typhimurium. This may be due to an overestimation of the extracted protein. However, DEPs in macrophages treated with heat-attenuated S. Typhimurium showed that macrophages may have adapted an anti-inflammatory M2 phenotype because the IFN-γ signalling pathway was downregulated. This may have contributed to non-expression of the chemokine IFN-γ in RAW264.7 cells. Moreover, proteins such as Hmox1 and Sqstm1 were upregulated, and this is also characteristic of M2 macrophages. This study provided new insights on the effect of solar irradiated Salmonella Typhimurium and Campylobacter jejuni on the proliferation and activation of macrophages in vitro.

Indiana University-Purdue University Indianapolis (IUPUI) / The immune system initiates tissue repair following injury. In response to sterile tissue injury, neutrophils infiltrate the tissue to remove tissue debris and subsequently undergo apoptosis. Proper clearance of apoptotic neutrophils in the tissue by recruited macrophages, in a process termed efferocytosis, is critical to facilitate the resolution of inflammation and tissue repair. However, the events leading to suppression of sterile inflammation following efferocytosis, and the contribution of other innate cell types are not clearly defined in an in vivo setting. Using a sterile mouse peritonitis model, we identified IL-4 production from efferocytosing macrophages in the peritoneum that activate invariant NKT cells to produce cytokines including IL-4 and IL-13. Importantly, IL-4 from macrophages functions in autocrine and paracrine circuits to promote alternative activation of peritoneal exudate macrophages and augment type-2 cytokine production from NKT cells to suppress inflammation. The increased peritonitis in mice deficient in IL-4, NKT cells, or IL-4Ra expression on myeloid cells suggested that each is a key component for resolution of sterile inflammation. The phagocyte NADPH oxidase, a multi-subunit enzyme complex we demonstrated to require a physical interaction between the Rac GTPase and the oxidase subunit gp91phox for generation of reactive oxygen species (ROS), is required for production of ROS within macrophage phagosomes containing ingested apoptotic cells. In mice with X-linked chronic granulomatous disease (X-CGD) that lack gp91phox, efferocytosing macrophages were unable to produce ROS and were defective in activating iNKT during sterile peritonitis, resulting in enhanced and prolonged inflammation. Thus, efferocytosis-induced IL-4 production and activation of IL-4-producing iNKT cells by macrophages are immunomodulatory events in an innate immune circuit required to resolve sterile inflammation and promote tissue repair.

Indiana University-Purdue University Indianapolis (IUPUI) / Vaccinia virus (VV) is the prototypical member of the orthopoxvirus genus of the Poxviridae family, and is currently being evaluated as a vector for vaccine development and cancer cell-targeting therapy. Despite the importance of studying poxvirus effects on the human immune system, reports of the direct interactions between poxviruses and primary human leukocytes (PHLs) are limited. We studied the specific molecular events that determine the VV tropism for major PHL subsets including monocytes, B cells, neutrophils, NK cells, and T cells. We found that VV exhibited an extremely strong bias towards binding and infecting monocytes among PHLs. VV binding strongly co-localized with lipid rafts on the surface of these cell types, even when lipid rafts were relocated to the cell uropods upon cell polarization. In humans, monocytic and professional antigen-presenting cells (APCs) have so far only been reported to exhibit abortive infections with VV. We found that monocyte-derived macrophages (MDMs), including granulocyte macrophage colony-stimulating factor (GM-CSF)-polarized M1 and macrophage colony-stimulating factor (M-CSF)-polarized M2, were permissive to VV replication. The majority of virions produced in MDMs were extracellular enveloped virions (EEV). Visualization of infected MDMs revealed the formation of VV factories, actin tails, virion-associated branching structures and cell linkages, indicating that infected MDMs are able to initiate de novo synthesis of viral DNA and promote virus release. Classical activation of MDMs by LPS plus IFN-γ stimulation caused no effect on VV replication, whereas alternative activation of MDMs by IL-10 or LPS plus IL-1β treatment significantly decreased VV production. The IL-10-mediated suppression of VV replication was largely due to STAT3 activation, as a STAT3 inhibitor restored virus production to levels observed without IL-10 stimulation. In conclusion, our data indicate that PHL subsets express and share VV protein receptors enriched in lipid rafts. We also demonstrate that primary human macrophages are permissive to VV replication. After infection, MDMs produced EEV for long-range dissemination and also form structures associated with virions which may contribute to cell-cell spread.