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TLR agonists on autoimmunity, cancer and M1/M2 macrophage polarization Skip to content Tez Künye Durumu TLR agonists on autoimmunity, cancer and M1/M2 macrophage polarization / TLR agonistlerinin otoimmünite, kanser ve M1/M2 makrofaj polarizasyonundaki etkileri Yazar:BEGÜM HAN HORULUOĞLU Danışman: PROF. DR. İHSAN GÜRSEL ; DR. DENNIS M. KLINMAN Yer Bilgisi: İhsan Doğramacı Bilkent Üniversitesi / Mühendislik ve Fen Bilimleri Enstitüsü / Moleküler Biyoloji ve Genetik Ana Bilim Dalı Konu:Biyoloji = Biology Dizin: Onaylandı Doktora İngilizce 2019 149 s. Makrofajlar, immün yanıtların başlamasında ve dengenin korunmasında önemli bir rol oynar. Makrofajlarin, fenotiplerindeki, fonksiyonlarındaki ve aktivasyon durumlarındaki değişikliklerin, otoimmün ve enflamatuar hastalıkların patogenezine etki ettiği gösterilmiştir. M1: M2 makrofaj oranındaki artış, Sistematik Lupus Eritematozus (SLE), vaskülit ve miyozit dahil olmak üzere birçok otoimmün hastalığın gelişimi ile ilişkilendirilmiştir. Daha önceki çalışmalar, TLR2 / 1 agonisti PAM3CSK4’ün (PAM3) normal insan monositlerini, enflamatuar M1-benzeri makrofajlardan ziyade immünosupresif M2-makrofaj değişimine desteklediği gösterdi. Bu çalışma, PAM3’ün hasta monositlerinden M2 makrofaj farklılaşmasını indükleyebilme yeteneğini araştırmayı ve PAM3’ün bir lupus fare modelinde terapötik etkinliğini değerlendirmeyi amaçlar. Çalışmalar sonucunda, belirtilen otoimmün hastalıklara sahip kişilerin enflamatuar tipte monositlerinde bir artış ile birlikte, anti- enflamatuvar olan klasik monositlerde ise bir azalma olduğunu göstermiştir. Ek olarak, bu monositler herhangi bir uyarıcı olmadan kültüre konulduğunda M1 tipi makrofajlara dönüşme eğiliminde oldukları gözlemlendi. PAM3 varlığında ise hasta ve sağlıklı monositleri herhangi bir fark olmadan fenotipik olarak M2 benzeri makrofajlarina değişti. Makrofajlarin fonksiyonlarını belirlemek için yapılan analizler, fenotipik analizleri destekleyici yonde olup, PAM3 ile geliştirilen makrofajlarin yüksek endositik yetenekleri olduğu göstermiş ve enflamatuar sitokinler yerine anti-enflamatuar sitokin sentezini indüklemişlerdir. Lupus eğilimli NZB x NZW F1 farelerine haftalık yapılan PAM3 tedavisi sonucunda insan bulgularına benzer sonuçlar elde edildi. PBS enjeksiyonu yapılan farelerde,gözlemlenen M1:M2 makrofaj oranındaki artış, PAM3 tedavisi ise normal seviyelere düştü. M2 makrofajlarinda görülen bu artışı, otoantikor ve enflamatuar sitokin seviyelerindeki düşüş eşlik etti. Ayrıca PAM3 tedavisi alan hayvanların böbreklerindeki hasar ise önemli ölçüde bastırıldı. PAM3 tedavisi sonucu 45 haftalık hayvanların %60’i hala hayatta iken, PBS alan kontrol grubunda bu yüzde %5’e kadar düştü. Sonuçlarımız PAM3’nin monositlerin M2 makrofajlarina değişimini sağladığını ve bu yöntemin otoimmun hastalıklar için bir yeni bir tedavi yöntemi olarak kullanılabileceğini göstermiştir. Bu tezin ikinci bölümü, TLR9 ligandı olan, CpG ODN’nin, lipozomlar içine yüklenerek kanser aşısı ajanları olarak kullanılmak üzere immünomodülatör etkilerini arttırmaya odaklanmıştır. D tipi ODN, pDC’yi IFNa üretmek için uyarırken, K tipi ODN pDC’lerin mature olmasını destekleyerek TNFa sekrete edilmesine sebep olur. Hücreler K ve D tipi ODN karışımı ile inkübe edildiğinde, K, D’nin aktivitesini maskelediği gözlemlenmiştir. Aşı ajanları hazırlanırken aktive ettikleri farklı yolakların birleşimi sayesinde hem K, hem de D tipi ODN’nin kullanılmasının yararlı olacağını düşünmekteyiz. Verilerimiz, nötral lipozomlara yüklenen D ODN ve katyonik lipozomlara yüklenen K ODN’nin, PBMC’ler tarafından TNFa’yı desteklemeye devam ederken IFNa üretimindeki engelin kalkarak, bu sitokinin sentezinde artış olduğunu göstermektedir. Lipozomal yükleme her iki ODN sınıfının da hücresel lokalizasyonunda değişime sebep olmadı, fakat hücre içine alim çalışmaları, katyonik lipozoma yüklenen K ODN’nin hücre alımını yavaşlayıp azaldığını, D’nin nötral lipozoma yüklenmesinin ise pDC’ler tarafından alımını arttırdığını gösterdi. K artı D lipozom kombinasyonlarının etkinliği, bir fare tümör aşı modelinde incelendi. Lipozom kombinasyonları, belirgin Thl yanlı anti- OV A yanıtını arttırarak, aşılamayı takiben B16-OV A tümörlerinde önemli bir azalmaya neden olmuştur. Bulgularımız, D ve K ODN’nin faydalı özelliklerinin uygun lipozamlara yüklenmesi sonucunda birleştirilerek, immunoterapi alanında faydalı olabileceğini göstermiştir. Macrophages play an important role in the initiation of immune responses and the maintenance of immune homeostasis. Alterations in their phenotype, function and activation state have been implicated in the pathogenesis of autoimmune and inflammatory diseases. An increased M1:M2 ratio is associated with the development of several autoimmune diseases including Systematic Lupus Erythematosus (SLE), vasculitis and myositis. Previous work showed that the TLR2/1 agonist PAM3CSK4 (PAM3) could stimulate normal human monocytes to preferentially differentiate into immunosuppressive M2-like rather than inflammatory M1-like macrophages. This work seeks to investigate the ability of PAM3 to induce M2 macrophage differentiation from patient monocytes and evaluate the therapeutic efficacy of PAM3 in a murine model of lupus. Our findings revealed that patients with indicated autoimmune diseases have a significant increase in monocytes of the inflammatory subtypes coupled with a decrease in non-inflammatory classical monocytes compared to healthy controls. Additionally, in the absence of stimulant patient monocytes differentiated into more M1-like macrophages. Nevertheless, phenotypic analysis of in vitro generated macrophages revealed that, PAM3 stimulation induced M2-like macrophage differentiation without any difference from patient and healthy monocytes. Phenotypic analysis was further supported by the high endocytic abilities and secretion of anti-inflammatory cytokines instead of pro-inflammatory cytokines by PAM3 generated macrophages. Lupus-prone NZB x NZW F1 mice responded similarly to weekly PAM3 treatment. Upon PAM3 treatment the increased M1:M2 ratio, which was observed in PBS treated group, was decreased to normal levels. The increase in M2 macrophages was accompanied by decreased autoantibody and inflammatory cytokines along with an,increase in anti-inflammatory cytokine production. Moreover, kidney damage was significantly suppressed and M2 macrophages were detected in the kidneys of PAM3 treated group. PAM3 treatment prolonged to survival of NZB/W significantly, at 45 weeks of age 60% of mice were still alive whereas in PBS group only 5% were. Our results indicate that, PAM3 induces immunosuppressive macrophages and thus could represent a novel approach to the therapy of autoimmune diseases. The second part of this thesis focused on enhancing the immunomodulatory effects of TLR9 ligands, CpG ODN upon encapsulation within liposomes as cancer vaccine adjuvants. Although both D and K ODN are strictly dependent on TLR9, K ODN trigger pDCs to mature and secrete TNFα while D ODN stimulate pDC to produce IFNα. When cells are incubated with a mixture of K and D ODN, K masks the activity of D. The use of both K and D ODN would be of benefit when preparing vaccine adjuvants and for immunotherapy. Our data indicate that simultaneous delivery of D ODN loaded into neutral liposomes plus K ODN loaded into cationic liposomes improved rather than masked IFNα production while continuing to support TNFα by PBMCs. Liposomal encapsulation did not alter the subcellular localization of either class of ODN, but internalization studies revealed that cationic liposome encapsulation slows and reduces the uptake of K ODN whereas neutral encapsulation of D increases their uptake by pDCs. The efficiency of K plus D liposome combinations was examined in a murine tumor vaccine model. The liposome combinations induced pronounced Th1-biased anti-OVA immunity and led to a significant reduction of B16-OVA tumors following inoculation. Our findings, demonstrate that the beneficial features of D and K ODN could be obtained simultaneously by appropriate liposomal formulation, further extending the breadth of CpG ODN-dependent immunotherapy.
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