Browsing by Subject "A151 ODN"

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    ItemOpen Access
    Immunomodulatory potential of human umbilical cord tissue-derived mesenchymal stromal cell (UCX®) exosomes in combination with immunosuppressive “A151” oligodeoxynucleotide
    (2019-07) Bulut, Özlem
    Mesenchymal stromal or stem cells (MSCs) modulate immune responses apart from their regenerative capacities. Accumulating evidence suggests that MSCs exert their paracrine effects through extracellular vesicles known as exosomes. In this study, we utilized a particular human umbilical cord tissue-derived MSC type termed as UCX®. UCX® is superior to the gold-standard bone marrow-derived MSCs in terms of immunosuppressive properties. We aimed to characterize and employ UCX® exosomes as cell-free immunosuppressive therapeutic agents. Another aim was to compare the functionalities of exosomes either isolated from 2-dimensional (2D) cultures or isolated from 3-dimensional (3D) spheroid cultures. 3D culture provides better cell-to-cell and cell-to-matrix interactions thereby mimics the in vivo environment better. A synthetic oligodeoxynucleotide called A151 ODN, which consists of 4 repeats of the mammalian telomeric TTAGGG motif, has broad immunosuppressive effects. Delivery of A151 ODN within liposomes or exosomes protects it from degradation by nucleases and improve the desired outcome. We also aimed to combine the immunomodulatory potentials of UCX® exosomes and A151 ODN through direct loading of A151 ODN into exosomes with ~95% efficiency via a dehydration-rehydration-based lyophilization method. First, we determined the binding and internalization kinetics of exosomes with different immune cells. 3D-exosomes interacted with the target cells much faster and more efficiently. Next, we investigated how UCX® exosomes influence Toll-like receptor (TLR) signaling in mouse splenocytes and bone marrow-derived macrophages (BMDMs). 3D-exosomes compared to 2D-exosomes were more potent to suppress IFNγ, IL6, IL12, and to a lesser extent TNFα production mediated by TLR1/2, TLR4, TLR7/8 and TLR9 but not by TLR3 triggering. A151 ODN-loading to either 2D- or 3D-exosomes improved the inhibition of all the above mentioned pro-inflammatory cytokines. Especially 3D-exosomes downregulated co-stimulatory molecules CD80 and CD86 along with MHC-II on BMDMs following TLR stimulation. Macrophage polarization experiments revealed that UCX® exosomes reprogram BMDMs to produce high amounts of nitric oxide and arginase-1 which are the key immunomodulatory factors induced by myeloid-derived suppressor cells (MDSCs) to inhibit T- and NK-cell activity. Besides shifting macrophages to an MDSC-like suppressive phenotype, exosomes also supported expansion of MDSC populations in vivo upon intraperitoneal injection. Next, we tested the therapeutic efficiency of UCX® exosomes with or without A151 ODN in zymosan-induced peritonitis and dextran sodium sulfate (DSS)-induced colitis models in mice. Exosomes could not alleviate zymosan-induced peritonitis which is an acute and severe inflammation. However, 3D-exosomes and A151 ODN-loaded versions of both 2D- and 3D-exosomes remarkably prevented DSS-induced colitis progression. A151 ODN itself was also therapeutic, albeit to a lesser degree. Standalone 3D-exosomes and A151 ODN-loaded exosomes prevented weight loss and colon shortening. All treatments except for 2D-exosomes could restore DSS-induced loss of T-cell numbers and cytokine-producing capacities in mesenteric lymph nodes and spleen. All treatments except for A151 ODN prevented DSS-induced macrophage accumulation in the lymph nodes. 3D-exosomes and A151 ODN-loaded versions of both exosomes normalized serum IL6 levels while only A151 ODN-loaded 3D-exosomes could impact the cytokine production capacities of macrophages. Finally, we tested the effects of UCX® exosomes with or without A151 ODN on wound healing. In vitro, 2D- and 3D-exosomes differentially upregulated the productions of wound healing-related cytokines and growth factors such as IL1α, TGFβ and VEGFα from fibroblast and keratinocyte cell lines. In vivo, in an excisional wound healing model, free or A151 ODN-loaded exosomes did not accelerate wound closure. However, they caused systemic immunosuppression at the late stages of wound healing. Systemic outcomes include reduced inflammatory capacity of macrophages and higher granulocytic MDSC numbers in spleen. A151 ODN-loaded 3D-exosomes also reduced T-cell numbers in spleen and pro-inflammatory cytokine levels in circulation. Taken together, this study revealed that 2D- but more importantly 3D-culturing of umbilical cord MSCs result in functionally different exosomes, 3D culture-derived exosomes display higher immunosuppressive potential, A151 ODN-loading into these exosomes improves immunosuppressive capacity and A151 ODN-loaded UCX® exosomes could be a valuable therapeutic agent for inflammatory and autoimmune disorders.
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    ItemOpen Access
    A suppressive oligodeoxynucleotide expressing TTAGGG motifs modulates cellular energetics through the mTOR signaling pathway
    (Oxford University Press, 2020) Yazar, Volkan; Kılıç, Gizem; Bulut, Özlem; Canavar-Yıldırım, Tuğçe; Yağcı, Fuat C.; Gamze, Aykut; Klinman, D. M.; Gürsel, M.; Gürsel, İhsan
    Immune-mediated inflammation must be down-regulated to facilitate tissue remodeling during homeostatic restoration of an inflammatory response. Uncontrolled or over-exuberant immune activation can cause autoimmune diseases, as well as tissue destruction. A151, the archetypal example of a chemically synthesized suppressive oligodeoxynucleotide (ODN) based on repetitive telomere-derived TTAGGG sequences, was shown to successfully down-regulate a variety of immune responses. However, the degree, duration and breadth of A151-induced transcriptome alterations remain elusive. Here, we performed a comprehensive microarray analysis in combination with Ingenuity Pathway Analysis (IPA) using murine splenocytes to investigate the underlying mechanism of A151-dependent immune suppression. Our results revealed that A151 significantly down-regulates critical mammalian target of rapamycin (mTOR) activators (Pi3kcd, Pdpk1 and Rheb), elements downstream of mTOR signaling (Rps6ka1, Myc, Stat3 and Slc2a1), an important component of the mTORC2 protein complex (Rictor) and Mtor itself. The effects of A151 on mTOR signaling were doseand time-dependent. Moreover, flow cytometry and immunoblotting analyses demonstrated that A151 is able to reverse mTOR phosphorylation comparably to the well-known mTOR inhibitor rapamycin. Furthermore, Seahorse metabolic assays showed an A151 ODN-induced decrease in both oxygen consumption and glycolysis implying that a metabolically inert state in macrophages could be triggered by A151 treatment. Overall, our findings suggested novel insights into the mechanism by which the immune system is metabolically modulated by A151 ODN.
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    Therapeutic potential of an immunosuppressive oligodeoxynucleotide encapsulated within liposomes on bleomycin-induced mouse model of lung inflammation and fibrosis
    (2019-05) Kılıç, Gizem
    Systemic sclerosis (SSc) is an autoimmune/autoinflammatory disease with unknown etiology. It is characterized by vascular dysfunction, inflammation and disseminated fibrosis of skin or internal organs. Although its prevalence is low, development of fibrosis on internal organs and lack of a curative treatment result in high morbidity. Current therapies targeting specific symptoms such as interstitial lung disease, Raynaud’s phenomenon and pulmonary arterial hypertension are inefficient, and at best, temporarily relieves the symptoms throughout the course of the treatment. Herein, we investigated the therapeutic potential of an immunosuppressive oligodeoxynucleotide expressing TTAGGG telomeric repeats which is known as the “A151 ODN” on bleomycin-induced mouse model of systemic sclerosis. A151 ODN is the single stranded synthetic form of the telomeric repeat sequence expressed on mammalian chromosome, and it contains four repeats of “TTAGGG” motif. In order to enhance the therapeutic effectivity while protecting its digestion from nuclease activity following administration, we encapsulated A151 ODN within anionic liposomes. Since pattern recognition receptors and their signaling pathways were demonstrated to initiate inflammation in SSc, we first explored the immunosuppressive capacity of A151 ODN by analyzing in vitro cytokine productions and surface marker expression levels. Similar with the previous findings, A151 ODN was highly potent to abolish cytokine production in response to TLR9 induction. Although A151 ODN by itself was not very effective to suppress cytokine secretion following TLR1/2 and TLR4 induction, encapsulation within anionic liposomes further improved the immunosuppressive potential in response to TLR engagement. Furthermore, flow cytometry analyses revealed that A151 ODN decreased antigen presentation capacity and activation of bone-marrow derived macrophages (BMDMs) in response to TLR stimulation which was demonstrated by the reduction in levels of surface MHCII and co-stimulatory molecules as well as proteins having role on macrophage adherence and migration. A151 ODN also inhibited transcription of two major genes known to play a critical role on the development of fibrosis, TGFβ and Col1a1, from fibroblasts. Following these promising results on A151 ODN’s immunosuppressive and anti-fibrotic potential, we tested its therapeutic role on bleomycin-induced lung inflammation and fibrosis on mice which reflects different phases of systemic sclerosis. First in vivo experiment that A151 ODN was used prior to bleomycin administration revealed that A151 ODN could prevent development of systemic sclerosis by reducing immune cell recruitment into alveolar space and suppressing the secretion of inflammatory cytokines. After that, we investigated if A151 ODN could abolish established lung inflammation triggered by bleomycin instillation. For that, we treated animals with an A151 ODN either in free form or encapsulated within anionic liposomes after lung inflammation was initiated following bleomycin instillation. Data indicated that A151 ODN reduced macrophage activation marker expressions, monocyte and neutrophil infiltration into alveolar space. Moreover, suppression on immune cells activation in bronchoalveolar lavage fluid (BALF) correlated with the inhibited cytokine production. As a result of reduced inflammation, pro-fibrotic gene expressions were less in A151 ODN-treated mice. Of note, liposomal encapsulation provided reduced gene expressions while failed to further enhance the immunosuppressive potential on surface marker expression or cytokine secretion of A151 ODN. Lastly, we tested whether treatment with liposome-encapsulated A151 ODN is still effective to regress fibrosis once it has been developed; therefore, we treated mice with single injection of liposomal A151 on different time points. Unfortunately, single instillation was insufficient to decrease fibrosis and macrophage activation as well as cytokine production. Taken together, our findings indicated that liposome-encapsulated A151 ODN is very potent to attenuate the lung inflammation whereas single injection was ineffective to regress established lung fibrosis.