Browsing by Subject "Innate immunity"

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Open Access
Biological properties of extracellular vesicles and their physiological functions
(Taylor & Francis, 2015) Yáñez-Mó, M.; Siljander, P. R. M.; Andreu, Z.; Zavec, A. B.; Borràs, F. E.; Buzas, E. I.; Buzas, K.; Casal, E.; Cappello, F.; Carvalho, J.; Colás, E.; Cordeiro-Da, S. A.; Fais, S.; Falcon-Perez, J. M.; Ghobrial, I. M.; Giebel, B.; Gimona, M.; Graner, M.; Gursel, I.; Gursel, M.; Heegaard, N. H. H.; Hendrix, A.; Kierulf, P.; Kokubun, K.; Kosanovic, M.; Kralj-Iglic, V.; Krämer-Albers, E. M.; Laitinen, S.; Lässer, C.; Lener, T.; Ligeti, E.; Line, A.; Lipps, G.; Llorente, A.; Lötvall, J.; Manček-Keber, M.; Marcilla, A.; Mittelbrunn, M.; Nazarenko, I.; Nolte-'t Hoen, E. N. M.; Nyman, T. A.; O'Driscoll, L.; Olivan, M.; Oliveira, C.; Pállinger, E.; Del Portillo, H. A.; Reventós, J.; Rigau, M.; Rohde, E.; Sammar, M.; Sánchez-Madrid, F.; Santarém, N.; Schallmoser, K.; Ostenfeld, M. S.; Stoorvogel, W.; Stukelj, R.; Grein V. D. S.G.; Helena,ü V. M.; Wauben, M. H. M.; De Wever, O.
In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells.While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.
Open Access
Chitosan polysaccharide suppress toll like receptor dependent immune response
(Turkish Society of Immunology, 2015) Tincer G.; Bayyurt, B.; Arıca, Y.M.; Gürsel İ.
Objectives: Chitosan is a widely used vaccine or anti-cancer delivery vehicle. In this study, we investigated the immunomodulatory effect of chitosan/pIC nanocomplexes on mouse immune cells. Materials and methods: Proliferative and cytotoxic features of chitosan were tested via CCK-8 assay on RAW 264. 7. IL-1β production was assessed via ELISA from PEC supernatants. TNF-α, and NO induction from chitosan treated RAW cells detected by ELISA and Griess assay, respectively. mRNA message levels of TLRs and cytokines on macrophages in response to chitosan/pIC nanocomplex treatments were evaluated by RT-PCR. Results: Results revealed that chitosan is non-toxic to cells, however, proliferative capacities of macrophages were reduced by chitosan administration. Mouse PECs treated with chitosan, led to NLRP3 dependent inflammasome activation as evidenced by dose-dependent IL-1β secretion. Chitosan/pIC nanocomplexes did not improve immunostimulatory action of pIC on RAW cells, since TNF-α and NO productions remained unaltered. Expression levels of several TLRs, CXCL-16 and IFN-α messages from mouse splenocytes were down regulated in response to chitosan/pIC nanocomplex treatment. Conclusion: Our results revealed that chitosan is an anti-proliferative and inflammasome triggering macromolecule on immune cells. Utilization of chitosan as a carrier system is of concern for immunotherapeutic applications. © 2015 Turkish Journal of Immunology.
Open Access
Elucidating immunomodulatory effects of telomeric repeat mimicking synthetic A151 oligodeoxynucleotide on immune cell transcriptome
(2019-09) Yazar, Volkan
Recent evidence revealed that DNA is beyond just the blueprint of life; it is also involved in immunomodulation. Unmethylated Cytosine-phosphate-Guanine (CpG) motifs of prokaryotic DNA stimulate immune response by interacting with Toll-like receptor 9 (TLR9). This interaction is mimicked using synthetic oligodeoxynucleotides (ODN) bearing similar DNA motifs to boost vaccinedriven immune response in human. Conversely, mammalian telomeric ends expressing TTAGGG repeats suppress immune response and contribute to fine-tuning of delicate immune balance. In this respect, suppressive ODN A151 with such G-rich telomeric repeats has proven useful in downregulating immune response; an overly active immune response is just as harmful to the host, as in the case of autoimmune disorders. Both CpG ODN and A151 are currently under preclinical/clinical trials with the aim of averting or medically treating a wide range of conditions from cancer to infectious disease or from autoimmune to autoinflammatory conditions. Contrary to CpG ODN, A151 literature is very limited and its modus operandi at gene level remains more of a mystery. Additionally, the degree, duration and breath of A151-induced alterations in immune transcriptome appear partially understood. Given the medical potential A151 holds for immunosuppressive therapy in human as a “self-molecule”, understanding the underlying molecular mechanisms via which A151 operates is invaluable. Toward this end, we attempted to uncover the unidentified features lying behind A151 ODNs immunosuppressive effects on immune cell transcriptome using a combined analysis approach of microarray data in this thesis. We demonstrated for the first time that A151 ODN deprives the cells energy by ceasing cellular uptake of fundamental molecules into the immune cells after derailing the entire intracellular trafficking. Putting it another way, A151 does not directly act on immune system cells but actually suffocates the cells by messing with intracellular trafficking, thereby blocking cellular uptake of fundamental molecules like glucose and glutamine. As such, immune suppression is just an indirect consequence of this larger cellular chaos. Our results indicated that this phenomenon occurs independent of CpG ODN stimulation of the cells and in a timely manner. Most, if not all, regulators of intracellular trafficking, vesicle signaling, and membrane protein transportation were found downregulated after incubation of cells with A151 at a physiologically relevant concentration, as well, implying full-blown entry to this intracellular turmoil at cellular level. The A151 effect on immune transcriptome was not just restricted to setting off a chaos for intracellular dynamics; novel long non-coding RNAs (lncRNAs) with immunometabolic activities were identified within the scope of this study among elements potentially regulated by A151, such as Lncpint, Malat1 and H2-T10 just to name a few. The involvement of lncRNAs in immune regulation is a well-documented phenomenon. Finally, our data showed that as an epiphenomenon of the intracellular turmoil mentioned above A151 has a deep impact in immune cells on mTOR network, the cardinal network of cellular energetics, growth, proliferation, and survival. A major shift in expression profile of relevant genes, i.e. downregulation of many activators of mTOR signaling along with core mTOR components, was validated on the benchtop after different layers of experimental validation using a wide range of marker genes and functional assays, reflecting A151’s ability to vastly shape dynamics of metabolism in favor of a metabolically inert state in macrophages and in B-cells. This knowledge will expand the breadth of A151 therapy in the clinics.
Open Access
Enhanced immunostimulatory activity of cyclic dinucleotides on mouse cells when complexed with a cell-penetrating peptide or combined with CpG
(Wiley - V C H Verlag GmbH & Co. KGaA, 2015) Yildiz, S.; Alpdundar, E.; Gungor, B.; Kahraman, T.; Bayyurt, B.; Gursel, I.; Gursel, M.
Recognition of pathogen-derived nucleic acids by immune cells is critical for the activation of protective innate immune responses. Bacterial cyclic dinucleotides (CDNs) are small nucleic acids that are directly recognized by the cytosolic DNA sensor STING (stimulator of IFN genes), initiating a response characterized by proinflammatory cytokine and type I IFN production. Strategies to improve the immune stimulatory activities of CDNs can further their potential for clinical development. Here, we demonstrate that a simple complex of cylic-di-GMP with a cell-penetrating peptide enhances both cellular delivery and biological activity of the cyclic-di-GMP in murine splenocytes. Furthermore, our findings establish that activation of the TLR-dependent and TLR-independent DNA recognition pathways through combined use of CpG oligonucleotide (ODN) and CDN results in synergistic activity, augmenting cytokine production (IFN-α/β, IL-6, TNF-α, IP-10), costimulatory molecule upregulation (MHC class II, CD86), and antigen-specific humoral and cellular immunity. Results presented herein indicate that 3′3′-cGAMP, a recently identified bacterial CDN, is a superior stimulator of IFN genes ligand than cyclic-di-GMP in human PBMCs. Collectively, these findings suggest that the immune-stimulatory properties of CDNs can be augmented through peptide complexation or synergistic use with CpG oligonucleotide and may be of interest for the development of CDN-based immunotherapeutic agents.
Open Access
Immunomodulatory effects of TLR ligands and polysaccharide combinations : strategies to augment innate immune response
(2007) Tincer, Gizem
Microbial infection initiates multiple TLR ligand mediated signaling cascade on innate immune cells. While some TLRs trigger a Th1 biased immune activation, others may lead to a Th2 dominant immune response. Extracellular (TLR1, 2, 4, 5, 6, 10, and 11) vs endosome-associated TLRs (TLR3, 7/8, and 9) display differential immune activation and cytokine milieu. Understanding contrasting and synergistic behaviors of these TLR subclasses when mixed together may lead to more potent formulations for immunotherapy. Delivery and retaining the stability of nucleic acid based labile TLR ligands to the site of immunologically relevant cells is also a challenge. In the first part of the thesis, optimum TLR combinations with differential immune effects will be brought into light. Next, immunomodulatory effect of a natural polysaccharide (PS) will be characterized. Finally the ability of a PS carrier to form complex with ligands of nucleic acid sensing TLRs and its potential as a controlled delivery vehicle to stimulate the immune cells will be documented. In brief, our results suggest that different PS types extracted from various mushroom sources are immunostimulatory and are targeted to TLR2/6 for delivery of other relevant stimulants. Moreover, certain TLR ligand combinations can be harnessed to induce more robust immune activation compared to their stand alone counterparts. This knowledge will pave the way for establishing an effective PS based carrier of DNA/RNA ligands thus, more effective immunotherapeutic strategies for treating infectious and other local or systemic diseases be possible.
Open Access
Immunotherapeutic applications of nucleic acid based tlr agonists and antagonists
(2014) Yağcı, Fuat Cem
Recent evidence suggests that genetic material is not just the blueprint of life. Depending on the type of the source that genomic material is extracted or the type of motif that DNA harbors DNA could be immunostimulatory or immunosuppressive to innate immune system cells. Unmethylated CpG motifs, from bacterial genome is recognized by TLR9 expressing cells as “non-self” and initiates an orchestrated immune activation characteristics of Th1-biased immunity. Conversely, mammalian genome rich in G-runs motifs, such as telomeric ends expressing TTAGGG repeats downregulate Th1-biased immune responses and contributes to restore over exuberant immune response. Several TLR agonists/antagonists candidates are currently under preclinical/clinical trials to prevent or treat cancer, infectious disease, allergic disorders and autoimmune or autoinflammatory diseases. In this thesis, we attempted to broaden the application of nucleic acid based TLR therapy by investigating their beneficial effects either as vaccine adjuvants against Foot and Mouse Disease or to test as immunosuppressive agents that may control autoimmune uveitis. We showed that an immunosuppressive ODN namely A151 downregulated severity of an established animal model of uveitis, endotoxin induced uveitis (EIU), up to more than 60% histologically, or more than 80% based on cytokine production such as MIP3α, IL6, IFNγ from uveitic animals. In order to compare effects of this biological immunosuppressive agent, next we studied effects of a broad-spectrum immunosuppressive drug, namely cyclosporine A in an emulsion form, commercially known as Restasis™. Our results indicated that Restasis™ had significantly lower capacity to reduce disease severity and downregulate in vivo chemokine or cytokine levels compared to A151. The second theme of this thesis was to demonstrate effective utilization of CpG ODN as an immunostimulatory agent. The adjuvant effect of CpG ODN 1555-PS in Foot and Mouth Disease (FMD) vaccine formulations were tested. In this study, CpG ODNs were formulated either with commercially used monovalent vaccine or mixed with free Serotype-O Ag. Data suggested that, in mice, inclusion of CpG ODN as an adjuvant, spared the Ag by 6 fold and the vaccine dependent virus neutralization titers were not only higher but also long lasting compared to commercial monovalent vaccine. CpG inclusion in the FMD vaccine helped to generate 1.5-2 fold more cell mediated immunity 24 h after booster injection. Implicating that virus infection could be more effectively controlled by the novel approach. In the last part of this thesis, effects of CpG ODNs as prophylactic agents for newborn broiler chicken were tested in vivo. Our results suggested that CpG ODN pretreatment not only significantly reduced mortality rates (> 2.0 %) but also contributed to growth performance of these industrially important animals. We demonstrated that nucleic acid based agonists and antagonists might be of great potential to be developed as therapeutic agents either in the clinic or forcontrolling health of industrially important animals.
Open Access
Impaired toll like receptor-7 and 9 induced immune activation in chronic spinal cord injured patients contributes to immune dysfunction
(Public Library of Science, 2017) Gucluler, G.; Adiguzel, E.; Gungor, B.; Kahraman, T.; Gursel, M.; Yilmaz, B.; Gursel, I.
Reduced immune activation or immunosuppression is seen in patients withneurological diseases. Urinary and respiratory infections mainly manifested as septicemia and pneumonia are the most frequent complications following spinal cord injuries and they account for the majority of deaths. The underlying reason of these losses is believed to arise due to impaired immune responses to pathogens. Here, we hypothesized that susceptibility to infections of chronic spinal cord injured (SCI) patients might be due to impairment in recognition of pathogen associated molecular patterns and subsequently declining innate and adaptive immune responses that lead to immune dysfunction. We tested our hypothesis on healthy and chronic SCI patients with a level of injury above T-6. Donor PBMCs were isolated and stimulated with different toll like receptor ligands and T-cell inducers aiming to investigate whether chronic SCI patients display differential immune activation to multiple innate and adaptive immune cell stimulants. We demonstrate that SCI patients' B-cell and plasmacytoid dendritic cells retain their functionality in response to TLR7 and TLR9 ligand stimulation as they secreted similar levels of IL6 and IFNα. The immune dysfunction is not probably due to impaired T-cell function, since neither CD4+ T-cell dependent IFNγ producing cell number nor IL10 producing regulatory T-cells resulted different outcomes in response to PMA-Ionomycin and PHA-LPS stimulation, respectively. We showed that TLR7 dependent IFNγ and IP10 levels and TLR9 mediated APC function reduced substantially in SCI patients compared to healthy subjects. More importantly, IP10 producing monocytes were significantly fewer compared to healthy subjects in response to TLR7 and TLR9 stimulation of SCI PBMCs. When taken together this work implicated that these defects could contribute to persistent complications due to increased susceptibility to infections of chronic SCI patients. © 2017 Gucluler et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Open Access
In vivo applications of liposomal vaccines encapsulating single or dual pathogenassociated molecular patterns
(2017-03) Bayyurt Kocabaş, Banu
Nucleic acid-based pattern recognition receptor (PRR) agonists are promising adjuvants and immunotherapeutic agents. Combination of PRR ligands potentiates immune response by providing synergistic immune activity via triggering different signaling pathways and may impact antigen dependent T-cell immune memory. However, the duration of short circulation due to nuclease attacks is hampering their clinical performance. Liposomes enable protein and nucleic acid based compounds to have high encapsulation efficiency. Herein, we aimed to develop liposomal carrier systems that co-encapsulating single TLR9 or combinations with TLR3 or STING ligands and assess their potential as adjuvants and immunostimulatory agents in in vivo applications. Liposomal dual nucleic acid formulations induced synergistic innate immune activation, enhanced cytokine production along with internalization capacity of ligands. In anti-cancer vaccine study, CpG ODN and poly(I:C) coencapsulation significantly increased OVA-specific Th1-biased immune even after eight months post-booster injection. Challenge with OVA-expressing tumor cell line, E.G7, demonstrated that mice immunized with liposomes co-encapsulating CpG ODN and poly(I:C) had significantly slower tumor progression dependent on OVAspecific cytotoxic memory T-cells. In our second in vivo application, liposomal CDN and TLR9 therapy led to 80% remission of established melanoma tumor. Increased IgG2c/IgG1 ratio in mice treated with liposomal formulations indicating the development of antigen specific Th1-biased immunity was observed. Furthermore, along with the treatment, IFN-dual ligands into liposomes enhanced the anti-tumor activity of single ligands. In the third part, immunization with CpG ODN loaded liposomal formulations together with antigens increased antigen-specific humoral response against FMDV and Helicobacter. In addition, the liposomal CpG ODN reduced bacterial gastric colonization by antigen-dependent Th1 and Th17 immune responses after helicobacter challenging.
Open Access
Purinergic regulation of the immune system
(Nature Publishing Group, 2016) Cekic, C.; Linden, J.
Cellular stress or apoptosis triggers the release of ATP, ADP and other nucleotides into the extracellular space. Extracellular nucleotides function as autocrine and paracrine signalling molecules by activating cell-surface P2 purinergic receptors that elicit pro-inflammatory immune responses. Over time, extracellular nucleotides are metabolized to adenosine, leading to reduced P2 signalling and increased signalling through anti-inflammatory adenosine (P1 purinergic) receptors. Here, we review how local purinergic signalling changes over time during tissue responses to injury or disease, and we discuss the potential of targeting purinergic signalling pathways for the immunotherapeutic treatment of ischaemia, organ transplantation, autoimmunity or cancer.
Open Access
Self-nanoparticle forming immunostimulatory DNA : structure-function relationship studies
(2009) Mammadov, Rashad
Toll-like receptors (TLRs) are one the most critical and widely studied members of the family of pattern recognition receptors expressed on innate immune cells. They recognize microbial signatures, such as bacterial/viral DNA, LPS, from gram negative bacteria, peptidoglycan from gram positive bacteria, zymosan from yeast, lipopeptides or profilin protein from parasites, and even single or double stranded RNA of viruses. Among several members of TLR family, TLR9, that recognizes microbial unmethylated dinucleotide motifs on DNA initiate a robust Th1- biased inflammatory response. Synthetic oligodeoxynucleotides expressing unmethylated CpG motifs, mimic bacterial DNA effect and can be harnessed for the treatment of health problems ranging from infectious diseases to cancer, or to allergy/asthma as well as stand alone immunoprotective agents and also as a vaccine adjuvants that improve protection against pathogens. To date, various classes of CpG ODNs have been identified and were shown to induce differential immune activation in mice and man. Distinct structure-function relationship analyses revealed that these single-stranded linear ODNs alter the immune milieu as they are formulated to form complex multimeric DNA aggregates. Recently, Guanosine-rich D type CpG ODNs has been reported to form complex aggregates, that are differentially regulating immune cells to mount an anti-viral immunity. However, the clinical trials of this type are hampered mainly due to batch to batch variation during large-scale synthesis. To the best of our knowledge, there is no report on self-nanoparticle forming DNA except G-rich sequences. This thesis project was designed to generate stable, selfnanoparticle forming, G-run free, CpG expressing ODNs. In this thesis, we designed a new generation CpG ODN, then characterized their structural and immunological properties. Our results suggest that dendrimeric structure confers higher immunostimulatory potential unparallel to conventional ODNs. Following four hours of in vivo ODN administration into mice indicated that nanoparticle-forming CpG ODNs initiated substantially high spleen and peritoneal exudate cell activation as evidenced by IFNγ and IL-12 production from culture medium. In order to shed light on the uptake and binding mechanisms, blocking experiments revealed that at least one type of scavenger receptor is critical for nanoparticle ODN internalization. Collectively, these data suggested that the improved stability to nucleases along with significantly higher binding to immune cells (no additional ODN formulation is required) seem to be the critical factors contributing to the nanoparticle CpG ODN mediated immune activation. The in vitro and in vivo performances implicated that these next generation immune stimulatory DNA molecules are promising candidates for various clinical applications.