CpG oligodeoxynucleotides (ODN) are short single-stranded synthetic DNA molecules that activate the immune system and have been found to be effective for preventing and treating infectious diseases, allergies, and cancers. Structurally distinct classes of synthetic ODN expressing CpG motifs differentially activate human immune cells. K-type ODN (K-ODN), which have progressed into human clinical trials as vaccine adjuvants and immunotherapeutic agents, are strong activators of B cells and trigger plasmacytoid dendritic cells (pDCs) to differentiate and produce tumor necrosis factor-α (TNFα). In contrast, D-type ODN (D-ODN) stimulate large amounts of interferon-α (IFNα) secretion from pDCs. This activity depends on the ability of D-ODN to adopt nanometer-sized G quadruplex-based structures, complicating their manufacturing and hampering their progress into the clinic. In search of a D-ODN substitute, we attempted to multimerize K-ODN into stable nanostructures using cationic peptides. We show that short ODN with a rigid secondary structure form nuclease-resistant nanorings after condensation with the HIV-derived peptide Tat(47-57). The nanorings enhanced cellular internalization, targeted the ODN to early endosomes, and induced a robust IFNα response from human pDCs. Compared to the conventional K-ODN, nanorings boosted T helper 1-mediated immune responses in mice immunized with the inactivated foot and mouth disease virus vaccine and generated superior antitumor immunity when used as a therapeutic tumor vaccine adjuvant in C57BL/6 mice bearing ovalbumin-expressing EG.7 thymoma tumors. These results suggest that the nanorings can act as D-ODN surrogates and may find a niche for further clinical applications.

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 dose- and 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.

Telomeric regions of mammalian chromosomes contain suppressive TTAGGG motifs that inhibit several proinflammatory and Th1-biased immune responses. Synthetic oligodeoxynucleotides (ODN) expressing suppressive motifs can reproduce the down-regulatory activity of mammalian telomeric repeats and have proven effective in the prevention and treatment of several autoimmune and autoinflammatory diseases. Endotoxin-induced uveitis (EIU) is an established animal model of acute ocular inflammation induced by LPS administration. Augmented expression of proinflammatory cytokines/chemokines such as TNF␣, IL-6, and MCP1 and bactericidal nitric oxide production mediated by LPS contribute to the development of EIU. Suppressing these mediators using agents that are devoid of undesirable systemic side effects may help prevent the development of EIU. This study demonstrates the selective down-regulatory role of suppressive ODN after (i) local or (ii) systemic treatment in EIU-induced rabbits and mice. Our results indicate that suppressive ODN down-regulate at both the transcript and protein levels of several proinflammatory cytokines and chemokines as well as nitric oxide and co-stimulatory surface marker molecules when administrated prior to, simultaneously with, or even after LPS challenge, thereby significantly reducing ocular inflammation in both rabbit and mouse eyes. These findings strongly suggest that suppressive ODN is a potent candidate for the prevention of uveitis and could be applied as a novel DNAbased immunoregulatory agent to control other autoimmune or autoinflammatory diseases.DNA and RNA are the essential components of all living organisms. Accumulated evidence strongly suggests that these nucleic acids have multiple and complex effects on the immune system and are more than a blueprint of life (1, 2). On one hand, due to their high unmethylated CpG motif frequency, bacterial DNAs are recognized as "non-self" via TLR9 (Toll-like receptor 9) and trigger an innate immune response characterized by the proliferation and maturation of B cells, natural killer cells, and plasmacytoid dendritic cells and the secretion of T-helper 1-type cytokines, chemokines, and/or multivalent immunoglobulins (3-8). On the other hand, telomeric regions of mammalian chromosomes contain suppressive TTAGGG motifs that can inhibit several TLR-dependent and TLR-independent Th1-mediated immune responses. Of note, these motifs are underrepresented in the prokaryotic genome. Synthetic singlestranded oligodeoxynucleotides (ODN) 3 containing repetitive TTAGGG motifs mimic this effect (1, 9 -11). Previous studies revealed that deleterious inflammatory responses to a host can be down-regulated by suppressive ODN. In vitro, suppressive ODN inhibits the production of several proinflammatory cytokines and chemokines induced by bacteria (1,(12)(13)(14). Furthermore, in vivo suppressive ODN administration reduces the frequency and severity of several autoimmune and inflammatory diseases such as arthritis, systemic lupus erythematosus, pulmonary inflam...

Some of the important properties of probiotics are the ability to survive during gastrointestinal transit and to modulate the immune functions. The objectives of the reported study were to assess in vivo gastrointestinal survival of orally administered Pediococcus pentosaceus OZF using an animal model BALB/c mice, and to examine its effects on the immune response. Following oral administration to mice, the ability of Pediococcus pentosaceus OZF to pass and survive through the mouse gastrointestinal system was investigated by analyzing the recovery of the strain in fecal samples. Microbiological and polymerase chain reaction (PCR) methods proved that the strain OZF could overcome specific conditions in the gastrointestinal tract of mice and reach the intestine alive after ingestion. To observe the effect of oral administration on immune response, IL-6, IL-12 and IFN-γ were measured by ELISA, and the strain OZF was found to cause increases in IL-6 synthesis in regularly fed mice. However, stimulation was carried out with various concentrations of bacterial ssDNA and heat killed cells of Pediococcus pentosaceus OZF. The heat killed cells of the strain OZF were shown to produce IFNγ independently from IL-12. On the other hand, a significant difference between control and experimental group was noticed when lipopolysaccharide, a TLR4 (toll like receptor) ligand, was used. Overall, Pediococcus pentosaceus OZF may be a valuable probiotic strain for therapeutic uses. Nevertheless, further studies on the mechanisms of immunomodulatory effect will allow for better clarification of the immune functions of this strain.

Immunostimulatory properties of mushroom derived polysaccharides (PS) as stand-alone agents were tested. Next, PS were nanocomplexed with polyI:C (pIC) to yield stable nanoparticles around 200 nm in size evidenced by atomic force microscopy and dynamic light scattering analyses. PSs were selectively engaged by cells expressing TLR2 and initiated NFκB dependent signaling cascade leading to a Th1-biased cytokine/chemokine secretion in addition to bactericidal nitric oxide (NO) production from macrophages. Moreover, cells treated with nanoparticles led to synergistic IL6, production and upregulation of TNFα, MIP3α, IFNγ and IP10 transcript expression. In mice, PS-Ovalbumin-pIC formulation surpassed anti-OVA IgG responses when compared to either PS-OVA or pIC-OVA mediated immunity. Our results revealed that signal transduction initiated both by TLR2 and TLR3 via co-delivery of pIC by PS in nanoparticle depot delivery system is an effective immunization strategy. The present work implicate that the PS and nucleic acid based nanoparticle approach along with protein antigens can be harnessed to prevent infectious diseases.

Behçet’s disease (BD) activity is characterised by sustained, over-exuberant immune activation, yet the underlying mechanisms leading to active BD state are poorly defined. Herein, we show that the human cathelicidin derived antimicrobial peptide LL37 associates with and directs plasma extracellular vesicles (EV) to immune cells, thereby leading to enhanced immune activation aggravating BD pathology. Notably, disease activity was correlated with elevated levels of circulating LL37 and EV plasma concentration. Stimulation of healthy PBMC with active BD patient EVs induced heightened IL1β, IFNα, IL6 and IP10 secretion compared to healthy and inactive BD EVs. Remarkably, when mixed with LL37, healthy plasma-EVs triggered a robust immune activation replicating the pathology inducing properties of BD EVs. The findings of this study could be of clinical interest in the management of BD, implicating LL37/EV association as one of the major contributors of BD pathogenesis. Abbreviations: BD: Behçet’s disease; EV: extracellular vesicle; BB: binding buffer; AnV: annexin V; autologEV: autologous extracellular vesicles; alloEV: allogeneic extracellular vesicles

Type I interferon inducers may potentially be engineered to function as antiviral and anticancer agents, or alternatively, vaccine adjuvants, all of which may have clinical applications. We recently described a simple strategy to convert a Toll-like receptor 9 (TLR9) agonist devoid of interferon α (IFNα) stimulating activity into a robust Type I interferon inducer with potent vaccine adjuvant activity.

Synthetic oligodeoxynucleotides (ODN) expressing suppressive TTAGGG motifs effectively down-regulate the production of proinflammatory and Th1 cytokines elicited either by Toll-Like Receptor (TLR) dependent or independent immune stimuli. Although initially identified by their ability to block CpG-induced immune activation, it was shown that suppressive ODN was able to block multiple forms of immune stimulation and to be effective in the prevention and treatment of pathologic autoimmune and autoinflammatory diseases. Endotoxin-induced uveitis (EIU) is an established animal model of acute ocular inflammation. It is induced by either systemic or intravitreal administration of lipopolysaccharide (LPS). Familial Mediaterranean Fever (FMF) is an autosomal recessive periodic fever disease characterized by recurrent, self-limiting, febrile, inflammatory attacks of the serosal membranes such as peritoneum, pleura, and synovia. Present study aims to demonstrate the downregulatory effect of the suppressive DNA on EIU and FMF as local autoimmune and systemic autoinflammatory diseases respectively. Results indicated that LPS induced EIU in mice, A151 treatment strongly downregulated either expression or protein levels of IL6, IP10, IL1β, MIP1β, MIP3a or iNOS. Moreover, FMF patient PBMCs were more responsive to TLR ligand stimulation compared to healthy subjects and A151 strongly reversed this activation and suppressed several key Th1-cytokine/chemokine levels