TLRs Ligands Induce Cell Proliferation In The Intact Rat Spinal Cord And Its Anti-neoplasmic Activity

Most of multicellular organisms has evolutionarily formed a highly effective set of defensive immune system that can identify and scavenge pathogens, The vertebrate immune system can be divided into innate and acquired immune system .During long-term evolutionary process, the ability of innate immune system to discriminate between self and foreign pathogens is highly developed.The microorganisms that are present in the environment have remained a conserved component which is so-called pathogen—associated molecular patterns (PAMPs). The PAMPs can be recognized by a family of evolutionarily conserved receptor, known as the pattern recognition receptors (PRRs). TLRs are the key PRRs which have a crucial role in early host defense against invading pathogens and allow mammals to detect the presence of infection by recognizing PAMPs. The release of PAMPs during an infection provides a "danger signal" and triggers the defensive innate immune response. Activation of TLR signalling pathways by different PAMPs that converged on the nuclear translocation of a transcription factor called nuclear factor-κB (NF-κB) and leads to the expression of many genes that function in host defence, including inflammatory cytokines, chemokines, the major histocompatibility complex, co-stimulatory molecules and multiple effector molecules, which is the first line of defense against pathogens. And indicates that TLR play an important role in inducing the defensive innate immune response and Inflammatory processes and thus is a prerequisite for the induction of acquired immunity.Synthetic oligodeoxynucleotides (ODNs) that contain unmethylated CpG motifs (CpG-ODN) that are similar to those found in bacterial DNA, The innate immune system detects these unmethylated CpG motifs using TLR9, ActivationTLR9 by CpG-ODN initiates a cascade of innate immune response, including inducing the activation of NF-κB, improving the expression of co-stimulatory molecules, stimulating the secrete of a wide array of cytokines, cellular adhesion molecules and chemokines that create a pro-inflammatory and can trigger Th1-polarized. (IL-12) immune milieu. These effects are highly CpG specific: DNA lacking unmethylated CpG motifs does not result in cytokine/chemokines secretion, Thus, CpG-ODN is another prototypic molecular pattern by which the immune system recognizes pathogens. Together, owing to their strong immunostimulatory activity, these immunomodulatory ODNs have various potential therapeutic uses, a number of CpG-ODN are at various stages of preclinical and clinical evaluation as anti-tumor, antiviral, antibacterial, anti-inflammatory agents, anti-allergia and as adjuvant in immunotherapy.The immune system is the major regulating system of organisms that defend pathogen and maintain the balance of homeostasis. There is an intricate interaction of phylogentically ancient cellular processes of the innate immune system. TLRs extensively express on the surface of both the innate immune cells and specific immune cells. The research of many directions of TLRs, including the recognition of ligands, signal transduction, activation of different immune factors, et al. provide a new view for recognition of immune system function and pattern, as well as for the development mechanism of different disease and the host susceptibility to disease. Along with the rapidly integrity development of immunology and life science, the study that lay stress on the evolutionarily origin and the interaction between host and environment are new source of vitality for accelerating the research on immunology, and thus is the objective background for innate immunity that bas become the hot spot to study and research again. Here we report the effects of peripheral administration of different stimulators of the innate immune system, TLR ligands on the proliferation of spinal cord neural progenitor cells and the antineoplasmic activity of CpG-ODN.Part One TLR ligands induce progenitor cell proliferation in the intact rat spinal cordObjection: Inflammation and regeneration are evolutionarily linked processes. Stem cells/progenitor cells contribute to local repair processes. The persistence of neural progenitor cells (NPCs) in the brain and spinal cord has raised hopes for restoring Central Nervous System (CNS) function lost through disease or injury. However, the mechanisms regulating NPC proliferation and differentiation remain elusive. In an attempt to find stimulators of local stem/progenitor cell proliferation, we have screened for agents that, after systemic injection, stimulate NPC proliferation in the spinal cord an organ shielded by the blood-brain barrier (BBB). Method: Here we report the effects of peripheral administration of different stimulators of the innate immune system, Toll-like receptor (TLR) ligands on the proliferation of spinal cord NPCs using 5-bromo-2'-deoxyuridine (BrdU) tracing. Results: Bolus injection of phosphorothioate oligonucleotides (ODN) containing CpG motifs had no prominent effects on spinal cord NPCs proliferation, whereas single intraperitoneal injection of polyinosine-polycytidylic acid (Poly I:C, TLR3 ligand), lipopolysaccharide (LPS, TLR4 ligand), or R848 (TLR7/8 ligand) temporarily increased the number of BrdU~+ cells in the spinal cord. For Poly I:C or R848 treated groups, the density of BrdU~+ cells reached maximal levels on Days 2-3 post injection, and then rapidly declined to baseline levels. Only a few of proliferating cells were of microglial origin .But BrdU~+ /nestin~+ cells were found, suggestive of a proliferation of local progenitor cells. In addition, stimulation of NPC proliferation correlated with activation of the innate immune system, i.e. microglial cells. Interestingly, activation of both cell compartments was inhibited by corticosteroid dexamethasone, but not by indomethacin. Conclusion: These findings suggest an intricate interaction of phylogentically ancient cellular processes of the innate immune system and regeneration. Recognition of PAMPs via TLR receptors appear to link inflammatory and regenerative processes and are thus valuable tools to develop pharmacological agents to promote tissue repair.Part Two The preparation of Chitosan- CpG-ODN nanoparticle and its enhancement of antineoplasmic activityObjection: The gene therapy of tumor can be attained through introducing exogenous oligonucleotides (ODN) to alter the gene expression of tumour cell, eliminate or retard the growth of tumor cells, stop the neovascularization, activate the organism anti- neoantigen immune response and prevent the diffusion of tumor cells. CpG-ODNs are the TLR-9 ligands, Activation TLR9 by CpG DNA initiates a cascade of innate immune response and can induced Th1-polarizing cytokines.Owing to strong immunostimulatory activity of CpG-ODNs, CpG-ODNs are widely used in preclinical and clinical research as anti-tumor, antiviral, antibacterial, anti-inflammatory agents and as adjuvant in immunotherapy. As a nonviral genetic carrier, Chitosan has extensive prospect for application, Till now there is no any report about the chitosan-CpG-ODNs nanoparticle complexes transfection in vivo, here we transfect the chitosan-plasmid nanoparticle complexes into rat and Validate the antineoplasmic activity of CpG-ODN. Methods: To prepare the chitosan-CpG-ODNs nanoparticle complexes by using chitosan as coating material, the complexes were Ip injected into rats, we detect the kill rate of CTL by MTT and Apoptosis rate by FACS, anti-tumor cytokine secretion induced by CpG-DNA was analysed using ELISA. Results: The index of anti-tumor of the of chitosan-CpG-ODNs nanoparticle group were superior in all the groups. Conclusion: Our results indicate that antineoplasmic activity of CpG-ODN can be enhanced by chitosan, TLR9 exerts an important role in anti-tumor immunity and tumor immunologic surveillance and provided a substantial basis for tumor gene therapy and immunotherapy.In summary, more and more studies indicate that the innate immunity play an significant role in tumor immunity, TLRs disclose a good perspective for investigate the innate and adaptive immunity and provide a new cure method for different disease. The agents that enhance the pathway signal might be a better adjuvant for anti-pathogenic organism or anti-tumor. Activation TLR9 signal pathway by CpG DNA is probably a new preventive and curative strategy for many kinds of diseases, especially for cancer. In the near future, to develop new pharmacological agents targeting TLRs as cure site will be a potential immunotherapy measure.