Pretreated Intranasal Administration Of TLR9 Ligand CpG Oligodeoxynucleiotides Prevent Development Of Allergic Rhinitis In Junvenile Guinea Pigs

Allergic airway diseases are prevailing in the world's population and allergic rhinitis is one of the most common in allergic airway inflammation. In a recent study involving individuals aged 16-60 years from 10 European countries, the prevalence of allergic respiratory disorders was estimated to be between 12 and 34%, and the average prevalence of allergic rhinitis was 11% in our country.Allergic rhinitis encompasses nasal symptoms such as sneezing, and running, congested or itchy nose.While the symptoms of allergic rhinitis are not life-threatening, they can have a substantial impact on Patients'quality of life (QoL) and daily functioning, causing daytime sleepiness, sleep disturbances, impaired social functioning, anddiminished productivity. Furthmore, emerging studies pointed out the link between allergic rhinitis and asthma,which was summarized"one airway, one disease". Allergen immunotherapy was initially reported to be effective for the desensitization of pollen allergic patients by Noon in 1911, and for many years IT was a first-line therapy for respiratory allergic diseases. However,in the last decade, IT has gradually fallen out of favor, in large part due to its limited scope of efficacy and its risk of eliciting rare but life-threatening anaphylactic reactions.Thus, it would be of interest to search a new method or modiffied route for immunotherapy. Studies of this question should be helpful for treatmeat of allergic rhinits.CpG oligonucleiotides (CpG ODN) is a unique synthesized DNA sequence with CpG motif which mimicry bacterial DNA. Synthetic oligodeoxynucleotides are known to be excellent immune adjuvants to drive Th1 immune responses in various animal disease models which includes allergic rhinitis and athma et al. Furthermore, Kenji Takabayashi,et al suggested intranasal CpG ODN immunotherapy is more effective than intradermal immunotherapy for the induction of airway allergen tolerance in Th2-sensitized mice. As far as we know, TLR9 is the only receptor of CpG DNAs. Expression of TLR9 in antigen presenting cells such as Dendritic cell have been proved while whether nasal epithelial cells express TLR9 have not been well understood.According to the hygiene hypopthesis, increasing exposure to infectious agents in juvenile individuals may reduce the risk of suffering of allergic rhinitis. CpG ODN is a functional Th1 reaction promoter which can also be regarded as infectious agents from the sources while may in turn supress allergic Th2 reaction. Can CpG ODN intronanal deliveration prevent the development of allergic rhinits at priming phase?So we aimed to evaluate the therapeutic effects of pretreated intransal administration of CpG ODN on prevetion of allgic rhinitis.First parts of my research focused on expression of TLR9 in human nasal epithelial cells by semi-quantitative reverse transcription-polymerase chain reaction, immunohistochemistry and flow cytometry. First, culture human primary nasal epithelial cells (HNECs). Then analyze expression of TLR9 in primary cultured HNECs was measured by semi-quantitative RT-PCR and immunohistochemistry and flow cytometry. Primary cultured HNECs were observed by 400×optical microscopes. Round or irregular cells stick to the bottom of cell culture plates. RT-PCR shows mRNA expressions of TLR9 were found in primary nasal epithelial cells by 1% agarose gel electrophoresis. Interestingly, through together analysis with gray level of GAPDH, expression of TLR9 mRNA in HNECs was higher than positive control PBMCs. Then expression of TLR9 in HNECs was proved by immunohistochemistry and flow cytometry. TLR9 is expressed by HNECs. Expression of TLR9 mRNA in nasal epithelial cells was higher than in PBMCs.In the second parts of this research, we evaluate the theraputic effect of intransal oligonucleotides with CpG motifs (CpG ODN) in prevention of alleric rhninitis in Juvenile guinea pigs.Juvenile guinea pigs, from 7 to 10 weeks of age were administrated with CpG ODN alone or combined with OVA at single dose concentration intranasally (on day 0, 5, 10, 15 sequencely) while control and blank group was administrated with saline. Both experimental and control animals were again sensitized by OVA (on day 18, 25) i.p., and 14 days after second sensitization animals were challenged by OVA intranasally (on day 39, 46). Two hours after challenge, animals were sacrificed. Then Hemotoxin and Eosin stain were carried out to analyze local eosinophilic reactions and nasal lesions. Local and sysmetic cytokines interleukin IL-5, leukotriene B4(LTB4), IFN-γlevels were examined by ELISA .In CpG ODN-administration or CpG ODN with OVA-administration group allergic rhinits symptoms were not as heavier as model control group.Compared to the model control group, CpG ODN-administration did not increase production of OVA-specific Th1 cytokine IFN-γbut decreased productions of ovalubmin-specific Th2 cytokines IL-5 both in serum and nasal specimen. Moreover, nasal lesions with infiltration of eosinophils and LTB4 levels were prominently reduced by the CpG ODN-treatment compared to the control animals.Immunoflourescence results shown lower ICAM-1 expression in nasal specimen of CpG group and CpG+OVA group animal compare to model control group.Finally, we can draw the conclusion that nasal epithelial cell expressed a certern level Toll like receptor 9 which ligand is CpG ODN and intransal deliveration of CpG oligonucleotides with or without allergen may be a effective way to prevent development of allergic rhinits .