Regulation Of TNF-alpha And IFN-Lambda 2 On Mast Cell Toll-like Receptors And The Expression Of Interferon-Lambda2 In Nasal Polyps From Allergic Rhinitis

Background Toll-like receptors(TLRs) are a highly conserved family of pattern recognition receptors(PAMP) that play an essential role in the induction of immunity. In recent years, studies have shown that TLRs may modulate the process of allergic diseases through multiple ways, which may provide an opportunity to study the mechanism of allergic disease and find a reasonable and effective treatment. As an important effector and regulator, Mast cells play an essential role not only in allergic disease, but also in the innate immunity of the host defense against pathogens. The latest studies show that TLRs can activate mast cell through TLR signaling pathways, and play an important role in the local production of cytokines, in the event of a variety of immune-related cells recruit and the involvement of the restructuring of the organization. This indicates that there may be a new pathogenic mechanism which is TLRs activated effector mechanisms of mast cell and it is different with the traditional mechanism, when mast cells in allergic diseases, particularly in the infected state. It will provide a new thinking for the role of TLRs in allergic diseases. Depth to explore the complex molecular mechanisms of mast cells and TLRs response diversity can open new avenues for the treatment of allergic diseases. In recent years, the incidence of allergic diseases has a significant increase in the world, not only affect the patient’s quality of life, and even endanger the lives of patients, this must arouse our attention. Further learn more about the mechanism of TLRs, it will help us to get a breakthrough in the treatment of allergic diseases.TNF-α(Tumor necrosis factor-alpha) is a cytokine which has multiple biological activities. It not only is involved in host immunity defense, but also as an important medium for the pathogenesis of inflammation, injury, and even the shock.Studies found that TNF-α released from activated mast cell could promote or aggravate the development of allergic diseases through various mechanisms. However, TNF-α whether or not affect the activation of mast cells by adjusting the TLRs, then play an important role in the regulation of the duration of allergic inflammation, has yet to be further studiedIFN-λ(Interferon-lambda) are a newly described member of the IFN family including IFN-λ 1(IL-29), IFN-λ 2(IL-28A) and IFN-λ 3(IL-28B), wherein IL-29 and IL-28 A have the stronger biological activity. The current research focuses on the anti-viral activity, and few reports of inflammation-related diseases. The latest studies found that IL-28 A could improve mice respiratory symptoms similar with allergic asthma. Previous studies in our laboratory also reported the serum levels of circulating IL-28 A were highly in the patients of asthma and allergic rhinitis, and mast cells could release IL-29 when stimulated with allergens. Exogenous IL-29 has a regulatory role of mast cells. All of these results suggest that IFN-lambda may be involved in the development of allergic disease. Since IL-28 A is 90% homologous with IL-29, IL-28 A may also have anti-inflammatory and anti-allergic effects, in addition to antiviral activity. Depth to explore the biological activity of IL-28 A and its relationship with inflammatory and allergic diseases, and whether IL-28 A was involved in host defense response by mast cells TLRs response, all of them will help to investigate the mechanism of allergic disease and provide a new choice for clinical therapy.The modulation of Toll-like receptor(TLR) in mast cell by TNF-α and IL-28 A Objective To investigate the effect of TNF-α and IL-28 A on the expression of TLRs from murine mast cell line(p815)Methods TNF-α and IL-28 A were used to challenge murine mast cells P815 at various concentration(0.1、1.0 、10、100 ng/ml)and cells were collected at different time point(2 h, 6 h and 16 h) respectively. Cells were fixed by 2% paraformaldehyde, resuspended by PBS and then added to respective TLRs antibody. Flow cytometry was used to detect the expression of TLRs in mast cell line P815 cells at the protein level. When positive result, we use the same method to stimulate cells. Reverse transcription polymerase chain reaction(RT-PCR), real-time PCR, and immunofluorescent staining were used to detect the expression of TLRs in mast cell line P815 cells at the levels of m RNA and protein. We also detected the changes of TLRs expression in P815 before and after TNF-a stimulated at various concentrations(1.0、3.0 ug/ml).Result 1. Using flow cytometry, we found that TNF-a upregulated expression of TLR4 in P815 cells in a concentration dependent manner. This result was further validated by Real-time PCR, and immunofluorescent staining. FCM results showed that different concentrations of TNF-alpha role for 2 h, 6 h, 16 h, only the expression of TLR4 has statistically difference compared with control group(P<0.05). TNF-α increase the expression of TLR4 protein starts at 2 h and in dose-dependent manner at 16 h. P815 was stimulated with TNF-α(100ng/ml)about 16 h, the expression of TLR4 was up-regulated about 2.6 fold compared with control group. Anti-TNF-α antibody could inhibit this effects, the result has statistically difference compared with control(P<0.050).2. Flow cytometry demonstrated that IL-28 A has no effect on the expression of TLRs in P815, and also no between different concentrations.Conclusion TNF-a has no effect on the expression of TLRs(1、2、3、5、6、7、8、9)in P815, but can up-regulate the expression of TLR4. Anti-TNF-a antibody can inhibit this effect. These results demonstrate TNF-α which was produced by mast cell can promote the expression of TLR4.TNF-α can promote the expression TLR4 to enhance the allergy reaction. IL-28 A has no effect on the expression of TLRs in P815.The expression of IL-28 A in nasal polyps from patients with allergic rhinitis Objective To detect the expression of IL-28 A in T cells, B cells, macrophages, neutrophils, eosinophils and fibroblasts of nasal polyps from patients with allergic rhinitisMethods 15 nasal polyps specimens were collected randomly from patients with allergic rhinitis. Specimens were fixed and embedded in paraffin wax. Paraffinized tissues were sectioned to 3μm thickness. Individually labeled by immunohistochemistry staining was used to detect the expression of IL-28 A in nasal polyps. To further determine which cell type expresses IL-28 A, double immunofluorescence staining was performed by confocal laser scanning to examine the co-expressed with T cells, B cells, macrophages, neutrophils, eosinophils and fibroblasts.Results IL-28 A is not expressed in T cells and macrophages but expressed in B cells, neutrophils, eosinophils and fibroblasts, especially in neutrophils.Conclusion IL-28 A is strongly expressed in nasal polyps from patients with allergic rhinitis. The mainly cell source of IL-28 A is B cells and eosinophils, and neutrophils also have a little weak expression. The above results show that IL-28 A may play an important role in allergic diseases through B cells and eosinophils.