Activation Of PKD1-NF-кB Signaling And Regulation Of Related Inflammatory Cytokines Expression Triggered By Aspergillums Fumigates

Background:Aspergillus fumigatus, one kind of fungus, is a saprophytic fungus that control the banlance of carbon and nitrogen in the environment.Aspergillus fumigatus is not the largest number of specises although the specises of fungus is abundant. It is widely survived in the soil and growed in the plants which is corrupted. The airborne conidia of Aspergillus fumigatus sporulates abundantly,and each conidial head producing thousands of conidia which could released to the air. The conidia is too small and could reach the lung of mammals.The mammals have two types of immunity, which including innate immunity and acquired immunity. Immuncompetent individuals rarely have any adverse effect because of the body can eliminated easily by innate immune mechanisms. However, the the use of wide-spectrum antibiotics and immunosuppressor after organ transplantation in recent years, The number of population who are infected by serious fungus is growing. The most common infection of fungus is Candida,but there is some evidences that the percentage of people infected by Aspergillus is increasing compared with Candida, and the clinical symptoms is more serious and even fatal. For most patients, the main site of infection for A.fumigatus is the respiratory tract. The invasion of mycelial colonization could be anywhere, According to the site of the disease which caused by A. fumigatus, We can classified it as Allergic diseases and Invasive Aspergillosis and so on. The invasive Aspergillosis is the most serious infection. The mortality rate is ofen about50%. The interaction of Aspergillosis fumigatus and the immune system is increasingly linked to the diseases.Currently, Taruna Madan et al found that the lung surfactant protein kinase D have an important immunological role in the early antifungal defense responses in the lung, through inhibiting infectivity of conidia by agglutinaytion and by enhancing uptake and killing of Aspergillus fumigatus by phagocytic cells. The protein kinase D(PKD) family of serine/threonine kinases has been implicated in the progression of avariety of diseases. The kinase family is composed of3isoforms:PKD1, PKD2, PKD3. There is some evidences that PKD1is one of the critical factors that plays a essential role in the development of hypersensitivity pneumonitis caused by Saccharopolyspora rectivirgula,Meanwhile, the inhibition of PKD1activation could be an effective way to control the hypersensitivity pneumonitis. The article is show that the PKD family might be involved in certain cytokine production mediated through TLR4and TLR5. The TLR superfamilly members use the molecule MyD88induce the activation of PKD1in human and murine macrophages, and eventually lead to the activation of NF-кB. It is not known whether Aspergillus fumigatus induces the activation of PKD1or whether PKD1plays a key role in the inflammatory responses induced by Aspergillus fumigatus.Nuclear factor-кB is a transcription factor by regulating the expression of inflammatory and immune genes.it plays critical role in the progression of a variety of cancer types. Many extracelluar stimuli, including viruses inflammatory cytokines, activate NF-кB. Accumulated evidences indicated that Mice which lack NF-кB proteins have serious influence on B-and T-cell proliferation, activation, isotype switching, and cytokine production.At the same time,the other evidence indicated that inhibitors of NF-кB activation have been shown to block the maturation of dendritic cells which has a central role in the immune responses. In the normal individuals, the Toll-like receptors (TLRs), a group of transmembrane proteins that mediate the activation of intracellular signaling pathway after recognizing extracellular pathogens, recruits MYD88, and then activate TNF-receptor-associated factor6(TRA6) phosphorylation and the NF-kB pathway activation. So far,the functional role of PKD1on the activation and the transcriptional activity of NF-кB signal pathway mediated by Aspergillums fumigates is unknown, so we want to explore the effective way to treat the disease which is caused by Aspergillums fumigates.Objective:To explore the functional role of PKD1and underlying mechanism on the activation and the transcriptional activity of NF-кB signal pathway mediated by Aspergillums fumigates.and to find an effective way to theat the disease which is caused by Aspergillums fumigates.Methods:A549cells or HEK293cells were transfected with GFP, GFP-PKD1, respectively. Transfected cells above were treated with1×105CFU/mL dose of Aspergillus fumigatus conidia for different time, phosphorylation of PKD1,IKB and p65(pS276) were measured by Western blot; Fourthermore, overexpression of GFP-PKD1or endogenous PKD1knockdown and phosphorylation of IKB, p65(pS276) in NF-кB pathway were also measured by Western blot. In addition, NF-кB-luc and renilla luciferase reporter pRL-SV40were cotransfected into A549cells containing GFP or GFP-PKD1and then treated with Aspergillus fumigatus conidia for24h, cells were harvested and NF-кB transcriptional activivty were determined by dual-luciferase reporter assay. Overexpression or knockdown of PKD1treatment whith Aspergillus fumigatus or not for24hours, and then extract the RNA from the A549cells with the way of Trizol.and the expression of IL-8、TNF-a was measured by the real-time PCR.Results:1、Overexpression of PKD1ehanced phosphorylation of Ser744/748and Ser916sites of PKD1in response to Aspergillus fumigatus in A549and HEK293cellsThe exogenous of GFP and GFP-PKD1were transfected to the A549cells, then we tested the overexpression of GFP and GFP-PKD1. We treated the A549cells with the Aspergillus fumigatus which is inactive for different time.The results showed that overexpression of PKD1ehances phosphorylation of Ser744/748and Ser916sites of PKD1in response to Aspergillus fumigatus in A549and HEK293cells.and it could enhance the phosphorylation in time-dependence manner. The phosphorylation of Ser744/748and Ser916sites of PKD1in response to aspergillus fumigatus in A549and HEK293cells were stable when the treatment time was30min.and when that time was60min,the phosphorylation is significantly decreased. And the same results were observed in HEK293cells. Which is conformed that overexpression of PKD1ehanced phosphorylation of Ser744/748and Ser916sites of PKD1in response to Aspergillus fumigatus in A549and HEK293cells. and it could enhanced the phosphorylation in time-dependence manner.2、Overexpression of PKD1increased phosphorylation of pIкB and p65(pS276) induced by Aspergillus fumigatus in A549cellsFunctional role of PKD1mediated the activate of NF-кB signaling pathway induced by Aspergillus fumigatus is still unknown. So the overexpression of PKD1in A549cells treatment with Aspergillus fumigatus or not was mearsured the phosphorylation of pIкcB and p65(pS276) to find out the influence to the NF-кB signaling pathway. The results showed that the overexpression of PKD1increased phosphorylation of pIкB and p65(pS276) induced by Aspergillus fumigatus in A549cells. And it did not change the phosphorylation of p65(pS536), which was confirmed that it may have selection to change the phosphorylation of p65.3、The PKD inhibitor GO6976reduced NF-кB signaling activation trigged by Aspergillus fumigatus in A549cellsThe A549cells was treated with the PKD inhibitor GO6976for1hour,and then the cell lysis solution was collected after it was treated with Aspergillus fumigatus for30mins, The results showed that the phosphorylation of pS744/748was decreased, and IкB expression was increased.That confirmed that The PKD specific inhibitor GO6976reduced NF-кB signaling activation trigged by Aspergillus fumigatus in A549cells.4、Knockdown of endogenous PKDl reduced NF-кB signaling activation trigged by Aspergillus fumigatus in A549cells Given that overexpression of PKD1increases phosphorylation of pIкB and p65(pS276) induced by Aspergillus fumigatus in A549cells. We think about if that knockdown of endogenous PKD1reduced NF-кB signaling activation trigged by Aspergillus fumigatus in A549cell is possible. To explore that conclusion, The siCTL(siRNA control) and siPKDl were transfected into the A549cells.after24hours,it were treated with serum-free medium for24hours. And then the cell lysis solution was collected after it was treated with Aspergillus fumigatus for30min. the results was conformed that knockdown of endogenous PKD1reduced NF-кB signaling activation trigged by Aspergillus fumigatus in A549cells. Which including decreased the phosphorylation of pIкB and p65(pS276),the expression of p65protin was not change.5、Overexpression of PKDl significantly enhanced NF-кB transcriptional activation induced by Aspergillus fumigatus in A549cells2xNF-кB-luc and renilla luciferase reporter pRL-SV40were cotransfected into A549cells containing GFP or GFP-PKD1.After12hours, it were treated with serum-free medium for12hours, and then theated the sample with Aspergillus fumigatus or not for24hours. The cell lysis solution was collected and NF-кB transcriptional activivty were determined by dual-luciferase reporter assay. The rate of the activities of firefly and Renilla among different groups represented with relative luciferease activity (RLU). Compared with the control group, overexpression of PKD1significantly enhanced NF-кB transcriptional activation induced by Aspergillus fumigatus in A549cells.6、The expression of cytokines was decreased after the knockdown of PKD1The siCTL(siRNA control) and siPKDl were transfected into the A549cells.After36hours, the cell lysis solution was collected after it was treated with Aspergillus fumigatus or not for24hours and then extracted the RNA from the A549cells with the way of Trizol.and the expression of IL-8、TNF-a was measured by the real-time PCR. We found that the expression of IL-8、TNF-a was decreased.Conclusion:PKD1may contribute to the activation and the transcriptional activity of NF-кB signal pathway induced by Aspergillums fumigates in A549cells. Overexpression of PKD1ehanced phosphorylation of Ser744/748and Ser916sites of PKD1in response to Aspergillus fumigatus in A549and HEK293cells. and it could enhanced the phosphorylation in time-dependence manner. Overexpression of PKD1increased phosphorylation of pIкB and p65(pS276) induced by Aspergillus fumigatus in A549cells. It did not change the phosphorylation of p56(pS536). which was confirmed that it may have selection to change the phosphorylation of p56. Knockdown of endogenous PKD1reduced NF-кB signaling activation trigged by Aspergillus fumigatus in A549cells. Overexpression of PKD1significantly enhanced NF-кB transcriptional activation induced by Aspergillus fumigatus in A549cells. The expression of cytokines was decreased after the knockdown of PKD1. And The PKD inhibitor GO6976reduced NF-кB signaling activation trigged by Aspergillus fumigatus in A549cells. In a word,we demonstrated that PKD1may contribute to the activation and the transcriptional activity of NF-кB signal pathway induced by Aspergillums fumigates in the development of the respiratory aspergillosis.