Innate Immunity Response To Influenza Virus In Human Organ Culture Model

PartⅠEstablishment of Precision-cut Human Lung Slice Model in Explant CultureObjective To explore the conditions and construct medium of explanted culture human lung slices, to optimize treatment and preserving human lung organ, to stabilize cells after slicing, to regulate equipment using in this model and select precision-cut slice thickness, to establish standard procedures, for an establishment a stable, robust, high-yield, high repeatability and practicality of multi-cells co-culture growing human lung organ model in explanted culture.Methods We select volunteers’ acceptation lung cancer surgery at the University of Oklahoma’s five hospitals according to the standard procedures of the normal lung tissue removal approved by University of Oklahoma Institutional Review Board, or traffic fatalities body organ donor lungs. Concerning improved methods of agarose filling lung tissue, we use mixed medium contain 1.5% low-melt-low-gel agarose, via inflating, Coring, slicing and culturing and so on, to create human precision-cut lung slices model. Then explanted culture human lung slices for a long time in vitro, selected slicing at different time points to observe the growth phase of structure and function under a microscope. Here, we evaluated cytotoxicity during the establishment of the model system by testing the release of lactate dehydrogenase.Results The system of establishment of human precision-cut lung slice model is stable, without significant cytotoxicity damage, easily repeated, high-yield preparation. According to this model preparation method, human lung slices cultured in vitro after a long period (> 14 days), we found that three-dimensional structure and structure of bronchial were clearly visible, lung slices’ structure were integrity and alveolar wall cells maintained the normal structure and function of lung organ with no change following culture time.Conclusion According to standard procedures, the system of established human precision-cut lung slices model in explant culture is robust, human lung slices in vitro could tolerate the long culture, structure clear, cell stability, could be used as a repeat of human lung organ culture model in vitro.PartⅡHuman Lung Slices Response to Influenza Virus in Explanted CultureObjective To use new establishment human precision-cut lung slice model in explant culture, we detected human lung slices after exposure to influenza virus whether or not supported influenza virus infection and virus replication.Methods According to the standard procedure of establishment human precision-cut lung slices model in explant culture by using low-melt-low-gel agarose inflating, Human lung slices were exposed to 6x106 PFU/ml influenza virus A/PR/8/34 (H1N1) (PR8 ) and A/Oklahoma/309/06 (H3N2) (OK/06), harvested after the different times’ culture of lung tissues in vitro. To detect human lung slices tissue response to influenza virus after exposure by semi-quantitative RT-PCR and confocal microscope.Results Human lung slices cultured and exposed to influenza virus PR8 and OK/06 for 24h in vitro. The results of immunohistochemistry demonstrated that the red fluorescence signal appeared in a variety of cells in human lung tissues under laser confocal microscope after human lung slices exposure to influenza virus. This suggested that anti-PR8-NP and anti--OK/06-NP were existed. However, there was only the nucleus stained blue in the absence of virus particles dilution group. These indicated human lung slices infected after exposure to influenza virus. In addition, NS1 mRNA expression of influenza virus PR8 and OK/06 both generated a significant peak at 8 hr after exposure to influenza viruses by semi-quantitative RT-PCR, which indicated that explant cultured human lung slices supported viral replication after influenza virus exposure.Conclusion Human lung slices exposure to influenza virus PR8 and OK/06, supported influenza virus infection and virus replication.PartⅢMulti-cells Sources from Human Lung Slices Response to Influenza Virus InfectionObjective To use human lung slices model infection by influenza virus A/PR/8/34 (H1N1) (PR8) in explanted culture and detects cytokine response induced by influenza virus in human lung slices and make sense cellular components in human lung how to play the role of the innate immune response to influenza virus.Methods In accordance with the standard process of establishment human lung slices model in explant culture, we used influenza virus PR8 stimulation human lung slices to detect mRNA expression of inflammatory cytokines at the transcriptional level and protein release of inflammatory factors by RNase protection assay (RPA) and ELISA, respectively, in order to detection innate immune response to influenza virus. Then, detect inflammation factors expression induced by influenza virus and make it clearly which cells are the sources of induction by immunohistochemistry under confocal microscope. Results Influenza virus induced a variety of inflammation factor components in lung slices after exposure to influenza virus. We found greatly expression of MIP-1αand IP-10 mRNA at the level of transcription and confirmed it that these cytokines proteins induced by influenza virus really increased release at the transcription level. Here, results of the immunohistochemistry demonstrated that cytokine response induced by the influenza virus were derived from a variety cell of sources in human lung, such as epithelial cells, alveolar macrophages, interstitial cells, and so on, they all involved in innate immune response to influenza virus.Conclusion Influenza virus activated a variety of innate cell sources from human lung to produce inflammatory cytokines and induced some cytokines like MIP-1α, IP-10 and others to involve in innate immune response to influenza virus in human lung slices.PartⅣCSE Suppressed Influenza Virus Triggered Innate Immunity Response in Human LungObjective To further clarify the innate immune response to influenza virus A/PR/8/34 (H1N1) (PR8) infection in explant cultured human lung slices and a variety of innate cell sources from human lung involved in the induced cytokines and antiviral factors response. To evaluate the impact of CSE on the influenza virus-induced cytokines and explore its possible mechanism in human lung organ culture model.Methods Here, we obtained cigarette smoke extract (CSE) with standard procedures and used it treatment human lung tissue slices, detected influenza virus PR8-induced protein expression of a variety of inflammatory cytokines and the impact of CSE on the expression of inflammatory cytokines by ELISA. We want to be explicit whether or not CSE suppressed human lung innate immunity to influenza virus exposure. Then, detected CSE treatment or with anti-oxidants on the influenza virus-induced inflammatory cytokines and the mRNA expression of PRRs using RT-PCR, further to determine RIG-I protein expression in human lung after CSE or with anti-oxidants treatment on the influenza virus using Western blot. In addition, we measured cytotoxicity of CSE on human lung slices by LDH release toxicity test. Results We found CSE attenuated influenza virus triggered cytokine response, and CSE suppressed the expression of RIG-I mRNA and protein in human lung at the level of transcription and translation. Treatment is safe with appropriate concentrations of CSE. Here, we also found CSE suppressed RIG-I and other PRRs signaling pathway can be protection by antioxidant.Conclusion CSE attenuated influenza viruses triggered cytokine response in human lung, mainly through RIG-I-mediated signaling pathway that influenza virus triggered innate immune response in human lung.