| NLRC5 (NLR family, CARD domain containing 5) has been found that it played an important role in the innate immunity and cellular immunity. Recently, this gene was proved that it directly regulated the transcript of MHCI moleculars in mammal immune cells and negatively regulated the function of NF-κB. Poultry Salmonella disease still frequently break out in China. It normally leads to severe lost of production and threats human health. Especially, the S. Pullorum depresses the expression of the MHC Class I molecular and induces the expression of the IL10, which is one reason that block the pathogen eliminating process, which leads to the forming of the suppressive carrier. The high throughput study on infection of S. Pullorum has not been reported yet, while other high throughput experiments on other strains that have different pathogenic mechanisms just could provide some reference. Thus it would be necessary to carry out the high thourghput experiment to investigate the immune response to S. Pullorum. Based on the results of expression profiles to investigate the the expression patterns of NRLC5 and its relevant genes and the activity of the promoter of NLRC5 by dual luciferase report experiments, which provide some novel discoveries about the modulation mechanism of chicken NLRC5 in the process of innate immune response and cellular immune response. These discoveries helpped us deeply understand how the SPI-2 III type secreting system of S. Pullorum influences the antigen presenting and finally the forming of the recessive carrier of S. Pullorum.1. We firstly investigated the difference of the expression profiles of chicken spleen after infected with two different strains of Salmonella. After comparing the expression profiles of chicken spleens infected with different Salmonella strains. The infection of S. Pullorum induced the typical immune response. According to the enrichment analysis of different expression genes, the lymphocyte proliferation and differentiation process were enriched, which illustrated that the adaptive immune system was activated. Furthermore, some genes such as IL15 and CD28 associated with the activation of NK cell and T cell were down regulated, but those genes such as IL7 corerlated with activation of B cell that was involved in the antibody response were up regulated. Those results suggested that the pathogen eliminating process were depressed but the humoral immunity were activated. The different expression genes in the ceacum of S. Pullorum were enriched in the anti-bacteria process. Most of those genes in the enriched GO terms were downregulated, which might be cuased by the enhancement of the antibody protection.Then time series expression microarray was used to identify the crucial molelcular events and relationship between different encriched pathways and genes in the infected chicken spleen. The body weight, Spleen Index, Bursa Index, Thymus Index and the concentration of IL-1α, IFN y, IgG and IgM were determined after chicks were sacrificed. The association analysis between phenotypes and gene expression data were investigated by using WGCNA package. MIC package calculated the linear and nonlinear correlations between DEGs. Venn’s Graph provided that some genes were frequently idenfied as DEGs at every time points. Finally the correlation between pathways were recruited to illustrate that some signal pathways and metabolic pathways were involved in innate and adaptive immune response at different stages. Finally, this analysis pipleine determinated the different expressive genes correlated with the recessive infection. The time series microarray results primarily revealed the changes of the splenic expression profiles at 2hpi,4hpi, 8hpi,24hpi,3dpi,5dpi,7dpi,12dpi and 21dpi after chicken were gavaged with S. Pullorum. The switch from innate immunity to humoral immunity were observed in the aspect of gene expression level. Some key regulation events including activation, proliferation and migration of lymphocytes during this stage in the spleen were identified such as MHC class IB-F, ANXA13, IL18, BF2, TNFRSF13B, BLB2, CD28 etc. Pathway enriched genes were divided into two highly negatively correlated groups. Some expression patterns of some hub genes of identified DEGs were related to chicken Spleen Index. The immune pathways had different correlations at different immune response stages with metabolic pathways and other signaling pathways. Some DEGs were localized in the QTL regions which associate with chicken antibody reaction. NLRC5 were identified as a DEG. The period from 3dpi to 5dpi was inferred that it is a key stage of transforming from depressing the innate immune response to activating the cellular immune response, in which the eliminating process were mainly carried out. The DEGs in these two time points might be correlated with the regulation mechanism of SPI-2 type III secreting system. The expression pattern of NLRC5 demonstrated that it might activate the expression of downstream moleculars and promote the cellular immune process, but be negative regulated by unknown factors.Subsequently, the co-expression analysis of NLRC5 with the other probes in the microarray by using mutual information method was recruited to investigate the co-regulated genes. In the microarray data, TAP1 and IL21R were identified as highly linear co-expression genes based on the mutual information algorithm, which suggested they might regulated by the same reglators we still not known and participated in related functions such as antigen presenting.2. Secondly, the complete CDS and the UTR of NLRC5 were coloned. The assembled sequence were well analyzed by using bioinformatics tools. Then qRT-PCR were used to deeply detect the expression pattern of NLRC5 and reported relevant genes in vivo and in vitro after stimulated with live bacteria and LPS separately. Based on the sequence we cloned, the expression vector and interfare vector were constructed and were transfected into the DFl cell line to build the stable expression cell strains. Then, immunocytofluorescent were carried out to illustrate the subcellular localization of the NLRC5. Based on these results and reported data we built a regulation model. The results of NLRC5 molecular cloning demonstrated that chicken NLRC5 was homologous with mammal NLRC5. They had the same domains which suggested they might have the same function in the immune system. Chicken NLRC5 probably could enter the cell nucleus and act as a transcription factor. The transcriptive regulation of NLRC5 were potentially influenced by epigenetic modification. The expression results of NLRC5 and relevant genes showed that NF-κB2 was significantly positive correlated with NLRC5. STAT1 were inferred that it was not involved in the regulation of the expression of NLRC5. STAT3 were observed it acted as a nagetive regulator depressing the expression of NLRC5. We assumpted that NLRC5 postively regulated the expression of STAT3 and IL18 besides the MHC I. We found the NLRC5 might have different modulation mechanism from mammal.3. Finally, if chicken NLRC5 played the same role as mammal in regulating the expression of MHCI, the SPI-2 III type of secreting system may suppress the expression of NLRC5 to control the function of MHCI to protect the Salmonella from eliminating mechanism. Therefore, the promoter region of NLRC5 should be clearly definded. In this study, the activity of different upstream segments before the initiation codon were detected in order to investigate the potential regulation factors and the binding sites. Firstly we predicted the transcription factor bind sites in the upstream of the ATG, then we investigated the activity of different fragments of this sequence, and tried to explain the potential mechanism of regulation. These results could help us well understand the function of NLRC5 and laid on a solid foundation on further investigating the mechanism of SPI-2 Ⅲ secreting system modulating the expression of MHCI. The series deletion fragments of NLRC5 promoter region revealed that NLRC5 had two promoters with high promoter activity. The second one promoter region from 1448 to 4372 probably was the main promoter taking in charge of the normal expression of NLRC5. The motif of NF-κB binding site were reliably identified in the core promoter region. The activity of the fragment from 2158 to 2213 was 80% of the whole length of the promoter. However, the longer downstream sequence of the transcription initial site showed decrease of the promoter activity, which suggest the downstream regulation mechanism is more complicated than we expected. But we still proved that the downstream elements contributed sigenifcantly to the expression activity of NLRC5. The core promoter of NLRC5 we identified was the fragment from 1828 to 2213, which had the most promoter activity. Some cis-elements were identified in this region, including STAT1, NF-κB, P300, MRF2, FAC1, PAX5, CREB, CPBP, NF-AT1, MRF-2, AML1, FAC1, CRX and RelA-p65.In the future, the first thing we should carried out was to provide the direct evidence that chicken NRLC5 directly regulate the expression of MHCI moleculars. Then, by utilizing the ChIP and ChIP-Seq technologies to investigate the sites of the genome could be binded by NLRC5. Those binding relation can further be validated by EMSA, point mutation, knock out, overexpression to make the regulation model we built in this work reliable. Then, we can start to work on discovering how the SPI-2 system regulate the expression of MHCI, which finally will explain how the suppressive infection form. |
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