| The blood cell in vertebrates consists of erythrocytes or red blood cells(RBCs) and leucocytes or white blood cells(WBCs). It is widely accepted that RBCs and WBCs perform distinct functions, with RBCs being responsible for gas exchange and WBCs being for immunity. Recent study on non-mammalian vertebrates suggested that nucleated RBC may be involved in immunity. This study focus on studied the immune roles of red blood cells in tilapia Oreochromis niloticus using molecular biological methods. The cell morphology of tilapia white blood cells(WBCs) and red blood cells(RBCs) and cell structure of tilapia RBCs. The average body mass of Nile tilapia was 200 + 10 g. Ficoll was used in this study to separate tilapia WBCs and RBCs. Smears of separated RBC and WBC samples were Giemsa-stained for standard light microscopy. Purities of separated RBCs and WBCs were examined by counting on 10 fields of smears on the basis of unique morphologies. For fluorescent staining of mitochondria and lysosomes, live RBCs immediately were washed with PBS and incubated in fluorochrome and 28°C for 10 min. RBCs were washed with phosphate buffered saline(PBS) and used to prepare smears. Microscopic observations and photographs were made on a Nikon Eclipse Ni-E. The results showed that WBCs displayed heterogeneity in staining, size and shape of the whole cells and their nuclei, whereas RBCs were homogeneous in size, exhibiting an oval shape and prominent nuclei residing in the cells’ center. Cell counting on the basis of distinct morphologies revealed that separated WBCs essentially lacked contaminating RBCs(less than 5%), and separated RBCs similarly contained few contaminating WBCs(less than 1%). Staining with fluorescent dyes MitoTracker Red CMXRos and LysoTracker? Green DND-26 revealed that the presence of numerous mitochondria and lysosomes in RBCs. Thus, gradient centrifugation provides a purity of both RBCs and RBCs satisfactory for analyzing gene expression profiles for RNA-seq and other examination experiments. Tilapia RBCs have not only prominent nuclei but also many mitochondria and lysosomes necessary for metabolism and gene transcription. Moreover, general PCR resules showed four types of immune genes tlr2、tlr3、irf1、irf3 were expressed at different extend in tilapia WBCs, RBCs and other organs.To study the transcriptomes of tilapia WBCs and RBCs, we adopted next-generation sequencing technology to analyze the gene expression profiles. RNA sequencing of RBCs and WBCs led to 20,876 annotatable genes. A pairwise comparison revealed 3,251 significantly differentially expressed genes(DEGs), with 707(21.75%) exhibiting high expression and 2544(78.25%) exhibiting low expression in RBCs than in WBCs. Interestingly, 144 DEGs were assigned into “immune system process” by GO annotation and 245 DEGs into “immune system” by KEGG pathway. Importantly, many immunity genes were expressed in not only WBCs but also RBCs. These include complement components, cytokines and their receptors, signal transducers and pattern recognition receptors such as Toll-like receptors(TLRs). Specifically, 42 genes of the TLR signaling pathway are expressed in both of tilapia WBCs and RBS. The expression of immune genes in both WBCs and RBCs was validated by RT-PCR analysis for seven representatives of the TLR pathway, namely interferon regulatory factors(irf) 1~5 and 9 as well as tlr3. All of them were upregulated in poly(I:C)-challenged WBCs by quantitative real-time PCR. Most importantly, three of them, namely irf1, irf3 and tlr3, displayed significant upregulation also in poly(I:C)-challenged RBCs. Therefore, fish RBCs express immune genes and response. This finding suggests that fish RBCs possess the potential to play a previously unrecognized role in host immunity.In order to study the expression and function of IRF1 in nile tilapia, the full length cDNA of interferon Regulatory Factor(IRF1) was cloned and applied for phylogenetic analysis, and the expression variation was checked by qRT-PCR after poly I:C stimulation. Cell transfection by eukaryotic expression vector was used to analyzing for subcellular localization. The opening reading frame of nile tilapia IRF1 contains 888 bp with deduced protein of 295 amino acids. Structurally, nile tilapia IRF1 protein typically shares the conserved characterizations with other species’ IRF1 homologues, displaying conserved DNA-binding domain, IRF association domain, serine-rich C terminal domain, and 6 tryptophan residues in the N terminal. The results of phylogenetic analysis showed that the IRF family was divided into four subfamily: IRF1, IRF3, IRF4 and IRF5. The nile tilapia IRF1 was found to be merged into the teleost subgroup, and amphibian and mammalian homologues are clustered into their corresponding subgroups. Real-time PCR revealed a broad expression pattern of nile tilapia IRF1 in various tissues. The expression of IRF1 was mainly in the head kidney, spleen, liver and gill, furthermore, which increased obviously after poly I:C stimulation, especially for the spleen and head kidney. Subcellular localization analysis showed that nile tilapia IRF1 mainly resides in the cytoplasm. These data supported the view that nile tilapia IRF1 is a potential molecule in IFN immune defense system against viral infection. |
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