Characterization of the chicken T cell receptor γ repertoire by high-throughput sequencing

Background As one of “γδ-high” species, chicken is an excellent model for the study of γδ T cells in non-mammalian animals. However, a comprehensive characterization of the TCRγδ repertoire is still missing in chicken. The objective of this study was to characterize the expressed TCRγ repertoire in chicken thymus using high-throughput sequencing. Methods In this study, we first obtained the detailed genomic organization of the TCRγ locus of chicken based on the latest assembly of the red jungle fowl genome sequences (GRCg6a) and then characterized the TCRγ repertoire in the thymus of four chickens by using 5′ Rapid Amplification of cDNA Ends (5′ RACE) along with high-throughput sequencing (HTS). Results The chicken TCRγ locus contains a single Cγ gene, three functional Jγ segments and 44 Vγ segments that could be classified into six subgroups, each containing six, nineteen, nine, four, three and three members. Dot-plot analysis of the chicken TCRγ locus against itself showed that almost all the entire zone containing Vγ segments had arisen through tandem duplication events, and the main homology unit, containing 9 or 10 Vγ gene segments, has tandemly duplicated for four times. For the analysis of chicken TCRγ repertoire, more than 100,000 unique Vγ-region nucleotide sequences were obtained from the thymus of each chicken. After alignment to the germline Vγ and Jγ segments identified above, we found that the four chickens had similar repertoire profile of TCRγ. In brief, four Vγ segments (including Vγ3.7, Vγ2.13, Vγ1.6 and Vγ1.3) and six Vγ-Jγ pairs (including Vγ3.7-Jγ3, Vγ2.13-Jγ1, Vγ2.13-Jγ3, Vγ1.6-Jγ3, Vγ3.7-Jγ1 and Vγ1.6-Jγ1) were preferentially utilized by all four individuals, and vast majority of the unique CDR3γ sequences encoded 4 to 22 amino acids with mean 12.90 amino acids, which exhibits a wider length distribution and/or a longer mean length than CDR3γ of human, mice and other animal species. Conclusions In this study, we present the first in-depth characterization of the TCRγ repertoire in chicken thymus. We believe that these data will facilitate the studies of adaptive immunology in birds.

Thymic transcriptome analysis after Newcastle disease virus inoculation in chickens and the influence of host small RNAs on NDV replication

Newcastle disease (ND), caused by virulent Newcastle disease virus (NDV), is a contagious viral disease affecting various birds and poultry worldwide. In this project, differentially expressed (DE) circRNAs, miRNAs and mRNAs were identified by high-throughput RNA sequencing (RNA-Seq) in chicken thymus at 24, 48, 72 or 96 h post LaSota NDV vaccine injection versus pre-inoculation group. The vital terms or pathways enriched by vaccine-influenced genes were tested through KEGG and GO analysis. DE genes implicated in innate immunity were preliminarily screened out through GO, InnateDB and Reactome Pathway databases. The interaction networks of DE innate immune genes were established by STRING website. Considering the high expression of gga-miR-6631-5p across all the four time points, DE circRNAs or mRNAs with the possibility to bind to gga-miR-6631-5p were screened out. Among DE genes that had the probability to interact with gga-miR-6631-5p, 7 genes were found to be related to innate immunity. Furthermore, gga-miR-6631-5p promoted LaSota NDV replication by targeting insulin induced gene 1 (INSIG1) in DF-1 chicken fibroblast cells. Taken together, our data provided the comprehensive information about molecular responses to NDV LaSota vaccine in Chinese Partridge Shank Chickens and elucidated the vital roles of gga-miR-6631-5p/INSIG1 axis in LaSota NDV replication.

Baicalin mitigated Mycoplasma gallisepticum-induced structural damage and attenuated oxidative stress and apoptosis in chicken thymus through the Nrf2/HO-1 defence pathway

The thymus is a primary lymphoid organ and plays a critical role in the immune response against infectious agents. Baicalin is a naturally derived flavonoid famous for its pharmacological properties, but the preventive effects of baicalin against immune impairment remain unclear. We examined this effect in the context of Mycoplasma gallisepticum (MG) infection-induced structural damage in the chicken thymus. Histopathological examination showed that the compact arrangement of cells in the thymus was lost in the MG-infected group. Inflammatory cell infiltration and nuclear debris accumulated, and the boundary between the cortex and medulla was not clearly visible. The mRNA and protein expression of apoptosis-related genes were significantly increased in the MG-infected group compared to the control group and the baicalin group. The number of positively stained nuclei in the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay were increased in the MG-infected group. In addition, electron microscopic examination showed chromatin condensation, mitochondrial swelling and apoptotic vesicles in the MG-infected group. However, baicalin treatment significantly alleviated the oxidative stress and apoptosis induced by MG infection. Importantly, the abnormal morphology was partially ameliorated by baicalin treatment. Compared to the MG-infected group, the baicalin-treated group showed significantly reduced expression of apoptosis-related genes at both the mRNA and protein levels. Meanwhile, the nuclear factor erythroid 2-related factor 2 (Nrf2) signalling pathway and downstream genes were significantly upregulated by baicalin to counteract MG-induced oxidative stress and apoptosis in the thymocytes of chickens. In summary, these findings suggest that baicalin treatment efficiently attenuated oxidative stress and apoptosis by activating the Nrf2 signalling pathway and could protect the thymus from MG infection-mediated structural and functional damage.