Transcriptomic Study On The Mechanism Of Thymus Injury Induced By Salmonella Lipopolysaccharide In Chicks

Thymus is the site for T cell development,the maintenance of its homeostasis is of great significance for the development of immune system.It has been defined as immune privilege organ for a long time,and neglected in the research,prevention and treatment of infectious diseases,as the existence of blood-thymus barrier.Recently accumulating evidences indicated that thymus is the target for multiple pathogens.Its infection could cause injury of multiple organs,even the death of chicks.However,the impact and mechanism of its infection on thymus of chicks were rarely reported.Its most toxic factor is LPS,which is commonly used to study its pathogenic mechanism.LncRNAs is a set of newly identified long RNAs which could not be translated into proteins.It could regulate life process directly with the form of RNAs.Transcriptomic study could reflect the expression patterns and functional attributes of RNAs,which has been applied increasingly in the research of poultry diseases.Therefore,we firstly evaluated the impact of stimulation of S.Typhimurium and LPS on thymus of 3-day-old chicks at the organ,tissue and cell levels,then systematically studied the mechanism of thymus injury induced by Salmonella LPS based on mRNAs and lncRNAs transcriptome analysis.The main results are listed as following:（1）S.Typhimurium infection induced thymus injury in chick,including increased cell death and organ atrophy.The numbers of dead cells in chick thymic cortex reached to the highest values at 36 h after infection of 5×10⁴ CFU S.Typhimurium,which significantly increased to 3.81 times（P<0.05）compared to that treated with saline.The thymus weight and index reached to the lowest values at 72 h after infection,which significantly reduced by 43%（P<0.01）and 26%（P<0.05）respectively compared to those treated with saline.During infection,chick thymus still maintained the integrity of tissue structure with distinctive border of cortex and medulla.（2）Salmonella LPS induced more rapid thymus injury in chicks than S.Typhimurium infection.The numbers of dead cells in chick thymic cortex reached to the highest values earlier at 12 h after stimulation of 50 mg/kg Salmonella LPS.At that time,the numbers of Formamide-MAb and TUNEL positive dead cells significantly increased to 2.95 and5.23 times（P<0.001）respectively compared to those treated with saline.The thymus weight and index reached to the lowest values at 36 h after stimulation of LPS,which significantly reduced by 35%（P<0.01）and 20%（P<0.05）respectively compared to those treated with saline.The structure of chick thymus was also sustained after treatment of LPS.Besides,the ratios of splenic chT1⁺cells,splenic and thymic CD4⁺/CD8⁺cells did not change significantly at 36 h after the stimulation of LPS.The numbers of mast cells in chick thymic cortico-medullary junction and medulla and expression of LEP100,marker for macrophages,in chick thymic cortex reached the highest value at 12 h after stimulation of Salmonella LPS,which significantly increased to 1.90 and 3.69 times（P<0.05）respectively compared with those treated with saline.（3）1767 mRNAs were significantly differentially expressed（DE,P<0.001,|log₂（fold change）|≥0.585）in chick thymus after treatment of Salmonella LPS.The Poly（A）+RNA-seq generated 158978794 of high-quality 50 bp single-end reads,77.05%of which could be mapped to chicken genome.Overall,873,536 and 856 mRNAs were differentially expressed in the comparisons of 12 h vs.0 h,36 h vs.0 h and72 h vs.0 h respectively after the stimulation of Salmonella LPS.The up-expressed mRNAs in comparison of 12 h vs.0 h were significantly enriched in biological processes such as“Inflammatory response”and“Immune response”,and involved in“Acute phase response signaling pathway”and so on;the down-expressed mRNAs（12 h vs.0h）were significantly enriched in processes such as“Cell division”and“Chromosome segregation”,involved in“Mitotic roles of Polo-like kinase”and so on.The up-expressed mRNAs in comparison of 36 h vs.0 h were significantly enriched in biological processes such as“Defense response to bacterium”and“Defense response to Gram-negative bacterium”,involved in“Acute phase response signaling pathway”and so on;the down-expressed mRNAs（36 h vs.0 h）were significantly enriched in biological processes such as“Kinetochore assembly”and“Chromosome segregation”.The up-expressed mRNAs in comparison of 72 h vs.0 h were mainly related to metabolic activities and the down-expressed mRNAs were mainly related with cell death,metabolic and immune activities.Therefore,during the time of thymus injury（12 h and 36 h）induced by LPS,the differentially expressed mRNAs in chick thymus were mainly related to immune response and cell division.The merged pathway analysis suggested that LPS may induce intrathymic inflammation by activating TLR4-MAPKs-FOS/JUN pathway,which caused DNA damage and cell cycle arrest.（4）5520 lncRNAs were identificated from chick thymus.The rRNA-depleted RNA-seq yielded 385393591 pairs of high-quality 150 bp pair-end reads,79.66%of them could also be mapped to chicken genome.After assembly of reads and screening of transcripts,2283 intergenic lncRNAs,2810 intronic lncRNAs and 427 antisense lncRNAs were identified.88.59%of lncRNAs are novel ones.Chick lncRNAs possess less and longer exon than mRNAs.（5）111 lncRNAs were significantly differentially expressed（DE,Q-value<0.05,|log₂（fold change）|≥1）in chick thymus after treatment of Salmonella LPS.There were47 DE lncRNAs in the comparison of 12 h vs.0 h,which were corresponded to 1260 neighbore mRNAs（cis-mRNAs）and 2811 co-expressed mRNAs（co-mRNAs）.There were 81 DE lncRNAs in the comparison of 36 h vs.0 h,which were corresponded to1766 cis-mRNAs and 3203 co-mRNAs.The cis-mRNAs of DE lncRNAs in the com-parison of 12 h vs.0 h were signigicantly enriched in processes such as“Chromatin si-lencing”and“Negative regulation of protein kinase activity”,involved in“Jak-STAT signaling pathway”and“MAPK signaling pathway”and so on;the co-mRNAs of DE lncRNAs（12 h vs.0 h）were significantly enriched in processes such as“Cell division”and“Response to lipopolysaccharide”,involed in“cell cycle”pathways and so on.The cis-mRNAs of DE lncRNAs in the comparison of 36 h vs.0 h were signigicantly enriched in processes such as“Immune response”and“Cell division”,involved in“Oocyte meiosis”and“Jak-STAT signaling pathway”and so on;the co-mRNAs of DE lncRNAs（36 h vs.0 h）were significantly enriched in processes such as“Inflam-matory response”and“Cell division”,involved in“Cell cycle”and“Cyto-kine-cytokine receptor interaction”pathways and so on.Therefore,during the time of thymus injury induced by LPS,the targeted genes of DE lncRNAs in chick thymus were also mainly related with inflammation response and cell division.The visualiza-tion analysis of pathways there were co-expressed or neighbore lncRNAs for most mRNAs in TLR-MAPKs-Cytokines and cell cycle pathways,thus lncRNAs may par-ticipate in thymus injury induced by Salmonella LPS through those pathways.（6）The expressions of 22 mRNAs and 6 lncRNAs were evaluated using real-time qPCR.To validate the data from RNA-seq,the expression patterns of 10 mRNAs detected by qPCR were compared with those detected by Poly（A）+RNA-seq.Their expression trends were similar and there was significant correlation between the results detected by these two methods（P=1.65e-8,r=0.828）.The expression patterns of 6lncRNAs detected by qPCR were also similar as thoses detected by rRNA-depleted RNA-seq,and there was also significant correlation between the results detected by these two methods（P=2.28e-4,r=0.871）.The expressions of 6 mRNAs significantly increased at 2⁶ h after stimulation of LPS,which indicated that transcriptional changes occurred more rapidly than other symptoms during thymus injury.S.Typhimurium infection did induce the similar changes of those 10 mRNAs,but the maximum changes occurred latter than those treated with LPS.Therefore,S.Typhimurium may impact the chick thymus mainly by its LPS,which changed intrathymic gene expressions.Thus,the present study concluded that S.Typhimurium and its LPS could induce thymus injury in chicks.TLR4-MAPKs-FOS/JUN pathway may play an important role in these processes and lncRNAs may also participate in these processes.