Lymphocyte Regeneration After Thymocyte Damage In Zebrafish

The thymus is an important source for the supply of T lymphocytes in the acquired immune system,as the body’s lymphoid organ,plays a central role in human immune system.Hematopoietic stem cells(HSCs)can differentiate to form blood cells of all lineages.After HSCs migrate to thymus through the bloodstream,it develops and differentiates to produce mature thymus-dependent lymphocytes or T lymphocytes under the thymic environment and hormone induction.T cells distribute to peripheral immune organs and can recirculate through lymphatic vessels,peripheral blood,tissue fluids and so on,which rapidly proliferate and differentiate under the stimulation of antigens and exert cellular immunity and immune regulation functions.In addition to defending against the invasion of pathogens,the immune system can also monitor and kill cancerous cells appearing in the body,remove aging and dead cells,and maintain the body’s environmental stability and physiological balance.Studies have shown that with the increase of human age,the thymus tissue shrinks,the number of thymus lymphocytes decreases and T cell function degenerates,especially in the elderly stage,susceptibility to infections such as cancer,autoimmune diseases,bacteria and viruses increases.Besides,radiation,chemotherapy,bacteria and DNA damage can also cause thymus atrophy and T lymphocyte defect,resulting in impaired immune function.Therefore,it is of great significance to explore how to promote the regeneration of T lymphocytes and the recovery of immune system in the case of damage to acquired immune system.In recent years,zebrafish has embryos developed in vitro and is transparent,which is convenient for live imaging,easy genetic manipulation and large-scale genetic screening.In addition,it contains a relatively complete hematopoietic and adaptive immune system and has a highly conservative mechanism compared to mammalian development.Therefore,it is widely used in the development of embryos,tissues,organs and regeneration of injuries,as well as in diseases and tumors.In this study,we took advantage of zebrafish to construct a thymocyte damage model through the Tg(coro1a:Dendra2-NTR)transgenic line based on the principle of Nitroreductase /Metronidazole(NTR/MTZ)system-mediated leukocyte elimination.First,the embryos were treated with2 m M metronidazole solution for 12 hours to induce thymic cell damage on the 5th day post fertilization(5pdf)and tested whether lymphocytes(marked by cmyb,rag1 and rag2)were specifically eliminated.Subsequently,we observed the damage,repair and regeneration process of thymocytes at different time windows on days 3,5,and 7 after treatment.It was found that lymphocytes were significantly reduced and apoptosis occurred in thymus,the entire thymic epithelium was atrophied on 1-2 days after MTZ treatment,but the number of precursors had no clear change in other parts such as caudal hematopoietic tissue through WISH and confocal microscopy observations.Then,T cells began to recover on the 3rd day after injury when the zebrafish had developed to 8dpf,and recovered completely at 12 dpf after observation and analysis.In order to further study the cause and source of regeneration after thymocyte damage,we used in vivo transient lineage tracing technology to mark the HSCs in kidney to track them.The results showed that the regeneration of thymocytes after MTZ treatment mainly depended on the differentiation of lymphocyte precursors migrated from kidney and partly on in situ thymus residual cell proliferation and development.After thymocyte damage,the migration time of most precursors from kidney to thymus occurred around 8dpf.Next,we examined the migration path of hematopoietic precursors during the regeneration of T cells and observed that the migration of such cells from kidney to thymus was uniform and regular.Through construction of double-transgenic strains combined with real-time live imaging,we found the migration of hematopoietic precursors to thymus after lymphocyte damage was through other paths than blood vessels or lymphatic vessels.It had been reported that in the mouse sepsis model,thymocytes were ablated in vivo and the m RNA levels of CC chemokine receptors Ccr7,Ccr9 and p-selectin glycoprotein ligand 1 were reduced,and the migration of bone marrow HSCs to thymus was impaired.In order to further explore the signal pathway or factor that mediate the migration of hematopoietic precursors after thymocyte damage in zebrafish.we screened chemokine receptors and ligands by their expression differences and positions after injury,and found that chemokine receptor cxcr4 b expressed in hematopoietic precursors and cxcl12 b expressed in thymic epidermal cells,both of which were clearly up-regulated during T cell regeneration,suggesting Cxcl12b/Cxcr4 b pathway may play an important role in this process.So as to confirm its function,we treated the fish with Cxcl12/Cxcr4 signaling pathway inhibitor.The result showed that the antagonist would hinder the migration of hematopoietic precursors from kidney to thymus after T cell damage,which in turn affected the recovery of lymphocytes at 12 dpf.Finally,combined with mutant phenotype analysis,we confirmed the Cxcl12b/Cxcr4 b pathway can guide the migration of precursors from kidney to thymus to participate in the repair and regeneration of lymphocytes.In summary,our work has initially shown that the Cxcl12b/Cxcr4 b chemokine signaling pathway plays a key role in its recovery process after using the NTR / MTZ system to cause thymic lymphocyte specific damage.The model of thymus damage in vivo has laid a foundation for the research of T cell regeneration,molecular mechanisms related to thymus atrophy and body aging.Targeting at CXCL12-CXCR4 signal axis may promote the recovery of T lymphocytes after thymus damage,which has certain guiding significance for the treatment of immunodeficiency.