| In mammalians,the spleen is a secondary lymphatic and reticuloendothelial organ that performs several functions related to hemocatheresis,blood cell storage,and immune response against infections as well as extramedullary hematopoiesis.As an immune related organ,the spleen is crucial in regulating immune homoeostasis through its ability to link innate and adaptive immunity and its function in protecting against infections.Congenital asplenia is a rare disorder characterized by the absence of a spleen at birth and has been referred to as a primary immune deficiency.Patients with congenital asplenia have increased susceptibility to invasive infections along with a high mortality rate despite aggressive therapy.As with the splenectomized patients,congenitally asplenic individuals are susceptible to overwhelming pneumococcal infection throughout life.In mice,several candidate transcription factors,including tlx1,wt1,sox11,tcf21,pbx1 and nkx2.5,expressed in the spleen primordium are essential for spleen development,and mice lacking the functions of these proteins exhibit spleen agenesis or hypoplasia.In fish,research on the early regulation of spleen development has not been reported.As one of the largest lymphoid and immunocompetent organs in fish,the spleen is enriched with several types of immune cells,including monocytes,lymphocytes,macrophages,and granulocytes,which participate in phagocytosis and antigen capture.Due to technological limitations,research on the roles of the spleen in fish immunity have largely focused on its roles in host-pathogen interactions after pathogen infection,and information on the immune response of fish without a spleen under pathogen challenge is scarce.In the present study,we characterized the expression pattern of mice spleen markers in zebrafish embryos.We also validated the functions of the identified gene in early spleen development by knocking it out using CRISPR/Cas9 technology.In addition,we analyzed the immunodeficiency of the mutant fish after pathogen infection.The main results are listed as follows:1.A congenitally asplenic zebrafish model is successfully established via CRISPR/Cas9.The WISH(whole-mount in situ hybridization)results showed that nkx3.2,wt1,tcf21,nkx2.5 and sox11 exhibited different expression patterns in zebrafish,and they were not expressed in the early developing spleen.However,tlx1 could be clearly detected on splenic primordium as early as 52 hpf(hours post fertilization),and the signal became progressively more prominent as the embryo develops.Tissue distribution analysis showed that nkx2.5,wt1a,tcf21 and tlx1 were highly expressed in the spleen of adult fish,of which tlx1 was specifically expressed in the spleen.The tlx1homozygous mutations with frameshifts due to a 26 bp deletion and an 8 bp insertion in the target site were obtained via CRISPR/Cas9 technology.No off-target effects was found in both mutations.The expression analysis of early liver marker gene(lfabp)and early pancreas marker gene(tryspin)showed that the early ontogenesis of pancreas and liver were not affected due to tlx1 mutation.The signal from the tlx1-/-cells on the splenic primordium persisted but decreased at 52 hpf and was no longer detectable after53 hpf in the tlx1-/-mutants in contrast to the observations in WT(wild-type)animals.At the same time,the TUNEL(Terminal Deoxynucleotidyl transferase-mediated d UTP-biotin Nick End Labeling)apoptosis assay results showed that the apoptotic signal in the tlx1-/-mutants was significantly increased than that in the WT at 52 hpf,and a high aggregation of apoptotic cells was observed at the splenic primordium,indicating tlx1 mutation might cause the programmed apoptosis in splenic primordium cells that,affects the normal development of the early spleen.Whole-mount preparations of abdominal organs in tlx1-/-mutants revealed that the entire spleen was absent,suggesting that tlx1 mutation resulted in congenital asplenia without other organogenesis defects in zebrafish.2.Congenital asplenia impairs the early immune response in zebrafish.Statistical analysis showed that the early survival rate of congenitally aspleen zebrafish larvae was significantly lower than that of WT.The expression of genes encoding complement components(c3a.4,c3a.6 and c4)and alkaline phosphatase gene(alpl)in tlx1-/-mutant larvae was significantly lower than that of WT;b7r,a marker of the MPS(mononuclear phagocyte system)was barely detected in tlx1-/-mutant larvae.In addition,the mortality of tlx1-/-mutant larvae was significantly higher than that of WT after Aeromonas hydrophila challenge.All these results revealed that congenital asplenia might impair the immune system and reduce the resistance to A.hydrophila infection in zebrafish.After A.hydrophila challenge and LPS(Lipopolysaccharide)treatment,the expression of pro-inflammatory cytokines(il1?,il6,tnf?,and tnf?)and NF-k B subunits(nfkb2)in tlx1-/-mutants was significantly higher than that of WT,while the expression of anti-inflammatory cytokine gene il10 was not significantly changed.In addition,the expression of b7r was also not detected and it failed to respond to A.hydrophila and LPS in the tlx1-/-mutants,suggesting the impairment of MPS.Enzyme-linked immunosorbent assay(ELISA)results showed that the protein levels of Il1?,Il6 and Tnf?were also significantly higher than that of WT,indicating the uncontrolled inflammatory response.In tlx1-/-mutant larvae,a large number of inflammatory cells migrated and accumulated in the tail of the intestine;meanwhile,the TUNEL apoptosis assay showed that a high aggregation of apoptotic cells clustered in the intestinal region after A.hydrophila challenge,suggesting the degree of apoptosis was significantly higher than that of WT.After exposure to LPS,a high aggregation of apoptotic cells was observed in the gill and trunk of congenitally asplenic zebrafish.In contrast,only a few TUNEL-positive cells were found in WT zebrafish after LPS exposure.The mortality and the number of inflammatory cells migrating to the lateral line were significantly increased in tlx1-/-mutants when compared with WT during the acute inflammatory response induced by CuSO4.In adult,asplenic zebrafish could survive and breed normally under standard laboratory conditions,but mortality rate was also significantly higher than that of WT after A.hydrophila challenge,indicating that congenital asplenia reduced the resistance to A.hydrophila infection in adult zebrafish.The expression of pro-inflammatory factors(il1?,Il6 and tnf?)in kidneys(head kidney and body kidney)of tlx1-/-mutants was significantly higher than that of WT,and the expression of b7r was also not detected in tlx1-/-mutants after A.hydrophila challenge.H&E(hematoxylin-eosin)staining revealed that the renal tubules exhibited obvious vacuolation and cell structure destruction in tlx1-/-mutants when compared with WT after injected with inactivated vaccine and LPS.In addition,the TUNEL apoptosis assay results showed that the number of apoptotic cells in renal tubular in tlx1-/-mutants was significantly increased when compared with WT.3.Congenital asplenia interrupts immune homeostasis and leads to an impaired innate and adaptive immune response in zebrafish.Global transcription profiles of whole kidneys(head-kidney and kidney)from both WT and tlx1-/-mutants were performed to gain a full understanding of how congenital asplenia affects the innate and adaptive immune response after injected with an inactivated A.hydrophila vaccine.In the stage of innate immune response,stress response–related biological processes(cell chemotaxis,response to bacteria,response to external biological stimuli,response to other organisms,response to biological stimuli,chemokines mediated signaling pathways,leukocyte chemotaxis,leukocyte migration,inflammatory response,etc.),inflammatory and apoptosis-associated pathways(TNF signaling pathway,RIG-1receptor signaling pathway and P53 signaling pathway)and pro-inflammatory cytokines/chemokines(il1?,il6,tnf?,tnf?,il13,il13ra2,cxcr4a,elf3,cxcl8a,cxcl18b,csf1b,ccl20a.3,ccl39.3,etc.)in congenitally asplenic zebrafish was significantly up-regulated in tlx1-/-mutants compared with WT after vaccination.Meanwhile,the expressions of pro-inflammatory related genes(il1?,il6,tnf?,tnf?and nfkb2)in liver and intestine were also significantly higher in tlx1-/-mutants when compared with WT.All these resultes indicated that congenital asplenia might disrupt immune homeostasis,leading to excessive systemic inflammation in zebrafish.In addition,complement components(c3a.1,c3a.6,c4,c6,c9),antigen processing and presentation and antibacterial-related genes(defbl1,hamp,tapbp.1,tap1)and haematopoiesis-related genes(tfr1a,gfi1b,nfil3,prg4b and sae1)were significantly reduced in tlx1-/-mutants.In the stage of adaptive immunity,adaptive immune response-related biological processes(T cell activation,regulation of T cell activation,positive regulation of T cell proliferation,T cell costimulation,T cell proliferation,lymphatic cell proliferation,etc.)and adaptive immune response-related signaling pathways(T cell receptor signaling pathway)were significantly down-regulated in tlx1-/-mutants.Immunoglobulin-related genes such as igl1c3,igl3v5,ighv1-4,ighv2-1 and ighv9-1 exhibited significantly reduced expression profiles in tlx1-/-mutants.In addition,q PCR analysis showed that the expression of MHCII/Ig M was also significantly reduced in tlx1-/-mutants when compared with WT.In summary,using CRISPR/Cas9-generated tlx1 mutations,the present study provides,for the first time,proof that tlx1 is a key regulator of early developing spleen in fish.Tlx1 mutation resulted in congenital asplenia in zebrafish.Congenital asplenia might partially impair the complement system and the mononuclear phagocyte system in zebrafish.In addition,congenital asplenia might interrupt immune homeostasis,inducing excessive cytokine/chemokine release and inflammatory damage that,in turn,resulted in an impaired immune response.Our study not only provided a model for the study of spleen dysfunction in non-mammalian vertebrates,but also provided insight into pathological response in splenectomized or congenitally asplenic patients after infections. |
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