Research On The Role Of AHR Signaling In The Development And Maturation Of Human Erythrocytes

Background and objectiveAHR is a ligand-dependent transcription factor,which belongs to basic-helix-loop-helix Per-ARN T-SIM(bHLH/PAS)family.AHR directly regulates genes transcription such as AHRR,CYPIB1,IL-22 and VEGF.In addition to various fundamental biological activities in cell differentiation and immune regulation,AHR plays an essential role in the process of hematopoiesis.Inhibition of AHR by SRI(StemRegenin-1,an AHR antagonist)not only promotes the development process from hemogenic endothelium(HE)to hematopoietic stem/progenitor cells(HSPCs),but also benefits the maintenance of HSPCs potential and marked expansion.On the other hand,AHR signaling drives some blood cell fate commitment and the differentiation and maturation of various blood cells,such as monocytes and megakaryocytes.However,little is known about the mechanism of AHR modulation on erythroid differentiation.RBCs(Red blood cells)are essential cells had important functions on oxygen transportation and immune,and the deficiency on erythroid development will cause anemia.Therefore,it is essential for the research on erythropoiesis.In clinic,frequent exposure to TCDD(an AHR agonist)could cause anemia,hinting that AHR might play an important role in erythroid development.In this study,SRI(AHR antagonist)and TCDD(AHR agonist)were used to assess the impact of AHR on erythroid development.Method1.SRI,TCDD,or DMSO vehicle control was added during H1/AGM-S3 co-culture,and flow cytometry was used to research the influence of AHR on hESC-derived erythroid cells.2.Different hematopoietic cells were sorted(hemogenic endothelium:CD34+KDR+GPA";hematopoietic progenitors:CD34+CD43+GPA-CD45-;co-D14 CD34+cells and UCB-derived CD34+HSPCs)and treated with SRI,and flow cytometry and colony forming unit assay were used to study the effect of AHR on erythrogenesis.3.Isolated UCB-derived erythroid precursors(BFU-E cells:CD34+CD123-GPA-CD71+CD36-,CFU-E cells:CD71+GPA-)were exposed to SRI.Flow cytometry and colonyforming-unit assay were used to study the influence of AHR on erythroid development.4.Sorted proerythroblasts(CD71+GPAlow)were dealt with AHR agonists or antagonists.Flow cytometry,immunofluorescence staining,and proteomics were used to research the effect of AHR on erythroid terminal differentiation.5.SRI-treated proerythroblasts cells were collected for RNA sequencing(RNA-seq),and GO enrichment and GSEA analysis were used to analyze the mechanism of SRI on promoting erythroid terminal diferentiation.6.UCB-derived proerythroblasts were transfected with shRNA to decrease SOX6(shSOX6)expression.Transfected cells were exposed to SR1,and the expression of GPA,hemoglobin-? and F-actin were examined by flow cytometry.On the other hand,to test the relationship between F-actin and AHR,F-actin assembly was disrupted by cytochalasin D(CytoD)and jasplakinolide(Jasp)in UCB-derived proerythroblasts,and the influence of SRI on proerythroblasts was tested by flow cytometry.7.SRI was added during erythroid terminal differentiation and membrane protein expression level,hemoglobin concentration,enucleation rate,deformation index,and P50 were tested on D12 or D21 respectively to study the influence of SRI on erythroid functional maturation,Results1.Hematopoiesis was markedly enhanced by SRI,and 3 times HSPCs and 1.6 times erythroid cells were produced in SRI treatment compared with control at co-D14.However,the number of HSPCs has no difference between TCDD treatment and control.Moreover,there was no difference on erythroid cell production between TCDD and control during co-culturing except co-D14,when erythroid cells was significant decreased in TCDD treatment compared to control.2.SRI did not increase CFU-E colonies generated from HEC.However,it could enhance the production of HSPC along with the ability of colony forming,including BFU-E colonies.Besides,(2-4)-day SRI added could increase UCB-derived erythroid cells proportion and CFU-E colonies,but the number of erythroid cells and BFU-E colonies have no difference between two groups.However,chronic SRI treatment could markedly decrease the number of UCB-derived erythroid cells,although increase the erythroid cell ratio and CFU-E colonies forming ability.3.UCB-derived BFU-E cells were exposed to SRI for 3 days.There was no significant difference on the ability of forming CFU-E colonies between SRI and control treatment at D1.However,more CFU-E colonies and CD71+GPA+cells were produced in SRI treatment at D2 and D3.4.UCB-derived CFU-E-enriched cells(CD71+GPA-)and proerythroblasts were exposed to SR1,TCDD,or DMSO for 3 days respectively.Compare with control,more GPA+cells were generated from SR1-treated CD71+GPA-and the expression of GPA was higher in SR1-treated proerythroblasts cells compared with DMSO-treated cells.However,TCDD-treated cells showed opposite results.MGG staining showed more mature erythroid cells in SR1 groups than control,but more immature cells under TCDD condition.Besides,proteomics results showed that the expression level of erythrocyte-related proteins was higher in SR1 treatment than control.In addition,co-D14-derived erythroid cells and early erythroblasts showed the similar results under SR1 condition.5.Co-D14 or UCB-derived Proerythroblasts cells after 3-day-SR1 treating were collected for RNA-seq.Analysis results showed that SR1 could induce oxidative stress response,and upregulate the expression of erythroid differentiation-associated genes,like AHSP,ALAS2,TMEM14C,SOX6,EPB42,SLC4A1,JAK2 and MFHAS1,and downregulate the expression of actin-related genes.6.The terminal differentiation of shSOX6 proerythroblasts was similarly promoted by SR1.Furthermore,the level of F-actin expression was lower in SR1 treatment compared with control.However,the expression of hemoglobin ? has no difference between SR1 and control in shSOX6 erythroid cells.7.Under SR1 condition,the expression level of ACTB was lower both in mRNA and protein level compared with control,but TCDD-treated cells showed a higher level.AHR inhibition could promote F-actin remodeling,and disrupting F-actin assembly by Cytochalasin D(CytoD)or Jasplakinolide(Jasp)could totally block the function of SR1 on erythroid terminal differentiation.8.SR1 was used during erythroid terminal differentiation.At D12,the expression of band3,CD47 and hemoglobin p in H1-derived erythroid cells was higher in SR1 treatment than control.At D21,hemoglobin concentration,enucleation ratio,and deformation index of both H1-and UCB-derived erythroid cells were higher in SR1 treatment than control.However,there was no difference on P50 between two groups in HI-derived erythroid cells.ConclusionAHR Inhibition markedly enhances hematopoiesis,and increases the production of hESC-derived HSPCs and erythroid cells.Although AHR inhibition could not directly promotes erythroid cells generation from hematopoietic cells,it could promote erythroid terminal differentiation and hemoglobin expression.This function of AHR inhibition is achieved by promoting cytoskeleton remodeling.Our study supplies the role of AHR on erythroid differentiation,and provides a new strategy for producing a number of erythroid cells in vitro.Besides,our study also provides theoretical principle for some anemia therapy.