Neutrophil Akt2 Plays a Critical Role In Heterotypic Neutrophil-Platelet Interactions During Vascular Inflammation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 321-321
Author(s):  
Jing Li ◽  
Kyungho Kim ◽  
Eunsil Hahm ◽  
Victor R. Gordeuk ◽  
Nissim Hay ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Vascular Inflammation ◽  
Critical Role ◽  
Specific Inhibitor ◽  
Microscopic Analysis ◽  
Neutrophil Recruitment ◽  
Neutrophil Adhesion ◽  
Inflammatory Conditions ◽  
Healthy Donors

Abstract Platelet-leukocyte interactions on activated endothelial cells play important roles in mediating pathological thrombosis and inflammation. Heterotypic platelet-leukocyte aggregation is mediated by the interaction of two crucial receptors and counter receptors; P-selectin-P-selectin glycoprotein ligand-1 and glycoprotein Ibalpha-alphaMbeta2 integrin. In spite of extensive understanding of receptor-counter receptor interactions, it remains unclear how heterotypic cell-cell interactions are regulated under thrombo-inflammatory conditions. Using real-time fluorescence intravital microscopic analysis of Akt isoform-specific knockout (KO) mice, we have demonstrated that Akt2, but not Akt1 or Akt3, plays an important role in neutrophil adhesion to the site of TNF-alpha-induced vascular inflammation. Further, heterotypic platelet-neutrophil interactions on the activated endothelium were markedly reduced in Akt2 but not Akt1 or Akt3 KO mice. Studies with chimeric mice generated from bone marrow transplants on wild-type and Akt2 KO mice revealed that hematopoietic but not endothelial cell Akt2 regulates neutrophil recruitment and platelet-neutrophil interactions during vascular inflammation. Using in vitro reconstituted systems in which platelets and neutrophils were treated with an Akt2 specific inhibitor or cells were isolated from WT and Akt KO mice, we observed that both platelet and neutrophil Akt2 play an important role in platelet-neutrophil aggregation under shear conditions. In particular, neutrophil Akt2 was critical for membrane translocation, activation, and adhesive function of alphaMbeta2 integrin. We found that the basal phosphorylation levels of Akt isoforms are significantly increased in neutrophils and platelets of patients with sickle cell disease (SCD), which is an inherited hematological disorder with vascular inflammation and occlusion. Also, SCD patients’ neutrophils show increased alphaMbeta2 integrin activation in the absence of an agonist, in comparison with healthy donors’ cells. Inhibition of Akt2 dose-dependently reduced heterotypic aggregation of patients’ neutrophils and platelets in vitro and inhibited neutrophil adhesion and neutrophil-platelet aggregation in SCD mice, thereby improving blood flow. Our results provide important genetic and pharmacologic evidence that neutrophil Akt2 regulates alphaMbeta2 integrin function and thus plays a critical role during neutrophil recruitment and neutrophil-platelet interactions under thrombo-inflammatory conditions such as SCD. Disclosures: No relevant conflicts of interest to declare.

Neutrophil DREAM Is a Novel Regulator for Beta2 Integrin Function through NF-Kappab Signaling and Modulates Neutrophil Recruitment during Vascular Inflammation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 888-888 ◽  
Author(s):  
Andrew Barazia ◽  
Jing Li ◽  
Kyungho Kim ◽  
Chinnaswamy Tiruppathi ◽  
Jaehyung Cho
Keyword(s):  
Target Genes ◽  
Conflicts Of Interest ◽  
Regulatory Element ◽  
Vascular Inflammation ◽  
Surface Expression ◽  
Neutrophil Recruitment ◽  
Β2 Integrin ◽  
Inflammatory Conditions ◽  
Tnf Α

Abstract The interaction between neutrophils and activated endothelial cells (ECs) is critical for the pathogenesis of vascular inflammation. However, it remains poorly understood how the cell-cell interactions are regulated under inflammatory conditions. Using intravital microscopy in mice lacking DREAM (downstream regulatory element antagonist modulator), a member of the neuronal Ca2+ sensors and a transcriptional repressor, we have found that DREAM plays an important role in neutrophil rolling and adhesion to the TNF-α-inflamed cremaster muscle venules. Studies with DREAM bone marrow chimeras revealed that both hematopoietic and EC DREAM are important for neutrophil recruitment. We found that neutrophils lacking DREAM exhibit reduced αMβ2 surface expression and decrease fibrinogen binding following stimulation with TNF-α, but not fMLF, implying the role for neutrophil DREAM in regulating β2 integrin function through a specific signaling pathway. Since recent studies demonstrated the important role of endothelial cell DREAM in NF-κB signaling, we further examined whether neutrophil DREAM regulates NF-κB signaling. Indeed, the protein expression of A20 (an inhibitor of NF-κB signaling) and p65 (a key subunit of the NF-κB complex) was significantly up- and down-regulated, respectively, in DREAM-deficient neutrophils, compared to WT neutrophils. Moreover, DREAM deletion impaired the phosphorylation of IKKa/β following TNF-a-stimulation, suggesting the role of neutrophil DREAM in NF-κB signaling. Consistently, we found that the transcription of the NF-κB target genes, such as IL-1β, TNF-α, and IL-6, was inhibited by DREAM deletion. Taken together, our results provide evidence that neutrophil DREAM is a novel regulator for β2 integrin function through NF-κB signaling and could be a therapeutic target for treatment of inflammatory disease. Disclosures No relevant conflicts of interest to declare.

Protein Disulfide Isomerase Plays An Important Role in Beta2 Integrin-Mediated Neutrophil Recruitment During Vascular Inflammation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 17-17
Author(s):  
Eunsil Hahm ◽  
Sungjin Huh ◽  
Snezna Rogelj ◽  
Jaehyung Cho
Keyword(s):  
Protein Disulfide Isomerase ◽  
Conflicts Of Interest ◽  
Vascular Inflammation ◽  
Neutrophil Recruitment ◽  
Intercellular Adhesion Molecule ◽  
Tnf Alpha ◽  
Neutrophil Adhesion ◽  
Disulfide Isomerase ◽  
Static Conditions ◽  
Protein Disulfide

Abstract Abstract 17 Cell surface protein disulfide isomerase (PDI), a prototypic thiol isomerase, plays an important role in regulating integrin-mediated cellular functions. Although surface PDI has been reported to regulate L-selectin shedding on neutrophils, its role in neutrophil recruitment during vascular inflammation has not been explored. In this paper, we present novel findings on how neutrophil PDI regulates neutrophil recruitment into the site of vascular inflammation. Using real-time microscopy with cell-impermeable inhibitors for PDI (bacitracin or function-blocking antibodies), we demonstrated that inhibition of PDI reduces adhesion of human blood neutrophils to TNF-alpha-stimulated human umbilical vein endothelial cells (HUVECs) under venous shear. There was no additive effect on neutrophil adhesion in a combination of blocking anti-PDI and anti-beta2 antibodies, suggesting that surface PDI regulates beta2 integrin-mediated neutrophil adhesion to TNF-alpha-activated HUVECs under shear. Further, bacitracin and blocking anti-PDI antibodies diminished alphaMbeta2 integrin-mediated neutrophil adhesion to intercellular adhesion molecule-1 (ICAM-1)-coated surfaces under static conditions. When PDI gene is knocked down in neutrophil-like HL60 cells by shRNA, alphaMbeta2 activation and cell adhesion to TNF-alpha-activated HUVECs under shear were significantly reduced. High resolution confocal microscopy and immunoprecipitation assay revealed that surface PDI interacts with alphaMbeta2 integrin and that such interaction was enhanced by neutrophil activation and inhibited by bacitracin, a thiol isomerase inhibitor. Exogenous PDI equivalently bound to CHO cells stably expressing alphaMbeta2 or alphaLbeta2 when those cells were treated with Mn2+, whereas PDI binding was significantly reduced when the active site CGHC residues on PDI were mutated. Using multi-channel fluorescence intravital microscopy, we demonstrated that inhibition of PDI by infusion of PDI inhibitors impairs stable adhesion of neutrophils to the TNF-alpha-inflamed cremaster muscle venule wall in living mice. These findings suggest the potentially fundamental but poorly understood role for surface PDI in the regulation of beta2 integrin function in neutrophils. Therefore, our results provide the first evidence that inhibition of PDI may be a therapeutic target for the neutrophil-mediated vascular inflammation. Disclosures: No relevant conflicts of interest to declare.

Abstract 47: FXR1 Regulates Inflammatory MRNA Stability and VSMC Proliferation by Modulation of HuR Activity

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Allison Herman ◽  
Ross England ◽  
Dale Haines ◽  
Sheri Kelemen ◽  
Mitali Ray ◽  
...  
Keyword(s):  
Mrna Stability ◽  
Fragile X ◽  
Vascular Inflammation ◽  
Critical Role ◽  
Vascular Diseases ◽  
Muscle Cells ◽  
Vsmc Proliferation ◽  
Inflammatory Conditions ◽  
Mass Spec ◽  
Inflammatory Proteins

Vascular smooth muscle cells (VSMC) play a critical role in the etiology and progression of many vascular diseases including atherosclerosis and restenosis. Our laboratory has found that one anti-inflammatory interleukin, IL-19, is atheroprotective and can decrease vascular inflammation by reduction in mRNA stability of inflammatory transcripts by reduction of activity of HuR, an mRNA stability protein. HuR translocates from the nucleus to the cytoplasm where it recognizes AU-rich elements present almost exclusively in the 3’UTR of pro-inflammatory genes. Proteins and pathways which limit HuR translocation are understudied, but may reduce inflammatory mRNA stability. Using MASS SPEC to identify HuR-interacting proteins under different inflammatory conditions, we identified one protein, Fragile X-related protein (FXR1), which interacts with HuR in inflammatory, but not basal conditions, a novel interaction. FXR1 mRNA expression is enhanced in muscle cells, but nothing has been reported on expression of FXR1 in VSMC or function for FXR1 in vascular disease. The FXR1 promoter contains multiple cholesterol-response elements, and in this study we demonstrate that FXR1 expression is increased in injured arteries and TNFα and oxLDL stimulated human VSMC, but also by IL-19. RNA EMSA demonstrates that FXR1 directly interacts with ARE in 3’UTR. SiRNA knock down of FXR1 in VSMC increases stability of inflammatory mRNA and protein abundance as well as VSMC proliferation, while overexpression of FXR1 reduces both their abundance and stability in addition to reducing proliferation. Since FXR1 appears to be a novel repressor of inflammatory proteins, and is also induced by IL-19, our overall hypothesis is that FXR1 expression and HuR interaction is an inflammation responsive, counter-regulatory mechanism to reduce abundance of pro-inflammatory proteins and therefore reduce inflammation.

scholarly journals Phosphorylated AKT preserves stallion sperm viability and motility by inhibiting caspases 3 and 7

Reproduction ◽  
2014 ◽  
Vol 148 (2) ◽  
pp. 221-235 ◽  
Author(s):  
Juan M Gallardo Bolaños ◽  
Carolina M Balao da Silva ◽  
Patricia Martín Muñoz ◽  
Antolín Morillo Rodríguez ◽  
María Plaza Dávila ◽  
...  
Keyword(s):  
Critical Role ◽  
Membrane Integrity ◽  
Specific Inhibitor ◽  
Akt Inhibitor ◽  
Akt Phosphorylation ◽  
Mitochondrial Superoxide ◽  
Phosphorylated Akt ◽  
Sperm Survival

AKT, also referred to as protein kinase B (PKB or RAC), plays a critical role in controlling cell survival and apoptosis. To gain insights into the mechanisms regulating sperm survival after ejaculation, the role of AKT was investigated in stallion spermatozoa using a specific inhibitor and a phosphoflow approach. Stallion spermatozoa were washed and incubated in Biggers–Whitten–Whittingham medium, supplemented with 1% polyvinyl alcohol (PVA) in the presence of 0 (vehicle), 10, 20 or 30 μM SH5, an AKT inhibitor. SH5 treatment reduced the percentage of sperm displaying AKT phosphorylation, with inhibition reaching a maximum after 1 h of incubation. This decrease in phosphorylation was attributable to either dephosphorylation or suppression of the active phosphorylation pathway. Stallion spermatozoa spontaneously dephosphorylated during in vitro incubation, resulting in a lack of a difference in AKT phosphorylation between the SH5-treated sperm and the control after 4 h of incubation. AKT inhibition decreased the proportion of motile spermatozoa (total and progressive) and the sperm velocity. Similarly, AKT inhibition reduced membrane integrity, leading to increased membrane permeability and reduced the mitochondrial membrane potential concomitantly with activation of caspases 3 and 7. However, the percentage of spermatozoa exhibiting oxidative stress, the production of mitochondrial superoxide radicals, DNA oxidation and DNA fragmentation were not affected by AKT inhibition. It is concluded that AKT maintains the membrane integrity of ejaculated stallion spermatozoa, presumably by inhibiting caspases 3 and 7, which prevents the progression of spermatozoa to an incomplete form of apoptosis.Free Spanish abstractA Spanish translation of this abstract is freely available at http://www.reproduction-online.org/content/148/2/221/suppl/DC1.

Mesenchimal Stroma Cells From Patients with Myelodysplastic Syndrome and Acute Myeloid Leukemia Show Distinct Cytogenetic and DNA-Mutation Data as Compared with Bone Marrow Leukemic Blasts.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4697-4697
Author(s):  
Olga Blau ◽  
Wolf-Karsten Hofmann ◽  
Claudia D Baldus ◽  
Gundula Thiel ◽  
Florian Nolte ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Control Analysis ◽  
Npm1 Mutation ◽  
Dna Mutation ◽  
Healthy Donors ◽  
Acute Myeloid

Abstract Abstract 4697 Bone marrow mesenchymal stroma cells (BMSC) are key components of the hematopoietic microenvironment. BMSC from patients with acute myeloid leukemia (AML) and myelodisplasic syndrome (MDS) display functional and quantitative alterations. To gain insight into these questions, we carried out cytogenetic analyses, FISH, FLT3 and NPM1 mutation examinations of both hematopoietic (HC) and BMSC derived from 53 AML and 54 MDS patients and 35 healthy donors after in vitro culture expansion. Clonal chromosomal aberrations were detectable in BMSC of 12% of patients. Using FISH we have assume that cytogenetic markers in BMSC were always distinct as the aberrations in HC from the same individual. 17% and 12% of AML patients showed FLT3 and NPM1 mutations in HC, respectively. In BMSC, we could not detect mutations of NPM1 and FLT3, independent from the mutation status of HC. For control analysis, BMSC cultures from 35 healthy donors were prepared under the same conditions. BMSC from healthy donors did show normal diploid karyotypes and absence of specific DNA-mutations of NPM1 and FLT3. Our data indicate that BMSC from MDS and AML patients are not a part of malignant clone and characterized by genetic aberrations. Lack of aberrations as detected in HC and appearance of novel clonal rearrangements in BMSC may suggest enhanced genetic susceptibility and potential involvement of BMSC in the pathogenesis of MDS and AML. Disclosures: No relevant conflicts of interest to declare.

A Novel Platelet Small-Molecule Agonist Targeting Glycoprotein Ibalpha.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4010-4010
Author(s):  
Jianfeng Yang ◽  
Zhi Chen ◽  
Weiliang Zhu ◽  
Changgeng Ruan
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Small Molecule ◽  
Platelet Adhesion ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Structural Basis ◽  
Terminal Fragment ◽  
Von Willebrand

Abstract Abstract 4010 Poster Board III-946 Introduction Interaction of glycoprotein (GP) Ibα with Von Willebrand factor (VWF) plays a critical role in platelet adhesion and signal transduction for αIIbβ3 activation under condition of high shear stress. Methods Based on the crystal structure of platelet GPIbα (PDB:1P9A), virtual screening was employed to identify active compounds. Compounds in SPECS database were docked to VWF binding site on the surface of GPIbα. The screening was carried out with the DOCK4.0 program. The 150 highest-scoring compounds were obtained for further bioassay and those with inhibitory activity of VWF binding to GPIbα were investigated the effect on platelet activation and aggregation. Results We found one compound, designated it as YC148, blocked ristocetin-induced plasma VWF binding to recombinant N-terminal fragment GPIbα (H1-V289) by ELISA method. More interestingly, YC148 did not inhibit ristocetin-induced platelet aggregation, on the contrary, it induced platelet aggregation itself in the absence of exogenous modulators such as ristocetin and botrocetin. A VWF A1 blocking antibody could not block platelet aggregation induced by YC148 despite it completely inhibited ristocetin-induced platelet agglutination. And YC148 also stimulated washed platelet aggregation where VWF was absent in the resuspension buffer. These indicated that the aggregation stimulated by YC148 could not the result from VWF binding. Flow cytomety also showed that YC148 increased P-selectin expression on platelet membrane and promoted monoclonal antibody PAC-1 binding to platelet. The platelet aggregation stimulated by YC148 was inhibited by anti-GPIbα monoclonal antibody AN51 and 6D1. Conclusion A novel exogenous small-molecule agonist was found to activate platelet through binding to GPIbα. It provides us a new tool for investigating platelet GPIb outside-in signaling pathway in platelet adhesion and aggregation. Furthermore, the structure of YC148 may provide a structural basis for developing new hemostatic drugs based on the inhibition of VWF-GPIb interaction. The effect of YC148 on platelet from Bernard-Soulier syndrome or GPIbα N-terminal fragment deficient platelet after in vitro cleavage will be further investigated. Disclosures: No relevant conflicts of interest to declare.

Quantitative Phosphoproteomics Identified a New Syk-Lyn-Axl Signalling Pathway Involved In Resistance to Nilotinib In Chronic Myeloid Leukemia Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3376-3376
Author(s):  
Romain Gioia ◽  
Cedric Leroy ◽  
Claire Drullion ◽  
Valérie Lagarde ◽  
Serge Roche ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Dependent Manner ◽  
Stable Isotope Labelling ◽  
Resistant Cells

Abstract Abstract 3376 Nilotinib has been developed to overcome resistance to imatinib, the first line treatment of chronic myeloid leukemia (CML). To anticipate resistance to nilotinib, we generate nilotinib resistant CML cell lines in vitro to characterize mechanisms and signaling pathways that may contribute to resistance. Among the different mechanisms of resistance identified, the overexpression of the Src-kinase Lyn was involved in resistance both in vitro, in a K562 cell line (K562-rn), and in vivo, in nilotinib-resistant CML patients. To characterize how Lyn mediates resistance, we performed a phosphoproteomic study using SILAC (Stable Isotope Labelling with Amino acid in Cell culture). Quantification and identification of phosphotyrosine proteins in the nilotinib resistant cells point out two tyrosine kinases, the spleen tyrosine kinase Syk and the UFO receptor Axl. The two tyrosine kinase Syk and Axl interact with Lyn as seen by coimmunopreciptation. Syk is phosphorylated on tyrosine 323 and 525/526 in Lyn dependent manner in nilotinib resistant cells. The inhibition of Syk tyrosine kinase by R406 or BAY31-6606 restores sensitivity to nilotinib in K562-rn cells. In parallel, the inhibition of Syk expression by ShRNA in K562-rn cells abolishes Lyn and Axl phosphorylation and then interaction between Lyn and Axl leading to a full restoration of nilotinib efficacy. In the opposite, the coexpression of Lyn and Syk in nilotinib sensitive K562 cells induced resistance to nilotinib whereas a Syk kinase dead mutant did not. These results highlight for the first time the critical role of Syk in resistance to tyrosine kinase inhibitors in CML disease emphasizing the therapeutic targeting of this tyrosine kinase. Moreover, Axl, which is already a target in solid tumor, will be also an interesting pathway to target in CML. Disclosures: No relevant conflicts of interest to declare.

STIM1 Deficiency Results In Impaired Platelet Procoagulant Activity and Protection From Arterial Thrombosis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 485-485
Author(s):  
Firdos Ahmad ◽  
Lucia Stefanini ◽  
Timothy Daniel Ouellette ◽  
Teshell K Greene ◽  
Stefan Feske ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Critical Role ◽  
Phosphatidyl Serine ◽  
Flow Chamber ◽  
Procoagulant Activity ◽  
Static Conditions

Abstract Abstract 485 Platelet activation is a central event in thrombosis and hemostasis. We recently demonstrated that most aspects of platelet activation depend on synergistic signaling by two signaling modules: 1) Ca2+/CalDAG-GEFI/Rap1 and 2) PKC/P2Y12/Rap1. The intracellular Ca2+ concentration of platelets is regulated by Ca2+ release from the endoplasmic reticulum (ER) and store-operated calcium entry (SOCE) through the plasma membrane. Stromal interaction molecule 1 (STIM1) was recently identified as the ER Ca2+ sensor that couples Ca2+ store release to SOCE. In this study, we compared the activation response of platelets lacking STIM1−/− or CalDAG-GEFI−/−, both in vitro and in vivo. To specifically investigate Ca2+-dependent platelet activation, some of the experiments were performed in the presence of inhibitors to P2Y12. The murine Stim1 gene was deleted in the megakaryocyte/platelet lineage by breeding Stim flox/flox mice with PF4-Cre mice (STIM1fl/fl). STIM1fl/fl platelets showed markedly reduced SOCE in response to agonist stimulation. aIIbβ3 activation in STIM1fl/fl platelets was significantly reduced in the presence but not in the absence of the P2Y12 inhibitor, 2-MesAMP. In contrast, aIIbb3 activation was completely inhibited in 2-MesAMP-treated CalDAG-GEFI−/− platelets. Deficiency in STIM1, and to a lesser extent in CalDAG-GEFI, reduced phosphatidyl serine (PS) exposure in platelets stimulated under static conditions. PS exposure was completely abolished in both STIM1fl/fl and CalDAG-GEFI−/− platelets stimulated in the presence of 2-MesAMP. To test the ability of platelets to form thrombi under conditions of arterial shear stress, we performed flow chamber experiments with anticoagulated blood perfused over a collagen surface. Thrombus formation was abolished in CalDAG-GEFI−/− blood and WT blood treated with 2-MesAMP. In contrast, STIM1fl/fl platelets were indistinguishable from WT platelets in their ability to form thrombi. STIM1fl/fl platelets, however, were impaired in their ability to express PS when adhering to collagen under flow. Consistently, when subjected to a laser injury thrombosis model, STIM1fl/fl mice showed delayed and reduced fibrin generation, resulting in the formation of unstable thrombi. In conclusion, our studies indicate a critical role of STIM1 in SOCE and platelet procoagulant activity, but not in CalDAG-GEFI mediated activation of aIIbb3 integrin. Disclosures: No relevant conflicts of interest to declare.

Innate Lymphoid Cell-Derived IL-22 Mediates Endogenous Thymic Repair Under the Control of IL-23

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 143-143
Author(s):  
Jarrod A Dudakov ◽  
Alan M Hanash ◽  
Lauren F. Young ◽  
Natalie V Singer ◽  
Mallory L West ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Critical Role ◽  
Lymphoid Cells ◽  
Thymic Epithelial Cells ◽  
Immune Competence ◽  
Strong Negative Correlation ◽  
Hematopoietic Stem ◽  
Stimulation Of

Abstract Abstract 143 Despite being exquisitely sensitive to insult, the thymus is remarkably resilient in young healthy animals. Endogenous regeneration of the thymus is a crucial function that allows for renewal of immune competence following infection or immunodepletion caused by cytoreductive chemotherapy or radiation. However, the mechanisms governing this regeneration remain poorly understood. Thymopoiesis is a highly complex process involving cross-talk between developing thymocytes and their supporting non-hematopoietic stromal microenvironment, which includes highly specialized thymic epithelial cells (TECs) that are crucial for T cell development. IL-22 is a recently identified cytokine predominantly associated with maintenance of barrier function at mucosal surfaces. Here we demonstrate for the first time a critical role for IL-22 in endogenous thymic repair. Comparing IL-22 KO and WT mice we observed that while IL-22 deficiency was redundant for steady-state thymopoiesis, it led to a pronounced and prolonged loss of thymus cellularity following sublethal total body irradiation (SL-TBI), which included depletion of both thymocytes (p=0.0001) and TECs (p=0.003). Strikingly, absolute levels of IL-22 were markedly increased following thymic insult (p<0.0001) despite the significant depletion of thymus cellularity. This resulted in a profound increase in the production of IL-22 on a per cell basis (p<0.0001). These enhanced levels of IL-22 peaked at days 5 to 7 after SL-TBI, immediately following the nadir of thymic cellularity. This was demonstrated by a strong negative correlation between thymic cellularity and absolute levels of IL-22 (Fig 1a). In mucosal tissues the regulation of IL-22 production has been closely associated with IL-23 produced by dendritic cells (DCs) and ex vivo incubation of cells with IL-23 stimulates the production of IL-22. Following thymic insult there was a significant increase in the amount of IL-23 produced by DCs (Fig 1b) resulting in similar kinetics of intrathymic levels of IL-22 and IL-23. We identified a population of radio-resistant CD3−CD4+IL7Ra+RORg(t)+ thymic innate lymphoid cells (tILCs) that upregulate both their production of IL-22 (Fig 1c) and expression of the IL-23R (p=0.0006) upon exposure to TBI. This suggests that they are responsive to IL-23 produced by DCs in vivo following TBI and, in fact, in vitro stimulation of tILCs by IL-23 led to upregulation of Il-22 production by these cells (Fig 1d). We found expression of the IL-22Ra on cortical and medullary TECs (cTECs and mTECs, respectively), and uniform expression across both mature MHCIIhi mTEC (mTEChi) and immature MHCIIlo mTECs (mTEClo). However, in vitro stimulation of TECs with recombinant IL-22 led to enhanced TEC proliferation primarily in cTEC and mTEClo subsets (p=0.002 and 0.004 respectively). It is currently unclear if IL-22 acts as a maturation signal for mTECs, however, the uniform expression of IL-22Ra between immature mTEClo and mature Aire-expressing mTEChi, together with the preferential promotion of proliferation amongst mTEClo and cTEC seem to argue against IL-22 as a maturational signal but rather as promoter of proliferation, which ultimately leads to terminal differentiation of TECs. Of major clinical importance, administration of exogenous IL-22 led to enhanced thymic recovery (Fig. 1e) following TBI, primarily by promoting the proliferation of TECs. Consistent with this, the administration of IL-22 also led to significantly enhanced thymopoiesis following syngeneic BMT. Taken together these findings suggest that following thymic insult, and specifically the depletion of developing thymocytes, upregulation of IL-23 by DCs induces the production of IL-22 by tILCs and regeneration of the supporting microenvironment. This cascade of events ultimately leads to rejuvenation of the thymocyte pool (Fig. 1f). These studies not only reveal a novel pathway underlying endogenous thymic regeneration, but also identify a novel regenerative strategy for improving immune competence in patients whose thymus has been damaged from infection, age or cytoreductive conditioning required for successful hematopoietic stem cell transplantation. Finally, these findings may also provide an avenue of study to further understand the repair and regeneration of other epithelial tissues such as skin, lung and breast. Disclosures: No relevant conflicts of interest to declare.

A Critical Role of Mir-9 in Myelopoesis and EVI1-Induced Leukemogenesis.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2332-2332
Author(s):  
Vitalyi Senyuk ◽  
Yunyuan Zhang ◽  
Yang Liu ◽  
Ming Ming ◽  
Jianjun Chen ◽  
...  
Keyword(s):  
Cell Transformation ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Ectopic Expression ◽  
Myeloid Differentiation ◽  
Dna Hypermethylation ◽  
Hematopoietic Stem

Abstract Abstract 2332 MicroRNA-9 (miR-9) is required for normal neurogenesis and organ development. The expression of miR-9 is altered in several types of solid tumors suggesting that it may have a function in cell transformation. However the role of this miR in normal hematopoiesis and leukemogenesis is unknown. Here we show that miR-9 is expressed at low levels in hematopoietic stem/progenitor cells (HSCs/HPCs), and that it is upregulated during hematopoietic differentiation. Ectopic expression of miR-9 strongly accelerates terminal myelopoiesis, while promoting apoptosis in vitro and in vivo. In addition, the inhibition of miR-9 in HPC with a miRNA sponge blocks myelopoiesis. EVI1, required for normal embryogenesis, and is considered an oncogene because inappropriate upregulation induces malignant transformation in solid and hematopoietic cancers. In vitro, EVI1 severely affects myeloid differentiation. Here we show that EVI1 binds to the promoter of miR-9–3 leading to DNA hypermethylation of the promoter as well as repression of miR-9. We also show that ectopic miR-9 reverses the myeloid differentiation block that is induced by EVI1. Our findings suggest that inappropriately expressed EVI1 delays or blocks myeloid differentiation, at least in part by DNA hypermethylation and downregulation of miR-9. It was previously reported that FoxOs genes inhibit myeloid differentiation and prevent differentiation of leukemia initiating cells. Here we identify FoxO3 and FoxO1 as new direct targets of miR-9 in hematopoietic cells, and we find that upregulation of FoxO3 in miR-9-positive cells reduces the acceleration of myelopoiesis. These results reveal a novel role of miR-9 in myelopoiesis and in the pathogenesis of EVI1-induced myeloid neoplasms. They also provide new insights on the potential chromatin-modifying role of oncogenes in epigenetic changes in cancer cells. Disclosures: No relevant conflicts of interest to declare.

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