Exploration On T-Cell Glucose Metabolism Disorders Mediated T Subsets Imbalances And Potential Therapeutic Role In ITP

Part Ⅰ:Empagliflozin modulates CD4⁺T cell differentiation via metabolic reprogramming in immune thrombocytopeniaBackground:Immune thrombocytopenia（ITP）is an acquired autoimmune disease characterized by low platelet counts due to immune-mediated platelet destruction and impaired platelet production.The pathogenesis of ITP is complicated and heterogenous,including multiple mechanisms.It has been reported that ITP is a T helper（Th）1 and Th17 polarized disease where proinflammatory cytokines IFN-γ and IL-17 are increased and suppressive T subsets regulatory T cells（Tregs）are decreased,leading to loss of tolerance to self-antigens.Recently,increasing evidence has demonstrated that metabolism is important for T cell functions.T cell activation is highly dependent on glycolysis even with normoxia.Furthermore,different T cell subsets showed distinct metabolic features.For example,T helper cells（Th1 and Th17）depend on aerobic glycolysis,whereas Tregs depend on oxidative phosphorylation（OXPHOS）.Previous studies showed that metabolic reprogramming was involved in the pathological mechanisms of autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus（SLE）.Nowadays,metabolic remodeling is becoming the emerging target of autoimmune diseases.Nevertheless,metabolism in CD4⁺T cells,particularly from different subsets,has not yet been investigated in ITP.Empagliflozin,a sodium-glucose cotransporter 2（SGLT2）inhibitor,has been developed as anti-diabetic agent by inhibiting renal reabsorption of glucose.SGLT2 inhibitors have more recently gained interest for potential anti-inflammatory effects beyond their glucose-lowing properties in the heart and kidney.Empagliflozin has been found to downregulate both lipopolysaccharide（LPS）-induced tumor necrosis factor（TNF）and inducible nitric oxide synthase（iNOS）expression in cardiomyocytes and macrophages.Further benefits of empagliflozin in the cardiovascular system include promoting macrophage polarization toward M2 via AMPK activation.However,the effect of SGLT2 inhibitor on T cells remains elusive.In this study,we revealed that metabolic reprogramming of CD4⁺T cell resulted in an imbalanced CD4⁺T cell subsets,with more Th1/Th17 cells and fewer Tregs in ITP.The SGLT2 inhibitor empagliflozin can restore metabolic balance and affect CD4⁺T subset differentiation,and can also treat thrombocytopenia in active ITP model.Objectives:（1）To explore the metabolic profile of CD4⁺T cells in ITP patients and to investigate the effect of empagliflozin on CD4⁺T subsets differentiation.（2）To describe the role of empagliflozin in influencing CD4⁺T cell differentiation through metabolic reprogramming and the confirmation of its role in an active ITP model.Methods:（1）ITP patients and controls:Patients with ITP and healthy controls were enrolled,their peripheral blood was collected,CD4⁺T cells were sorted and cultured in vitro for flow cytometry,Seahorse energy analysis,RT-PCR,Western-blot and other experiments.（2）Seahorse metabolic assay:glycolytic stress assay and mitochondrial stress assay were used to detect oxidative phosphorylation and glycolysis levels in CD4⁺T cells from ITP patients and healthy controls as well as to detect oxidative phosphorylation and glycolysis levels in CD4⁺T cells from ITP patients after empagliflozin treatment.（3）Flow cytometry was performed to detect the proportion of CD4⁺T subsets:Th1,Th 17,Tregs.（4）RT-PCR was assessed of glycolysis-related genes Glut1,HK2,PFKB1,PKM2 mRNA levels.（5）Western blotting was used for detecting mTOR,PI3K,protein levels of mTORrelated signaling pathway.（6）Active ITP mice model:To verify the effect of empagliflozin in the ITP model and to observe the recovery of platelet count and changes in CD4⁺T subpopulation in the spleen of the active ITP mice model after empagliflozin drug treatment.Results:（1）CD4⁺T cells in ITP patients underwent metabolic reprogrammingSeahorse glycolytic stress and mitochondrial stress assays showed that CD4⁺T cell glycolysis levels were significantly upregulated and oxidative phosphorylation levels were significantly decreased in ITP patients compared to healthy controls.（2）The SGLT2 inhibitor,empagliflozin regulated CD4⁺T subpopulation differentiation.In vitro cell culture showed that empagliflozin could inhibit the ratio of Thl and Th17 and up-regulate the ratio of Tregs.（3）Empagliflozin regulated CD4⁺T subsets differentiation by affecting metabolic reorganization（4）RT-PCR showed that empagliflozin could inhibit mRNA levels of glycolysisrelated genes.（5）Empagliflozin inhibited mTOR signaling pathway to exert metabolic regulation.Western blot showed that empagliflozin could inhibit the phosphorylation of mTOR and PI3K.（6）Empagliflozin ameliorated thrombocytopenia in a mouse model of active ITP.Empagliflozin attenuated the decrease in platelet count and normalized platelet levels earlier in the active ITP mouse model,and normalized the ratio of imbalanced T subpopulation cells in the spleen of ITP mice.Conclusions:（1）For the first time,we demonstrated that CD4⁺T cells from ITP patients underwent a metabolic reorganization shift from oxidative phosphorylation to glycolysis and the metabolic reprogramming affected the proportion of T cell subpopulations.（2）For the first time,empagliflozin,an SGLT2 inhibitor,was shown to regulate the differentiation of CD4⁺T subpopulations by suppressing Th1 and Th17 cell populations,while increasing the proportion of Tregs.（3）Empagliflozin regulated CD4⁺T cell differentiation in immune thrombocytopenia through metabolic reprogramming.（4）Empagliflozin regulated CD4⁺T cell differentiation by inhibiting the mTOR signaling pathway.（5）Empagliflozin had a therapeutic effect on active ITP mice and is expected to be a potential drug for the treatment of ITP.Part Ⅱ:Investigation of the Immunomodulatory Effect of Bitter Taste Receptor on CD4⁺T cells in immune thrombocytopeniaBackground:Immune thrombocytopenia（ITP）is a common hemorrhagic autoimmune disease clinically characterized with skin and mucosa bleeding or bleeding tendency caused by thrombocytopenia.It is characterized by the platelet membrane proteins become antigens and stimulate the immune system,resulting in thrombocytopenia.The main point of view is immune-mediated platelet destruction and/or inhibition of platelet production.The pathogenesis of ITP is involved in many factors and links.T-cellmediated immune abnormalities are considered to be an important part of the pathogenesis of ITP.Panitsas et al found that the pathogenesis of adult ITP is centered on cellular immune disorder and is a cellular immune response polarized to Th1 and Th17 cells.The complex disorders of cellular immunity include the transfer to the dominance of Th1 and Th17,the decrease of the ratio of Th2 and Tregs,the increase of the proportion of Th1/2 and Th17/Tregs,the loss of tolerance caused by T subsets imbalance and functional deficiency,and the destruction of platelets by cytotoxic T lymphocytes（CTLs）.Bitter taste receptors（TAS2Rs or T2Rs）are cell surface receptors closely associated to the transmission and expression of bitterness perception.They were first identified in the taste cells of taste buds in 2000.Bitter taste perception is one of the body’s effective self-protection mechanisms against the ingestion of harmful substances.Bitter taste receptors belong to the G protein-coupled receptor superfamily.In human,TAS2Rs belong to a family of 25 functional members clustered on chromosomes 5,7 and 12.There is growing evidence that T2Rs are expressed in organs other than the gustatory system,including the gastrointestinal tract,thyroid,respiratory organs,kidney,central system and skin.In addition to bitter taste perception,bitter taste receptors perform different functions in different cells,such as innate immunity,muscle contraction,secretion,and regeneration.The research on bitter taste receptor in immunity is mainly focused on innate immunity,but there are few reports on adaptive immunity.Agne,Malki,Julia et al.in 2015 first identified the presence of bitter taste receptors in blood cells,including NK cells,monocytes and lymphocytes;Hoai T.T.Tran et al.in 2018 reported that T2R38（bitter taste receptor member 38）expression was higher in T cells after activation than in resting T cells.However,it has not been reported of the function of bitter taste receptors in T cell differentiation.In order to explore the function of bitter taste receptor in T cell differentiation,we studied the immunomodulatory effect of bitter taste receptor in CD4⁺T cells of ITP patients and its mechanism in the context of ITP disease.In this study,we explored the role and mechanism of bitter taste receptor in T cell differentiation.Through screening,we found that T2R14 blockers could regulate the differentiation of CD4⁺T cell subsets.,The expression of T2R14 was elevated in CD4⁺T cells from ITP patients compared to healthy controls and its regulation of differentiation was associated with modulating metabolism.Here,we demonstrated the immunomodulatory role of T2R14 in CD4⁺ T cells of ITP patients.Methods:（1）Cell culture:CD4⁺T cells from ITP patients were treated with different bitter taste receptor blockers in an in vitro induction assay to screen for bitter taste receptors that may have a role in T cell differentiation.（2）Calcium ion assay:to assess whether T2R14 agonists and blockers act on bitterness receptors.（3）Flow cytometry:to compare the expression levels of T2R14 and T2R39 between ITP patients and healthy controls;to detect the ratios of CD4⁺T subset,Th1,Th2,Th17;to detect the expression level of T2R14 in different Th subsets.（4）siRNA-T2R14 transfection:to further validate the immunomodulatory role of T2R14 and exclude the effect of T2R39.（5）Seahorse metabolic assay:glycolysis stress assay and mitochondrial stress assay were performed to detect the effects of T2R14 inhibitors and agonists on metabolism.（6）RT-PCR:it was used to assess the mRNA levels of T2R14 and T2R39 in ITP patients and healthy controls and the mRNA levels of glycolysis-related genes Glut1,HK2,PFKB1,PKM2 and different subunits of mTOR after treatment with T2R14 blockers and agonists.Results:（1）Screening with bitter taste receptor blockers revealed that 6-methoxyflavanone（6-MeOF）-T2R14 and T2R39 blockers suppressed Thl and Th17 cell ratios and elevated the ratio of Th2.（2）Calcium ion assays confirmed that T2R14 agonist and blocker acted on bitter receptors.（3）Flow cytometry was used to compare the expression of T2R14 and T2R39 in the CD4⁺T cells of ITP patients and healthy controls.It revealed T2R14 expression was statistically higher in CD4⁺T cells of ITP patients than in healthy controls and T2R39 expression was also elevated in the ITP group without reaching statistical differences;T cells were cultured in vitro with T2R14 agonist and T2R14 blocker and we found that T2R14 agonist and T2R14 blocker had opposite effects on CD4⁺T cell differentiation:T2R14 blocker inhibited the ratio of Th1 and Th17 cells and upregulated the ratio of Th2 cells,while T2R14 agonist upregulated the ratio of Thl and Th17 cells and downregulated the ratio of Th2 cells;T2R14 expression was highest in Th2 cells,followed by Th17 cells,and Th1 cells.（4）siRNA-T2R14 transfection:consistent with the regulation of T cell differentiation by T2R14 blocker,it inhibited Th1 and Th17 cells and up-regulated Th2 cells.（5）Seahorse Metabolic assays revealed that T2R14 affected metabolism,with its agonist up-regulating glycolysis and oxidative phosphorylation levels in CD4⁺T cells and its blocker down-regulating glycolysis and oxidative phosphorylation levels,with the combined effect mainly affecting glycolysis.（6）RT-PCR results revealed that in CD4⁺T cells,ITP patients had elevated mRNA expression levels of T2R14 compared to healthy controls;T2R14 agonist and blockers affected mRNA levels of glycolysis-related genes,with opposite effects on the two subunits of mTOR:T2R14 blockers inhibited mRNA levels of Raptor and elevated mRNA levels of Rictor mRNA levels,and T2R14 agonists elevated mRNA levels of Raptor and decreased mRNA levels of Rictor.Conclusion:（1）T2R14 blocker can regulate CD4⁺T cell differentiation in ITP patients.（2）T2R14 expression was elevated in CD4⁺T cells of ITP patients.（3）T2R14 exerted its regulatory effects on CD4⁺T cells by regulating metabolism,with a predominant effect on glycolysis.（4）T2R14 exerted immunomodulatory effects by acting differently on the two subunits of mTOR.（5）T2R14 could be a potential target for the treatment of ITP.Part Ⅲ:Investigation of the role of the bitter taste receptor agonist denatonium benzoate in hematopoietic reconstitution after hematopoietic stem cell transplantation in miceBackground:In the hematopoietic system,hematopoietic stem cells（HSCs）are located at the top of the hematopoietic layer and have the capacity of self-renewal and multidirectional differentiation,which are essential for the lifelong maintenance of stem cell pool and the production of all types of blood cells.Most hematopoietic stem cells are in a quiescent state in niches within the bone marrow,a process that is tightly regulated to maintain a stable number of white blood cells,platelets and red blood cells in the blood.Individual hematopoietic stem cell undergo one of several destinies:quiescence,apoptosis,recycling for self-renewal,or production of multipotent progenitors（MPPs）,or migration.These fates are regulated by a combination of intrinsic and extrinsic factors.HSCs respond to stimuli when specific local microenvironmental（niche）changes lead to a disruption of the balance of the HSCs pool.Hematopoietic stem cell transplantation is currently the most effective treatment for malignancies of the hematopoietic system.The successful restoration of hematopoietic function after transplantation depends on the effective homing of hematopoietic stem cells.The homing of stem cells to specific niches within the bone marrow is a rapid and coordinated process that is tightly regulated,with a variety of adhesion molecules,chemokines,glycoproteins,and integrins,all involved in the homing of hematopoietic stem cells after transplantation.Bitter taste receptors（TAS2Rs/T2Rs）belong to the G protein-coupled receptors（GPCR）family,which have 25 different members in human and 35 members in mice.T2Rs was first found in the taste bud cells and is responsible for perception the taste of bitterness.In recent years,accumulating evidence have found that it is widely expressed in the extraoral system.The functions of bitter taste receptors are also increasingly being identified,including muscle contraction,secretion,regeneration and so on.We have already investigated the role of bitter taste receptors in T-cell immunomodulation in the second part.In 2020,it has been reported acute myeloid leukemia cells expressed T2R subtypes and were involved in regulating the biological functions of leukemia cells participated in the regulation of biological function of leukemic cells.However,the study of bitter taste receptors in hematopoiesis and hematopoietic reconstitution has not been reported.Denatonium benzoate（DB）is one of the most bitter substances known and has been widely demonstrated to be a bitter agonist that can be used to activate bitter receptors in many different cell types,but its role in hematopoiesis has not been reported.In this study,denatonium benzoate was used as a bitter taste receptor agonist to study the role of bitter taste receptors in the hematopoietic reconstitution of bone marrow transplantation（BMT）mice for the first time.Methods:（1）In this study,a homogeneous bone marrow transplantation model was used to explore the effect of bitter taste receptor agonist denatonium benzoate in the hematopoietic reconstitution after bone marrow transplantation in lethally irradiated mice.（2）RT-PCR verified the mRNA level of bitter taste receptor related genes.（3）Calcium ion assay was performed to analyze whether denatonium benzoate can activate the bitter receptor and cause an increase of calcium ion.（4）Complete blood cell count was used to assess the effect of denatonium benzoate in hematopoietic reconstruction in BMT mice.（5）Flow cytometric assay assessed the proportion and pedigree changes of hematopoietic stem cells and the effects of terminal differentiation in BMT mice.（6）Colony-Forming Unit（CFU）of mouse hematopoietic stem cells reflected the proliferative and differentiation ability of hematopoietic stem/progenitor cells to multiple lineages after DB treatment.（7）Cycle detection was performed to detect the effect of denatonium benzoate on the proportion of bone marrow LSK cycle ratio in BMT mice.Results:（1）RT-PCR detected and demonstrated the presence of bitter taste receptors in bone marrow cells,and mRNA expression of bitter taste receptor subtypes was elevated after DB treatment.（2）Calcium ion assays confirmed that stimulation of bone marrow mononuclear cells by the T2R agonist DB induced an increase in intracellular Ca²⁺concentration,demonstrating its function of activation of the T2R.（3）Complete blood cell counts showed that DB could promote recovery of blood cell levels after bone marrow transplantation in lethally irradiated mice.（4）Flow cytometry results confirmed that DB increased the proportion of hematopoietic stem progenitor cells in the bone marrow of BMT mice.（5）CFU test revealed that DB facilitated the formation of different colonies in vitro,which is beneficial to the maintenance of hematopoietic progenitor cell pool.（6）The cycle assay showed that DB promoted the proportion of bone marrow HSC entering the cycle after BMT in lethally irradiated mice.Conclusion:Our study expanded the research scope of bitter taste receptor to the field of hematopoiesis for the first time,and clarified the function of T2R in hematopoietic stem cell differentiation and its regulatory role in hematopoietic stem cell transplantation.Denatonium benzoate,a bitter taste receptor agonist,could promote the differentiation of hematopoietic stem cells into cells of various pedigree groups,improved hematopoietic reconstitution in mice after bone marrow transplantation.