Ion channel Piezo1 activation aggravates the endothelial dysfunction under a high glucose environment.

BACKGROUND: Vasculopathy is the most common complication of diabetes. Endothelial cells located in the innermost layer of blood vessels are constantly affected by blood flow or vascular components; thus, their mechanosensitivity plays an important role in mediating vascular regulation. Endothelial damage, one of the main causes of hyperglycemic vascular complications, has been extensively studied. However, the role of mechanosensitive signaling in hyperglycemic endothelial damage remains unclear. METHODS: Vascular endothelial-specific Piezo1 knockout mice were generated to investigate the effects of Piezo1 on Streptozotocin-induced hyperglycemia and vascular endothelial injury. In vitro activation or knockdown of Piezo1 was performed to evaluate the effects on the proliferation, migration, and tubular function of human umbilical vein endothelial cells in high glucose. Reactive oxygen species production, mitochondrial membrane potential alternations, and oxidative stress-related products were used to assess the extent of oxidative stress damage caused by Piezo1 activation. RESULTS: Our study found that in VECreERT2;Piezo1flox/flox mice with Piezo1 conditional knockout in vascular endothelial cells, Piezo1 deficiency alleviated streptozotocin-induced hyperglycemia with reduced apoptosis and abscission of thoracic aortic endothelial cells, and decreased the inflammatory response of aortic tissue caused by high glucose. Moreover, the knockout of Piezo1 showed a thinner thoracic aortic wall, reduced tunica media damage, and increased endothelial nitric oxide synthase expression in transgenic mice, indicating the relief of endothelial damage caused by hyperglycemia. We also showed that Piezo1 activation aggravated oxidative stress injury and resulted in severe dysfunction through the Ca2+-induced CaMKII-Nrf2 axis in human umbilical vein endothelial cells. In Piezo1 conditional knockout mice, Piezo1 deficiency partially restored superoxide dismutase activity and reduced malondialdehyde content in the thoracic aorta. Mechanistically, Piezo1 deficiency decreased CaMKII phosphorylation and restored the expression of Nrf2 and its downstream molecules HO-1 and NQO1. CONCLUSION: In summary, our study revealed that Piezo1 is involved in high glucose-induced oxidative stress injury and aggravated endothelial dysfunction, which have great significance for alleviating endothelial damage caused by hyperglycemia.

Obesity is associated with poor outcomes of corticosteroid treatment in patients with primary immune thrombocytopenia.

Emerging evidence has demonstrated that obesity impacts multiple immune-related diseases. It remains unclear whether and how obesity alters treatment outcomes in patients with primary immune thrombocytopenia (ITP). Thus, we retrospectively investigated 214 treatment-naïve patients who received standard high-dose dexamethasone therapy in Qilu Hospital. Patients with obesity showed significantly lower overall initial response (underweight vs. normal vs. overweight vs. obese: 85.7% vs. 85.2% vs. 72.0% vs. 52.3%, p = 0.001) and initial complete response ([CR], 71.4% vs. 70.4% vs. 53.3% vs. 27.3%, p < 0.001) rates. The same trend was observed in the 6-month sustained response (63.6% vs. 52.3% vs. 35.6% vs. 22.7%, p = 0.03) and sustained CR (36.4% vs. 44.6% vs. 24.4% vs. 9.1%, p = 0.01). The Kaplan-Meier analysis revealed a shortened duration of remission in the obese group (median duration of remission, not reached vs. 16 months vs. 2 months vs. 1 month, p = 0.002). In multivariate regression analysis, obesity was independently associated with poor initial and sustained responses, and an increased risk for relapse. In conclusion, obesity is a negative predictor for outcomes of corticosteroid treatment. A stratified strategy according to body mass index status may facilitate the precision management of ITP.

Plasma long noncoding RNAs lncDC and THRIL as potential diagnostic markers of adult primary immune thrombocytopenia.

INTRODUCTION: Immune thrombocytopenia (ITP) is a common acquired hemorrhagic disease without "gold standard" for the diagnosis, long non-coding RNA (lncRNAs) can participate in regulating gene expression through various mechanisms and may play a role in immune intolerance in ITP. Several previous studies have confirmed that lncRNA lncDC and THRIL are involved in the development of autoimmune diseases. This study investigates the relationship between expression levels of two plasma lncRNAs (lncDC and THRIL) and clinical characteristics of adult primary ITP patients, ascertain their potential applications as diagnostic markers of ITP. METHODS: We recruited 102 subjects, including 41 ITP patients, 41 healthy controls (HCs) and 20 patients under myelosuppression phase after chemotherapy (MS). qRT-PCR was used to detect the expression of two lncRNAs in the peripheral blood plasma of the three groups. Receiver operating characteristic (ROC) curves were used to test the diagnostic efficacy of lncDC and THRIL in ITP. RESULTS: The expression level of lncDC was downregulated in ITP patients compared with HCs (p = . 012) and MS (p = .035), whereas THRIL was significantly upregulated (p = .0005, p < . 0001). We further revealed that lncDC has a high sensitivity (78. 05%), while THRIL has a high specificity (97. 56%). The area under the curve (AUC) (0.869, 95% CI: 0.795-0.943, p < .0001) of the ROC curve for this combination increased significantly. CONCLUSIONS: THRIL and lncDC expression levels were changed in adult ITP patients. The lncRNAs lncDC and THRIL can serve as potential diagnostic markers for adult primary ITP.

Identification of novel NFKB1 and ICOS frameshift variants in patients with CVID.

Common variable immunodeficiency (CVID) is a 'late-onset' primary immunodeficiency characterized by variable manifestations and genetic heterogeneity. A monogenic cause of CVID has been reported in 10% of patients. In this study, we identified two novel pathogenic variants implicated in monogenic CVID by whole exome sequencing (WES) analysis: a heterozygous nuclear factor κB subunit 1 (NFKB1) p.G686fs mutation and a homozygous inducible T-cell co-stimulator (ICOS) p.L96Sfs mutation. The predicted crystal models indicated premature truncation of the two mutated proteins. Both variants were demonstrated as loss-of-function mutations and were associated with overlapped manifestations of respiratory fungal infection and splenomegaly. We further performed a detailed assessment of immunologic phenotypes and impaired lymphocyte functions in patients. Moreover, we discovered an association between monoclonal T-large granular lymphocyte proliferation and ICOS-deficient CVID for the first time. These observations lead to a new perspective on the underlying genetic heterogeneity of CVID.

Deciphering transcriptome alterations in bone marrow hematopoiesis at single-cell resolution in immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune disorder, in which megakaryocyte dysfunction caused by an autoimmune reaction can lead to thrombocytopenia, although the underlying mechanisms remain unclear. Here, we performed single-cell transcriptome profiling of bone marrow CD34+ hematopoietic stem and progenitor cells (HSPCs) to determine defects in megakaryopoiesis in ITP. Gene expression, cell-cell interactions, and transcriptional regulatory networks varied in HSPCs of ITP, particularly in immune cell progenitors. Differentially expressed gene (DEG) analysis indicated that there was an impaired megakaryopoiesis of ITP. Flow cytometry confirmed that the number of CD9+ and HES1+ cells from Lin-CD34+CD45RA- HSPCs decreased in ITP. Liquid culture assays demonstrated that CD9+Lin-CD34+CD45RA- HSPCs tended to differentiate into megakaryocytes; however, this tendency was not observed in ITP patients and more erythrocytes were produced. The percentage of megakaryocytes differentiated from CD9+Lin-CD34+CD45RA- HSPCs was 3-fold higher than that of the CD9- counterparts from healthy controls (HCs), whereas, in ITP patients, the percentage decreased to only 1/4th of that in the HCs and was comparable to that from the CD9- HSPCs. Additionally, when co-cultured with pre-B cells from ITP patients, the differentiation of CD9+Lin-CD34+CD45RA- HSPCs toward the megakaryopoietic lineage was impaired. Further analysis revealed that megakaryocytic progenitors (MkP) can be divided into seven subclusters with different gene expression patterns and functions. The ITP-associated DEGs were MkP subtype-specific, with most DEGs concentrated in the subcluster possessing dual functions of immunomodulation and platelet generation. This study comprehensively dissects defective hematopoiesis and provides novel insights regarding the pathogenesis of ITP.

Ion channel Piezo1 activation promotes aerobic glycolysis in macrophages.

Altered microenvironmental stiffness is a hallmark of inflammation. It is sensed by the mechanically activated cation channel Piezo1 in macrophages to induce subsequent immune responses. However, the mechanism by which the mechanosensitive signals shape the metabolic status of macrophages and tune immune responses remains unclear. We revealed that Piezo1-deficient macrophages exhibit reduced aerobic glycolysis in resting or liposaccharide (LPS)-stimulated macrophages with impaired LPS-induced secretion of inflammatory cytokines in vitro. Additionally, pretreatment with the Piezo1 agonist, Yoda1, or cyclical hydrostatic pressure (CHP) upregulated glycolytic activity and enhanced LPS-induced secretion of inflammatory cytokines. Piezo1-deficient mice were less susceptible to dextran sulfate sodium (DSS)-induced colitis, whereas Yoda1 treatment aggravated colitis. Mechanistically, we found that Piezo1 activation promotes aerobic glycolysis through the Ca2+-induced CaMKII-HIF1α axis. Therefore, our study revealed that Piezo1-mediated mechanosensitive signals Piezo1 can enhance aerobic glycolysis and promote the LPS-induced immune response in macrophages.

Empagliflozin modulates CD4+ T-cell differentiation via metabolic reprogramming in immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an acquired autoimmune disease, in which the imbalance of CD4+ T cell subsets play a key role in the pathogenesis. Since T cells highly depend on metabolism for their function, we hypothesized that T cell dysfunction may be due to intracellular metabolic reprogramming. We found that in ITP, T cell metabolism shifts from oxidative phosphorylation to glycolysis. Empagliflozin, a sodium-glucose cotransporter 2 inhibitor, has shown regulatory metabolic effects on proximal tubular epithelial cells and cardiac cells beyond glucose lowering. However, the effects of empagliflozin on T cells remain unknown. To further investigate the metabolic dysfunction of CD4+ T cells in ITP, we explored the effect of empagliflozin on CD4+ T-cell differentiation in ITP. Our results are the first to show that increased glycolysis in CD4+ T cells resulted in an unbalanced CD4+ T-cell population. Furthermore, empagliflozin can affect the differentiation of CD4+ T-cell subsets by inhibiting Th1 and Th17 cell populations while increasing Tregs. Empagliflozin appears to regulate CD4+ T cells through inhibiting the mTOR signal pathway. Considering these results, we propose that empagliflozin could be used as a potential therapeutic option for ITP by modulating metabolic reprogramming in CD4+ T cells.

Glucocorticoid receptor modulates myeloid-derived suppressor cell function via mitochondrial metabolism in immune thrombocytopenia.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature cells and natural inhibitors of adaptive immunity. Intracellular metabolic changes in MDSCs exert a direct immunological influence on their suppressive activity. Our previous study demonstrated that high-dose dexamethasone (HD-DXM) corrected the functional impairment of MDSCs in immune thrombocytopenia (ITP); however, the MDSC population was not restored in nonresponders, and the mechanism remained unclear. In this study, altered mitochondrial physiology and reduced mitochondrial gene transcription were detected in MDSCs from HD-DXM nonresponders, accompanied by decreased levels of carnitine palmitoyltransferase-1 (CPT-1), a rate-limiting enzyme in fatty acid oxidation (FAO). Blockade of FAO with a CPT-1 inhibitor abolished the immunosuppressive function of MDSCs in HD-DXM responders. We also report that MDSCs from ITP patients had lower expression of the glucocorticoid receptor (GR), which can translocate into mitochondria to regulate the transcription of mitochondrial DNA (mtDNA) as well as the level of oxidative phosphorylation. It was confirmed that the expression of CPT-1 and mtDNA-encoded genes was downregulated in GR-siRNA-treated murine MDSCs. Finally, by establishing murine models of active and passive ITP via adoptive transfer of DXM-modulated MDSCs, we confirmed that GR-silenced MDSCs failed to alleviate thrombocytopenia in mice with ITP. In conclusion, our study indicated that impaired aerobic metabolism in MDSCs participates in the pathogenesis of glucocorticoid resistance in ITP and that intact control of MDSC metabolism by GR contributes to the homeostatic regulation of immunosuppressive cell function.

Lineage Tracing Reveals the Dynamic Contribution of Id2+ Progenitor Cells to Branching Morphogenesis.

Branching morphogenesis is an important process in shaping the arborized structures of several organs. However, the driving force that directs this process from progenitor pools remains incompletely understood. In this lineage tracing study, we investigated the role of Id2+ embryonic progenitor cells in branching organs such as the pancreas, kidney, mammary gland, thyroid gland, and salivary gland. We found that a subset of Id2+ distal progenitor cells in the embryonic pancreas and kidney can give rise to multiple lineages of progeny cells during branching morphogenesis. Id2-labeled cells also supported the postnatal development of the mammary glands. However, Id2+ cells did not contribute to the development of the salivary and thyroid glands. We found the Id2+ cells located in the tip progenitor pools of pancreas and kidney have self-renewal potential and contribute descendants to multiple epithelial cell lineages. Our findings enrich the current model of distal progenitor pools driving branching morphogenesis and provide a new marker to investigate the regularity of branching in these organs.

Novel factor VII gene mutations in six families with hereditary coagulation factor VII deficiency.

INTRODUCTION: Hereditary human coagulation factor VII (FVII) deficiency is an inherited autosomal recessive hemorrhagic disease involving mutations in the F7 gene. The sites and types of F7 mutations may influence the coagulation activities of plasma FVII (FVII: C) and severity of hemorrhage symptoms. However, the specific mutations that impact FVII activity are not completely known. METHODS: We tested the coagulation functions and plasma activities of FVII in seven patients recruited from six families with hereditary FVII deficiency and sequenced the F7 gene of the patients and their families. Then, we analyzed the genetic information from the six families and predicted the structures of the mutated proteins. RESULTS: In this study, we detected 11 F7 mutations, including four novel mutations, in which the mutations p.Phe84Ser and p.Gly156Cys encoded the Gla and EGF domains of FVII, respectively, while the mutation p.Ser339Leu encoded the recognition site of the enzymatic protein and maintained the conformation of the catalytic domain structure. Meanwhile, the mutation in the 5' untranslated region (UTR) was closely associated with the mRNA regulatory sequence. CONCLUSION: We have identified novel genetic mutations and performed pedigree analysis that shed light on the pathogenesis of hereditary human coagulation FVII deficiency and may contribute to the development of treatments for this disease.

SIRT1 single-nucleotide polymorphisms are associated with corticosteroid sensitivity in primary immune thrombocytopenia patients.

BACKGROUND: Primary immune thrombocytopenia (ITP) is an autoimmune disorder characterized by decreased platelet count. While corticosteroids are a useful first-line therapy for ITP patients, their long-term effectiveness is limited, and the determinants of corticosteroid sensitivity in ITP patients remain largely unknown. Sirtuin 1 (SIRT1), a member of the mammalian sirtuin family, is related to the anti-inflammatory effects of corticosteroids. Here, we investigate the contribution of the SIRT1 single-nucleotide polymorphisms (SNPs) rs12778366 and rs4746720 to ITP susceptibility. METHODS: We recruited 330 ITP patients and 309 healthy controls from Han population, and performed genotyping of SIRT1 rs12778366 and rs4746720 using a MassARRAY system. The results were validated in another 55 ITP patients from ethnic minorities. RESULTS: Using clinical data of patients and controls from Han polulation, including corticosteroid sensitivity, susceptibility, refractoriness, and severity, our results revealed that the CC/TC genotypes of SIRT1 rs12778366 were associated with a 2.034-fold increased risk of corticosteroid resistance compared to the homozygous major TT genotype (dominant, CC/TC vs. TT, OR = 2.034, 95% CI = 1.039-3.984, p = 0.038). In contrast, the CC/CT genotype of SIRT1 rs4746720 showed a 0.560-fold decreased risk of corticosteroid resistance (dominant, 95% CI = 0.321-0.976, OR = 0.560, p = 0.041). The C allele substitute in SIRT1 rs12778366 was significantly associated with the corticosteroid sensitivity of ITP patients (p = 0.021). The similar results were obtained in minority ITP patients. CONCLUSION: This study indicates that SIRT1 rs12778366 and rs4746720 may be genetic factors related to corticosteroid sensitivity in ITP patients.

Immune Checkpoint-Related Gene Polymorphisms Are Associated With Primary Immune Thrombocytopenia.

Cancer immunotherapy by immune checkpoint blockade has been effective in the treatment of certain tumors. However, the association between immune checkpoints and autoimmune diseases remains elusive and requires urgent investigation. Primary immune thrombocytopenia (ITP), characterized by reduced platelet count and a consequent increased risk of bleeding, is an autoimmune disorder with a hyper-activated T cell response. Here, we investigated the contribution of immune checkpoint-related single-nucleotide polymorphisms (SNPs), including CD28, ICOS, PD1, TNFSF4, DNAM1, TIM3, CTLA4, and LAG3 to the susceptibility and therapeutic effects of ITP. In this case-control study, 307 ITP patients and 295 age-matched healthy participants were recruited. We used the MassARRAY system for genotyping immune checkpoint-related SNPs. Our results revealed that rs1980422 in CD28 was associated with an increased risk of ITP after false discovery rate correction (codominant, CT vs. TT, OR = 1.788, 95% CI = 1.178-2.713, p = 0.006). In addition, CD28 expression at both the mRNA and protein levels was significantly higher in patients with CT than in those with the TT genotype (p = 0.028 and p = 0.001, respectively). Furthermore, the T allele of PD1 rs36084323 was a risk factor for ITP severity and the T allele of DNAM1 rs763361 for corticosteroid-resistance. In contrast, the T allele of LAG3 rs870849 was a protective factor for ITP severity, and the T allele of ICOS rs6726035 was protective against corticosteroid-resistance. The TT/CT genotypes of PD1 rs36084323 also showed an 8.889-fold increase in the risk of developing refractory ITP. This study indicates that immune checkpoint-related SNPs, especially CD28 rs1980422, may be genetic factors associated with the development and treatment of ITP patients. Our results shed new light on prognosis prediction, disease severity, and discovering new therapeutic targets.