MST4 kinase regulates immune thrombocytopenia by phosphorylating STAT1-mediated M1 polarization of macrophages.

Primary immune thrombocytopenia (ITP) is an autoimmune hemorrhagic disorder in which macrophages play a critical role. Mammalian sterile-20-like kinase 4 (MST4), a member of the germinal-center kinase STE20 family, has been demonstrated to be a regulator of inflammation. Whether MST4 participates in the macrophage-dependent inflammation of ITP remains elusive. The expression and function of MST4 in macrophages of ITP patients and THP-1 cells, and of a macrophage-specific Mst4-/- (Mst4ΔM/ΔM) ITP mouse model were determined. Macrophage phagocytic assays, RNA sequencing (RNA-seq) analysis, immunofluorescence analysis, coimmunoprecipitation (co-IP), mass spectrometry (MS), bioinformatics analysis, and phosphoproteomics analysis were performed to reveal the underlying mechanisms. The expression levels of the MST4 gene were elevated in the expanded M1-like macrophages of ITP patients, and this elevated expression of MST4 was restored to basal levels in patients with remission after high-dose dexamethasone treatment. The expression of the MST4 gene was significantly elevated in THP-1-derived M1 macrophages. Silencing of MST4 decreased the expression of M1 macrophage markers and cytokines, and impaired phagocytosis, which could be increased by overexpression of MST4. In a passive ITP mouse model, macrophage-specific depletion of Mst4 reduced the numbers of M1 macrophages in the spleen and peritoneal lavage fluid, attenuated the expression of M1 cytokines, and promoted the predominance of FcγRIIb in splenic macrophages, which resulted in amelioration of thrombocytopenia. Downregulation of MST4 directly inhibited STAT1 phosphorylation, which is essential for M1 polarization of macrophages. Our study elucidates a critical role for MST4 kinase in the pathology of ITP and identifies MST4 kinase as a potential therapeutic target for refractory ITP.

Decreased cyclooxygenase-2 associated with impaired megakaryopoiesis and thrombopoiesis in primary immune thrombocytopenia.

BACKGROUND: Cyclooxygenase (COX)-2 is a rate-limiting enzyme in the biosynthesis of prostanoids, which is mostly inducible by inflammatory cytokines. The participation of COX-2 in the maturation of megakaryocytes has been reported but barely studied in primary immune thrombocytopenia (ITP). METHODS: The expressions of COX-2 and Caspase-1, Caspase-3 and Caspase-3 p17 subunit in platelets from ITP patients and healthy controls (HC), and the expressions of COX-2 and CD41 in bone marrow (BM) of ITP patients were measured and analyzed for correlations. The effects of COX-2 inhibitor on megakaryopoiesis and thrombopoiesis were assessed by in vitro culture of Meg01 cells and murine BM-derived megakaryocytes and in vivo experiments of passive ITP mice. RESULTS: The expression of COX-2 was decreased and Caspase-1 and Caspase-3 p17 were increased in platelets from ITP patients compared to HC. In platelets from ITP patients, the COX-2 expression was positively correlated with platelet count and negatively correlated to the expression of Caspase-1. In ITP patients BM, the expression of CD41 was positively correlated with the expression of COX-2. COX-2 inhibitor inhibited the count of megakaryocytes and impaired the maturation and platelet production in Meg01 cells and bone marrow-derived megakaryocytes. COX-2 inhibitor aggravated thrombocytopenia and damaged megakaryopoiesis in ITP murine model. CONCLUSION: COX-2 plays a vital role in the physiologic and pathologic conditions of ITP by intervening the survival of platelets and impairing the megakaryopoiesis and thrombopoiesis of megakaryocytes.

Low-dose decitabine promotes M2 macrophage polarization in patients with primary immune thrombocytopenia via enhancing KLF4 binding to PPARγ promoter.

BACKGROUND: The first-line therapy is effective for the treatment of primary immune thrombocytopenia (ITP); however, maintaining the long-term responses remains challenging. Low-dose decitabine (DAC) has been adopted to treat refractory ITP, while its role in macrophage polarization has not been fully understood. We aimed to investigate the mechanistic role of DAC in M2 macrophage polarization and evaluated its therapeutic effect in ITP. METHODS: The M2 monocytes were identified by flow cytometry from peripheral blood mononuclear cells in healthy controls (HCs) and ITP patients. The expression of PPARγ, Arg-1, DNMT3b and NLRP3, together with IL-10 plasma levels was measured to examine its function. Bisulfite-sequencing PCR was used to evaluate the methylation status of PPARγ promoter, and the binding affinity of KLF4 was measured by Cut&Tag. A sh-PPARγ THP-1 cell line was created to verify if low-dose DAC-modulated M2 macrophage polarization was PPARγ-dependent. The passive ITP models were used to investigate the therapeutic effects of low-dose DAC and its role in modulating polarization and immunomodulatory function of macrophages. NLRP3 inflammasome and reactive oxygen species were also tested to understand the downstream of PPARγ. RESULTS: The M2 monocytes with impaired immunoregulation were observed in ITP. After high-dose dexamethasone (HD-DXM) treatment, M2 monocytes increased significantly with the elevated expression of PPARγ, Arg-1 and IL-10 in CR patients. Low-dose DAC promoted M2 macrophage polarization in a PPARγ-dependent way via demethylating the promoter of PPARγ, especially the KLF4 binding sites. Low-dose DAC alleviated ITP mice by restoring the M1/M2 balance and fine-tuning immunomodulatory function of macrophages. The downstream of the PPARγ modulation of M2 macrophage polarization might physiologically antagonize NLRP3 inflammasome. CONCLUSIONS: Low-dose DAC promoted M2 macrophage polarization due to the demethylation within the promoter of PPARγ, thus enhanced the KLF4 binding affinity in ITP.

Proinflammatory plasticity towards Th17 paradigm of regulatory T cells consistent with elevated prevalence of TGFBR2 variants in elderly patients with primary immune thrombocytopenia.

BACKGROUND: Primary immune thrombocytopenia (ITP) is characterized for the skewed Th differentiation towards Th1 and Th17 cells as well as the impaired number and function of regulatory T cells (Tregs). Tregs are capable of co-expressing effector Th markers in different inflammatory milieu, which probably indicates Treg dysfunction and incompetence to counter over-activated immune responses. METHODS: Ninety-two primary ITP patients from March 2013 to December 2018 were included, and proinflammatory plasticity in different Treg compartments, age groups, and TGFBR2 variant carrier status were investigated. RESULTS: Patients were categorized into elderly (n = 44) and younger (n = 48) groups according to an age of 50 years at disease onset. The overall remission rate was 82.6% after first-line regimens, including 47.8% complete remission. TGFBR2 variants were found in 7 (7.6%) patients with three V216I and four T340M heterozygote carriers. ITP patients demonstrated elevated co-expression of IL-17 and decreased co-expression of both IFN-γ and IL-13 than health control (all p < 0.01). The elderly group demonstrated elevated prevalence of TGFBR2 variants (p = 0.037) and elevated co-expression of IL-17 (p = 0.017) in Tregs, while female predominance was found in the younger group (p = 0.037). In the elderly group, TGFBR2 variant carriers demonstrated further elevated co-expression of IL-17 (p = 0.023) and decreased co-expression of both IFN-γ (p = 0.039) and IL-13 (p = 0.046) in the aTreg compartment. CONCLUSIONS: Our findings revealed additional aberrations of Treg proinflammatory plasticity in elderly primary ITP patients, and highlighted the potential role of Treg dysfunction and senescence in the pathogenesis and management among these patients.

TCR CDR3 Sequencing as a Clue to Elucidate the Landscape of Dysimmunity in Patients with Primary Immune Thrombocytopenia.

Background: Primary immune thrombocytopenia (ITP) is an autoimmune disorder. The existence of autoreactive T cells has long been proposed in ITP. Yet the identification of autoreactive T cells has not been achieved, which is an important step to elucidate the pathogenesis of ITP. Methods: ITP patients' peripheral blood was collected prior to the treatment and one month after initiating dexamethasone treatment per related therapeutic guideline. Serum cytokines were profiled to examine T cell subtypes imbalance using a protein chip. TCR Vß analysis in CD8+T cells of ITP patients, and TCR CDR3 DNA sequencing of CD4+T and CD8+T cells were performed to determine the autoreactive T cells' clones. Results: Cytokine profiling revealed imbalanced distribution of T cells subtypes, which was confirmed by CD4+T and CD8+T cells' oligoclonal expansion of TCR Vß analysis and TCR CDR3 DNA sequencing. VDJ segments were found to be more frequently presented in ITP patients, when compared with health controls. There was an individualized CD4+T cell or CD8+T cell CDR3 sequence in each ITP patient. Conclusions: The present study revealed that T cell clones expanded in ITP patients' peripheral blood, and each clone had an individualized TCR CDR3 sequence. The expanded T cell clones preferred to use some specific VDJ segment. Further studies are warranted to get access to individualized treatment such as Car-T in patients with ITP.

Recombinant Thrombopoietin Effectively Shortens the Time to Response and Increases Platelet Counts in Elderly Patients with Severe Immune Thrombocytopenia.

BACKGROUND: This study was conducted to investigate the short-term efficacy and safety of rhTPO for the management of severe ITP in the elderly as first-line treatment. METHODS: A total of 54 elderly patients with severe ITP were studied, including 39 patients treated with a combination regimen of rhTPO plus standard treatment (glucocorticoid; rhTPO group) and 15 patients treated with glucocorticoid treatment alone (control group). The response rate, time to initial response, peak platelet counts, and time to peak platelet counts were compared, and clinical characteristics correlated with the efficacy of rhTPO were analyzed. The efficacy of rhTPO in the elderly is comparable to the non-elderly in terms of the OR, CR, time to initial response, and peak platelet counts. RESULTS: There were no differences in the overall response (OR) and the complete response (CR) in the rhTPO group compared to the control group. The time to initial response in the rhTPO group was shorter than that in the control group (p = 0.032). In patients without intravenous immunoglobulin (IVIg) and platelet transfusion, the peak platelet counts in the rhTPO group were higher than those in the control group (p = 0.003). CONCLUSIONS: Standard glucocorticoid treatment plus rhTPO effectively shortens the time to response and increases platelet counts in the elderly with severe ITP.

Impaired mitochondria of Tregs decreases OXPHOS-derived ATP in primary immune thrombocytopenia with positive plasma pathogens detected by metagenomic sequencing.

BACKGROUND: Primary immune thrombocytopenia (ITP) is an autoimmune disease. Some ITP patients are associated with pathogen infection undetected with conventional technologies. Investigating the changes of T cells and potential metabolic mechanism are important for better understanding of ITP. METHODS: The study enrolled 75 newly diagnosed ITP patients. The pathogens of patients were detected by metagenomic next-generation sequencing (mNGS). Plasma lipids were measured by liquid chromatography-mass spectrometry (LC-MS). CD4 T cell and CD8 T cell were analyzed using flow cytometry. Mitochondrial reactive oxygen species (ROS) and mitochondrial membrane potential were measured by flow cytometry. Seahorse XF real-time ATP rate assay was used to investigate the change of cellular metabolism. RESULTS: Positive plasma pathogens were detected in seven ITP patients. Of them, 5 (71.4%) positive pathogen-ITP patients were no response (NR) after first-line treatment with corticosteroids. Regulatory T cells (Tregs) increased significantly in positive pathogen-ITP patients compared to negative pathogen-ITP patients and healthy controls (HC). Mitochondrial membrane potential of Th17 and Tregs were decreased in positive pathogen-ITP and negative pathogen-ITP patients, compared to HC (all p < 0.05). The overall metabolism flux of positive pathogen-ITP patients was decreased, as compared to HC (p = 0.004), of them a higher proportion of glycolysis-derived ATP and a smaller proportion of oxidative phosphorylation (OXPHOS)-derived ATP were found in Tregs. The ATP rate index of Tregs was decreased significantly in positive pathogen-ITP patients compared to negative pathogen-ITP patients and HC (p < 0.05). CONCLUSIONS: Impaired mitochondria function of Tregs in positive pathogen-ITP patients caused a decrease of OXPHOS-derived ATP and overall metabolism flux that might be the cause of steroid resistance in ITP patients.

Exosomal circCARM1 from spheroids reprograms cell metabolism by regulating PFKFB2 in breast cancer.

Cancer stem cells (CSC) are the major obstacle for cancer therapy in clinic. Exosomes are one type of vesicles that containing circular RNA (circRNAs) involved in cell-cell communication. However, the roles of breast CSC (BCSC) exosomes are still unclear, and the purpose of the study was to investigate breast cancer cell metabolism reprogramming by circRNAs from BCSC exosomes. The circRNA array was performed in the exosomes secreted from spheroids of MDA-231 cells. circCARM1 was higher in BCSC exosomes than it in the parent breast cancer cells. Further investigation demonstrated that BCSC exosomes circCARM1 played an important role in breast cancer cell glycolysis by miR-1252-5p/PFKFB2. In a conclusion, BCSC exosome-derived circCARM1 played an important role in breast cancer cell glycolysis by sponging miR-1252-5p which regulated PFKFB2 expression.

Carcinoma associated fibroblasts small extracellular vesicles with low miR-7641 promotes breast cancer stemness and glycolysis by HIF-1α.

Fibroblasts play an important role in cancer development and progression. Small extracellular vesicles (sEVs) are one type of extracellular vesicles, which mediate the interaction between cancer-associated fibroblasts and cancer cells by transferring their contents. However, the roles of sEVs from cancer-associated fibroblasts on breast cancer stem cell properties are largely unraveled. The purpose of this study was to explore the roles of sEVs from cancer-associated fibroblasts on breast cancer progression. The miRNA array data showed a different miRNA profile between CAFs sEVs and normal fibroblasts sEVs. By verification using real-time RT-PCR, the data analysis indicated that miR-7641 levels were lower in sEVs from CAFs compared with NFs. The cellular functions were assayed and the results indicated that CAFs derived sEVs with low miR-7641 levels suppressed breast cancer cell survival, glycolysis, and stem cell properties via the HIF-1α pathway. Collectively, these findings indicated that sEVs from CAFs promoted breast cancer stem cell properties and glycolysis via miR-7641/HIF-1α, which was a possible new way for targeting breast cancer.

Cytokine Consistency Between Bone Marrow and Peripheral Blood in Patients With Philadelphia-Negative Myeloproliferative Neoplasms.

Background: Inflammation might play a critical role in the pathogenesis and progression of Philadelphia-negative myeloproliferative neoplasms (Ph-MPNs) with elevated inflammatory cytokines in peripheral blood (PB). However, the inflammatory status inside the bone marrow (BM), which is the place of malignancy origin and important microenvironment of neoplasm evolution, has not yet been elucidated. Methods: Inflammatory cytokine profiles in PB and BM of 24 Ph-MPNs patients were measured by a multiplex quantitative inflammation array. Cytokines that correlated between PB and BM were selected and then validated by ELISA in a separate cohort of 52 MPN patients. Furthermore, a panel of cytokines was identified and examined for potential application as non-invasive markers for the diagnosis and prediction of fibrosis progress of MPN subtypes. Results: The levels of G-CSF, I-309, IL-1ß, IL-1ra, IL-12p40, IL-15, IL-16, M-CSF, MIG, PDGF-BB, and TIMP-1 in BM supernatants were significantly higher than those in PB (all p < 0.05). Linear correlations between BM and PB levels were found in 13 cytokines, including BLC, Eotaxin-2, I-309, sICAM-1, IL-15, M-CSF, MIP-1α, MIP-1δ, RANTES, TIMP-1, TIMP-2, sTNFRI, and sTNFRII (all R > 0.4 and p < 0.05). Levels of BLC, Eotaxin-2, M-CSF, and TIMP-1 in PB were significantly different from those in health controls (all p < 0.05). In PB, levels of TIMP-1 and Eotaxin-2 in essential thrombocythemia (ET) group were significantly lower than those in groups of prefibrotic primary myelofibrosis (pre-PMF) [TIMP-1: 685.2 (322.2-1,229) ng/ml vs. 1,369 (1,175-1,497) ng/ml, p = 0.0221; Eotaxin-2: 531.4 (317.9-756.6) pg/ml vs. 942.4 (699.3-1,474) pg/ml, p = 0.0393] and primary myelofibrosis (PMF) [TIMP-1: 685.2 (322.2-1229) ng/ml vs. 1,365 (1,115-1,681) ng/ml, p = 0.0043; Eotaxin-2: 531.4 (317.9-756.6) pg/ml vs. 1,010 (818-1,556) pg/ml, p = 0.0030]. The level of TIMP-1 in myelofibrosis (MF) >1 group was significantly higher than that in MF ≤ 1 group. Conclusion: Abnormal inflammatory status is present in MPN, especially in its BM microenvironment. Consistency between PB and BM levels was found in multiple inflammatory cytokines. Circulating cytokine levels of BLC, M-CSF, Eotaxin-2, and TIMP-1 reflected inflammation inside BM niche, suggesting potential diagnostic value for MPN subtypes and prognostic value for fibrosis progression.

Decreased levels of immune-regulatory cytokines in patients with immune thrombocytopenia and long-lasting overexpression of these cytokines in the splenectomized patients.

Immune thrombocytopenia (ITP) is an autoimmune-mediated disease characterized by decreased platelet counts. Cytokines play important roles in modulating the immune response and are involved in the pathogenesis of many autoimmune diseases. This study aimed at exploring the serum levels of a core set of cytokines that exert immune-regulatory functions in newly diagnosed ITP patients (both before and after treatment) and splenectomized ITP patients. Using the Bio-Plex suspension array system and ELISA, the serum levels of IL-10, IL-21, IL-27, IL-33, IL-35, IL-37, and TGF-ß1 were detected. The data showed that the serum levels of the immune regulatory cytokines IL-10, IL-35, and TGF-ß1 were significantly lower in newly diagnosed ITP patients. Decreased cytokine levels could be improved in patients with a complete response or a response after steroid-based treatment(s). The serum concentrations of TGF-ß1 were positively correlated with the platelet counts both before and after treatment. All the detected immune-regulatory cytokines, except IL-37, showed significantly higher levels in splenectomized ITP patients than pretreatment ITP patients and healthy controls. In conclusion, these data suggest that lower levels of immune-regulatory cytokines are involved in the pathogenesis of ITP and that there is a long-lasting overexpression of immune-regulatory cytokines in ITP patients with splenectomy.

Interleukin (IL)-1 family cytokines could differentiate primary immune thrombocytopenia from systemic lupus erythematosus-associated thrombocytopenia.

BACKGROUND: Primary immune thrombocytopenia (ITP) is an autoimmune-mediated disorder characterized by a decreased platelet count. Systemic lupus erythematosus (SLE) is also an autoimmune disease in which thrombocytopenia is a common hematologic manifestation. Interleukin (IL)-1 family cytokines are major proinflammatory and immunoregulatory mediators. This study aimed to investigate the role of IL-1 cytokines in patients with ITP and SLE and the potential pathophysiologic mechanism to differentiate SLE-associated thrombocytopenia (SLE-TP) from ITP. METHODS: Multiplex cytokine assay and real-time polymerase chain reaction (RT-PCR) were used to measure IL-1 cytokines in 17 newly diagnosed ITP patients, 17 SLE-TP patients, 19 SLE patients without thrombocytopenia (SLE-NTP), and 10 healthy controls. RESULTS: The serum levels of IL-1ß, IL-18, IL-36α, IL-36ß, IL-36γ, and IL-33 were decreased significantly in ITP patients compared with SLE-TP patients, SLE-NTP patients, and healthy controls (P<0.05). While there was no significant difference in the serum level of IL-37 between ITP and SLE-TP patients, there was a positive correlation between the platelet count and IL-37 level in ITP patients. Our data suggested that serum IL-1ß, IL-18, IL-36α, IL-36ß, IL-36γ, IL-33, and IL-37 could be considered biomarkers in the diagnosis of ITP. CONCLUSIONS: Serum IL-1ß, IL-18, IL-36α, IL-36ß, IL-36γ, and IL-33 could be considered biomarkers to differentiate SLE-TP from ITP patients.

Circulating CX3CR1+CD163+ M2 monocytes markedly elevated and correlated with cardiac markers in patients with acute myocardial infarction.

BACKGROUND: Vulnerable plaques have been generally recognized to play a role in the pathogenesis of acute myocardial infarction (AMI), however, the role of circulating CX3CR1+CD163+ M2 monocytes has not been studied properly. We aim to evaluate the features of CX3CR1+CD163+ M2 monocytes and its relationship with cardiac specific markers in AMI patients. METHODS: The circulating M2 monocytes were identified in AMI patients (n=35) and healthy controls (HCs, n=10) by flow cytometry using two staining methods: CD68+CD163+ (cytoplasmic staining) and CX3CR1+CD163+ (surface staining). CX3CR1+ monocytes were purified by magnetic cell sorting. The expression level of peroxisome proliferator-activated receptor γ (PPARγ) and arginase-1 (Arg-1) were measured by real-time quantitative PCR and Western Blot in CX3CR1+ monocytes. RESULTS: Circulating M2 monocytes extremely expanded in AMI patients compared with HCs (P<0.01). Positive linear correlation was confirmed between CD68+CD163+ and CX3CR1+CD163+ cell populations in AMI patients (r=0.39, P=0.02). The percentage of circulating CX3CR1+CD163+ M2 monocytes positively correlated with cardiac specific markers (cTNT, CK-MB) and acute phase markers (glucose, hs-CRP) (cTNT, r=0.63, P<0.01, CK-MB, r=0.54, P<0.01, glucose, r=0.62, P<0.01, hs-CRP, r=0.58, P<0.01). CX3CR1+ monocytes in AMI patients expressed higher levels of PPARγ and Arg-1 than those in HCs (P<0.01). CONCLUSIONS: Circulating M2 monocytes increased in AMI patients and positively correlated with the elevation of both cardiac specific and acute phase markers. CX3CR1+CD163+ M2 monocytes might have application value for the early diagnosis of AMI.

Intestinal microbiota dysbiosis play a role in pathogenesis of patients with primary immune thrombocytopenia.

BACKGROUND: The intestinal microbiota is essential for the maintenance of the physiology of immune homeostasis. Dysbiosis has been described in some autoimmune diseases, however its role is still elusive in primary immune thrombocytopenia (ITP), which is one kind of autoimmune diseases. This study aimed to characterize the phylogenetic diversity of the fecal microbiota and its relationship with the platelet activation status in patients with ITP. METHODS: The platelet activation status was assessed by 2 platelet markers, PAC-1 (antibody that recognizes the activated GPIIb/IIIa complex) and CD62p (Platelet surface P-selectin) by flow cytometry. Total DNA was extracted from fecal samples of ITP patients and healthy controls (HC). Sequencing the V4 hypervariable region of bacterial 16S rRNA genes was used to identify the changes in phylogenetic diversity and composition of the intestinal flora. The obtained sequencing reads were assigned to operational taxonomic units (OTUs, 97% sequence identity) and taxonomically classified to assess composition and diversity. RESULTS: The percentage of PAC-1+ platelets in ITP patients was higher than that in control group (p < 0.001), The percentage of CD62p+ and PAC-1+CD62p+ platelets in ITP patients both higher than those in control group (p < 0.001). At the phylum level, eight different phyla were identified in ITP individuals, with a majority of Bacteroidetes (45.96%) and Firmicutes (38.59%), followed by Proteobacteria (11.43%), Fusobacteria(1.29%), and Actinobacteria (1.22%). While in the Healthy volunteers, ten phyla were detected, with a predominance of Firmicutes (50.92%) and Bacteroidetes (34.26%), came before Proteobacteria (13.60%), and Actinobacteria (0.90%). The gut microbiota was skewed in ITP, with an increased proportion of Proteobacteria, Bacteroidetes and Bacteroidetes/Firmicutes ratio, a decreased proportion of Firmicutes compared with HC. Disease specific alterations in diversity was also identified, especially the potential markers (Anaerorhabdus, sutterella, Peptostreptococcaceae, Clostridium_XI and carnobacteriaceae, p < 0.05) for ITP. CONCLUSIONS: The results suggested that the distinct microbiota dysbiosis in ITP characterized by alterations in biodiversity and composition, which could provide insights for diet therapy and fecal microbiota transplantation treatment to cure ITP. There might be somehow compensatory enhancement of platelet activation in ITP patients. And there is associate between platelet activation and intestinal microbiota in patients with ITP.

Transcription Factor IRF4 Dysfunction Affects the Immunosuppressive Function of Treg Cells in Patients with Primary Immune Thrombocytopenia.

BACKGROUND: Th17/Treg balance skews towards Th17 in ITP patient. IRF4 has been highlighted for its close relationship to the immunosuppressive function of Treg cells and the IL-17 synthesis in CD4+ T cells. This study was aimed at examining the effects of IRF4 to the Th17/Treg cells in patients with ITP. METHODS: Treg and Teff cells were isolated from PBMCs of newly diagnosed ITP patients. The percentages of CD4+CD25hiFoxp3+Treg cells and the CD3+CD4+IL-17+Th17 cells were detected by flow cytometry. After being cultured, the supernatants of Tregs were collected for IL-10 concentration test. The IRF4 levels of Tregs were measured. Teffs were cultured alone or with Tregs for 24 hours. Then the supernatants were collected for IL-17 concentration test. The binding intensity of IRF4 to the gene IL-10 in Treg cells was detected by ChIP-qPCR. Metabolic assays for Teffs and Tregs were performed with Agilent Seahorse XF96 Analyzer. RESULTS: The secretion of IL-10 by Tregs was decreased in ITP patients. The intensity of IRF4 binding to IL-10 DNA of Tregs in patients was higher than that of normal controls and Teffs in ITP patients. The expressions of IRF4 of Tregs in ITP patients were remarkably lower than that of healthy controls. The percentage of Th17 cells in healthy controls was significantly increased after IRF4 mRNA silencing. Abnormal metabolism of Treg and Teff cells was found in ITP patients. CONCLUSION: The skewed ratio of Th17/Treg cells and dysfunction of Treg cells in newly diagnosed ITP patients was at least partly caused by IRF4 dysfunction. The underlying mechanism might be the impact of IRF4 on the metabolism of Treg and Teff cells.

The prediction value of Treg cell subtype alterations for glucocorticoid treatment in newly diagnosed primary immune thrombocytopenia patients.

BACKGROUND: Primary immune thrombocytopenia (ITP) is an autoimmune heterogeneous disorder of which Treg cells are numerically or functionally deficient. It is known that human FoxP3+CD4+ T cells were composed of 3 phenotypically and functionally distinct subpopulations (resting Treg, rTreg; activated Treg, aTreg; and non-suppressive Treg, n-sTreg). The current study was aimed to determine whether these Treg subtypes are altered in ITP patients and the related potential clinical applications. METHOD: Normal control volunteers and newly diagnosed ITP patients were enrolled in the study. The percentage of Treg cells' subtypes in peripheral blood was examined by flow cytometry before and after the glucocorticoid treatment. The IL-10 production by Treg subtypes was also examined. RESULTS: Treg cell subtypes of aTreg increased, rTreg decreased, and n-s Treg increased in newly diagnosed ITP patients' peripheral blood. The IL-10 production by respective Treg subtype didn't change after the treatment, and aTreg cells had the highest IL-10 yield. Patients who gained remission during follow-up had a higher aTreg cells' percentage than those who did not at the disease diagnosis. CONCLUSION: Tregs cell subtypes percentage was altered when ITP occurred. The increased aTreg cells at disease diagnosis might predict a better glucocorticoid treatment efficacy.

Clinical Significance of Platelet Volume and Other Platelet Parameters in Acute Myocardial Infarction and Stable Coronary Artery Disease / Significado Clínico do Volume Plaquetário e Outros Parâmetros Plaquetários no Infarto Agudo do Miocárdio e Doença Arterial Coronariana Estável

Abstract Background: Platelets are important in the initiation of thrombosis, and their morphological and functional changes are closely related with the occurrence and development of coronary artery thrombosis. Platelet parameters might be valuable in distinguishing between acute myocardial infarction (AMI) and stable coronary artery disease (SCAD). Objective: This study was designed to detect and compare changes in platelet parameters, such as mean platelet volume (MPV) in patients with acute myocardial infarction (AMI) and stable coronary artery disease (SCAD) and to investigate their roles in these diseases. Methods: Specimen collection: Between January 2011 and December 2013, 2 mL of elbow vein blood was drawn from each of 31 patients primarily diagnosed with AMI, 34 SCAD patients and 50 healthy subjects; and placed in EDTA-K2 anticoagulant tubes. Platelet count (PLT), MPV, plateletcrit (PCT), platelet distribution width (PDW), white blood cell (WBC) and neutrophil (NEU) counts were determined using an STKS automated hematology analyzer (Beckman Courter). Results: Compared with the control group, MPV levels were significantly higher in the AMI and SCAD groups (p < 0.05), while PLT was significantly lower (p < 0.05). Conclusion: These results suggest that MPV and other related parameters have a certain value in the diagnosis of SCAD and AMI.

Resumo Fundamento: As plaquetas são importantes no início da trombose e suas alterações morfológicas e funcionais estão intimamente relacionadas com a ocorrência e o desenvolvimento de trombose da artéria coronária. Os parâmetros plaquetários podem ser valiosos na distinção entre infarto agudo do miocárdio (IAM) e doença arterial coronariana estável (DACE). Objetivo: O objetivo desse estudo foi detectar e comparar alterações nos parâmetros plaquetários, como o volume plaquetário médio (VPM) em pacientes com infarto agudo do miocárdio (IAM) e doença arterial coronariana estável (DACE) e investigar seu papel nessas doenças. Métodos: Coleta de amostras: Entre janeiro de 2011 e dezembro de 2013, foram retirados 2 mL de sangue da veia do antebraço de cada um dos 31 pacientes diagnosticados principalmente com IAM, 34 pacientes com DACE e 50 indivíduos saudáveis; e colocado em tubos com anticoagulante EDTA-K2. As contagens de plaquetas (PQT), VPM, massa total de plaquetas (MTP), Amplitude de Distribuição de Plaquetas (PDW, do inglês platelet distribution width), contagem de glóbulos brancos (WBC, do inglês white blood cells) e neutrófilos (NEU) foram determinadas utilizando-se um analisador de hematologia automatizado STKS (Beckman Courter). Resultados: Comparado com o grupo controle, os níveis de VPM foram significativamente maiores nos grupos IAM e DACE (p < 0,05), enquanto os níveis de PQT foram significativamente menores (p < 0,05). Conclusão: Esses resultados sugerem que o VPM e outros parâmetros associados têm um certo valor no diagnóstico de DACE e IAM.

Clinical Significance of Platelet Volume and Other Platelet Parameters in Acute Myocardial Infarction and Stable Coronary Artery Disease.

BACKGROUND: Platelets are important in the initiation of thrombosis, and their morphological and functional changes are closely related with the occurrence and development of coronary artery thrombosis. Platelet parameters might be valuable in distinguishing between acute myocardial infarction (AMI) and stable coronary artery disease (SCAD). OBJECTIVE: This study was designed to detect and compare changes in platelet parameters, such as mean platelet volume (MPV) in patients with acute myocardial infarction (AMI) and stable coronary artery disease (SCAD) and to investigate their roles in these diseases. METHODS: Specimen collection: Between January 2011 and December 2013, 2 mL of elbow vein blood was drawn from each of 31 patients primarily diagnosed with AMI, 34 SCAD patients and 50 healthy subjects; and placed in EDTA-K2 anticoagulant tubes. Platelet count (PLT), MPV, plateletcrit (PCT), platelet distribution width (PDW), white blood cell (WBC) and neutrophil (NEU) counts were determined using an STKS automated hematology analyzer (Beckman Courter). RESULTS: Compared with the control group, MPV levels were significantly higher in the AMI and SCAD groups (p < 0.05), while PLT was significantly lower (p < 0.05). CONCLUSION: These results suggest that MPV and other related parameters have a certain value in the diagnosis of SCAD and AMI.