Although glucagon-like peptide 1- (GLP-1-) based therapy of hyperglycemia in burn injury has shown great potential in clinical trials, its safety is seldom evaluated. We hypothesize that exendin-4, a GLP-1 analogue, might affect the immune response via the activation of the sympathetic nervous system in burn injury. in vitro was examined in cultured splenocytes in the presence of β-adrenoceptor antagonist propranolol (1 nmol/L) or GLP-1R antagonist exendin (9-39) (1 μmol/L), whereas its in vivo effect was determined by i.p. injection of exendin-4 (2.4 nmol/kg) in mice. To further elucidate the sympathetic mechanism, propranolol (30 mg/kg) or vehicle was applied 30 min prior to injury.  Male Balb/c mice were subjected to sham or thermal injury of 15% total body surface area. Exendin-4 on T cell function in vitro could be restored by propranolol instead of exendin (9-39). However, a Th2 switch by exendin-4 in vitro could only be reversed by exendin (9-39). Likewise, the inhibition of splenic T cell function and NFAT activity by exendin-4 in vivo was restored by propranolol. By contrast, the increased splenic NF-κB translocation by exendin-4 in vivo was potentiated by propranolol in sham mice but suppressed in burn mice. Accordingly, propranolol abrogated the heightened inflammatory response in the lung and the accelerated organ injuries by exendin-4 in burn mice. On the contrary, a Th2 switch and higher serum levels of inflammatory mediators by exendin-4 were potentiated by propranolol in burn mice. Lastly, exendin-4 raised serum stress hormones which could be remarkably augmented by propranolol.  Although the exacerbated burn-induced mortality by exendin-4 was worsened by propranolol pretreatment, the inhibition of T cell proliferation by exendin-4  Exendin-4 suppresses T cell function and promotes organ inflammation through the activation of the sympathetic nervous system, while elicits Th2 switch via GLP-1R in burn injury. Moreove T cells , 18, but T cells . Ex-4 th T cells , but cou T cells . The imm T cells , 22 and  T cells . In inna T cells . Meanwhirophages  and in trophages  through ges [κB) . Additioges [κB)  and adipges [κB)  in mice.β-hydroxylase, a key enzyme in noradrenaline synthesis in the brain of mice [Furthermore, as a neuropeptide released from both the brain and the intestine, GLP-1 could activate the sympathetic nervous system (SNS) both peripherally and centrally. In human, infusion of GLP-1 significantly increased sympathetic activity systemically , 27 or l of mice , 31, and of mice . However of mice , indicat+ T cell was associated with SNS hyperactivity [β-adrenoceptor (β-AR) on lymphocytes leads to inhibition of inflammatory cytokine production, lymphocyte killing [+ T lymphocyte proliferation [β2-ARs could intersect with the NF-κB signaling cascade at different levels in a cell type and gene-specific manner [β-AR antagonist that mitigates the catecholamine response associated with burns. However, results on the immunomodulatory role of β-blockade from burn injury [Increasing evidence suggests that the SNS be involved in the modulation of the cellular immune system via adrenoceptor-dependent mechanism , 35. It activity . In an aactivity . Moreove killing , and CD4feration . Intriguc manner . Regardic manner , IL-6 prc manner , myelopoc manner , and chec manner . Proprann injury , 47 and n injury , 49 are Although GLP-1 and its analogues have shown great potential in managing hyperglycemia in critical illness, literature remains sparse as to the immunomodulatory property of Ex-4 in critical illness. Likewise, few studies have passed out the indirect effect of SNS stimulation by Ex-4 versus direct effect via GLP-1R-cAMP stimulation on immune response. We thereby pursued the GLP-1 action on the immune response and the underlying SNS-dependent mechanism in thermal-injured mice. We now demonstrate that T cell function, organ inflammation, and organ injury are controlled by exogenous Ex-4 in a SNS-dependent manner, whereas T cell differentiation and systemic inflammation are promoted by Ex-4 via GLP-1R activation. Elucidation of its potential mechanism for immunoregulation of GLP-1R agonist in critical illness would help to avoid the side effect of incretin-based therapy .Guide for the Care and Use of Laboratory Animals, with the approval of the Scientific Investigation Board of the Chinese PLA General Hospital, Beijing, China.Male Balb/c mice  were acclimatized for one week before exposed to 94°C thermal injury for 8 seconds of 15% total body surface area (TBSA) after anesthetized with diethyl ether as previously reported . Sham-in5 splenocytes separated from both sham and burn mice 24 h following injury were cultivated in the presence of T cell mitogen concanavalin A  in 96-well plates containing 200 μL/well of complete RMPI 1640 medium at 37°C with a humidified 5% CO2/air atmosphere. Consecutive concentrations of Ex-4  [μmol/L; Ex-9, E7269, Sigma, MD, USA) pretreatment for 1 h [For ex vivo study, aliquots of 4 × 10, Sigma)  were add, Sigma)  or exend for 1 h .in vivo study, the burn mice were separated into four subgroups: vehicle, Ex-4, Ex-4 plus Prop, and Prop (n = 25 − 28/subgroup). Peripheral adrenoceptor blockade with Prop (30 mg/kg) [n = 20/subgroup) and underwent the same procedures. They were killed at 6 h and 24 h (n = 6 − 8/group/time point), respectively, and after injury, blood and tissues were collected as follows.For 0 mg/kg)  or vehic0 mg/kg)  or vehic0 mg/kg) , 54. TheμL heparin at 6 h and 24 h after injury. The samples were centrifuged at 1000g for 10 min at 4°C. In the meantime, another investigator simultaneously removed the spleen and lung. All tissue samples were flash frozen on liquid nitrogen and stored at -80°C.Blood was drained from the posterior orbital vein of ether-anesthetized mice into the tubes containing with or without 10 μm mesh  with a syringe and were purified in lysis buffer (Applygen Technologies Inc.) to remove the red cells. Cell suspensions were then washed and resuspended in complete culture medium . Cells were then allowed to adhere on a plastic Petri dishes  at 5 × 106 cells/mL for 2 h to remove the adherent myeloid cells at 37°C in a 5% CO2. Nonadherent cells, mainly the lymphocytes, were resuspended in complete culture medium and used in subsequent studies [6 separated cells in staining buffer (1% BSA in 0.01 M PBS) were incubated with PE-anti-mouse CD3ε  for 30 min on ice. Cells were washed with 2 mL staining buffer and centrifuged at 1500 rpm for 5 min at 4°C before analyzed on a FACScan cytometer .The spleens were harvested 24 h after injury and put into ice-cold RPMI1640 culture media. Splenocyte suspensions were obtained by flushing through 70  studies , 56. To + T cells were fixed in 4% polyformaldehyde for 20 min and were blocked in 3% bovine serum albumin  for 1 h at room temperature and stained overnight at 4°C with GLP-1R antibody  and rat anti-mouse CD3  in PBS including 1% BSA. After washed 3 times with PBS, sections were incubated in DyLight™ 549-conjugated AffiniPure goat anti-rabbit and anti-mouse IgG (H+L) antibodies  for 2 h at room temperature in dark box. Slides were rinsed and coverslips were affixed with VECTASHIELD mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) for nucleus staining . Control experiments were performed with unspecific isotype antibodies of rabbit instead of the primary antibody. Fluorescent cell images were obtained on a confocal laser scanning microscope .Sections with purified CD3γ and Th2 cytokine secretion of IL-4. T cell function was represented by cell proliferation using a CCK-8 method  in a multiplate spectrophotometer  and IL-2 secretion by ELISA (ExCell Biology Inc.).Splenocytes were cultured in complete medium stimulated by ConA  for 48 h. Cell-free supernatant fractions were collected and stored at -80°C until cytokines analysis by ELISA . T cell differentiation was represented by Th1 cytokine secretion of IFNμL per well (2-10 μg/well) as instructed . Protein content was determined by bicinchoninic acid assay (Applygen Technologies Inc.).The calcineurin/nuclear factor of activated T cells (NFATc1) is activated upon T cell receptor activation followed by intracytoplasmatic calcium influx  and subsκB p65 analysis, nuclear protein of splenocyte (50 μg/lane) from both groups at 6 h and 24 h postburn was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis in Mini-PROTEAN® Tetra electrophoresis cell  and transferred onto Immobilon-P polyvinylidene difluoride (PVDF) membranes  by Mini Trans-Blot® Electrophoretic Transfer Cell (Bio-Rad). PVDF membranes were incubated overnight at 4°C with the NF-κB p65 antibody  followed by incubation with peroxidase-conjugated secondary antibody . The blots were exposed to the super signal luminescence reagent (Applygen Technologies Inc.), and the signals were scanned by ImageQuant LAS 4000 . Then, the membranes were stripped and reprobed with lamin B1 antibody  and the signals were scanned again. NF-κB p65 densitometry was calculated by ImageJ software, normalized to lamin B1.For NF-Lung tissues 24 h after injury were emulsified with a mechanical homogenizer  in a lysis buffer (0.5% Triton with 1% PMSF in 0.01 M PBS) and subjected to two rounds of freeze thaw. After being incubated at 4°C for 1 h, the homogenate was centrifuged and the supernatants were collected. The data were expressed at pg/mg protein.Serum samples collected 24 h after injury were subjected to cytokines measurement by ELISA . Serum alanine aminotransferase (ALT), aspartate transaminase (AST), urea, and creatinine were measured by commercial kits  in the clinical laboratory of our hospital. Serum and plasma samples collected at 6 h and 24 h after injury were for stress hormone measurement by ELISA .P < 0.05 was considered significant for all analyses. The survival rate in the life table was analyzed by the Wilcoxon test. Duration of survivals has been presented as Kaplan-Meier curves with the log-rank tests comparing equality of strata. The significance of difference among groups was evaluated by the Student's t-test for two groups or one-way ANOVA with a post hoc LSD test for multiple comparisons when comparing three or more groups.All data are expressed as group mean ± SEM. A value of P = 0.054, P = 0.399). Single pretreatment of Prop could significantly deteriorate the survival rate of burn injury , when in combination of Ex-4, the survival rate was further worsened .The survival rate analyzed by life table revealed marginally difference in the survival rate among the groups with burn injury , Prop pretreatment could significantly reduce burn-induced survival in PBS-treated  and Ex-4-treated mice . Intriguingly, there was no difference of the survival time between single Prop pretreatment and that in combination with Ex-4 application .However, there was a significant difference in the mean survival time among the groups with burn injury  and burn mice . Additionally, the effect of Ex-4 on T cell proliferation was dependent on β-AR, as Prop pretreatment could restore the inhibitory effects of Ex-M on cell proliferation in both groups. Unexpectedly, blocking GLP-1R by competitive antagonist Ex-9 could further potentiate the inhibitory effects of Ex-M on cell proliferation  and burn-injured mice , whereas increasing IFNγ secretion in sham  and burn-injured mice . The increased secretions of IL-4 instead of IFNγ induced by Ex-4 could be reversed by Prop pretreatment in burn cells  other than in sham cells , indicating Th2 differentiation promoted by Ex-4 might be dependent on β-AR. Propranolol alone could inhibit T cell proliferation mildly, but promote both IFNγ and IL-4 secretions from both groups significantly. On the contrary, GLP-1R blockade by Ex-9 could reverse the increased IL-4 levels by Ex-M in both groups of cells  (γ level by Ex-M could only be reversed by Ex-9 in cells from sham mice (P = 0.601 vs. PBS).Regarding T cell differentiation, burn injury could elicit a Th2-type response with higher IL-4 secretion and lower IFNecretion . Further t-test) . The incin vitro findings, Ex-4 in vivo (2.4 nmol/kg) could exert profound effect on T cell function and differentiation in both groups of mice. In detail, in sham mice, Ex-4 could mildly enhance spleen cellularity  .Considering the robust activation of the SNS by Ex-4 as referred –29, 32, Paradoxically, serum levels of hormones were profoundly lower in burn-injured mice compared to that in sham-injured mice at 6 h postburn . Again iκB translocation, and consequent alleviated lung inflammation in burn-injured mice. However, Ex-4-induced Th2 switch was further potentiated by Prop pretreatment with increased systemic levels of both pre- and anti-inflammatory cytokines. Therefore, the immunosuppressive effects of Ex-4 likely override its anti-inflammatory actions in burn injury, resulting in suppressed T cell function in contrast with heightened inflammatory response in response to Ex-4 in burn injury.This study illustrates for the first time that modulation of the SNS by Ex-4 might deteriorate the morbidity of thermal-injured mice; however, the exacerbated mortality by Ex-4 was not dependent on the activation of the SNS by Ex-4. These data suggest that Ex-4 might regulate T cell function through two distinct pathways in burn injury. First, exogenous Ex-4 directly suppresses splenic T cell function after burn injury depending on SNS activation. Secondly, exogenous Ex-4 acts as a Th2 switch signal for splenic T cells via GLP-1R, which is further potentiated by SNS blockade. That is, Th2 switch by Ex-4 was independent on SNS activation. Removal of these two immunosuppressive functions of Ex-4 by Prop resulted in an improved T cell function with elevated NFATc1 activity, reduced splenic nuclear NF-+ T cell, suggesting that GLP-1 or its analogue Ex-4 could exert direct effect on T cells. As expected, Ex-4 could inhibit T cell function from sham and burn mice in vitro depending on β-AR, whereas promoted Th2 switch via the activation of GLP-1R.The distinct effects between T cell function and differentiation indicated that the two events be driven by two different pathways. Preclinical studies have provided conflicting results about the effect of Ex-4 on lymphocyte function. It was reported that Ex-4 in vitro could not affect the proliferation of primary thymocytes, splenocytes, lymph node cells [+CD8+ T cells in lymph nodes in vivo [in vitro reduced the production of cytokines from activated intestinal intraepithelial lymphocyte, acute exendin-4 administration in vivo robustly induced the expression of genes encoding cytokines and chemokines in the normal and injured intestine [We found the fluorescent GLP-1R on CD3de cells , or splede cells ; however in vivo  that lea in vivo . Moreoventestine . These mβ-AR blockade that prevented the hormones from binding to the receptor, so the hormones were accumulated in the serum.Considering the potency of Ex-4 in neuroendocrine activation, we went on to examine the stress hormones after Ex-4 application. Although the plasma levels of stress hormones were shortly elevated in burn as opposed to sham mice owing to the increased sympathetic tone in our study, Ex-4 could not affect the plasma levels of hormones neither in sham nor in burn mice. It has been found that GLP-1 alone could not elevate plasma catecholamine in humans or in humans during exercise ; howeverin vitro [γ by Ex-4 or in combination with Prop relative to vehicle-treated burn mice was possibly due to the elevated levels of serum norepinephrine in these mice.Then, we wonder whether Ex-4 could modulate T cell immunity via a neuroendocrine pathway. As expected, the alterations of T cell function by Ex-4 could be reversed by Prop pretreatment in both groups, indicating a SNS-dependent mechanism. In accordance, Ex-4-treated burn mice exhibited reduced cellularity of the spleen which could be restored by Prop pretreatment. It has been reported that the spleen atrophy observed in stroke may result from immune cell migration from the spleen to the cerebral injury site . In our in vitro . CoincidκB was consistently upregulated in Ex-4-treated burn mice compared to that in vehicle-treated burn mice depending on β-AR. These unexpected findings were not consistent with the consensus regarding the anti-inflammatory effect of Ex-4 in vitro [in vivo [α proteins in the jejunum of mice 4 h following injection [β mRNA in the brain 90 min following injection [β2-AR/NF-κB interactions in the context of NF-κB-driven gene expression can be either positive or negative depending on the cell type and the context [β-blockers for severe burn injury be associated with enhanced production of inflammatory mediators [α release was not significantly induced by 15% TBSA burn injury, MCP-1 level was greatly elevated, indicating an inflammatory response evoked. In the meanwhile, plasma stress hormone levels were greatly raised in this animal model. In this context, Ex-4 could induce higher levels of TNF-α, IL-10, and MCP-1 in burn mice either alone or in combination with Prop pretreatment. It is possible that the genes for these cytokines undergo synergistic expression upon coactivation of β2-ARs and NF-κB pathway by catecholamine in the setting of burn injury.Actually, increased levels of cytokines from both the lung and serum were identified in Ex-4-treated as opposed to vehicle-treated mice at 24 h postburn. However, only the increased cytokines by Ex-4 in the lung other than those in the serum could be reversed by Prop pretreatment in burn mice, reinforcing an organ-exclusive SNS-dependent effect of Ex-4. To make things worse, the increased serum levels of cytokines by Ex-4 were further potentiated by Prop pretreatment, indicating a SNS-independent effect of Ex-4 in systemic immune response. It could be explained by the compartment effect of immune response that results in distinct inflammatory response among different tissues and organs, even different segments of the intestine . Followiin vitro  and in v[in vivo , 22. Thi[in vivo . The enh[in vivo . Secondlnjection , and cennjection . The thi context . Severalediators –67 and tediators  in burn Another two critical issues should be mentioned in our study. One is that the hormones in serum were significantly lower than that in plasma. It is asserted that serum differs from plasma only in lacking of the fibrinogen and the clotting factors . The cirIn summary, we have demonstrated a novel aspect of neural suppression of immune response by Ex-4, namely, T cell function, an effect that was robust and distinct between sham- and burn-injured mice. Our results showed that a response to GLP-1R activation be mediated by the SNS that could suppress splenic T cell function accompanied by more pronounced inflammation and severe organ injuries. However, systemic inflammation was augmented by application of GLP-1R agonist, independently on SNS activation. Taken together, our findings raise important considerations for incretin-based therapy of stress or infection-induced hyperglycemia and whether those immunoregulatory effects could be related to adverse effects of drugs with GLP-1R activity.


Epoxy resins are commonly used to manufacture the molding compounds, reinforced plastics, coatings, or adhesives required in various industries. However, the demand for new epoxy resins has increased to satisfy diverse industrial requirements such as enhanced mechanical properties, thermal stability, or electrical properties. Therefore, in this study, we synthesized new epoxy resin (PPME) by modifying phosphorous-containing polyol. The prepared resin was analyzed and added to epoxy compositions in various quantities. The compositions were cured at high temperatures to obtain plastics to further test the mechanical and thermal properties of the epoxy resin. The measured tensile and flexural strength of epoxy compositions were similar to the composition without synthesized epoxy resin. However, the heat release rates of the compositions exhibited tendencies of a decrease proportional to the amount of PPME.  Epoxy resins are commonly used to manufacture molding composites, reinforced plastics, coatings, and adhesives required for various industrial applications due to their high moisture resistance, mechanical properties, chemical resistance, or thermal properties ,3,4,5,6.Traditionally, inorganic additives are often used to improve thermal stability such as aluminum hydroxide, magnesium hydroxide, or ammonium polyphosphate (APP). However, for a few applications such as those involving the resin transfer molding system, the modification of epoxy resins is preferred for the uniform distribution of the resin in the composites . An examHere, we synthesized a thermally stable epoxy modified resin and studied the variations in its physical properties for different epoxy compositions. To this end, tensile and flexural strength, and impact strength were measured to test the mechanical properties of the epoxy resins. Differential scanning calorimetry (DSC) and micro combustion calorimetry (MCC) were used to analyze the thermal properties of the epoxy resins.Diglycidyl ether of bisphenol F , 177 g/eq), and diglycidyl ether of bisphenol A  were obtained from Momentive Co. . D-230 (average molecular weight: 230 g/mol) was obtained from Kukdo Chemicals . Ethylene glycol (g/mol), tetrabutylammonium iodide (TBAI), trimethylamine (g/mol), phenylphosphonic dichloride (PPD) (g/mol), methylene chloride, and diethyl ether were purchased from Sigma-Aldrich .1H-NMR : δ = 7.8~7.4 (aromatic H), 4.2~4 , 3.9~3.8 , 3.6~3.4 .Ethylene glycol (20 g), and trimethylamine (60 g) were added in a 250 mL three neck round bottom flask filled with 30 mL of methylene chloride. PPD (55 g) in 100 mL of methylene chloride was slowly added using a dropping funnel into the flask and stirred for 24 h at room temperature , and the1H-NMR  and FT-I1H-NMR  to confiMW. 370 g, 56 g, 0.15 mol) with the presence of tetrabutylammonium iodide in 250 mL of the reactor (P-Polyol Modified Epoxy (PPME) was synthesized by reacting P-polyol  with bisphenol F epoxy resin ). The amount of synthesized PPME in the compositions was varied in the range from 5 to 15 parts by weight per 100 parts of epoxy resin (phr). The components of the epoxy compositions are summarized in The compositions were prepared by mixing DGEBA, PPME, and D-230 and stirred with mechanical stirrer for 30 min at 40 °C under vacuum. To prepare the cured plastic test specimen, the paste was poured into a metal mold. The polymerization between epoxy resin and D-230 was preceded by heating the mold for 1 h at 80 °C in a heating oven.1H-NMR, Bruker Avance 300 MHz spectrometer, Bruker, MA, USA) spectroscopy in the DMSO-d6 solvent. The molecular weight of P-polyol was obtained by titrating the –OH value with a potentiometric titrator  with the ASTM E 1899-08 method. For the experiment, the hydroxyl group in the polyol was reacted with an excess amount of toluene-4-sulfonyl-isocyanate to form carbamate. The carbamate was titrated with tetrabutylammonium hydroxide to obtain the hydroxyl value. Furthermore, P-polyol, PPME, and F epoxy were analyzed using Fourier transform-infrared spectrometry . The thermal properties of the compositions were observed using differential scanning calorimetry  by running DSC from 25 °C to 250 °C at a heating rate of 10 °C/min. The heat resistance was studied with pyrolysis combustion flow calorimetry  with the ASTM D7309 method. A sample loaded in the instrument is heated at a rate of 1 °C/min from 100 to 900 °C to obtain heat release rate (HRR) and total heat release (THR). The mechanical properties of the cured epoxy compositions in the metal mold were tested by processing the plastics to achieve a 60 × 25 × 3 mm test specimen for flexural strength tests by the ASTM D790M standard and of 150 × 13 × 3 mm for the tensile strength test by the ASTM D638 method. Both experiments were conducted using a universal testing machine . The impact strength was measured with an Izod impact tester  by the ASTM D256 method, while viscoelastic properties of the cured compositions were evaluated by dynamic mechanical analysis  processed at a size of 60 × 12 × 3 mm. DMA experiments were conducted by mounting the test samples on a dual cantilever probe and tested at a heating rate of 5 °C/min from 25 to 200 °C at a frequency of 1 Hz. The fractured surface was observed with the obtained samples during the impact test by FE-SEM . Epoxy equivalent weight (EEW) was measured by the ASTM D 1652 method.The synthesized P-polyol was analyzed using proton nuclear magnetic resonance  of 77 °C and peak temperature (Tpeak) of 116 °C. As there is no significant variation due to the added P-polyol, all samples were cured at 80 °C, which is slightly higher than Tonset to provide sufficient energy for the polymerization.The reaction scheme of bisphenol A epoxy resin, PPME, and D-230 is displayed in peak), and total heat release  b by measase THR, d. The obMechanical properties such as tensile and flexural strengths of the cured epoxy compositions were analyzed using the UTM . Though For the Izod impact strength measurement, though the test values of the test specimen exhibited a decreasing tendency, the degree of impact strength was not as noticeable .g) for the samples with PPME by 20 °C (gs was similar for all samples, irrespective of the amount of PPME. Furthermore, the storage modulus of the samples with PPME increased from 2035 to over 3330 MPa (The viscoelastic properties of the cured polymers were analyzed with DMA as shown in by 20 °C a. The re3330 MPa b, indicaThe fractured images of the binder and epoxy compositions with PPME obtained from the Izod impact test were observed with FE-SEM . The surg of epoxy compositions tends to be lowered from 108.6 °C to 79.8 °C by the increased amount of PPME. The effect of thermal resistance by adding PPME was monitored with MCC. HRR decreased from 629 W/g with the binder to 290 W/g with PPME-15, while the total heat release decreased from 26.1 kJ/g with the binder to 19.2 kJ/g with PPME-15.In this study, a phosphor-containing polyol was reacted with epoxy resin to provide a modified epoxy resin, PPME. Various amounts of PPME were blended with the mixture of DGEBA and D-230 to afford epoxy compositions. The prepared epoxy compositions were cured at high temperatures to form the epoxy polymer. The variation in the physical properties of the cured plastics with PPME were determined via the tensile, flexural, and impact strength measurements. The cure behavior was monitored using DSC to set cure temperature to 80 °C. The results of tensile and flexural strength demonstrate that the epoxy compositions with PPME remained the same, irrespective of the contents of PPME. Tensile strength was maintained at approximately 68 MPa while flexural strength was at approximately 101 MPa for all test samples in the range of standard deviation. However, the impact strength of the compositions proportionally decreased from 44.5 J/m with the binder to 39.7 J/m with PPME-15. Furthermore, the storage modulus of the cured samples increased from 2035 MPa up to 3330 MPa with the increase in PPME. However, the T


Gfi1b is a transcriptional repressor expressed in hematopoietic stem cells (HSCs) and megakaryocytes (MKs). Gfi1b deficiency leads to expansion of both cell types and abrogates the ability of MKs to respond to integrin. Here we show that Gfi1b forms complexes with β-catenin, its co-factors Pontin52, CHD8, TLE3 and CtBP1 and regulates Wnt/β-catenin-dependent gene expression. In reporter assays, Gfi1b can activate TCF-dependent transcription and Wnt3a treatment enhances this activation. This requires interaction between Gfi1b and LSD1 and suggests that a tripartite β-catenin/Gfi1b/LSD1 complex exists, which regulates Wnt/β-catenin target genes. Consistently, numerous canonical Wnt/β-catenin target genes, co-occupied by Gfi1b, β-catenin and LSD1, have their expression deregulated in Gfi1b-deficient cells. When Gfi1b-deficient cells are treated with Wnt3a, their normal cellularity is restored and Gfi1b-deficient MKs regained their ability to spread on integrin substrates. This indicates that Gfi1b controls both the cellularity and functional integrity of HSCs and MKs by regulating Wnt/β-catenin signaling pathway. Gfi1b regulates cellularity of haematopoietic stem cells (HSCs) and megakaryocytes (MKs) as well as spreading of MKs on matrix. Here the authors show that Gfi1b regulates this behaviour by recruiting LSD1 and β-catenin to Wnt/β-catenin signalling targets. Gfi1b is expressed in HSCs, myeloid/erythroid precursors (MEPs), megakaryocytes (MKs) and to varying levels during erythrocyte maturation2. Both Gfi1 and Gfi1b have an N-terminal Snail/Gfi1 (SNAG) domain, which enables transcriptional repression through the recruitment of cofactors Lysine (K)-specific demethylase 1A (LSD1/KDM1A) and CoREST/Rcor15. Interestingly, LSD1 seems to play an essential structural rather than enzymatic role as part of the Gfi1 repressive complex6. LSD1’s loss disrupts Gfi1’s association with and repression of target loci. Gfi1 and Gfi1b also both interact with co-repressors, such as histone lysine methyltransferase 2 (EHMT2/G9a) and histone deacetylases (HDAC1/2)8. While germline deletion of Gfi1b in mice causes lethality at around day 14.5 of embryonic development, conditional knockout mice have been generated and show that Gfi1b controls HSC and MK expansion10. While Gfi1b-deficient HSCs remain functional and give rise to all hematopoietic lineages upon transplantation, MKs that lack Gfi1b cannot produce platelets and are unable to respond with spreading and membrane ruffling to integrin receptor stimulation due to defects in cytoskeletal organization11.G15. Overactive Wnt/β-catenin signaling leads to exhaustion of HSCs, but insufficient activation is equally detrimental17. β-catenin acts as a transcriptional co-activator in complexes with transcription factors, such as the T-cell factor/lymphoid enhancer factor (TCF/LEF) family members to regulate gene expression. The canonical Wnt signaling is under negative regulation at various levels. For instance, GRG/TLE (Groucho/transducin-like enhancer) proteins associate with TCF molecules in the nucleus to switch off expression of Wnt target genes in the absence of nuclear β-catenin18. CtBP1 and HDACs are other negative regulators of canonical Wnt signaling. Multiple non-canonical Wnt signaling pathways also exist and although these pathways all function in a β-catenin independent manner, crosstalk exists between canonical and non-canonical signaling pathways in various contexts20. Several studies have shown that non-canonical Wnt signaling antagonizes the canonical Wnt pathway through different mechanisms22; one example being NFAT5, which is a transcription factor downstream of the non-canonical Wnt/Ca2+ pathway that inhibits canonical Wnt signaling via inhibition of β-catenin acetylation21.Wnt/β-catenin signaling also plays a crucial role in early hematopoiesis, notably in HSCs. Loss- and gain-of-function studies demonstrated that tight control of Wnt signaling and β-catenin activity is necessary for proper function and cellularity control of hematopoietic cells including HSCs and MKsAxin2 promoter. We show that Gfi1b can enhance transcription of TCF/LEF dependent promoters and reporter genes in vitro and in vivo and we present evidence that Gfi1b does this by recruiting LSD1 via its SNAG domain to β-catenin containing complexes. In agreement with this, we show that Gfi1b-deficient HSCs and MKs have decreased levels of canonical Wnt signaling in vivo, which can be reversed when Wnt/β-catenin signaling is stimulated externally by Wnt3A treatment.Here we present evidence that Gfi1b controls HSC and MK cellularity and MK spreading in response to integrin substrates by regulating Wnt/β-catenin signaling. Our results show that Gfi1b interacts with β-catenin as well as regulators of Wnt/β-catenin signaling pathway and that loss of Gfi1b affects the expression of Wnt target genes in both MKs and HSCs. We also reveal a tripartite Gfi1b/LSD1/β-catenin complex that co-occupies key Wnt/β-catenin signaling target regions like the Rosa-CreER transgene into Gfi1bfl/fl mice11. Floxed Gfi1b alleles were deleted by tamoxifen injections , CD48−, CD150+, CD41high/CD9high or CD41low/CD9low to eliminate potential contamination by MK precursors  mice we introduced a ons Fig.  and confons Fig. . MKs andod, Fig.  confirmiion Fig.  supporti11. On such substrates, Gfi1b KO MKs are smaller, stay completely round, and do not form protrusions like WT cells or form platelets  of cultured cells clearly showed that Gfi1b-deficient MKs maintained significantly low RIs, indicating that they are unable to spread and form protrusions, confirming a poor integrin ligands response , a c-Myc interacting protein involved in chromatin remodeling and transcription24, APC, a tumor suppressor and antagonist of the Wnt signaling pathway18, PP2A, a phosphatase that regulates Wnt signaling at several levels and CtBP1 , a repressor of Wnt signaling that associates with LSD126  a histone acetyltransferase that binds to β-catenin29 . This suggested that GFI1B activates TCF/LEF-dependent transcription without binding DNA directly. To test this, we generated TOP reporter genes with GFI1B binding motifs (AAATCTCTGCA) before and after the TCF/LEF binding sites and observed that GFI1B was now able to inhibit transcription of this modified TOP reporter Fig. . This inAxin2LacZ knockin reporter mouse, which enables monitoring of TCF/LEF dependent transcription by measuring β-galactosidase (LacZ) activity in mouse primary cells  activity was reduced in HSCs and MKs with Gfi1b deletion 41, Egr1 (transcription factor)42 and Ptgs2 (enzyme in prostaglandin biosynthesis)43 22, Nfat521, Zfp467 44, and Rock2 (serine/threonine kinase downstream of PTK7)45 were up-regulated in Gfi1b-deficient MKs  and increased the expression of NFAT5, ROCK2 and PTK7 genes   Fig. , similarely Fig. . This su25. Especially, two canonical Wnt gene sets were significantly down-regulated in Gfi1b KO MK samples  and conversely, β-catenin peaks are more likely found at a TSS when they overlap a Gfi1b peak, as compared to peaks with more than 1 kb distance to the other factor  and KD (K562-S23) levels of GFI1B. Levels of GFI1B enrichment confirmed the decrease in GFI1B protein at target genes within GFI1B KD K562 cells Fig. . Importaers Fig. . Other lSince LSD1 is a histone demethylase, specifically for histone 3, lysine 4, and 9 (H3K4 and H3K9), we assessed H3K4me1 and H3K9me2 levels by ChIP-seq in K562 cells before and after Wnt3a treatment. The majority of genes have high H3K4me1 enrichment if targeted by one or more of β-catenin, GFI1B and LSD1 upon treatment (60–90%). Only when none of the three factors were detected at promoters did similar proportions of genes experience increases or decreases in H3K4me1 levels , while LSD1 was located primarily within intergenic (~37%) or intronic (~41%) regions   Fig. . After 7nd) Fig. . Howevernd) Fig. . To valiThe concept that Gfi1b regulates Wnt/β-catenin target genes is supported by several, independent lines of experimental evidence from this study: Gfi1b is found in β-catenin protein complexes found in immune-precipitations analyzed by mass spectrometry as well as BioID experiments and western blotting; Gfi1b, LSD1, and β-catenin co-target promoters and enhancers at endogenous levels in the erythroleukemic K562 cell line; β-catenin binds significantly less at specific target regions in K562 cells upon GFI1B depletion; GFI1B activates TCF-dependent transcription in a TOP/FOP reporter assay and Gfi1b deficiency leads to lower Wnt/β-catenin signaling in MK and HSCs in vivo when measured by two different Wnt/β-catenin dependent reporter mice. Also, Gfi1b- deficient murine HSCs and MKs show deregulated expression of Wnt target genes and knockdown of GFI1B in K562 cells reproduces this deregulation of Wnt target genes seen in cells from Gfi1b knockout mice. And lastly, cellular defects associated with Gfi1b deficiency can be in part restored by overexpressing β-catenin or by stimulating the Wnt/β-catenin signaling pathway.YAF2 and CCRL2, upon Wnt signaling activation. Other loci, like AXIN2 and SP5, did not show a significant change in β-catenin levels upon GFI1B depletion, despite the tripartite complex being detected at these loci in K562 cells. This indicates that β-catenin may also bind to some loci independently of GFI1B.Our biochemical analyses suggest that a tripartite Gfi1b/LSD1/β-catenin complex exists occupying promoter and enhancer regions. This is supported by published ChIP-seqs from transfected cells over-expressing these factors and our own ChIP-seqs on K562 cells in which this complex exists endogenously at target sites. One important line of evidence suggesting the importance of GFI1B for β-catenin recruitment to DNA is the fact that almost all β-catenin binding sites significantly overlap with GFI1B binding sites see Fig. . NotablyInterestingly, the majority of β-catenin targets are co-occupied by GFI1B and LSD1, while β-catenin only co-occupies a very small fraction of GFI1b/LSD1 targets , Wnt stimulation does not alter the majority of GFI1B and LSD1 binding and β-catenin generally binds to promoters where GFI1B and LSD1 are already present. The fact that GFI1B binding motifs are less frequent at such sites compared to sites bound by GFI1B but not β-catenin suggests that GFI1B plays a role at these sites which is specific to the regulation of β-catenin targets. It should be noted that consensus motifs are not strictly required for binding to DNA. It is possible for transcription factors to bind in the absence of their established motif, and to have their nucleotide sequence preference altered by DNA shape, genomic context, DNA modifications and coding and noncoding (genetic) variation51. Our data suggest that GFI1B recruitment to TCF/LEF sites can be independent of its consensus element. An alternative possibility is that GFI1B can be recruited in a complex with β-catenin to TCF at sites where TCF binds DNA. In this situation GFI1B does not necessarily have to contact DNA directly and thus would not require a DNA binding motif to be present at this site. Independently of this, we also suggest an additional potential mechanism whereby GFI1B could activate TCF-dependent transcription by sequestering negative regulators without binding DNA or occupying a specific site via another factor. This mechanism is also independent of DNA binding. At the moment, we have evidence supporting these models without excluding one or the other. It is also possible that GFI1B is present at sites prior to the recruitment of β-catenin as a form of priming, and that once β-catenin is recruited to these sites, it forms a new complex with GFI1B that is responsible for the regulation of the gene. The fact that recruitment of β-catenin to many of these sites is reduced in the context of GFI1B knockdown supports this hypothesis.Analysis of consensus motifs at GFI1B and β-catenin binding sites also supports a regulatory interaction between the two proteins but do not reveal the mechanism of this interaction. While there are promoters where GFI1B and β-catenin bind only after Wnt treatment  or TCF/Lef:H2B-GFP  transgene reporter line. PF4-cre mice were used to delete Gfi1b in MKs for immunofluorescence microscopy analysis. Age and sex-matched mice were used in all experiments. For BM transplantation C57B6 CD45.1 mice were used. All mice were housed under SPF conditions and the IRCM Institutional Review Board approved all animal protocols and experimental procedures were performed in compliance with IRCM and CCAC  guidelines.Gfi1bSuboptimal doses of tamoxifen were injected at 100 mg per kg at day 0 followed by 50 mg per kg the day after, and mice were analyzed at day 10.Hematopoietic cells were analyzed with LSR, or LSR Fortessa flow cytometers  and analyzed using BD FACS Diva software (BD Biosciences) or FlowJo . For cell sorting, lineage negative BM cells were first depleted using mouse lineage cell depletion kit (Miltenyi Biotec) then applied to five-laser FACSAria II sorter (BD Biosciences).K562 (ATCC CRL-3344), HEL (ATCC TIB-180) cells were maintained in RPMI media (Multicell) supplemented with 10% Bovine Growth Serum  and 100 IU Penicillin and 100 μg/ml Streptomycin (Multicell). HEK-293 (ATCC CRL-1573) and U2OS (ATCC HTB-96) cells were maintained DMEM media (Multicell) with above mentioned supplements. We verified that none of the cell lines used in this study were found in the Register of Misidentified Cell Lines maintained by the International Cell Line Authentication Committee.The following  antibodies were used for western blots analysis at 1/1000 dilution: β-catenin , TLE1 (sc-9121), TLE3 (sc-13374), Pontin52 (ab133513), CHD8 , LSD1 , Gfi1b (ARP30094 and sc-28356), Tubulin , G6PD (ab993), HDAC1 (ab702A). Uncropped and unprocessed scans of the western blots are provided in Supplementary Data 2 for 3–6 h with or without Wnt3A (R&D systems) allowing megakaryocytes to attach and spread. After incubation cells were fixed with 4% formalin, permeabilized with 0.1% Triton-X100 in PBS, and blocked with FcBlock . Cells were then labeled with FITC-CD41 (BD Biosciences), AF555-β-tubulin  and covered with Vector Shield containing DAPI (Vector Laboratories). Immunofluorescence imaging of the slides was done using a DCX-950P DP72 camera (Sony) mounted on a Leitz DMRB microscope (Leica). Images were analyzed using ImageJ v1.46r (NIH).Lineage depleted bone marrow from PF4-Cre Gfi1b-flox/flox and Gfi1b-wt/flox mice were cultured in Stemspan/2.6% FBS supplemented with 1% L-Glutamine and SCF (20 ng per ml). TPO (50 ng per ml) was added to fresh medium at day 2 and cells were cultured for 4 more days. MKs were enriched on a BSA gradient and plated on fibronectin  coated 12-µ-chamber slide (ibidi). Cells were incubated at 37 °C, 5% CO55. The Proteowizard4 tool was used to convert RAW files to.mzXML files. Peptide search and identification were performed by using Human RefSeq version 57 and the iProphet tool integrated in ProHits56.The BioID-MS data were analyzed using the ProHits software57. Biological process or molecular function of prey proteins identified in GFI1B-BirA*-Flag BioID/MS or β-catenin-BirA*-Flag BioID/MS with the indicated constructs are shown in heat map analyses. Reviewed UniProtKB entries of the prey proteins analyzed in Significance Analysis of INTeractome (SAINT) results generated in ProHits were entered in the Query field on g:Profiler for GO term analysis58. Contaminant proteins identified in GFI1B-Flag AP/MS were filtered by using the Contaminant Repository for Affinity Purification (CRAPome)59 repository prior to the GO term analysis on g:Profiler. The enrichment score of GO terms is shown as the −log10 of corrected P values.Functional annotation of GO terms was analyzed by using the g:Profiler toolβ-catenin-BirA*-Flag BioID/MS data analyzed in ProHits were submitted in ProHits-viz to perform dot plot analyses60.SAINT output files of GFI1B-BirA*-Flag BioID/MS or 61.293T Cells were transfected with TOP/FOP Flash reporters, β-galactosidase, and effector plasmids using Lipofectamine® (ThermoFisher) and luciferase activity was normalized by β-galactosidase activityβ-D-galactopyranoside (FDG) substrate (AAT Bioquest) by hypotonic shock at 37 °C for 5 min, prior to cell surface antibody staining. β-galactosidase reaction was stopped with 1 mM Phenylethyl β-D-thiogalactopyranoside .Lineage negative BM cells were loaded with 2 mM Fluorescein di-fl/fl and 10 Rosa-Cre Gfi1bwt/fl mice treated with tamoxifen 2 weeks prior the experiment) was harvested in PBS/2.5% FBS and pooled prior to lineage negative depletion using autoMACS Pro separator (Miltenyi Biotec). Cells were incubated with a lineage antibody cocktail  and then labeled with PE/Cy5- streptavidin, PE-anti-CD41, AF700- anti-CD9, APC- anti-CD150, BV421-anti-cKit, BV510-anti-CD48 and PE/Cy7-anti-Sca1 antibodies. MKs (Lin− cKit+ CD41+ CD9+), HSCs (Lin− cKit+ Sca1+ Cd48− CD150+), and HSCs CD41low CD9 low were sorted on FACSAria II sorter (BD Biosciences). RNA was extracted using MagMax-96 Total RNA Isolation kit (Ambion) and quality-checked with RNA 6000 Pico kit (Agilent). RNA-seq libraries were prepared from the RNA extracts using the Illumina TruSeq Stranded mRNA Kit according to the manufacturer’s instructions, and sequenced using the TruSeq PE Clusterkit v3-cBot-HS on an Illumina HiSEq 2000 system. Sequencing reads were aligned to the mm10 genome using Tophat v2.0.1062. Reads were processed with Samtools63 and then mapped to Ensembl transcripts using HTSeq64. Differential expression was tested using the DESeq R package65. A genome coverage file was generated and scaled to RPM using Bedtools66. RNA-seq data are available under accession number GSE71310 (MKs) and GSE85737 (HSCs). Total read numbers and aligned read numbers for each experiment is shown in Supplementary Data Bone marrow from 2 tibiae, 2 femora and 2 humeri .The enrichment of selected biological functions of interest  or CHIR99021  for 4 or 8 h, respectively. The cells were cross-linked with 1.5 mM EGS for 20 min and 1% formaldehyde for 8 min before quenching with 125 mM glycine. Cells were lysed in lysis buffer and sonicated using a Covaris E220 to generate 200–600 bp fragments69. Samples were immuno-precipitated with 2–5 µg of either anti- GFI1B , anti-LSD1 , anti-β-catenin antibody , anti-H3K4me1 , anti-H3K4me2 , anti-H3K4me3  or anti-H3K9me2 . Libraries were generated according to Illumina’s instructions. Libraries were sequenced on the Illumina Hi-seq 2000 following the manufacturer’s protocols to obtain 50 bp paired end reads. ChIP-seq results for Gfi1b binding in HPC7 cells38 and CTCF in ESCs46 were obtained from the laboratory website of Dr. Goettgens (http://hscl.cimr.cam.ac.uk/). Results for β-catenin39 and LSD140 binding in ESCs were obtained from GEO accessions GSE43597 and GSE22557, respectively. External datasets were obtained in the form of.bed files of peaks and.wig visualization tracks, aligned to the mm9 build, with the exception of LSD1, which only included the.bed peak file.GFI1B, LSD1, β-catenin and histone modification ChIPs were performed on 1–20 × 10https://useast.ensembl.org/index.html) For enhancer regions, we used the Fantom5 human_permissive_enhancers_phase_1_and_2 enhancers (February 2015) dataset (http://fantom.gsc.riken.jp).For gene promoters, we used the Ensembl Genes 92 database, dataset GRCh38.p12. . Cells were then labeled with a biotinylated lineage antibody cocktail  and anti-biotin magnetic beads and separated on an AutoMACS. The negative fraction was washed with DMEM/10% FBS and suspended in StemSpan SFEM (StemCell Technologies) that contained 2.6% FBS, 1% L-Glutamine and SCF (20 ng per ml). Cells were then cultured two days at 37 °C and 5% CO2. The media was then replaced with fresh StemSpan SFEM that contained 2.6% FBS, 1% L-Glutamine, SCF (20 ng per ml) and TPO (50 ng per ml) and cells were cultured for 4 more days. On the 7th day, mature megakaryocytes were enriched on a BSA gradient and plated in fibronectin coated 12-well μ-Chamber glass slides (ibidi). Cells were then allowed to attach and to spread for 3–8 h at 37 °C under 5% CO2. To quantify the spreading of MKs on coated slides, we compared the periphery (P) of each cell to the circumference of a perfect circle (2πr) having the same area as this cell and calculated a roundness index (RI) resulting in a numerical value that increases as the complexity of a spreading cell increases, starting at RI = 1 for a perfectly round cell11.Primary megakaryocytic cultures were prepared by flushing bone marrow into DMEM/10% FBS and suspended in 1x ACK red blood cell lysis buffer . The sample size of data points for each assay is shown in Supplementary Data The paired Student Further information on experimental design is available in the Supplementary InformationPeer Review FileReporting SummaryDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4