Journal: Haematologica

Article Title: Enhancing regulatory T-cell function via inhibition of high mobility group box 1 protein signaling in immune thrombocytopenia

doi: 10.3324/haematol.2022.281557

Figure Lengend Snippet: 18β-GA inhibited the expression of HMGB1 in CD4 + T cells from patients with immune thrombocytopenia by suppressing NF-KB signaling. (A) The levels of HMGB1 in plasma of patients with immune thrombocytopenia (ITP) were higher than those in healthy controls (unpaired Student t test, * P =0.0335, 69.8793±20.7887 vs. 114.8579±60.2011, healthy controls n=10, ITP patients n=15). Immunoprecipitation of plasma HMGB1 with anti-HMGB1 antibodies in ITP patients. Western blotting with the anti-human HMGB1 antibody and acetylated-lysine antibody. Plasma HMGB1 in ITP patients was obviously acetylated. (B) The percentage of Treg stimulated with different redox states of HMGB1. 18β-GA inhibited the effect of HMGB1 with different redox states on Treg. a: DMSO; b: HC-HMGB1 (HMGB1 from healthy controls); c: ITP-HMGB1 (HMGB1 from ITP patients); d: ITP-frHMGB1 (ITP-fully reduced HMGB1); e: ITP-oxiHMGB1 (ITP-oxidized HMGB1); f: ITP-HMGB1+18β-GA; g: ITP-frHMGB1+18β-GA; h: ITP-oxiHMGB1+18β-GA. n=6. Paired t test, ** P ab =0.0013; *** P ac =0.0001; *** P bc =0.0006; ** P ae =0.0023; **** P cf <0.0001; * P dg =0.0109; * P eh =0.0303. (C) The level of HMGB1 in plasma correlated negatively with the platelet count in routine blood tests in ITP patients, n=15, Pearson correlation, r=-0.5796, R2=0.3359, * P =0.0236. (D) HMGB1 mRNA expression in CD4 + T cells from ITP patients at the time of enrollment were higher than that in healthy controls (unpaired Student t test, ** P =0.0014, 0.0314±0.0203 vs. 0.0852±0.0449, healthy controls n=10, ITP patients n=18). (E) Relative integrated density of HMGB1 of isolated CD4 + T cells from ITP patients and healthy controls after 3 days cultured by western blotting (unpaired t test, * P =0.0367, healthy controls n=3, ITP patients n=4). (F) 18β-GA reduced HMGB1 expression (* P =0.0182) and phosphorylated NF-KB expression (* P =0.0476) of CD4 + T cells from ITP patients after 3 days (paired t test, n=3). IP: immunoprecipitation; HC: healthy controls; DMSO: dimethylsulfoxide; ns: not significant.

Article Snippet: Splenocytes from these immunized mice were engrafted into CD61 + severe combined immunodeficient (SCID) mice to construct the active ITP murine model. ITP mice were then randomly separated into groups that received the same volume of either 18β-GA (30 mg/kg, intraperitoneal injection, CAS471-53-4, Sigma-Aldrich, USA), the HMGB1 inhibitor lycopene (5 mg/kg, intragastric administration, S3943, Selleck, USA), or 3% dimethylsulfoxide (DMSO, intraperitoneal injection, Sigma-Aldrich, USA) every 2 days from day 1 after radiation and splenocyte infusion.

Techniques: Expressing, Clinical Proteomics, Immunoprecipitation, Western Blot, Isolation, Cell Culture

Journal: Haematologica

Article Title: Enhancing regulatory T-cell function via inhibition of high mobility group box 1 protein signaling in immune thrombocytopenia

doi: 10.3324/haematol.2022.281557

Figure Lengend Snippet: 18β-GA inhibited the expression of HMGB1 by increasing the number and restoring the immunosuppressive capacity of Treg from patients with immune thrombocytopenia. (A) Compared to dimethylsulfoxide (DMSO), 18β-GA increased the generation of Treg in CD4 + T cells from patients with immune thrombocytopenia (ITP), and this change was attenuated in the rhHMGB1 (100 ng/mL) group, n=11, paired t test, *** P DMSO vs . rhHMGB1 =0.0007, ** P rhHMGB1 vs . rhHMGB1+18β-GA =0.0036, ** P DMSO vs . 18β-GA =0.0069. (B) HMGB1 shRNA transfection successfully silenced the HMGB1 gene in Treg of ITP patients after 72 hours; the knockdown efficiency of HMGB1 gene was ≥78.7% as determined by real-time quantitative polymerase chain reaction (paired t test, n=4, * P =0.0191). (C) Representative histogram of proliferation of CD4 + CFSE + effector T cells. (D) After treatment with DMSO or 18β-GA, HMGB1 or control shRNA-transfected Treg were co-cultured with CD4 + CD25 - effector T cells. The graph shows the division index of the proliferation of effector T cells. Effector T cells co-cultured with HMGB1 shRNA-Treg had a lower level of proliferation, compared with those co-cultured with control shRNA-Treg ( * P cd =0.0209, * P ce =0.0146). There was no statistically significant difference in the immuno-suppression of Treg transfected with HMGB1 shRNA after treatment with DMSO or 18β-GA ( P ef =ns). Paired t test, n=4. (E) Flow chart of the lentiviral interference. T eff : T effector cells; CFSE: 5(6)-carboxyfluorescein diacetate N-succinimidyl ester; MFI: mean fluorescence intensity.

Article Snippet: Splenocytes from these immunized mice were engrafted into CD61 + severe combined immunodeficient (SCID) mice to construct the active ITP murine model. ITP mice were then randomly separated into groups that received the same volume of either 18β-GA (30 mg/kg, intraperitoneal injection, CAS471-53-4, Sigma-Aldrich, USA), the HMGB1 inhibitor lycopene (5 mg/kg, intragastric administration, S3943, Selleck, USA), or 3% dimethylsulfoxide (DMSO, intraperitoneal injection, Sigma-Aldrich, USA) every 2 days from day 1 after radiation and splenocyte infusion.

Techniques: Expressing, shRNA, Transfection, Knockdown, Real-time Polymerase Chain Reaction, Control, Cell Culture, Fluorescence

Journal: Haematologica

Article Title: Enhancing regulatory T-cell function via inhibition of high mobility group box 1 protein signaling in immune thrombocytopenia

doi: 10.3324/haematol.2022.281557

Figure Lengend Snippet: 18β-GA ameliorated thrombocytopenia in a murine model of active immune thrombocytopenia with increased Treg. (A) Immune thrombocytopenia (ITP) was established in radiated severe combined immunodeficient (SCID) mice with infusion of 5×10 4 splenocytes from CD61-knockout mice immunized against wildtype C57BL/6 mice platelets. Platelet counts were monitored weekly for 5 weeks. We defined the day of splenocyte infusion as day 0. The drug intervention was administered on day 1, and repeated every 2 days thereafter. (B-F) Treatment with 18β-GA (30 mg/kg) or control (3% dimethylsulfoxide [DMSO] in phosphate-buffered saline) was administered on day 1 and repeated every 2 days; n=5 for the control group and n=5 for the 18β-GA group. (B) On day 21, a significantly higher platelet count was observed in the 18β-GA-treated group than in the control group. The lines denote medians of platelet counts in each group as the data were not normally distributed. Significance among groups was determined by two-way analysis of variance, **** P Time <0.0001, *** P platelets =0.0002 (multiple comparisons: * P 21 =0.0158). (C) The level of plasma HMGB1 in mice with ITP was significantly higher than that in SCID mice. Unpaired t test, * P =0.0142. (D) Relative mRNA expression of HMGB1 in the spleen at day 35 was higher after 18β-GA treatment than after DMSO treatment. Unpaired t test, * P =0.0482. Peripheral blood, spleens, thymuses, inguinal lymph nodes and livers were harvested from ITP mice at day 35 after splenocyte transfer. (E) The gating strategy and representative density plots for identification of CD4 + CD25 + Foxp3 + Treg (F) 18β-GA-treated ITP mice had a higher percentage of Treg in spleen ( * P =0.0340), thymus ( ** P =0.0075), inguinal lymph nodes ( ** P =0.0017), and peripheral blood ( P =0.1796), compared with the control ITP mice. Differences between two groups were determined by an unpaired t test. KO: knockout.

Article Snippet: Splenocytes from these immunized mice were engrafted into CD61 + severe combined immunodeficient (SCID) mice to construct the active ITP murine model. ITP mice were then randomly separated into groups that received the same volume of either 18β-GA (30 mg/kg, intraperitoneal injection, CAS471-53-4, Sigma-Aldrich, USA), the HMGB1 inhibitor lycopene (5 mg/kg, intragastric administration, S3943, Selleck, USA), or 3% dimethylsulfoxide (DMSO, intraperitoneal injection, Sigma-Aldrich, USA) every 2 days from day 1 after radiation and splenocyte infusion.

Techniques: Knock-Out, Control, Saline, Clinical Proteomics, Expressing

Journal: Haematologica

Article Title: Enhancing regulatory T-cell function via inhibition of high mobility group box 1 protein signaling in immune thrombocytopenia

doi: 10.3324/haematol.2022.281557

Figure Lengend Snippet: 18β-GA exerted a therapeutic effect by inhibiting HMGB1 in mice with immune thrombocytopenia. (A-E) Treatment with 18β-GA (30 mg/kg), lycopene (5 mg/kg), or control (3% dimethylsulfoxide in phosphate-buffered saline) was administered on day 1, and repeated every 2 days after murine models of active immune thrombocytopenia had been established, n=4 for each group. a: control, b: 18β-GA, c: lycopene, d: 18β-GA+lycopene. (A) On day 21, a significantly higher platelet count was observed in the 18β-GA-treated group than in the control group (* P c ont ro l vs 18Β-GA=0.0128). On day 28, platelet counts increased significantly with treatment with 18β-GA (* P co ntrol vs 18Β-GA=0.0101) or lycopene (* P co ntrol vs ly co ene=0.0160). The lines denote the mean (± standard deviation [SD]) of platelet counts in each group as the data are normally distributed. Statistical significance among groups was determined by two-way analysis of variance (ANOVA), **** P Time <0.0001, ** P l at elets=0.0010. (B) The percentage of Treg in the spleen, thymus, inguinal lymph nodes, and peripheral blood on day 35. Statistical significance among groups was determined by one-way ANOVA, n=4 for each group. For spleens, ** P ab =0.0028, ** P ac =0.0098, ** P ad =0.0012. For thymuses, * P ab =0.0168, * P ac =0.0243, * P ad =0.0109. For inguinal lymph nodes, * P ab =0.0346, * P ad =0.0144. For peripheral blood, * P ab =0.0155, * P ac =0.0167, ** P ad =0.0016. (C) The gating strategy and representative plots for anti-CD61 antibody (%). (D) The level of serum antiplatelet CD61-specific antibodies decreased after day 14. The lines denote the mean (±SD) of % anti-CD61 antibody in each group as the data are normally distributed. * Significant differences among groups emerging on day 14. Two-way ANOVA. *** P ab =0.0003, ** P ac =0.0037, *** P ad =0.0001 at day 14. At day 35, ** P ab =0.0063, * P ac =0.0329, ** P ad =0.0038. (E) Plasma HMGB1 levels in ITP mice at day 35. Statistical significance among groups was determined by one-way ANOVA ** P b=0 0049 ** P =0 0016 ** P d=0 0012 (F) Thrombocytopenia was exacerbated in ITP mice receiving Treg-depleted splenocytes, where 18β-GA failed to raise platelet counts. The lines denote median (with range) of platelets in each group as the data are not normally distributed, n=4 for each group. Statistical significance among groups was determined by two-way ANOVA, and emerged on day 21: * P Control vs 18Β GA=0.0367. P Tre deleted Control vs Tre deleted 18β GA =not significant (ns).

Article Snippet: Splenocytes from these immunized mice were engrafted into CD61 + severe combined immunodeficient (SCID) mice to construct the active ITP murine model. ITP mice were then randomly separated into groups that received the same volume of either 18β-GA (30 mg/kg, intraperitoneal injection, CAS471-53-4, Sigma-Aldrich, USA), the HMGB1 inhibitor lycopene (5 mg/kg, intragastric administration, S3943, Selleck, USA), or 3% dimethylsulfoxide (DMSO, intraperitoneal injection, Sigma-Aldrich, USA) every 2 days from day 1 after radiation and splenocyte infusion.

Techniques: Control, Saline, Standard Deviation, Clinical Proteomics

Journal: Haematologica

Article Title: Enhancing regulatory T-cell function via inhibition of high mobility group box 1 protein signaling in immune thrombocytopenia

doi: 10.3324/haematol.2022.281557

Figure Lengend Snippet: 18β-GA alleviated the phagocytosis of macrophages in patients with immune thrombocytopenia. (A) Representative plots of intracellular fluorescence of monocyte-derived macrophages in patients with immune thrombocytopenia (ITP) after treatment with DMSO or 18β-GA. Intracellular 5-chloromethylfluorescein diacetate (CMFDA) fluorescence-positive scatters indicate platelets phagocytosed by monocyte-derived macrophages. CD61 + scatters indicate adhered but not-phagocytosed platelets. (B) 18β-GA (25 mM) inhibited phagocytosis of monocyte-derived macrophages from ITP patients towards anti-human CD41 antibody-coated platelets in vitro . Wilcoxon matched-pairs signed rank test, n=8, * P =0.0234. (C) HMGB1 shRNA transfection in monocyte-derived macrophages. HMGB1 shRNA transfection successfully silenced the HMGB1 gene in monocyte-derived macrophages from ITP patients after 72 hours; the knockdown efficiency of the HMGB1 gene was ≥81%, as determined by real-time quantitative polymerase chain reaction (paired t test, n=6, *** P =0.0003). (D) 18β-GA reduced phagocytic indices of monocyte-derived macrophages by blocking HMGB1 in ITP. Statistical significance among groups was determined by one-way analysis of variance, n=6, ** P =0.0037. Multiple comparisons: * P ab =0.0457; * P ac =0.0107. (E) Microscopy images comparing phagocytosis towards CMFDA-labeled CD41-opsonized platelets by monocyte-derived macrophages of ITP patients in the presence of DMSO or 18β-GA. Bound but external platelets (CMFDA+APC-CD61: green and red) were distinguished from internalized platelets (CMFDA: green) using APC-anti human-CD61 antibody. Cell nuclei were labeled with DAPI (blue). Ratio of endocytosed platelets to macrophages: the numbers of endocytic platelets and macrophages were calculated by Imagepro Plus 6.0 software, under 200× magnification. Differences between two groups were determined by a paired t test, n=4. (F) Diagram to show the effect of 18β-GA acting on Treg and macrophages via HMGB1 signaling. Mϕ: macrophages; ns: not significant.

Article Snippet: Splenocytes from these immunized mice were engrafted into CD61 + severe combined immunodeficient (SCID) mice to construct the active ITP murine model. ITP mice were then randomly separated into groups that received the same volume of either 18β-GA (30 mg/kg, intraperitoneal injection, CAS471-53-4, Sigma-Aldrich, USA), the HMGB1 inhibitor lycopene (5 mg/kg, intragastric administration, S3943, Selleck, USA), or 3% dimethylsulfoxide (DMSO, intraperitoneal injection, Sigma-Aldrich, USA) every 2 days from day 1 after radiation and splenocyte infusion.

Techniques: Fluorescence, Derivative Assay, In Vitro, shRNA, Transfection, Knockdown, Real-time Polymerase Chain Reaction, Blocking Assay, Microscopy, Labeling, Software