Journal: Advanced Science

Article Title: Exosome‐Mediated Lectin Pathway and Resistin‐MIF‐AA Metabolism Axis Drive Immune Dysfunction in Immune Thrombocytopenia

doi: 10.1002/advs.202412378

Figure Lengend Snippet: Landscape of BM mononuclear cells in patients with ITP. A). Uniform manifold approximation projection (UMAP) visualization of single‐cell transcriptional states obtained from BM mononuclear cells of patients with ITP compared with HDs. Distinct colors represent individual cell types determined based on gene expression: HSPC, hematopoietic stem and progenitor cells; T/NK, T and NK cells; pDC, plasmacytoid dendritic cells; DC, dendritic cells; Mono, monocytes. B). Analysis of proportions of different cell types in each sample. C). Selected lists of DE genes for ITP patient‐derived BMMC compared to HD‐derived BMMC were analyzed by GO analysis and annotated using GO terms. Graphs depicting selected GO terms, including complement‐associated genes. D). UMAP of BMMC showing the scores of “UCell: HALLMARK‐COMPLEMENT.” E). Bubble plot of complement pathway associated gene expression in myeloid population, including CD14 + CD16 − monocytes, CD16 + monocytes, and DC from patients with ITP versus healthy donors.

Article Snippet: [ ] Cells were incubated with different combinations of antibody mixtures (PE anti‐human CD284 (TLR4) Antibody (Clone No. HTA125, Cat. No. 312 805, BioLegend), APC Anti‐Human CD14 Antibody (Clone No. M5E2, Cat. No. E‐AB‐F1209E, Elabscience), APC/Cyanine7 anti‐human CD16 Antibody (Clone No. 3G8, Cat. No. 302 018, BioLegend), PE Anti‐Human CD16 Antibody (Clone No. 3G8, Cat. No. E‐AB‐F1236D, Elabscience), APC/Cyanine7 anti‐human CD46 Antibody (Clone No. TRA‐2‐10, Cat. No. 352 410, BioLegend), PE/Cyanine7 anti‐human CD88 (C5aR) Antibody (Clone No. S5/1, Cat. No. 344 308, BioLegend), Biotin anti‐human CD35 Antibody (Clone No. E11, Cat. No. 333 414, BioLegend), FITC anti‐human CD55 Antibody (Clone No. JS11, Cat. 311 306, BioLegend), PE anti‐human CD59 Antibody (Clone No. p282 (H19), Cat. 304 708, BioLegend), PerCP/Cyanine5.5 anti‐mouse/human CD11b Antibody (Clone No. M1/70, Cat. No. 101 228, BioLegend)), Biotin anti‐human CD11c Antibody (Clone No. Bu15, Cat. No. 337 232, BioLegend), APC anti‐human C3AR Antibody (Clone No. hC3aRZ8, Cat. No. 345 806, BioLegend), FITC anti‐human CD18 Antibody (Clone No. TS1/18, Cat. No. 302 106, BioLegend), Biotin anti‐human/mouse CXCR7 Antibody (Clone No. 8F11‐M16, Cat. No. 331 111, BioLegend), APC/Cyanine7 anti‐mouse/human CD44 Antibody (Clone No. IM7, Cat. No. 103 028, BioLegend), PE anti‐human CD184 (CXCR4) Recombinant Antibody (Clone No. QA18A64, Cat. No. 304 504, BioLegend), APC anti‐human CD74 Antibody (Clone No. LN2, Cat. No. 326 812, BioLegend) for 45 min in the dark at 4 °C.

Techniques: Gene Expression, Derivative Assay

Journal: Inflammation

Article Title: FTO-dependent m 6 A Demethylation Activates Mxd1 To Enhance Vitamin D-induced Suppression of Neuroinflammation Via PTEN/AKT/PGC-1α Signaling Pathways in Microglia

doi: 10.1007/s10753-026-02450-5

Figure Lengend Snippet: VitD promotes M2 microglial polarization in LPS-stimulated BV-2 cells and mice. A-B CCK-8 assays of BV-2 cell viability following treatment with various concentrations of VitD, with or without LPS stimulation. Data are representative of three independent experiments and shown as mean ± SD. C Western blot analysis of CD16, CD18 (M1 markers), CD206 and ARG1 (M2 markers) in LPS- or LPS + VitD (VitD)-treated BV-2 cells and primary mouse microglia. Data are representative of three independent experiments and shown as mean ± SD. n = 3 ** P < 0.01. One-way ANOVA and Tukey’s test. D Immunofluorescence analysis of the levels of CD16 and CD206 in LPS- or LPS + VitD (VitD)-treated BV-2 cells. Scale bar: 50 μm. Right panel: Quantification of CD16 and CD206 IF intensity. Six regions were randomly selected for each group, and the IF intensities of CD16 and CD206 were quantified using the ImageJ software. Data are shown as mean ± SD. ** P < 0.01. One-way ANOVA and Tukey’s test. Red arrows indicate CD16-positive microglia and green arrows indicate CD206-positive microglia. E ELISA analysis of supernatant levels of TNF-α, IL1β, IL-6, IL-4 and IL-10 in LPS- or LPS + VitD (VitD)-treated BV-2 cells. n = 6. Data are shown as mean ± SD. ** P < 0.01. One-way ANOVA and Tukey’s test. F Immunofluorescence analysis of the levels of CD16 and CD206 in cerebral cortex and hippocampal CA1 area of LPS- or LPS + VitD(VitD)-treated mice. Scale bar: 20 μm. n = 6 mice per group. Data are shown as mean ± SD. ** P < 0.01. One-way ANOVA and Tukey’s test. Red arrows indicate CD16-positive microglia and green arrows indicate CD206-positive microglia.

Article Snippet: For immunofluorescence analysis, brain sections were cut at 7 μm, washed with 0.1 M PBS, permeabilized with 0.3% (v/v) Triton X-100-PBS, blocked with 10% (v/v) donkey serum for 1 h, and then incubated with primary antibodies against CD16 (1:200, ProteinTech, Wuhan, China), CD206 (1:200, RecordBio) at 4 °C overnight.

Techniques: CCK-8 Assay, Western Blot, Immunofluorescence, Software, Enzyme-linked Immunosorbent Assay

Journal: Inflammation

Article Title: FTO-dependent m 6 A Demethylation Activates Mxd1 To Enhance Vitamin D-induced Suppression of Neuroinflammation Via PTEN/AKT/PGC-1α Signaling Pathways in Microglia

doi: 10.1007/s10753-026-02450-5

Figure Lengend Snippet: Mxd1 is essential for VitD-induced microglial M2 polarization in LPS-stimulated conditions. A Immunohistochemistry analysis of Mxd1 expression in cerebral cortex and hippocampal CA1 area hippocampal CA1 area of mice receiving LPS or LPS + VitD (VitD) treatment. Scale bar: 20 μm. Quantification of Mxd1-positive area is shown in the right panel. n = 6 mice per group. Data are shown as mean ± SD. ** P < 0.01. One-way ANOVA and Tukey’s test. B Western blot analysis of the protein levels of Mxd1 and c-MYC in BV-2 cells treated with LPS or LPS + VitD (VitD). Data are representative of three independent experiments and shown as mean ± SD. n = 3. ** P < 0.01. One-way ANOVA and Tukey’s test. C Immunofluorescence analysis of the levels of Mxd1 in in BV-2 cells treated with LPS or LPS + VitD (VitD). Scale bar: 100 μm. Right panel: Quantification of Mxd1 IF intensity. Six regions were randomly selected for each group, and the IF intensity of Mxd1 was quantified using the ImageJ software. Data are shown as mean ± SD. ** P < 0.01. One-way ANOVA and Tukey’s test. D Western blot analysis of the protein levels of CD16, CD18, CD206, ARG1 and Mxd1 in LPS or LPS + VitD (VitD)-treated BV-2 cells with either negative control siRNA (siNC), or Mxd1 siRNA (siMxd1). Data are representative of three independent experiments and shown as mean ± SD. n = 3. ns: not significant; * P < 0.05, ** P < 0.01. Two-way ANOVA and Tukey’s test. E ELISA analysis of supernatant levels of TNF-α, IL1β, IL-6, IL-4 and IL-10 in LPS or VitD-treated BV-2 cells with siNC or siMxd1. Data are representative of three independent experiments and shown as mean ± SD. n = 3 ** P < 0.01. Two-way ANOVA and Tukey’s test.

Article Snippet: For immunofluorescence analysis, brain sections were cut at 7 μm, washed with 0.1 M PBS, permeabilized with 0.3% (v/v) Triton X-100-PBS, blocked with 10% (v/v) donkey serum for 1 h, and then incubated with primary antibodies against CD16 (1:200, ProteinTech, Wuhan, China), CD206 (1:200, RecordBio) at 4 °C overnight.

Techniques: Immunohistochemistry, Expressing, Western Blot, Immunofluorescence, Software, Negative Control, Enzyme-linked Immunosorbent Assay

Journal: Inflammation

Article Title: FTO-dependent m 6 A Demethylation Activates Mxd1 To Enhance Vitamin D-induced Suppression of Neuroinflammation Via PTEN/AKT/PGC-1α Signaling Pathways in Microglia

doi: 10.1007/s10753-026-02450-5

Figure Lengend Snippet: Effect of FTO on VitD-induced microglial M2 polarization. A Global m 6 A RNA methylation analysis of total mRNAs in LPS- and LPS + VitD(VitD)-treated BV-2 cells. Data are representative of three independent experiments and shown as mean ± SD. * P < 0.05; ** P < 0.01. One-way ANOVA and Tukey’s test. B Western blot of m 6 A methylation regulator (METTL3, METTL14, WTAP, ALKBH5, and FTO) in the same treatment conditions. Data are representative of three independent experiments and shown as mean ± SD. ns: not significant; ** P < 0.01. One-way ANOVA and Tukey’s test. C m 6 A quantification in BV-2 cells transfected with negative control siRNA (siNC) or FTO siRNA (siFTO) and treated with LPS or LPS + VitD (VitD). Data are representative of three independent experiments and shown as mean ± SD. * P < 0.05; ** P < 0.01. Two-way ANOVA and Tukey’s test. D Western blot of CD16, CD18, CD206, ARG1 and Mxd1 in the same treatment conditions. Data are representative of three independent experiments and shown as mean ± SD. ns: not significant; * P < 0.05; ** P < 0.01. Two-way ANOVA and Tukey’s test. E ELISA of cytokine levels in corresponding supernatants. Data are representative of three independent experiments and shown as mean ± SD. ns: not significant; ** P < 0.01. Two-way ANOVA and Tukey’s test. F Immunohistochemistry for Iba-1 in the cerebral cortex and hippocampal CA1 area of mice treated with vehicle, LPS, LPS + VitD, and LPS + VitD + FB23-2. Scale bar: 20 μm. Quantification data are shown in the right panel. n = 6 mice per group. Data are shown as mean ± SD. ** P < 0.01. One-way ANOVA and Tukey’s test.

Article Snippet: For immunofluorescence analysis, brain sections were cut at 7 μm, washed with 0.1 M PBS, permeabilized with 0.3% (v/v) Triton X-100-PBS, blocked with 10% (v/v) donkey serum for 1 h, and then incubated with primary antibodies against CD16 (1:200, ProteinTech, Wuhan, China), CD206 (1:200, RecordBio) at 4 °C overnight.

Techniques: Methylation, Western Blot, Transfection, Negative Control, Enzyme-linked Immunosorbent Assay, Immunohistochemistry

Journal: Inflammation

Article Title: FTO-dependent m 6 A Demethylation Activates Mxd1 To Enhance Vitamin D-induced Suppression of Neuroinflammation Via PTEN/AKT/PGC-1α Signaling Pathways in Microglia

doi: 10.1007/s10753-026-02450-5

Figure Lengend Snippet: PGC-1α is required for VitD-induced M2 microglial polarization. A Immunofluorescence analysis of PGC-1α nuclear localization in LPS- or LPS + VitD-treated BV-2 cells. Scale bar: 100 μm. Right panel: Quantification of PGC-1α IF intensity. Six regions were randomly selected for each group, and the IF intensity of PGC-1α was quantified using the ImageJ software. Data are shown as mean ± SD. ** P < 0.01. One-way ANOVA and Tukey’s test. B Western blot analysis of CD16, CD18, CD206 and ARG1 in LPS- or LPS + VitD(VitD)-treated BV-2 cells with vehicle or SR-18,292. Data are representative of three independent experiments and shown as mean ± SD. ns: not significant; ** P < 0.01. Two-way ANOVA and Tukey’s test. C ELISA quantification of TNF-α, IL1β, IL-6, IL-4 and IL-10 in culture supernatants from the same experimental groups. Data are representative of three independent experiments and shown as mean ± SD. ns: not significant; ** P < 0.01. Two-way ANOVA and Tukey’s test. D Immunohistochemical staining and quantification of Iba-1-positive cells in cerebral cortex and hippocampal CA1 area of LPS- or VitD-treated mice with vehicle or SR-18,292, and the quantification of Iba-1 positive cells in each group was shown in the low panel. Scale bar: 20 μm. n = 6 mice per group. Data are shown as mean ± SD. ** P < 0.01. One-way ANOVA and Tukey’s test.

Article Snippet: For immunofluorescence analysis, brain sections were cut at 7 μm, washed with 0.1 M PBS, permeabilized with 0.3% (v/v) Triton X-100-PBS, blocked with 10% (v/v) donkey serum for 1 h, and then incubated with primary antibodies against CD16 (1:200, ProteinTech, Wuhan, China), CD206 (1:200, RecordBio) at 4 °C overnight.

Techniques: Immunofluorescence, Software, Western Blot, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining

Journal: PLoS ONE

Article Title: FcγRIIIa Expression on Monocytes in Rheumatoid Arthritis: Role in Immune-Complex Stimulated TNF Production and Non-Response to Methotrexate Therapy

doi: 10.1371/journal.pone.0028918

Figure Lengend Snippet: Example flow cytometric analysis of peripheral blood monocytes from a single healthy control subject (A) and a long-standing RA patient (B). Monocytes were initially gated based on light forward and side scatter (gated on monocytes and the upper portion of lymphocytes). Within this population, monocyte subsets were distinguished by their staining with antibodies to CD14 and CD16. Examples of the separate gates created around the CD14 low /CD16 ++ populations (dashed boxes) and the CD14 ++ /CD16 neg/low population (solid boxes) are shown. The same samples stained with anti-CD14-PerCP and IgG1-phycoerythrin (PE) isotype control in place of anti-CD16-PE were used to set gates. CD14 neg /CD16 ++ cells were excluded from the CD14 low/ CD16 ++ based on gates set using staining with IgG2a-PerCP isotype control. (C) CD16 expression levels (MFI units) on CD14 ++ monocytes in control (n = 40), early RA (n = 42) and long-standing RA patients (n = 38). (D) Percentage of CD14 ++ monocytes expressing CD16 in the same healthy control, early RA and long-standing RA patients. Box plots represent median and upper and lower quartiles and dots represent outliers. Statistics; Mann-Whitney U test.

Article Snippet: Cells were stained (30 min, ice) with anti-CD16-PE (clone 3G8, Caltag-Medsystems, Buckingham, UK) and anti-CD14-PerCP (clone MφP9, BD Biosciences, Oxford, UK) or irrelevant isotype controls.

Techniques: Staining, Expressing, MANN-WHITNEY

Journal: PLoS ONE

Article Title: FcγRIIIa Expression on Monocytes in Rheumatoid Arthritis: Role in Immune-Complex Stimulated TNF Production and Non-Response to Methotrexate Therapy

doi: 10.1371/journal.pone.0028918

Figure Lengend Snippet: (A) Healthy control subjects across a range of ages (x-axis) were tested for CD16 expression levels (MFI units, y-axis) on the CD14 ++ monocyte subset. (B) Healthy control subjects were stratified according to gender and levels of CD16 expression examined. (C) CRP levels in long-standing RA patients (closed points) and early RA patients (open points) were tested at the time of donating blood sample and examined for correlation with CD16 expression levels (MFI units, y-axis). (D) ESR values (x-axis) were measured in early RA patients only and examined for correlation with CD16 expression level (MFI units, y-axis). Statistics; Spearman's correlation.

Article Snippet: Cells were stained (30 min, ice) with anti-CD16-PE (clone 3G8, Caltag-Medsystems, Buckingham, UK) and anti-CD14-PerCP (clone MφP9, BD Biosciences, Oxford, UK) or irrelevant isotype controls.

Techniques: Expressing

Journal: PLoS ONE

Article Title: FcγRIIIa Expression on Monocytes in Rheumatoid Arthritis: Role in Immune-Complex Stimulated TNF Production and Non-Response to Methotrexate Therapy

doi: 10.1371/journal.pone.0028918

Figure Lengend Snippet: A) Thawed A) Cryopreserved PBMC from control and RA patients were stimulated for 4hrs with LPS (1 µg/ml), heat-aggregated Ig [HAG] (100 µg/ml) or the absence of stimulation (NS). Representative dot plots/histograms are shown for all treatments in a HC individual and for HAG stimulation in a RA patient (bottom panel). Intracellular staining was performed using anti-human TNF-PE or isotype control-PE antibodies and MFI units of expression measured. B) Intracellular staining for TNF in response to LPS (left panel) and HAG (right panel) in healthy control (HC, n = 8) and RA (n = 15) subjects. C) Correlation between HAG-stimulated TNF intracellular staining and % CD16 positive monocytes in RA patients. The percentage of CD14 ++ monocytes expressing CD16 (x-axis) and the MFI of anti-TNF-PE expression (y-axis) were determined by flow cytometry. Open diamonds represent early RA patient and shaded diamonds represent long-standing RA patients. Correlation coefficient spearman's rho = 0·813, p<0.001.

Article Snippet: Cells were stained (30 min, ice) with anti-CD16-PE (clone 3G8, Caltag-Medsystems, Buckingham, UK) and anti-CD14-PerCP (clone MφP9, BD Biosciences, Oxford, UK) or irrelevant isotype controls.

Techniques: Staining, Expressing, Flow Cytometry

Journal: PLoS ONE

Article Title: FcγRIIIa Expression on Monocytes in Rheumatoid Arthritis: Role in Immune-Complex Stimulated TNF Production and Non-Response to Methotrexate Therapy

doi: 10.1371/journal.pone.0028918

Figure Lengend Snippet: Early RA patient baseline demographics and associations with response to steroid/methotrexate therapy.

Article Snippet: Cells were stained (30 min, ice) with anti-CD16-PE (clone 3G8, Caltag-Medsystems, Buckingham, UK) and anti-CD14-PerCP (clone MφP9, BD Biosciences, Oxford, UK) or irrelevant isotype controls.

Techniques: Expressing

Journal: PLoS ONE

Article Title: FcγRIIIa Expression on Monocytes in Rheumatoid Arthritis: Role in Immune-Complex Stimulated TNF Production and Non-Response to Methotrexate Therapy

doi: 10.1371/journal.pone.0028918

Figure Lengend Snippet: CD16 expression levels were examined on CD14 ++ monocytes for correlations with baseline characteristics in a cohort of early RA patients prior to methotrexate therapy. Demographics examined were gender (A), age at baseline (B), smoking status (C), CCP autoantibody-positivity (D), RF-positivity (E), baseline DAS28ESR (F), disease duration at initiation of therapy (G), foot erosions (H), hand erosions (I) and FCGR3A genotype (J). Relationships between these baseline characteristics and EULAR responses are shown in .

Article Snippet: Cells were stained (30 min, ice) with anti-CD16-PE (clone 3G8, Caltag-Medsystems, Buckingham, UK) and anti-CD14-PerCP (clone MφP9, BD Biosciences, Oxford, UK) or irrelevant isotype controls.

Techniques: Expressing

Journal: PLoS ONE

Article Title: FcγRIIIa Expression on Monocytes in Rheumatoid Arthritis: Role in Immune-Complex Stimulated TNF Production and Non-Response to Methotrexate Therapy

doi: 10.1371/journal.pone.0028918

Figure Lengend Snippet: A) The percentage of CD16 positive CD14 ++ monocytes was measured at baseline (y-axis) and correlated with patient responses to methotrexate treatment. Patients were classified at week 14 post-initiation of therapy as non-responders (NR, n = 12), moderate (MOD, n = 12) or good (GOOD, n = 15) according to EULAR criteria and compared using Mann Whitney U test. Dots represent outliers. (B) The percentage reduction in DAS28-ESR between 0 and 14 weeks post-initiation of therapy (x-axis) and the % of CD14 ++ cells expressing CD16 at baseline (y-axis). Negative levels indicate increased DAS28ESR between baseline and week 14. Statistics; Spearman's correlation. C) The percentage of CD16 positive CD14 ++ monocytes was measured at baseline (wk0), 2 and 14 weeks after initiation of steroid/methotrexate treatment in early DMARD-naïve RA patients. Samples at 2 weeks mainly reflect influence of steroid treatment whereas week 14 samples will mainly reflect the effect of methotrexate therapy. Wilcoxon signed ranks test used to compare paired samples pre- and post- therapy.

Article Snippet: Cells were stained (30 min, ice) with anti-CD16-PE (clone 3G8, Caltag-Medsystems, Buckingham, UK) and anti-CD14-PerCP (clone MφP9, BD Biosciences, Oxford, UK) or irrelevant isotype controls.

Techniques: MANN-WHITNEY, Expressing