Journal: Science Advances

Article Title: Inflammatory arthritis irAE may represent a unique autoimmune disease primarily driven by T cells but likely not autoantibodies

doi: 10.1126/sciadv.aea4262

Figure Lengend Snippet: ( A ) Expression of CD27 and IgD on CD19 + B cells. Right: Percentage of IgD + CD27 − naïve B cells and IgD − CD27 − DN B cells. ( B ) Expression of CD27 and CD38 on CD19 + B cells. Right: Percentages of CD27 hi CD38 hi ASCs and CD27 + CD38 − memory B cells. ( C ) Expression of CD138 on CD27 hi CD38 hi ASCs. Right: Percentage of CD138 + ASCs and CD138 − ASCs. ( D ) Expression of CD11c and CD21 on CD19 + B cells. Right: Percentage of CD11c + CD21 − CD19 + B cells. [(A) to (D)] HC ( n = 63), irAE ( n = 33), RAC ( n = 46), and ICI ( n = 20). ( E to G ) GSEA was performed on the B cells between irAE and ICI. (E) Significantly enriched pathways in B cells from irAE and ICI. GSEA plots of IFN-α and IFN-γ response (F), and oxidative phosphorylation (G). ( H ) The volcano plots of the citrullinated or noncitrullinated relative IgG or IgM isotype autoantigen levels comparing RA versus HC, irAE versus ICI, irAE versus HC, or irAE versus RA. The autoantigens were labeled when P < 0.01. TNF-α reactivities resulted from the administration of anti–TNF-α therapy for the treatment of RA. ( I ) Immunoglobulin isotype levels in the plasma were measured by multiplex assay. HC ( n = 22), irAE ( n = 34), RAC ( n = 46), and ICI ( n = 26). ( J ) CXCL13 levels in the plasma were measured by enzyme-linked immunosorbent assay (ELISA). HC ( n = 20), irAE ( n = 34) RAC ( n = 47), and ICI ( n = 23). ( K ) B-cell activating factor (BAFF) levels in the plasma were measured by multiplex assay. HC ( n = 21), irAE ( n = 34), RAC ( n = 47), and ICI ( n = 18). Data in graphs represent mean ± SEM. Significance was tested by one-way ANOVA [(A) to (D) and (I) to (K)] and logistic regression (H). [(A) to (D) and (F) to (K)] ICI, ICI control.

Article Snippet: For CXCL13, IL-21, and CX3CL1 measurements, the following kits were used: Human CXCL13/BLC/BCA-1 Quantikine Enzyme-Linked Immunosorbent Assay (ELISA) (R&D Systems, catalog no. DCX130), Human IL-21 DuoSet ELISA (R&D Systems, catalog no. DY8879-05), and Human CX3CL1/Fractalkine DuoSet ELISA (R&D Systems, DY365); all steps were performed according to the manufacturer’s instructions.

Techniques: Expressing, Phospho-proteomics, Immunopeptidomics, Labeling, Clinical Proteomics, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Cell Research

Article Title: P2RX 7 sensitizes Mac-1/ICAM-1-dependent leukocyte-endothelial adhesion and promotes neurovascular injury during septic encephalopathy

doi: 10.1038/cr.2015.61

Figure Lengend Snippet: Sepsis increases brain endothelial chemokine expression. The chemokine CXCL1 , CXCL5 , CXCL10 , CX3CL1 , CCL2 and CCL3 mRNA levels from mouse brain endothelial cells were measured. The chemokine mRNAs were determined by real-time PCR analysis 4 h after CLP. Data are presented as a percentage change (%) compared to the sham control (means ± SEM, n = 4). ** P < 0.01. NS, not significant.

Article Snippet: CXCL1 and CX3CL1 levels in serum from sham and CLP animals were measured using the mouse ELISA kit (Elabscience Biotechnology), according to the manufacturer's instructions.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control

Journal: Cell Research

Article Title: P2RX 7 sensitizes Mac-1/ICAM-1-dependent leukocyte-endothelial adhesion and promotes neurovascular injury during septic encephalopathy

doi: 10.1038/cr.2015.61

Figure Lengend Snippet: CXCL1 promotes leukocyte adhesion via β2 integrin/ICAM-1 signaling. (A) Flow cytometry analysis of the surface expression of CD11a/CD18, CD11b/CD18 and CD18 on leukocytes (labeled by Ly6G staining) from peripheral blood of Sham mice and CLP (4 h) mice. (B) Surface staining of ICAM-1 on vascular endothelial cells in Sham and CLP mice. Data are representative of three independent experiments with three mice per group. (C, D) CXCL1-neutralizing antibody treatment significantly inhibited leukocytes adhesion (C) and endothelial ICAM-1 elevation (D) in CLP mice. IgG treatment (Control IgG) serves as control. The results are shown as means ± SEM ( n = 4). ** P < 0.01. Flow cytometry data are representative of three independent experiments with four mice per group. (E) CLP-induced CXCL1 and CX3CL1 mRNA elevation in brain vessels were abrogated after CXCL1-neutralizing antibody treatment. Data are expressed as means ± SEM ( n = 4). ** P < 0.01. (F, G) CLP induced leukocyte-endothelial cell adhesion (F) and the cumulative frequency of rolling velocities of leukocytes (G) was reduced in CD18 hypomorphic mutant (CD18 hypo ) mice. Two-photon imaging results are shown as means ± SEM ( n = 4). ** P < 0.01. (H) CLP-induced leukocyte β2 integrin elevation was abrogated in CD18 hypo mice. Flow cytometry data are representative of three independent experiments with four mice per group. (I) The CX3CL1 mRNA level was reduced in CD18 hypo mice 4 h after CLP. Data are expressed as means ± SEM ( n = 4). ** P < 0.01.

Article Snippet: CXCL1 and CX3CL1 levels in serum from sham and CLP animals were measured using the mouse ELISA kit (Elabscience Biotechnology), according to the manufacturer's instructions.

Techniques: Flow Cytometry, Expressing, Labeling, Staining, Control, Mutagenesis, Imaging

Journal: Cell Research

Article Title: P2RX 7 sensitizes Mac-1/ICAM-1-dependent leukocyte-endothelial adhesion and promotes neurovascular injury during septic encephalopathy

doi: 10.1038/cr.2015.61

Figure Lengend Snippet: P2RX 7 knockdown ameliorates leukocyte-endothelial cell adhesion in septic mice. (A) Representative images from TPLSM analysis of leukocyte adhesion to brain vessels 4 h after CLP. The mice were treated intracerebroventricularly with either a scrambled shRNA (LV shScrambled) or P2RX 7 shRNA two weeks before CLP. Scale bar, 30 μm. (B) Quantitative analysis of adherent leukocytes in A . Results are shown as means ± SEM ( n = 4). ** P < 0.01. NS, not significant. (C) In lenti- P2RX 7 shRNA group, the cumulative frequency of rolling velocities of leukocytes showed a significant reduction compared with lenti-shScramble-treated group after CLP. Rolling velocities of 138 cells per group ( n = 4 mice) were measured. (D) The representative changes of morphological parameters of microglial cells by lenti- P2RX 7 shRNA transfection were observed at regions I, II and III by using intravital TPLSM. Scale bar, 100 μm (upper panel). Microglias in regions I, II and III were mapped at 4 h after CLP (lower panel). (E, F) Microglia numbers (E) and mean values of microglia soma size (F) in regions I, II and III were measured after CLP. Data are expressed as means ± SEM ( n = 6). * P < 0.05, ** P < 0.01 versus region I; ## P < 0.01 versus LV shScramble + CLP mice. (G) The immunohistochemical staining for ICAM (red) in sham, lenti-shScramble and P2RX 7 shRNA group at 8 h after CLP. Scale bar, 20 μm. (H) Quantification of ICAM fluorescent signals in brain microvessels from G (mean ± SEM, n = 4 mice). ** P < 0.01. (I) Effect of lenti- P2RX 7 shRNA transfection on endothelial ICAM-1 expression in CLP mice. Flow cytometry analysis shows the surface staining of ICAM-1 in the indicated groups. Data are representative of three independent experiments with four mice per group. (J) P2RX 7 knockdown decreased CXCL1 and CX3CL1 mRNA levels in brain microvasculature of CLP mice. The upregulation of chemokine CXCL1 and CX3CL1 mRNA level was inhibited after P2RX 7 shRNA treatment in CLP mice. Data are expressed as means ± SEM ( n = 4). * P < 0.05.

Article Snippet: CXCL1 and CX3CL1 levels in serum from sham and CLP animals were measured using the mouse ELISA kit (Elabscience Biotechnology), according to the manufacturer's instructions.

Techniques: Knockdown, shRNA, Transfection, Immunohistochemical staining, Staining, Expressing, Flow Cytometry

Journal: Cell Research

Article Title: P2RX 7 sensitizes Mac-1/ICAM-1-dependent leukocyte-endothelial adhesion and promotes neurovascular injury during septic encephalopathy

doi: 10.1038/cr.2015.61

Figure Lengend Snippet: P2RX 7 inhibition reduces the adhesion molecules in brain microvasculature of CLP mice. (A) Adherent leukocytes were decreased in A438079-treated mice versus saline-treated mice 4 h after CLP. The results are shown as the means ± SEM ( n = 4). ** P < 0.01. (B) The Scatter plots show the cumulative frequency of rolling velocities of leukocytes in each treatment conditions. (C, D) Changes of brain endothelial ICAM-1 and VCAM-1 levels from CLP mice with or without A438079 treatment. Flow cytometry analysis shows the surface staining of ICAM-1 and VCAM-1 in the indicated groups. Data are representative of three independent experiments with four mice per group. (E) The chemokine CXCL1 and CX3CL1 mRNA levels were decreased in CLP mice after A438079 treatment. Data are expressed as means ± SEM ( n = 4). * P < 0.05, ** P < 0.01.

Article Snippet: CXCL1 and CX3CL1 levels in serum from sham and CLP animals were measured using the mouse ELISA kit (Elabscience Biotechnology), according to the manufacturer's instructions.

Techniques: Inhibition, Saline, Flow Cytometry, Staining

Journal: Cell Research

Article Title: P2RX 7 sensitizes Mac-1/ICAM-1-dependent leukocyte-endothelial adhesion and promotes neurovascular injury during septic encephalopathy

doi: 10.1038/cr.2015.61

Figure Lengend Snippet: Effect of IL-1β neutralization on expression of adhesion molecules in CLP mice. (A) Adherent leukocytes were decreased in IL-1β-neutralizing antibody-treated mice versus IgG-treated mice (Control IgG) 4 h after CLP. The results are shown as means ± SEM ( n = 4). ** P < 0.01. (B) IL-1β-neutralizing antibody treatment decreased the cumulative frequency of rolling velocities of leukocytes after CLP. Rolling velocities of 128 cells per group ( n = 4) were measured. (C , D) The brain endothelial cells were isolated from sham and CLP mice and labeled with ICAM-1 and VCAM-1, respectively. IL-1β-neutralizing antibody treatment inhibits the expression of ICAM-1 4 h after CLP. Data are representative of three independent experiments with three mice per group. (E) IL-1β-neutralizing antibody treatment reduced chemokine CXCL1 and CX3CL1 mRNA levels in brain endothelial cells of CLP mice. Data are expressed as means ± SEM ( n = 4). * P < 0.05; ** P < 0.01.

Article Snippet: CXCL1 and CX3CL1 levels in serum from sham and CLP animals were measured using the mouse ELISA kit (Elabscience Biotechnology), according to the manufacturer's instructions.

Techniques: Neutralization, Expressing, Control, Isolation, Labeling