Fig 2: Effect of IL-1ß neutralization on expression of adhesion molecules in CLP mice. (A) Adherent leukocytes were decreased in IL-1ß-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-1ß-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-1ß-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-1ß-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.

Fig 3: CXCL1 promotes leukocyte adhesion via ß2 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 (CD18hypo) mice. Two-photon imaging results are shown as means ± SEM (n = 4). **P < 0.01. (H) CLP-induced leukocyte ß2 integrin elevation was abrogated in CD18hypo mice. Flow cytometry data are representative of three independent experiments with four mice per group. (I) The CX3CL1 mRNA level was reduced in CD18hypo mice 4 h after CLP. Data are expressed as means ± SEM (n = 4). **P < 0.01.

Fig 4: P2RX7 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.

Fig 5: P2RX7 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 P2RX7 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-P2RX7 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-P2RX7 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 P2RX7 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-P2RX7 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) P2RX7 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 P2RX7 shRNA treatment in CLP mice. Data are expressed as means ± SEM (n = 4). *P < 0.05.