P2RX7 sensitizes Mac-1/ICAM-1-dependent leukocyte-endothelial adhesion and promotes neurovascular injury during septic encephalopathy

Abstract

Septic encephalopathy (SE) is a critical factor determining sepsis mortality. Vascular inflammation is known to be involved in SE, but the molecular events that lead to the development of encephalopathy remain unclear. Using time-lapse in vivo two-photon laser scanning microscopy, we provide the first direct evidence that cecal ligation and puncture in septic mice induces microglial trafficking to sites adjacent to leukocyte adhesion on inflamed cerebral microvessels. Our data further demonstrate that septic injury increased the chemokine CXCL1 level in brain endothelial cells by activating endothelial P2RX7 and eventually enhanced the binding of Mac-1 (CD11b/CD18)-expressing leukocytes to endothelial ICAM-1. In turn, leukocyte adhesion upregulated endothelial CX3CL1, thereby triggering microglia trafficking to the injured site. The sepsis-induced increase in endothelial CX3CL1 was abolished in CD18 hypomorphic mutant mice. Inhibition of the P2RX7 pathway not only decreased endothelial ICAM-1 expression and leukocyte adhesion but also prevented microglia overactivation, reduced brain injury, and consequently doubled the early survival of septic mice. These results demonstrate the role of the P2RX7 pathway in linking neurovascular inflammation to brain damage in vivo and provide a rationale for targeting endothelial P2RX7 for neurovascular protection during SE.

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“…After DAPI counterstaining, the slices were rinsed in 3% BSA-PBS and mounted onto glass slides with Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA). Images were acquired using a Zeiss LSM 510 confocal microscope [20]. …”

“…Slices were mounted with VectaShield (Vector Labs) on glass slides. Images were acquired using a Zeiss LSM 510 confocal microscope [20]. …”

Cathepsin B inhibition ameliorates leukocyte‐endothelial adhesion in the BTBR mouse model of autism

“…Mice were anesthetized using chloral hydrate (400 mg/kg body weight ip) and subjected to in vivo imaging. A craniotomy window, centered by stereotaxic coordinates (2.5 mm later bregma, 2.5 mm lateral), was prepared for studying leukocyte adhesion. The custom‐made metal frame (1 cm diameter), with a removable GOMN glass lid, was carefully fixed to the skull using glue and dental cement .…”

“…Leukocyte attachment to the vascular endothelium is a hallmark of the inflammatory process; it facilitates transendothelial migration of leukocytes into the CNS and thereby elicits neuroinflammation in different brain diseases, as previously reported, for example, multiple sclerosis, atherosclerosis, and septic encephalopathy . In particular, the modulation pattern of leukocyte‐endothelium adhesion in autistic patients has remained controversial.…”

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“…The purified nuclei were resuspended with buffer C containing 50 mM Tris-HCl (pH 7.4), 4 mM EGTA, 10 mM EDTA, 0.5% Triton X-100, 0.5 M NaCl, 50 mM NaF, 30 mM sodium pyrophosphate, 1 mM Na 3 VO 4 and protease inhibitors [19]. The immunoblotting was carried out by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after determination of protein concentrations using Bradford's solution [21,22]. FKBP25 (1:1000; Santa Cruz Biotechnology, Dallas, Texas, USA), β-actin (1:5000; Sigma Aldrich, St. Louis, MO, USA), lamin B1 (1:1000; Santa Cruz Biotechnology, Dallas, Texas, USA) primary antibodies were used, followed by a secondary antibody used for detection.…”