Systemic inhibition of tissue-nonspecific alkaline phosphatase alters the brain-immune axis in experimental sepsis

Abstract

Tissue-nonspecific alkaline phosphatase (TNAP) is a ubiquitous enzyme present in many cells and tissues, including the central nervous system. Yet its functions at the brain-immune axis remain unclear. The goal of this study was to use a novel small molecular inhibitor of TNAP, SBI-425, to interrogate the function of TNAP in neuroimmune disorders. Following intraperitoneal (IP) administration of SBI-425, mass spectrometry analysis revealed that the SBI-425 does not cross the blood-brain barrier (BBB) in healthy mice. To elucidate the role of TNAP at the brain-immune axis, mice were subjected to experimental sepsis and received either vehicle or SBI-425 (25 mg/kg, IP) daily for 7 days. While SBI-425 administration did not affect clinical severity outcomes, we found that SBI-425 administration suppressed CD4 + Foxp3+ CD25− and CD8 + Foxp3+ CD25− splenocyte T-cell populations compared to controls. Further evaluation of SBI-425’s effects in the brain revealed that TNAP activity was suppressed in the brain parenchyma of SBI-425-treated mice compared to controls. When primary brain endothelial cells were treated with a proinflammatory stimulus the addition of SBI-425 treatment potentiated the loss of barrier function in BBB endothelial cells. To further demonstrate a protective role for TNAP at endothelial barriers within this axis, transgenic mice with a conditional overexpression of TNAP were subjected to experimental sepsis and found to have increased survival and decreased clinical severity scores compared to controls. Taken together, these results demonstrate a novel role for TNAP activity in shaping the dynamic interactions within the brain-immune axis.

Brain endothelial cell tissue-nonspecific alkaline phosphatase (TNAP) activity promotes maintenance of barrier integrityviathe ROCK pathway

“…Brain microvascular endothelial cells (BMECs) were cultured from male and female mice as previously described [28]. Briefly, 6-8 week old Alpl fl/fl (n = 5) and VE-cKO (n = 5) were perfused with 0.01 M phosphate buffered saline (PBS).…”

“…Barrier function assays were carried out as previously described for D3 cells [19] and in pBMECs [28].…”

“…Brain sections and cell cultures were immunostained using standard immunohistochemistry techniques [19,28]. Briefly, tissue sections and cells were washed three times, permeabilized, and blocked for 30 min on a shaker.…”

“…The cecal ligation and puncture (CLP) model of polymicrobial sepsis was employed as previously described [19,28]. Briefly, C57BL/6J mice were anesthetized by the inhalation of 1-2% isoflurane and abdominal access was obtained via a midline incision.…”

Experimental Stroke Induces Chronic Gut Dysbiosis and Neuroinflammation in Male Mice

“…Mouse brains were analyzed as free-floating sections to localize immunoreactivity for GFAP (Z0334, Dako/Agilent; 1:10,000 primary, 1:10,000 secondary), Iba-1 (019-19741, Wako; 1:2,000 primary, 1:1,000 secondary), ALPL (702454, Invitrogen, 1:100 primary, 1:1,000 secondary), and CD11b (ab 133357 Abcam, 1:500 primary, 1:1,000 secondary) using a modified ABC procedure (Vector Laboratories, Burlingame, CA) as previously described [28,29]. Briefly, sections were treated to remove endogenous peroxidases followed by permeabilization.…”