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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.

SUBMITTER: Brichacek AL

PROVIDER: S-EPMC6906465 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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

Brichacek Allison L AL Benkovic Stanley A SA Chakraborty Sreeparna S Nwafor Divine C DC Wang Wei W Jun Sujung S Dakhlallah Duaa D Geldenhuys Werner J WJ Pinkerton Anthony B AB Millán José Luis JL Brown Candice M CM

Scientific reports 20191211 1

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 ...[more]

PMID: 31827139

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Project description:Tissue-nonspecific alkaline phosphatase (TNAP) is necessary for skeletal mineralization by its ability to hydrolyze the mineralization inhibitor inorganic pyrophosphate (PPi), which is mainly generated from extracellular ATP by ectonucleotide pyrophosphatase phosphodiesterase 1 (NPP1). Since children with TNAP deficiency develop bone metaphyseal auto-inflammations in addition to rickets, we hypothesized that TNAP also exerts anti-inflammatory effects relying on the hydrolysis of pro-inflammatory adenosine nucleotides into the anti-inflammatory adenosine. We explored this hypothesis in bone metaphyses of 7-day-old Alpl+/- mice (encoding TNAP), in mineralizing hypertrophic chondrocytes and osteoblasts, and non-mineralizing mesenchymal stem cells (MSCs) and neutrophils, which express TNAP and are present, or can be recruited in the metaphysis. Bone metaphyses of 7-day-old Alpl+/- mice had significantly increased levels of Il-1? and Il-6 and decreased levels of the anti-inflammatory Il-10 cytokine as compared with Alpl+/+ mice. In bone metaphyses, murine hypertrophic chondrocytes and osteoblasts, Alpl mRNA levels were much higher than those of the adenosine nucleotidases Npp1, Cd39 and Cd73. In hypertrophic chondrocytes, inhibition of TNAP with 25 ?M of MLS-0038949 decreased the hydrolysis of AMP and ATP. However, TNAP inhibition did not significantly modulate ATP- and adenosine-associated effects in these cells. We observed that part of TNAP proteins in hypertrophic chondrocytes was sent from the cell membrane to matrix vesicles, which may explain why TNAP participated in the hydrolysis of ATP but did not significantly modulate its autocrine pro-inflammatory effects. In MSCs, TNAP did not participate in ATP hydrolysis nor in secretion of inflammatory mediators. In contrast, in neutrophils, TNAP inhibition with MLS-0038949 significantly exacerbated ATP-associated activation and secretion of IL-1?, and extended cell survival. Collectively, these results demonstrate that TNAP is a nucleotidase in both hypertrophic chondrocytes and neutrophils, and that this nucleotidase function is associated with autocrine effects on inflammation only in neutrophils.

Project description:Tissue-nonspecific alkaline phosphatase (TNALP) is a glycoprotein expressed by osteoblasts that promotes bone mineralization. TNALP catalyzes the hydrolysis of the mineralization inhibitor inorganic pyrophosphate and ATP to provide inorganic phosphate, thus controlling the inorganic pyrophosphate/inorganic phosphate ratio to enable the growth of hydroxyapatite crystals. N-linked glycosylation of TNALP is essential for protein stability and enzymatic activity and is responsible for the presence of different bone isoforms of TNALP associated with functional and clinical differences. The site-specific glycosylation profiles of TNALP are, however, elusive. TNALP has 5 potential N-glycosylation sites located at the asparagine (N) residues 140, 230, 271, 303, and 430. The objective of this study was to reveal the presence and structure of site-specific glycosylation in TNALP expressed in osteoblasts. Calvarial osteoblasts derived from Alpl+/- expressing SV40 Large T antigen were transfected with soluble epitope-tagged human TNALP. Purified TNALP was analyzed with a lectin microarray, matrix-assisted laser desorption/ionization-time of flight mass spectrometry, and liquid chromatography with tandem mass spectrometry. The results showed that all sites (n = 5) were fully occupied predominantly with complex-type N-glycans. High abundance of galactosylated biantennary N-glycans with various degrees of sialylation was observed on all sites, as well as glycans with no terminal galactose and sialic acid. Furthermore, all sites had core fucosylation except site N271. Modelling of TNALP, with the protein structure prediction software ColabFold, showed possible steric hindrance by the adjacent side chain of W270, which could explain the absence of core fucosylation at N271. These novel findings provide evidence for N-linked glycosylation on all 5 sites of TNALP, as well as core fucosylation on 4 out of 5 sites. We anticipate that this new knowledge can aid in the development of functional and clinical assays specific for the TNALP bone isoforms.

Project description:Hypophosphatasia is a rare heritable disorder caused by inactivating mutations in the gene (Alpl) that encodes tissue nonspecific alkaline phosphatase (TNAP). Hypophosphatasia with onset in infants and children can manifest as rickets. How TNAP deficiency leads to bone hypomineralization is well explained by TNAP's primary function of pyrophosphate hydrolysis when expressed in differentiated bone forming cells. How TNAP deficiency leads to abnormalities within endochondral growth plates is not yet known. Previous studies in hypophosphatemic mice showed that phosphate promotes chondrocyte maturation and apoptosis via MAPK signaling. Alpl-/- mice are not hypophosphatemic but TNAP activity does increase local levels of inorganic phosphate. Therefore, we hypothesize that TNAP influences endochondral bone development via MAPK. In support of this premise, here we demonstrate cranial base bone growth deficiency in Alpl-/- mice, utilize primary rib chondrocytes to show that TNAP influences chondrocyte maturation, apoptosis, and MAPK signaling in a cell autonomous manner; and demonstrate that similar chondrocyte signaling and apoptosis abnormalities are present in the cranial base synchondroses of Alpl-/- mice. Micro CT studies revealed diminished anterior cranial base bone and total cranial base lengths in Alpl-/- mice, that were prevented upon injection with mineral-targeted recombinant TNAP (strensiq). Histomorphometry of the inter-sphenoidal synchondrosis (cranial base growth plate) demonstrated significant expansion of the hypertrophic chondrocyte zone in Alpl-/- mice that was minimized upon treatment with recombinant TNAP. Alpl-/- primary rib chondrocytes exhibited diminished chondrocyte proliferation, aberrant mRNA expression, diminished hypertrophic chondrocyte apoptosis and diminished MAPK signaling. Diminished apoptosis and VEGF expression were also seen in 15 day-old cranial base synchondroses of Alpl-/- mice. MAPK signaling was significantly diminished in 5 day-old cranial base synchondroses of Alpl-/- mice. Together, our data suggests that TNAP is essential for the later stages of endochondral bone development including hypertrophic chondrocyte apoptosis and VEGF mediated recruitment of blood vessels for replacement of cartilage with bone. These changes may be mediated by diminished MAPK signaling in TNAP deficient chondrocytes due to diminished local inorganic phosphate production.

Project description:Sepsis is a host response to systemic inflammation and infection that may lead to multi-organ dysfunction and eventual death. While acute brain dysfunction is common among all sepsis patients, chronic neurological impairment is prevalent among sepsis survivors. The brain microvasculature has emerged as a major determinant of sepsis-associated brain dysfunction, yet the mechanisms that underlie its associated neuroimmune perturbations and behavioral deficits are not well understood. An emerging body of data suggests that inhibition of tissue-nonspecific alkaline phosphatase (TNAP) enzyme activity in cerebral microvessels may be associated with changes in endothelial cell barrier integrity. The objective of this study was to elucidate the connection between alterations in cerebrovascular TNAP enzyme activity and brain microvascular dysfunction in late sepsis. We hypothesized that the disruption of TNAP enzymatic activity in cerebral microvessels would be coupled to the sustained loss of brain microvascular integrity, elevated neuroinflammatory responses, and behavioral deficits. Male mice were subjected to cecal ligation and puncture (CLP), a model of experimental sepsis, and assessed up to seven days post-sepsis. All mice were observed daily for sickness behavior and underwent behavioral testing. Our results showed a significant decrease in brain microvascular TNAP enzyme activity in the somatosensory cortex and spinal cord of septic mice but not in the CA1 and CA3 hippocampal regions. Furthermore, we showed that loss of cerebrovascular TNAP enzyme activity was coupled to a loss of claudin-5 and increased perivascular IgG infiltration in the somatosensory cortex. Analyses of whole brain myeloid and T-lymphoid cell populations also revealed a persistent elevation of infiltrating leukocytes, which included both neutrophil and monocyte myeloid derived suppressor cells (MDSCs). Regional analyses of the somatosensory cortex, hippocampus, and spinal cord revealed significant astrogliosis and microgliosis in the cortex and spinal cord of septic mice that was accompanied by significant microgliosis in the CA1 and CA3 hippocampal regions. Assessment of behavioral deficits revealed no changes in learning and memory or evoked locomotion. However, the hot plate test uncovered a novel anti-nociceptive phenotype in our septic mice, and we speculate that this phenotype may be a consequence of sustained GFAP astrogliosis and loss of TNAP activity in the somatosensory cortex and spinal cord of septic mice. Taken together, these results demonstrate that the loss of TNAP enzyme activity in cerebral microvessels during late sepsis is coupled to sustained neuroimmune dysfunction which may underlie, in part, the chronic neurological impairments observed in sepsis survivors.

Dataset Information

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

Publications

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

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