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Loss of tissue-nonspecific alkaline phosphatase (TNAP) enzyme activity in cerebral microvessels is coupled to persistent neuroinflammation and behavioral deficits in late sepsis.

ABSTRACT: 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.

SUBMITTER: Nwafor DC

PROVIDER: S-EPMC7010562 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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:Overexpression of tissue-nonspecific alkaline phosphatase (TNAP) in endothelium leads to arterial calcification in mice. The purpose of this study was to examine the effect of elevated endothelial TNAP on coronary atherosclerosis. In addition, we aimed to examine endogenous TNAP activity in human myocardium.A vascular pattern of TNAP activity was observed in human non-failing, ischemic, and idiopathic dilated hearts (5 per group); no differences were noted between groups in this study. Endothelial overexpression of TNAP was achieved in mice harboring a homozygous recessive mutation in the low density lipoprotein receptor (whc allele) utilizing a Tie2-cre recombinase (WHC-eTNAP mice). WHC-eTNAP developed significant coronary artery calcification at baseline compared WHC controls (4312 vs 0?m2 alizarin red area, p<0.001). Eight weeks after induction of atherosclerosis, lipid deposition in the coronary arteries of WHC-eTNAP was increased compared to WHC controls (121633 vs 9330?m2 oil red O area, p<0.05). Coronary lesions in WHC-eTNAP mice exhibited intimal thickening, calcifications, foam cells, and necrotic cores. This was accompanied by the reduction in body weight and left ventricular ejection fraction (19.5 vs. 23.6g, p<0.01; 35% vs. 47%, p<0.05). In a placebo-controlled experiment under atherogenic conditions, pharmacological inhibition of TNAP in WHC-eTNAP mice by a specific inhibitor SBI-425 (30mg*kg-1*d-1, for 5 weeks) reduced coronary calcium (78838 vs.144622?m2) and lipids (30754 vs. 77317?m2); improved body weight (22.4 vs.18.8g) and ejection fraction (59 vs. 47%). The effects of SBI-425 were significant in the direct comparisons with placebo but disappeared after TNAP-negative placebo-treated group was included in the models as healthy controls.Endogenous TNAP activity is present in human cardiac tissues. TNAP overexpression in vascular endothelium in mice leads to an unusual course of coronary atherosclerosis, in which calcification precedes lipid deposition. The prevalence and significance of this mechanism in human atherosclerosis requires further investigations.

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.

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Loss of tissue-nonspecific alkaline phosphatase (TNAP) enzyme activity in cerebral microvessels is coupled to persistent neuroinflammation and behavioral deficits in late sepsis.

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