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Neutrophils provide first line of host defense against bacterial infections utilizing glycolysis for their effector functions. How glycolysis and its major byproduct lactate are triggered in bone marrow (BM) neutrophils and their contribution to neutrophil mobilization in acute inflammation is not clear. Here we report that bacterial lipopolysaccharides (LPS) or Salmonella Typhimurium triggers lactate release by increasing glycolysis, NADPH-oxidase-mediated reactive oxygen species and HIF-1α levels in BM neutrophils. Increased release of BM lactate preferentially promotes neutrophil mobilization by reducing endothelial VE-Cadherin expression, increasing BM vascular permeability via endothelial lactate-receptor GPR81 signaling. GPR81−/− mice mobilize reduced levels of neutrophils in response to LPS, unless rescued by VE-Cadherin disrupting antibodies. Lactate administration also induces release of the BM neutrophil mobilizers G-CSF, CXCL1 and CXCL2, indicating that this metabolite drives neutrophil mobilization via multiple pathways. Our study reveals a metabolic crosstalk between lactate-producing neutrophils and BM endothelium, which controls neutrophil mobilization under bacterial infection., Lactate is a by-product of glycolysis that can function via its G protein receptor GPR81. Here the authors show that LPS or Salmonella infection enhances glycolytic metabolism in bone marrow neutrophils, resulting in lactate production, which increases endothelial barrier permeability and mobilization of these neutrophils by targeting endothelial GPR81.

Hematopoietic stem and progenitor cells (HSPCs) tightly couple maintenance of the bone marrow (BM) reservoir, including undifferentiated long-term repopulating hematopoietic stem cells (LT-HSCs), with intensive daily production of mature leukocytes and blood replenishment. We found two daily peaks of BM HSPC activity that are initiated by onset of light and darkness providing this coupling. Both peaks follow transient elevation of BM norepinephrine and TNF secretion, which temporarily increase HSPC reactive oxygen species (ROS) levels. Light-induced norepinephrine and TNF secretion augments HSPC differentiation and increases vascular permeability to replenish the blood. In contrast, darkness-induced TNF increases melatonin secretion to drive renewal of HSPCs and LT-HSC potential through modulating surface CD150 and c-Kit expression, increasing COX-2/αSMA+ macrophages, diminishing vascular permeability, and reducing HSPC ROS levels. These findings reveal that light- and darkness-induced daily bursts of norepinephrine, TNF, and melatonin within the BM are essential for synchronized mature blood cell production and HSPC pool repopulation.

Innate immune neutrophils provide the first line of host defense against bacterial infections. Neutrophils under steady state rely almost entirely on glycolysis and exhibit very low levels of oxidative phosphorylation. The metabolite lactate has long been considered a "waste byproduct" of cell metabolism which accumulates during inflammation and sepsis. Increased plasma lactate levels in human patients is used as a marker for sepsis diagnosis. However, the direct effector actions of lactate, particularly in regulating neutrophil mobilization and function during inflammation has remained obscure. To better understand the metabolic consequences of BM neutrophil activation during the onset of inflammation, we tested how bacterial lipopolysaccharides (mimicking gram negative bacterial inflammation) introduced intraperitoneally (i.p.) affect neutrophil metabolism and mobilization. RNAseq of sorted BM neutrophils revealed that LPS-activated neutrophils upregulate enzymes catalyzing the first part of glycolysis (hexokinase and PFKL) and downregulate the expression of TCA cycle enzymatic genes. In addition, LPS enhanced neutrophil lactate production and release as indicated by higher levels of BM lactate and higher expression of LDHA and MCT4. In addition, LPS increased NADPH oxidase (NOX)-mediated reactive oxygen species and HIF-1α levels in BM neutrophils, which are up-stream of glycolytic enzymes and lactate production and release. Recently, we reported that i.p. lactate administration rapidly activated and mobilized neutrophils from BM to the circulation (ASH, 2017). To test if lactate acts preferentially on neutrophils, we also examined other types of hematopoietic cells. Interestingly, we found that lactate specifically and rapidly (i.e., within 4 hrs) mobilized neutrophils to the circulation whereas the levels of peripheral blood (PB) monocytes, lymphocytes, granulocyte monocyte progenitors (GMPs) and hematopoietic progenitor stem cells (LSK) were reduced following lactate administration. LPS treatment failed to mobilize activated ROShigh neutrophils to the PB in NOX-/- mice, while lactate administration partially rescued this defect following LPS treatment. Our data also reveal that the NOX/ROS axis operates upstream of lactate production in BM neutrophils since abnormal metabolic rates were found in NOX-/- neutrophils during the onset of the acute inflammatory responses. Moreover, we found that BM endothelial cells (BMEC) abundantly express the highly selective lactate receptor GPR81, and that neutrophil-released lactate increased BM vascular permeability via BMEC GPR81 signaling (ASH, 2017). Consistent with a role of the lactate/GPR81 axis in enhanced vascular permeability, we find that i.p. injected LPS reduced VE-Cadherin expression on highly permeable sBMECs in GPR81 dependent manner. Notably, neutralizing VE-Cadherin in GPR81-/- mice can rescue and elevate PB neutrophil levels, similarly to wild-type (WT) mice, suggesting that VE-Cadherin is downstream of GPR81 signaling and plays a role in neutrophil mobilization. Finally, to examine the potential clinical relevance of our findings, we infected WT, NOX-/- and GPR81-/- mice with Salmonella Typhimurium and found out that this pathogen drove high generation of ROS, elevated HIF-1αlevels, and triggered lactate production and release in WT BM neutrophils. In contrast, BM neutrophils of infected NOX-/- mice exhibited significantly lower HIF-1αand impaired lactate production and release. Consequently, WT mice infected with Salmonella had a higher levels of neutrophils in the blood, as compared to their NOX-/- or GPR81-/- mice counterparts. Altogether, our data reveal that the same regulatory mechanisms by which neutrophils respond to LPS challenges are used during bacterial infection with Salmonella. Our study highlights lactate released by BM neutrophils as a key pro-inflammatory stimulus of a novel immune-metabolic crosstalk which is triggered by infection and locally opens the BM vascular barrier to facilitate neutrophil mobilization and recruitment to sites of inflammation. Targeting this immune-metabolic crosstalk between lactate-producing neutrophils and the BM endothelium could be useful for the control of pathological neutrophil activation and mobilization during bacterial infections and help treatments of neutrophil related immune disorders. Disclosures No relevant conflicts of interest to declare.

Bone marrow (BM) residing hematopoietic stem and progenitor cells (HSPC) replenish the blood with mature cells with a finite life span on a daily basis while maintaining the reservoir of undifferentiated stem cells. We recently showed that light/darkness onset induce two different BM HSPC peaks. Morning-induced norepinephrine and TNF secretion metabolically facilitate HSPC differentiation and egress to replenish the circulation with new mature leukocytes. Night augmented BM melatonin renews BM CD150+ hematopoietic stem cell (HSC) reservoir and their long-term repopulation potential (Golan et al, Cell Stem Cell, In Press). How melatonin primes BM HSPC to change their phenotype and function to re-acquire an undifferentiated and primitive state, is poorly understood. The hormone melatonin is an important mediator of bone formation and mineralization, and ultimately regulates the balance of bone remodeling (Cardinali DP et al, J. Pineal Res., 2003). The cross talk between HSPC and their BM stromal microenvironment is tightly regulated and determines HSPC fate. Therefore, we examined whether melatonin plays a role in regulation of murine BM mesenchymal stem and progenitor cells (MSPC, CD45-/Sca-1+/PDGFRα+), known to support HSPC maintenance in their BM niches. Mice treated with melatonin for 5h during the morning had increased levels of BM MSPC endowed with higher colony-forming unit fibroblast (CFU-F) potential in vitro. Interestingly, the metabolic state of these progenitor cells was altered by melatonin demonstrating reduced glucose uptake ability and lower mitochondria content. To test if differences in stromal cells content exist between day and night, we examined BM MSPC and found increased levels at 11PM, the time of melatonin BM peak, with higher Sca-1high surface expression levels, as compared to daylight 11AM. These changes were associated with augmented CFU-F levels by MSPC harvested at 11PM and accompanied by reduced glucose uptake levels and mitochondria content. Our preliminary results suggest that melatonin at night increases BM MSPC levels and reduces their metabolic activity to maintain them in a primitive and undifferentiated state. Moreover, we found that melatonin-elevated HSPC at 11PM also share lower glucose uptake ability with reduced mitochondria content and lower mitochondrial membrane potential (evaluated by TMRE). We hypothesize that melatonin reprograms the metabolic state of both HSPC and their stromal MSPC microenvironment to renew and maintain a primitive state of both populations at night. One of the factors inhibited by melatonin is the bioactive lipid Sphingosine 1-Phosphate (S1P), which in turn inhibits melatonin production. We found that mice with low S1P levels (S1Plow) due to lack of the SPHK1 enzyme have high BM melatonin levels also during the day in contrast to wild type (WT) mice. S1Plow mice had higher levels of primitive stromal progenitor cells including CFU-F and lower levels of differentiating osteoblast precursors compared to WT mice. In addition, these mice had less BM Reactive Oxygen Species (ROS)high committed hematopoietic progenitor cells, but more primitive ROSlow EPCR+ HSC endowed with higher long-term repopulation capacity in both primary and serially transplanted recipients. Next, we examined how light/dark cues affect the homing of transplanted BM HSPC into the BM of irradiated hosts 18h after transplantation. We found that donor HSPC harvested at 11PM have elevated homing ability compared to 11AM harvested cells. Importantly, MSPC also better homed to the BM of irradiated recipients when we transplanted donor BM cells obtained at 11PM compared with 11AM. As a result, accelerated BM repopulation kinetics was documented one week post transplantation in mice transplanted with BM cells harvested at 11 PM. Taken together, our results reveal that in vivo melatonin renews and maintains the BM reservoir and function of primitive MSPC and HSPC by metabolically reprogramming these cells during the night on a daily basis. Since the primed HSPC and MSPC at night showed improved function and BM homing potential, these features might be mimicked by human BM cells in order to harness them for improved clinical transplantation protocols. Disclosures No relevant conflicts of interest to declare.