Leukotriene Pathway Research Articles

Potential Effects of Leukotriene Receptor Antagonist Montelukast in Treatment of Neuroinflammation in Parkinson's Disease.

Parkinson’s disease (PD) is a neurodegenerative disorder where misfolded alpha-synuclein-enriched aggregates called Lewy bodies are central in pathogenesis. No neuroprotective or disease-modifying treatments are currently available. Parkinson’s disease is considered a multifactorial disease and evidence from multiple patient studies and animal models has shown a significant immune component during the course of the disease, highlighting immunomodulation as a potential treatment strategy. The immune changes occur centrally, involving microglia and astrocytes but also peripherally with changes to the innate and adaptive immune system. Here, we review current understanding of different components of the PD immune response with a particular emphasis on the leukotriene pathway. We will also describe evidence of montelukast, a leukotriene receptor antagonist, as a possible anti-inflammatory treatment for PD.

In Vitro Effects of 5-Lipoxygenase Pathway Inhibition on Rhinovirus-Associated Bronchial Epithelial Inflammation.

IntroductionThe leukotriene pathway may be implicated in the induction of virus-induced inflammation. Respiratory epithelial cells may express low levels of 5-lipoxygenase (5-LO) and release leukotrienes (LTs) C4, D4, and E4, upon exposure to viruses or other stimuli. Enhanced expression of 5-LO pathway proteins after rhinovirus (RV) infection has previously been described. We hypothesized that anti-leukotriene treatment of epithelial cells, with or without exposure to RV-infected peripheral blood mononuclear cells (PBMCs)-conditioned media, may inhibit RV-induced up-regulation of inflammatory cytokines.MethodsPBMCs from a healthy donor were exposed to RV1B and supernatants were harvested at 48 h post infection. BEAS-2B cells were infected with RV, with or without conditioning with the PBMC supernatant. Treatment with anti-LT agents was performed either on both PBMCs and BEAS-2B or at the bronchial epithelial level only, with varying concentrations of montelukast (CysLT receptor antagonist) or MK-886 [FLAP(5-lipoxygenase-activating-protein) inhibitor]. Evaluation of the inflammatory cytokines IL-8, RANTES, IL-11, IL-6, and IP-10 was performed using ELISA.ResultsOur results show that anti-LT treatment of RV-infected bronchial epithelial cells suppresses epithelial RV-mediated cytokine production, independent of conditioning.ConclusionsThis observation may represent an indirect mode of action of the anti-leukotrienes in virus-induced asthma.Supplementary InformationThe online version contains supplementary material available at 10.1007/s41030-021-00152-x.

Alkaloids of Adhatoda vasica Nees. as potential inhibitors of cyclooxygenases – an in-silico study

Eicosanoid pathways play a crucial role in the progression and resolution of inflammation. NSAIDs act as anti-inflammatory agents by inhibiting both the isoforms of cyclooxygenases (COXs) whereas, COXIBs act as specific COX-2 inhibitors. Excessive usage of the same is linked with gastrointestinal bleeding and increased cardiovascular risk, respectively. The current in-silico study was aimed at evaluating the potential of major alkaloids of A. vasica (vasicine (VAS), vasicinone (VAE), and Deoxyvasicine (DOV)) as inhibitors of COXs. The results of the computed binding energy (ΔG) indicate that Celecoxib (CEL), DOV, and VAS have a higher affinity to COX-2, while VAE has a higher affinity to COX-1, and Mefenamic acid (MEF) was not selective. Among the alkaloids, VAE exhibited the best ΔG (of −8.2 kcal/mol) with COX-1, while VAS exhibited the best ΔG (of −8.2 kcal/mol) with COX-2. This was comparable to the ΔG exhibited by Mefenamic acid (-8.7 kcal/mol with both the COXs). With their potential to remain gastroprotective while having the ability to inhibit enzymes of both the prostaglandin and leukotriene pathways, the alkaloids of A. vasica could be promising leads for the design of Eicosanoid pathway modulators/inhibitors. Communicated by Ramaswamy H. Sarma

Lipidomics Profiling of Hidradenitis Suppurativa Skin Lesions Reveals Lipoxygenase Pathway Dysregulation and Accumulation of Proinflammatory Leukotriene B4

Hidradenitis suppurativa (HS) is a chronic, recurring inflammatory dermatosis characterized by abscesses, deep-seated nodules, sinus tracts, and fibrosis in skin lesions around hair follicles of the axillary, inguinal, and anogenital regions. Whereas the exact pathogenesis remains poorly defined, clear evidence suggests that HS is a multifactorial inflammatory disease characterized by innate and adaptive immune components. Bioactive lipids are important regulators of cutaneous homeostasis, inflammation, and resolution of inflammation. Alterations in the lipid mediator profile can lead to malfunction and cutaneous inflammation. We used targeted lipidomics to analyze selected omega-3 and omega-6 polyunsaturated fatty acids in skin of patients with HS and of healthy volunteers. Lesional HS skin displayed enrichment of 5-lipoxygenase (LO)‒derived metabolites, especially leukotriene B4. In addition, 15-LO‒derived metabolites were underrepresented in HS lesions. Changes in the lipid mediator profile were accompanied by transcriptomic dysregulation of the 5-LO and 15-LO pathways. Hyperactivation of the 5-LO pathway in lesional macrophages identified these cells as potential sources of leukotriene B4, which may cause neutrophil influx and activation. Furthermore, leukotriene B4-induced mediators and pathways were elevated in HS lesions, suggesting a contribution of this proinflammatory lipid meditator to the pathophysiology of HS.

Production of leukotriene signaling mediators is limited by ORMDL3/serine palmitoyltransferase/5-lipoxygenase crosstalk

Abstract Leukotrienes and sphingolipids are critical lipid mediators participating in cellular signal transduction and development of various diseases. Metabolic pathways initiating production of these lipid mediators involve 5-lipoxygenase (5-LO)-mediated conversion of arachidonic acid to leukotrienes and serine palmitoyltransferase (SPT) de novo synthesis of sphingolipids. Previous studies showed that endoplasmic reticulum membrane protein ORMDL3 inhibits the activity of SPT and sphingolipid synthesis. However, the role of ORMDL3 in synthesis of leukotrienes is not known. In this study, we used peritoneal-derived mast cells (PDMCs) isolated from mice with ORMDL3 knockout (KO) or control mice and examined their properties. We found that PDMCs with ORMDL3 KO exhibited increased calcium response and ß-glucuronidase release when activated with antigen. These events were accompanied by increased phosphorylation of IκB-α and TNF-α production. Lipid analysis showed that ORMDL3-deficient cells exhibited not only enhanced production of sphingolipids, but also increased production of leukotriene inflammatory mediators, such as LTB4 and LTC4. These data were supported by the finding that ORMDL3 physically interacts with 5-LO. Further studies showed that 5-LO interacts with the SPT long-chain (LC)1 and SPTLC2 subunits and decreases the ceramide levels. In line with these findings, 5-LO knockdown increased the ceramide levels, and silencing of SPTLC1 decreased transition of arachidonic acid to leukotrienes. These results demonstrate physical and functional crosstalk between leukotriene and sphingolipid metabolism pathways leading to production of lipid signaling mediators.

Antagonism of cysteinyl leukotrienes and their receptors as a neuroinflammatory target in Alzheimer's disease.

Alzheimer's disease is a complex multifaceted neurodegenerative disorder. It is characterized by the deposition of extracellular amyloid senile plaques and intracellular neurofibrillary tangles leading to progressive dementia and death in aged adult population. Recent emerging research has highlighted a potential pharmacological role of 5-lipoxyenase-cysteinyl leukotriene pathway in molecular pathogenesis of Alzheimer's disease. Although cysteinyl leukotrienes and their receptors have a major clinical role in chronic respiratory inflammation, their roles in chronic neuroinflammation in Alzheimer's disease need a detailed and careful exploration. This review article highlights a novel role of cysteinyl leukotrienes and their receptors in pathophysiology of Alzheimer's disease in order to understand the underlying molecular mechanism. In addition, it summarizes the recent advances in various pre-clinical and clinical strategies used to modulate this pathway for therapeutic targeting of Alzheimer's disease.

Leukotriene Pathway Activation Associates with Poor Glycemic Control and with Cardiovascular Autonomic Neuropathy in Type 1 Diabetes.

Background and Aims Since hyperglycemia promotes inflammation by different pathways and inflammation participates in the development of chronic diabetes complications, we investigated the association between the leukotriene (LT) pathway and microvascular diabetes complications. Methods and Results Quantitative polymerase chain reaction was employed to quantify the expression of ALOX5 (encodes 5-lipoxygenase), LTB4R (encodes one of the LTB4 receptors), and MYD88 in peripheral blood mononuclear cells from 164 type 1 diabetes (T1D) individuals presenting or not diabetes kidney disease, retinopathy, peripheral neuropathy, and cardiovascular autonomic neuropathy (CAN); 26 nondiabetic subjects were included as controls. LTB4 plasmatic concentrations were also evaluated. The expression of LTB4R was significantly higher in T1D individuals than in controls. T1D individuals with microvascular complications presented lower MYD88 mRNA expression when compared to those without microvascular complications. Higher LTB4 concentrations were found in individuals with CAN versus without CAN. The observation of two distinct subgroups of T1D individuals in the correlation analyses motivated us to evaluate the characteristics of each one of these groups separately. The group presenting higher expression of ALOX5 and of LTB4R also presented higher values of HbA1C, of fructosamine, and of plasmatic LTB4. Conclusion In the diabetes setting, the LT pathway is not only activated by hyperglycemia but is also modulated by the status of the autonomic nervous system.

Establishing Urinary Leukotriene E4 as a Diagnostic Biomarker for Chronic Rhinosinusitis with Comorbid Asthma and Atopy.

While urinary leukotriene E4 (uLTE4) is a validated biomarker for the cysteinyl leukotriene pathway, which is central to the pathophysiology of asthma, atopy, and chronic rhinosinusitis (CRS), the contributions of comorbid asthma and atopy to uLTE4 levels in various CRS subtypes have not been previously characterized. We sought to (1) identify reference values for uLTE4 in subjects with and without CRS and (2) determine how the presence of comorbid atopy and asthma affects uLTE4 levels in CRS. Tertiary referral medical center. A prospective case-control study was conducted to compare uLTE4 levels between patients with CRS and healthy controls. Urinary LTE4 levels were measured by enzyme immunoassay and were adjusted for urinary creatinine concentrations (pg/mg Cr). Patients with CRS were stratified by the clinical comorbidities to determine normative uLTE4 values for patients with CRS with and without comorbid asthma or atopy. A total of 153 patients (mean age, 47.3; 47.1% female) were included in the study. Patients with CRS demonstrated significantly higher concentrations of uLTE4 than healthy controls (1652 vs 1065 pg/mg Cr, P = .032). Within the group of patients with CRS, comorbid asthma also individually correlated with elevated uLTE4 levels (1597 pg/mg Cr, P = .0098). Patients with CRS who did not have comorbid allergy and asthma, in contrast, did not have statistically higher uLTE4 levels than healthy controls (1142 pg/mg Cr, P = .61). Urinary LTE4 serves as a noninvasive measure of the inflammatory state in CRS. Comorbid asthma and atopy contribute to elevated uLTE4 levels in CRS.

Montelukast Prevents Early Diabetic Retinopathy in Mice.

Chronic inflammation and oxidative stress are critical components in the pathogenic cascade of early diabetic retinopathy, characterized by neuronal and vascular degeneration. We investigated pharmacologic inhibition of the proinflammatory leukotriene cascade for therapeutic benefit in early diabetic retinopathy. Using the streptozotocin-induced diabetes mouse model, we administered montelukast, a leukotriene receptor antagonist, and diabetes-related retinal pathology was assessed. Early biochemical and cellular function measures were evaluated at 3 months’ diabetes duration and included vascular permeability, superoxide production, leukotriene generation, leukocyte-induced microvascular endothelial cell death, and retinal function by electroretinography. Histopathology assessments at 9 months’ diabetes duration included capillary degeneration and retinal ganglion cell loss. Leukotriene receptor antagonism resulted in a significant reduction of early, diabetes-induced retinal capillary leakage, superoxide generation, leukocyte adherence, and leukotriene generation. After 9 months of diabetes, the retinal microvasculature from untreated diabetic mice demonstrated a nearly threefold increase in capillary degeneration compared with nondiabetic mice. Montelukast inhibited the diabetes-induced capillary and neuronal degeneration, whether administered as a prevention strategy, immediately after induction of diabetes, or as an intervention strategy starting at 4.5 months after confirmation of diabetes. Pharmacologic blockade of the leukotriene pathway holds potential as a novel therapy to prevent or slow the development of diabetic retinopathy.

Involvement of leukotriene pathway in the development of sevoflurane-induced pica in rats.

We previously reported that sevoflurane-induced pica, kaolin ingestion behavior, in rats has the potential to reflect postoperative nausea and vomiting (PONV) in humans. It is well-known that corticosteroids, which inhibit both prostaglandin and leukotriene syntheses due to phospholipase A2 inhibition, are effective for reducing PONV; however, the precise mechanisms remain unclear. We investigated the involvement of the prostaglandin or leukotriene pathway in the development of sevoflurane-induced pica. We found that sevoflurane-induced pica was effectively inhibited by pretreatment with a leukotriene receptor antagonist (montelukast) or an inhibitor of 5-lipoxygenase (zileuton), rather than an inhibitor of cyclooxygenase (flurbiprofen). Furthermore, we observed that sevoflurane significantly increased urinary leukotriene excretion and 5-lipoxygenase mRNA expression in the spleen, but not hypothalamus. These results suggest that the production of leukotriene may lead to the development of sevoflurane-induced pica in rats, and that inhibition of the leukotriene pathway could be potentially useful for the treatment of PONV.

Genetic variation in the leukotriene pathway is associated with myocardial infarction in the Chinese population

BackgroundGenetic variation in the genes ALOX5 (arachidonate 5-lipoxygenase), ALOX5AP (arachidonate 5-lipoxygenase-activating protein) and LTA4H (leukotriene A4 hydrolase) has previously been shown to contribute to the risk of MI (myocardial infarction) in Caucasian and African American populations. All genes encode proteins playing a role in the synthesis of the pro-inflammatory leukotriene B mediators, possibly providing a link between MI and inflammation. The aim of the present study was to investigate whether these associations could be confirmed in the study of China MI patients. The study included 401 Han Chinese MI patients and 409 controls. Six tag single nucleotide polymorphisms (SNPs)—ALOX5 rs12762303 and rs12264801, ALOX5AP rs10507391, LTA4H rs2072512, rs2540487 and rs2540477—were selected. SNP genotyping was performed by an improved multiplex ligation detection reaction assay.ResultsThe rs2540487 genotype was associated with the risk of MI in overdominant model (P = 0.008). rs12762303 and rs10507391 SNPs were significantly associated with lipid levels in MI patients (P < 0.006–0.008). Several SNPs interacted with alcohol consumption, cigarette smoking, and hypertension to modify TC, TG, LDL-C and CRE levels, and the risk of MI (P < 0.0017 for all). No association between the SNPs of LT pathway and susceptibility to MI was found (P > 0.05 for all).ConclusionsTaken together, this study provides additional evidence that functional genetic variation of the LT pathway can mediate atherogenic processes and the risk of MI in Chinese.

A novel class of tyrosine derivatives as dual 5-LOX and COX-2/mPGES1 inhibitors with PGE2 mediated anticancer properties

Leukotriene and prostaglandin pathways are controlled by the enzymes, LOX and COX/mPGES1 respectively and are responsible for inflammatory responses.

The Pharmacogenomics of Asthma Beyond its Endotypes

While endotype-driven therapeutic strategies are increasingly successful for asthma, the issues of dissociated effect and drug efficacy at the target sites remain unresolved. This review is to provide a comprehensive overview of the pharmacogenomics of asthma along with its endotype subsets. We undertook a search in NCBI All Databases by key words "pharmacogenomics AND allergen", "pharmacogenomics AND asthma AND rhinitis", etc. The remaining papers were reviewed without bias after the exclusion of redundant papers and non-English papers (except for Chinese papers). Papers regarding similar issues or items were then pooled together to support the corresponding viewpoints and subtitles, conclusions were thus drawn. Eighty of the retrieved 139 papers were included in the review. Immunopathogenesis of asthma found that the induction of asthma involves genetic variation, TLR gene-activated immune responses, cell-mediated inflammation, and other immunological changes. Human lung mast cell has a crucial effect on the symptoms of asthma. JAK-3 mediates cytokine signaling, while DNA methylation in the ADRB2 gene decreases asthma symptom severity. Pharmacotherapeutics of asthma has focused on fewer candidate genes, relating to glucocorticoid, leukotriene, and β2-adrenergic receptor pathways, and other pathways involving IgE, IL-5, IL-4, IL-13 and IL-17. The genetic profiles of gene variants can predict individual disease susceptibility and risk for disease progression. Fewer genes, relating to glucocorticoid, leukotriene, histamine, and β2-adrenergic receptor pathways, have been concentrated. A personalized, tailored approach is necessary for health care delivery according to the individual variability in genes, environment and lifestyle of each individual.

Pathogenesis of NSAID-induced reactions in aspirin-exacerbated respiratory disease.

It is well-established that following ingestion of aspirin or any other inhibitor of cyclooxygenase-1, patients with Samter's disease, or aspirin-exacerbated respiratory disease (AERD) develop the sudden onset of worsening respiratory clinical symptoms, which usually involves nasal congestion, rhinorrhea, wheezing and bronchospasm. Gastrointestinal distress, nausea, a pruritic rash and angioedema can also occasionally develop. However, the underlying pathologic mechanism that drives these clinical reactions remains elusive. Pretreatment with medications that inhibit the leukotriene pathway decreases the severity of clinical reactions, which points to the involvement of cysteinyl leukotrienes (cysLTs) in the pathogenesis of these aspirin-induced reactions. Furthermore, studies of aspirin challenges in carefully-phenotyped patients with AERD have confirmed that both proinflammatory lipid mediators, predominantly cysLTs and prostaglandin (PG) D2, and the influx of effector cells to the respiratory tissue, contribute to symptom development during aspirin-induced reactions. Mast cells, which have been identified as the major cellular source of cysLTs and PGD2, are likely to be major participants in the acute reactions, and are an attractive target for future pharmacotherapies in AERD. Although several recent studies support the role of platelets as inflammatory effector cells and as a source of cysLT overproduction in AERD, it is not yet clear whether platelet activation plays a direct role in the development of the aspirin-induced reactions. To further our understanding of the pathogenesis of aspirin-induced reactions in AERD, and to broaden the pharmacotherapeutic options available to these patients, additional investigations with targeted clinical trials will be required.

Lactobacillus rhamnosus GG increases cyclooxygenase-2 expression and prostaglandin E2 secretion in colonic myofibroblasts via a MyD88-dependent mechanism during homeostasis.

Prostaglandin E2 (PGE2 ) plays a critical role in intestinal mucosal tolerance and barrier integrity. Cyclooxygenase-2 (COX-2)-dependent PGE2 production involves mobilisation of arachidonic acid. Lactobacillus rhamnosus GG (LbGG) is one of the most widely used probiotics reported to colonise the colonic mucosa. LbGG contributes to the protection of the small intestine against radiation injury through the repositioning of mucosal COX-2 expressing cells. However, it is unknown if LbGG modulates PGE2 production in the colonic mucosa under homeostasis and the major cellular elements involved in these processes. Colonic epithelial and CD90+ mesenchymal stromal cells, also known as (myo) fibroblasts (CMFs), are abundant innate immune cells in normal colonic mucosa able to produce PGE2 . Herein, we tested the hypothesis that under colonic mucosal homeostasis, LbGG modulates the eicosanoid pathway resulting in increased PGE2 production in both epithelial and stromal cells. Among the five tested human colonic epithelial cell lines, only exposure of Caco-2 to LbGG for 24hr led to the mobilisation of arachidonic acid with concomitant increase in the components within the leukotriene and COX-2-dependent PGE2 pathways. By contrast, CMFs isolated from the normal human colonic mucosa responded to LbGG with increased expression of COX-2 and PGE2 in the prostaglandin pathway, but not 5-LO in the leukotriene pathway. Oral gavage of C57BL/6 mice for 5days with LbGG (5×108 Colony-Forming Unit (CFU)/dose) increased COX-2 expression in the colonic mucosa. The majority of cells upregulating COX-2 protein expression were located in the colonic lamina propria and colocalised with α-SMA+ cells corresponding to the CMF phenotype. This process was myeloid differentiation factor-88-dependent, because silencing of myeloid differentiation factor-88 expression in CMFs abrogated LbGG-induced upregulation of COX-2 in culture and in vivo. Taken together, our data suggest that LbGG increases release of COX-2-mediated PGE2 , contributing to the maintenance of mucosal homeostasis in the colon and CMFs are among the major contributors to this process.

Learning Impairments, Memory Deficits, and Neuropathology in Aged Tau Transgenic Mice Are Dependent on Leukotrienes Biosynthesis: Role of the cdk5 Kinase Pathway.

Previous studies showed that the leukotrienes pathway is increased in human tauopathy and that its manipulation may modulate the onset and development of the pathological phenotype of tau transgenic mice. However, whether interfering with leukotrienes biosynthesis is beneficial after the behavioral deficits and the neuropathology have fully developed in these mice is not known. To test this hypothesis, aged tau transgenic mice were randomized to receive zileuton, a specific leukotriene biosynthesis inhibitor, or vehicle starting at 12months of age for 16weeks and then assessed in their functional and pathological phenotype. Compared with baseline, we observed that untreated tau mice had a worsening of their memory and spatial learning. By contrast, tau mice treated with zileuton had a reversal of these deficits and behaved in an undistinguishable manner from wild-type mice. Leukotriene-inhibited tau mice had an amelioration of synaptic integrity, lower levels of neuroinflammation, and a significant reduction in tau phosphorylation and pathology, which was secondary to an involvement of the cdk5 kinase pathway. Taken together, our findings represent the first demonstration that the leukotriene biosynthesis is functionally involved at the later stages of the tau pathological phenotype and represents an ideal target with viable therapeutic potential for treating human tauopathies.

5-Lipoxygenase: Its involvement in gastrointestinal malignancies.

Lipoxygenases (LOXs) are dioxygenases that catalyze the peroxidation of linoleic acid (LA) or arachidonic acid (AA), in the presence of molecular oxygen. The existence of inflammatory component in the tumor microenvironment intimately links the LOXs to gastrointestinal (GI) cancer progression. Amongst the six-different human LOX-isoforms, 5-LOX is the most vital enzyme for leukotriene (LT) biosynthesis, which is the main inflammation intermediaries. As recent investigations have shown the association of 5-LOX with tumor metastasis, there has also been significant progress in discovering the function of 5-LOX pathway in GI cancer. Studies on GI cancer cells using the pharmacological drugs targeting 5-LOX pathway have shown antiproliferative and proapoptotic effects. Pharmacogenetic discoveries in other diseases have revealed strong heritable basis for the leukotriene pathway, which helps in exploring the mechanistic source of genetic alteration within the leukotriene pathway and offer insights into GI cancer pathogenesis and future prospects for treatment and prevention. This review recapitulates the current research status of 5-LOX activity in GI malignancies.

Frontline Science: Pathological conditioning of human neutrophils recruited to the airway milieu in cystic fibrosis.

Recruitment of neutrophils to the airways, and their pathological conditioning therein, drive tissue damage and coincide with the loss of lung function in patients with cystic fibrosis (CF). So far, these key processes have not been adequately recapitulated in models, hampering drug development. Here, we hypothesized that the migration of naïve blood neutrophils into CF airway fluid in vitro would induce similar functional adaptation to that observed in vivo, and provide a model to identify new therapies. We used multiple platforms (flow cytometry, bacteria-killing, and metabolic assays) to characterize functional properties of blood neutrophils recruited in a transepithelial migration model using airway milieu from CF subjects as an apical chemoattractant. Similarly to neutrophils recruited to CF airways in vivo, neutrophils migrated into CF airway milieu in vitro display depressed phagocytic receptor expression and bacterial killing, but enhanced granule release, immunoregulatory function (arginase-1 activation), and metabolic activities, including high Glut1 expression, glycolysis, and oxidant production. We also identify enhanced pinocytic activity as a novel feature of these cells. In vitro treatment with the leukotriene pathway inhibitor acebilustat reduces the number of transmigrating neutrophils, while the metabolic modulator metformin decreases metabolism and oxidant production, but fails to restore bacterial killing. Interestingly, we describe similar pathological conditioning of neutrophils in other inflammatory airway diseases. We successfully tested the hypothesis that recruitment of neutrophils into airway milieu from patients with CF in vitro induces similar pathological conditioning to that observed in vivo, opening new avenues for targeted therapeutic intervention.

Defining murine organogenesis at single-cell resolution reveals a role for the leukotriene pathway in regulating blood progenitor formation.

During gastrulation, cell types from all three germ layers are specified and the basic body plan is established1. However, molecular analysis of this key developmental stage has been hampered by limited cell numbers and a paucity of markers. Single cell RNA sequencing circumvents these problems, but has so far been limited to specific organ systems2. Here we report single-cell transcriptomic characterisation of over 20000 cells immediately following gastrulation at E8.25 of mouse development. We identify 20 major cell types, which frequently contain sub-structure, including three distinct signatures in early foregut cells. Pseudospace ordering of somitic progenitor cells identifies dynamic waves of transcription and candidate regulators, which are validated by molecular characterisation of spatially resolved regions of the embryo. Within the endothelial population, cells that transition from haemogenic endothelial to erythro-myeloid progenitors specifically express Alox5 and its co-factor Alox5ap, which control leukotriene production. Functional assays using mouse embryonic stem cells demonstrate that leukotrienes promote haematopoietic progenitor cell generation. This comprehensive single cell map therefore can be exploited to reveal previously unrecognised pathways contributing to tissue development.

Unique regulation of Na-glutamine cotransporter SN2/SNAT5 in rabbit intestinal crypt cells during chronic enteritis.

The only Na‐nutrient cotransporter described in mammalian small intestinal crypt cells is SN2/SNAT5, which facilitates glutamine uptake. In a rabbit model of chronic intestinal inflammation, SN2 stimulation is secondary to an increase in affinity of the cotransporter for glutamine. However, the immune regulation of SN2 in the crypt cells during chronic intestinal inflammation is unknown. We sought to determine the mechanism of regulation of Na‐nutrient cotransporter SN2 by arachidonic acid metabolites in crypt cells. The small intestines of New Zealand white male rabbits were inflamed via inoculation with Eimeria magna oocytes. After 2‐week incubation, control and inflamed rabbits were subjected to intramuscular injections of arachidonyl trifluoromethyl ketone (ATK), piroxicam and MK886 for 48 hrs. After injections, the rabbits were euthanized and crypt cells from small intestines were harvested and used. Results: Treatment of rabbits with ATK prevented the release of AA and reversed stimulation of SN2. Inhibition of cyclooxygenase (COX) with piroxicam did not affect stimulation of SN2. However, inhibition of lipoxygenase (LOX) with MK886, thus reducing leukotriene formation during chronic enteritis, reversed the stimulation of SN2. Kinetic studies showed that the mechanism of restoration of SN2 by ATK or MK886 was secondary to the restoration of the affinity of the cotransporter for glutamine. For all treatment conditions, Western blot analysis revealed no change in SN2 protein levels. COX inhibition proved ineffective at reversing the stimulation of SN2. Thus, this study provides evidence that SN2 stimulation in crypt cells is mediated by the leukotriene pathway during chronic intestinal inflammation.