Intestinal Tuft-2 cells exert antimicrobial immunity via sensing bacterial metabolite N-undecanoylglycine.

Tuft cells are a type of intestinal epithelial cells that exist in epithelial barriers and play a critical role in immunity against parasite infection. It remains insufficiently clear whether Tuft cells participate in bacterial eradication. Here, we identified Sh2d6 as a signature marker for CD45+ Tuft-2 cells. Depletion of Tuft-2 cells resulted in susceptibility to bacterial infection. Tuft-2 cells quickly expanded in response to bacterial infection and sensed the bacterial metabolite N-undecanoylglycine through vomeronasal receptor Vmn2r26. Mechanistically, Vmn2r26 engaged with N-undecanoylglycine activated G-protein-coupled receptor-phospholipase C gamma2 (GPCR-PLCγ2)-Ca2+ signaling axis, which initiated prostaglandin D2 (PGD2) production. PGD2 enhanced the mucus secretion of goblet cells and induced antibacterial immunity. Moreover, Vmn2r26 signaling also promoted SpiB transcription factor expression, which is responsible for Tuft-2 cell development and expansion in response to bacterial challenge. Our findings reveal an additional function of Tuft-2 cells in immunity against bacterial infection through Vmn2r26-mediated recognition of bacterial metabolites.

Mutant KRAS Enhances Tumor Cell Fitness by Upregulating Stress Granules.

There is growing evidence that stress-coping mechanisms represent tumor cell vulnerabilities that may function as therapeutically beneficial targets. Recent work has delineated an integrated stress adaptation mechanism that is characterized by the formation of cytoplasmic mRNA and protein foci, termed stress granules (SGs). Here, we demonstrate that SGs are markedly elevated in mutant KRAS cells following exposure to stress-inducing stimuli. The upregulation of SGs by mutant KRAS is dependent on the production of the signaling lipid molecule 15-deoxy-delta 12,14 prostaglandin J2 (15-d-PGJ2) and confers cytoprotection against stress stimuli and chemotherapeutic agents. The secretion of 15-d-PGJ2 by mutant KRAS cells is sufficient to enhance SG formation and stress resistance in cancer cells that are wild-type for KRAS. Our findings identify a mutant KRAS-dependent cell non-autonomous mechanism that may afford the establishment of a stress-resistant niche that encompasses different tumor subclones. These results should inform the design of strategies to eradicate tumor cell communities.

Mast cell-derived prostaglandin D2 limits the subcutaneous absorption of honey bee venom in mice.

Mast cells play pivotal roles in innate host defenses against venom. Activated mast cells release large amounts of prostaglandin D2 (PGD2). However, the role of PGD2 in such host defense remains unclear. We found that c-kit-dependent and c-kit-independent mast cell-specific hematopoietic prostaglandin D synthase (H-pgds) deficiency significantly exacerbated honey bee venom (BV)-induced hypothermia and increased mortality rates in mice. BV absorption via postcapillary venules in the skin was accelerated upon endothelial barrier disruption resulting in increased plasma venom concentrations. These results suggest that mast cell-derived PGD2 may enhance host defense against BV and save lives by inhibiting BV absorption into circulation.

Mast cell maturation is driven via a group III phospholipase A2-prostaglandin D2-DP1 receptor paracrine axis.

Microenvironment-based alterations in phenotypes of mast cells influence the susceptibility to anaphylaxis, yet the mechanisms underlying proper maturation of mast cells toward an anaphylaxis-sensitive phenotype are incompletely understood. Here we report that PLA2G3, a mammalian homolog of anaphylactic bee venom phospholipase A2, regulates this process. PLA2G3 secreted from mast cells is coupled with fibroblastic lipocalin-type PGD2 synthase (L-PGDS) to provide PGD2, which facilitates mast-cell maturation via PGD2 receptor DP1. Mice lacking PLA2G3, L-PGDS or DP1, mast cell-deficient mice reconstituted with PLA2G3-null or DP1-null mast cells, or mast cells cultured with L-PGDS-ablated fibroblasts exhibited impaired maturation and anaphylaxis of mast cells. Thus, we describe a lipid-driven PLA2G3-L-PGDS-DP1 loop that drives mast cell maturation.

Mycobacterium tuberculosis-Induced Prostaglandin J2 and 15-Deoxy-Prostaglandin J2 Inhibit Inflammatory Signals in Human M1 Macrophages via a Negative Feedback Loop.

Tuberculosis caused by Mycobacterium tuberculosis is a leading cause of death globally and a major health concern. In humans, macrophages are the first line invaded by M. tuberculosis. Upon infection, macrophages upregulate cyclooxygenase-2 (COX-2) expression and consequently elevate the formation of PGs, including PGE2 and PGD2. Although the role of proinflammatory PGE2 in M. tuberculosis infection has been reported, the roles of PGJ2 and 15-deoxy-PGJ2 (collectively named J2-PGs), the metabolites of PGD2 with anti-inflammatory features, remain elusive. In this study, we show that M. tuberculosis (H37Rv strain)-conditioned medium stimulates human monocyte-derived macrophages (MDMs) to elevate COX-2 expression along with robust generation of PGJ2, exceeding PGD2 formation, and to a minor extent also of 15-deoxy-PGJ2. Of interest, in M1-MDM phenotypes, PGJ2 and 15-deoxy-PGJ2 decreased M. tuberculosis (H37Rv strain)-conditioned medium-induced COX-2 expression and related PG formation by a negative feedback loop. Moreover, these J2-PGs downregulated the expression of the proinflammatory cytokines IL-6, IL-1ß, and IFN-γ, but elevated the anti-inflammatory cytokine IL-10 and the M2 markers arginase-1 and CD163. These anti-inflammatory effects of J2-PGs in M1-MDM correlated with impaired activation of TGF-ß-activated kinase 1/NF-κB/MAPK pathways. Finally, we found that J2-PGs regulate COX-2 expression, at least partially, via PGD2 receptor (DP1) and chemoattractant receptor homologue expressed on Th2 cells/DP2 receptors, but independent of the J2-PG receptor peroxisome proliferator-activated receptor-γ. Together, our findings reveal that M. tuberculosis induces COX-2 expression in human M1-MDMs, along with robust formation of J2-PGs that mediates anti-inflammatory effects via a negative feedback loop.

Molecular basis for lipid recognition by the prostaglandin D2 receptor CRTH2.

Prostaglandin D2 (PGD2) signals through the G protein-coupled receptor (GPCR) CRTH2 to mediate various inflammatory responses. CRTH2 is the only member of the prostanoid receptor family that is phylogenetically distant from others, implying a nonconserved mechanism of lipid action on CRTH2. Here, we report a crystal structure of human CRTH2 bound to a PGD2 derivative, 15R-methyl-PGD2 (15mPGD2), by serial femtosecond crystallography. The structure revealed a "polar group in"-binding mode of 15mPGD2 contrasting the "polar group out"-binding mode of PGE2 in its receptor EP3. Structural comparison analysis suggested that these two lipid-binding modes, associated with distinct charge distributions of ligand-binding pockets, may apply to other lipid GPCRs. Molecular dynamics simulations together with mutagenesis studies also identified charged residues at the ligand entry port that function to capture lipid ligands of CRTH2 from the lipid bilayer. Together, our studies suggest critical roles of charge environment in lipid recognition by GPCRs.

Dual activation of estrogen receptor alpha and glucocorticoid receptor upregulate CRTh2-mediated type 2 inflammation; mechanism driving asthma severity in women?

BACKGROUND: Type 2-high asthma is characterized by elevated levels of circulating Th2 cells and eosinophils, cells that express chemoattractant-homologous receptor expressed on Th2 cells (CRTh2). Severe asthma is more common in women than men; however, the underlying mechanism(s) remain elusive. Here we examined whether the relationship between severe asthma and type 2 inflammation differs by sex and if estrogen influences Th2 cell response to glucocorticoid (GC). METHODS: Type 2 inflammation and the proportion of blood Th2 cells (CD4+ CRTh2+ ) were assessed in whole blood from subjects with asthma (n = 66). The effects of GC and estrogen receptor alpha (ERα) agonist on in vitro differentiated Th2 cells were examined. Expression of CRTh2, type 2 cytokines and degree of apoptosis (Annexin V+ , 7-AAD) were determined by flow cytometry, qRT-PCR, western blot and ELISA. RESULTS: In severe asthma, the proportion of circulating Th2 cells and hospitalizations were higher in women than men. Women with severe asthma also had more Th2 cells and serum IL-13 than women with mild/moderate asthma. Th2 cells, eosinophils and CRTh2 mRNA correlated with clinical characteristics associated with asthma control in women but not men. In vitro, GC and ERα agonist treated Th2 cells exhibited less apoptosis, more CRTh2 as well as IL-5 and IL-13 following CRTh2 activation than Th2 cells treated with GC alone. CONCLUSION: Women with severe asthma had higher levels of circulating Th2 cells than men, which may be due to estrogen modifying the effects of GC, enhancing Th2 cell survival and type 2 cytokine production.

Interleukin-4 treatment reduces leukemia burden in acute myeloid leukemia.

Interleukin-4 (IL-4) is a signature cytokine pivotal in Type 2 helper T cell (Th2) immune response, particularly in allergy and hypersensitivity. Interestingly, IL-4 increases endogenous levels of prostaglandin D2 (PGD2 ) and its metabolites, Δ12 -prostaglandin J2 (Δ12 -PGJ2 ) and 15-deoxy-Δ12,14 -prostaglandin J2 (15d-PGJ2 ), collectively called cyclopentenone PGs (CyPGs). However, the therapeutic role of IL-4 in hematologic malignancies remains unclear. Here, we employed a murine model of acute myeloid leukemia (AML), where human MLL-AF9 fusion oncoprotein was expressed in hematopoietic progenitor cells, to test the effect of IL-4 treatment in vivo. Daily intraperitoneal treatment with IL-4 at 60 µg/kg/d significantly alleviated the severity of AML, as seen by decreased leukemia-initiating cells (LICs). The effect of IL-4 was mediated, in part, by the enhanced expression of hematopoietic- PGD2  synthase (H-PGDS) to effect endogenous production of CyPGs, through autocrine and paracrine signaling mechanisms. Similar results were seen with patient-derived AML cells cultured ex vivo with IL-4. Use of GW9662, a peroxisome proliferator-activated receptor gamma (PPARγ) antagonist, suggested endogenous CyPGs-PPARγ axis mediated p53-dependent apoptosis of LICs by IL-4. Taken together, our results reveal a beneficial role of IL-4 treatment in AML suggesting a potential therapeutic regimen worthy of clinical trials in patients with AML.

Increased prostaglandin-D2 in male STAT3-deficient hearts shifts cardiac progenitor cells from endothelial to white adipocyte differentiation.

Cardiac levels of the signal transducer and activator of transcription factor-3 (STAT3) decline with age, and male but not female mice with a cardiomyocyte-specific STAT3 deficiency conditional knockout (CKO) display premature age-related heart failure associated with reduced cardiac capillary density. In the present study, isolated male and female CKO-cardiomyocytes exhibit increased prostaglandin (PG)-generating cyclooxygenase-2 (COX-2) expression. The PG-degrading hydroxyprostaglandin-dehydrogenase-15 (HPGD) expression is only reduced in male cardiomyocytes, which is associated with increased prostaglandin D2 (PGD2) secretion from isolated male but not female CKO-cardiomyocytes. Reduced HPGD expression in male cardiomyocytes derive from impaired androgen receptor (AR)-signaling due to loss of its cofactor STAT3. Elevated PGD2 secretion in males is associated with increased white adipocyte accumulation in aged male but not female hearts. Adipocyte differentiation is enhanced in isolated stem cell antigen-1 (SCA-1)+ cardiac progenitor cells (CPC) from young male CKO-mice compared with the adipocyte differentiation of male wild-type (WT)-CPC and CPC isolated from female mice. Epigenetic analysis in freshly isolated male CKO-CPC display hypermethylation in pro-angiogenic genes (Fgfr2, Epas1) and hypomethylation in the white adipocyte differentiation gene Zfp423 associated with up-regulated ZFP423 expression and a shift from endothelial to white adipocyte differentiation compared with WT-CPC. The expression of the histone-methyltransferase EZH2 is reduced in male CKO-CPC compared with male WT-CPC, whereas no differences in the EZH2 expression in female CPC were observed. Clonally expanded CPC can differentiate into endothelial cells or into adipocytes depending on the differentiation conditions. ZFP423 overexpression is sufficient to induce white adipocyte differentiation of clonal CPC. In isolated WT-CPC, PGD2 stimulation reduces the expression of EZH2, thereby up-regulating ZFP423 expression and promoting white adipocyte differentiation. The treatment of young male CKO mice with the COX inhibitor Ibuprofen or the PGD2 receptor (DP)2 receptor antagonist BAY-u 3405 in vivo increased EZH2 expression and reduced ZFP423 expression and adipocyte differentiation in CKO-CPC. Thus, cardiomyocyte STAT3 deficiency leads to age-related and sex-specific cardiac remodeling and failure in part due to sex-specific alterations in PGD2 secretion and subsequent epigenetic impairment of the differentiation potential of CPC. Causally involved is the impaired AR signaling in absence of STAT3, which reduces the expression of the PG-degrading enzyme HPGD.

Low 15d-PGJ2 status is associated with oxidative stress in chronic obstructive pulmonary disease patients.

BACKGROUND: Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-dependent nuclear receptor and highly expressed in human and rodent lungs. 15-Deoxy-delta-12,14-prostaglandin J2 (15d-PGJ2), known for cyclopentenone prostaglandin, is the endogenous ligand of PPARγ. However, the associations among PPARγ, 15d-PGJ2 and chronic obstructive pulmonary disease (COPD) were unclear. METHODS: All 130 fasting blood samples and 40 lung specimens were obtained from COPD patients and control subjects. Serum 15d-PGJ2 was detected by ELISA. The expressions of oxidative stress indicators were measured using western blotting and PPARγ nuclei were evaluated with immunohistochemistry in lungs. The associations among serum 15d-PGJ2, pulmonary PPARγ and oxidative stress indicators, and COPD were estimated. RESULTS: Serum 15d-PGJ2 was reduced in COPD patients compared with healthy volunteers. Linear and logistic regression analysis indicated that serum 15d-PGJ2 was positively associated with pulmonary function in COPD patients. In addition, PPARγ-positive nuclei were reduced and oxidative stress indicators, included HO-1 and NOX-4, were increased in lungs of COPD patients. Further correlative analysis suggested that pulmonary function parameters was positively correlated with serum 15d-PGJ2 and pulmonary PPARγ-positive nuclei, inversely related to oxidative stress indicators in lungs of COPD patients. Pretreatment with 15d-PGJ2 obviously attenuated TNFα-induced oxidative stress in BEAS-2B cells. CONCLUSIONS: Serum 15d-PGJ2 and pulmonary PPARγ are reduced, and oxidative stress is elevated in COPD patients. Serum 15d-PGJ2 is inversely associated with oxidative stress in COPD patients.

L-PGDS Attenuates Acute Lung Injury by Prostaglandin D2 in Both Dependent and Independent Ways.

Lipocalin-type PG D synthase (L-PGDS) has two roles: it can be a PGD synthase, or it can be a carrier protein of hydrophobic small molecules. In this study, we investigated the dual roles of L-PGDS in acute lung injury by using L-PGDS-deficient and point-mutated mice, which lack PGD2 producibility but maintain lipocalin ability. Hydrochloride (HCl) administration (0.1 M intratracheally for 6 h) caused hemorrhage and dysfunction in the wild-type (WT) mouse lung. These symptoms were accompanied by an increase in PGD2 production. Both deficiency and point mutation of L-PGDS aggravated the HCl-induced hemorrhage and dysfunction. Although both the gene modifications decreased PGD2 production, only L-PGDS-deficient mice, but not point mutation mice, lacked protein expressions of L-PGDS in the lungs. In the WT mice, HCl administration caused pulmonary edema, indexed as an increase in lung water content and protein leakage in bronchoalveolar lavage fluid. L-PGDS deficiency and point mutation similarly aggravated edema formation. HCl administration also stimulated mucin production and bronchoalveolar lavage fluid leukocyte infiltration in the WT mouse lungs. Of interest, L-PGDS deficiency, but not point mutation, exacerbated these manifestations. Consistently, only L-PGDS deficiency increased the mRNA expression of IL-33, which stimulates mucin production in the inflamed lung. These results show that L-PGDS attenuated HCl-induced acute lung injury progresses in two different ways: L-PGDS produced PGD2, which inhibited pulmonary edema formation, whereas its lipocalin ability decreased mucin formation and inflammatory cell infiltration in the inflamed lung.

Prostaglandin D2 signaling and cardiovascular homeostasis.

Cardiovascular diseases are the leading cause of death worldwide. A chronic inflammatory response is a common pathological alteration in diverse cardiovascular diseases. Prostaglandin (PG) D2, a key lipid mediator derived from arachidonic acid metabolism, promotes resolution of inflammation and regulated T cell function through its receptors. Accumulated evidence has shown that dysregulated PGD2 signaling is involved in the pathogenesis of cardiovascular diseases, including atherosclerosis, hypertension, pulmonary hypertension, abdominal aortic aneurysm, and myocardial ischemia. Here, we summarized the recent progresses on PGD2 in cardiovascular homeostasis and discussed potential therapeutic translation by targeting PGD2 signaling.

Epo receptor signaling in macrophages alters the splenic niche to promote erythroid differentiation.

Anemic stress induces stress erythropoiesis, which rapidly generates new erythrocytes to restore tissue oxygenation. Stress erythropoiesis is best understood in mice where it is extramedullary and occurs primarily in the spleen. However, both human and mouse stress erythropoiesis use signals and progenitor cells that are distinct from steady-state erythropoiesis. Immature stress erythroid progenitors (SEPs) are derived from short-term hematopoietic stem cells. Although the SEPs are capable of self-renewal, they are erythroid restricted. Inflammation and anemic stress induce the rapid proliferation of SEPs, but they do not differentiate until serum erythropoietin (Epo) levels increase. Here we show that rather than directly regulating SEPs, Epo promotes this transition from proliferation to differentiation by acting on macrophages in the splenic niche. During the proliferative stage, macrophages produce canonical Wnt ligands that promote proliferation and inhibit differentiation. Epo/Stat5-dependent signaling induces the production of bioactive lipid mediators in macrophages. Increased production of prostaglandin J2 (PGJ2) activates peroxisome proliferator-activated receptor γ (PPARγ)-dependent repression of Wnt expression, whereas increased production of prostaglandin E2 (PGE2) promotes the differentiation of SEPs.

PGD2 /CRTH2 signaling promotes acquired immunity against bee venom by enhancing IgE production.

IgE-dependent/independent activation of mast cell (MC) has been assumed to play a host defensive role against venom injection in skin. However, its detailed mechanisms remain unknown. We aimed to investigate the contribution of MC-derived prostaglandin D2 (PGD2 )-mediated signaling in host defense against bee venom (BV). To achieve this, we utilized gene-deficient mice of a PGD2 receptor, chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). We first confirmed that subcutaneous injection of BV produced PGD2 equally in wild-type (WT) and CRTH2-deficient (Crth2-/- ) mice skins. The BV injection dropped body temperature and impaired kidney equally in both lines of mice. In WT mice, pre-injection of BV (3 weeks) significantly inhibited the hypothermia and kidney impairment caused by second BV injection. In contrast, this pre-injection was not effective for the second BV injection in Crth2-/- mice. We also found that BV injections increased serum BV-specific IgE levels in WT mice, and its serum transfused mice improved the BV-induced hypothermia in naïve WT mice. In contrast, serum BV-specific IgE level was significantly lower in Crth2-/- mice. FACS analysis showed the BV injection stimulate migration of dendritic cells (DCs) into regional lymph nodes in WT mice. In Crth2-/- mice, its number was significantly smaller than that of WT mice. In conclusion, PGD2 /CRTH2 signaling plays defensive role against second BV injection. This signaling promotes BV-specific IgE production at least partially by promoting DCs migration into regional lymph node.

Loss of Endothelial FTO Antagonizes Obesity-Induced Metabolic and Vascular Dysfunction.

RATIONALE: Increasing prevalence of obesity and its associated risk with cardiovascular diseases demands a better understanding of the contribution of different cell types within this complex disease for developing new treatment options. Previous studies could prove a fundamental role of FTO (fat mass and obesity-associated protein) within obesity; however, its functional role within different cell types is less understood. OBJECTIVES: We identify endothelial FTO as a previously unknown central regulator of both obesity-induced metabolic and vascular alterations. METHODS AND RESULTS: We generated endothelial Fto-deficient mice and analyzed the impact of obesity on those mice. While the loss of endothelial FTO did not influence the development of obesity and dyslipidemia, it protected mice from high-fat diet-induced glucose intolerance and insulin resistance by increasing AKT (protein kinase B) phosphorylation in endothelial cells and skeletal muscle. Furthermore, loss of endothelial FTO prevented the development of obesity-induced hypertension by preserving myogenic tone in resistance arteries. In Fto-deficient arteries, microarray analysis identified upregulation of L-Pgds with significant increases in prostaglandin D2 levels. Blockade of prostaglandin D2 synthesis inhibited the myogenic tone protection in resistance arteries of endothelial Fto-deficient mice on high-fat diet; conversely, direct addition of prostaglandin D2 rescued myogenic tone in high-fat diet-fed control mice. Myogenic tone was increased in obese human arteries with FTO inhibitors or prostaglandin D2 application. CONCLUSIONS: These data identify endothelial FTO as a previously unknown regulator in the development of obesity-induced metabolic and vascular changes, which is independent of its known function in regulation of obesity.

Niacin Attenuates Pulmonary Hypertension Through H-PGDS in Macrophages.

RATIONALE: Pulmonary arterial hypertension (PAH) is characterized by progressive pulmonary vascular remodeling, accompanied by varying degrees of perivascular inflammation. Niacin, a commonly used lipid-lowering drug, possesses vasodilating and proresolution effects by promoting the release of prostaglandin D2 (PGD2). However, whether or not niacin confers protection against PAH pathogenesis is still unknown. OBJECTIVE: This study aimed to determine whether or not niacin attenuates the development of PAH and, if so, to elucidate the molecular mechanisms underlying its effects. METHODS AND RESULTS: Vascular endothelial growth factor receptor inhibitor SU5416 and hypoxic exposure were used to induce pulmonary hypertension (PH) in rodents. We found that niacin attenuated the development of this hypoxia/SU5416-induced PH in mice and suppressed progression of monocrotaline-induced and hypoxia/SU5416-induced PH in rats through the reduction of pulmonary artery remodeling. Niacin boosted PGD2 generation in lung tissue, mainly through H-PGDS (hematopoietic PGD2 synthases). Deletion of H-PGDS, but not lipocalin-type PGDS, exacerbated the hypoxia/SU5416-induced PH in mice and abolished the protective effects of niacin against PAH. Moreover, H-PGDS was expressed dominantly in infiltrated macrophages in lungs of PH mice and patients with idiopathic PAH. Macrophage-specific deletion of H-PGDS markedly decreased PGD2 generation in lungs, aggravated hypoxia/SU5416-induced PH in mice, and attenuated the therapeutic effect of niacin on PAH. CONCLUSIONS: Niacin treatment ameliorates the progression of PAH through the suppression of vascular remodeling by stimulating H-PGDS-derived PGD2 release from macrophages.

Lipocalin-Type Prostaglandin D2 Synthase Protein- A Central Player in Metabolism.

Lipocalin-type prostaglandin D synthase was previously known as ß-trace protein (BTP), a low-molecular-weight glycoprotein that is heavily expressed in human cerebrospinal fluid. Nevertheless, it is also seen to be expressed in numerous other tissues including the kidney, liver, lung, heart, adipose, muscle, and pancreas. Functionally, L-PGDS behaves like a lipocalin type protein where it helps in binding and transportation of small lipophilic substances, such as steroids, retinoids, and other lipophilic ligands. Enzymatically, L-PGDS functions as a prostaglandin synthase where it helps in the production of PGD2 by catalyzing the isomerization of PGH2, a common precursor of the two series of prostaglandins. PGD2 regulates its physiological function through two individual receptors named DP1 and DP2. L-PGDS has been a central player in many diseases, its role in metabolism including diabetes, fatty liver disease, and obesity has gathered a large attention. In this review, we summarize the current state of knowledge about L-PGDS and it's signaling in adipose, hepatic, skeletal muscle, and pancreas tissues, which are core targets for metabolic studies. Modulation of L-PGDS signaling can be considered as a potential future therapeutic target for the treatment of insulin resistance as well as fatty liver disease.

The Prostaglandin D2 Receptor CRTH2 Promotes IL-33-Induced ILC2 Accumulation in the Lung.

Group 2 innate lymphoid cells (ILC2s) are rare innate immune cells that accumulate in tissues during allergy and helminth infection, performing critical effector functions that drive type 2 inflammation. ILC2s express ST2, the receptor for the cytokine IL-33, and chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), a receptor for the bioactive lipid prostaglandin D2 (PGD2). The IL-33-ST2 and the PGD2-CRTH2 pathways have both been implicated in promoting ILC2 accumulation during type 2 inflammation. However, whether these two pathways coordinate to regulate ILC2 population size in the tissue in vivo remains undefined. In this study, we show that ILC2 accumulation in the murine lung in response to systemic IL-33 treatment was partially dependent on CRTH2. This effect was not a result of reduced ILC2 proliferation, increased apoptosis or cell death, or differences in expression of the ST2 receptor in the absence of CRTH2. Rather, data from adoptive transfer studies suggested that defective accumulation of CRTH2-deficient ILC2s in response to IL-33 was due to altered ILC2 migration patterns. Whereas donor wild-type ILC2s preferentially accumulated in the lungs compared with CRTH2-deficient ILC2s following transfer into IL-33-treated recipients, wild-type and CRTH2-deficient ILC2s accumulated equally in the recipient mediastinal lymph node. These data suggest that CRTH2-dependent effects lie downstream of IL-33, directly affecting the migration of ILC2s into inflamed lung tissues. A better understanding of the complex interactions between the IL-33 and PGD2-CRTH2 pathways that regulate ILC2 population size will be useful in understanding how these pathways could be targeted to treat diseases associated with type 2 inflammation.

DP1 Activation Reverses Age-Related Hypertension Via NEDD4L-Mediated T-Bet Degradation in T Cells.

BACKGROUND: Blood pressure often rises with aging, but exact mechanisms are still not completely understood. With aging, the level of proinflammatory cytokines increases in T lymphocytes. Prostaglandin D2, a proresolution mediator, suppresses Type 1 T helper (Th1) cytokines through D-prostanoid receptor 1 (DP1). In this study, we aimed to investigate the role of the prostaglandin D2/DP1 axis in T cells on age-related hypertension. METHODS: To clarify the physiological and pathophysiological roles of DP1 in T cells with aging, peripheral blood samples were collected from young and older male participants, and CD4+ T cells were sorted for gene expression, prostaglandin production, and Western blot assays. Mice blood pressure was quantified by invasive telemetric monitor. RESULTS: The prostaglandin D2/DP1 axis was downregulated in CD4+ T cells from older humans and aged mice. DP1 deletion in CD4+ T cells augmented age-related hypertension in aged male mice by enhancing Th1 cytokine secretion, vascular remodeling, CD4+ T cells infiltration, and superoxide production in vasculature and kidneys. Conversely, forced expression of exogenous DP1 in T cells retarded age-associated hypertension in mice by reducing Th1 cytokine secretion. Tumor necrosis factor α neutralization or interferon γ deletion ameliorated the age-related hypertension in DP1 deletion in CD4+ T cells mice. Mechanistically, DP1 inhibited Th1 activity via the PKA (protein kinase A)/p-Sp1 (phosphorylated specificity protein 1)/neural precursor cell expressed developmentally downregulated 4-like (NEDD4L) pathway-mediated T-box-expressed-in-T-cells (T-bet) ubiquitination. T-bet deletion or forced NEDD4L expression in CD4+ T cells attenuated age-related hypertension in CD4+ T cell-specific DP1-deficient mice. DP1 receptor activation by BW245C prevented age-associated blood pressure elevation and reduced vascular/renal superoxide production in male mice. CONCLUSIONS: The prostaglandin D2/DP1 axis suppresses age-related Th1 activation and subsequent hypertensive response in male mice through increase of NEDD4L-mediated T-bet degradation by ubiquitination. Therefore, the T cell DP1 receptor may be an attractive therapeutic target for age-related hypertension.

Tuft Cells Inhibit Pancreatic Tumorigenesis in Mice by Producing Prostaglandin D2.

BACKGROUND & AIMS: Development of pancreatic ductal adenocarcinoma (PDA) involves acinar to ductal metaplasia and genesis of tuft cells. It has been a challenge to study these rare cells because of the lack of animal models. We investigated the role of tuft cells in pancreatic tumorigenesis. METHODS: We performed studies with LSL-KrasG12D/+;Ptf1aCre/+ mice (KC; develop pancreatic tumors), KC mice crossed with mice with pancreatic disruption of Pou2f3 (KPouC mice; do not develop tuft cells), or mice with pancreatic disruption of the hematopoietic prostaglandin D synthase gene (Hpgds, KHC mice) and wild-type mice. Mice were allowed to age or were given caerulein to induce pancreatitis; pancreata were collected and analyzed by histology, immunohistochemistry, RNA sequencing, ultrastructural microscopy, and metabolic profiling. We performed laser-capture dissection and RNA-sequencing analysis of pancreatic tissues from 26 patients with pancreatic intraepithelial neoplasia (PanIN), 19 patients with intraductal papillary mucinous neoplasms (IPMNs), and 197 patients with PDA. RESULTS: Pancreata from KC mice had increased formation of tuft cells and higher levels of prostaglandin D2 than wild-type mice. Pancreas-specific deletion of POU2F3 in KC mice (KPouC mice) resulted in a loss of tuft cells and accelerated tumorigenesis. KPouC mice had increased fibrosis and activation of immune cells after administration of caerulein. Pancreata from KPouC and KHC mice had significantly lower levels of prostaglandin D2, compared with KC mice, and significantly increased numbers of PanINs and PDAs. KPouC and KHC mice had increased pancreatic injury after administration of caerulein, significantly less normal tissue, more extracellular matrix deposition, and higher PanIN grade than KC mice. Human PanIN and intraductal papillary mucinous neoplasm had gene expression signatures associated with tuft cells and increased expression of Hpgds messenger RNA compared with PDA. CONCLUSIONS: In mice with KRAS-induced pancreatic tumorigenesis, loss of tuft cells accelerates tumorigenesis and increases the severity of caerulein-induced pancreatic injury, via decreased production of prostaglandin D2. These data are consistent with the hypothesis that tuft cells are a metaplasia-induced tumor attenuating cell type.