Prolonged sleep deprivation induces a cytokine-storm-like syndrome in mammals.

Most animals require sleep, and sleep loss induces serious pathophysiological consequences, including death. Previous experimental approaches for investigating sleep impacts in mice have been unable to persistently deprive animals of both rapid eye movement sleep (REMS) and non-rapid eye movement sleep (NREMS). Here, we report a "curling prevention by water" paradigm wherein mice remain awake 96% of the time. After 4 days of exposure, mice exhibit severe inflammation, and approximately 80% die. Sleep deprivation increases levels of prostaglandin D2 (PGD2) in the brain, and we found that elevated PGD2 efflux across the blood-brain-barrier-mediated by ATP-binding cassette subfamily C4 transporter-induces both accumulation of circulating neutrophils and a cytokine-storm-like syndrome. Experimental disruption of the PGD2/DP1 axis dramatically reduced sleep-deprivation-induced inflammation. Thus, our study reveals that sleep-related changes in PGD2 in the central nervous system drive profound pathological consequences in the peripheral immune system.

Parallel neural pathways control sodium consumption and taste valence.

The hedonic value of salt fundamentally changes depending on the internal state. High concentrations of salt induce innate aversion under sated states, whereas such aversive stimuli transform into appetitive ones under sodium depletion. Neural mechanisms underlying this state-dependent salt valence switch are poorly understood. Using transcriptomics state-to-cell-type mapping and neural manipulations, we show that positive and negative valences of salt are controlled by anatomically distinct neural circuits in the mammalian brain. The hindbrain interoceptive circuit regulates sodium-specific appetitive drive , whereas behavioral tolerance of aversive salts is encoded by a dedicated class of neurons in the forebrain lamina terminalis (LT) expressing prostaglandin E2 (PGE2) receptor, Ptger3. We show that these LT neurons regulate salt tolerance by selectively modulating aversive taste sensitivity, partly through a PGE2-Ptger3 axis. These results reveal the bimodal regulation of appetitive and tolerance signals toward salt, which together dictate the amount of sodium consumption under different internal states.

Mast cells control lung type 2 inflammation via prostaglandin E2-driven soluble ST2.

Severe asthma and sinus disease are consequences of type 2 inflammation (T2I), mediated by interleukin (IL)-33 signaling through its membrane-bound receptor, ST2. Soluble (s)ST2 reduces available IL-33 and limits T2I, but little is known about its regulation. We demonstrate that prostaglandin E2 (PGE2) drives production of sST2 to limit features of lung T2I. PGE2-deficient mice display diminished sST2. In humans with severe respiratory T2I, urinary PGE2 metabolites correlate with serum sST2. In mice, PGE2 enhanced sST2 secretion by mast cells (MCs). Mice lacking MCs, ST2 expression by MCs, or E prostanoid (EP)2 receptors by MCs showed reduced sST2 lung concentrations and strong T2I. Recombinant sST2 reduced T2I in mice lacking PGE2 or ST2 expression by MCs back to control levels. PGE2 deficiency also reversed the hyperinflammatory phenotype in mice lacking ST2 expression by MCs. PGE2 thus suppresses T2I through MC-derived sST2, explaining the severe T2I observed in low PGE2 states.

Tumor-derived prostaglandin E2 programs cDC1 dysfunction to impair intratumoral orchestration of anti-cancer T cell responses.

Type 1 conventional dendritic cells (cDC1s) are critical for anti-cancer immunity. Protective anti-cancer immunity is thought to require cDC1s to sustain T cell responses within tumors, but it is poorly understood how this function is regulated and whether its subversion contributes to immune evasion. Here, we show that tumor-derived prostaglandin E2 (PGE2) programmed a dysfunctional state in intratumoral cDC1s, disabling their ability to locally orchestrate anti-cancer CD8+ T cell responses. Mechanistically, cAMP signaling downstream of the PGE2-receptors EP2 and EP4 was responsible for the programming of cDC1 dysfunction, which depended on the loss of the transcription factor IRF8. Blockade of the PGE2-EP2/EP4-cDC1 axis prevented cDC1 dysfunction in tumors, locally reinvigorated anti-cancer CD8+ T cell responses, and achieved cancer immune control. In human cDC1s, PGE2-induced dysfunction is conserved and associated with poor cancer patient prognosis. Our findings reveal a cDC1-dependent intratumoral checkpoint for anti-cancer immunity that is targeted by PGE2 for immune evasion.

Microglial activation elicits a negative affective state through prostaglandin-mediated modulation of striatal neurons.

Microglia are activated in many neurological diseases and have been suggested to play an important role in the development of affective disorders including major depression. To investigate how microglial signaling regulates mood, we used bidirectional chemogenetic manipulations of microglial activity in mice. Activation of microglia in the dorsal striatum induced local cytokine expression and a negative affective state characterized by anhedonia and aversion, whereas inactivation of microglia blocked aversion induced by systemic inflammation. Interleukin-6 signaling and cyclooxygenase-1 mediated prostaglandin synthesis in the microglia were critical for the inflammation-induced aversion. Correspondingly, microglial activation led to a prostaglandin-dependent reduction of the excitability of striatal neurons. These findings demonstrate a mechanism by which microglial activation causes negative affect through prostaglandin-dependent modulation of striatal neurons and indicate that interference with this mechanism could milden the depressive symptoms in somatic and psychiatric diseases involving microglial activation.

Antagonistic Inflammatory Phenotypes Dictate Tumor Fate and Response to Immune Checkpoint Blockade.

Inflammation can support or restrain cancer progression and the response to therapy. Here, we searched for primary regulators of cancer-inhibitory inflammation through deep profiling of inflammatory tumor microenvironments (TMEs) linked to immune-dependent control in mice. We found that early intratumoral accumulation of interferon gamma (IFN-γ)-producing natural killer (NK) cells induced a profound remodeling of the TME and unleashed cytotoxic T cell (CTL)-mediated tumor eradication. Mechanistically, tumor-derived prostaglandin E2 (PGE2) acted selectively on EP2 and EP4 receptors on NK cells, hampered the TME switch, and enabled immune evasion. Analysis of patient datasets across human cancers revealed distinct inflammatory TME phenotypes resembling those associated with cancer immune control versus escape in mice. This allowed us to generate a gene-expression signature that integrated opposing inflammatory factors and predicted patient survival and response to immune checkpoint blockade. Our findings identify features of the tumor inflammatory milieu associated with immune control of cancer and establish a strategy to predict immunotherapy outcomes.

TRPV4 Channel Signaling in Macrophages Promotes Gastrointestinal Motility via Direct Effects on Smooth Muscle Cells.

Intestinal macrophages are critical for gastrointestinal (GI) homeostasis, but our understanding of their role in regulating intestinal motility is incomplete. Here, we report that CX3C chemokine receptor 1-expressing muscularis macrophages (MMs) were required to maintain normal GI motility. MMs expressed the transient receptor potential vanilloid 4 (TRPV4) channel, which senses thermal, mechanical, and chemical cues. Selective pharmacologic inhibition of TRPV4 or conditional deletion of TRPV4 from macrophages decreased intestinal motility and was sufficient to reverse the GI hypermotility that is associated with chemotherapy treatment. Mechanistically, stimulation of MMs via TRPV4 promoted the release of prostaglandin E2 and elicited colon contraction in a paracrine manner via prostaglandin E receptor signaling in intestinal smooth muscle cells without input from the enteric nervous system. Collectively, our data identify TRPV4-expressing MMs as an essential component required for maintaining normal GI motility and provide potential drug targets for GI motility disorders.

Mitochondrial Membrane Potential Regulates Nuclear Gene Expression in Macrophages Exposed to Prostaglandin E2.

Metabolic engagement is intrinsic to immune cell function. Prostaglandin E2 (PGE2) has been shown to modulate macrophage activation, yet how PGE2 might affect metabolism is unclear. Here, we show that PGE2 caused mitochondrial membrane potential (Δψm) to dissipate in interleukin-4-activated (M(IL-4)) macrophages. Effects on Δψm were a consequence of PGE2-initiated transcriptional regulation of genes, particularly Got1, in the malate-aspartate shuttle (MAS). Reduced Δψm caused alterations in the expression of 126 voltage-regulated genes (VRGs), including those encoding resistin-like molecule α (RELMα), a key marker of M(IL-4) cells, and genes that regulate the cell cycle. The transcription factor ETS variant 1 (ETV1) played a role in the regulation of 38% of the VRGs. These results reveal ETV1 as a Δψm-sensitive transcription factor and Δψm as a mediator of mitochondrial-directed nuclear gene expression.

An Aspirin a Day: New Pharmacological Developments and Cancer Chemoprevention.

Chemoprevention refers to the use of natural or synthetic agents to reverse, suppress, or prevent the progression or recurrence of cancer. A large body of preclinical and clinical data suggest the ability of aspirin to prevent precursor lesions and cancers, but much of the clinical data are inferential and based on descriptive epidemiology, case control, and cohort studies or studies designed to answer other questions (e.g., cardiovascular mortality). Multiple pharmacological, clinical, and epidemiologic studies suggest that aspirin can prevent certain cancers but may also cause other effects depending on the tissue or disease and organ site in question. The best-known biological targets of aspirin are cyclooxygenases, which drive a wide variety of functions, including hemostasis, inflammation, and immune modulation. Newly recognized molecular and cellular interactions suggest additional modifiable functional targets, and the existence of consensus molecular cancer subtypes suggests that aspirin may have differential effects based on tumor heterogeneity. This review focuses on new pharmacological developments and innovations in biopharmacology that clarify the potential role of aspirin in cancer chemoprevention.

Adipose triglyceride lipase promotes prostaglandin-dependent actin remodeling by regulating substrate release from lipid droplets.

Lipid droplets (LDs), crucial regulators of lipid metabolism, accumulate during oocyte development. However, their roles in fertility remain largely unknown. During Drosophila oogenesis, LD accumulation coincides with the actin remodeling necessary for follicle development. Loss of the LD-associated Adipose Triglyceride Lipase (ATGL) disrupts both actin bundle formation and cortical actin integrity, an unusual phenotype also seen when the prostaglandin (PG) synthase Pxt is missing. Dominant genetic interactions and PG treatment of follicles indicate that ATGL acts upstream of Pxt to regulate actin remodeling. Our data suggest that ATGL releases arachidonic acid (AA) from LDs to serve as the substrate for PG synthesis. Lipidomic analysis detects AA-containing triglycerides in ovaries, and these are increased when ATGL is lost. High levels of exogenous AA block follicle development; this is enhanced by impairing LD formation and suppressed by reducing ATGL. Together, these data support the model that AA stored in LD triglycerides is released by ATGL to drive the production of PGs, which promote the actin remodeling necessary for follicle development. We speculate that this pathway is conserved across organisms to regulate oocyte development and promote fertility.

Esrrγa regulates nephron and ciliary development by controlling prostaglandin synthesis.

Cilia are essential for the ontogeny and function of many tissues, including the kidney. Here, we report that transcription factor ERRγ ortholog estrogen related receptor gamma a (Esrrγa) is essential for renal cell fate choice and ciliogenesis in zebrafish. esrrγa deficiency altered proximodistal nephron patterning, decreased the multiciliated cell populace and disrupted ciliogenesis in the nephron, Kupffer's vesicle and otic vesicle. These phenotypes were consistent with interruptions in prostaglandin signaling, and we found that ciliogenesis was rescued by PGE2 or the cyclooxygenase enzyme Ptgs1. Genetic interaction revealed that peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (Ppargc1a), which acts upstream of Ptgs1-mediated prostaglandin synthesis, has a synergistic relationship with Esrrγa in the ciliogenic pathway. These ciliopathic phenotypes were also observed in mice lacking renal epithelial cell (REC) ERRγ, where significantly shorter cilia formed on proximal and distal tubule cells. Decreased cilia length preceded cyst formation in REC-ERRγ knockout mice, suggesting that ciliary changes occur early during pathogenesis. These data position Esrrγa as a novel link between ciliogenesis and nephrogenesis through regulation of prostaglandin signaling and cooperation with Ppargc1a.

Structures of the Human PGD2 Receptor CRTH2 Reveal Novel Mechanisms for Ligand Recognition.

The signaling of prostaglandin D2 (PGD2) through G-protein-coupled receptor (GPCR) CRTH2 is a major pathway in type 2 inflammation. Compelling evidence suggests the therapeutic benefits of blocking CRTH2 signaling in many inflammatory disorders. Currently, a number of CRTH2 antagonists are under clinical investigation, and one compound, fevipiprant, has advanced to phase 3 clinical trials for asthma. Here, we present the crystal structures of human CRTH2 with two antagonists, fevipiprant and CAY10471. The structures, together with docking and ligand-binding data, reveal a semi-occluded pocket covered by a well-structured amino terminus and different binding modes of chemically diverse CRTH2 antagonists. Structural analysis suggests a ligand entry port and a binding process that is facilitated by opposite charge attraction for PGD2, which differs significantly from the binding pose and binding environment of lysophospholipids and endocannabinoids, revealing a new mechanism for lipid recognition by GPCRs.

Prostaglandin F2α drives female pheromone signaling in cichlids, revealing a basis for evolutionary divergence in olfactory signaling.

Pheromones play essential roles in reproduction in many species. Prostaglandin F2α (PGF2α) acts as a female reproductive hormone and as a sex pheromone in some species. An olfactory receptor (OR) for PGF2α was recently discovered in zebrafish, but this signaling pathway is evolutionarily labile. To understand the evolution of signals that attract males to fertile females, we used the African cichlid Astatotilapia burtoni and found that adult males strongly prefer fertile female odors. Injection of a prostaglandin synthesis inhibitor abolishes this attractivity of fertile females, indicating these hormones are necessary for pheromonal signaling. Unlike zebrafish, A. burtoni males are insensitive to PGF2α, but they do exhibit strong preference for females injected with PGF2α. This attractiveness is independent of the PGF2α hormonal receptor Ptgfr, indicating that this pheromone signaling derives from PGF2α metabolization into a yet-undiscovered pheromone. We further discovered that fish that are insensitive to PGF2α lack an ortholog for the OR Or114 that zebrafish use to detect PGF2α. These results indicate that PGF2α itself does not directly induce male preference in cichlids. Rather, it plays a vital role that primes females to become attractive via an alternative male OR.

PGE2 alters chromatin through H2A.Z-variant enhancer nucleosome modification to promote hematopoietic stem cell fate.

Prostaglandin E2 (PGE2) and 16,16-dimethyl-PGE2 (dmPGE2) are important regulators of hematopoietic stem and progenitor cell (HSPC) fate and offer potential to enhance stem cell therapies [C. Cutler et al. Blood 122, 3074-3081(2013); W. Goessling et al. Cell Stem Cell 8, 445-458 (2011); W. Goessling et al. Cell 136, 1136-1147 (2009)]. Here, we report that PGE2-induced changes in chromatin at enhancer regions through histone-variant H2A.Z permit acute inflammatory gene induction to promote HSPC fate. We found that dmPGE2-inducible enhancers retain MNase-accessible, H2A.Z-variant nucleosomes permissive of CREB transcription factor (TF) binding. CREB binding to enhancer nucleosomes following dmPGE2 stimulation is concomitant with deposition of histone acetyltransferases p300 and Tip60 on chromatin. Subsequent H2A.Z acetylation improves chromatin accessibility at stimuli-responsive enhancers. Our findings support a model where histone-variant nucleosomes retained within inducible enhancers facilitate TF binding. Histone-variant acetylation by TF-associated nucleosome remodelers creates the accessible nucleosome landscape required for immediate enhancer activation and gene induction. Our work provides a mechanism through which inflammatory mediators, such as dmPGE2, lead to acute transcriptional changes and modify HSPC behavior to improve stem cell transplantation.

Single hormone or synthetic agonist induces Gs/Gi coupling selectivity of EP receptors via distinct binding modes and propagating paths.

To accomplish concerted physiological reactions, nature has diversified functions of a single hormone at at least two primary levels: 1) Different receptors recognize the same hormone, and 2) different cellular effectors couple to the same hormone-receptor pair [R.P. Xiao, Sci STKE 2001, re15 (2001); L. Hein, J. D. Altman, B.K. Kobilka, Nature 402, 181-184 (1999); Y. Daaka, L. M. Luttrell, R. J. Lefkowitz, Nature 390, 88-91 (1997)]. Not only these questions lie in the heart of hormone actions and receptor signaling but also dissecting mechanisms underlying these questions could offer therapeutic routes for refractory diseases, such as kidney injury (KI) or X-linked nephrogenic diabetes insipidus (NDI). Here, we identified that Gs-biased signaling, but not Gi activation downstream of EP4, showed beneficial effects for both KI and NDI treatments. Notably, by solving Cryo-electron microscope (cryo-EM) structures of EP3-Gi, EP4-Gs, and EP4-Gi in complex with endogenous prostaglandin E2 (PGE2)or two synthetic agonists and comparing with PGE2-EP2-Gs structures, we found that unique primary sequences of prostaglandin E2 receptor (EP) receptors and distinct conformational states of the EP4 ligand pocket govern the Gs/Gi transducer coupling selectivity through different structural propagation paths, especially via TM6 and TM7, to generate selective cytoplasmic structural features. In particular, the orientation of the PGE2 ω-chain and two distinct pockets encompassing agonist L902688 of EP4 were differentiated by their Gs/Gi coupling ability. Further, we identified common and distinct features of cytoplasmic side of EP receptors for Gs/Gi coupling and provide a structural basis for selective and biased agonist design of EP4 with therapeutic potential.

Prostaglandin E2 activates melanin-concentrating hormone neurons to drive diet-induced obesity.

Hypothalamic inflammation reduces appetite and body weight during inflammatory diseases, while promoting weight gain when induced by high-fat diet (HFD). How hypothalamic inflammation can induce opposite energy balance outcomes remains unclear. We found that prostaglandin E2 (PGE2), a key hypothalamic inflammatory mediator of sickness, also mediates diet-induced obesity (DIO) by activating appetite-promoting melanin-concentrating hormone (MCH) neurons in the hypothalamus in rats and mice. The effect of PGE2 on MCH neurons is excitatory at low concentrations while inhibitory at high concentrations, indicating that these neurons can bidirectionally respond to varying levels of inflammation. During prolonged HFD, endogenous PGE2 depolarizes MCH neurons through an EP2 receptor-mediated inhibition of the electrogenic Na+/K+-ATPase. Disrupting this mechanism by genetic deletion of EP2 receptors on MCH neurons is protective against DIO and liver steatosis in male and female mice. Thus, an inflammatory mediator can directly stimulate appetite-promoting neurons to exacerbate DIO and fatty liver.

Reactive oxygen species are regulated by immune deficiency and Toll pathways in determining the host specificity of honeybee gut bacteria.

Host specificity is observed in gut symbionts of diverse animal lineages. But how hosts maintain symbionts while rejecting their close relatives remains elusive. We use eusocial bees and their codiversified gut bacteria to understand host regulation driving symbiotic specificity. The cross-inoculation of bumblebee Gilliamella induced higher prostaglandin in the honeybee gut, promoting a pronounced host response through immune deficiency (IMD) and Toll pathways. Gene silencing and vitamin C treatments indicate that reactive oxygen species (ROS), not antimicrobial peptides, acts as the effector in inhibiting the non-native strain. Quantitative PCR and RNAi further reveal a regulatory function of the IMD and Toll pathways, in which Relish and dorsal-1 may regulate Dual Oxidase (Duox) for ROS production. Therefore, the honeybee maintains symbiotic specificity by creating a hostile gut environment to exotic bacteria, through differential regulation of its immune system, reflecting a co-opting of existing machinery evolved to combat pathogens.

Prostaglandin E2 impacts multiple stages of the natural killer cell antitumor immune response.

Tumor immune escape is a major factor contributing to cancer progression and unresponsiveness to cancer therapies. Tumors can produce prostaglandin E2 (PGE2 ), an inflammatory mediator that directly acts on Natural killer (NK) cells to inhibit antitumor immunity. However, precisely how PGE2 influences NK cell tumor-restraining functions remains unclear. Here, we report that following PGE2 treatment, human NK cells exhibited altered expression of specific activating receptors and a reduced ability to degranulate and kill cancer targets. Transcriptional analysis uncovered that PGE2 also differentially modulated the expression of chemokine receptors by NK cells, inhibiting CXCR3 but increasing CXCR4. Consistent with this, PGE2-treated NK cells exhibited decreased migration to CXCL10 but increased ability to migrate toward CXCL12. Using live cell imaging, we showed that in the presence of PGE2 , NK cells were slower and less likely to kill cancer target cells following conjugation. Imaging the sequential stages of NK cell killing revealed that PGE2 impaired NK cell polarization, but not the re-organization of synaptic actin or the release of perforin itself. Together, these findings demonstrate that PGE2 affects multiple but select NK cell functions. Understanding how cancer cells subvert NK cells is necessary to more effectively harness the cancer-inhibitory function of NK cells in treatments.

PGE2-EP4 signaling steers cDC2 maturation toward the induction of suppressive T-cell responses.

Dendritic cells (DCs) shape adaptive immunity in response to environmental cues such as cytokines or lipid mediators, including prostaglandin E2 (PGE2). In cancer, tumors are known to establish an enriched PGE2 microenvironment. Tumor-derived PGE2 primes regulatory features across immune cells, including DCs, facilitating tumor progression. PGE2 shapes DC function by providing signaling via its two so-called E-prostanoid receptors (EPs) EP2 and EP4. Although studies with monocyte-derived DCs have shown the importance of PGE2 signaling, the role of PGE2-EP2/EP4 on conventional DCs type 2 (cDC2s), is still poorly defined. In this study, we investigated the function of EP2 and EP4 using specific EP antagonists on human cDC2s. Our results show that EP2 and EP4 exhibit different functions in cDC2s, with EP4 modulating the upregulation of activation markers (CD80, CD86, CD83, MHC class II) and the production of IL-10 and IL-23. Furthermore, PGE2-EP4 boosts CCR type 7-based migration as well as a higher T-cell expansion capacity, characterized by the enrichment of suppressive rather than pro-inflammatory T-cell populations. Our findings are relevant to further understanding the role of EP receptors in cDC2s, underscoring the benefit of targeting the PGE2-EP2/4 axis for therapeutic purposes in diseases such as cancer.

Unraveling the role of lipid droplets and perilipin 2 in bovine luteal cells.

Steroidogenic tissues contain cytosolic lipid droplets that are important for steroidogenesis. Perilipin 2 (PLIN2), a structural coat protein located on the surface of lipid droplets in mammalian cells, plays a crucial role in regulating lipid droplet formation and contributing to various cellular processes such as lipid storage and energy homeostasis. Herein, we examine the role that PLIN2 plays in regulating progesterone synthesis in the bovine corpus luteum. Utilizing gene array databases and Western blotting, we have delineated the expression pattern of PLIN2 throughout the follicular to luteal transition. Our findings reveal the presence of PLIN2 in both ovarian follicular and steroidogenic luteal cells, demonstrating an increase in its levels as follicular cells transition into the luteal phase. Moreover, the depletion of PLIN2 via siRNA enhanced progesterone production in small luteal cells, whereas adenovirus-mediated overexpression of both PLIN2 and Perilipin 3 (PLIN3) induced an increase in cytosolic lipid droplet accumulation and decreased hormone-induced progesterone synthesis in these cells. Lastly, in vivo administration of the luteolytic hormone prostaglandin F2α resulted in an upregulation of PLIN2 mRNA and protein expression, accompanied by a decline in serum progesterone. Our findings highlight the pivotal role of PLIN2 in regulating progesterone synthesis in the bovine corpus luteum, as supported by its dynamic expression pattern during the follicular to luteal transition and its responsiveness to luteotropic and luteolytic hormones. We suggest PLIN2 as a potential therapeutic target for modulating luteal function.