G-Protein Coupled Receptor 30 (GPR30): A Novel Regulator of Endothelial Inflammation

Estrogen, the female sex hormone, is known to exert anti-inflammatory and anti-atherogenic effects. Traditionally, estrogen effects were believed to be largely mediated through the classical estrogen receptors (ERs). However, there is increasing evidence that G-protein coupled receptor 30 (GPR30), a novel estrogen receptor, can mediate many estrogenic effects on the vasculature. Despite this, the localization and functional significance of GPR30 in the human vascular endothelium remains poorly understood. Given this background, we examined the subcellular location and potential anti-inflammatory roles of GPR30 using human umbilical vein endothelial cells as a model system. Inflammatory changes were induced by treatment with tumor necrosis factor (TNF), a pro-inflammatory cytokine involved in atherogenesis and many other inflammatory conditions. We found that GPR30 was located predominantly in the endothelial cell nuclei. Treatment with the selective GPR30 agonist G-1 partially attenuated the TNF induced upregulation of pro-inflammatory proteins such as intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). This effect was completely abolished by the selective GPR30 antagonist G-15, suggesting that it was indeed mediated in a GPR30 dependent manner. Interestingly, estrogen alone had no effects on TNF-treated endothelium. Concomitant activation of the classical ERs blocked the anti-inflammatory effects of G-1, indicating opposing effects of GPR30 and the classical ERs. Our findings demonstrate that endothelial GPR30 is a novel regulator of the inflammatory response which could be a potential therapeutic target against atherosclerosis and other inflammatory diseases.     

DHA/AA alleviates LPS-induced Kupffer cells pyroptosis via GPR120 interaction with NLRP3 to inhibit inflammasome complexes assembly

Pyroptosis is a novel type of programmed cell death associated with the pathogenesis of many inflammatory diseases. Docosahexaenoic acid (DHA) and Arachidonic acid (AA) is widely involved in inflammatory pathological processes. However, the effect and mechanism of DHA and AA on pyroptosis in Kupffer cells are poorly understood. The present study demonstrated that DHA and AA ameliorated lipopolysaccharide (LPS)-induced Kupffer cells pyroptosis by reversing the increased expression of NLRP3 inflammasome complex, GSDMD, IL-1β, IL-18, and PI-stained positive rate. Next, the study revealed that GPR120 silencing eliminated the anti-pyroptosis of DHA and AA in LPS-induced Kupffer cells, suggesting that DHA and AA exerted their effect through GPR120 signaling. Importantly, GPR120 endocytose and binds to NLRP3 under LPS stimulation. Furthermore, co-immunoprecipitation showed that DHA and AA promoted the interaction between GPR120 and NLRP3 in LPS-exposed Kupffer cells, thus inhibiting the self-assembly of NLRP3 inflammasome complex. Finally, the study verified that DHA and AA alleviated hepatic injury through inhibiting Kupffer cells pyroptosis in vivo. The findings indicated that DHA and AA alleviated LPS-induced Kupffer cells pyroptosis via GPR120 interaction with NLRP3, it might become a potential therapeutic approach hepatic injury.Subject terms: Cell death, Immunology

DHA/AA alleviated LPS-induced Kupffer cells pyroptosis via GPR120 interaction with NLRP3 to inhibit NLRP3 inflammasome assembly.     

Involvement of adenosine A2A receptors in engulfment-dependent apoptotic cell suppression of inflammation

Efficient execution of apoptotic cell death followed by efficient clearance mediated by professional macrophages is a key mechanism in maintaining tissue homeostasis. Removal of apoptotic cells usually involves three central elements: 1) attraction of phagocytes via soluble "find me" signals, 2) recognition and phagocytosis via cell surface-presenting "eat me" signals, and 3) suppression or initiation of inflammatory responses depending on additional innate immune stimuli. Suppression of inflammation involves both direct inhibition of proinflammatory cytokine production and release of anti-inflammatory factors, which all contribute to the resolution of inflammation. In the current study, using wild-type and adenosine A(2A) receptor (A2AR) null mice, we investigated whether A2ARs, known to mediate anti-inflammatory signals in macrophages, participate in the apoptotic cell-mediated immunosuppression. We found that macrophages engulfing apoptotic cells release adenosine in sufficient amount to trigger A2ARs, and simultaneously increase the expression of A2ARs, as a result of possible activation of liver X receptor and peroxisome proliferators activated receptor δ. In macrophages engulfing apoptotic cells, stimulation of A2ARs suppresses the NO-dependent formation of neutrophil migration factors, such as macrophage inflammatory protein-2, using the adenylate cyclase/protein kinase A pathway. As a result, loss of A2ARs results in elevated chemoattractant secretion. This was evident as pronounced neutrophil migration upon exposure of macrophages to apoptotic cells in an in vivo peritonitis model. Altogether, our data indicate that adenosine is one of the soluble mediators released by macrophages that mediate engulfment-dependent apoptotic cell suppression of inflammation.     

Lipopolysaccharide inhibits GPR120 expression in macrophages via Toll‐like receptor 4 and p38 MAPK activation

Free fatty acid receptor G protein‐coupled receptor 120 (GPR120) is highly expressed in macrophages and was reported to inhibit lipopolysaccharide (LPS)‐stimulated cytokine expression. Under inflammation, macrophages exhibit striking functional changes, but changes in GPR120 expression and signaling are not known. In this study, the effects of LPS treatment on macrophage GPR120 expression and activation were investigated. The results showed that LPS inhibited GPR120 expression in mouse macrophage cell line Ana‐1 cells. Moreover, LPS treatment inhibited GPR120 expression in mouse alveolar macrophages both in vitro and in vivo. The inhibitory effect of LPS on GPR120 expression was blocked by Toll‐like receptor 4 (TLR4) inhibitor TAK242 and p38 mitogen‐activated protein kinase inhibitor LY222820, but not by ERK1/2 inhibitor U0126 and c‐Jun N‐terminal kinase inhibitor SP600125. LPS‐induced inhibition of GPR120 expression was not attenuated by GPR120 agonists TUG891 and GW9508. TUG891 inhibited the phagocytosis of alveolar macrophages, and LPS treatment counteracted the effects of TUG891 on phagocytosis. These results indicate that pretreatment with LPS inhibits GPR120 expression and activation in macrophages. It is suggested that LPS‐induced inhibition of GPR120 expression is a reaction enhancing the LPS‐induced pro‐inflammatory response of macrophages.     

Programmed cell death 1 positive lymphocytes at palate tonsils in the elder patients with chronic tonsillitis

Circulating lymphocytes infiltrate into local foci at the inflammatory phase of acute wound healing for activation of the immune system and express an immune checkpoint protein programmed cell death 1 (PD-1) at the resolution phase for inactivation of the immune system. Conversely, the PD-1 expression was still found even on circulating lymphocytes of the elder patients with chronic tonsillitis at the palliative stage. Recently, an adhesion G protein coupled receptor 56 (GPR56) was reported to at least work as a proliferation factor for infiltrated lymphocytes into local foci at the resolution phase of acute wound healing. To preliminary examine a similar role of PD-1 and GPR56 at local foci at chronic inflammation, palate tonsils were prepared from small amounts of patients with chronic tonsillitis and tonsillar hypertrophy. A positive relationship of RNA expression might be observed between PD-1 and GPR56 in the elder patients with chronic tonsillitis. In regard to immunohistopathological findings, there were huge and small amounts of PD-1 and GPR56 expression at the marginal zone of lymphoid follicles of palate tonsils with chronic tonsillitis. Moreover, the positive relationship of RNA expression between PD-1 and GPR56 confirmed in large numbers of the elder patients with chronic tonsillitis. Probably, GPR56 participates in a supplement of PD-1⁺ lymphocytes to circulating bloods of the elder patients with chronic tonsillitis through a lymphocyte cell maintenance system at the marginal zone of the lymphoid follicles of palate tonsils.     

GPR65 (TDAG8) inhibits intestinal inflammation and colitis-associated colorectal cancer development in experimental mouse models

GPR65 (TDAG8) is a proton-sensing G protein-coupled receptor predominantly expressed in immune cells. Genome-wide association studies (GWAS) have identified GPR65 gene polymorphisms as an emerging risk factor for the development of inflammatory bowel disease (IBD). Patients with IBD have an elevated risk of developing colorectal cancer when compared to the general population. To study the role of GPR65 in intestinal inflammation and colitis-associated colorectal cancer (CAC), colitis and CAC were induced in GPR65 knockout (KO) and wild-type (WT) mice using dextran sulfate sodium (DSS) and azoxymethane (AOM)/DSS, respectively. Disease severity parameters such as fecal score, colon shortening, histopathology, and mesenteric lymph node enlargement were aggravated in GPR65 KO mice compared to WT mice treated with DSS. Elevated leukocyte infiltration and fibrosis were observed in the inflamed colon of GPR65 KO when compared to WT mice which may represent a cellular mechanism for the observed exacerbation of intestinal inflammation. In line with high expression of GPR65 in infiltrated leukocytes, GPR65 gene expression was increased in inflamed intestinal tissue samples of IBD patients compared to normal intestinal tissues. Moreover, colitis-associated colorectal cancer development was higher in GPR65 KO mice than WT mice when treated with AOM/DSS. Altogether, our data demonstrate that GPR65 suppresses intestinal inflammation and colitis-associated tumor development in murine colitis and CAC models, suggesting potentiation of GPR65 with agonists may have an anti-inflammatory therapeutic effect in IBD and reduce the risk of developing colitis-associated colorectal cancer.     

MKP-1 coordinates ordered macrophage-phenotype transitions essential for stem cell-dependent tissue repair

Re-establishing tissue homoeostasis in response to injury requires infiltration of inflammatory cells and activation of resident stem cells. However, full tissue recovery also requires that the inflammation is resolved. While it is known that disturbing the interactions between inflammatory cells and tissue resident cells prevents successful healing, the molecular mechanisms underlying the paracrine interactions between these cell types are practically unknown. Here, and in a recent study, we provide mechanistic evidence that macrophages control stem cell-dependent tissue repair. In particular, we found that the temporal spacing of the pro- to anti-inflammatory macrophage polarization switch is controlled by the balance of p38 MAPK (termed here p38) and the MAPK phosphatase MKP-1 during the muscle healing process. Moreover, we demonstrate a new function for MKP-1-regulated p38 signaling in deactivating macrophages during inflammation resolution after injury. Specifically, at advanced stages of regeneration, MKP-1 loss caused an unscheduled “exhaustion-like” state in muscle macrophages, in which neither pro- nor anti-inflammatory cytokines are expressed despite persistent tissue damage, leading to dysregulated reparation by the tissue stem cells. Mechanistically, we demonstrate that p38 and MKP-1 control the AKT pathway through a miR-21-dependent PTEN regulation. Importantly, both genetic and pharmacological interference with the individual components of this pathway restored inflammation-dependent tissue homeostasis in MKP-1-deficient mice and delayed inflammation resolution and tissue repair dysregulation in wild-type mice. Because the process of tolerance to bacterial infection involves a progressive attenuation of pro-inflammatory gene expression, we discuss here the potential similarities between the mechanisms underlying inflammation resolution during tissue repair and those controlling endotoxin tolerance.     

Crosstalk between ferroptosis and the epithelial-mesenchymal transition: Implications for inflammation and cancer therapy

Both genomic instability and the presence of chronic inflammation are involved in carcinogenesis and tumor progression. These alterations predispose the cancer cells to undergo metabolic reprogramming as well as the epithelial-mesenchymal transition (EMT). These pathways allow cancer cells to avoid apoptosis and stimulate tumor progression. EMT is an important early event in tumor cell invasion, which can be regulated through inflammatory signaling pathways. Cancer cells undergoing EMT are vulnerable to cell death by the process of ferroptosis. Ferroptosis is a form of regulated cell death involving iron-dependent lipid peroxidation, designed to maintain cellular homeostasis. Several reports have linked ferroptosis, inflammation, and cancer. Ferroptosis inhibitors and EMT inducers have been used to understand the anti-inflammatory and anticancer effects in experimental models. A better understanding of the crosstalk between ferroptosis and EMT, and the involvment of inflammatory mediators may accelerate the discovery of therapeutic strategies to eradicate cancer cells and overcome drug-resistance.     

Identification of a key pathway required for the sterile inflammatory response triggered by dying cells

Dying cells stimulate inflammation, and this response is thought to contribute to the pathogenesis of many diseases. Very little has been known, however, about how cell death triggers inflammation. We found here that the acute neutrophilic inflammatory response to cell injury requires the signaling protein myeloid differentiation primary response gene 88 (Myd88). Analysis of the contribution of Myd88-dependent receptors to this response revealed only a minor reduction in mice doubly deficient in Toll-like receptor 2 (Tlr2) and Tlr4 and normal responses in mice lacking Tlr1, Tlr3, Tlr6, Tlr7, Tlr9, Tlr11 or the interleukin-18 receptor (IL-18R). However, mice lacking IL-1R showed a markedly reduced neutrophilic inflammatory response to dead cells and tissue injury in vivo as well as greatly decreased collateral damage from inflammation. This inflammatory response required IL-1alpha, and IL-1R function was required on non-bone-marrow-derived cells. Notably, the acute monocyte response to cell death, which is thought to be important for tissue repair, was much less dependent on the IL-1R-Myd88 pathway. Also, this pathway was not required for the neutrophil response to a microbial stimulus. These findings suggest that inhibiting the IL-1R-Myd88 pathway in vivo could block the damage from acute inflammation that occurs in response to sterile cell death, and do so in a way that might not compromise tissue repair or host defense against pathogens.     

Functional GPR37 trafficking protects against toxicity induced by 6‐OHDA,MPP+ or rotenone in a catecholaminergic cell line

G protein‐coupled receptor 37 (GPR37) is suggested to be implicated in the pathogenesis of Parkinson's disease and is accumulating in Lewy bodies within afflicted brain regions. Over‐expressed GPR37 is prone to misfolding and aggregation, causing cell death via endoplasmic reticulum stress. Although the cytotoxicity of misfolded GPR37 is well established, effects of the functional receptor on cell viability are still unknown. An N2a cell line stably expressing green fluorescent protein (GFP)‐tagged human GPR37 was created to study its trafficking and effects on cell viability upon challenge with the toxins 1‐methyl‐4‐phenylpyridinium (MPP+), rotenone and 6‐hydroxydopamine (6‐OHDA). Neuronal‐like differentiation into a tyrosine hydroxylase expressing phenotype, using dibutyryl‐cAMP, induced trafficking of GPR37 to the plasma membrane. 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) cell viability and lactate dehydrogenase (LDH) cell death assays revealed that GPR37 was protective against all three toxins in differentiated cells. In undifferentiated cells, the majority of GPR37 was cytoplasmic and the protective effects were more variable: GPR37 expression protected against rotenone and MPP+ but not against 6‐OHDA in MTT assays, while it protected against 6‐OHDA but not against MPP+ or rotenone in lactate dehydrogenase (LDH) assays. These results suggest that GPR37 functionally trafficked to the plasma membrane protects against toxicity.     

Epithelial cells as phagocytes: apoptotic epithelial cells are engulfed by mammary alveolar epithelial cells and repress inflammatory mediator release

Clearance of apoptotic cells is critical to tissue homeostasis and resolution of inflammatory lesions. Macrophages are known to remove dying cells and release anti-inflammatory mediators in response; however, many cells traditionally thought of as poor phagocytes can mediate this function as well. In the lactating mammary gland following weaning, alveolar epithelial cell death is massive, yet the gland involutes rapidly, attaining its prepregnancy state in a matter of days. We found histologic evidence of apoptotic cell phagocytosis by viable mammary epithelial cells (MEC) in the involuting mouse mammary gland. Cultured MEC were able to engulf apoptotic cells in vitro, utilizing many of the same receptors used by macrophages, including the phosphatidylserine receptor (PSR), CD36, the vitronectin receptor alpha(v)beta3, and CD91. In addition, MEC, like macrophages, produced TGFbeta in response to stimulation of the PSR by apoptotic cells or the anti-PSR ab 217G8E9, and downregulated endotoxin-stimulated proinflammatory cytokine production. These data support the hypothesis that amateur phagocytes play a significant role in apoptotic cell clearance and its regulation of inflammation.     

Acidosis Activation of the Proton-Sensing GPR4 Receptor Stimulates Vascular Endothelial Cell Inflammatory Responses Revealed by Transcriptome Analysis

Acidic tissue microenvironment commonly exists in inflammatory diseases, tumors, ischemic organs, sickle cell disease, and many other pathological conditions due to hypoxia, glycolytic cell metabolism and deficient blood perfusion. However, the molecular mechanisms by which cells sense and respond to the acidic microenvironment are not well understood. GPR4 is a proton-sensing receptor expressed in endothelial cells and other cell types. The receptor is fully activated by acidic extracellular pH but exhibits lesser activity at the physiological pH 7.4 and minimal activity at more alkaline pH. To delineate the function and signaling pathways of GPR4 activation by acidosis in endothelial cells, we compared the global gene expression of the acidosis response in primary human umbilical vein endothelial cells (HUVEC) with varying level of GPR4. The results demonstrated that acidosis activation of GPR4 in HUVEC substantially increased the expression of a number of inflammatory genes such as chemokines, cytokines, adhesion molecules, NF-κB pathway genes, and prostaglandin-endoperoxidase synthase 2 (PTGS2 or COX-2) and stress response genes such as ATF3 and DDIT3 (CHOP). Similar GPR4-mediated acidosis induction of the inflammatory genes was also noted in other types of endothelial cells including human lung microvascular endothelial cells and pulmonary artery endothelial cells. Further analyses indicated that the NF-κB pathway was important for the acidosis/GPR4-induced inflammatory gene expression. Moreover, acidosis activation of GPR4 increased the adhesion of HUVEC to U937 monocytic cells under a flow condition. Importantly, treatment with a recently identified GPR4 antagonist significantly reduced the acidosis/GPR4-mediated endothelial cell inflammatory response. Taken together, these results show that activation of GPR4 by acidosis stimulates the expression of a wide range of inflammatory genes in endothelial cells. Such inflammatory response can be suppressed by GPR4 small molecule inhibitors and hold potential therapeutic value.     

Quantitative expression profiling of G-protein-coupled receptors (GPCRs) in metastatic melanoma: the constitutively active orphan GPCR GPR18 as novel drug target

G-protein-coupled receptors (GPCRs) have been implicated in the tumorigenesis and metastasis of human cancers and are considered amongst the most desirable targets for drug development. Utilizing a robust quantitative PCR array, we quantified expression of 94 human GPCRs, including 75 orphan GPCRs and 19 chemokine receptors, and 36 chemokine ligands, in 40 melanoma metastases from different individuals and benign nevi. Inter-metastatic site comparison revealed that orphan GPR174 and CCL28 are statistically significantly overexpressed in subcutaneous metastases, while P2RY5 is overexpressed in brain metastases. Comparison between metastases (all three metastatic sites) and benign nevi revealed that 16 genes, including six orphan receptors (GPR18, GPR34, GPR119, GPR160, GPR183 and P2RY10) and chemokine receptors CCR5, CXCR4, and CXCR6, were statistically significantly differentially expressed. Subsequent functional experiments in yeast and melanoma cells indicate that GPR18, the most abundantly overexpressed orphan GPCR in all melanoma metastases, is constitutively active and inhibits apoptosis, indicating an important role for GPR18 in tumor cell survival. GPR18 and five other orphan GPCRs with yet unknown biological function may be considered potential novel anticancer targets in metastatic melanoma.     

白细胞介素-6在酒精性肝病中的作用

酒精性肝病(alcoholic liver disease,ALD)是由于长期过量饮酒导致肝的内部组织发生炎症损伤的慢性肝病.乙醇及其衍生物在代谢过程中直接或间接诱导引起的肝炎症反应可能是ALD发病的重要机制.然而,该过程内在的细胞分子机制尚不明确.最新研究发现,白细胞介素-6(interleukin-6,IL-6)对...     

Macrophage Dysfunction Impairs Resolution of Inflammation in the Wounds of Diabetic Mice

Background

Chronic inflammation is a characteristic feature of diabetic cutaneous wounds. We sought to delineate novel mechanisms involved in the impairment of resolution of inflammation in diabetic cutaneous wounds. At the wound-site, efficient dead cell clearance (efferocytosis) is a pre-requisite for the timely resolution of inflammation and successful healing.

Methodology/Principal Findings

Macrophages isolated from wounds of diabetic mice showed significant impairment in efferocytosis. Impaired efferocytosis was associated with significantly higher burden of apoptotic cells in wound tissue as well as higher expression of pro-inflammatory and lower expression of anti-inflammatory cytokines. Observations related to apoptotic cell load at the wound site in mice were validated in the wound tissue of diabetic and non-diabetic patients. Forced Fas ligand driven elevation of apoptotic cell burden at the wound site augmented pro-inflammatory and attenuated anti-inflammatory cytokine response. Furthermore, successful efferocytosis switched wound macrophages from pro-inflammatory to an anti-inflammatory mode.

Conclusions/Significance

Taken together, this study presents first evidence demonstrating that diabetic wounds suffer from dysfunctional macrophage efferocytosis resulting in increased apoptotic cell burden at the wound site. This burden, in turn, prolongs the inflammatory phase and complicates wound healing.     

Phagocytosis of apoptotic cells

Removal of apoptotic cells by phagocytes plays an important role in many biological processes, including embryological development and tissue remodelling. In addition, it has become apparent that one of the key mechanisms for the successful resolution of inflammation is the orchestrated clearance of apoptotic inflammatory cells by phagocytes (e.g., macrophages and dendritic cells) and other cells known to have phagocytic capacity (e.g., hepatocytes, endothelial cells, epithelial cells, etc.). Furthermore, phagocytosis of apoptotic cells is an active and highly regulated process that not only serves to remove potentially histotoxic cells from the inflammatory milieu, but also directs the phenotype of the phagocytic cell to be anti-inflammatory. Convincing evidence has been presented that reduced or dysregulated phagocytosis of apoptotic cells contributes to the development and propagation of inflammatory disorders. Conversely, enhanced phagocytosis of apoptotic cells may be exploited for therapeutic gain. Indeed, powerful anti-inflammatory drugs such as the glucocorticoids have been shown to augment clearance of apoptotic cells which may contribute to their therapeutic effectiveness. In this chapter, we describe methods for studying phagocytosis of apoptotic cells.     

PGE2 Promotes Apoptosis Induced by Cytokine Deprivation through EP3 Receptor and Induces Bim in Mouse Mast Cells

Increased mast cell numbers are observed at sites of allergic inflammation and restoration of normal mast cell numbers is critical to the resolution of these responses. Early studies showed that cytokines protect mast cells from apoptosis, suggesting a simple model in which diminished cytokine levels during resolution leads to cell death. The report that prostaglandins can contribute both to recruitment and to the resolution of inflammation together with the demonstration that mast cells express all four PGE₂ receptors raises the question of whether a single PGE₂ receptor mediates the ability of PGE₂ to regulate mast cell survival and apoptosis. We report here that PGE₂ through the EP3 receptor promotes cell death of mast cells initiated by cytokine withdrawal. Furthermore, the ability of PGE₂ to limit reconstitution of tissues with cultured mast cells is lost in cell lacking the EP3 receptor. Apoptosis is accompanied by higher dissipation of mitochondrial potential (ΔΨ_m), increased caspase-3 activation, chromatin condensation, and low molecular weight DNA cleavage. PGE₂ augmented cell death is dependent on an increase in intracellular calcium release, calmodulin dependent kinase II and MAPK activation. Synergy between the EP3 pathway and the intrinsic mitochondrial apoptotic pathway results in increased Bim expression and higher sensitivity of mast cells to cytokine deprivation. This supports a model in which PGE₂ can contribute to the resolution of inflammation in part by augmenting the removal of inflammatory cells in this case, mast cells.