Platelet-activating Factor Receptor Activation Research Articles

Platelet-Activating Factor Receptor (PAFR) Regulates Retinal Progenitor/Stem Cells Profile in Ciliary Epithelium Cells.

The retina is a central nervous tissue essential to visual perception and highly susceptible to environmental damage. Lower vertebrate retinas activate intrinsic regeneration mechanisms in response to retinal injury regulated by a specialized population of progenitor cells. The mammalian retina does not have populations of progenitor/stem cells available to activate regeneration, but contains a subpopulation of differentiated cells that can be reprogrammed into retinal stem cells, the ciliary epithelium (CE) cells. Despite the regenerative potential, stem cells derived from CE exhibit limited reprogramming capacity probably associated with the expression of intrinsic regulatory mechanisms. Platelet-activating factor (PAF) is a lipid mediator widely expressed in many cells and plays an important role in stem cell proliferation and differentiation. During mammalian development, PAF receptor signaling showed important effects on retinal progenitors' cell cycle regulation and neuronal differentiation that need to be further investigated. In this study, our findings suggested a dynamic role for PAF receptor signaling in CE cells, impacting stem cell characteristics and neurosphere formation. We showed that PAF receptors and PAF-related enzymes are downregulated in retinal progenitor/stem cells derived from PE cells. Blocking PAFR activity using antagonists increased the expression of specific progenitor markers, revealing potential implications for retinal tissue development and maintenance.

Platelet-activating factor and protease-activated receptor 2 cooperate to promote neutrophil recruitment and lung inflammation through nuclear factor-kappa B transactivation

Although it is well established that platelet-activated receptor (PAF) and protease-activated receptor 2 (PAR2) play a pivotal role in the pathophysiology of lung and airway inflammatory diseases, a role for a PAR2-PAFR cooperation in lung inflammation has not been investigated. Here, we investigated the role of PAR2 in PAF-induced lung inflammation and neutrophil recruitment in lungs of BALB/c mice. Mice were pretreated with the PAR2 antagonist ENMD1068, PAF receptor (PAFR) antagonist WEB2086, or aprotinin prior to intranasal instillation of carbamyl-PAF (C-PAF) or the PAR2 agonist peptide SLIGRL-NH2 (PAR2-AP). Leukocyte infiltration in bronchoalveolar lavage fluid (BALF), C-X-C motif ligand 1 (CXCL)1 and CXCL2 chemokines, myeloperoxidase (MPO), and N-acetyl-glycosaminidase (NAG) levels in BALF, or lung inflammation were evaluated. Intracellular calcium signaling, PAFR/PAR2 physical interaction, and the expression of PAR2 and nuclear factor-kappa B (NF-КB, p65) transcription factor were investigated in RAW 264.7 cells stimulated with C-PAF in the presence or absence of ENMD1068. C-PAF- or PAR2-AP-induced neutrophil recruitment into lungs was inhibited in mice pretreated with ENMD1068 and aprotinin or WEB2086, respectively. PAR2 blockade impaired C-PAF-induced neutrophil rolling and adhesion, lung inflammation, and production of MPO, NAG, CXCL1, and CXCL2 production in lungs of mice. PAFR activation reduced PAR2 expression and physical interaction of PAR2 and PAFR; co-activation is required for PAFR/PAR2 physical interaction. PAR2 blockade impaired C-PAF-induced calcium signal and NF-κB p65 translocation in RAW 264.7 murine macrophages. This study provides the first evidence for a cooperation between PAFR and PAR2 mediating neutrophil recruitment, lung inflammation, and macrophage activation.

Platelet-activating factor and microvesicle particles as potential mediators for the toxicity associated with intoxicated thermal burn injury.

Thermal burn injuries (TBIs) in patients who are alcohol-intoxicated result in greater morbidity and mortality. The systemic toxicity found in human patients, which includes both immediate systemic cytokine generation with multiple organ failure and a delayed systemic immunosuppression, has previously been replicated in mouse models combining ethanol and localized TBI. Though considerable insights have been provided with these models, the exact mechanisms for these pathologic effects are unclear. In this review, we highlight the roles of the lipid mediator platelet-activating factor (PAF) and subcellular microvesicle particle (MVP) release in response to intoxicated thermal burn injury (ITBI) as effectors in the pathology. Particularly, MVP is released from keratinocytes in response to PAF receptor (PAFR) activation due to excess PAF produced by ITBI. These subcellular particles carry and thus protect the metabolically labile PAF which enable binding of this potent lipid mediator to several key sites. We hypothesize that PAF carried by MVP can bind to PAFR within the gut, activating myosin light chain kinase (MLCK). The subsequent gut barrier dysfunction in response to MLCK activation then allows bacteria to invade the lymphatic system and, eventually, the bloodstream, resulting in sepsis and resultant dysregulated inflammation in multiple organs. PAF in MVP also activate the skin mast cell PAFR resulting in migration of this key effector cell to the lymph nodes to induce immunosuppression. This review thus provides a mechanism and potential therapeutic approaches for the increased toxicity and immunosuppressive outcomes of TBI in the presence of acute ethanol exposure.

Effects of miRNA-149-5p and Platelet-Activating Factor-Receptor Signaling on the Growth and Targeted Therapy Response on Lung Cancer Cells

Accumulating evidence indicates that microRNAs (miRs) play critical roles in essentially all biological processes and their altered expression has been documented in various disease conditions, including human malignancies. Although several cellular mechanisms have been identified in mediating the effects of miRs, the involvement of G-protein-coupled, platelet-activating factor-receptor (PAFR) signaling in miR-149-5p-induced effects on lung cancer growth and therapeutic potential has not been studied. To that end, we first evaluated the functional significance of PAFR and miR-149-5p in A549 and H1299 human non-small cell lung cancer (NSCLC) cell lines. We observed that these tumor lines express endogenous PAFR and miR-149-5p and that PAFR activation by PAF agonist (CPAF) significantly increased, whereas miR-149-5p mimic transfection inhibited cell proliferation in a dose-dependent manner. Interestingly, miR-149-5p mimic significantly attenuated CPAF-mediated increased proliferation of NSCLC cells, as confirmed by miR-149-5p, cyclin D1, and forkhead box protein M1 (FOXM1) expression analysis via qPCR. Our next studies examined PAFR- and miR-149-5p-mediated effects on targeted therapy (i.e., erlotinib and gefitinib) responses. We observed that erlotinib and gefitinib inhibited A549 and H1299 cell survival in a dose- and time-dependent manner, and CPAF significantly blocked this effect. These findings indicate that miR-149-5p blocks PAFR-mediated increased cell proliferation, and PAFR activation attenuates the cytotoxic effects of targeted therapy.

Cathepsin G-induced malignant progression of MCF-7 cells involves suppression of PAF signaling through induced expression of PAFAH1B2

Breast cancer is primarily classified into ductal and lobular types, as well as into noninvasive and invasive cancer. Invasive cancer involves lymphatic and hematogenous metastasis. In breast cancer patients with distant metastases, a neutrophil-derived serine protease; cathepsin G (Cat G), is highly expressed in breast cancer cells. Cat G induces cell migration and multicellular aggregation of MCF-7 human breast cancer cells; however, the mechanism is not clear. Recently, platelet-activating factor (PAF)-acetylhydrolase (PAF-AH), the enzyme responsible for PAF degradation, was reported to be overexpressed in some tumor types, including pancreatic and breast cancers. In this study, we investigated whether PAF-AH is involved in Cat G-induced aggregation and migration of MCF-7 cells. We first showed that Cat G increased PAF-AH activity and elevated PAFAH1B2 expression in MCF-7 cells. The elevated expression of PAFAH1B2 was also observed in human breast cancer tissue specimens by immunohistochemical analysis. Furthermore, knockdown of PAFAH1B2 in MCF-7 cells suppressed the cell migration and aggregation induced by low concentrations, but not high concentrations, of Cat G. Carbamoyl PAF (cPAF), a nonhydrolyzable PAF analog, completely suppressed Cat G-induced migration of MCF-7 cells. In addition, PAF receptor (PAFR) inhibition induced cell migration of MCF-7 cells even in the absence of Cat G, suggesting that Cat G suppresses the activation of PAFR through enhanced PAF degradation due to elevated expression of PAFAH1B2 and thereby induces malignant phenotypes in MCF-7 cells. Our findings may lead to a novel therapeutic modality for treating breast cancer by modulating the activity of Cat G/PAF signaling.

Antagonizing astrocytic platelet activating factor receptor-neuroinflammation for total flavone of epimedium in response to cuprizone demyelination

Demyelinating diseases of the central nervous system are characterized by recurrent demyelination and progressive neurodegeneration, but there are no clinical drugs targeting myelin regeneration or improving functional disability in the treatment of multiple sclerosis. Total flavone of Epimedium (TFE) is the main active components of Epimedium, which exhibits the beneficial biological activities in the treatment of diseases, but there is no report in the treatment of demyelinating disorder. The purpose of this study was to explore the therapeutic potential and possible mechanism of TFE in the treatment of demyelination. The results showed that TFE efficiently improved the behavioural performance and histological demyelination in cuprizone (CPZ)-induced demyelinating model. In terms of action, TFE increased astrocytes enrichment in corpus callosum, striatum and cortex, and promoted astrocytes to express neurotrophic factors. Furthermore, the expression of platelet-activating factor receptor (PAFR) in astrocytes was induced by CPZ feeding and LPS stimulation, accompanied by the increase of inflammatory cytokines TNF-α,IL-6 and IL-1β. TFE declined the expression of PAFR, and inhibited inflammatory response. At the same time, TFE also antagonized PAFR activation and inflammatory response triggered by PAF, which further confirmed that TFE, as a new PAFR antagonist, inhibited the astrocyte-derived inflammatory response by antagonizing PAFR-neuroinflammation axis, thus contributing to myelin protection and regeneration.

Simultaneous silencing of lysophosphatidylcholine acyltransferases 1-4 by nucleic acid nanoparticles (NANPs) improves radiation response of melanoma cells

Radiation induces the generation of platelet-activating factor receptor (PAF-R) ligands, including PAF and oxidized phospholipids. Alternatively, PAF is also synthesized by the biosynthetic enzymes lysophosphatidylcholine acyltransferases (LPCATs) which are expressed by tumor cells including melanoma. The activation of PAF-R by PAF and oxidized lipids triggers a survival response protecting tumor cells from radiation-induced cell death, suggesting the involvement of the PAF/PAF-R axis in radioresistance. Here, we investigated the role of LPCATs in the melanoma cell radiotherapy response. LPCAT is a family of four enzymes, LPCAT1-4, and modular nucleic acid nanoparticles (NANPs) allowed for the simultaneous silencing of all four LPCATs. We found that the in vitro simultaneous silencing of all four LPCAT transcripts by NANPs enhanced the therapeutic effects of radiation in melanoma cells by increasing cell death, reducing long-term cell survival, and activating apoptosis. Thus, we propose that NANPs are an effective strategy for improving radiotherapy efficacy in melanomas.

Keratinocyte-derived microvesicle particles mediate ultraviolet B radiation-induced systemic immunosuppression.

A complete carcinogen, ultraviolet B (UVB) radiation (290-320 nm), is the major cause of skin cancer. UVB-induced systemic immunosuppression that contributes to photocarcinogenesis is due to the glycerophosphocholine-derived lipid mediator platelet-activating factor (PAF). A major question in photobiology is how UVB radiation, which only absorbs appreciably in the epidermal layers of skin, can generate systemic effects. UVB exposure and PAF receptor (PAFR) activation in keratinocytes induce the release of large numbers of microvesicle particles (MVPs; extracellular vesicles ranging from 100 to 1000 nm in size). MVPs released from skin keratinocytes in vitro in response to UVB (UVB-MVPs) are dependent on the keratinocyte PAFR. Here, we used both pharmacologic and genetic approaches in cells and mice to show that both the PAFR and enzyme acid sphingomyelinase (aSMase) were necessary for UVB-MVP generation. Our discovery that the calcium-sensing receptor is a keratinocyte-selective MVP marker allowed us to determine that UVB-MVPs leaving the keratinocyte can be found systemically in mice and humans following UVB exposure. Moreover, we found that UVB-MVPs contained bioactive contents including PAFR agonists that allowed them to serve as effectors for UVB downstream effects, in particular UVB-mediated systemic immunosuppression.

PLATELET-ACTIVATING FACTOR (PAF) RECEPTOR ANTAGONIST TREATMENT INCREASES PROLIFERATION AND PROGENITOR MARKERS EXPRESSION IN RETINAL STEM CELLS

<h3>Background</h3> Retinal degenerative diseases are a worldwide public health problem and the main cause of visual loss in humans. Considering stem cell-based therapy as a possible medical approach, quiescent retinal stem cells were identified in the Ciliary Epithelium (CE) of adult mammals; however, regulatory mechanisms inhibit CE stemness, limiting its regenerative potential. It is known that PAF receptor (PAFR) activation suppresses retinal progenitor cell proliferation, suggesting that PAF/PAFR signaling regulates eye stem cells and development. Here we investigated the expression of PAF regulatory enzymes in CE-derived stem cells and the effect of PAFR modulation on proliferation, progenitor, and epithelial markers expression. <h3>Methods</h3> Pigmented epithelium was dissected from mice CE tissue, cultured in DMEN-F12 with 10% FBS for 30 days. Later we add growth factors (EGF/FGF2) to form neurospheres. We analyzed CE and neurospheres transcriptional expression of Pafr, Lpcat1, Pafah1 by real-time PCR. PAFR was modulated during neurosphere forming assay with cPAF (agonist) or PCA4248 (antagonist), and epithelial markers (E-cadherin, N-cadherin), proliferation (Ki67), and progenitor (Pax6 and Nestin) transcripts were analyzed by real-time PCR. An immunohistochemistry assay was performed to investigate proliferation and progenitor markers expression (Ki67 and Sox2). <h3>Results</h3> CE cells downregulated PAF-related enzymes and PAFR expression during neurospheres reprogramming. PAFR agonist inhibited neurosphere formation and controlled epithelial transcriptional expression and morphological phenotype. Oppositely, PAFR antagonist upregulated progenitor transcripts, enhancing CE stemness through increased Ki67, Sox2, Nestin, and Pax6 expression. <h3>Conclusion</h3> Together, our data strongly suggest that PAFR controls CE stemness and can be a promising target for retinal regenerative approaches. <h3>Financial support</h3> FAPESP grant number 2015/24001-1 and 2019/08544-6.

New Insights Into the Pathologic Roles of the Platelet-Activating Factor System.

Described almost 50 years ago, the glycerophosphocholine lipid mediator Platelet-activating factor (PAF) has been implicated in many pathologic processes. Indeed, elevated levels of PAF can be measured in response to almost every type of pathology involving inflammation and cell damage/death. In this review, we provide evidence for PAF involvement in pathologic processes, with focus on cancer, the nervous system, and in photobiology. Importantly, recent insights into how PAF can generate and travel via bioactive extracellular vesicles such as microvesicle particles (MVP) are presented. What appears to be emerging from diverse pathologies in different organ systems is a common theme where pro-oxidative stressors generate oxidized glycerophosphocholines with PAF agonistic effects, which then trigger more enzymatic PAF synthesis via the PAF receptor. A downstream consequence of PAF receptor activation is the generation and release of MVP which provide a mechanism to transmit PAF as well as other bioactive agents. The knowledge gaps which when addressed could result in novel therapeutic strategies are also discussed. Taken together, an enhanced understanding of the PAF family of lipid mediators is essential in our improved comprehension of the relationship amongst the diverse cutaneous, cancerous, neurologic and systemic pathologic processes.

LB916 Effects of the platelet-activating factor-receptor and microRNA-149 in lung cancer growth and therapy effectiveness

Lung cancer remains the leading cause of cancer-related deaths, with low response rates to the current treatment options, indicating the need to explore potential factors, involved in lung cancer growth or impeding the efficacy of therapeutic agents. Studies, including ours, have shown the critical roles of a G-protein coupled, platelet-activating factor-receptor (PAF-R) signaling in augmenting tumor growth or limiting therapy effectiveness in various experimental cancer models. While several mechanisms of the PAF-R pathway have been proposed, its effect with microRNAs (miRs) has not been studied. In particular, while miR-149 has been shown to play oncogenic roles in other cancer types, it functions as a tumor suppressor in lung cancer. The current study aimed to determine the effects of PAF-R and miR-149 in lung cancer growth and therapy effectiveness. We first evaluated the functional significance of PAF-R and miR-149 using A549 and H1299 human non-small cell lung cancer (NSCLC) cell lines as tools. These tumor lines express endogenous PAF-R and miR-149, and PAF-R activation by PAF agonist (CPAF) significantly increased-, whereas miR-149 mimic transfection inhibited cell proliferation in a dose-dependent manner. Interestingly, miR-149 mimic significantly attenuated CPAF-mediated increased proliferation of A549 cells, as also confirmed by miR-149 expression analysis via qPCR. We then examined PAF-R and miR-149 effects on currently used targeted therapy (i.e., erlotinib and gefitinib) responses. Both these agents inhibited the survival of A549 and H1299 cell lines in a dose- and time-dependent manner. While CPAF significantly blocked erlotinib and gefitinib (at ∼IC50 dose)-mediated decreased cell proliferation, PAF-R antagonist and miR-149 mimic did not exert any effects. While additional studies are needed, these findings indicate that miR-149 overcomes PAF-R-mediated increased cell proliferation effect, and PAF-R activation attenuates cytotoxic response of targeted therapy.

Structural basis for signal recognition and transduction by platelet-activating-factor receptor.

Platelet-activating-factor receptor (PAFR) responds to platelet-activating factor (PAF), a phospholipid mediator of cell-to-cell communication that exhibits diverse physiological effects. PAFR is considered an important drug target for treating asthma, inflammation and cardiovascular diseases. Here we report crystal structures of human PAFR in complex with the antagonist SR 27417 and the inverse agonist ABT-491 at 2.8-Å and 2.9-Å resolution, respectively. The structures, supported by molecular docking of PAF, provide insights into the signal-recognition mechanisms of PAFR. The PAFR-SR 27417 structure reveals an unusual conformation showing that the intracellular tips of helices II and IV shift outward by 13 Å and 4 Å, respectively, and helix VIII adopts an inward conformation. The PAFR structures, combined with single-molecule FRET and cell-based functional assays, suggest that the conformational change in the helical bundle is ligand dependent and plays a critical role in PAFR activation, thus greatly extending knowledge about signaling by G-protein-coupled receptors.

Cigarette smoke promotes COPD by activating platelet-activating factor receptor and inducing neutrophil autophagic death in mice.

Neutrophils are the most important effector cells during the development of chronic obstructive pulmonary disease (COPD). Although neutrophil elastase is critical in cigarette smoke (CS)-induced lung parenchyma, the mechanism by which CS triggers elastase release from neutrophils remains unclear. Here we report that CS induction of autophagy in neutrophils by activating platelet- activating factor receptor (PAFR) promotes COPD progression in mouse. We found that the dead neutrophils were increased in bronchoalveolar lavage fluid from CS-exposed mice. Blocking PAFR suppressed the CS-induced autophagy in neutrophils, protected neutrophils from death, and reduced elastase release. Mechanistically, CS enhanced ROS production and High mobility group box 1 (HMGB1) expression through activation of PAFR. The elevated HMGB1 interacted with beclin1, which promoted the dissociation of Bcl-2 from beclin1 and the assembly of autophagy core complex. Moreover, the antagonism of PAFR by rupatadine, a prescription PAFR inhibitor, protected against the development of emphysema, and reduced the autophagic death of neutrophils after CS exposure. These results suggest that CS contributes to the pathogenesis of COPD partly by inducing a PAFR-dependent autophagic death of neutrophils. Therefore, PAFR may be a therapeutic target for COPD and inhibition of PAFR may provide potential therapeutic benefits in the treatment of patients with COPD.

Acetylsalicylic acid inhibits the growth of melanoma tumors via SOX2-dependent-PAF-R-independent signaling pathway.

Acquired resistance to standard therapies remains a serious challenge, requiring novel therapeutic approaches that incorporate potential factors involved in tumor resistance. As cancers including melanoma express inflammatory cyclooxygenases generating prostaglandins implicated in tumor growth, we investigated mechanism of anti-inflammatory drug, acetylsalicylic acid (ASA) which has been shown to inhibit various tumor types, however, its effects against highly aggressive melanoma model are unclear. Given our reports that an activation of platelet-activating factor-receptor (PAF-R) augments the growth and impede efficacies of therapeutic agents in experimental melanoma, we also sought to determine if PAF-R mediates anti-melanoma activity of ASA. The current studies using stably PAF-R-positive (B16-PAFR) and negative (B16-MSCV) murine melanoma cells and PAF-R-expressing and deficient mice, demonstrate that ASA inhibits the in-vitro and in-vivo growth of highly aggressive B16F10 melanoma via bypassing tumoral or stromal PAF-R signaling. Similar ASA-induced effects in-vitro were seen in human melanoma and nasopharyngeal carcinoma cells positive or negative in PAF-R. Mechanistically, the ASA-induced decrease in cell survival and increase in apoptosis were significantly blocked by prostaglandin F2 alpha (PGF2α) agonists. Importantly, PCR array and qRT-PCR analysis of B16-tumors revealed significant downregulation of sry-related high-mobility-box-2 (SOX2) oncogene by ASA treatment. Interestingly, modulation of SOX2 expression by PGF2α agonists and upregulation by fibroblast growth factor 1 (FGF-1) rescued melanoma cells from ASA-induced decreased survival and increased apoptosis. Moreover, PGF2α-receptor antagonist, AL8810 mimics ASA-induced decreased melanoma cells survival which was significantly blocked by PGF2α and FGF-1. These findings indicate that ASA inhibits the growth of aggressive melanoma via SOX2-dependent-PAF-R-indepedent pathway.

Platelet-Activation Factor Receptor Induces Interleukin 10 Production through STAT3 Activation in Dendritic Cells

The activation of the platelet-activating factor receptor (PAFR) is associated to a suppressor phenotype in macrophages and dendritic cells (DCs). In the present study, we investigated mechanisms underlying the production of the interleukin 10 (IL-10) through PAFR activation in murine DCs. For this purpose, BALB/c mice bone marrow-derived DCs were differentiated by GM-CSF treatment and stimulated with LPS in the presence of the PAFR antagonist WEB2086. Signalling pathways downstream to TLR4 activation were investigated. We found that LPS stimulus induced PAFR ligands generation by DCs, but it did not affect the PAFR expression. The LPS-induced IL-10 production was found to be partially dependent of PAFR, since it was reduced in the presence of WEB2086. The IL-10 production through PAFR activation was independent on CREB and PPARγ, as the treatment with selective inhibitors of these pathways did not affect the IL-10 production. TLR4 adaptor molecules (MyD88 and TRIF) expression, MAPK, or NF-κB (p105/50 and p65 subunits) activation pathways were also excluded, since they were not affected by the treatment with WEB2086. The blockage of PAFR by WEB2086 downregulated the STAT3 (Tyr705) phosphorylation induced by LPS. Additionally, DCs treated with STAT3 inhibitor (S3I-201) showed reduced IL-10 production to the same levels observed in DCs treated with WEB2086. The requirement of STAT3 was confirmed in PAFR-KO DCs, since the STAT3 inhibition did not affect IL-10 production by these cells. Our data show an additional molecular mechanism whereby PAFR contributes to IL-10 production in DCs and support the importance of the PAFR activation in DCs phenotype and function.

Boosting Adaptive Immunity: A New Role for PAFR Antagonists.

We have previously shown that the Platelet-Activating Factor Receptor (PAFR) engagement in murine macrophages and dendritic cells (DCs) promotes a tolerogenic phenotype reversed by PAFR-antagonists treatment in vitro. Here, we investigated whether a PAFR antagonist would modulate the immune response in vivo. Mice were subcutaneously injected with OVA or OVA with PAFR-antagonist WEB2170 on days 0 and 7. On day 14, OVA–specific IgG2a and IgG1 were measured in the serum. The presence of WEB2170 during immunization significantly increased IgG2a without affecting IgG1 levels. When WEB2170 was added to OVA in complete Freund’s adjuvant, enhanced IgG2a but not IgG1 production was also observed, and CD4+ FoxP3+ T cell frequency in the spleen was reduced compared to mice immunized without the antagonist. Similar results were observed in PAFR-deficient mice, along with increased Tbet mRNA expression in the spleen. Additionally, bone marrow-derived DCs loaded with OVA were transferred into naïve mice and their splenocytes were co-cultured with fresh OVA-loaded DCs. CD4+ T cell proliferation was higher in the group transferred with DCs treated with the PAFR-antagonist. We propose that the activation of PAFR by ligands present in the site of immunization is able to fine-tune the adaptive immune response.

PAFR activation of NF-κB p65 or p105 precursor dictates pro- and anti-inflammatory responses during TLR activation in murine macrophages.

Platelet-activating factor receptor (PAFR) is a G protein-coupled receptor (GPCR) implicated in many diseases. Toll-like receptors (TLRs) play a critical role in shaping innate and adaptive immune responses. In this study, we investigated whether PAFR signaling changes the macrophages responsiveness to agonists of TLR2 (Pam3Cys), TLR4 (LPS), and TLR3 agonist Poly(I:C). Exogenous PAF inhibited the production of pro-inflammatory cytokines (IL-12p40, IL-6, and TNF-α) and increased anti-inflammatory IL-10 in macrophages challenged with Pam3Cys and LPS, but not with Poly (I:C). PAF did not affect mRNA expression of MyD88, suggesting that PAF acts downstream the adaptor. PAF inhibited LPS-induced phosphorylation of NF-κB p65 and increased NF-κB p105 phosphorylation, which is processed in the proteasome to generate p50 subunit. The PAF potentiation of IL-10 production was dependent on proteasome processing but independent of NF-κB transactivation domain. Inhibition of p50 abolished the PAF-induced IL-10 production. These findings indicate that the impaired transcriptional activity of the p65 subunit and the enhanced p105 phosphorylation induced by PAF are responsible for down regulation of pro-inflammatory cytokines and up regulation of IL-10, respectively, in LPS-challenged macrophages. Together, our data unveil a heretofore unrecognized role for PAFR in modulating activation of NF-κB in macrophages.

Platelet Activating Factor (PAF) Receptor Deletion or Antagonism Attenuates Severe HSV-1 Meningoencephalitis.

Herpes simplex virus type 1 (HSV-1) is a human pathogen that may cause severe encephalitis. The exacerbated immune response against the virus contributes to the disease severity and death. Platelet activating factor (PAF) is a mediator capable of inducing increase in vascular permeability, production of cytokines on endothelial cells and leukocytes. We aimed to investigate the activation of PAF receptor (PAFR) and its contribution to the severity of the inflammatory response in the brain following HSV-1 infection. C57BL/6 wild-type (WT) and PAFR deficient (PAFR-/-) mice were inoculated intracranially with 104 plaque-forming units (PFU) of HSV-1. Visualization of leukocyte recruitment was performed using intravital microscopy. Cells infiltration in the brain tissue were analyzed by flow cytometry. Brain was removed for chemokine assessment by ELISA and for histopathological analysis. The pharmacological inhibition by the PAFR antagonist UK-74,505 was also analyzed. In PAFR-/- mice, there was delayed lethality but no difference in viral load. Histopathological analysis of infected PAFR-/- mice showed that brain lesions were less severe when compared to their WT counterparts. Moreover, PAFR-/- mice showed less TCD4+, TCD8+ and macrophages in brain tissue. This reduction of the presence of leukocytes in parenchyma may be mechanistically explained by a decrease in leukocytes rolling and adhesion. PAFR-/- mice also presented a reduction of the chemokine CXCL9 in the brain. In addition, by antagonizing PAFR, survival of C57BL/6 infected mice increased. Altogether, our data suggest that PAFR plays a role in the pathogenesis of experimental HSV-1 meningoencephalitis, and its blockade prevents severe disease manifestation.

Platelet-activating factor receptor (PAFR) plays a crucial role in experimental global cerebral ischemia and reperfusion.

Stroke is one of the most frequent causes of death and disability worldwide leading to a significant clinical and socioeconomic burden. Although different mechanisms are involved in the pathogenesis of stroke, inflammatory response occurs after ischemia and contributes to the expansion of brain injury. Platelet-activating factor receptor (PAF) plays crucial roles in both physiological and pathological conditions in the brain. PAF receptor (PAFR) may be expressed on cellular and nuclear membranes of various cell types, especially leukocytes, platelets, endothelial cells, neuronal cells and microglia. Herein, using mice lacking the PAFR receptor (PAFR(-/-)), we investigate a potential role for this receptor during experimental transient global cerebral ischemia and reperfusion (BCCAo). In PAFR deficiency, we observed a significant improvement in the neurological deficits, which were associated with a reduction of brain infarcted area as evaluated by triphenyltetrazolium chloride (TTC). Moreover, a decrease in the percentage of necrotic cavities areas and in the frequency of ischemic neurons was also found by employing histometric analysis. In addition, in PAFR(-/-) mice there was prevention of caspase-3 activation and decreased vascular permeability and brain edema. Decreased brain levels of the cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and the chemokine (C-X-C motif) ligand 1 (CXCL1) by ELISA were also detected in PAFR(-/-) BCCAo animals. Taken together, our results suggest that PAFR activation might be crucial for the global brain ischemia and reperfusion injury.

Mast cell-derived histamine is necessary for platelet-activating factor mediated systemic immunosuppression (IRC4P.606)

Abstract Platelet-activating Factor (PAF) activates many cell types via the G-protein coupled PAF-receptor (PAF-R). Systemic PAF-R activation exerts acute pro-inflammatory, as well as delayed systemic immunosuppressive effects in vivo. PAF enzymatic synthesis and degradation is tightly regulated, but reactive oxygen species from oxidative stressors, such as UVB, chemotherapy and cigarette smoke, generate PAF-like species via the unregulated oxidation of membrane lipids. The systemic immunosuppressive effects of these pro-oxidative stressors are due to PAF-R signaling. This work establishes the role of the mast cell (MC) and the soluble mediators involved in PAF-R-mediated immunosuppression. Using a contact hypersensitivity model, we show that PAF loses its immunosuppressive effects in MC-deficient (Wsh) mice, a phenotype rescued by dermal reconstitution of Wsh mice with WT bone marrow derived MCs. Use of PAF-R-deficient Wsh mice suggests that the MC PAF-R is necessary and sufficient to mediate PAF-R systemic suppression, in a process attenuated by COX-2 and TGF-β inhibitors. Finally, by transplanting histamine (histidine decarboxylase-null) deficient MCs, we demonstrate that this pathway requires MC-derived histamine release. These studies fit with the hypothesis that MC PAF-R activation with resultant histamine release mediates PAF-R systemic immunosuppression.