Implication of the Annexin 1/FPR axis in leishmanial exosome-mediated Leishmania major skin hyperpathogenesis.

Introduction: Exosomes produced by the protozoan parasite Leishmania (LeishEXO) are well-established drivers of virulence, though mechanisms underlying their exacerbation of experimental leishmaniasis remain elusive. Expression of Annexin A1 (ANXA1), a protein implicated in exosome-mediated pathologies and viral internalization, has been shown to correlate with cutaneous leishmaniasis severity. Given ANXA1's regulation of myeloid cells - the canonical hosts for Leishmania - we studied the potential role of ANXA1 and its receptors FPR1/2 in exerting LeishEXO's effects. Methods: Murine and in vitro ANXA1-/- models were used to study the generation of protective TH1 responses during experimental L. major infection with and without LeishEXO. Recruitment of inflammatory cells was assessed using a peritoneal cell recruitment assay and immunophenotyping, and production of inflammatory mediators was measured using a cytokine and chemokine array. Treatment of experimental models with FPR2 antagonist WRW4 and FPR1/2 agonist WKYMVm was used to delineate the role of the FPR/ANXA1 axis in LeishEXO-mediated hyperpathogenesis. Results: We established that ANXA1 deficiency prohibits LeishEXO-mediated pathogenesis and myeloid cell infection, with minimal alterations to adaptive and innate immune phenotypes. FPR2 blockade with WRW4 similarly inhibited leishmanial hyperpathogenesis, while direct activation of FPRs with WKYMVm enhanced infection and recapitulated the LeishEXO-mediated phenotype. This research describes LeishEXO's utilization of the ANXA1/FPR axis to facilitate parasitic internalization and pathogenesis, which may be leveraged in the development of therapeutics for leishmaniasis.

Cancer stem cells release interleukin-33 within large oncosomes to promote immunosuppressive differentiation of macrophage precursors.

In squamous cell carcinoma (SCC), macrophages responding to interleukin (IL)-33 create a TGF-ß-rich stromal niche that maintains cancer stem cells (CSCs), which evade chemotherapy-induced apoptosis in part via activation of the NRF2 antioxidant program. Here, we examined how IL-33 derived from CSCs facilitates the development of an immunosuppressive microenvironment. CSCs with high NRF2 activity redistributed nuclear IL-33 to the cytoplasm and released IL-33 as cargo of large oncosomes (LOs). Mechanistically, NRF2 increased the expression of the lipid scramblase ATG9B, which exposed an "eat me" signal on the LO surface, leading to annexin A1 (ANXA1) loading. These LOs promoted the differentiation of AXNA1 receptor+ myeloid precursors into immunosuppressive macrophages. Blocking ATG9B's scramblase activity or depleting ANXA1 decreased niche macrophages and hindered tumor progression. Thus, IL-33 is released from live CSCs via LOs to promote the differentiation of alternatively activated macrophage, with potential relevance to other settings of inflammation and tissue repair.

LL37/FPR2 regulates neutrophil mPTP promoting the development of neutrophil extracellular traps in diabetic retinopathy.

Diabetic retinopathy (DR) is characterized by chronic, low-grade inflammation. This state may be related to the heightened production of neutrophil extracellular traps (NETs) induced by high glucose (HG). Human cathelicidin antimicrobial peptide (LL37) is an endogenous ligand of G protein-coupled chemoattractant receptor formyl peptide receptor 2 (FPR2), expressed on neutrophils and facilitating the formation and stabilization of the structure of NETs. In this study, we detected neutrophils cultured under different conditions, the retinal tissue of diabetic mice, and fibrovascular epiretinal membranes (FVM) samples of patients with proliferative diabetic retinopathy (PDR) to explore the regulating effect of LL37/FPR2 on neutrophil in the development of NETs during the process of DR. Specifically, HG or NG with LL37 upregulates the expression of FPR2 in neutrophils, induces the opening of mitochondrial permeability transition pore (mPTP), promotes the increase of reactive oxygen species and mitochondrial ROS, and then leads to the rise of NET production, which is mainly manifested by the release of DNA reticular structure and the increased expression of NETs-related markers. The PI3K/AKT signaling pathway was activated in neutrophils, and the phosphorylation level was enhanced by FPR2 agonists in vitro. In vivo, increased expression of NETs markers was detected in the retina of diabetic mice and in FVM, vitreous fluid, and serum of PDR patients. Transgenic FPR2 deletion led to decreased NETs in the retina of diabetic mice. Furthermore, in vitro, inhibition of the LL37/FPR2/mPTP axis and PI3K/AKT signaling pathway decreased NET production induced by high glucose. These results suggested that FPR2 plays an essential role in regulating the production of NETs induced by HG, thus may be considered as one of the potential therapeutic targets.

Gene Regulation of Neutrophils Mediated Liver and Lung Injury through NETosis in Acute Pancreatitis.

Acute pancreatitis (AP) is one of the most common gastrointestinal emergencies, often resulting in self-digestion, edema, hemorrhage, and even necrosis of pancreatic tissue. When AP progresses to severe acute pancreatitis (SAP), it often causes multi-organ damage, leading to a high mortality rate. However, the molecular mechanisms underlying SAP-mediated organ damage remain unclear. This study aims to systematically mine SAP data from public databases and combine experimental validation to identify key molecules involved in multi-organ damage caused by SAP. Retrieve transcriptomic data of mice pancreatic tissue for AP, lung and liver tissue for SAP, and corresponding normal tissue from the Gene Expression Omnibus (GEO) database. Conduct gene differential analysis using Limma and DEseq2 methods. Perform enrichment analysis using the clusterProfiler package in R software. Score immune cells and immune status in various organs using single-sample gene set enrichment analysis (ssGSEA). Evaluate mRNA expression levels of core genes using reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Validate serum amylase, TNF-α, IL-1ß, and IL-6 levels in peripheral blood using enzyme-linked immunosorbent assay (ELISA), and detect the formation of neutrophil extracellular traps (NETs) in mice pancreatic, liver, and lung tissues using immunofluorescence. Differential analysis reveals that 46 genes exhibit expression dysregulation in mice pancreatic tissue for AP, liver and lung tissue for SAP, as well as peripheral blood in humans. Functional enrichment analysis indicates that these genes are primarily associated with neutrophil-related biological processes. ROC curve analysis indicates that 12 neutrophil-related genes have diagnostic potential for SAP. Immune infiltration analysis reveals high neutrophil infiltration in various organs affected by SAP. Single-cell sequencing analysis shows that these genes are predominantly expressed in neutrophils and macrophages. FPR1, ITGAM, and C5AR1 are identified as key genes involved in the formation of NETs and activation of neutrophils. qPCR and IHC results demonstrate upregulation of FPR1, ITGAM, and C5AR1 expression in pancreatic, liver, and lung tissues of mice with SAP. Immunofluorescence staining shows increased levels of neutrophils and NETs in SAP mice. Inhibition of NETs formation can alleviate the severity of SAP as well as the levels of inflammation in the liver and lung tissues. This study identified key genes involved in the formation of NETs, namely FPR1, ITGAM, and C5AR1, which are upregulated during multi-organ damage in SAP. Inhibition of NETs release effectively reduces the systemic inflammatory response and liver-lung damage in SAP. This research provides new therapeutic targets for the multi-organ damage associated with SAP.

Action and therapeutic targets of myosin light chain kinase, an important cardiovascular signaling mechanism.

The global incidence of cardiac diseases is increasing, imposing a substantial socioeconomic burden on healthcare systems. The pathogenesis of cardiovascular disease is complex and not fully understood, and the physiological function of the heart is inextricably linked to well-regulated cardiac muscle movement. Myosin light chain kinase (MLCK) is essential for myocardial contraction and diastole, cardiac electrophysiological homeostasis, vasoconstriction of vascular nerves and blood pressure regulation. In this sense, MLCK appears to be an attractive therapeutic target for cardiac diseases. MLCK participates in myocardial cell movement and migration through diverse pathways, including regulation of calcium homeostasis, activation of myosin light chain phosphorylation, and stimulation of vascular smooth muscle cell contraction or relaxation. Recently, phosphorylation of myosin light chains has been shown to be closely associated with the activation of myocardial exercise signaling, and MLCK mediates systolic and diastolic functions of the heart through the interaction of myosin thick filaments and actin thin filaments. It works by upholding the integrity of the cytoskeleton, modifying the conformation of the myosin head, and modulating innervation. MLCK governs vasoconstriction and diastolic function and is associated with the activation of adrenergic and sympathetic nervous systems, extracellular transport, endothelial permeability, and the regulation of nitric oxide and angiotensin II. Additionally, MLCK plays a crucial role in the process of cardiac aging. Multiple natural products/phytochemicals and chemical compounds, such as quercetin, cyclosporin, and ML-7 hydrochloride, have been shown to regulate cardiomyocyte MLCK. The MLCK-modifying capacity of these compounds should be considered in designing novel therapeutic agents. This review summarizes the mechanism of action of MLCK in the cardiovascular system and the therapeutic potential of reported chemical compounds in cardiac diseases by modifying MLCK processes.

Post-treatment with Resolvin D1 attenuates pulmonary hypertension by inhibiting endothelial-to-mesenchymal transition.

Pulmonary hypertension (PH) is a life-threatening disease characterized by pulmonary vascular remodeling. Endothelial-to-mesenchymal transition (EndMT) is an important manifestation and mechanism of pulmonary vascular remodeling. Resolvin D1 (RvD1) is an endogenous lipid mediator promoting the resolution of inflammation. However, the role of RvD1 on EndMT in PH remains unknown. Here, we aimed to investigate the effect and mechanisms of RvD1 on the treatment of PH. We showed that RvD1 and its receptor FPR2 expression were markedly decreased in PH patients and both chronic hypoxia-induced PH (CH-PH) and sugen 5416/hypoxia-induced PH (SuHx-PH) mice models. RvD1 treatment decreased right ventricular systolic pressure (RVSP) and alleviated right ventricular function, and reduced pulmonary vascular remodeling and collagen deposition in the perivascular of both two PH mice models. Then, RvD1 inhibited EndMT in both the lungs of PH mice models and primary cultured human umbilical vein endothelial cells (HUVECs) treated with TGF-ß and IL-1ß. Moreover, RvD1 inhibited EndMT by downregulating Smad2/3 phosphorylation in vivo and in vitro via FPR2. In conclusion, our date suggest that RvD1/FPR2 axis prevent experimental PH by inhibiting endothelial-mensenchymal-transition and may be a therapeutic target for PH.

FPR1, as a Potential Biomarker of Diagnosis and Infliximab Therapy Responses for Crohn's Disease, is Related to Disease Activity, Inflammation and Macrophage Polarization.

Purpose: Crohn's disease (CD) represents a multifaceted inflammatory gastrointestinal condition, with a profound significance placed on unraveling its molecular pathways to enhance both diagnostic capabilities and therapeutic interventions. This study focused on identifying a robust macrophage-related signatures (MacroSig) for diagnosing CD, emphasizing the role of FPR1 in macrophage polarization and its implications in CD. Patients and Methods: Expression profiles from intestinal biopsies and macrophages of 1804 CD patients were retrieved from the Gene Expression Omnibus (GEO). Utilizing CIBERSORTx, differential expression analysis, and weighted correlation network analysis to to identify macrophage-related genes (MRGs). By unsupervised clustering, distinct clusters of CD were identified. Potential biomarkers were identified via using four machine learning algorithms, leading to the establishment of MacroSig which combines insights from 12 machine learning algorithms. Furthermore, the expression of FPR1 was verified in intestinal biopsies of CD patients and two murine experimental colitis models. Finally, we further explored the role of FPR1 in macrophage polarization through single-cell analysis as well as through the study of RAW264.7 cells and peritoneal macrophages. Results: Two distinct clusters with differential levels of macrophage infiltration and inflammation were identified. The MacroSig, which included FPR1 and LILRB2, exhibited high diagnostic accuracy and outperformed existing biomarkers and signatures. Clinical analysis demonstrated a strong correlation of FPR1 with disease activity, endoscopic inflammation status, and response to infliximab treatment. The expression levels of FPR1 were validated in our CD cohort by immunohistochemistry and confirmed in two colitis mouse models. Single-cell analysis indicated that FPR1 is predominantly expressed in macrophages and monocytes. In vitro studies demonstrated that FPR1 was upregulated in M1 macrophages, and its activation promoted M1 polarization. Conclusion: We developed a promising diagnostic signature for CD, and targeting FPR1 to modulate macrophage polarization may represent a novel therapeutic strategy.

ARAP3 protects from excessive formylated peptide-induced microvascular leakage by acting on endothelial cells and neutrophils.

Vascular permeability is temporarily heightened during inflammation, but excessive inflammation-associated microvascular leakage can be detrimental, as evidenced in the inflamed lung. Formylated peptides regulate vascular leakage indirectly via formylated peptide receptor-1 (FPR1)-mediated recruitment and activation of neutrophils. Here we identify how the GTPase-activating protein ARAP3 protects against formylated peptide-induced microvascular permeability via endothelial cells and neutrophils. In vitro, Arap3-/- endothelial monolayers were characterised by enhanced formylated peptide-induced permeability due to upregulated endothelial FPR1 and enhanced vascular endothelial cadherin internalisation. In vivo, enhanced inflammation-associated microvascular leakage was observed in Arap3-/- mice. Leakage of plasma protein into the lungs of Arap3-/- mice increased within hours of formylated peptide administration. Adoptive transfer experiments indicated this was dependent upon ARAP3 deficiency in both immune and non-immune cells. Bronchoalveolar lavages of formylated peptide-challenged Arap3-/- mice contained neutrophil extracellular traps (NETs). Pharmacological inhibition of NET formation abrogated excessive microvascular leakage, indicating a critical function of NETs in this context. The observation that Arap3-/- mice developed more severe influenza suggests these findings are pertinent to pathological situations characterised by abundant formylated peptides. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

FPR3 reprograms glycolytic metabolism and stemness in gastric cancer via calcium-NFATc1 pathway.

Aerobic glycolysis accelerates tumor proliferation and progression, and inhibitors or drugs targeting abnormal cancer metabolism have been developing. Cancer stem-like cells (CSCs) significantly contribute to tumor initiation, metastasis, therapy resistance, and recurrence. Formyl peptide receptor 3 (FPR3), a member of FPR family, involves in inflammation, tissue repair, and angiogenesis. However, studies in exploring the regulatory mechanisms of aerobic glycolysis and CSCs by FPR3 in gastric cancer (GC) remain unknown. Here, we demonstrated that overexpressed FPR3 suppressed glycolytic capacity and stemness of tumor cells, then inhibited GC cells proliferation. Mechanistically, FPR3 impeded cytoplasmic calcium ion flux and hindered nuclear factor of activated T cells 1 (NFATc1) nuclear translocation, leading to the transcriptional inactivation of NFATc1-binding neurogenic locus notch homolog protein 3 (NOTCH3) promoter, subsequently obstructing NOTCH3 expression and the AKT/mTORC1 signaling pathway, and ultimately downregulating glycolysis. Additionally, NFATc1 directly binds to the sex determining region Y-box 2 (SOX2) promoter and modifies stemness in GC. In conclusion, our work illustrated that FPR3 played a negative role in GC progression by modulating NFATc1-mediated glycolysis and stemness in a calcium-dependent manner, providing potential insights into cancer therapy.

Differential gene expression and miRNA regulatory network in coronary slow flow.

Coronary slow flow (CSF) is characterized by slow progression of coronary angiography without epicardial stenosis. The aim of this study was to explore the potential biomarkers and regulatory mechanism for CSF. Peripheral blood mononuclear cells from 3 cases of CSF and 3 healthy controls were collected for high-throughput sequencing of mRNA and miRNA, respectively. The differentially expressed mRNAs (DE-mRNAs) and miRNAs (DE-miRNAs) was identified. A total of 117 DE-mRNAs and 32 DE-miRNAs were obtained and they were mainly enriched in immune and inflammatory responses. Twenty-six DE-mRNAs were the predicted target genes for miRNAs by RAID, and then the regulatory network of 15 miRNAs were constructed. In addition, through the PPI network, we identified the three genes (FPR1, FPR2 and CXCR4) with larger degrees as hub genes. Among them, FPR1 was regulated by hsa-miR-342-3p, hsa-let-7c-5p and hsa-miR-197-3p and participated in the immune response. Finally, we validated the differential expression of hub genes and key miRNAs between 20 CSF and 20 control. Moreover, we found that miR-342-3p has a targeted regulatory relationship with FPR1, and their expression is negatively correlated. Then we established a hypoxia/reoxygenation (H/R) HUVEC model and detected FPR1, cell proliferation and apoptosis. Transfection with miR-342-3p mimics can significantly promote the proliferation of HUVEC under H/R conditions. FPR1 were associated with CSF as a biomarker and may be regulated by miR-342-3p potential biomarkers.

The time-dependent expression of FPR2 and ANXA1 in murine deep vein thrombosis model and its relation to thrombus age.

Thrombus age determination in fatal venous thromboembolism cases is an important task for forensic pathologists. In this study, we investigated the time-dependent expressions of formyl peptide receptor 2 (FPR2) and Annexin A1 (ANXA1) in a stasis-induced deep vein thrombosis (DVT) murine model, with the aim of obtaining useful information for thrombus age timing. A total of 75 ICR mice were randomly classified into thrombosis group and control group. In thrombosis group, a DVT model was established by ligating the inferior vena cava (IVC) of mice, and thrombosed IVCs were harvested at 1, 3, 5, 7, 10, 14, and 21 days after modeling. In control group, IVCs without thrombosis were taken as control samples. The expressions of FPR2 and ANXA1 during thrombosis were detected using immunohistochemistry and double immunofluorescence staining. Their protein and mRNA levels in the samples were determined by Western blotting and quantitative real-time PCR. The results reveal that FPR2 was predominantly expressed by intrathrombotic neutrophils and macrophages. ANXA1 expression in the thrombi was mainly distributed in neutrophils, endothelial cells of neovessels, and fibroblastic cells. After thrombosis, the expressions of FPR2 and ANXA1 were time-dependently up-regulated. The percentage of FPR2-positive cells and the level of FPR2 protein significantly elevated at 1, 3, 5 and 7 days after IVC ligation as compared to those at 10, 14 and 21 days after ligation (p < 0.05). Moreover, the mRNA level of FPR2 were significantly higher at 5 days than that at the other post-ligation intervals (p < 0.05). Besides, the levels of ANXA1 mRNA and protein peaked at 10 and 14 days after ligation, respectively. A significant increase in the mRNA level of ANXA1 was found at 10 and 14 days as compared with that at the other post-ligation intervals (p < 0.01). Our findings suggest that FPR2 and ANXA1 are promising as useful markers for age estimation of venous thrombi.

FPR1: A critical gatekeeper of the heart and brain.

G protein-coupled receptors (GPCRs) are currently the most widely focused drug targets in the clinic, exerting their biological functions by binding to chemicals and activating a series of intracellular signaling pathways. Formyl-peptide receptor 1 (FPR1) has a typical seven-transmembrane structure of GPCRs and can be stimulated by a large number of endogenous or exogenous ligands with different chemical properties, the first of which was identified as formyl-methionine-leucyl-phenylalanine (fMLF). Through receptor-ligand interactions, FPR1 is involved in inflammatory response, immune cell recruitment, and cellular signaling regulation in key cell types, including neutrophils, neural stem cells (NSCs), and microglia. This review outlines the critical roles of FPR1 in a variety of heart and brain diseases, including myocardial infarction (MI), ischemia/reperfusion (I/R) injury, neurodegenerative diseases, and neurological tumors, with particular emphasis on the milestones of FPR1 agonists and antagonists. Therefore, an in-depth study of FPR1 contributes to the research of innovative biomarkers, therapeutic targets for heart and brain diseases, and clinical applications.

The role of FPR2-mediated ferroptosis in formyl peptide-induced acute lung injury against endothelial barrier damage and protective effect of the mitochondria-derived peptide MOTS-c.

BACKGROUND: Acute lung injury (ALI) has garnered significant attention in the field of respiratory and critical care due to its high mortality and morbidity, and limited treatment options. The role of the endothelial barrier in the development of ALI is crucial. Several bacterial pathogenic factors, including the bacteria-derived formyl peptide (fMLP), have been implicated in damaging the endothelial barrier and initiating ALI. However, the mechanism by which fMLP causes ALI remains unclear. In this study, we aim to explore the mechanisms of ALI caused by fMLP and evaluate the protective effects of MOTS-c, a mitochondrial-derived peptide. METHODS: We established a rat model of ALI and a human pulmonary microvascular endothelial cell (HPMVEC) model of ALI by treatment with fMLP. In vivo experiments involved lung histopathology assays, assessments of inflammatory and oxidative stress factors, and measurements of ferroptosis-related proteins and barrier proteins to evaluate the severity of fMLP-induced ALI and the type of tissue damage in rats. In vitro experiments included evaluations of fMLP-induced damage on HPMVEC using cell activity assays, assessments of inflammatory and oxidative stress factors, measurements of ferroptosis-related proteins, endothelial barrier function assays, and examination of the key role of FPR2 in fMLP-induced ALI. We also assessed the protective effect of MOTS-c and investigated its mechanism on the fMLP-induced ALI in vivo and in vitro. RESULTS: Results from both in vitro and in vivo experiments demonstrate that fMLP promotes the expression of inflammatory and oxidative stress factors, activates ferroptosis and disrupts the vascular endothelial barrier, ultimately contributing to the development and progression of ALI. Mechanistically, ferroptosis mediated by FPR2 plays a key role in fMLP-induced injury, and the Nrf2 and MAPK pathways are involved in this process. Knockdown of FPR2 and inhibition of ferroptosis can attenuate ALI induced by fMLP. Moreover, MOTS-c could protect the vascular endothelial barrier function by inhibiting ferroptosis and suppressing the expression of inflammatory and oxidative stress factors through Nrf2 and MAPK pathways, thereby alleviating fMLP-induced ALI. CONCLUSION: Overall, fMLP disrupts the vascular endothelial barrier through FPR2-mediated ferroptosis, leading to the development and progression of ALI. MOTS-c demonstrates potential as a protective treatment against ALI by alleviating the damage induced by fMLP.

Anti-inflammatory actions of aspirin-triggered resolvin D1 (AT-RvD1) in bronchial epithelial cells stimulated by cigarette smoke extract.

Smoking causes several diseases such as chronic obstructive pulmonary disease (COPD). Aspirin-triggered-resolvin D1 (AT-RvD1) is a lipid mediator produced during the resolution of inflammation and demonstrates anti-inflammatory and pro-resolution effects in several inflammatory experimental models including in the airways. Here we evaluated the role of AT-RvD1 (100 nM) in bronchial epithelial cells (BEAS-2B) stimulated by cigarette smoke extract (CSE; 1%; 1 cigarette) for 24 h. CSE induced the productions of IL-1ß, TNF-α, IL-10, IL-4 and IFN-γ as well as the activations of NF-κB and STAT3 and the expression of ALX/FPR2 receptor. AT-RvD1 reduced the IL-1ß and TNF-α production and increased the production of IFN-γ. These effects were reversed BOC2, an antagonist of ALX/FPR2 receptor for AT-RvD1. The production of IL-4 and IL-10 were not altered by AT-RvD1. In addition, AT-RvD1 reduced the phosphorylation of NF-κB and STAT3 when compared to CSE-stimulated BEAS-2B cells. No alteration of ALX/FPR2 expression was observed by AT-RvD1 when compared to CSE group. In the human monocytic leukemia cell line, the relative number of copies of IL-1ß and IL-4 was significantly higher in CSE + AT-RvD1 group compared CSE group, however, the expression of M1 cytokine was more pronounced than M2 profile. AT-RvD1 could be an important target for the reduction of inflammation in the airways associated with smoking.

Enhancement of LC3-associated efferocytosis for the alleviation of intestinal inflammation.

LC3-associated phagocytosis (LAP) is an instrumental machinery for the clearance of extracellular particles including apoptotic cells for the alleviation of inflammation. While pharmacological approaches to modulate LAP for inflammation regulation have been poorly explored, in our study we identified a novel compound, columbamine (COL), which can trigger LAP and enhance efferocytosis in an animal model of colitis to attenuate inflammation. We found that COL directly binds to and biasedly activates FPR2 (formyl peptide receptor 2) to promote efferocytosis and alleviate colitis. Biochemically, COL induces an interaction between RAC1 and the PIK3C3/VPS34-RUBCN/RUBICON complex, stimulating LC3-associated efferocytosis. These findings provide a novel interpretation of the potential roles of LAP in regulating inflammatory bowel disease (IBD), reveal the relationship between G protein-coupled receptors (GPCRs) and LAP, and highlight the role of RAC1 in regulating the PIK3C3/VPS34-RUBCN complex in LAP.

Formylated Peptide Receptor-1-Mediated Gut Inflammation as a Therapeutic Target in Inflammatory Bowel Disease.

Background: Formylated peptide receptor (FPR)-1 is a G-coupled receptor that senses foreign bacterial and host-derived mitochondrial formylated peptides (FPs), leading to innate immune system activation. Aim: We sought to investigate the role of FPR1-mediated inflammation and its potential as a therapeutic target in inflammatory bowel disease (IBD). Methods: We characterized FPR1 gene and protein expression in 8 human IBD (~1000 patients) datasets with analysis on disease subtype, mucosal inflammation, and drug response. We performed in vivo dextran-sulfate sodium (DSS) colitis in C57/BL6 FPR1 knockout mice. In ex vivo studies, we studied the role of mitochondrial FPs and pharmacological blockade of FPR1 using cyclosporin H in human peripheral blood neutrophils. Finally, we assess mitochondrial FPs as a potential mechanistic biomarker in the blood and stools of patients with IBD. Results: Detailed in silico analysis in human intestinal biopsies showed that FPR1 is highly expressed in IBD (n = 207 IBD vs 67 non-IBD controls, P < .001), and highly correlated with gut inflammation in ulcerative colitis (UC) and Crohn's disease (CD) (both P < .001). FPR1 receptor is predominantly expressed in leukocytes, and we showed significantly higher FPR1+ve neutrophils in inflamed gut tissue section in IBD (17 CD and 24 UC; both P < .001). Further analysis in 6 independent IBD (data available under Gene Expression Omnibus accession numbers GSE59071, GSE206285, GSE73661, GSE16879, GSE92415, and GSE235970) showed an association with active gut inflammation and treatment resistance to infliximab, ustekinumab, and vedolizumab. FPR1 gene deletion is protective in murine DSS colitis with lower gut neutrophil inflammation. In the human ex vivo neutrophil system, mitochondrial FP, nicotinamide adenine dinucleotide dehydrogenase subunit-6 (ND6) is a potent activator of neutrophils resulting in higher CD62L shedding, CD63 expression, reactive oxygen species production, and chemotactic capacity; these effects are inhibited by cyclosporin H. We screened for mitochondrial ND6 in IBD (n = 54) using ELISA and detected ND6 in stools with median values of 2.2 gg/mL (interquartile range [IQR] 0.0-4.99; range 0-53.3) but not in blood. Stool ND6 levels, however, were not significantly correlated with paired stool calprotectin, C-reactive protein, and clinical IBD activity. Conclusions: Our data suggest that FPR1-mediated neutrophilic inflammation is a tractable target in IBD; however, further work is required to clarify the clinical utility of mitochondrial FPs as a potential mechanistic marker for future stratification.

Formyl Peptide Receptor 2-Dependent cPLA2 and 5-LOX Activation Requires a Functional NADPH Oxidase.

Phospholipases (PL) A2 catalyzes the hydrolysis of membrane phospholipids and mostly generates arachidonic acid (AA). The enzyme 5-lipoxygenase (5-LOX) can metabolize AA to obtain inflammatory leukotrienes, whose biosynthesis highly depends on cPLA2 and 5-LOX activities. Formyl Peptide Receptor 2 (FPR2) belongs to a subfamily of class A GPCRs and is considered the most versatile FPRs isoform. Signaling triggered by FPR2 includes the activation of several downstream kinases and NADPH oxidase (NOX)-dependent ROS generation. In a metabolomic analysis we observed a significant increase in AA concentration in FPR2-stimulated lung cancer cell line CaLu-6. We analyzed cPLA2 phosphorylation and observed a time-dependent increase in cPLA2 Ser505 phosphorylation in FPR2-stimulated cells, which was prevented by the MEK inhibitor (PD098059) and the p38MAPK inhibitor (SB203580) and by blocking NOX function. Similarly, we demonstrated that phosphorylation of 5-LOX at Ser271 and Ser663 residues requires FPR2-dependent p38MAPK and ERKs activation. Moreover, we showed that 5-LOX Ser271 phosphorylation depends on a functional NOX expression. Our overall data demonstrate for the first time that FPR2-induced ERK- and p38MAPK-dependent phosphorylation/activation of cPLA2 and 5-LOX requires a functional NADPH oxidase. These findings represent an important step towards future novel therapeutic possibilities aimed at resolving the inflammatory processes underlying many human diseases.

New evidence for fractional pressure ratio prediction by pulsatility index from transcranial Doppler in patients with symptomatic cerebrovascular stenosis disease.

Background: The pulsatility index (PI) derived from transcranial Doppler (TCD) assessment may represent the cerebral resistance and altered cerebral blood flow. The purpose of this study was to assess the performance of the TCD PI in correlation with wire-based fractional pressure ratio (FPR). Methods: This study included 33 patients with symptomatic atherosclerotic lesions of the extracranial and intracranial large arteries, specifically the internal carotid artery, middle cerebral artery (MCA), vertebral artery (VA) V4 segment, and basilar artery (BA), all of which exhibited luminal stenosis ranging from 50% to 70%. TCD was performed prior to angiography in order to determine the flow distal to the lesion. We performed cerebrovascular angiography with a pressure wire to measure the FPR of vessels with stenotic lesions. Bland-Altman analysis and ordinal least square (OLS) linear regression were used to quantify the correlation between PI and FPR. Results: A total of 42 TCD data points were analyzed. At the TCD locations distal to the lesions, the correlation coefficients were no less than 0.90%, with almost all P values <0.001, which indicated very strong positive correlations; the exception to this was the distal TCD for MCA segment lesions (r=0.897; P=0.015) and VA V4 segment (r=0.964; P=0.036). The Bland-Altman plot demonstrated a small difference (0.003) between the distal TCD PI and the FPR, with an acceptable 95% confidence interval [95% confidence interval (CI): 0.06-0.12]. Conclusions: The PI obtained through TCD assessment distal to the stenotic lesion exhibited a correlation with the FPR computed using pressure wire measurements.

Lipoxin-mediated signaling: ALX/FPR2 interaction and beyond.

In the aftermath of tissue injury or infection, an efficient resolution mechanism is crucial to allow tissue healing and preserve appropriate organ functioning. Pro-resolving bioactive lipids prevent uncontrolled inflammation and its consequences. Among these mediators, lipoxins were the first described and their pro-resolving actions have been mainly described in immune cells. They exert their actions mostly through formyl-peptide receptor 2 (ALX/FPR2 receptor), a G-protein-coupled receptor whose biological function is tremendously complex, primarily due to its capacity to mediate variable cellular responses. Moreover, lipoxins can also interact with alternative receptors like the cytoplasmic aryl hydrocarbon receptor, the cysteinyl-leukotrienes receptors or GPR32, triggering different intracellular signaling pathways. The available information about this complex response mediated by lipoxins is addressed in this review, going over the different mechanisms used by these molecules to stop the inflammatory reaction and avoid the development of dysregulated and chronic pathologies.

Enhancement of efferocytosis through biased FPR2 signaling attenuates intestinal inflammation.

Efficient clearance of dying cells (efferocytosis) is an evolutionarily conserved process for tissue homeostasis. Genetic enhancement of efferocytosis exhibits therapeutic potential for inflammation resolution and tissue repair. However, pharmacological approaches to enhance efferocytosis remain sparse due to a lack of targets for modulation. Here, we report the identification of columbamine (COL) which enhances macrophage-mediated efferocytosis and attenuates intestinal inflammation in a murine colitis model. COL enhances efferocytosis by promoting LC3-associated phagocytosis (LAP), a non-canonical form of autophagy. Transcriptome analysis and pharmacological characterization revealed that COL is a biased agonist that occupies a part of the ligand binding pocket of formyl peptide receptor 2 (FPR2), a G-protein coupled receptor involved in inflammation regulation. Genetic ablation of the Fpr2 gene or treatment with an FPR2 antagonist abolishes COL-induced efferocytosis, anti-colitis activity and LAP. Taken together, our study identifies FPR2 as a potential target for modulating LC3-associated efferocytosis to alleviate intestinal inflammation and highlights the therapeutic value of COL, a natural and biased agonist of FPR2, in the treatment of inflammatory bowel disease.