Formyl Peptide Research Articles

Turn-Adopting Peptidomimetic as a Formyl Peptide Receptor-1 Antagonist

Turn-Adopting Peptidomimetic as a Formyl Peptide Receptor-1 Antagonist

The impact of trauma relevant concentrations of prostaglandin E2 on the anti-microbial activity of the innate immune system

BackgroundThe mechanisms underlying the state of systemic immune suppression that develops following major trauma are poorly understood. A post-injury increase in circulating levels of prostaglandin E2 (PGE2) has been proposed as a contributory factor, yet few studies have addressed how trauma influences PGE2 biology.MethodsBlood samples from 95 traumatically-injured patients (injury severity score ≥8) were collected across the pre-hospital (≤2 hours), acute (4-12 hours) and subacute (48-72 hours) post-injury settings. Alongside ex vivo assessments of lipopolysaccharide (LPS)-induced cytokine production by monocytes, neutrophil reactive oxygen species production and phagocytosis, serum concentrations of PGE2 and its scavenger albumin were measured, and the expression of enzymes and receptors involved in PGE2 synthesis and signalling analysed. Leukocytes from trauma patients were treated with cyclooxygenase (COX) inhibitors (indomethacin or NS-398), or the protein kinase A inhibitor H89, to determine whether injury-induced immune suppression could be reversed by targeting the PGE2 pathway. The effect that trauma relevant concentrations of PGE2 had on the anti-microbial functions of neutrophils, monocytes and monocyte-derived macrophages (MDMs) from healthy controls (HC) was examined, as was the effect of PGE2 on efferocytosis. To identify factors that may trigger PGE2 production post-trauma, leukocytes from HC were treated with mitochondrial-derived damage associated molecular patterns (mtDAMPs) and COX-2 expression and PGE2 generation measured.ResultsPGE2 concentrations peaked in blood samples acquired ≤2 hours post-injury and coincided with significantly reduced levels of albumin and impaired LPS-induced cytokine production by monocytes. Significantly higher COX-2 and phospholipase A2 expression was detected in neutrophils and/or peripheral blood mononuclear cells isolated from trauma patients. Treatment of patient leukocytes with indomethacin, NS-398 or H89 enhanced LPS-induced cytokine production and neutrophil extracellular trap generation. Exposure to physiological concentrations of PGE2 suppressed the anti-microbial activity of monocytes, neutrophils and MDMs of HC, but did not influence efferocytosis. In a formyl-peptide receptor-1 dependent manner, mtDAMP treatment significantly increased COX-2 protein expression in neutrophils and monocytes, which resulted in increased PGE2 production.ConclusionsPhysiological concentrations of PGE2 suppress the anti-microbial activities of neutrophils, monocytes and MDMs. Targeting the PGE2 pathway could be a therapeutic approach by which to enhance innate immune function post-injury.

Pan-cancer analysis and experimental validation of FPR3 as a prognostic and immune infiltration-related biomarker for glioma.

Formyl peptide receptor 3 (FPR3) is known to have implications in the progression of various cancer types. Despite this, its biological significance within pan-cancer datasets has yet to be investigated. In this investigation, we scrutinized FPR3's expression profiles, genetic alterations, prognostic significance, immune-related characteristics, methylation status, tumor mutation burden (TMB), and microsatellite instability (MSI) across different types of cancer. We utilized TISCH's single-cell data to identify immune cells closely associated with FPR3. The predictive significance of FPR3 was evaluated independently in gliomas using data from TCGA and CGGA datasets, leading to the development of a prognostic nomogram. Immunohistochemistry and Western blot analysis confirmed FPR3 expression in gliomas. Lastly, the CCK-8 and wound-healing assays were employed to assess the impact of FPR3 on the proliferation and metastasis of GBM cell lines. In numerous cancer types, heightened FPR3 expression correlated with adverse outcomes, immune cell infiltration, immune checkpoints, TMB, and MSI. In glioma, FPR3 emerged as a notable risk factor, with the prognostic model effectively forecasting patient results. The potential biological relevance of FPR3 was confirmed in glioma, and it was shown to have significant involvement in the processes of glioma growth, immune infiltration, and metastasis. Our results imply a potential association of FPR3 with tumor immunity, indicating its viability as a prognostic indicator in glioma.

Differential requirement of formyl peptide receptor 1 in macrophages and neutrophils in the host defense against Mycobacterium tuberculosis Infection

Formyl peptide receptors (FPR), part of the G-protein coupled receptor superfamily, are pivotal in directing phagocyte migration towards chemotactic signals from bacteria and host tissues. Although their roles in acute bacterial infections are well-documented, their involvement in immunity against tuberculosis (TB) remains unexplored. Here, we investigate the functions of Fpr1 and Fpr2 in defense against Mycobacterium tuberculosis (Mtb), the causative agent of TB. Elevated levels of Fpr1 and Fpr2 were found in the lungs of mice, rabbits and peripheral blood of humans infected with Mtb, suggesting a crucial role in the immune response. The effects of Fpr1 and Fpr2 deletion on bacterial load, lung damage, and cellular inflammation were assessed in a murine TB model utilizing hypervirulent strain of Mtb from the W-Beijing lineage. While Fpr2 deletion had no impact on disease outcome, Fpr1-deficient mice demonstrated improved bacterial control, especially by macrophages. Bone marrow-derived macrophages from these Fpr1−/− mice exhibited an enhanced ability to contain bacterial growth over time. Contrarily, treating genetically susceptible mice with Fpr1-specific inhibitors caused impaired early bacterial control, corresponding with increased Mtb persistence in necrotic neutrophils. Furthermore, ex vivo assays revealed that Fpr1−/− neutrophils were unable to restrain Mtb growth, indicating a differential function of Fpr1 among myeloid cells. These findings highlight the distinct and complex roles of Fpr1 in myeloid cell-mediated immunity against Mtb infection, underscoring the need for further research into these mechanisms for a better understanding of TB immunity.

P2X7R-primed keratinocytes are susceptible to apoptosis via GPCR-Gβγ-pERK signal pathways

BackgroundCell death constitutes a pivotal biological phenomenon essential for the preservation of homeostasis within living organisms. In the context of maintaining a functional skin barrier, keratinocytes exert positively and negatively control cell death signals. However, in patients with severe drug eruptions, anomalous overexpression of the formyl peptide receptor 1 (FPR1) in keratinocytes elicits a distinctive mode of cell death known as necroptosis, thereby suffering a loss of the skin barrier. The precise molecular mechanisms connecting FPR1 activation to this cell death remain unclear. ObjectiveWe have investigated the intracellular signal transduction cascade governing FPR1-mediated cell death in cultured keratinocytes. MethodsWe used HaCaT cells as a model keratinocyte. The expression of FPR1 was detected with qPCR. The presence of cell death events was monitored through live-cell fluorescent staining and LDH release assays. Furthermore, the phosphorylation of ERK was assessed via Western blot analysis. Intracellular signal pathways were investigated using specific inhibitors. ResultsLigand stimulation of an endogenous ion channel, purinergic receptor P2X7 (P2X7R), increased the FPR1 expression level. This upregulated FPR1 demonstrated functional competence in the phosphorylation of downstream MAP kinase and the initiation of cell death. Notably, this cell death was ameliorated upon the administration of inhibitors targeting Gβγ, ERK, and caspases. ConclusionThe induction and stimulation of FPR1 initiated apoptosis in keratinocytes via the Gβγ-pERK signaling pathway. Our findings postulate that the downstream components of FPR1 represent an alternative therapeutic target for preventing unintended keratinocyte cell death.

Structural basis for the access and binding of resolvin D1 (RvD1) to formyl peptide receptor 2 (FPR2/ALX), a class A GPCR.

Inflammation is essential to the body's defense against tissue injury and microbial invasion. However, uncontrolled inflammation is highly detrimental and can result in chronic inflammatory diseases such as asthma, cancer, obesity, and diabetes. An increasing body of evidence suggests that specialized pro-resolving lipid mediators (SPMs), such as resolvins, are actively involved in critical cellular events that drive the resolution of inflammation and a return to homeostasis. An imbalance caused by insufficient SPMs can result in the unsuccessful resolution of inflammation. The D-series resolvins (metabolites of docosahexaenoic acid), such as resolvin D1 (RvD1) and resolvin D2 (RvD2), carry out their pro-resolving functions by directly binding to class A G protein-coupled receptors FPR2/ALXR and GPR32, and GPR18, respectively. We recently demonstrated that RvD1 and RvD2 preferentially partition and accumulate at the polar headgroup regions of the membrane. However, the mechanistic detail of how RvD1 gains access to the FPR2 binding site from a surrounding membrane environment remains unknown. In this study, we used classical MD and well-tempered metadynamics simulations to examine the structural basis for the access and binding of RvD1 to its target receptor from aqueous and membrane environments. The results offer valuable insights into the access path, potential binding pose, and key residue interactions essential for the access and binding of RvD1 to FPR2/ALXR and may help in identifying small molecule therapeutics as a possible treatment for inflammatory disorders.

Comparative analysis of formyl peptide receptor 1 and formyl peptide receptor 2 reveals shared and preserved signalling profiles.

The pattern recognition receptors, formyl peptide receptors, FPR1 and FPR2, are G protein-coupled receptors that recognize many different pathogen- and host-derived ligands. While FPR1 conveys pro-inflammatory signals, FPR2 is linked with pro-resolving outcomes. To analyse how the two very similar FPRs exert opposite effects in modulating inflammatory responses despite their high homology, a shared expression profile on immune cells and an overlapping ligand repertoire, we questioned whether the signalling profile differs between these two receptors. We deduced EC50 and Emax values for synthetic, pathogen-derived and host-derived peptide agonists for both FPR1 and FPR2 and analysed them within the framework of biased signalling. We furthermore investigated whether FPR isoform-specific agonists affect the ex vivo lifespan of human neutrophils. The FPRs share a core signature across signalling pathways. Whereas the synthetic WKYMVm and formylated peptides acted as potent agonists at FPR1, and at FPR2, only WKYMVm was a full agonist. Natural FPR2 agonists, irrespective of N-terminal formylation, displayed lower activity ratios, suggesting an underutilized signalling potential of this receptor. FPR2 agonism did not counteract LPS-induced neutrophil survival, indicating that FPR2 activation per se is not linked with a pro-resolving function. Activation of FPR1 and FPR2 by a representative agonist panel revealed a lack of a receptor-specific signalling texture, challenging assumptions about distinct inflammatory profiles linked to specific receptor isoforms, signalling patterns or agonist classes. These conclusions are restricted to the specific agonists and signalling pathways examined.

In vitro and in silico therapeutic properties of Artepillin C and Aromadendrin as collagenase and elastase inhibitors and investigation of anti-Ovarian cancer effects and antioxidant potential

In this investigation, the enzymes collagenase and elastase were inhibited by artepillin.C and aromadendrin molecules with excellent to good IC50 values of 16.65, and 0.79 μM for artepillin.C and 7.31, 19.38, and 10.56 μM for aromadendrin. Using the molecular modeling study, the chemical activity of these compounds against the enzymes, collagenase, and elastase were evaluated. The anti-cancer properties of the compounds were evaluated using the following cell lines: NIH: OVCAR-3, ES-2, UACC-1598, Hs 832(C).T [Hs832.Tc], TOV-21G, UWB1.289. The DPPH (1,1-diphenyl-2-pricrylhydrazyl) free radical scavenging assay was used to measure antioxidant activity, and the spectrophotometric method was used in this work. The chemical activities of artepillin.C and aromadendrin against collagenase and elastase were investigated utilizing the molecular docking study. The anti-cancer activities of the compounds were evaluated against NIH: OVCAR-3, ES-2, UACC-1598, Hs 832(C).T [Hs832.Tc], TOV-21G, UWB1.289 cell lines. Also, some cell lines had best results for aromadendrin like NIH: OVCAR-3, ES-2, UACC-1598, Hs 832(C).T [Hs832.Tc], TOV-21G, UWB1.289 (10.54 ± 0.94, 22.11 ± 2.52, 31.85 ± 4.73, 8.14 ± 1.52, 17.94 ± 1.88, and 24.31 ± 2.64 μM). The chemical activities of artepillin.C and aromadendrin against some of the expressed surface receptor proteins (folate receptor, CD44, EGFR, Formyl Peptide Receptor–Like 1, M2 muscarinic receptor, and estrogen receptors) in the mentioned cell lines were assessed using the molecular docking calculations. The outcomes provided information on potential interactions and their atomic-level properties. According to the docking scores, the compounds show a high affinity for binding to proteins and enzymes. Furthermore, these substances made good contact with the receptors and enzymes. As a result, these substances may have the ability to suppress cancer cells and enzymes.

Hepatic Proteomic Changes Associated with Liver Injury Caused by Alcohol Consumption in Fpr2-/- Mice.

Alcohol-associated liver disease (ALD) is a prevalent medical problem with limited effective treatment strategies. Although many biological processes contributing to ALD have been elucidated, a complete understanding of the underlying mechanisms is still lacking. The current study employed a proteomic approach to identify hepatic changes resulting from ethanol (EtOH) consumption and the genetic ablation of the formyl peptide receptor 2 (FPR2), a G-protein coupled receptor known to regulate multiple signaling pathways and biological processes, in a mouse model of ALD. Since previous research from our team demonstrated a notable reduction in hepatic FPR2 protein levels in patients with alcohol-associated hepatitis (AH), the proteomic changes in the livers of Fpr2-/- EtOH mice were compared to those observed in patients with AH in order to identify common hepatic proteomic alterations. Several pathways linked to exacerbated ALD in Fpr2-/- EtOH mice, as well as hepatic protein changes resembling those found in patients suffering from AH, were identified. These alterations included decreased levels of coagulation factors F2 and F9, as well as reduced hepatic levels of glutamate-cysteine ligase catalytic subunit (GCLC) and total glutathione in Fpr2-/- EtOH compared to WT EtOH mice. In conclusion, the data suggest that FPR2 may play a regulatory role in hepatic blood coagulation and the antioxidant system, both in a pre-clinical model of ALD and in human AH, however further experiments are required to validate these findings.

Annexin A1 activates the G protein-coupled formyl peptide receptor type 2-dependent endothelial nitric oxide synthase pathway to alleviate sepsis associated acute lung injury

To investigate whether annexin A1 (ANXA1) improves sepsis-induced lung injury by activating G protein-coupled formyl peptide receptor type 2 (FPR2)-dependent endothelial nitric oxide synthase (eNOS) pathway. Twenty-four male SD rats were randomly divided into normal group (Control group), lipopolysaccharide (LPS) induced lung injury model group (LPS group), LPS+ANXA1 mimetic peptide group (LPS+Ac2-26 group) and LPS+ANXA1 mimetic peptide+FPR2 inhibitor group (LPS+Ac2-26+WRW4 group), with 6 rats in each group. On the third day before modeling, rats of the LPS+Ac2-26 group were injected with 1 mg/kg Ac2-26 by the tail vein and rats of LPS+Ac2-26+WRW4 group were injected with 1 mg/kg Ac2-26 and 2.2 mg/kg WRW4 by the tail vein. The rats of control group and LPS group were injected same volume of physiological saline. After 48 hours of modeling, the rats were anesthetized and the carotid blood was taken to detect the oxygenation index (OI). Lung tissue was taken from the euthanized rats. The wet/dry (W/D) ratio was determined. The pathological changes of lung tissue were observed under light microscope and pathological score was performed. The levels of tumor necrosis factor-α (TNF-α), interleukins (IL-1β, IL-6, IL-10), malondialdehyde (MDA) and myeloperoxidase (MPO) were detected by enzyme-linked immunosorbent assay (ELISA). The protein expressions of eNOS, inducible nitric oxide synthase (iNOS) and nuclear factor-κB (NF-κB) were detected by Western blotting. Under light microscope, compared with LPS group, the infiltration degree of inflammatory cells in the lung tissue of LPS+Ac2-26 group was reduced, and the thickness of the alveolar septum was improved. The degree of inflammatory cell infiltration in the lung tissue of LPS+Ac2-26+WRW4 group was more severe than that of LPS+Ac2-26 group, and the thickness of the alveolar septum increased. These findings suggested that ANXA1 significantly inhibited inflammatory cell infiltration and improved alveolar septal thickness, WRW4 reversed the lung improvement effects of ANXA1. Compared with control group, OI in LPS group was significantly decreased, and W/D ratio, pathological score and TNF-α, IL-1β, IL-6, MDA and MPO levels in lung tissue were significantly increased. Compared with LPS group, OI and IL-10 levels in lung tissue were significantly increased in LPS+Ac2-26 group, while W/D ratio, pathological score, TNF-α, IL-1β, IL-6, MDA and MPO levels in lung tissue were significantly decreased. These results indicated that ANXA1 can improve the oxygenation capacity, improve lung tissue leakage, reduce edema, and inhibit lung tissue inflammation in rats with lung injury. Compared with LPS+Ac2-26 group, the LPS+Ac2-26+WRW4 group showed significant decreases in OI and lung tissue IL-10 level [OI (mmHg, 1 mmHg ≈ 0.133 kPa): 132.16±24.00 vs. 248.67±18.70, IL-10 (ng/L): 27.30±3.04 vs. 36.10±3.92, both P < 0.05], the lung tissue W/D ratio, pathological score and levels of TNF-α, IL-1β, IL-6, MDA and MPO were significantly increased [W/D ratio: 5.29±0.02 vs. 4.83±0.02, pathological score: 5.00±0.28 vs. 2.67±0.52, TNF-α (ng/L): 39.80±4.36 vs. 32.10±2.15, IL-1β (ng/L): 200.00±15.68 vs. 152.60±9.74, IL-6 (ng/L): 181.50±18.02 vs. 148.50±7.34, MDA (mmol/mg): 82.01±8.22 vs. 70.43±5.69, MPO (pg/mg): 6.50±0.32 vs. 4.60±0.56, all P < 0.05]. These results suggested that WRW4 could block the above improvement of ANXA1. Western blotting results showed that compared with control group, the expression of eNOS, iNOS and NF-κB in LPS group was significantly up-regulated. Compared with LPS group, the protein expression of eNOS in LPS+Ac2-26 group was significantly up-regulated (eNOS/β-actin: 0.25±0.01 vs. 0.14±0.01, P < 0.05), and the protein expression of iNOS and NF-κB was significantly down-regulated (iNOS/β-actin: 0.09±0.02 vs. 0.12±0.02, NF-κB/β-actin: 0.35±0.06 vs. 0.59±0.13, both P < 0.05). These findings suggested that ANXA1 might activate the eNOS pathway and down-regulate the expression of NF-κB. Compared with LPS+Ac2-26 group, the protein expression of eNOS in LPS+Ac2-26+WRW4 group was significantly down-regulated (eNOS/β-actin: 0.17±0.02 vs. 0.25±0.01, P < 0.05), while the protein expression of iNOS and NF-κB was significantly up-regulated (iNOS/β-actin: 0.12±0.02 vs. 0.09±0.02, NF-κB/β-actin: 0.52±0.10 vs. 0.35±0.06, both P < 0.05). These results suggested that WRW4 blocked the activation of the eNOS pathway by ANXA1. ANXA1 can improve lung injury associated with sepsis by activating FPR2-dependent eNOS pathway.

Treatment with lipoxin A4 improves influenza A infection outcome, induces macrophage reprogramming, anti-inflammatory and pro-resolutive responses.

Influenza A is a virus from theOrthomixoviridaefamily responsible for high lethality rates and morbidity, despite clinically proven vaccination strategies and some anti-viral therapies. The eicosanoid Lipoxin A4 (LXA4) promotes the resolution of inflammation by decreasing cell recruitment and pro-inflammatory cytokines release, but also for inducing activation of apoptosis, efferocytosis, and macrophage reprogramming. Here, we evaluated whether a synthetic lipoxin mimetic, designated AT-01-KG, would improve the course of influenza A infection in a murine model. Mice were infected with influenza A/H1N1 and treated with AT-01-KG (1.7μg/kg/day, i.p.) at day 3 post-infection. AT-01-KG attenuated mortality, reducing leukocyte infiltration and lung damage at day 5 and day 7 post-infection. AT-01-KG is a Formyl Peptide Receptor 2 (designated FPR2/3 in mice) agonist, and the protective responses were not observed in fpr2/3-/-animals. In mice treated with LXA4(50μg/kg/day, i.p., days 3-6 post-infection), at day 7, macrophage reprogramming was observed, as seen by a decrease in classically activated macrophages and an increase in alternatively activated macrophages in the lungs. Furthermore, the number of apoptotic cells and cells undergoing efferocytosis was increased in the lavage of treated mice. Treatment also modulated the adaptive immune response, increasing the number of T helper 2 cells (Th2) and regulatory T (Tregs) cells in the lungs of the treated mice. Therefore, treatment with a lipoxin A4analog was beneficial in a model of influenza A infection in mice. The drug decreased inflammation and promoted resolution and beneficial immune responses, suggesting it may be useful in patients with severe influenza.

The differential expression of adipose tissue genes in short, medium and long-term periods after bariatric surgery

Bariatric surgery is an approved treatment for obesity that consistently improves metabolic syndrome, with well-documented beneficial effects on dyslipidemia, cardiovascular risk, nonalcoholic fatty liver disease and glucose homeostasis. In this study, we determined the differential expression genes in three periods after bariatric surgery: short-term (4-months), medium-term (1- and 2-years), and long-term (5-years) periods. Two microarray profiles were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were identified by comparing the expression of adipose tissue genes before surgery compared to short, medium and long-term periods following surgery. Shared DEGs for the medium-term were evaluated by comparing the DEGs for both 1 and 2 years. 165, 65, and 59 DEGs were identified in short–medium–long periods. The protein–protein interactions were analyzed by STRING. A co-expression network was constructed by mapping the DEGs onto the GeneMANIA plugin of Cytoscape. Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) and wikipathway analysis were done for each group of DEGs. Interleukin-8 receptor activity, complement receptor activity and opsonin receptor activity/N-formyl peptide receptor activity in GO Function enrichment and cellular response to interleukin-8, positive regulation of hippocampal neuron apoptotic process, and positive regulation of hippocampal neuron apoptotic process in GO Process showed the best scores in short-, medium-, and long-term periods, respectively. Eight genes, including CCL2 (Chemokine ligand 2), CXCR4 (CXC motif chemokine receptor 4), EGR2 (Early Growth Response 2), FPR1 (Formyl Peptide Receptor 1), IL6 (interleukin-6), RGS2 (regulator of gene protein signaling2), SELPLG (Selectin P Ligand), and THBS1 (Thrombospondin 1) were identified as shared DEGs in the three periods after surgery. Importantly, results of DAVID database analysis showed 7, 6, 4, and 4 of these genes have roles in immune/ cancer/cardiovascular diseases, type 2 diabetes, myocardial infarct, and atherosclerosis, respectively.

GPCRs overexpression and impaired fMLP-induced functions in neutrophils from chronic kidney disease patients.

G-protein coupled receptors (GPCRs) expressed on neutrophils regulate their mobilization from the bone marrow into the blood, their half-live in the circulation, and their pro- and anti-inflammatory activities during inflammation. Chronic kidney disease (CKD) is associated with systemic inflammatory responses, and neutrophilia is a hallmark of CKD onset and progression. Nonetheless, the role of neutrophils in CKD is currently unclear. Blood and renal tissue were collected from non-dialysis CKD (grade 3 - 5) patients to evaluate GPCR neutrophil expressions and functions in CKD development. CKD patients presented a higher blood neutrophil-to-lymphocyte ratio (NLR), which was inversely correlated with the glomerular filtration rate (eGFR). A higher frequency of neutrophils expressing the senescent GPCR receptor (CXCR4) and activation markers (CD18+CD11b+CD62L+) was detected in CKD patients. Moreover, CKD neutrophils expressed higher amounts of GPCR formyl peptide receptors (FPR) 1 and 2, known as neutrophil pro- and anti-inflammatory receptors, respectively. Cytoskeletal organization, migration, and production of reactive oxygen species (ROS) by CKD neutrophils were impaired in response to the FPR1 agonist (fMLP), despite the higher expression of FPR1. In addition, CKD neutrophils presented enhanced intracellular, but reduced membrane expression of the protein Annexin A1 (AnxA1), and an impaired ability to secrete it into the extracellular compartment. Secreted and phosphorylated AnxA1 is a recognized ligand of FPR2, pivotal in anti-inflammatory and efferocytosis effects. CKD renal tissue presented a low number of neutrophils, which were AnxA1+. Together, these data highlight that CKD neutrophils overexpress GPCRs, which may contribute to an unbalanced aging process in the circulation, migration into inflamed tissues, and efferocytosis.

Biased receptor signalling and intracellular trafficking profiles of structurally distinct formylpeptide receptor 2 agonists.

There is increasing interest in developing FPR2 agonists (compound 43, ACT-389949 and BMS-986235) as potential pro-resolving therapeutics, with ACT-389949 and BMS-986235 having entered phase I clinical development. FPR2 activation leads to diverse downstream outputs. ACT-389949 was observed to cause rapid tachyphylaxis, while BMS-986235 and compound 43 induced cardioprotective effects in preclinical models. We aim to characterise the differences in ligand-receptor engagement and downstream signalling and trafficking bias profile. Concentration-response curves to G protein dissociation, β-arrestin recruitment, receptor trafficking and second messenger signalling were generated using FPR2 ligands (BMS-986235, ACT-389949, compound 43 and WKYMVm), in HEK293A cells. Log(τ/KA) was obtained from the operational model for bias analysis using WKYMVm as a reference ligand. Docking of FPR2 ligands into the active FPR2 cryoEM structure (PDBID: 7T6S) was performed using ICM pro software. Bias analysis revealed that WKYMVm and ACT-389949 shared a very similar bias profile. In comparison, BMS-986235 and compound 43 displayed approximately 5- to 50-fold bias away from β-arrestin recruitment and trafficking pathways, while being 35- to 60-fold biased towards cAMP inhibition and pERK1/2. Molecular docking predicted key amino acid interactions at the FPR2 shared between WKYMVm and ACT-389949, but not with BMS-986235 and compound 43. In vitro characterisation demonstrated that WKYMVm and ACT-389949 differ from BMS-986235 and compound 43 in their signalling and protein coupling profile. This observation may be explained by differences in the ligand-receptor interactions. In vitro characterisation provided significant insights into identifying the desired bias profile for FPR2-based pharmacotherapy.

Discovery and Optimization of Aryl Piperidinone Ureas as Selective Formyl Peptide Receptor 2 Agonists.

We report the discovery and optimization of aryl piperidinone urea formyl peptide receptor 2 (FPR2) agonists from a weakly active high-throughput screening (HTS) hit to potent and selective agonists with favorable efficacy in acute in vivo models. A basis for the selectivity for FPR2 over FPR1 is proposed based on docking molecules into recently reported FPR2 and FPR1 cryoEM structures. Compounds from the new scaffold reported in this study exhibited superior potency and selectivity and favorable ADME profiles. Furthermore, select compounds were evaluated in an acute rat lipopolysaccharide (LPS) inflammation model and demonstrated robust dose-dependent induction of IL10, a marker for inflammation resolution, providing a valuable proof of concept for this class of FPR2 agonists.

Removal of circulating mitochondrial N-formyl peptides via immobilized antibody therapy restores sepsis-induced neutrophil dysfunction.

During recovery from septic shock, circulating mitochondrial N-formyl peptides (mtFPs) predispose to secondary infection by occupying formyl peptide receptor 1 (FPR1) on the neutrophil (polymorphonuclear leukocyte, PMN) membrane, suppressing cytosolic calcium ([Ca2+]i)-dependent responses to secondarily encountered bacteria. However, no study has yet investigated therapeutic clearance of circulating mtFPs in clinical settings. Thus, we studied how to remove mtFPs from septic-shock plasma and whether such removal could preserve cell-surface FPR1 and restore sepsis-induced PMN dysfunction by normalizing [Ca2+]i flux. In in vitro model systems, mtFP removal rescued PMN FPR1-mediated [Ca2+]i flux and chemotaxis that had been suppressed by prior mtFP exposure. However, PMN functional recovery occurred in a stepwise fashion over 30 - 90 minutes. Intracellular Ca2+-calmodulin appears to contribute to this delay. In ex vivo model systems using blood samples obtained from patients with septic shock, anti-mtFP antibodies alone failed to eliminate mtFPs from septic-shock plasma or inhibit mtFP activity. We therefore created a beads-based anti-mtFP antibody cocktail (bb-AMfpA) by combining protein A/sepharose with antibodies specific for the most potent human mtFP chemoattractants. The bb-AMfpA treatment successfully removed those active mtFPs from septic-shock plasma. Furthermore, the bb-AMfpA treatment significantly restored chemotactic and bactericidal dysfunction of PMNs obtained from patients with septic shock who developed secondary infections. By clearing circulating mtFPs, the immobilized anti-mtFP antibody therapy prevented mtFP interactions with surface FPR1, thereby restoring [Ca2+]i-dependent PMN antimicrobial function in clinical septic-shock environments. This approach may help prevent the development of secondary, nosocomial infections in patients recovering from septic shock.

The analgesic effects of Yu-Xue-Bi tablet (YXB) on mice with inflammatory pain by regulating LXA4-FPR2-TRPA1 pathway

BackgroundOxylipins including lipoxin A4 (LXA4) facilitate the resolution of inflammation and possess analgesic properties by inhibiting macrophage infiltration and transient receptor potential (TRP) protein expression. Yu-Xue-Bi Tablet (YXB) is a traditional Chinese patent medicine used to relieve inflammatory pain. Our previous research has shown that the analgesic effect of YXB is related to inhibiting peripheral inflammation and regulating macrophage infiltration, but the mechanism is not yet clear. The purpose of this study is to explore the mechanisms of YXB on mice models with Complete Freund’s Adjuvant (CFA)-induced inflammatory pain from the perspective at the resolution of inflammation.MethodsMechanical allodynia thresholds and heat hypersensitivity were measured using the Von Frey test and the hot plate test respectively. The open field test and the tail suspension test were employed to measure anxiety and depressive behaviors respectively. The expression of CD68+ and the proportion of F4/80+CD11b+ cells were measured by immunofluorescence staining and flow cytometry. The expression of transient receptor potential ankyrin 1(TRPA1) was measured by immunofluorescence staining and western blotting. Oxylipins omics analysis provided quantitative data on oxylipins in the paws, and enzyme linked immunosorbent assay (ELISA) was used to measure the levels of LXA4 there. Immunofluorescence staining was used to perform the expression of Leukotriene A4 hydroxylase (LTA4H) in the paws of mice. The impact of injecting the formyl peptide receptor 2(FPR2) antagonist WRW4 and the TRPA1 agonist AITC into the left paws was observed, focusing on the expression of mechanical allodynia thresholds, the expression of CD68+, TRPA1 in the paws, and Calcitonin gene-related peptide (CGRP) in the L5 spinal dorsal horn.ResultsYXB elevated mechanical allodynia thresholds, alleviated heat hypersensitivity and anxiety and depressive behaviors in CFA mice. It significantly reduced the number of CD68+ and proportion of F4/80+CD11b+ within the paws, thereby decreasing macrophage infiltration. Additionally, it diminished the expression of TRPA1 in the paws and TRPV1 in the DRG, leading to an inhibition of peripheral sensitization. Through quantitative analysis, it was found that YXB could modulate DHA-derived oxylipins and LXA4. ELISA results indicated that YXB elevated the levels of LXA4 and inhibited the expression of LAT4H in the paws. Furthermore, the pro-resolution and analgesic effects of YXB were hindered after administration of the FPR2 antagonist. Compared with the AITC group, YXB showed no significant improvement in anti-inflammatory and analgesic effects.ConclusionsYXB can regulate the oxylipins of paws in CFA mice to promote the resolution of inflammation. The LXA4-FPR2-TRPA1 pathway is a key mechanism for the resolution of inflammation and analgesic effects.

Sphingosine-1-Phosphate–Cathelicidin Axis Plays a Pivotal Role in the Development of Cutaneous Squamous Cell Carcinoma

Cutaneous squamous cell carcinoma (cSCC) is a common skin cancer, caused by mutagenesis resulting from excess ultraviolet radiation or other types of oxidative stress. These stressors also upregulate production of a cutaneous innate immune element, cathelicidin antimicrobial peptide (CAMP), via endoplasmic reticulum (ER) stress-initiated, sphingosine-1-phosphate (S1P) signaling pathway. While CAMP has beneficial antimicrobial activities, it also can be pro-inflammatory and pro-carcinogenic. We addressed whether and how S1P-induced CAMP production leads to cSCC development. Our study demonstrated that: 1) CAMP expression is increased in cSCC cells and skin from cSCC patients; 2) S1P levels are elevated in cSCC cells, while inhibition of S1P production attenuates CAMP-stimulated cSCC growth; 3) exogenous CAMP stimulates cSCC, but not normal human keratinocyte growth; 4) blockade of formyl peptide receptor-like (FPRL) 1 protein, a CAMP receptor, attenuates cSCC growth as well as the growth and invasion of cSCC cells mediated by CAMP into an extracellular matrix-containing fibroblast substrate; 5) Foxp3+ regulatory T cell (which decreases anti-tumor immunity) levels increase in cSCC skin; and 6) CAMP induces ER stress in cSCC cells. Together, the ER stress-S1P-CAMP axis forms a vicious circle, creating a favorable environment for cSCC development, i.e., cSCC growth and invasion impedes anti-cancer immunity.

Tumor-derived mitochondrial formyl peptides suppress tumor immunity through modification of the tumor microenvironment.

Mitochondrial N-formylpeptides are released from damaged or dead cells to the extracellular spaces and cause inflammatory responses. The role of mitochondrial N-formylpeptides in aseptic systemic inflammatory response syndromes induced by trauma or cardiac surgery has been well investigated. However, there are no reports regarding the role of mitochondrial N-formylpeptides in cancer. In this study, we investigated the role of tumor cell-derived mitochondrial N-formylpeptides in anti-tumor immunity using knockout murine tumor cells of mitochondrial methionyl-tRNA formyltransferase (MTFMT), which catalyze N-formylation of mitochondrial DNA-encoded proteins. There was no apparent difference among the wild-type and MTFMT-knockout clones of E.G7-OVA cells with respect to morphology, mitochondrial dynamics, glycolysis and oxidative phosphorylation, oxygen consumption rate, or in vitro cell growth. In contrast, in vivo tumor growth of MTFMT-knockout cells was slower than that of wild-type cells. A reduced number of myeloid-derived suppressor cells and an increase of cytotoxic T-lymphocytes in the tumor tissues were observed in the MTFMT-knockout tumors. These results suggested that tumor cell-derived mitochondrial N-formylpeptides had a negative role in the host anti-tumor immunity through modification of the tumor microenvironment.

Formylpeptide Receptor Agonism as a Novel Approach for Limiting Abdominal Aortic Aneurysm: Impact on Smooth Muscle Cell Phenotypic Switching

Formylpeptide Receptor Agonism as a Novel Approach for Limiting Abdominal Aortic Aneurysm: Impact on Smooth Muscle Cell Phenotypic Switching