Formyl Peptide Research Articles

Targeting the Annexin A1-FPR2/ALX pathway for host-directed therapy in dengue disease.

Host immune responses contribute to dengue's pathogenesis and severity, yet the possibility that failure in endogenous inflammation resolution pathways could characterise the disease has not been contemplated. The pro-resolving protein Annexin A1 (AnxA1) is known to counterbalance overexuberant inflammation and mast cell (MC) activation. We hypothesised that inadequate AnxA1 engagement underlies the cytokine storm and vascular pathologies associated with dengue disease. Levels of AnxA1 were examined in the plasma of dengue patients and infected mice. Immunocompetent, interferon (alpha and beta) receptor one knockout (KO), AnxA1 KO, and formyl peptide receptor 2 (FPR2) KO mice were infected with dengue virus (DENV) and treated with the AnxA1 mimetic peptide Ac2-26 for analysis. In addition, the effect of Ac2-26 on DENV-induced MC degranulation was assessed in vitro and in vivo. We observed that circulating levels of AnxA1 were reduced in dengue patients and DENV-infected mice. Whilst the absence of AnxA1 or its receptor FPR2 aggravated illness in infected mice, treatment with AnxA1 agonistic peptide attenuated disease manifestationsatteanuated the symptoms of the disease. Both clinical outcomes were attributed to modulation of DENV-mediated viral load-independent MC degranulation. We have thereby identified that altered levels of the pro-resolving mediator AnxA1 are of pathological relevance in DENV infection, suggesting FPR2/ALX agonists as a therapeutic target for dengue disease.

The Molecular Structure and Role of Humanin in Neural and Skeletal Diseases, and in Tissue Regeneration.

Humanin (HN) belongs to a member of mitochondrial-derived peptides (MDPs) which are encoded by mitochondrial genes. HN shares sequence homology with thirteen HN-like proteins, named MTRNR2L1 to MTRNR2L13, which encompass 24–28 amino acid residues in length. HN mediates mitochondrial status and cell survival by acting via an intracellular mechanism, or as a secreted factor via extracellular signals. Intracellularly, it binds Bcl2-associated X protein (BAX), Bim and tBid, and IGFBP3 to inhibit caspase activity and cell apoptosis. When released from cells as a secreted peptide, HN interacts with G protein-coupled formyl peptide receptor-like 1 (FPRL1/2) to mediate apoptosis signal-regulating kinase (ASK) and c-Jun N-terminal kinase (JNK) signalling pathways. Additionally, it interacts with CNTFR-α/gp130/WSX-1 trimeric receptors to induce JAK2/STA3 signalling cascades. HN also binds soluble extracellular proteins such as VSTM2L and IGFBP3 to modulate cytoprotection. It is reported that HN plays a role in neuronal disorders such as Alzheimer’s disease, as well as in diabetes mellitus, infertility, and cardiac diseases. Its roles in the skeletal system are emerging, where it appears to be involved with the regulation of osteoclasts, osteoblasts, and chondrocytes. Understanding the molecular structure and role of HN in neural and skeletal diseases is vital to the application of HN in tissue regeneration.

Selective Display of a Chemoattractant Agonist on Cancer Cells Activates the Formyl Peptide Receptor 1 on Immune Cells.

Current immunotherapeutics often work by directing components of the immune system to recognize biomarkers on the surface of cancer cells to generate an immune response. However, variable changes in biomarker distribution and expression can result in inconsistent patient response. The development of a more universal tumor-homing strategy has the potential to improve selectivity and extend therapy to cancers with decreased expression or absence of specific biomarkers. Here, we designed a bifunctional agent that exploits the inherent acidic microenvironment of most solid tumors to selectively graft the surface of cancer cells with a formyl peptide receptor ligand (FPRL). Our approach is based on the pH(Low) insertion peptide (pHLIP), a unique peptide that selectively targets tumors in vivo by anchoring to cancer cells in a pH-dependent manner. We establish that selectively remodeling cancer cells with a pHLIP-based FPRL activates formyl peptide receptors on recruited immune cells, potentially initiating an immune response towards tumors.

Design, Synthesis, Biological Evaluation, and Computational Studies of Novel Ureidopropanamides as Formyl Peptide Receptor 2 (FPR2) Agonists to Target the Resolution of Inflammation in Central Nervous System Disorders.

Formyl peptide receptor 2 (FPR2) agonists can boost the resolution of inflammation and can offer alternative approaches for the treatment of pathologies with underlying chronic neuroinflammation, including neurodegenerative disorders. Starting from the FPR2 agonist 2 previously identified in our laboratory and through fine-tuning of FPR2 potency and metabolic stability, we have identified a new series of ureidopropanamide derivatives endowed with a balanced combination of such properties. Computational studies provided insights into the key interactions of the new compounds for FPR2 activation. In mouse microglial N9 cells and in rat primary microglial cells stimulated with lipopolysaccharide, selected compounds inhibited the production of pro-inflammatory cytokines, counterbalanced the changes in mitochondrial function, and inhibited caspase-3 activity. Among the new agonists, (S)-11l stands out also for the ability to permeate the blood-brain barrier and to accumulate in the mouse brain in vivo, thus representing a valuable pharmacological tool for studies in vivo.

Annexin-A1: The culprit or the solution?

Annexin‐A1 has a well‐defined anti‐inflammatory role in the innate immune system, but its function in adaptive immunity remains controversial. This glucocorticoid‐induced protein has been implicated in a range of inflammatory conditions and cancers, as well as being found to be overexpressed on the T cells of patients with autoimmune disease. Moreover, the formyl peptide family of receptors, through which annexin‐A1 primarily signals, has also been implicated in these diseases. In contrast, treatment with recombinant annexin‐A1 peptides resulted in suppression of inflammatory processes in murine models of inflammation. This review will focus on what is currently known about annexin‐A1 in health and disease and discuss the potential of this protein as a biomarker and therapeutic target.

Molecular recognition of formylpeptides and diverse agonists by the formylpeptide receptors FPR1 and FPR2

The formylpeptide receptors (FPRs) mediate pattern recognition of formylated peptides derived from invading pathogens or mitochondria from dead host cells. They can also sense other structurally distinct native peptides and even lipid mediators to either promote or resolve inflammation. Pharmacological targeting of FPRs represents a novel therapeutic approach in treating inflammatory diseases. However, the molecular mechanisms underlying FPR ligand recognition are elusive. We report cryo-EM structures of Gi-coupled FPR1 and FPR2 bound to a formylpeptide and Gi-coupled FPR2 bound to two synthetic peptide and small-molecule agonists. Together with mutagenesis data, our structures reveal the molecular mechanism of formylpeptide recognition by FPRs and structural variations of FPR1 and FPR2 leading to their different ligand preferences. Structural analysis also suggests that diverse FPR agonists sample a conserved activation chamber at the bottom of ligand-binding pockets to activate FPRs. Our results provide a basis for rational drug design on FPRs.

Efferocytosis and enhanced FPR2 expression following apoptotic cell instillation attenuate radiation-induced lung inflammation and fibrosis

Lung inflammation and fibrosis are common side effects of radiotherapy that can lead to serious reduction in the quality of life of patients. However, no effective treatment is available, and the mechanisms underlying its pathophysiology are poorly understood. Irradiation increases formyl peptide receptor 2 (FPR2) expression in lung tissue, and FPR2 agonists are known to promote the uptake of apoptosis cells, referred to as efferocytosis that is a hallmark of the resolution of inflammation. Herein, in a mouse model of radiation-induced lung injury (RILI), efferocytosis was induced by injecting apoptotic cells into the lung through the trachea, and its correlation with FPR expression and the effect of efferocytosis and FPR expression on RILI were assessed. Interestingly, when apoptotic cells were injected into the lung, the radiation-induced increase in FPR2 expression was further amplified. In the mouse model of RILI, apoptotic cell instillation reduced the volume of the damaged lung and prevented the decrease in lung function. Additionally, the expression of inflammatory cytokines, fibrosis-related markers, and oxidative stress-related markers was reduced by apoptotic cell instillation. Co-administration of apoptotic Jurkat cells and WRW4, the FPR2 antagonist, reversed these effects. These findings suggest that efferocytosis induced by apoptotic cell instillation and enhanced FPR2 expression attenuate RILI, thereby alleviating lung inflammation and fibrosis.

Abstract P5-08-06: MDX-124, a novel annexin-A1 antibody, shows anti-tumor efficacy in several preclinical models of triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) accounts for 10-15% of all breast cancer cases and is a particularly aggressive form of the disease, with poorer prognosis compared to other breast cancer subtypes. Advances in TNBC treatment have been made with the introduction of checkpoint inhibitors, PARP inhibitors and antibody drug conjugates. However, these therapies still have several limitations including low patient response rates, treatment resistance or relapse, and considerable side effects. Therefore, a significant unmet clinical need remains to develop novel targeted agents to treat TNBC. Annexin-A1 (ANXA1) is a phospholipid binding protein secreted in response to several physiological stimuli where it activates formyl peptide receptors (FPR1/2) triggering multiple oncogenic cell signaling pathways. Overexpression of ANXA1 by TNBC cells has been shown to influence several cancer-related processes including cell growth, cell cycle progression, angiogenesis, migration and invasion. In addition, ANXA1 has been shown to have immunomodulatory effects on T-cells, macrophages and dendritic cells. High ANXA1 expression in TNBC patients correlates with both poor overall survival and progression-free survival, indicating ANXA1 is a therapeutic target in TNBC. MDX-124 is a novel humanized antibody targeting ANXA1, and we have previously demonstrated its significant anti-proliferative activity. Here we present additional data showing the efficacy of MDX-124 in preclinical models of TNBC. Methods: The impact of MDX-124 on cell cycle progression was evaluated by measuring changes in DNA content using flow cytometry and analyzed with FlowJo™ software.The modulation of key oncogenic cell signalling pathways by MDX-124 was assessed using human phospho-kinase and XL Oncology Proteome Profiler™ antibody arrays. The effect of MDX-124 in combination therapy with chemotherapy was studied in-vitro using MTT cell proliferation assays, whilst synergy with anti-PD-1 therapy was evaluated using the EMT6 syngeneic mouse model of TNBC. Results: MDX-124 treatment dramatically decreased the proportion of HCC1806 TNBC cells in S-phase by 29.1% with a concomitant increase in G1 of 33.5% versus untreated cells. This occurred in a dose-dependent manner and is consistent with an MDX-124 mediated increase in cell cycle arrest.Treatment of HCC1806 TNBC cells with MDX-124 altered the expression of key oncogenic proteins and the phosphorylation of several kinases that regulate cell signaling pathways involved in proliferation, survival and migration. Notably, MDX-124 substantially reduced ERK and AKT phosphorylation by 84% and 72% respectively versus untreated control cells. In the HCC1806 TNBC cell line, proliferation was significantly reduced after 72 h treatment with MDX-124 and chemotherapy versus control.In the EMT6 syngeneic mouse model of TNBC, the murine analogue of MDX-124 (MDX-001) potentiated mean tumor growth inhibition of single agent anti-PD-1 treatment by 15%. Additionally, 30% of treated mice showed tumor regression in the MDX-001 combination therapy group versus 10% in the single agent anti-PD-1 group. This suggest that anti-ANXA1 antibody therapy acts synergistically with anti-PD-1 immunotherapy.Conclusion: MDX-124 binds to secreted and extracellular ANXA1 disrupting interactions with FPR1/2. This results in altered expression levels of several key cancer-related proteins preventing the activation of oncogenic signaling pathways that promote cancer progression. MDX-124 has demonstrated anti-cancer activity in several TNBC cell line and mouse models, as both a single agent and in combination with other drugs, including anti-PD-1 immunotherapy. Medannex plan to initiate a First-In-Human study in Q4 2021 to evaluate MDX-124 in solid malignancies, including TNBC. Citation Format: Fiona C Dempsey, Hussein Al-Ali, Scott J Crichton, Charlene Fabian, Chris Pepper, Christopher N Parris. MDX-124, a novel annexin-A1 antibody, shows anti-tumor efficacy in several preclinical models of triple-negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-08-06.

Pepducin-mediated G Protein-Coupled Receptor Signaling in the Cardiovascular System.

Pepducins are small-lipidated peptides designed from the intracellular loops of G protein-coupled receptors (GPCRs) that act in an allosteric manner to modulate the activity of GPCRs. Over the past 2 decades, pepducins have progressed initially from pharmacologic tools used to manipulate GPCR activity in an orthosteric site-independent manner to compounds with therapeutic potential that have even been used safely in phase 1 and 2 clinical trials in human subjects. The effect of pepducins at their cognate receptors has been shown to vary between antagonist, partial agonist, and biased agonist outcomes in various primary and clonal cell systems, with even small changes in amino acid sequence altering these properties and their receptor selectivity. To date, pepducins designed from numerous GPCRs have been studied for their impact on pathologic conditions, including cardiovascular diseases such as thrombosis, myocardial infarction, and atherosclerosis. This review will focus in particular on pepducins designed from protease-activated receptors, C-X-C motif chemokine receptors, formyl peptide receptors, and the β2-adrenergic receptor. We will discuss the historic context of pepducin development for each receptor, as well as the structural, signaling, pathophysiologic consequences, and therapeutic potential for each pepducin class.

Method Matters: Effect of Purification Technology on Neutrophil Phenotype and Function.

Neutrophils are the most abundant leukocytes in human blood and the first cells responding to infection and injury. Due to their limited ex vivo lifespan and the impossibility to cryopreserve or expand them in vitro, neutrophils need to be purified from fresh blood for immediate use in experiments. Importantly, neutrophil purification methods may artificially modify the phenotype and functional characteristics of the isolated cells. The aim of this study was to expose the effects of ‘classical’ density-gradient purification versus the more expensive but faster immunomagnetic isolation on neutrophil phenotype and functionality. We found that in the absence of inflammatory stimuli, density-gradient-derived neutrophils showed increased polarization responses as well as enhanced release of reactive oxygen species (ROS), neutrophil extracellular traps (NETs) and granular proteins compared to cells derived from immunomagnetic isolation, which yields mostly quiescent neutrophils. Upon exposure to pro-inflammatory mediators, immunomagnetic isolation-derived neutrophils were significantly more responsive in polarization, ROS production, phagocytosis, NETosis and degranulation assays, in comparison to density-gradient-derived cells. We found no difference in chemotactic response in Multiscreen and under-agarose migration assays, but Boyden assays showed reduced chemotaxis of immunomagnetic isolation-derived neutrophils. Finally, we confirmed that density-gradient purification induces artificial activation of neutrophils, evidenced by e.g. higher expression of CD66b, formyl peptide receptor 1 (FPR1) and CD35, and the appearance of a separate neutrophil population expressing surface molecules atypical for neutrophils (e.g. CXCR3, MHC-II and CD14). Based on these results, we recommend using immunomagnetic separation of neutrophils for studying neutrophil polarization, phagocytosis, ROS production, degranulation and NETosis, whereas for Boyden chemotaxis assays, the density-gradient purification is more suitable.

Multipathway In Vitro Pharmacological Characterization of Specialized Proresolving G Protein-Coupled Receptors.

Specialized proresolving mediators (SPMs) and their cognate G protein-coupled receptors are implicated in autoimmune disorders, including chronic inflammation, rheumatoid arthritis, systemic scleroderma, and lupus erythematosus. To date, six G protein-coupled receptors (GPCRs) have been paired with numerous endogenous and synthetic ligands. However, the function and downstream signaling of these receptors remains unclear. To address this knowledge gap, we systematically expressed each receptor in a human embryonic kindney 293 (HEK293)-Flp-In-CD8a-FLAG cell system. Each receptor was pharmacologically characterized with both synthetic and putative endogenous ligands across different signaling assays, covering both G protein-dependent (Gs, Gi, and Gq) and independent mechanisms (β-arrestin2 recruitment). Three orphan GPCRs previously identified as SPM receptors (GPR 18, GPR32 and GPR37) failed to express in HEK 293 cells. Although we were unsuccessful in identifying an endogenous ligand for formyl peptide receptor 2 (FPR2)/lipoxin A4 receptor (ALX), with only a modest response to N-formylmethionine-leucyl-phenylalanine (fMLP), we did reveal clear signaling bias away from extracelluar signal-related kinase (ERK) 1/2 phosphorylation for the clinically tested agonist N-(2-{[4-(1,1-difluoroethyl)-1,3-oxazol-2-yl]methyl}-2H-1,2,3-triazol-4-yl)-2-methyl-5-(3-methylphenyl)-1,3-oxazole-4-carboxamide (ACT-389949), adding further evidence for its poor efficacy in two phase I studies. We also identified neuroprotectin D1 as a new leukotriene B4 receptor 1 (BLT1) agonist, implying an alternative target for the neuroprotective effects of the ligand. We confirmed activity for resolvin E1 (RvE1) at BLT1 but failed to observe any response at the chemerin1 receptor. This study provides some much-needed clarity around published receptor-ligand pairings but indicates that the expression and function of these SPM GPCRs remains very much context-dependent. In addition, the identification of signaling bias at FPR2/ALX may assist in guiding design of new FPR2/ALX agonists for the treatment of autoimmune disorders. SIGNIFICANCE STATEMENT: To our knowledge, this is the first study to comprehensibly show how several natural mediators and synthetic ligands signal through three specialized proresolving mediator GPCRs using multiple ligands from different classes across four-six endpoint signaling assays. This study discovers new ligand pairings, refutes others, reveals poly-pharmacology, and identifies biased agonism in formyl peptide receptor 2/lipoxin A4 receptor pharmacology. This study highlights the potential of these receptors in treating specific autoimmune diseases, including rheumatoid arthritis, systemic scleroderma, and systemic lupus erythematosus.

Overriding impaired FPR chemotaxis signaling in diabetic neutrophil stimulates infection control in murine diabetic wound.

Infection is a major co-morbidity that contributes to impaired healing in diabetic wounds. Although impairments in diabetic neutrophils have been blamed for this co-morbidity, what causes these impairments and whether they can be overcome, remain largely unclear. Diabetic neutrophils, isolated from diabetic individuals, exhibit chemotaxis impairment but this peculiar functional impairment has been largely ignored because it appears to contradict the clinical findings which blame excessive neutrophil influx as a major impediment to healing in chronic diabetic ulcers. Here, we report that exposure to glucose in diabetic range results in impaired chemotaxis signaling through the formyl peptide receptor (FPR) in neutrophils, culminating in reduced chemotaxis and delayed neutrophil trafficking in the wound of Leprdb (db/db) type two diabetic mice, rendering diabetic wound vulnerable to infection. We further show that at least some auxiliary receptors remain functional under diabetic conditions and their engagement by the pro-inflammatory cytokine CCL3, overrides the requirement for FPR signaling and substantially improves infection control by jumpstarting the neutrophil trafficking toward infection, and stimulates healing in diabetic wound. We posit that CCL3 may have therapeutic potential for the treatment of diabetic foot ulcers if it is applied topically after the surgical debridement process which is intended to reset chronic ulcers into acute fresh wounds.

Neutrophils discriminate live from dead bacteria by integrating signals initiated by Fprs and TLRs.

The mechanisms whereby neutrophils respond differentially to live and dead organisms are unknown. We show here that neutrophils produce 5- to 30-fold higher levels of the Cxcl2 chemokine in response to live bacteria, compared with killed bacteria or isolated bacterial components, despite producing similar levels of Cxcl1 or pro-inflammatory cytokines. Secretion of high levels of Cxcl2, which potently activates neutrophils by an autocrine mechanism, requires three signals. The first two signals are provided by two different sets of signal peptides released by live bacteria, which selectively activate formylated peptide receptor 1 (Fpr1) and Fpr2, respectively. Signal 3 originates from Toll-like receptor activation by microbial components present in both live and killed bacteria. Mechanistically, these signaling pathways converge at the level of the p38 MAP kinase, leading to activation of the AP-1 transcription factor and to Cxcl2 induction. Collectively, our data demonstrate that the simultaneous presence of agonists for Fpr1, Fpr2, and Toll-like receptors represents a unique signature associated with viable bacteria, which is sensed by neutrophils and induces Cxcl2-dependent autocrine cell activation.

Methylprednisolone up-regulates annexin A1 (ANXA1) to inhibit the inflammation, apoptosis and oxidative stress of cigarette smoke extract (CSE)-induced bronchial epithelial cells, a chronic obstructive pulmonary disease in vitro model, through the formyl peptide receptor 2 (FPR2) receptors and the adenosine 5’-monophosphate (AMP)-activated protein kinase (AMPK) pathway

ABSTRACT Chronic obstructive pulmonary disease (COPD) is a progressive degenerative disease, of which smoking is the main causer. We carried out this study with the aim of exploring the underlying mechanism of methylprednisolone (MP) treating the COPD. To stimulate COPD in vitro, cigarette smoke extract (CSE)was employed to induce human bronchial epithelial cells BEAS-2B. With the help of MTT and Tunel assays, the viability and apoptosis of BEAS-2B cells after indicated treatment were assessed. The levels of inflammatory response and oxidative stress were determined by the changes of markers basing on their commercial kits. Additionally, annexin A1 (ANXA1) expressions at both protein and mRNA levels were assessed with Western blot and Reverse transcription‑quantitative PCR (RT-qPCR). Moreover, the expressions of apoptosis- and formyl peptide receptor 2 (FPR2) receptors and the adenosine 5’-monophosphate (AMP)-activated protein kinase (AMPK) pathway-related proteins were determined with Western blot., related proteins and proteins. As a result, MP up-regulated the ANXA1 expression in CSE-induced BEAS-2B cells. MP enhanced the viability but suppressed the apoptosis, inflammatory response and oxidative stress of CSE-induced BEAS-2B cells via regulating FPR2/AMPK pathway, while ANXA1 knockdown exhibited oppositive effects on them. In conclusion, MP up-regulated ANXA1 to inhibit the inflammation, apoptosis and oxidative stress of BEAS-2B cells induced by CSE, alleviating COPD through suppressing the FPR2/AMPK pathway.

Combined screening analysis of aberrantly methylated-differentially expressed genes and pathways in hepatocellular carcinoma.

Methylation plays an important role in hepatocellular carcinoma (HCC) by altering the expression of key genes. The aim of this study was to screen the aberrantly methylated-differentially expressed genes (DEGs) in HCC and elucidate their underlying molecular mechanism. Gene expression microarrays (GSE101685) and gene methylation microarrays (GSE44909) were selected. DEGs and differentially methylated genes (DMGs) were screened. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed using the Database for Annotation, Visualization, and Integrated discovery (DAVID). The Search Tool for the Retrieval of Interacting Genes (STRING) database was used to analyze the functional protein-protein interaction (PPI) network. Molecular Complex Detection (MCODE) analysis was performed using the Cytoscape software. Hub genes were verified in The Cancer Genome Atlas (TCGA) database. A total of 80 hypomethylation-high expression genes (Hypo-HGs) were identified. Pathway enrichment analysis showed DNA replication, cell cycle, viral carcinogenesis, and the spliceosome. The top 5 hub genes were minichromosome maintenance complex component 3 (MCM3), checkpoint kinase 1 (CHEK1), kinesin family member 11 (KIF11), PDZ binding kinase (PBK), and Rac GTPase activating protein 1 (RACGAP1). In addition, 189 hypermethylation-low expression genes (Hyper-LGs) were identified. Pathway enrichment analysis indicated enrichment in metabolic pathways, drug metabolism-other enzymes, and chemical carcinogenesis. The top 5 hub genes were leukocyte immunoglobulin like receptor B2 (LILRB2), formyl peptide receptor 1 (FPR1), S100 calcium binding protein A9 (S100A9), S100 calcium binding protein A8 (S100A8), and myeloid cell nuclear differentiation antigen (MNDA). The methylation status and mRNA expression of MCM3, CHEK1, KIF11, PBK, and S100A9 were consistent in the TCGA database and significantly correlated with the prognosis of patients. Combined screening of aberrantly methylated-DEGs based on bioinformatic analysis may provide new clues for elucidating the epigenetic mechanism in HCC. Hub genes, including MCM3, CHEK1, KIF11, PBK, and S100A9, may serve as biomarkers for the precise diagnosis of HCC.

INKT cells are enriched in inflammatory infiltrates of chronic rhinosinusitis with nasal polyposis

iNKT cells, a subset of T cells, are potent effectors of Th2-inflammation that have been associated with a range of allergic diseases, but their involvement in chronic rhinosinusitis with nasal polyposis (CRSwNP) has not been well studied. We investigated their presence and expression of prostaglandin D2 and lipoxin-A4 receptors, Chemoattractant receptor-homologue-Th2 (CRTH2) and formyl-peptide Receptor-2 respectively, in nasal polyp tissue of patients with CRSwNP, as inflammatory regulator, in the presence of commensal bacteria, Staphylococcus aureus.

Efficacy of MDX-124, a novel anti-annexin-A1 antibody, in preclinical models of pancreatic cancer.

590 Background: Pancreatic cancer is a highly fatal disease with poor survival and response to both chemotherapy and immunotherapy. Novel approaches to treat this disease are urgently required. Annexin-A1 (ANXA1) is secreted in response to several physiological stimuli where it activates formyl peptide receptors (FPR1/2) triggering multiple oncogenic processes. High ANXA1 expression in pancreatic cancer patients is associated with poor overall survival, and influences cancer progression, drug sensitivity, migration and invasion. MDX-124 is a novel humanized antibody targeting ANXA1 and we have previously presented data demonstrating its significant antiproliferative activity. Here we present further data showing the efficacy of MDX-124 in several preclinical models of pancreatic cancer. Methods: In-vitro models utilized MIA PaCa-2, PANC-1 or BxPC-3 human pancreatic cancer cell lines. Cell cycle progression was evaluated by measuring changes in DNA content via flow cytometry. Pancreatic cancer cell viability following incubation with MDX-124 (0-10 µM) and 5FU (IC50) was assessed via MTT assay. A transwell migration assay was used to evaluate the effect of MDX-124 (0-50 µM) on pancreatic cancer cell migration. In-vivo efficacy was evaluated using an orthotopic mouse model of metastatic pancreatic cancer (FC1242luc/zsGreen; KPC-derived cell line) with bioluminescent imaging used to quantify the incidence and burden of lung metastases. Results: When compared to untreated MIA PaCa-2 pancreatic cancer cells, MDX-124 treatment decreased the proportion of cells in S-phase by 29% and G2 phase by 9.1%, with a concomitant increase in G1 of 38.1%. This occurred in a dose-dependent manner and is consistent with an MDX-124 mediated increase in cell cycle arrest. MDX-124 significantly reduced the viability of MIA PaCa-2 and PANC-1 cell lines versus an IgG control in a dose-dependent manner. Additionally in these two cell lines, combination of MDX-124 with 5FU (IC50) had a significant synergistic impact reducing cancer cell viability by 99.8% and 91.2% respectively. Furthermore, MDX-124 significantly reduced the migratory ability of MIA PaCa-2 and BxPC-3 pancreatic cancer cells. In the orthotopic model of metastatic pancreatic cancer, the murine analog of MDX-124 (MDX-001), markedly reduced both the incidence and size of lung metastases. Conclusions: MDX-124 demonstrated significant anti-tumor efficacy in several preclinical models of pancreatic cancer as a single agent, with increased potency observed when used in combination with 5FU. Medannex will initiate a First-In-Human study in Q4 2021 to evaluate MDX-124 in solid malignancies, including pancreatic cancer.

Formyl peptide receptor 2 determines sex-specific differences in the progression of nonalcoholic fatty liver disease and steatohepatitis

Nonalcoholic fatty liver disease (NAFLD) is an important health concern worldwide and progresses into nonalcoholic steatohepatitis (NASH). Although prevalence and severity of NAFLD/NASH are higher in men than premenopausal women, it remains unclear how sex affects NAFLD/NASH pathophysiology. Formyl peptide receptor 2 (FPR2) modulates inflammatory responses in several organs; however, its role in the liver is unknown. Here we show that FPR2 mediates sex-specific responses to diet-induced NAFLD/NASH. NASH-like liver injury was induced in both sexes during choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) feeding, but compared with females, male mice had more severe hepatic damage. Fpr2 was more highly expressed in hepatocytes and healthy livers from females than males, and FPR2 deletion exacerbated liver damage in CDAHFD-fed female mice. Estradiol induced Fpr2 expression, which protected hepatocytes and the liver from damage. In conclusion, our results demonstrate that FPR2 mediates sex-specific responses to diet-induced NAFLD/NASH, suggesting a novel therapeutic target for NAFLD/NASH.

Behavioral, Anti-Inflammatory, and Neuroprotective Effects of a Novel FPR2 Agonist in Two Mouse Models of Autism.

Autism spectrum disorders (ASD) are a group of heterogeneous neurodevelopmental conditions characterized by social deficits, repetitive stereotyped behaviors, and altered inflammatory responses. Accordingly, children with ASD show decreased plasma levels of lipoxin A4 (LXA4), a mediator involved in the resolution of inflammation, which is the endogenous ligand of the formyl peptide receptor 2 (FPR2). To investigate the role of FPR2 in ASDs, we have used a new ureidopropanamide derivative able to activate the receptor, named MR-39. The effects of MR-39 (10 mg/kg, for 8 days) on hippocampal pro-inflammatory profile, neuronal plasticity, and social behavior were evaluated in two validated animal models of ASD: BTBR mouse strain and mice prenatally exposed to valproic acid (VPA). Primary cultures of hippocampal neurons from BTBR mice were also used to evaluate the effect of MR-39 on neurite elongation. Our results show that MR-39 treatment reduced several inflammatory markers, restored the low expression of LXA4, and modulated FPR2 expression in hippocampal tissues of both ASD animal models. These findings were accompanied by a significant positive effect of MR-39 on social behavioral tests of ASD mice. Finally, MR-39 stimulates neurite elongation in isolated hippocampal neurons of BTBR mice. In conclusion, these data indicate FPR2 as a potential target for an innovative therapeutical approach for the cure of ASD.

Effects of Formyl Peptide Receptor Agonists Ac9-12 and WKYMV in In Vivo and In Vitro Acute Inflammatory Experimental Models.

Formyl peptide receptors (Fprs) are a G-protein-coupled receptor family mainly expressed on leukocytes. The activation of Fpr1 and Fpr2 triggers a cascade of signaling events, leading to leukocyte migration, cytokine release, and increased phagocytosis. In this study, we evaluate the effects of the Fpr1 and Fpr2 agonists Ac9-12 and WKYMV, respectively, in carrageenan-induced acute peritonitis and LPS-stimulated macrophages. Peritonitis was induced in male C57BL/6 mice through the intraperitoneal injection of 1 mL of 3% carrageenan solution or saline (control). Pre-treatments with Ac9-12 and WKYMV reduced leukocyte influx to the peritoneal cavity, particularly neutrophils and monocytes, and the release of IL-1β. The addition of the Fpr2 antagonist WRW4 reversed only the anti-inflammatory actions of WKYMV. In vitro, the administration of Boc2 and WRW4 reversed the effects of Ac9-12 and WKYMV, respectively, in the production of IL-6 by LPS-stimulated macrophages. These biological effects of peptides were differently regulated by ERK and p38 signaling pathways. Lipidomic analysis evidenced that Ac9-12 and WKYMV altered the intracellular lipid profile of LPS-stimulated macrophages, revealing an increased concentration of several glycerophospholipids, suggesting regulation of inflammatory pathways triggered by LPS. Overall, our data indicate the therapeutic potential of Ac9-12 and WKYMV via Fpr1 or Fpr2-activation in the inflammatory response and macrophage activation.