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Commensal bacteria and/or their products engender beneficial effects to the mammalian gut, including stimulating physiological cellular turnover and enhancing wound healing, without activating overt inflammation. In the present study, we observed commensal bacteria-mediated activation of the noninflammatory extracellular signal-regulated kinase[ERK]/mitogen-activated protein kinase and Akt signaling pathways in gut epithelial cells and delineated a mechanism for this bacterially activated signaling. All tested strains of commensal bacteria induced ERK phosphorylation without stimulating pro-inflammatory phospho-IκB or pro-apoptotic phospho-c-Jun NH(2)-terminal kinase, with Lactobacillus species being most potent. This pattern of signaling activation was recapitulated using the peptide N-formyl-Met-Leu-Phe, a bacterial product known to stimulate signaling events in mammalian phagocytes. Sensing of N-formyl-Met-Leu-Phe by gut epithelial cells occurs via recently characterized formyl peptide receptors located in the plasma membrane. Both commensal bacteria and N-formyl-Met-Leu-Phe application to the apical surface of polarized gut epithelial cells resulted in specific formyl peptide receptor activation. In addition, pretreatment of model epithelia and murine colon with Boc2 (a specific peptide antagonist) or pertussis toxin (a G(i)-protein inhibitor) abolished commensal-mediated ERK phosphorylation. Taken together, these data show that commensal bacteria specifically activate the ERK/mitogen-activated protein kinase pathway in an formyl peptide receptor-dependent manner, delineating a mechanism by which commensal bacteria contribute to cellular signaling in gut epithelia.
The detection of invasive pathogens is critical for host immune defense. Cell surface receptors play a key role in the recognition of diverse microbe-associated molecules, triggering leukocyte recruitment, phagocytosis, release of antimicrobial compounds, and cytokine production. The intense evolutionary forces acting on innate immune receptor genes have contributed to their rapid diversification across plants and animals. However, the functional consequences of immune receptor divergence are often unclear. Formyl peptide receptors (FPRs) comprise a family of animal G protein-coupled receptors which are activated in response to a variety of ligands including formylated bacterial peptides, pathogen virulence factors, and host-derived antimicrobial peptides. FPR activation in turn promotes inflammatory signaling and leukocyte migration to sites of infection. Here we investigate patterns of gene loss, diversification, and ligand recognition among FPRs in primates and carnivores. We find that FPR1, which plays a critical role in innate immune defense in humans, has been lost in New World primates. Amino acid variation in FPR1 and FPR2 among primates and carnivores is consistent with a history of repeated positive selection acting on extracellular domains involved in ligand recognition. To assess the consequences of FPR divergence on bacterial ligand interactions, we measured binding between primate FPRs and the FPR agonist Staphylococcus aureus enterotoxin B, as well as S. aureus FLIPr-like, an FPR inhibitor. We found that few rapidly evolving sites in primate FPRs are sufficient to modulate recognition of bacterial proteins, demonstrating how natural selection may serve to tune FPR activation in response to diverse microbial ligands.
The formyl peptide receptor (Fpr) family is well known for its contribution to immune defense against pathogens in human and rodent leukocytes. Recently, several structurally related members of these receptors were discovered in sensory neurons of the mouse vomeronasal organ (VNO), key detectors of pheromones and related semiochemicals. Although the biological role of vomeronasal Fprs is not yet clear, the known contribution of other Fprs to host immune defense suggested that they could contribute to vomeronasal pathogen sensing. Precise knowledge about the agonist properties of mouse Fprs is required to determine their function. We expressed all seven mouse and three human Fprs using an in vitro system and tested their activation with 32 selected compounds by conducting high throughput calcium measurements. We found an intriguing functional conservation between human and mouse immune Fprs that is most likely a consequence of closely similar biological constraints. By contrast, our data suggest a neofunctionalization of the vomeronasal Fprs. We show that the vomeronasal receptor mFpr-rs1 can be activated robustly by W-peptide and structural derivatives but not by other typical ligands of immune Fprs. mFpr-rs1 exhibits a stereo-selective preference for peptides containing d-amino acids. The same peptide motifs are contained in pathogenic microorganisms. Thus, the ligand profile of mFpr-rs1 is consistent with a role in vomeronasal pathogen sensing.
Annexin A1 (ANXA1, lipocortin-1) is a glucocorticoid-regulated 37-kDa protein, so called since its main property is to bind (i.e. to annex) to cellular membranes in a Ca(2+)-dependent manner. Although ANXA1 has predominantly been studied in the context of immune responses and cancer, the protein can affect a larger variety of biological phenomena, including cell proliferation and migration. Our previous results show that endogenous ANXA1 positively modulates myoblast cell differentiation by promoting migration of satellite cells and, consequently, skeletal muscle differentiation. In this work, we have evaluated the hypothesis that ANXA1 is able to exert effects on myoblast cell migration acting through formyl peptide receptors (FPRs) following changes in its subcellular localization as in other cell types and tissues. The analysis of the subcellular localization of ANXA1 in C2C12 myoblasts during myogenic differentiation showed an interesting increase of extracellular ANXA1 starting from the initial phases of skeletal muscle cell differentiation. The investigation of intracellular Ca(2+) perturbation following exogenous administration of the ANXA1 N-terminal derived peptide Ac2-26 established the engagement of the FPRs which expression in C2C12 cells was assessed by qualitative PCR. Wound healing assay experiments showed that Ac2-26 peptide is able to increase migration of C2C12 skeletal muscle cells and to induce cell surface translocation and secretion of ANXA1. Our results suggest a role for ANXA1 as a highly versatile component in the signaling chains triggered by the proper calcium perturbation that takes place during active migration and differentiation or membrane repair since the protein is strongly redistributed onto the plasma membranes after an rapid increase of intracellular levels of Ca(2+). These properties indicate that ANXA1 may be involved in a novel repair mechanism for skeletal muscle and may have therapeutic implications with respect to the development of ANXA1 mimetics.
We investigated the effects of Crotoxin (CTX), the main toxin of South American rattlesnake (Crotalus durissus terrificus) venom, on Walker 256 tumor growth, the pain symptoms associated (hyperalgesia and allodynia), and participation of endogenous lipoxin A4. Treatment with CTX (s.c.), daily, for 5 days reduced tumor growth at the 5th day after injection of Walker 256 carcinoma cells into the plantar surface of adult rat hind paw. This observation was associated with inhibition of new blood vessel formation and decrease in blood vessel diameter. The treatment with CTX raised plasma concentrations of lipoxin A4 and its natural analogue 15-epi-LXA4, an effect mediated by formyl peptide receptors (FPRs). In fact, the treatment with Boc-2, an inhibitor of FPRs, abolished the increase in plasma levels of these mediators triggered by CTX. The blockage of these receptors also abolished the inhibitory action of CTX on tumor growth and blood vessel formation and the decrease in blood vessel diameter. Together, the results herein presented demonstrate that CTX increases plasma concentrations of lipoxin A4 and 15-epi-LXA4, which might inhibit both tumor growth and formation of new vessels via FPRs.
The signaling of membrane receptors is modified in obesity characterized by low-grade inflammation. The obesity-resistant state of organisms is poorly understood. We analyzed the generation of reactive oxygen species (ROS) initiated though membrane formyl peptide receptors (Fpr1, Fpr2) in bone-marrow granulocytes of obesity-resistant mice (ORM). A chemiluminescence assay was used to assess NADPH-oxidase-related intensity of ROS generation. ORM were chosen from animals that received high-fat diets and had metric body parameters as controls (standard diet). High spontaneous ROS production was observed in ORM cells. The EC50 for responses to bacterial or mitochondrial peptide N-formyl-MLF was higher in ORM with and without inflammation vs. the same control groups, indicating an insignificant role of high-affinity Fpr1. Increased responses to synthetic peptide WKYMVM (Fpr2 agonist) were observed in controls with acute inflammation, but they were similar in other groups. Fpr2 was possibly partially inactivated in ORM owing to the inflammatory state. Weakened Fpr1 and Fpr2 signaling via MAPKs was revealed in ORM using specific inhibitors for p38, ERK1/2, and JNK. P38 signaling via Fpr2 was lower in ORM with inflammation. Thus, a high-fat diet modified FPRs' role and suppressed MAPK signaling in NADPH-oxidase regulation in ORM. This result can be useful to understand the immunological features of obesity resistance.
An important hallmark of Alzheimer's disease (AD) is the increase of Aβ1-42 burden and its accumulation to senile plaques, leading the reactive gliosis and neurodegeneration. The modulation of glia cell function represents an attractive therapeutic strategy, but is currently limited by an incomplete understanding of its relevance for AD. The chemotactic G-protein coupled formyl peptide receptor (FPR), which is known to modulate Aβ1-42 uptake and signal transduction, might be one candidate molecule regulating glia function in AD. Here, we investigate whether the modulation of FPR exerts beneficial effects in an AD preclinical model.
A dogma of innate immunity is that neutrophils use G-protein-coupled receptors (GPCRs) for chemoattractant to chase bacteria through chemotaxis and then use phagocytic receptors coupled with tyrosine kinases to destroy opsonized bacteria via phagocytosis. Our current work showed that G-protein-coupled formyl peptide receptors (FPRs) directly mediate neutrophil phagocytosis. Mouse neutrophils lacking formyl peptide receptors (Fpr1/2-/-) are defective in the phagocytosis of Escherichia coli and the chemoattractant N-formyl-Met-Leu-Phe (fMLP)-coated beads. fMLP immobilized onto the surface of a bead interacts with FPRs, which trigger a Ca2+ response and induce actin polymerization to form a phagocytic cup for engulfment of the bead. This chemoattractant GPCR/Gi signaling works independently of phagocytic receptor/tyrosine kinase signaling to promote phagocytosis. Thus, in addition to phagocytic receptor-mediated phagocytosis, neutrophils also utilize the chemoattractant GPCR/Gi signaling to mediate phagocytosis to fight against invading bacteria.
Formyl peptide receptors (FPRs) are G-protein-coupled receptors that function as chemoattractant receptors in innate immune responses. Here we perform systematic structure-function analyses of FPRs from six mammalian species using structurally diverse FPR peptide agonists and identify a common set of conserved agonist properties with typical features of pathogen-associated molecular patterns. Guided by these results, we discover that bacterial signal peptides, normally used to translocate proteins across cytoplasmic membranes, are a vast family of natural FPR agonists. N-terminally formylated signal peptide fragments with variable sequence and length activate human and mouse FPR1 and FPR2 at low nanomolar concentrations, thus establishing FPR1 and FPR2 as sensitive and broad signal peptide receptors. The vomeronasal receptor mFpr-rs1 and its sequence orthologue hFPR3 also react to signal peptides but are much more narrowly tuned in signal peptide recognition. Furthermore, all signal peptides examined here function as potent activators of the innate immune system. They elicit robust, FPR-dependent calcium mobilization in human and mouse leukocytes and trigger a range of classical innate defense mechanisms, such as the production of reactive oxygen species, metalloprotease release, and chemotaxis. Thus, bacterial signal peptides constitute a novel class of immune activators that are likely to contribute to mammalian immune defense against bacteria. This evolutionarily conserved detection mechanism combines structural promiscuity with high specificity and enables discrimination between bacterial and eukaryotic signal sequences. With at least 175,542 predicted sequences, bacterial signal peptides represent the largest and structurally most heterogeneous class of G-protein-coupled receptor agonists currently known for the innate immune system.
Non-responsiveness to anti-TNF-α therapies presents relevant rates in inflammatory bowel disease patients, presenting the need to find biomarkers involved in therapeutic efficacy. Herein, we demonstrate that higher levels of colonic formyl peptide receptor 1 and annexin A1 correlate with histological recovery in Crohn's disease patients under remission. Using the dextran sulfate sodium colitis model in mice, we suggest that infliximab induces annexin A1 expression and secretion in activated intestinal leukocytes. Conversely, this mechanism might stimulate epithelial formyl peptide receptors, inducing wound healing and consequent histological remission. Our data indicate that assessing intestinal expressions of formyl peptide receptors and annexin A1 might provide precious information on the disease activity and responsiveness to infliximab in inflammatory bowel disease patients.
The Crotalus durissus terrificus rattlesnake venom, its main toxin, crotoxin (CTX), and its crotapotin (CA) and phospholipase A2 (CB) subunits modulate the immune system. Formyl peptide receptors (FPRs) and lipoxin A4 (LXA4) are involved in CTX's effect on macrophages and neutrophils. Dendritic cells (DCs) are plasticity cells involved in the induction of adaptive immunity and tolerance maintenance. Therefore, we evaluated the effect of CTX, CA or CB on the maturation of DCs derived from murine bone marrow (BM). According to data, CTX and CB-but not CA-induced an increase of MHC-II, but not costimulatory molecules on DCs. Furthermore, CTX and CB inhibited the expression of costimulatory and MHC-II molecules, secretion of proinflammatory cytokines and NF-κBp65 and p38/ERK1/2-MAPK signaling pathways by LPS-incubated DCs. Differently, CTX and CB induced IL-10, PGE2 and LXA4 secretion in LPS-incubated DCs. Lower proliferation and IL-2 secretion were verified in coculture of CD3+ cells and DCs incubated with LPS plus CTX or CB compared with LPS-incubated DCs. The effect of CTX and CB on DCs was abolished in cultures incubated with a FPRs antagonist. Hence, CTX and CB exert a modulation on functional activity of DCs; we also checked the involvement the FPR family on cell activities.
Mesoglycan is a mixture of glycosaminoglycans (GAG) with fibrinolytic effects and the potential to enhance skin wound repair. Here, we have used endothelial cells isolated from wild-type (WT) and Syndecan-4 null (Sdc4-/-) C57BL/6 mice to demonstrate that mesoglycan promotes cell motility and in vitro angiogenesis acting on the co-receptor Syndecan-4 (SDC4). This latter is known to participate in the formation and release of extracellular vesicles (EVs). We characterized EVs released by HUVECs and assessed their effect on angiogenesis. Particularly, we focused on Annexin A1 (ANXA1) containing EVs, since they may contribute to tube formation via interactions with Formyl peptide receptors (FPRs). In our model, the bond ANXA1-FPRs stimulates the release of vascular endothelial growth factor (VEGF-A) that interacts with vascular endothelial receptor-2 (VEGFR2) and activates the pathway enhancing cell motility in an autocrine manner, as shown by wound healing/invasion assays, and the induction of endothelial to mesenchymal transition (EndMT). Thus, we have shown for the first time that mesoglycan exerts its pro-angiogenic effects in the healing process triggering the activation of the three interconnected molecular axis: mesoglycan-SDC4, EVs-ANXA1-FPRs, and VEGF-A-VEGFR2.
A pepducin is a lipopeptide containing a peptide sequence that is identical to one of the intracellular domains of the G-protein coupled receptor (GPCR) assumed to be the target. Neutrophils express two closely related formyl peptide receptors belonging to the family of GPCRs; FPR1 and FPR2 in human and their respective orthologue Fpr1 and Fpr2 in mouse. By applying the pepducin concept, we have earlier identified FPR2 activating pepducins generated from the third intracellular loop of FPR2. The third intracellular loop of FPR2 differs in two amino acids from that of FPR1, seven from Fpr2 and three from Fpr1. Despite this, we found that pepducins generated from FPR1, FPR2, Fpr1 and Fpr2 all targeted FPR2 in human neutrophils and Fpr2 in mouse, but with different modulating outcomes. Whereas the FPR1/Fpr1 derived pepducins inhibited the FPR2 function in human neutrophils, they activated Fpr2 in mouse. The FPR2 derived pepducin activated FPR2/Fpr2, whereas the pepducin generated from Fpr2 inhibited both FPR2 and Fpr2. In summary, our data demonstrate that pepducins generated from the third intracellular loop of human FPR1/2 and mouse Fpr1/2, all targeted FPR2 in human and Fpr2 in mouse. With respect to the modulating outcomes, pepducin inhibitors identified for FPR2 are in fact activators for Fpr2 in mouse neutrophils. Our data thus questions the validity of pepducin concept regarding their receptor selectivity but supports the notion that FPR2/Fpr2 may recognize a lipopeptide molecular pattern, and highlight the differences in ligand recognition profile between FPR2 and its mouse orthologue Fpr2.
The functional activity of the phagocytes, as well as the development and resolution of the inflammation, is determined by formylpeptide receptors (FPRs) signaling. There is a growing data on the signaling pathways from two major types of formylpeptide receptors, FPR1 and FPR2, which could be activated by different sets of ligands to provide certain defense functions. Generation of reactive oxygen species (ROS) by the membrane enzyme NADPH oxidase is the most important among them. One of the most studied and significant mechanism for the regulation of activity of NADPH oxidase is phosphorylation by a variety of kinases, including MAP kinases. The question arose whether the role of MAPKs differ in the activation of NADPH oxidase through FPR1 and FPR2. We have studied Fpr1- and Fpr2-induced phosphorylation of p38, ERK, and JNK kinases and their role in the activation of the respiratory burst in isolated mice bone marrow granulocytes. Data has shown distinct patterns of MAP kinase activity for Fpr1 and Fpr2: JNK was involved in both Fpr1 and Fpr2 mediated activation of ROS production, while p38 MAPK and ERK were involved in Fpr1 induced ROS generation only.
The soluble urokinase plasminogen activator receptor (suPAR) has been implicated in a wide range of pathological conditions including primary nephrotic syndromes and acute kidney injuries. suPAR can trigger transduction cascades in podocytes by outside-in activation of αVβ3-integrin, but there is evidence that the functional cell surface response element is actually a complex of different types of receptors, which may also include the receptor for advanced glycation end-products (RAGE) and formyl peptide receptors (FPRs). Here we observed that ROS accumulation and Src activation could be evoked by continuous 24 h exposure to either suPAR or the FPR agonist fMLF. Responses to suPAR and fMLF were completely blocked by either the FPR antagonist WRW4 or by the αV-integrin inhibitor cilengitide. Moreover, endogenous podocyte mouse Fpr1 co-immunoprecipitates with β3-integrin, suggesting that these receptors occur as a complex on the cell surface. suPAR- and fMLF-evoked activation of Src and ROS differed in time course. Thus, robust pertussis toxin (PTX)-sensitive responses were evoked by 60 min exposures to fMLF but not to suPAR. By contrast, responses to 24 h exposures to either suPAR or fMLF were PTX-resistant and were instead abolished by knockdown of β-arrestin-1 (BAR1). FPRs, integrins, and RAGE (along with various Toll-like receptors) can all function as pattern-recognition receptors that respond to "danger signals" associated with infections and tissue injury. The fact that podocytes express such a wide array of pattern-recognition receptors suggests that the glomerular filter is designed to change its function under certain conditions, possibly to facilitate clearance of toxic macromolecules.
As the immune cells of the brain, microglia are crucial for the maintenance of brain function. The aims of the present study were to determine whether and how annexin-1 is able to affect microglial phenotype and migration in the lesion microenvironment. In the current experiment, we enhanced the interaction between annexin-1 and formyl peptide receptors in microglia and analyzed the function. We found that annexin-1 could polarize microglia to a beneficial phenotype and promote microglial migration to protect neurons from ischemia-like injury, and the annexin-1-mediated neuroprotective effect was dependent on the release of glutamate and ATP from the injured neurons.
N-formyl peptide receptors (FPR1, FPR2, and FPR3) play key roles in the regulation of inflammatory processes, and recently, it was demonstrated that FPR1 and FPR2 have a dual role in the progression/suppression of some cancers. Therefore, FPRs represent an important therapeutic target for the treatment of both cancer and inflammatory diseases. Previously, we identified selective or mixed FPR agonists with pyridazinone or pyridinone scaffolds showing a common 4-(bromophenyl)acetamide fragment, which was essential for activity. We report here new pyrazole and pyrazolone derivatives as restricted analogues of the above 6-membered compounds, all exhibiting the same 4-bromophenylacetamide side chain. Most new products had low or absent FPR agonist activity, suggesting that the pyrazole nucleus was not appropriate for FPR agonists. This hypothesis was confirmed by molecular modeling studies, which highlighted that the five-membered scaffold was responsible for a worse arrangement of the molecules in the receptor binding site.
Neutrophils express several G-protein coupled receptors (GPCRs) and they cross regulate each other. We described a novel cross-talk mechanism in neutrophils, by which signals generated by the receptor for ATP (P2Y2) reactivate desensitized formyl peptide receptors (FPRs) so that these ligand-bound inactive FPRs resume signaling. At the signaling level, the cross-talk was unidirectional, i.e., P2Y2 ligation reactivated FPR, but not vice versa and was sensitive to the phosphatase inhibitor calyculinA. Further, we show that the cross talk between P2Y2 and FPR bypassed cytosolic Ca(2+) transients and did not rely on the actin cytoskeleton. In summary, our data demonstrate a novel cross-talk mechanism that results in reactivation of desensitized FPRs and, an amplification of the neutrophil response to ATP.
This study aimed to determine whether formyl peptide receptors (FPRs) regulated the differentiation of neural stem cells (NSCs). FPRs promote the migration of NSCs both in vitro and in vivo. However, the role of FPRs during differentiation of NSCs is unknown. Analysis by Western blot showed significantly increased expression of FPR1 and FPR2 during differentiation of NSCs. The activation of FPRs promotes NSCs to differentiate into neurons with more primary neurites and branch points and longer neurites per cell. Meanwhile, this activation also inhibits the differentiation of NSC into astrocytes. This bidirectional effect can be inhibited by the FPRs-specific inhibitor. Moreover, it was found that the activation of FPRs increased the generation of reactive oxygen species (ROS) and phosphorylation of AKT in the NSCs, while N-acetylcysteine and LY294002 inhibited the FPRs-stimulated increase in ROS generation and AKT phosphorylation, and blocked the FPRs-stimulated neural differentiation into neurons. Therefore, FPRs-stimulated neural differentiation was mediated via ROS and PI3K-AKT signaling pathways. Collectively, the present findings provided a novel insight into the functional role of FPRs in neurogenesis, with important implications for its potential use as a candidate for treating brain or spinal cord injury.
In inflammation, pain is regulated by a balance of pro- and analgesic mediators. Analgesic mediators include opioid peptides which are secreted by neutrophils at the site of inflammation, leading to activation of opioid receptors on peripheral sensory neurons. In humans, local opioids and opioid peptides significantly downregulate postoperative as well as arthritic pain. In rats, inflammatory pain is induced by intraplantar injection of heat inactivated Mycobacterium butyricum, a component of complete Freund's adjuvant. We hypothesized that mycobacterially derived formyl peptide receptor (FPR) and/or toll like receptor (TLR) agonists could activate neutrophils, leading to opioid peptide release and inhibition of inflammatory pain. In complete Freund's adjuvant-induced inflammation, thermal and mechanical nociceptive thresholds of the paw were quantified (Hargreaves and Randall-Selitto methods, respectively). Withdrawal time to heat was decreased following systemic neutrophil depletion as well as local injection of opioid receptor antagonists or anti-opioid peptide (i.e. Met-enkephalin, beta-endorphin) antibodies indicating an increase in pain. In vitro, opioid peptide release from human and rat neutrophils was measured by radioimmunoassay. Met-enkephalin release was triggered by Mycobacterium butyricum and formyl peptides but not by TLR-2 or TLR-4 agonists. Mycobacterium butyricum induced a rise in intracellular calcium as determined by FURA loading and calcium imaging. Opioid peptide release was blocked by intracellular calcium chelation as well as phosphoinositol-3-kinase inhibition. The FPR antagonists Boc-FLFLF and cyclosporine H reduced opioid peptide release in vitro and increased inflammatory pain in vivo while TLR 2/4 did not appear to be involved. In summary, mycobacteria activate FPR on neutrophils, resulting in tonic secretion of opioid peptides from neutrophils and in a decrease in inflammatory pain. Future therapeutic strategies may aim at selective FPR agonists to boost endogenous analgesia.
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