The Role of Formyl Peptide Receptors in Permanent and Low-Grade Inflammation: Helicobacter pylori Infection as a Model

Formyl peptide receptors (FPRs) are cell surface pattern recognition receptors (PRRs), belonging to the chemoattractant G protein-coupled receptors (GPCRs) family. They play a key role in the innate immune system, regulating both the initiation and the resolution of the inflammatory response. FPRs were originally identified as receptors with high binding affinity for bacteria or mitochondria N-formylated peptides. However, they can also bind a variety of structurally different ligands. Among FPRs, formyl peptide receptor-like 1 (FPRL1) is the most versatile, recognizing N-formyl peptides, non-formylated peptides, and synthetic molecules. In addition, according to the ligand nature, FPRL1 can mediate either pro- or anti-inflammatory responses. Hp(2-20), a Helicobacter pylori-derived, non-formylated peptide, is a potent FPRL1 agonist, participating in Helicobacter pylori-induced gastric inflammation, thus contributing to the related site or not-site specific diseases. The aim of this review is to provide insights into the role of FPRs in H. pylori-associated chronic inflammation, which suggests this receptor as potential target to mitigate both microbial and sterile inflammatory diseases.


Introduction
The human body is continuously exposed to millions of pathogens, which can enter it through different ways. The immune system, and in particular, innate immunity, has an important role in controlling the infections, representing the front-line defense against invading pathogens [1][2][3].
Innate immunity is mediated by anatomic barriers (skin and mucosa), physiological functions (pH values, temperature, chemical mediators), and cellular elements [4]. Skin and mucosal surfaces-including the gastrointestinal tract, the respiratory tract, the urogenital tract, and ocular surface-are the earliest sites of host-pathogen interactions, separating the body from the external environment [3,5]. This feature makes them directly involved in preventing the entry of invading microorganisms [3], acting as chemical-physical barriers [6]. Nevertheless, mechanical or chemical cleansing mechanisms provided by anatomic barriers can fail, promoting the action of other innate immune elements-specifically, immune cells, which participate to potentiate the protective role of the mucosal surface. The mucosal immune system, indeed, comprises approximately 80% of all the immune cells (macrophages, monocytes, neutrophils, dendritic cells, B and T cells, and natural killer cells) [6,7], which express specific receptors called pattern recognition receptors (PRRs).
Pattern recognition receptors are germline-encoded receptors, evolutionary selected and conserved. They mediate innate immune recognition by detecting conserved molecular structures associated with pathogens or host tissue/cell damage, also called pathogen-

Formyl Peptide Receptors: Cell Distribution and Classification
Formyl peptide receptors are a family of classical chemoattractant receptors. Human cells present three different isoforms of FPRs: FPR1, FPR2, or formyl peptide receptorlike 1 (FPRL1), and FPR3 or Formyl peptide receptor-like 2 (FPRL2). Each receptor is encoded by a specific gene of the human FPR gene family (FPR1, FPR2, FPR3) clustered on the chromosomal region 19q13.3 [14]. FPR1, FPR2, and FPR3 genes share high level of sequence homology [9], resulting in receptor similarity. Compared to FPR1, FPRL1 has one additional amino acid, for a total of 351 residues, and a sequence homology corresponding to 69% between the two receptors. Meanwhile, compared to FPR1, FPRL2 has two additional amino acids, for a total of 352, and a sequence homology of 56% [14]. Despite the high sequence identity among the three FPRs, they differ in cell expression, role (Table 1), and ligand recognition ( Table 2). Phagocytic cells were the first in which FPRs were identified. Monocytes express the three FPR genes, but their expression can change in differentiating monocytes. Specifically, monocytes differentiating in macrophages maintain FPRL1 expression, while the transition from immature to mature dendritic cells determines the loss of FPR1. The role of FPRs in macrophage and dendritic cell differentiation has been widely investigated, demonstrating that WKYMV (an FPR ligand) negatively regulates LPS-induced dendritic cell differentiation [15,16], while Lipoxin-A4, LL-37, and serum amyloid A protein (SAA) (FPRL1 agonists) mediate macrophage polarization to the antiinflammatory form M2 [16]. These findings suggest that FPRs play a regulatory function in immune cell differentiation and activation. Unlike monocytes, neutrophils express both FPR1 and FPRL1, but they lack the FPRL2 gene [14]. Recently, adaptative immune cells were discovered to express FPRs. FPRL1 was found in human tonsillar follicular helper T cells, Th1 cells, Th2 cells, Th17 cells, and in naïve CD4 T cells (CD3+, CD4+, CD45RA+, CD45RO-, and CCR7+) [16,17].
In addition, FPR expression has also been described in platelets, microglial cells, epithelial cells, hepatocytes, fibroblasts, and endothelial cells, as well as spleen, brain, placenta, liver, and further tissues, demonstrating the almost ubiquitous distribution of these receptors [18]. The wide expression of formyl peptide receptors in immune and non-immune cells, including various organs and tissues, suggests their involvement in multiple biological functions, with inflammation as the core.
Among the three receptors, formyl peptide receptor-like 1 is the most ubiquitous one. It has been found in different non-myeloid-endothelial cells, epithelial cells, as well as the hepatocyte-and tissue-gastrointestinal tract, brain, spleen, female organ tissues, pancreas, and endocrine glands [19,20]. Furthermore, compared to FPR1 and FPRL2, FPRL1 can interact with the highest variety of chemically different ligands, including non-formylated peptides, organic molecules, and lipid mediators [19]. These properties make FPRL1 the most promiscuous formyl peptide receptor, and more generally, G-protein coupled receptor (GPCR), able to elicit different cell responses, with inflammation as the common feature.

FPRs: G-Protein Coupled Receptors
FPRs belong to the family of G-protein coupled receptors (GPCRs), the largest class of cell surface seven-transmembrane proteins. Conventionally, GPCRs are located on the cell surface. However, they can also be expressed in the cell nucleus [46]. Recent studies have demonstrated the nuclear localization of FPRL1 in cancer cell lines [46], providing a further element confirming the versatility of this receptor.
More than 3% of the human genome encodes 800 GPCRs, mainly involved in regulating physiological processes by responding to endogenous ligands, such as hormones, neurotransmitters, chemokines, and calcium ions, or to exogenous ligands, such as odorant molecules and light photons [47,48]. Like hormones and neurotransmitter receptors, GPCRs regulate the communication between the inside and outside of the cells [49], transducing the extracellular signals in cellular response.
GPCRs are coupled to heterotrimeric G proteins, consisting of Gα, Gβ, and Gγ subunits, which initiate the downstream signaling associated with the receptor. Ligand binding stimulates GPCR conformational change and the activation of the heterotrimeric G proteins, resulting in guanosine triphosphate (GTP)-bound Gα and Gβ-γ subunit dissociation. In the active state, G proteins activate specific enzymes, which in turn generate second messengers, leading to cellular response [50].
Chemiotaxis are a common cellular response initiated by GPCRs, expressed on chemotactic cells. This subfamily of G protein-coupled receptors plays an important role in sensing chemoattractant molecules [51], coordinating cell migration at the damaged site, and initiation of the immune response.
N-formylated peptides are one of the first identified chemotactic stimuli [14], exhibiting high affinity for G protein-coupled receptors [51]. This has been demonstrated by the observation that the pertussis toxin inhibits formyl peptide-induced chemotaxis, altering their binding affinity for the receptor [52]. More specifically, the pertussis toxin acts by inducing ADP-ribosylation of the α subunit of a Gi-heterotrimeric G protein [53], affecting the molecular shifts responsible for the cell signal translation pathway and specifically inhibiting cell migration.
Based on this evidence, FPRs are Gi protein-coupled receptors-belonging to the γ-group of rhodopsin-like receptors-able [54] to recognize the presence of bacteria or host cell damage and promote the immune response by activating the chemiotaxis.

N-Formylated Peptides: Signal Peptides Detected by FPRs
The capability to distinguish between self and non-self is fundamental for the wellness of the host. The immune system has the essential role of initiating a defensive response against pathogens, by detecting microbial chemical elements: pathogen-associated molecular patterns (PAMPs). Bacterial signal peptides represent an important class of PAMPs, responsible for innate immune system activation by interacting with specific pattern recognition receptors (PRRs). They play a critical role in nascent protein translocation from the cytoplasm to other sites inside or outside the cell [55,56]. Chemotaxis, pro-inflammatory activity [16] Macrophages FPR1, FPRL1, FPRL2 Chemotaxis, pro-inflammatory activity [16] Adaptative Immune Cell Expression Interferon-γ production [16,17] Th1 cells FPRL1 - [16,17] Th2 FPRL1 - [16,17] Th17 FPRL1 - [16,17] Non-Immune Cells, Organ/Tissue Expression Epithelial cells FPRL1 Chemotaxis [18,24] Endothelial cells FPRL1 Chemotaxis, angiogenesis, and cell proliferation [25] Microglial cells FPRL1, FPRL2 Inflammation and neurogenerative activity [18,26] Keratinocytes FPRL1 Cell proliferation and pro-inflammatory activity [27] Fibroblasts FPRL1 Chemotaxis and innate immune response stimulation [28] Astrocytes FPRL1 Inflammation and neurogenerative activity [18,26] Hepatocytes FPRL1 Chemotaxis, angiogenesis [18,29] Intestinal epithelial cells FPR1, FPRL1 Cell proliferation, inflammation, and tumorigenesis [30] Brain FPRL1 Inflammation and neurodegenerative activity [16][17][18]    Signal peptides at the N-terminal of the newly synthesized proteins are recognized by specific particles responsible for locating the proteins at the correct site. After translocation, signal peptides are removed by specific enzymes and the mature protein is released [56,57]. Prokaryotic and eukaryotic signal peptides present three highly conserved domains, containing the correct information for protein translocation and removal: an N-terminal region, starting with a methionine, a hydrophobic core, and a C-terminal region [58]. However, all prokaryotes start the protein synthesis with an N-formyl methionine [59], resulting in the fact that bacterial signal peptides are "N-formylated peptides". Mitochondria, as bacterial ancestry [60], also initiate proteins synthesis with a formylated methionine, revealing that, besides bacteria, damaged host cells also release N-formylated peptides as damage-associated molecular patterns (DAMPs) [61].
These peptides thus represent a host danger signal, which induces innate immunity activation via specific pattern recognition receptors, corresponding to formyl peptide receptors-named according to their high affinity for N-formylated peptides.

More Than N-Formylated Peptides: Other Ligands Detected by FPRL1
FPRs were originally discovered as receptors with high binding affinity for bacteria or mitochondria N-formylated peptides. In addition to N-formyl-methionine-leucylphenylalanine (fMLF), the prototype for N-formylated peptides [18,21], a variety of structurally different ligands have been shown to interact with formyl peptide receptors.
Compared to FPR1, FPRL1 exhibits a lower affinity for formylated peptides. On the contrary, FPRL1 shows the greatest ligand promiscuity, also detecting non-formylated peptides, synthetic molecules, and the eicosanoid Lipoxin A4 (LXA4) [20]. WKYMV is a synthetic, non-formylated peptide recognized by FPRL1. Figure 1 shows the interaction between FPRL1 and WKYMV, displaying the FPRL1 binding site. According to the nature of its ligand, FPRL1 modifies its conformation and consequently its biological function, eliciting a pro-or anti-inflammatory response. In addition, anti-inflammatory ligands may cause receptor homodimerization or heterodimerization with other FPRs, resulting in the activation of the pro-resolving pathway p38/MAPKAPK/Hsp27/IL10, and in the resolution of the inflammation [20,62,63]. The ability of FPRL1 to bind ligands, mediating opposite biological effects, makes it a potential target to control inflammation and inflammatory-associated diseases. resolution of the inflammation [20,62,63]. The ability of FPRL1 to bind ligands, mediating opposite biological effects, makes it a potential target to control inflammation and inflammatory-associated diseases.

Helicobacter pylori and Chronic Inflammatory Response
Helicobacter pylori is a Gram-negative bacterium colonizing the gastric mucosa of over 50% of the population worldwide [64]. The bacterium colonizes the stomach and infects gastric epithelial cells, promoting chronic inflammation, leading to chronic gastritis, which can eventually degenerate in peptic ulcer and then gastric carcinoma [65]. Several studies have reported the relationship between H. pylori infection and gastric cancer, classifying the bacterium as the primary cause of gastric carcinoma [66]. Our recent work demonstrates the capability of H. pylori to interfere also with biological processes outside the stomach, contributing to the development of neurodegenerative diseases, such as Alzheimer, or metabolic pathologies (type 2 diabetes, obesity, and cardiovascular diseases) [67]. Although the mechanisms responsible for the extra-gastric manifestations of H. pylori remain unclear, a plausible explanation may reside in the H. pylori-associated, low-grade inflammatory state. Therefore, the clinical outcome of H. pylori infection is related to the severity of the inflammatory response, influenced by both host characteristics and bacterial virulence factors [65].
H. pylori utilizes various virulence factors responsible for its pathogenicity by inducing different pathways, resulting in cytokine and chemokine release, as well as the production of oxygen/nitrogen species (ROS/RNS) and growth factors [68]. These factors-especially in chronic conditions-contribute to tissue damage and severe disease progression.

Helicobacter pylori and Chronic Inflammatory Response
Helicobacter pylori is a Gram-negative bacterium colonizing the gastric mucosa of over 50% of the population worldwide [64]. The bacterium colonizes the stomach and infects gastric epithelial cells, promoting chronic inflammation, leading to chronic gastritis, which can eventually degenerate in peptic ulcer and then gastric carcinoma [65]. Several studies have reported the relationship between H. pylori infection and gastric cancer, classifying the bacterium as the primary cause of gastric carcinoma [66]. Our recent work demonstrates the capability of H. pylori to interfere also with biological processes outside the stomach, contributing to the development of neurodegenerative diseases, such as Alzheimer, or metabolic pathologies (type 2 diabetes, obesity, and cardiovascular diseases) [67]. Although the mechanisms responsible for the extra-gastric manifestations of H. pylori remain unclear, a plausible explanation may reside in the H. pylori-associated, low-grade inflammatory state. Therefore, the clinical outcome of H. pylori infection is related to the severity of the inflammatory response, influenced by both host characteristics and bacterial virulence factors [65].
H. pylori utilizes various virulence factors responsible for its pathogenicity by inducing different pathways, resulting in cytokine and chemokine release, as well as the production of oxygen/nitrogen species (ROS/RNS) and growth factors [68]. These factors-especially in chronic conditions-contribute to tissue damage and severe disease progression.
Lipopolysaccharide (LPS), peptidoglycan, and the cytotoxic-associated gene A (CagA) are the most studied H. pylori pathogenicity factors [68,69]. The gene CagA encodes the cytotoxic protein CagA, whose intracellular signaling results in the activation of inflammatory genes and in the modification of the cell scaffold, thus promoting neoplastic transformation [70]. LPS and peptidoglycan instead are directly involved in the bacterium's adhesion to the gastric epithelium [64]. As these structures are unique to the pathogen, known as pathogen-associated molecular patterns (PAMPs), they are recognized by pattern recognition receptors (PRRs) displayed by eukaryotic cells. Specifically, Toll-like receptor 4 (TLR4) binds LPS, while peptidoglycan is recognized by gastric cells through nucleotide oligomerization domain 1 (NOD1) [68].
FPRs are one of the most relevant class of PRRs. They play an important role in H. pylori infection by interacting with Hp(2-20), an H. pylori-released, non-formylated peptide with the greatest affinity for formyl peptide receptor like-1. Hp(2-20) is a chemotactic factor [34], contributing to Helicobacter pylori-induced gastric inflammation [71,72].

Helicobacter pylori Hp(2-20) Modulated the Host Immune Response by Interacting with FPRL1
Hp(2-20) is a cecropin-like peptide released during H. pylori growth and possessing different functional characteristics. As an antimicrobial peptide (AMP), Hp (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) plays an important role in H. pylori gastric mucosa persistence by acting as bactericidal molecule [73], thus conferring an advantage over other microorganisms. Moreover, it orchestrates the host inflammatory response by interacting with FPRs [34]. When H. pylori colonizes the human stomach, the epithelial cells of the gastric mucosa represent the first component of the innate immune response to be encountered [74]. Hp (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) participates to activate the innate immune response by interacting with the FPRL1 expressed on epithelial gastric cells. In particular, the interaction between Hp(2-20) and FPRL1 favors H. pylori gastric mucosa inflammation [71] by initiating a cell signaling cascade, which leads to cytokine release, the recruitment of inflammatory cells, and stimulation of NADPH oxidase-dependent superoxide generation [75]. This bacterial peptide also stimulates gastric epithelial cell migration and proliferation, and increases the expression level of vascular endothelial growth factor (VEGF), whose role is to restore the gastric mucosa after induced injury [7,34]. These events, if contained, favor the host defense and the restoration of the homeostasis. However, in the case of H. pylori infection, the inflammatory response is sustained and exacerbated, thus resulting in the worsening of the clinical outcome due to the onset of severe gastric and extra-gastric diseases [67,76]. Interestingly, despite the massive inflammatory response, Helicobacter pylori has developed several strategies to manipulate the host immune system and persist within the gastric mucosa. H. pylori is able to both send and integrate signals from the gastric epithelium, allowing the host and bacteria to become linked in a dynamic equilibrium [77]. The long-term persistence of H. pylori promotes a permanent and low-grade inflammation, which is a risk factor for severe diseases, including cancer.
Given this scenario, Hp(2-20) has a critical role in the pathological processes associated with H. pylori infection by its interaction with FPRL1.
Phospholipase Cβ hydrolyzes phosphatidylinositol 4,5-biphosphate (PIP2) in diacylglycerol (DAG)-which remains at the membrane level-and inositol trisphosphate (IP3). This latter moves to the endoplasmic reticulum, regulating Ca++ release and increasing cytosolic Ca++ levels. In addition, both of the second messengers activate protein kinase C (PKC), resulting in NADPH oxidase activation and ROS production [78].

Conclusions and Future Perspectives
The FPR family is one of the most important class of cell surface receptors, playing important roles in innate immunity and host defense by mediating key events during the inflammatory response. Nevertheless, detrimental effects can result from FPR activation. In recent years, several studies have demonstrated the link between FPR activation and the pathogenesis of inflammatory diseases, including cancer [13].
Based on these considerations, FPR inhibition could represent a promising therapeutical strategy for treating various chronic inflammatory pathologies. Of particular interest is FRPL1, expressed by a variety of cells and tissues and able to bind a broad range of structurally different ligands, exerting pro-or anti-inflammatory actions. Thus, FPRL1 represents a potential target to inhibit inflammatory responses by using agonists with anti-inflammatory properties or antagonists.
This drew attention in searching novel natural molecules able to antagonize FPRL1 or elicit anti-inflammatory responses, paving the way for the discovery of new drugs for the treatment of inflammatory diseases.
Such an approach could also be extended to inflammatory disorders associated with viral infections. Our predictive studies (data not shown) indicate that FPRL1-showing an effective interaction with various coronavirus peptides-could also contribute to the coronavirus disease (COVID)-19 pathogenesis.