Introduction

Inflammation plays a dual role in human health. Proper inflammatory response contributes to the clearance of pathogens, whereas excessive or incontrollable inflammation could lead to diseases. Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn’s disease (CD), is a group of idiopathic colorectal inflammatory disorders with a progressive and unpredictable course, impairing the quality of life among patients [1]. The incidence and prevalence of IBD begin to increase markedly at the turn of this century [2]. It has become a major health burden worldwide [3]. Currently, treatment strategies for IBD make use of anti-inflammatory agents such as aminosalicylates, corticosteroids, small-molecule immunosuppressants, and therapeutic antibodies. Some patients eventually need surgery to remove intestinal lesions [4, 5]. However, these existing drugs have limited clinical efficacy, significant adverse events and an unwanted therapeutic ceiling [6].

Approximately 30% of IBD patients develop drug resistance and allergic reactions during long-term therapy with anti-tumor necrosis factor-α (TNF-α) antibody, although it gives relief to patients with refractory IBD [7]. In patients with moderate to severe IBD, the rate of mucosal healing was only about 50% despite intensive anti-inflammatory treatment [8]. In addition, more than 50% of patients with IBD were at risk of experiencing a suboptimal response to anti-TNF-α therapy in a 2-year trial [9]. These results reveal an unmet medical demand and a therapeutic ceiling associated with anti-inflammatory approaches.

Resolution of inflammation is an active process initiated after the onset of acute inflammatory response. Some studies suggest that promoting resolution was beneficial to mucosal healing of chronic diseases like IBD [10]. Despite the goal of complete mucosal healing is difficult to achieve, gastroenterologists have turned their attention from colitis symptom control to mucosal healing and pro-resolution of inflammation in an effort to treat IBD and deal with the therapeutic ceiling [11]. Furthermore, recent reports highlight the role of FPR2/ALX [combining nomenclature of formyl peptide receptor 2 (FPR2) and receptor for aspirin-triggered-lipoxins (ALX)], a human G protein-coupled receptor (GPCR), in resolution mechanisms [12]. Some FPR2/ALX agonists exhibited pro-resolving effects and therapeutic potential in mouse models of IBD [13].

Key triggers of inflammation and resolution

Four phases of inflammation development

Inflammation as a protective host response, manifesting as pathophysiological tissue dysfunction or homeostasis damage, occurs when a variety of infections, toxins or trauma activate the immune system. These harmful triggers are subsequently neutralized in a coordinated and active process of resolution that repairs and restores tissue integrity and function [14, 15]. Classical symptoms associated with this process include pain, fever, redness, swelling, dysfunction and organ damage [16]. At molecular and cellular levels, a series of inflammatory responses lead to increased blood flow, telangiectasia, leukocyte infiltration and release of chemical mediators. This course can be divided into four phases: (i) acute or chronic injury and barrier defect; (ii) onset of acute inflammation; (iii) triggering resolution mechanisms; and (iv) chronic inflammation [10]. Several reports described that these four phases do not progress sequentially but rather overlap; in other words, the activation and resolution of inflammation can occur simultaneously [17]. As a rapid and self-limiting process, acute inflammation lasts for a few days generally, and once harmful factors are eliminated, it resolves without causing major damage to the body. Properly controlling inflammation prevents the spread of infection or injury, followed by resolution in which the affected tissue returns to its original structural and functional state [18]. However, inadequate resolution may cause intractable injury and persistent inflammation leading to chronic inflammation [10].

Resolution of inflammation is a multi-stage and complex process, characteristics of limitation of blood-borne cells, regulation of chemokines and cytokines, alteration of leukocyte-survive-related pathways, and moderation of macrophage functions [19]. Resolution was once considered a passive process without regulators and a subsequent course that occurred only when pro-inflammatory lipid catabolism and chemokine modification were completed [20, 21]. It was the discovery of specialized pro-resolving mediators (SPMs) that has reshaped our understanding of resolution as an active process of healing or repair [21]. Three key mediators are thought important for resolution: lipids, neutrophils and phagocytes. Failure of one or more resolution steps may result in pathogenesis of long-term inflammation and chronic inflammatory diseases, triggered by continuous activation of the immune system, excessive production of pro-inflammatory cytokines, oxidative stress, tissue damages and dysfunction of homeostasis [19, 22]. In 2015, the concept of “Resolution Pharmacology” was proposed by Perretti and colleagues based on the comparison of pro-resolving vs. standard anti-inflammatory therapies [23]. Pro-resolving approaches were described as suppressing inflammation with fewer unwanted side-effects and better pathogen clearance, tissue repair and function recovery [6, 23].

Lipid mediators associated with resolution

Pro-resolving mediators are endogenous substances that promote resolution actions, including lipids, proteins, gaseous molecules, purines, neuromodulators and reactive oxygen species (ROS). Among them, lipids were demonstrated to act crucially in the inflammation processes starting from onset through progression to resolution [21, 24]. Under physiological conditions, lipid biosynthesis is reprogramed from pro-inflammatory signals to produce SPMs. Derived from polyunsaturated fatty acids (PUFAs), SPMs timely stop tissue damage caused by inflammatory response to avoid lasting impact that may lead to inflammation albeit of a primitive nature [25]. Most of SPMs are metabolites of ω−3 PUFA. It was found that long-term Western diets containing abundant saturated fat and ω−6 PUFA as well as scarce long-chain ω−3 PUFA were associated with an increased risk of IBD [26, 27]. Adherence to the consumption of ω−3 PUFA showed a positive role of evading pathology related to CD [28] and has been strongly recommended for the prevention of IBD [29]. Thus far, attention has been focused on the roles of PUFA-derived SPMs in the transition of inflammation from acute onset to active resolution [19]. Through their biosynthesis in injury tissues and later interactions with GPCRs on infiltrative neutrophils and resident epithelial cells, SPMs trigger three key cellular events at the inflammatory site: (i) weakening activation of epithelial cells; (ii) promoting apoptosis and limiting extravasation of neutrophils; and (iii) inducing M2 phenotype of macrophages and initiating efferocytosis by phagocytes [30]. SPMs additionally exhibit immunomodulatory actions on T and B cells and act on stem cells responsible for tissue repair or wound healing [30].

Regarding the question whether specific SPMs are preferentially secreted by a cell type [31], recent studies on endogenous active lipids revealed a main SPM biosynthesis pathway. Generally, SPMs, including lipoxins (LXs) and aspirin-triggered (AT)-LXs, are synthesized from arachidonic acid (AA) or docosahexaenoic acid (DHA) and catalyzed by 15-lipooxidase (15-LOX). They can be transformed to D series resolvins (RvDs), protectins and maresins. Biosynthesis of E series resolvins (RvEs) comes from eicosapentaenoic acid (EPA) and ω−3 docosapentaenoic acid (DPA) via aspirin acetylation of cyclooxygenase-2 (COX-2-ASA) that can be transformed to T series resolvins (RvTs). Resolvin, protectin and maresin conjugates in tissue regeneration come from their epoxy precursors [19, 31]. LXs are the most typical type of lipid mediators that exhibit pro-resolving properties and can be generated by transcellular metabolism at the inflammatory site [32, 33]. Despite precursor AA as a ω−6 pro-inflammatory lipid, the metabolites LXs mainly act to resolve inflammatory responses. From acute inflammation to resolution phase, the action of LXs on neutrophil infiltration and efferocytosis facilitates resolution and repairs damaged tissues [34, 35]. Ongoing studies on lipid biosynthesis demonstrate the presence of several ω−3 SPMs including resolvins [36], maresins [37] and protectins (neuroprotectins) [38] in inflammation. Resolvins of D, E and T series show effects of controlling the duration and resolution of inflammation [36]. RvE1 was the first identified SPM derived from EPA and isolated from resolving exudates and disease models that reduced neutrophil infiltration [39]. RvD1 and AT-RvD1 are potent regulators of phagocytes, stimulating the phagocytosis of microbes and dying cells [40]. In a recent RvT study, Chiang et al. revealed the role of RvTs in modulating phagocyte functions and neutrophil extracellular traps (NETs) [41]. Additionally, DHA-derived protectin D1 (PD1) is biosynthesized through 15-LOX-initiated mechanisms in exudates and neural tissues and shows strong neuroprotective effects in stroke models [42]. Existing protectin studies focused on resolution and repair processes of traumatic brain injury, such as PD1 and AT-PD1 actions in controlling neutrophil and macrophage functions to attenuate experimental stroke [43, 44]. As DHA-derived and 12/15-LOX-catalyzed macrophage mediators, maresins play an essential role in tissue repair and regeneration [45]. The complete stereochemistry was determined for maresin 1 (MaR1) possessing robust regenerative, repairing and neuroprotective capability. Meanwhile, synthetic MaR1 was shown to promote planarian regeneration after head resection in freshwater flatworm models [46, 47]. Together, existing data highlight that SPMs exert an essential action in resolution and repair (Summary in Table 1).

Table 1 Pro-resolving lipids and their biological characteristics
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Neutrophil death-related inflammation

Neutrophil death is finely regulated under physiological conditions [48]. Response to infection or injury is characterized by early and sustained release of the ‘Go’ signals, consisting of pro-inflammatory cytokines and cell adhesion molecules, thereby promoting migration of leukocytes to tissues. Such events are offset by parallel discharge of the ‘Stop’ signals (IL-10, prostaglandin E2, and factors controlling Toll-like receptor and NF-κB signaling) [49]. Neutrophils associated with these signals possess different attacking strategies to invading microorganisms: phagocytosis, release of ROS, generation of pro-inflammatory mediators and NETs [50]. Neutrophils are the first responder (followed by macrophages) to acute inflammatory reaction. Infiltration to the inflammatory site and subsequent cytotoxicity are essential to host defense against infection or injury [51]. Taking advantage of depolymerized DNA structure as their skeleton, NETs contain histone, myeloperoxidase (MPO), cathepsin G, and other bactericidal and pro-inflammatory mediators released to the extracellular space [52]. Clinical observations showed that a handful of NETs were detected in the intestinal tract of IBD patients, suggesting that with the activation of neutrophils in IBD, more NETs could be released to kill pathogens [53, 54]. An increased neutrophil life-span contributes to the effective clearance of invading pathogens [55], but excessively delayed neutrophil death may lead to chronic inflammation [51]. When neutrophils are not cleared in time, some of their proteases would cause tissue damage and amplification of the inflammatory response via release of pro-inflammatory cytokines and chemokines from the extracellular matrix (ECM) [56]. Therefore, the action of neutrophils should be properly regulated such as cell death including apoptosis, autophagy, pyroptosis, necroptosis, NET-related cell death (NETosis), and necrosis [48]. Dead neutrophils are recruited to an efferocytosis process by phagocytes [57]. Abnormally increased neutrophil life-span due to reduced apoptosis could elevate disease severity of chronic inflammation such as asthma [58], chronic obstructive pulmonary disease (COPD) [59], and IBD [60]. It appears that neutrophil death may serve as a therapeutic strategy for inflammatory diseases [61].

Efferocytosis by phagocytes

Carried out by macrophages and other phagocytes like monocytes, dendritic cells (DCs), and intestinal epithelial cells (IECs), efferocytosis is a critical component of the resolution processes, and its failure prolongs the inflammatory response and may lead to chronic inflammation [57, 62]. The M2 macrophages exhibit higher phagocytosis capacity and undertake host defense and wound healing tasks, with contributions to the inhibition of pro-inflammatory cytokines and the production of anti-inflammatory cytokines to down-regulate inflammation [63]. Phagocytes (mainly macrophages) can recruit dying cells (e.g., apoptotic neutrophils) to a phagocytosis process called efferocytosis, which in general consists of four steps: recruitment, binding, internalization/engulfment and degradation [64]. Apoptotic neutrophils release ‘Find-me’ signals (modified membrane lipids [65], nucleotides [66] and chemokines [67]), which guide macrophage recruitment to the site of apoptotic neutrophils [68]. In contrast to apoptosis, plasma membrane integrity of non-apoptotic cells is compromised, and these ruptured dying cells liberate inflammatory signals allowing phagocytes to recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) [57, 68], and then bind to targeted neutrophils via ‘Eat-me’ signals (phosphatidylserine [69] and calreticulin [70]) present in these dying cells. Actin remodeling will follow to facilitate phagosome formation and dying-cell internalization/engulfment [71]. Rac1, as the Rho family of small GTPases, is a key regulator in the reorganization of actin. Its activation is mediated through two different mechanisms [57], namely, LDL receptor-related protein 1 (LRP1) and adapter protein (e.g., GULP96), or the assembly of guanine nucleotide exchange factor (e.g., Dock180) and phagocytic regulatory protein [e.g., engulfment and cell motility protein (ELMO)]. To date, the precise mechanism of activating Rac1 by LRP1 and GULP is still elusive, but its activation by the Dock180-ELMO complex to trigger cytoskeleton rearrangement and internalization for phagocytosis has been documented [72]. Degradation of these internalized cells occurs via lysosomes with subsequent actions of membrane recovery and processing of dying cell-derived metabolites. Additionally, phagocytes that carry out efferocytosis are regulated by ‘Good-bye’ signals (spermidine, guanosine, fructose-1,6-bis-phosphate, etc.) and some metabolites from internalized dying cells, which effects include increased release of anti-inflammatory molecules, cytoskeleton rearrangement that promotes phagocytosis, promoting energy production and preventing apoptosis of phagocytes [73].

Aberration in efferocytosis pathways has been linked recently with autoimmune diseases [74, 75], wound healing [76], atherosclerosis [77, 78], arthritis [79], neurodegeneration [80], and organ inflammation or injury [81,82,83], including systemic lupus erythematosus (SLE). Non-cleared dead cells were detected in the blood, skin, and lymph nodes of SLE patients, and the severity of the disease was positively correlated with defective efferocytosis and dead cell accumulation [84]. IECs possess a high turnover of cells in the intestinal tissue, and efferocytosis is constantly required to prevent unnecessary inflammation. It was reported that defects in the binding mechanism of efferocytosis (upon cleaning dying IECs) caused by ablation of CD300f (a phosphatidylserine receptor as an ‘Eat-me’ signal) were associated with IBD and colon cancer [85]. In the dextran sulfate sodium (DSS)-induced colitis model, mice with genetic deletion of G2A (a lipid product receptor as a ‘Find-me’ signal) exhibited worsened colitis and had fewer CD4+ lymphocytes recruited to the inflamed colon that impaired efferocytosis [81]. Besides, other mouse models revealed that blockade of phosphatidylserine recognition or ablation of MerTK (an ‘Eat-me’ signal) triggered efferocytosis defects and then impaired tissue repair in lung and intestinal injury [86]. Obviously, efferocytosis dysfunction can contribute to failed resolution of intestinal inflammation in IBD.

FPR2/ALX: Towards a pro-resolving strategy

GPCRs in the resolution phase

SPMs exert pro-resolving actions through a variety of GPCRs, capable of identifying PAMPs and DAMPs, interacting with a large number of ligands to process extracellular signals [87], and regulating pro-inflammatory mediators (IL-1, IL-6, IL-7, IL-8, TNF-α, etc.). Reduction of leukocyte aggregation in the inflammatory tissue, promotion of apoptosis, and efferocytosis of neutrophils will follow [88]. In the gastrointestinal (GI) tract, GPCRs participate in key functional processes including digestion, immune cell infiltration, pain perception, and tissue repair. Certain studies highlighted the role of GPCRs in intestinal mucosal repair [88]. Expression of GPCRs in the GI epithelial membrane is up-regulated by pro-inflammatory cytokines, while SPMs can bind to GPCRs at orthosteric or allosteric sites and promote the migration and proliferation of IECs aiming at restoring homeostasis and GI wound repair [89]. It is known that resolution-related GPCRs may include FPR2/ALX [12], GPR32 [90], GPR18 [91], chemerin1 (ChemR23) [92], GPR37 [93], GPR101 [94], EP4 [95], LGR6 [96] and BLT1 [97] (Table 1), although definite receptors for some SPMs have yet to be identified [88]. These GPCRs have been studied as pro-resolving targets for IBD. An overview of the association [98,99,100] between pro-resolving GPCRs and their clinical application in IBD [95, 99,100,101,102,103,104] is summarized in Fig. 1. Among them, FPR2/ALX, a high-affinity receptor for AA-derived LXA4 and DHA-derived RvD1, mediates the actions of SPMs by inhibiting and resolving a wide range of inflammatory reactions [12]. The following parts of review give an overview of FPR2/ALX as a potential pro-resolving target to treat inflammatory diseases including IBD.

Fig. 1: Typical studies regarding IBD treatment using pro-resolving strategies.
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BLT1 B-type leukotriene 1, CAC colitis-associated cancer, CD Crohn’s disease, Chemerin1 (ChemR23) chemokine receptor-like 1, DSS dextran sulfate sodium, EP4 E-type prostanoid receptor 4, FPR2/ALX formyl peptide receptor 2, GPCR G protein-coupled receptor, IBD inflammatory bowel disease, IECs intestinal epithelial cells, LGR6 leucine-rich repeat-containing G-protein coupled receptor 6, LXA4 lipoxin A4, mAb monoclonal antibody, MaR1 maresin 1, PD1n-3 n-3 protectin D1, PGE2 prostaglandin E2, RvD1 D series resolvin 1, TNBS 2,4,6-trinitrobenzene sulfonic acid, UC ulcerative colitis, SPMs specialized pro-resolving mediators. This figure was generated by an open-type platform FigDraw (http://www.figdraw.com/).

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Formyl peptide receptor 2

FPRs belong to class A GPCR family and are expressed in different cell types [105]. Earlier understanding was that FPRs cause a series of cellular signaling events resulting in myeloid cell migration and inflammatory mediator release [12]. More recent studies indicate that FPRs not only control inflammation but also participate in many key pathophysiological processes [106]. The human FPRs include FPR1, FPR2/ALX, and FPR3 with their genes located in chromosome 19q13.3, while the mouse FPR gene family has 8 members (Fpr1 – Fpr3 and Fpr-rs3 – Fpr-rs7) that localize to chromosome 17A3.2 [105]. In human non-immune organs and tissues, FPR1 and FPR2/ALX show a broader distribution than FPR3 and are expressed in a variety of non-myeloid cells, including astrocytoma cells, epithelial cells, hepatocytes, microvascular endothelial cells, and neuroblastoma cells. In the immune system, they are expressed mainly in innate immune cells like neutrophils, monocytes/macrophages, DCs, etc. [107]. They bind to a large number of structurally diverse ligands in the inflammatory environment [105, 108], such as PAMP-derived formylated bacterial peptides (e.g., f-MLF), PAMP-derived non-formylated microbe peptides [e.g., Hp(2–20)], DAMP-derived formylated mitochondria peptides (e.g., f-MMYALF), host-derived molecules (e.g., LXA4 and RvD1) and some synthetic compounds (e.g., WKYMVm and Quin-C1). The effects of these ligands are mediated by FPR signaling causing Ca2+ mobilization, superoxide production, transcriptional regulation, chemotaxis, and phagocytosis [108]. Neutrophils, macrophages, and other leukocytes are important participants of inflammatory responses that are partially regulated through FPRs and other GPCRs on the cell surface [88]. It was found that mitochondrial N-formyl peptide induced aggregation of pulmonary neutrophils via FPRs and damaged lung tissues through hemorrhagic shock in rats [109]. In a mouse acute lung injury model, FPR1 antagonism was demonstrated to reduce lipopolysaccharide (LPS)-induced neutrophil aggregation and to improve acute pulmonary edema and alveolar injury [110]. FPRs can recognize formylated peptides and guide chemotactic neutrophils to phagocytose bacterial pathogens or damaged tissues. In the Fpr1/2−/− (gene deletions of Fpr1 and Fpr2) mouse model, chemotactic phagocytosis of neutrophils against bacterial pathogens was impaired [111]. Similarly, FPR1 [112] and FPR2/ALX [113] are implicated in the migration of macrophages and phagocytosis (efferocytosis) of apoptotic neutrophils.

Based on the recommendation of the International Union of Basic and Clinical Pharmacology (IUPHAR), the nomenclature of human FPRs follows these descriptions: FPR1 as a cognate receptor for N-formylated peptides of bacterial and mammalian origins, FPR2/ALX to which binds N-formylated peptides with low affinity as well as LXA4 and AT-LXs (ALX as a receptor for LXA4 and AT-LXs), and FPR3 as another receptor that binds and responds to the mitochondrial peptide fMMYALF [105]. Among them, FPR2/ALX is recognized as a resolution receptor [12]. Although it remains unclear how FPR2/ALX binds to different ligands and changes its conformation to activate different signaling pathways [114], current studies have implied a few potential mechanisms of receptor agonism, including signal bias either towards G protein activation (pro-inflammation; Fig. 2a) or towards β-arrestin recruitment (pro-resolution; Fig. 2b) [114, 115]. FPR2/ALX can also be phosphorylated to mediate heterologous desensitization through inhibition of PKA/PKC pathway [116] (Fig. 2c). For example, ACT-389949 was found to cause FPR2/ALX desensitization in a phase I clinical trial despite its elusive mechanism of action [117]. In addition, FPR2/ALX internalization is associated with LXA4/AnxA1 stimulation and desensitization process [118] (Fig. 2d).

Fig. 2: Potential mechanisms of FPR2/ALX mediated responses induced by various ligands.
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Like other chemoattractant receptors, FPR2/ALX couples to the Gi class of heterotrimeric G proteins. Bifurcation of FPR2/ALX signaling exists. a Some ligands like amyloid β1-42 (Aβ1-42) and serum amyloid A (SAA) can activate FPR1 to trigger pro-inflammatory signals directly, or they activate FPR2/ALX by coupling to Gαi resulting in Ca2+ mobilization and ERK phosphorylation, thereby showing biased pro-inflammation. b Most FPR2/ALX agonists (LXA4, AnxA1, Ac2-26, ATL, RvD1 and Quin-C1) induce biased pro-resolving signals via β-arrestin 2 recruitment, cAMP production and p38 MAPK phosphorylation. c Heterologous desensitization of FPR2/ALX is probably mediated by β-arrestins. FPR2/ALX is phosphorylated by G protein-coupled receptor kinases (GRKs) to recruit β-arrestins, which then inhibit protein kinase A/C (PKA/PKC) signaling pathways. d FPR2/ALX internalization is essential to LXA4- and AnxA1-stimulated pro-resolution as the internalized receptor inhibits NF-κB activity. Some desensitized FPR2/ALX receptors are also internalized into cells. This figure was generated by an open-type platform FigDraw (http://www.figdraw.com/).

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FPR2/ALX dysfunction and diseases

Owing to its critical roles in lipid metabolism, neutrophil functions, and efferocytosis-related resolution processes, FPR2/ALX dysfunction could develop inflammation and cause failure in resolution [35]. Pro-resolving studies highlighted a partial contribution of SPMs via FPR2/ALX to key immune cell events [10, 119,120,121] during inflammation and injury (Fig. 3). Another piece of evidence suggested that rs11666254 polymorphism that decreases FPR2/ALX expression was associated with compromised immune responses in patients with severe trauma [122]. The gene products of both Fpr2 and Fpr3 (previously named Fpr-rs1) are mouse orthologs of human FPR2/ALX. They facilitated the use of Fpr2/3 knockout (Fpr2/3−/−) mice to characterize human FPR2/ALX dysfunction [123,124,125]. In 2010, Dufton et al. [124] and Chen et al. [126] reported the creation of a mouse colony in which Fpr2 was deleted, but Dufton et al. made a correction in 2011 that their targeting strategy would have also resulted in the deletion of Fpr3 because this gene incorporated an exon found in Fpr2 [125].

Fig. 3: FPR2/ALX in the resolution of inflammation.
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Taking the gastrointestinal tract as a model, when injury or infection occurs, neutrophils migrate to the inflamed site attracted by chemokines. DAMPs and PAMPs invade via IECs to the damaged tissues and the production of pro-inflammatory cytokines is up-regulated to clear pathogens after activating T helper (Th) cells, resulting in inflammatory response. In the resolution phase, an increase in SPMs is accompanied by a decrease in pro-inflammatory cytokines. After recognizing LL-37 (an anti-microbial peptide) and SPMs via FPR2/ALX, DCs are activated with elevated phagocytosis and CD40 expression to clear PAMP and DAPM signals. Upon completing their mission of resisting pathogen invasion, neutrophils enter into the apoptosis process associated with SPMs and FPR2/ALX. SPMs reprogram macrophages towards M2 pro-resolving phenotypes, and apoptotic neutrophils are phagocytosed by M2 macrophages thereafter. FPR2/ALX expression is related to that of CCR6 in monocytes thereby facilitating the latter to the inflamed site and the epithelial remodeling in the wound. CCR chemokine receptor; DAMP damage-associated molecular pattern; DC dendritic cell; FPR2/ALX formyl peptide receptor 2; IEC intestinal epithelial cell; PAMP pathogen-associated molecular pattern; SPM specialized pro-resolving mediator. This figure was generated by an open-type platform FigDraw (http://www.figdraw.com/).

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Essential roles of FPR2/ALX in regulating inflammation of different diseases are summarized in Table 2. Machado et al. demonstrated that Fpr2/3−/− mice were highly susceptible to infection, displaying uncontrolled inflammation, increased bacterial dissemination, and pulmonary dysfunction, associated with the loss of lung barrier integrity and increased neutrophil activation upon Streptococcus pneumoniae stimulation [127]. This suggests that FPR2/ALX signals control inflammation and bacterial dissemination during pneumococcal pneumonia by promoting host defense. Another study found that Fpr2/3−/− mice showed phagocytosis impairment in macrophages with the expansion of neutrophils and reduced SPM levels in the infarcted heart and spleen. Lack of murine Fpr2/3 led to obesity and leukocyte dysfunction, and facilitated profound inter-organ non-resolving inflammation in mice, i.e., obesogenic cardiomyopathy and renal inflammation [128]. Since FPR2/ALX was thought to be protective against inflammation and tissue injury [129], the role of Fpr2/3 in orchestrating intestinal resolution and repair was explored. It was found that Fpr2/3 knockout mice were susceptible to experimental colitis. In the DSS-induced mouse model of colitis, such a role in mucosal homeostasis and resolution was revealed by impaired epithelial restitution in the colon and delayed mucosal restoration after injury of Fpr2/3−/− mice compared to that of the wild-type [130]. Birkl et al. showed a similar impairment in Fpr2/3−/− colitis mice and suggested the contribution of FPR2/ALX in facilitating monocyte recruitment to mucosal injury sites for intestinal wound repair [120].

Table 2 Disease-associated FPR2/ALX dysfunction in mouse models and humans
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Clinical studies revealed the relationship between FPR2/ALX and inflammatory diseases showing different receptor expression profiles depending on the disease stages [10]. FPR2/ALX (FPR2) down-regulation was found in advanced stages with failed resolution, while its up-regulation appeared in early stages with active resolution (Table 2). In children with severe asthma (SA), FPR2/ALX expression was reduced in sputum cells compared to healthy controls [131]. In the placenta of patients with chorioamnionitis (CAM), RvD1 decreased while FPR2/ALX increased, accompanied by inhibition of PPARγ and NF-κB nuclear translocation [132]. FPR2 expression was associated with clinical outcomes in trauma patients, and those had uncomplicated recoveries displayed significantly higher FPR2 expression and lower gene expression ratio of leukotriene compared to that seen among patients experiencing complicated recoveries [133]. Colonic FPR2/ALX mRNA level was also positively correlated with the histology scores of IBD patients and the intestinal stricture of CD patients [98], exhibiting a 6-fold increase in the inflamed region of patients with CD [134]. In early-stage tendon pathology, expression of FPR2/ALX vs. healthy tendons was elevated, suggesting tendons mount a counter-resolution response to inflammation, while that in intermediate-advanced disease tendons dropped indicating a failure of the resolution process [135].

Pharmacology of FPR2/ALX agonists

Agonism at FPR2/ALX has been reported in animal models of renal fibrosis [136], rheumatoid arthritis (RA) [137, 138], IBD [101], myocardial ischemia-reperfusion [139], diabetic complications [140], sepsis [141], COPD [142], neuroinflammation [143] and cancer [144]. An overview of key findings is provided in Table 3.

Table 3 Characteristics of FPR2/ALX agonists reported in the literature
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SPMs and lipid analogs

Typical SPMs, such as LXA4 and RvD1, are FPR2/ALX agonists with pro-resolving capability. Interaction between LXA4 and FPR2/ALX activates several intracellular signaling pathways and the conformational changes induced by LXA4 prevent the binding of pro-inflammatory amyloid β or serum amyloid A (SAA) [12]. Current data suggest that LXs exert strong endogenous pro-resolving effects. However, chemical and metabolic liability of them hampers the therapeutic development. Particularly, LXA4 is metabolized by prostaglandin dehydrogenase (PGDH) at C15 and omega-oxidation at C20 [145]. Some synthetic LX analogs are less susceptible to PGDH with a longer half-life and better pro-resolving properties [13]. Thus, they are under development with several showing the potential in treating inflammatory disorders such as colitis and IBD. ZK-192 as a LXA4 analog was protective in trinitrobenzene sulphonate (TNBS)-induced colitis mice [146]. Another LXA4 analog BML111 was found to rescue CD-like intestinal inflammation in cyclooxygenase-2 (COX-2) myeloid knockout (MKO) mice [147]. Meanwhile, NAP1051 showed LXA4-like in vitro features and anti-tumor activity in colorectal cancer xenograft models via inhibiting neutrophils, reducing NETosis and stimulating T cell recruitment in the tumor microenvironment (TME) [148]. In a non-colitis inflammation study, synthetic dimethyl-imidazole-containing LXA4 mimetic AT-01-KG was effective in RA animal models by decreasing the number of neutrophils in the knee exudate [137]. It also reduced tissue damage and hyper nociception in MSU-induced gout model [149]. Such effects were not observed in Fpr2/3−/− gout mice [149]. Additionally, a recent phase 1 clinical trial (NCT02342691) demonstrated that benzo-LXA4 analog BLXA4 was safe and efficacious in periodontitis patients showing an increased level of pro-resolving mediators systemically [150].

Unlike LXs, DHA-derived RvDs not only bind to FPR2/ALX but also GPR32 and GPR18 [13, 90, 91, 151,152,153,154]. RvD1 and RvD2 were shown to prevent DSS- or TNBS-induced colitis and improve colon epithelial damage and macrophage infiltration in mice [155]. Several RvD1 analogs were developed, such as 17-(R/S)-methyl-RvD1 methyl ester, 17R-hydroxy-19-para-fluorophenoxy-RvD1 methyl ester and benzo-diacetylenic-17R-RvD1-methyl ester (BDA-RvD1). Although they have yet to be evaluated in animal models of colitis or IBD, their therapeutic potential was demonstrated in lung injury via neutrophil suppression and phagocytosis stimulation [156,157,158].

Proteins and peptides

Some endogenous proteins, their peptide analogs and synthetic peptides are considered as FPR2/ALX agonists as well. Annexin A1 (AnxA1) is a calcium-dependent phospholipid-binding protein widely seen in eukaryotic cells and regulates inflammatory response through FPR2/ALX [140]. In DSS-induced colitis mice, exogenous AnxA1 activated the FPR2/STAT3 pathway and enhanced the therapeutic effect of anti-TNF nanobody V7 [159]. AC2-26, as an AnxA1 mimetic, was found to modulate the function of human mast cells (MCs) and capable of interfering with intestinal MC degranulation via FPR2/ALX [160]. Apolipoprotein A-I (APOA1) is the main structural protein of high-density lipoprotein (HDL) associated with inflammation [161]. Its mimetic peptides 4F and Tg6F either mitigated COX-2-MKO/CCHF and piroxicam-accelerated IL-10−/− models of IBD or effectively reduced intestinal inflammation in COX-2-MKO/CCHF model [147]. FAM3D is a newly found endogenous protein for FPRs, essential to colon homeostasis and host defense against inflammation, as FAM3D−/− mice showed increased spontaneous colitis, impaired colonic mucosal integrity, and excessive sensitivity to chemically induced colitis-associated cancers [162]. Unlike natural AnxA1 and APOA1, WKYMVm (Trp-Lys-Met-Val-D-Met) is a modified hexapeptide with high potency for both FPR1 and FPR2/ALX [163]. WKYMVm displayed therapeutic value in mouse models of ischemia [164, 165], diabetic wounds [166], pneumosepsis [167], and colitis [101]. Other synthetic peptides LESIFRSLLLFRVM (MMK1) [168] and TIPMFVPESTSKLQKFTSWFTSWFM-Amide (CGEN-855A) [169] are also FPR2/ALX agonists exhibiting pro-resolving effects on neutrophil infiltration in an air pouch model.

Small molecule modulators

A number of small molecule FPR2/ALX agonists with promising therapeutic potential have been developed. Their structural types include phenyl urea (compound 17), ureidopropanamide (MR-39), pyridazinone (compound 43), pyridinone/pyrimidindione (compound 47), quinazolinone (Quin-C1), aminotriazole (ACT-389949), pyrrolidinone (BMS-986235), etc. Some of them were evaluated in mouse inflammatory models. Compounds 17 blocked neutrophil infiltration and promoted punch dermal wound healing [170]. MR-39 exerted a pro-resolving action in LPS-stimulated microglia [171]. In myocardial infarction models, compound 43 showed a positive effect on viable myocardium and induced phagocytic resolution [172]. In rat models of RA, compound 47 displayed the potential of decreasing pain hypersensitivity [173]. In addition, Quin-C1 induced FPR2/ALX-mediated intracellular Ca2+ mobilization and showed therapeutic efficacy in bleomycin-induced lung injury [174]. Phase I clinical trials are underway with respect to two FPR2/ALX agonists. ACT-389949 was found to be as potent as WKYMVm [175] and relevant trial results of which (NCT02099071 and NCT02099201) showed its safety and tolerability in healthy subjects despite rapid receptor desensitization [117]. Phase I clinical trial of BMS-986235 (NCT03335553) was concluded but the results have not been disclosed. However, BMS-986235 was protective against experimental heart failure [176].

Clinical translation of FPR2/ALX agonism

Given the solid evidence that FPR2/ALX dysfunction is associated with IBD, FPR2/ALX agonists may be used as therapeutic candidates for IBD. Biased FPR2/ALX agonism towards pro-resolution is likely to induce mild-to-moderate suppression of inflammation without immunosuppression, thus suitable to patients intolerant to immunosuppressants or resistant to antibody treatment. Major challenges for clinical translation of the pro-resolving strategy that harnesses FPR2/ALX activation include: (i) receptor desensitization as seen with a few agonists (e.g., ACT-389949); (ii) application of biased signaling property by guiding receptor-mediated response towards pro-resolving outcomes; (iii) available biomarkers to accurately monitor mucosal healing [6]; and (iv) unintended consequences of such a therapeutic approach.

Conclusion

Failed resolution of inflammation can underpin the pathogenesis of chronic inflammatory diseases including IBD. Existing data revealed the resolution mechanisms in the early stage (neutrophil functions, cytokines, and SPMs) and intermediate to advanced stages (efferocytosis and adaptive immunity). Resolution or repair agents for patients with IBD are not available at present but potential therapeutic targets like FPR2/ALX may fill the gap. FPR2/ALX centric pro-resolving strategy will address a key issue on the recurrence of inflammation and mucosal injury in IBD. Ongoing studies cover a variety of FPR2/ALX agonists including lipids, peptides and small molecules. Of which, the latter may offer advantages such as oral bioavailability, easy to use, better compliance, and low cost.