Formyl Peptide Receptors in Bone Research

Abstract

Purpose/Aim of the Study

The formyl peptide receptor (FPR) participates in the immune response, with roles in infection and inflammation. In this review article, we summarize the current literature on these roles before discussing the function of FPRs in the pathogenesis of musculoskeletal disorders including osteoarthritis (OA), degenerative disc disease (DDD), and rheumatoid arthritis (RA). Additionally, we discuss potential diagnostic and therapeutic roles of FPRs in these domains.

Methods

PubMed and Ovid MEDLINE searches were performed from 1965 through March 2022. Keywords included: ‘FPR, tissue expression, inflammation, infection, musculoskeletal disorder, bone, rheumatoid arthritis, osteoarthritis, degenerative disc disease, mitochondria’.

Results

69 studies were included in this review article. FPRs appear to be ubiquitous in the pathogenesis, diagnosis, and treatment of common musculoskeletal disorders. They can potentially be utilized for the earlier diagnosis of OA and DDD. They may be employed with mesenchymal stem cells (MSCs) to reverse OA and DDD pathologies. With anti-inflammatory, anti-osteolytic, and pro-angiogenic functions, they may broaden treatment options in RA.

Conclusions

FPRs appear to be heavily involved in the pathogenesis of common musculoskeletal conditions, including arthritis, degenerative disc disease, and rheumatoid arthritis. Furthermore, they demonstrate much promise in the diagnosis and treatment of these conditions. Their roles should continue to be explored.

Introduction

N-formyl peptide receptors (FPRs) are G-protein coupled receptors (GPCRs) that function as chemoattractant receptors¹. There are three subtypes in humans, known as FPR1, FPR2/ALX, and FPR3¹, which are encoded by FPR1, FPR2, and FPR3 on the chromosomal region 19q13.3, respectively².

These receptors were first identified and investigated via a series of experiments studying the response to infection. Because of pus accumulation at sites of infection, it was hypothesized that pathogen-derived molecules could attract immune cells to these areas. Early in vitro studies demonstrated chemotactic activity for phagocytes in the presence of filtrates from both Gram-positive and Gram-negative organisms³, while subsequent Escherichia coli (E. coli) studies showed that these factors were low-molecular weight peptides with blocked amino groups⁴. Eventually, formyl-Met-Leu-Phe (fMLP), an endogenous E. coli peptide, was isolated, and its potent chemotactic effects on neutrophils was demonstrated⁵. These results, in addition to work with N-formylmethionine from Staphylococcus aureus (S. aureus) cultures⁶ and studies on synthetically synthesized N-formylmethionine peptides⁷, provided support to the theory that N-formylmethionine peptides originating from bacterial sources are strong mediators of neutrophil chemotaxis to sites of infection, driving an innate immune response through FPRs.

FPRs have also been shown to function in mitochondrial distress and inflammation. Similar to prokaryotic protein synthesis, mitochondrial protein synthesis is initiated with N-formyl methionine. As a consequence, injured or dead mitochondria release several danger signals, among them mitochondrial formyl peptides (mtFPs)⁸. These act as ligands for FPRs and stimulate leukocyte chemotaxis to the site of injury, causing production of reactive oxygen species and degranulation⁸. Thus, activation of FPR signaling can contribute to inflammatory conditions.

Dysregulation of the inflammatory pathway and progression to chronic inflammation is implicated in various disease processes. As such, much research has been aimed at investigating the process of inflammation and avenues by which disease processes can be ameliorated, resulting in an expansion of our understanding of both FPRs and their ligands. FPRs are mostly commonly expressed in cells of the immune system, and in particular those of myeloid lineage. These cells, as well as FPR expression patterns and significance, are summarized in Table 1, while several roles of FPRs in non-immune cells are described in Table 2. Select FPR ligands, which are diverse in both structure and function and include agonist and antagonist peptides, lipid molecules, and synthetic non-peptide compounds, are summarized in Table 3.

Table 1:

Formyl peptide receptors (FPRs) in immune cells, including their patterns of expression and significance.

FPR Subtype	Cell Type	Expression Pattern	Significance	Reference
1	Neutrophil			9
	Dendritic	elevated mRNA and protein levels seen in immature, but not mature, DCs	differential expression supports the role of FPR1 in the initiation of the immune response via trafficking	10
	Natural Killer		elicits cytolytic activity	11
2	Neutrophil			12
	Natural Killer		elicits cytolytic activity	11
	Monocyte	mRNA and protein levels decreased with differentiation to iDCs	supports the role of FPR2 in the innate, not adaptive, immune response	13
	T-cell	seen in CD4+, Th1, Th2, Th17, Helper T-cells. ANXA1 binding induces FPR2 expression.	assists with T-cell activation and differentiation, drives Th1/Th2 shift	14,15,16
	Follicular DC		promotes B-cell proliferation and activation in Peyer’s Patches, assisting with mucosal immunity	17
	B-cell	FPR2 upregulated, IgG and IgM downregulated with ALX binding	further supports the variable expression and effects of FPR2/ALX	18
3	Dendritic	present on mDCs	FPR3 is the predominant FPR on mDCs, with a role in DC trafficking	19,20
	Macrophage/Eosinophil	found in subpopulations of lung, colon, skin	demonstrates a role of FPR3 in innate immunity, immunoregulation, and allergic pathologies	21

Abbreviations: DC – dendritic cell, iDC – immature dendritic cell, mDC – mature dendritic cell, ANXA1 – Annexin A1.

Table 2:

Formyl peptide receptors (FPRs) in non-immune cells, including individual organs/tissues and their functions.

Cell/Organ	Role	Reference
Liver parenchyma	regulates acute phase protein synthesis in concentration- and time-dependent manner	22
Astrocytes and microglia	express both FPR1 and FPR2/ALX	23,24
Coronary arteries	FPR2/ALX and FPR3 found in tunica intima and adventitia, functions in TXA2 and PGI2 synthase expression with potential role in coronary vasospasm	25

Abbreviations: TXA2 – Thromboxane A₂, PGI₂ – Prostaglandin A₂.

Table 3:

Ligands for formyl peptide receptors (FPRs) include both peptides and lipids, with agonist and antagonist functions.²⁶

Agonist/Antagonist	Type	Molecule	Role/Function
Agonist	Peptide	fMLP	Pro-inflammatory chemotaxis, cytokine production, calcium flux, with roles in inflammatory bowel disease
		gp41, gp120	HIV-1
		Hp2-20	H. pylori
		PrP	prion disease
		SAA	acute phase protein
		LL-37	migration of neutrophils, monocutes, T-lympchocyes, and fibroblasts, with a role in wound healing and angiogenesis
		uPAR	fibrinolysis
		FAM3D	gut inflammation and homeostasis
	Lipid	Lipoxin A4	anti-inflammatory
		oxLDL	stimulation of macrophages
Antagonist	Peptide	t-Boc-MLF	inhibits neutrophil NADPH oxidase activity
		cyclosporin H	reduces acute inflammation via FPR1
		uteroglobin	airway epithelium of mammals

Overall, these studies demonstrates that the expression and function of FPRs are variable, with myriad downstream effects depending on cell type. Furthermore, FPRs may have more ubiquitous function than previously thought, with both inflammatory and anti-inflammatory roles in several common musculoskeletal disorders. The goal of this review article is to summarize the current literature on the involvement of FPRs in several of these conditions: arthritis, degenerative disc disease (DDD), and rheumatoid arthritis (RA) (Figure 1). The potential diagnostic and therapeutic applications of FPRs in these conditions will be discussed.

Figure 1: Select roles of formyl peptide receptors (FPRs) in the pathogenesis and diagnosis and treatment of many common musculoskeletal diseases.

A1: role of FPRs in arthritis pathogenesis. A2: role of FPRs in arthritis diagnosis and treatment. B1: role of FPRs in degenerative disc disease (DDD) diagnosis and treatment. C1: role of FPRs in rheumatoid arthritis (RA) pathogenesis. C2: role of FPRs in RA diagnosis and treatment.

Search Strategy and Selection Criteria

Searches were performed using PubMed and Ovid MEDLINE encompassing literature published from 1 January 1965 to 1 March 2022. Key search words were: ‘FPR, tissue expression, inflammation, infection, musculoskeletal disorder, bone, RA, osteoarthritis (OA), DDD, mitochondria.’ FPR was searched in combination with each additional key search word, yielding 797 articles. Eligible articles included reviews, in vivo and in vitro studies, studies performed upon humans and animals, and studies published in English. After removing duplicate articles, abstracts, and case reports, we reviewed all remaining article abstracts. Those not related to the topic of formyl peptide receptors were removed, yielding 69 unique articles.

FPRs in Musculoskeletal Disorders

Arthritis

Arthritis is classically characterized by degeneration of the joint complex, including the articular cartilage, subchondral bone, and synovium. Weight-bearing and heavily used joints are most frequently affected, with typical signs and symptoms including pain, morning stiffness and crepitus²⁷. The initiation and progression of arthritis remains incompletely understood and much research continues to be conducted in an attempt to elucidate its pathophysiology, diagnosis, and treatment.

FPRs in Arthritis Pathobiology

FPRs have been implicated in arthritis primarily through their role in immune cell migration into diseased joints in both the infection and trauma settings. A previous mice study demonstrated that bacterial formylated peptides, ligands for FPRs, were sufficient in mediating S. aureus-induced arthritis²⁸. Unlike mice inoculated with an isogenic mutant strain lacking the ability to produce formylated peptides, mice inoculated with wild-type S. aureus experienced increased infiltration of neutrophils with subsequent development of arthritis and severe joint destruction. Thus, formylated peptides from S. aureus can mediate extensive infiltration and activation of neutrophils beyond what is necessary to clear infection, causing severe inflammation and destruction of the joints.

A separate study used a rodent knee model of OA induced by surgical anterior cruciate ligament transection (ACLT)²⁹. On histology, there was significant damage to the synovial membrane, cartilage, meniscus, and subchondral bone as well as osteophyte formation. Expression of FPR1 mRNA as well as the inflammatory markers iNOS, TNFα and IL-1β were increased in LPS-activated mice peritoneal macrophages and bone marrow-derived macrophages.

FPRs for OA Diagnosis

There are currently no well-established diagnostic tools that aid in the early diagnosis of OA³⁰. However, because of the previously discussed role of FPRs in inflammatory responses and the subsequent development of arthritis, imaging studies utilizing FPR ligands for the evaluation of FPR expression represent a potential mechanism for the earlier diagnosis of OA. In a study using a MIA-induced rat knee arthritis model, FPR1 expression was significantly enhanced in synovial membrane of the affected knee joint. This correlated with PET signals from the fluorescent tracer cFLFLF, a FPR1-specific antagonist³¹. Similarly, the previously discussed ACLT study was able to correlate inflammation with increased SPECT signals attained from a modified cFLFLF tracer binding to FPR11 expressed on immune cells that had infiltrated into the affected knees²⁹.

FPRs in OA Therapeutics

The treatment of OA is typically conservative with symptomatic management until disease progression provides an indication for surgery³². However, there are still no available treatments addressing the underlying disease processes of OA, making the role of FPRs as possible therapeutic targets the subject of active research. Much of this has focused on the therapeutic potential of mesenchymal stem cells (MSCs) which are able to differentiate into chondrocytes, adipocytes, and osteoblasts³³ and express FPR1 and FPR2 at the mRNA and protein levels³⁴.

Prior studies have demonstrated that stimulation by fMLP³⁴ and ANXA1, a ligand of FPR1 and FPR2³⁵, causes adhesion and migration of hMSCs³⁴. Corticosteroids have also been shown to cause increased expression of FPR1 mRNA and protein in bone-marrow derived MSCs and endothelial progenitor cells (EPCs)³⁶, as well as to increase fMLP-mediated chemotaxis in MSCs and EPCs. These findings indicate FPR signaling could be a possible mechanism by which hMSCs can be directed towards sites of inflammation, promoting healing. Another recent study found FPRs can play a major role in osteoblast differentiation from MSCs³⁷. FPR1 expression significantly increased as MSCs differentiated into osteoblasts, while fMLP was able to suppress differentiation of bone marrow-derived monocytes into osteoclasts. This indicates that FPR1 may create osteogenesis-dominant conditions when activated. These in vitro studies were further supported by in vivo studies in zebrafish and rabbit models of bone formation which demonstrated that fMLP induced osteogenesis in a FPR1-dependent mechanism³⁷. This is significant because it suggests there is an endogenous mechanism by which FPRs are involved in osteogenesis.

Additional studies should be conducted to further examine these results and to explore the potential roles of FPRs in addressing and even reversing the underlying disease processes of OA. In fact, studies demonstrated that intra-articular injections of synovial MSCs inhibited OA progression in an ACLT-induced rat OA model³⁸, while a recent clinical trial demonstrated that intra-articular injections of synovial MSCs prevented cartilage loss and improved patient clinical scores³⁹.

Degenerative Disc Diseases

Intervertebral discs (IVDs) are fibrocartilaginous tissues that connect adjacent vertebra, enabling their motion and functioning as shock absorbers. DDD occurs because of degenerative changes to intervertebral discs, decreasing their ability to withstand compression, tension, shear, and torque stresses. The exact mechanism behind DDD remains unclear, but it is evident that persistent inflammation exacerbates DDD^40,41.

Diagnosis and Treatment of DDD

In general, the gold standard for the diagnosis of DDD is MRI⁴². However, it is important to note that imaging abnormalities do not always correlate with disease states⁴³, making clinical context critical to the diagnosis. Similar to OA, DDD treatments range from conservative therapy to surgical intervention, and there are no currently approved medications that treat the underlying disease⁴¹. Therefore, FPRs have been investigated as a possible target for diagnostic testing and therapeutic delivery.

Previous research has shown that macrophages infiltrate herniated IVDs in humans⁴⁴ and contribute to inflammation⁴⁵. In a needle puncture model of IVD herniation, it was demonstrated that radiolabeled cFLFLF could be used to identify ongoing inflammation at a specific site of injury due to neutrophil and macrophage infiltration. This has the potential for improved diagnostic yield⁴⁶. A separate study using a murine model of DDD demonstrated that the conjugated fullerene nanoparticle FT-C₆₀ preferentially binds to FPR1 on activated macrophages, resulting in significantly decreased mRNA expression of the proinflammatory molecules interleukin-6 (IL-6) and IL-1. Hyperalgesia testing demonstrated that systemic FT-C₆₀ decreased mechanical hyperalgesia at similar levels to that of local injection. Furthermore, histological staining demonstrated that FT-C₆₀ accumulated at the site of injury and dramatically reduced inflammation, demonstrating the targeting, diagnostic, and therapeutic moieties of this nanoparticle⁴⁶. Altogether, these studies provide evidence that FPR expression can be exploited for the development of non-invasive diagnostic tests to track disease progression and treatments for DDD.

Rheumatoid Arthritis

RA is a chronic inflammatory autoimmune disorder characterized by the progressive destruction of joints as well as systemic symptoms including fever, osteoporosis, and muscle weakness. Although complex and not entirely understood, RA pathogenesis is in part attributed to high levels of inflammatory signals produced by resident and infiltrating cells of joint tissue^47,48. Several are briefly discussed below before outlining the potential diagnostic and therapeutic roles of FPRs in RA.

Cellular Biology in RA

Neutrophils.

The most abundant cell infiltrating joint tissue is the neutrophil, which has been implicated in the initiation and progression of RA^49,50. Importantly, elevated markers of neutrophil activation have been shown to predict erosive disease and joint space narrowing in RA⁵¹. Neutrophils from RA patients have been shown to exhibit higher rates of oxidative burst upon activation by fMLP when compared to healthy controls⁵², demonstrating neutrophil contribution to excessive chronic inflammation. Inhibition of FPR1 by cyclosporin H attenuated neutrophil ROS-induction by plasma from patients in RA remission but not those with active RA, suggesting that FPR1 involvement is primarily in active disease⁵³.

Mitochondria.

Levels of platelet-derived extracellular mitochondria are consistently elevated in the synovial fluid of RA patients⁵⁴. These intact mitochondria may be the source of the elevated mtFP levels in human plasma of patients with RA⁵³, though this is not yet completely understood. These levels have been shown to be predictive of current and future disease activity. Furthremore, they are also associated with increased neutrophil activation markers like calprotectin (S100A8/A9), peroxidase, and neutrophil extracellular traps (NETs)⁵³, all of which are reported to be elevated in RA patients⁵¹.

Synoviocytes.

The role of fibroblast-like synoviocytes (FLS) in RA is significant due to their role as local synovial cells that promote RA by causing invasive synovial hyperplasia and increased inflammatory conditions within joints⁵⁵.

Osteoclasts.

Osteoclasts are formed through the fusion of monocytes and macrophages⁵⁶; this process can be induced by stimulating macrophages with receptor activator of nuclear factor κB ligand (RANKL) in the presence of macrophage colony-stimulating factor (M-CSF)⁵⁷. These bone-dissolving multinucleated phagocytes function in bone metabolism and homeostasis, and much evidence suggests that they play an important role in bone destruction in RA⁵⁸. Due to the involvement of osteoclasts in bone diseases, several studies have attempted to identify molecules that can inhibit osteoclast differentiation.

Diagnosis and Treatment of RA

Due to the heterogeneous and often insidious onset of the disease⁵⁹, the diagnosis of RA remains one of clinical judgement. There are no pathognomonic studies for RA, and the goal of treatment is to prevent disease progression using long-term disease-modifying antirheumatic drug (DMARD) therapy. The role of FPRs in the diagnosis of RA is still being elucidated, with potential avenues including expansion of the previously discussed PET cFLFLF tracer studies for OA. The literature does demonstrate several potential therapeutic avenues of FPRs for RA, including anti-inflammatory^60,61,62,63, anti-osteolytic^64,65, and pro-angiogenic roles^66,67,68,69.

In a K/BxN serum transfer-induced arthritis murine study, compound 43 (Cpd43), a synthetic agonist for FPR2/ALX, demonstrated reduced histologic evidence of inflammation⁶⁰. The same study found decreased production of IL-6 in RA tissue-derived FLS and decreased osteoclast differentiation. A separate study using T-cell driven models of RA found Cpd43 caused decreased joint swelling as well as reduced joint tissue inflammation, synovitis, and cartilage damage in CIA and AIA mice⁶¹. In the same study, an increased proportion of regulatory T-cells, responsible for many anti-inflammatory effects, were observed in the spleen of AIA mice. It was also observed that RA FLS ligation of Cpd43 resulted in downregulation of FLS stimulated by TNF. A second study demonstrated that a novel anti-microbial peptide (AMP), scolopendrasin IX, functions via FPR2/ALX signaling to reduce both neutrophil infiltration into joint synovium on histology and production of the inflammatory cytokines IL-1β, CCL2, TNF- α, and IL-6 from the synovial joints of arthritic mice⁶², while a third has examined the importance of pyridinone derivatives in these roles⁶³. Altogether, these results demonstrate that FPR2/ALX can suppress disease severity by acting on neutrophils, T-cells, and FLS, highlighting a potential therapeutic avenue.

The protective role of FPR2/ALX in inflammatory osteolysis is also related to its effects on osteoclastogenesis. RANKL-induced osteoclastogenesis has been shown to be inhibited via Family with sequence similarity 19 member A (FAM19A)⁶⁴ in the presence of FPR2/ALX. Additionally, WKYMVm, a synthetic FPR2 agonist peptide, inhibits RANKL- and LPS-induced osteoclast differentiation and maturation in murine bone marrow-derived macrophages (BMMs)⁶⁵. Elucidating the mechanism by which FPR2/ALX signaling can modulate bone homeostasis may prove important to further therapeutic research.

In a separate study, WKYMVm caused increased exosome secretion by murine bone marrow-derived MSCs (BMSCs) in a FPR2/ALX-dependent manner⁶⁶, indirectly promoting angiogenesis and local vascularization^67,68. As early angiogenesis and vascularization are key steps in the process of osteogenesis and successful bone repair⁶⁹, further research into FPR signaling and its involvement in angiogenesis should be pursued, as well as the role of FPR2/ALX in mediating M2 macrophage activity as a possible vascularization strategy in bone repair.

Conclusions and Future Perspectives

In this review, we have discussed the physiological expression of FPRs. With infectious, inflammatory, and anti-inflammatory functions, it is evident that FPR signaling has an important role in the pathogenesis, diagnosis, and treatment of many common musculoskeletal conditions including arthritis, degenerative disc disease, and rheumatoid arthritis.

FPRs have been shown to drive the migration of immune cells in both the infection and trauma settings, contributing to arthritis pathobiology, while emerging research indicates that FPR ligands like cFLFLF may be utilized not only for earlier and improved imaging and diagnosis of OA, but direction of hMSCs to sites of OA. This has the potential to drive osteogenesis and promote disease reversal. Similar cFLFLF findings are reported in DDD, in addition to the potential for other FPR ligands like FT-C₆₀ to be utilized in anti-inflammatory and analgesic roles. Furthermore, the literature strongly supports a role for FPRs in the treatment of rheumatoid arthritis, with potential anti-inflammatory, anti-osteolytic, and pro-angiogenic roles. In fact, it is still incompletely understood how mitochondrial disruption occurs in RA. Given that mtFPs activate neutrophils, stimulate chemotaxis, and drive inflammation via FPRs and in doing so contribute to RA pathogenesis, it would be prudent to investigate both how mitochondrial disruption occurs in the setting of RA and how plasma levels of mtFPs become elevated. There is also emerging evidence showing a pivotal role of FPR1 in regulation differentiation of both ADSCs and BMSCs.

Indeed, the wide distribution and activity of FPRs presents a rich territory for research. Their expression in cells of the immune system can be used for novel modalities of imaging and tracking disease activity. Considering the regulative roles of FPRs in differentiation of BMSCs and osteoclasts, more intensive and extensive investigations will be needed to explore FPRs in other bone diseases such as osteonecrosis, osteoporosis and bone fracture.