Lipid Mediators in Neutrophil biology: Inflammation, Resolution and Beyond

Abstract

Purpose of review:

Acute inflammation is the body’s first defense in response to pathogens or injury. Failure to efficiently resolve the inflammatory insult can severely affect tissue homeostasis, leading to chronic inflammation. Neutrophils play a pivotal role in eradicating infectious pathogens, orchestrating the initiation and resolution of acute inflammation, and maintaining physiological functions. The resolution of inflammation is a highly orchestrated biochemical process, partially modulated by a novel class of endogenous lipids mediators known as specialized pro-resolving mediators (SPMs). SPMs mediate their potent bioactions via activating specific cell-surface G protein-coupled receptors (GPCR).

Recent Findings:

This review focuses on recent advances in understanding the multifaceted functions of SPMs, detailing their roles in expediting neutrophil apoptosis, promoting clearance by macrophages, regulating their excessive infiltration at inflammation sites, orchestrating bone marrow deployment, also enhances neutrophil phagocytosis and tissue repair mechanisms under both physiological and pathological conditions. We also focus on the novel role of SPMs in regulating bone marrow neutrophil functions, differentiation, and highlight open questions about SPMs functions in neutrophil heterogeneity.

Summary:

SPMs play a pivotal role in mitigating excessive neutrophil infiltration and hyperactivity within pathological milieus, notably in conditions such as sepsis, cardiovascular disease, ischemic events, and cancer. This significant function highlights SPMs as promising therapeutic agents in the management of both acute and chronic inflammatory disorders.

Introduction

Neutrophils, the most abundant leukocytes in human peripheral blood, contain an arsenal of enzymes and antimicrobial peptides that enable them to be frontline responders during pathogen invasions, while maintaining plasticity for adaptation to the environments to preserve physiological functions (1). Neutrophils are also crucial for orchestrating the body’s acute inflammatory response, utilizing their biochemical armory to produce cytokines (i.e., IL-1β, IL-8), vasoactive products (e.g., histamine and complement factor 5a), and lipid mediators (LM) to dictate the tempo and tone of the inflammatory response (2, 3). These include the classic eicosanoids, prostaglandins, and leukotrienes, which contribute to inflammation by increasing vasodilation, vascular permeability, and enhancing neutrophil recruitment to the site of inflammation. Importantly, neutrophils also play a pivotal role in initiating the resolution phase of inflammation, an active biochemical process that is place for the removal of cellular debris and return of tissue to homeostasis (2).

The resolution of inflammation is partially governed in vivo by the biosynthesis of a novel class of lipid mediators, coined specialized pro-resolving mediators (SPMs). These molecules are composed of structurally distinct bioactive chemical mediators including arachidonic acid (AA) derived lipoxins, eicosapentaenoic acid (EPA) derived E-series Resolvins, and docosahexaenoic acid (DHA) derived D-series resolvins, protectins, and maresins (2). Other molecules also reported to regulate resolution of inflammation include proteins (e.g., Annexin A1, Del-1, melanocortins), gasses (e.g., H₂S, CO), microRNAs (e.g., miR-21, miR −146, miR-33) and nucleotides (2, 4). This review focuses on recent advances describing the multifaceted role of SPMs (D and E-series) in controlling neutrophil functions, such as accelerating neutrophil apoptosis, enhancing their clearance by macrophages, regulating excessive infiltration at sites of inflammation and deployment from the bone marrow (BM), while also promoting neutrophil phagocytosis and tissue repair mechanisms in physiological and pathological conditions. These mediators are biosynthesized by neutrophils via cell-to-cell interactions with endothelia, epithelia, macrophages, and platelets to activate resolution circuits. We particularly focus on the newly discovered roles of resolvins in controlling neutrophil BM differentiation and deployment during stress induced granulopoiesis (5).

How SPMs are Made: Cell to Cell Interactions Between Neutrophils and Collaborators

SPMs are biosynthesized via cell-to-cell communication, a process termed transcellular biosynthesis (2). This process begins where one cell, such as an endothelial cell, coverts DHA or EPA into an intermediate molecule. This intermediate is then transferred to a neighboring cell type, such as neutrophils, which can further metabolize it into the final bioactive product. Neither cell type alone can produce the final molecule without this cooperative interaction. We summarize the transcellular biosynthesis of selected SPMs by neutrophils and neighboring cells in Figure 1a–d. The first SPM elucidated from EPA was the resolution-phase interaction product, Resolvin E1 (RvE1), which resulted from interactions between human peripheral blood neutrophils and vascular endothelial cells in hypoxic conditions and was found in pus-containing exudate from the resolution phase of acute inflammation (6) (Figure 1a). This pathway also yields Resolvin E2 (RvE2) (7), the vicinal diol Resolvin E3 (RvE3) (8), and Resolvin E4 (RvE4). This molecule is biosynthesized during physiological hypoxia via interactions between macrophages and neutrophils (9) or by macrophages during erythrophagocytosis (Figure 1c) (9, 10). RvE4 biosynthesis was confirmed by independent investigators (11). Human neutrophils convert the bioactive RvE4 to the inactive product ω−20 hydroxylated metabolite (20-OH-RvE4) (12). RvE4 has been identified in human cerebrospinal fluid (13), and in plasma from COVID-19 patients (14).

Figure 1. Transcellular biosynthesis of D and E series Resolvins and Maresins and Receptors.

(a-b) In physiological hypoxia, neutrophil-endothelial interactions result in the biosynthesis of both E and D series resolvins. Unesterified EPA is converted to 18-hydroxyeicosapentaenoic acid (18-HEPE) by either acetylated cyclooxygenase-2 or the microbial cytochrome P450 in vascular endothelial cells and then released. Next, neutrophils uptake 18-HEPE and sequentially convert it to 5S-hydroperoxy-18R-HEPE, followed by 5(6)-epoxy-18R-HEPE via 5-LO. This compound is further metabolized to RvE1 by LTA₄H (6); this step was confirmed using human recombinant LTA₄H (7, 20). DHA can also be is converted via 15-LO to produce 17S-hydroxydocosahexaenoic acid (17S-HpDHA) by endothelial cells or epithelia cells (29) This bioactive product is transferred by neutrophils, where it undergoes a second lipoxygenation via 5-LO at the C-7 position, giving rise to the intermediate 7S,8S-epoxide Resolvin. Enzymatic hydrolysis of this epoxy-Resolvin generates the bioactive mediators RvD1 and RvD2 (29, 34). Alternative, 17S-HpDHA can also be converted by 5-LO at the C-4 position forms 4S, 5S-epoxy-Resolvin that selectively that RvD3 and RvD4 by neutrophils (28, 29).

(c) Macrophages in physiological hypoxia convert EPA to 15S-HEPE via 15-LO. Subsequently this intermediate, it undergoes further lipoxygenation via either 5-LO or 15-LO, leading to the production of 15S-hydroxy-5S-HpEPE in macrophages. Neutrophils then convert 15S-HpEPE into RvE4 via 5-LO.

(d) Platelets convert DHA to 14S-HDHA, which is up-taken by neutrophils and further converted into MaR1 via 12 LOX.

(e) D and E series resolvins and Maresins stimulate neutrophil functions via activating specific cell surface GPCRs to increase neutrophil phagocytosis. RvD1 activates both ALX/FPR2 and GPR32. RvD2 binds to GPR18. In addition, RvE1 binds to chemR23, whereas Maresin 1 binds to LGR6.

DHA-derived SPMs include D-series resolvins, such as Resolvin D1 (RvD1), RvD2, RvD3, RvD4 and RvD5, as well as protectins and maresins (15). The complete stereochemistry and biosynthetic pathway of each SPM, total organic synthesis and stereochemical has been established for each molecule has been reviewed in detail (16–18). SPMs mediate their actions via activation of G-protein coupled surface receptor (GPCR) found on neutrophils (Figure 1e), macrophages, monocytes, and dendritic cells (2, 7, 19, 20). For example, both RvE1 and RvE2 selectively activate the receptor ChemR23, RvD1 activates both human GPR32 (21) and mouse ALX/FPR2 and receptors, and RvD2 binds to GPR18 (22) (Figure 1e). Of interest, ChemR23 agonist antibody activates receptor mediated signaling to promote macrophage efferocytosis, reduced neutrophil infiltration at the site of inflammation, and accelerates resolution in a model inflammatory bowel disease (23). In receptor-deficient mice, the RvD1 and RvD2 receptor axis has been proven to regulate neutrophil phagocytosis and control neutrophil diapedesis. The heightened expression of GPR18 in human peripheral blood neutrophils correlates positively with lower mortality rates in sepsis patients (24). Notably, in studies using GPR18 knockout mice, the protective effects of RvD2 against neutrophil-mediated injury during sepsis, reperfusion after second organ injury, and hind limb ischemia were lost (25, 26). These studies suggest a crucial role for GPR18 in mediating the beneficial effects of RvD2 in mitigating sepsis and neutrophil mediated tissue damage (22, 25–27). (28, 29).

Of interest, Maresin 1 (MaR1) is also biosynthesized during neutrophil interactions with platelets (30) (Figure 1d). This molecule binds to leucine-rich repeat containing G protein-coupled receptor 6 (LGR6) on neutrophils to increase phagocytosis of E. coli (31) (Figure 1e). MaR1, in addition to RvD1 and RvD2, helps reduce the activation of cold-stored platelets, which is crucial for preserving platelets over extended periods for transfusions (32). Each SPM displays potent actions in many animal disease models and in vitro at picomolar to nanomolar concentrations is summarized in Table 1 and 2. SPMs have proven to impact disease models, encompassing infection (bacterial and viral) (33), sepsis (34, 35), lupus (36), vascular (37, 38), airway (39, 40), dermal (41), ocular (42), pain (43, 44), fibrosis (45), tissue healing (46), and cancer (47). Reduced SPM levels are identified in the peripheral blood of patients with sepsis (48, 49), SARS-COV2 (50, 51), lupus (52), ischemic stroke (53), cystic fibrosis (54, 55), allergic lung inflammation (56) and Alzheimer’s disease (57). SPM biosynthetic pathways are evolutionarily conserved from invertebrates to humans (2, 58, 59) and were recently identified in humans in areas including the spleen (58), placenta (60), and intestinal mucosal (61), bone marrow (59), liver (62), brain (63), breast milk (64), and lymph nodes (58).

Table 1:

In vivo SPM Functions

Name, abbreviation, stereochemical name, and receptor	Animal Model	Dose	Function in vivo	Receptor
Resolvin E1 (RvE1) 5S,12R,18R-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid (EPA) ChemR23 Receptor	Mice with polymicrobial sepsis	[1μg/mouse]	Attenuated cardiac dysfunction and improved clinical scores. Increased recruitment of CD64lowLy6Chigh monocytes and CD64highLy6Clow macrophages, and cardiac phosphorylation of Akt on Ser473 (150).
	Murine dorsal air pouch model	[50 μg/kg/day]	Accelerated resolution of acute inflammation. Increased neutrophil apoptosis and efferocytosis by macrophages. Decreased excessive neutrophil infiltration (23).	Infiltrating neutrophil have increased expression of ChemR23 (23).
	Severe aplastic anemia (SAA) murine model	[250 ng]	Decreased expression of SIRPα on monocytes and macrophages in the bone marrow. Increased efferocytosis by monocytes (135).	ChemR23 is upregulated in SAA bone marrow monocytes, neutrophils, and T cells (135).
Resolvin E2 (RvE2) 5S,18R-dihydroxy-6E,8Z,11Z,14Z,16E- EPA	BALB/c newborn mice with asthma	[300 ng/mouse]	Reduced airway inflammation by decreasing IL-5 (BALF and serum), IL-13 (BALF), and eosinophil infiltration into lung tissue (98).
Resolvin E3 (RvE3) 17R,18R-dihydroxy-5Z,8Z,11Z,13E,15E- EPA	High fat diet (HFD)-induced type 2 diabetic mice	[1.2 ng/g body weight, 3x/week] [50, 150, 300 ng/mouse 4x days]	Improved glucose tolerance and phosphorylation of Akt. Prevented hyperglycemia of HFD-fed mice in a dose dependent manner (154).
Resolvin E4 (RvE4) 5S,15S-dihydroxy-6E,8Z,11Z,13E,17Z-EPA	Hemorrhagic peritonitis in mice	[100 ng]	Reduced neutrophils and increased macrophages in hemorrhagic exudates. Increased macrophage efferocytosis of neutrophils and red blood cells (RBCs) (9).
Resolvin D1 (RvD1) 7S,8R,17S-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid (DHA) ALX/FPR2 Receptor GPR32 Human Receptor	Murine ischemia/reperfusion brain injury (IRI)	[15 μg/kg/day]	Reduced Ly6G+ neutrophil accumulation and NET formation in ischemic stroke via decreasing IL-1β, TNF-α, IFN-γ, IL-6, and iNOS. Increased neutrophil phagocytosis by CD68+ microglia (143).	ALX/FPR2 was expressed on TMEM119+ microglia. Knockdown of FPR2 via siRNA blocked RvD1 mediated phagocytosis of neutrophil (143).
	Murine lung hilar ligation IRI and orthotopic lung transplant model	[100 ng/kg]	IRI: Protected against early pulmonary dysfunction and organ injury. Increased IL-10 and co-expression of CD11c+SiglecF+ macrophages with intracellular Ly6G+ neutrophils. Decreased neutrophil infiltration, edema, MPO expression, TNF-α, HMGB1, IL-17, CXCL1, IL-6, MIP-α, and RANTES. Transplant: Decreased lung infiltrating neutrophils and the production of CXCL1, TNF-α, IL-17A, MCP-1, and HMGB1. Increased IL-10 secretion (118).	ALX/FPR2−/− KO mice: Increased lung dysfunction, excessive neutrophil infiltration and MPO expression after IR. Decreased TNF-α and HMGB1 secretion in MH-S cells (118).
	IRI in murine lung grafts	[1.2 μg]	Decreased neutrophil infiltration and swarming, ratios of neutrophil to classical monocytes, CXCL2, and TNF-α (119).	ALX/FPR2 is increased on neutrophil, alveolar macrophages, and nonclassical monocytes after reperfusion (119).
	Acute-on-chronic murine liver failure	[0.3 μg/kg]	Decreased Tregs and foxp3/RORγt cells, IL-17a mRNA in liver, and serum alanine aminotransferase (147).
	Murine peritonitis	[100 ng/mouse]	Increased reparative Ly6Clow monocytes and dendritic cells (5).	Phosphorylation of ERK1/2 and STAT3 is blocked in neutrophils from FPR2 deficient mice (5).
	Murine liver injury	[500 ng/mouse]	Decreased TNFα, IFNγ, hepatic neutrophil accumulation, and hepatic pyroptosis via downregulation of Irgb10 and Gbp2 (71).
Aspirin Triggered Resolvin D1 (AT-RvD1) 7S,8R,17R-trihydroxy-4Z,9E,11E,13Z,15E,19Z-DHA ALX/FPR2 Receptor	Murine dorsal skin fold window chamber Tail skin transplant graft	[100 μl] [4 μg/ml]	Increased CD49d+ angiogenic neutrophil, M2 macrophages, IL10-high dendritic cells, and accelerated wound closure (139).	CD49d+Ly6G+ neutrophils have increased expression of FPR2 (139).
	Murine IRI		Decreased IL-1β, IL-6, TNF-α, edema, red blood cells, and excessive neutrophil infiltration (159).	ALX/FPR2 antagonist WRW4 blocked pro-resolving effects of AT-RvD1 (159).
	Subclinical acute kidney injury model in sepsis-surviving mice	[5 μg/kg]	Decreased kidney tubulointerstitial injury, collagen deposition, Iba1+ cells and cleaved casp-3 expression in renal tissue, TNF-α, IL-10, CCL2, TNF-α, IL-4, and IL-10 (151).
Resolvin D2 (RvD2) 7S,16R,17S-trihydroxy-4Z,8E,10Z,12E,14E,19Z-DHA GPR18 Receptor	Apolipoprotein E deficient hyperlipidemic mice	[100 ng/mouse]	Decreased atherosclerosis, necrotic core area, and iNOS expression. Increased macrophage phagocytosis of zymosan (152).	GPR18 antagonist, O-1918, blocked RvD2 actions in atherosclerosis (152).
	Angiotensin II (AngII) infused mice	[100 ng/mouse, every second day]	AngII differentially regulates cardiac lipid mediators: Decreased 17R-RvD1, RvD5, RvD6, RvE3, LXA4, LXB4, and 22-COOH-MaR1, as well as increased the expression of RvD1n-3 DPA, PD2n-3 DPA, MaR1 and TxB2. Prevented cardiac hypertrophy, fibrosis, and dysfunction. Decreased Cd11b cells, monocyte, neutrophils, and reduced aortic expression of Cd3, Runx1, and ICAM-1. Increased PGI2, nitric oxide, Hmox-1, and Cd163 (160).	Mice infused with AngII had increased mRNA levels of Alox5, Alox15, and GPR18 in the aorta and heart (160).
	Murine peritonitis and secondary lung infection	[100 ng/mouse]	Reduced bacterial load (blood and lungs) and IL-23. Increased IL-1B, efficiency of splenic neutrophils, and non-inflammatory alveolar macrophages (CD11b-SiglecF+) (95).
	Murine model of allergic lung inflammation	[1 mg/kg in 100 μl]	Identified two eosinophil subsets: CD101low Eos localized to lung vascular niche, and CD101high Eos located in bronchoalveolar lavage (BAL). Reduced total eosinophil numbers, and the phenotypic conversion of CD101low Eos to CD101high Eos (56).	O-1918 blocked the pro-resolving actions of RvD2 on CD101low Eos in lung, and CD101high Eos in lung and BAL (56).
Resolvin D3 (RvD3) 4S,11R,17S-trihydroxy- 5Z,7E,9E,13Z,15E,19Z-DHA	Lipogenic hepatic steatosis in murine liver	[10 μg/kg, every two days]	Attenuated hepatic steatosis via reversing aggravated glucose tolerance and impaired insulin sensitivity (161).
Resolvin D4 (RvD4) 4S,5R,17S-trihydroxydocosa-6E,8E,10Z,13Z,15E,19Z-DHA	Self-limited E. coli murine peritonitis	[1–100 ng/mice]	Decreased G-CSF, CXCL12, neutrophil infiltration and deployment, Increased efferocytosis of bacteria and apoptotic neutrophil in bone marrow and exudate (5).
	Murine deep vein thrombosis (DVT)	[3 μg]	Decreased macrophage recruitment, neutrophil infiltration, and NET formation. Increased reparative monocytes and apoptotic neutrophils. Decreased neutrophil NET formation (91).

Table 2:

In vitro SPM Functions

Name, abbreviation, and stereochemical name	Dose	Cell Type	Functions in vitro
Resolvin E1 (RvE1) 5S,12R,18R-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid (EPA)	[10 nM]	Human nasal epithelial cells from cystic fibrosis donors	Restored epithelium cilia beating dynamics (68).
		Monocytes from blood of atherosclerosis patients	Elevated ratios of LTB4 to RvE1, and increased binding of the high-affinity ligand LTB4 to BLT-1, are associated with both prolonged inflammation and atherosclerosis (69).
	[10 nM]	Murine bone marrow derived macrophages	Reduced IL-6, IL-1β, CCL2, and TNFα mRNA expression. Increased phagocytosis of E. coli (150).
		Human peripheral blood, bone marrow, and colon mononuclear cells.	ChemR23 is overexpressed in inflamed colon tissue of severe inflammatory bowel disease (IBD) patients. Increased mucosal neutrophil infiltration and ChemR23 expression is associated with anti-TNF and anti-α4β7 immunotherapy resistance in IBD (23).
Resolvin E2 (RvE2) 5S,18R-dihydroxy-6E,8Z,11Z,14Z,16E-EPA	[10–6 M] Itraconazole induces RvE2	Human cervical cancer cells	Incubation with Itraconazole increased RvE2 production. Co-incubation with ML351 (12/15-LOX inhibitor) did not lead to increased RvE2 production (162).
Resolvin E3 (RvE3) 17R,18R-dihydroxy-5Z,8Z,11Z,13E,15E- EPA	[10–6 M] Itraconazole induces RvE3	Human cervical squamous carcinoma cells	Co-incubation with ML351 negatively impacted itraconazole effects on RvE3 production and cancer cell death (162).
	[100 nM]	3T3L1 adipocytes	Increased glucose uptake via the PI3K/Akt pathway (154).
Resolvin E4 (RvE4) 5S,15S-dihydroxy-6E,8Z,11Z,13E,17Z-EPA	[1–10 nM]	Human neutrophil and macrophages	Increased M2 macrophage efferocytosis of sRBCs and apoptotic neutrophils (10).
	[1, 100 ng/mL, 0.3 μM]	Human neutrophil and peripheral blood mononuclear cells (PBMCs).	Neutrophil convert RvE4 to ω-20 hydroxylated metabolite, an inactive product (12).
Resolvin D1 (RvD1) 7S,8R,17S-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid (DHA)	[1–100 nM]	Neutrophil and monocytes from sepsis patients	Three neutrophil subsets identified in sepsis: mature CD16bright neutrophil, immature CD16dim, and CD16– neutrophil. Enhanced phagocytic capacity of monocytes and neutrophil. Increased GPR32 and GPR18 on neutrophil and monocytes (141).
	[250 nM]	Microglia neutrophil	Enhanced microglial phagocytosis of neutrophils. Increased oxidative phosphorylation metabolism and ATP production in microglia after oxygen glucose deprivation/reoxygenation and enhanced glutaminolysis in an AMPK-dependent manner (143).
	[100 ng/kg]	Alveolar macrophages	Decreased secretion of TNF-α and HMGB1 in hypoxia/reoxygenation. Increased expression MerTK in murine IR injury.(118)
	[100nM]	Coronary arteries segments from heart transplantation surgery patients	Reduced human coronary artery ring contractions induced by PGE2 (72).
		Human pulmonary graft samples	SPM levels increased within human lung grafts following reperfusion (119).
Resolvin D2 (RvD2) 7S,16R,17S-trihydroxy-4Z,8E,10Z,12E,14E,19Z-DHA	[100 nM]	Neutrophil from peripheral blood of sepsis patients	Increased neutrophil subsets (mature, immature, and CD16-), phagocytosis of bacteria, maturation, activation, and GPR18 expression. Increased intermediate monocytes and their phagocytic capacity (141).
		Human coronary arteries from atherosclerotic patients	Increased RvD2 and CD68 expression in coronary arteries (152).
Resolvin D3 (RvD3) 4S,11R,17S-trihydroxy-5Z,7E,9E,13Z,15E,19Z-DHA	[50–200 nM]	Mouse primary hepatocytes and C2C12 skeletal muscle cell	Decreased hepatic steatosis, phospho-eIF2, phospho-IRE1, phospho-JNK and CHOP. Increased AMPK expression, LC3 conversion, p62 degradation, and autophagosome formation in C2C12 myocytes and primary hepatocytes (161).
Resolvin D4 (RvD4) 4S,5R,17S-trihydroxydocosa-6E,8E,10Z,13Z,15E,19Z-DHA	[10 nM]	Human bone marrow aspirates and peripheral blood neutrophil	Increased S6 protein and pERK1/2 phosphorylation, STAT3, and neutrophil phagocytosis of live E coli (5).
		Human neutrophil and macrophages	4S,5S-epoxy-17S-hydroxy-6E,8E,10Z,13Z,15E,19Z-docosahexaenoic methyl ester incubated with human neutrophil or M2 macrophages resulted in the production of RvD4 (28).
	[3 μg]	Murine neutrophil	Reduced NET formation (91).

Each SPM exhibits potent pro-resolving actions that are essential for resolution of inflammation (2). These include limiting or halting excessive neutrophil infiltration into the inflamed tissue, regulating chemokines and cytokines, promoting neutrophil apoptosis, and facilitating their clearance by macrophages through a process termed efferocytosis (Figure 2). Controlled neutrophil transmigration is essential for host defense, but their persistent transmigration is maladaptive and can lead to excessive collateral tissue damage. SPMs directly impact neutrophil migration and chemotaxis behavior during inflammation by downregulating the surface expression of CD11b/CD18 and inhibiting their responsiveness to chemotactic gradients, such IL-8 and LTB₄, while preventing the shedding of CD62L (65) (Figure 2). SPMs are also potent agonists that enhance neutrophil mediated pathogen (microbes and viruses) containment, killing, and clearance (33, 34, 66) (Figure 2). For example, SPMs at the single-cell level regulate phosphorylation of ERK1/2 and S6 protein in neutrophils to enhance bacterial containment in human peripheral whole blood (66). SPMs also increases IL-10, downregulates early initiators of acute inflammation, (e.g., prostaglandins, COX-2 expression, leukotrienes) and pro-inflammatory cytokines to promote tissue repair in a manner that does not compromise host defense (2, 18, 33, 34). SPMs are proven to potently limit excessive neutrophil infiltration increase neutrophil phagocytosis in various tissues and regulate neutrophil mediated damage in abdominal aortic aneurysms (67), cystic fibrosis (68), atherosclerosis (69) asthma (70), liver injury (71), and UV induced inflammation, coronary artery contractions (72), infections (73–77), cornea (73), and gingivitis (78, 79) and during second organ ischemia reperfusion (59).

Figure 2. SPMs control neutrophil deployment, infiltration and phagocytic ability.

Neutrophil development begins in the bone marrow and are deployed into circulation in response to circadian mechanisms, such as the clock gene Bmal1, and receptors CXCR2 and CXCR4, along with oscillating levels of CXCL12. In response to pathogens or trauma, neutrophils are recruited to the site of inflammation via chemo-attractants, cytokines, vasoactive mediators, and lipid mediators. An increase in neutrophil demands activates the bone marrow to accelerate granulopoiesis and deploy neutrophils to the site of inflammation. At the post-capillary venules, neutrophils roll and adhere to the activated endothelial vessel wall, and then diapedeses through the vasculature towards the inflammatory stimuli along a chemokine gradient. The pro-inflammatory mediator, LTB4, is amplified in neutrophil swarming, which is essential for the containment, killing, and clearance of pathogens. SPMs block excessive neutrophil transmigration, adhesion, and rolling on activated endothelial cells. These lipid mediators also counter-regulate LTB4-mediated actions, as well as increase neutrophil phagocytosis and their efferocytosis by macrophages. SPMs also block neutrophil-mediated NET formation and the production of inflammatory cytokines.

During inflammation, the lifespan of neutrophils is significantly prolonged, often by several fold, as they undergo activation and delay their clearance from the tissue. For example, RvE1 increases expression of CCR5 on apoptotic neutrophils and activates apoptotic T cells during the resolution phase of murine peritonitis facilitating the scaffolding of CCL3 and CCL5 by macrophages (80). In human neutrophils, RvE1 mitigate AKT and ERK mediated survival signals enhancing their apoptosis (81) and their clearance by macrophages via activating its receptor, ChemR23 (19, 20, 82, 83). Beyond their role in phagocytosis, neutrophils release their DNA content, forming NETs that are crucial for capturing and destroying invading pathogens (84). However, excessive NET release and ineffective clearance can lead to sepsis, arthritis, deep vein thrombosis, age-related organ fibrosis, complications associated with COVID-19 infections, and cancer (84–86). Chronic stress can foster a pro-metastatic lung microenvironment for breast cancer cells by inducing NET formation via glucocorticoid release (87). Studies on acute and chronic stress reveal alterations in neutrophils in the spleen and BM, along with decreased levels of SPMs (88). RvD2 and T series resolvins reduce NETosis and enhance their clearance by macrophages (89, 90). RvD4 has shown to accelerate thrombus resolution by decreasing NETs and myeloperoxidase (MPO) formation, while also protecting against neutrophil-mediated secondary organ injury (91). RvD1 also decreased neutrophil accumulation and NETs formation following ischemic stroke, along with decreasing levels of IL-1β, TNF-alpha, IL-6, and iNOS (92). Importantly, we recently uncovered a novel function for RvD4, in controlling neutrophil apoptosis, deployment, and emergency-induced stress granulopoiesis, while also blocking LTB₄-mediated mobilization of neutrophils (5) (Figure 2).

Resolvins are also potent regulators of myeloid functions. Recently, RvD2 was demonstrated to reduce liver steatosis and hepatic fibrosis mediated by increased of both monocyte-macrophage progenitors and differentiation of reparative macrophages (93). In a murine double hit model of cecal ligation puncture-induced sepsis and secondary lung infection with Pseudomonas aeruginosa, RvD2 reduced bacterial load in both blood and lungs, along with decreased lung lavage levels of IL-23 (94, 95). Importantly, RvD2 increased the numbers of non-inflammatory macrophages and enhanced neutrophil reactive oxygen species (ROS) production. Of interest, RvD2 accelerated the resolution of allergic lung inflammation by decreasing the recruitment, activation, and production of pro-inflammatory genes (Chi3l3, Tlr8, and Cysltr1) in CD101^low eosinophils. RvD2 decreased PGD₂ release by CD101^high eosinophils and their production of IL-5, as this population is present only during lung inflammation (56). Human mast cells release RvD1 after IgE mediated activation, thus highlighting the role of resolvins in the potential regulation of allergic inflammation (96). MaR1 and RvD1 both increase the anti-inflammatory and antimicrobial properties of human macrophages infected with M. tuberculosis (97). RvE2 also reduces airway inflammation in newborn mice with asthma by decreasing eosinophil infiltration into lung tissue, IL-5, and IL-13 levels in BALF (98). Overall, these studies highlight the potential for SPMs in regulating neutrophil behavior and stress-induced responses, shedding light on their therapeutic potential for managing inflammatory disorders.

Leukotriene B4 and SPMs are Controllers of Neutrophil Swarming

Neutrophil vascular recruitment, arrest, and transendothelial migration are regulated by LTB₄ during inflammation. LTB₄ is one of the most potent in vivo neutrophil chemoattractants and serves as the primary orchestrator of neutrophil swarming, amplification, and activation (99). LTB₄ regulates neutrophil cell polarization and adhesion responses during neutrophil arrest in vivo by mediating the redistribution of non-muscle myosin IIA and β2-integrin, subsequently leading to their extravasation into the inflamed tissue (100). Human neutrophils biosynthesize LTB₄ by converting AA to LTA₄, which is further converted to LTB₄ (101) (Figure 3). LTA₄ can also serve as the substrate for 12-LO found in platelets to biosynthesize SPMs, Lipoxin-A₄ (LXA₄), and the positional isomer Lipoxin-B₄ (LXB₄) (102) (Figure 3). Both LXA₄ and LXB₄ are potent agonists that control excessive neutrophil infiltration at the site of inflammation (102). During the early stages of swarming, neutrophils biosynthesize LTB₄ either via neutrophil-neutrophil interactions (i.e., transcellular biosynthesis) (103) or independently (101) (Figure 3). This biochemical process was demonstrated via co-incubation of neutrophils from 5-LO and LTA₄H deficient mice. Importantly, LTB₄-mediated swarming was impaired in neutrophils from either 5-LO or LTA₄H deficient mice and was restored upon co-culturing (103).

Figure 3. Transcellular biosynthesis of Leukotriene B₄ (LTB₄) and the pro-resolving mediator Lipoxin A₄ (LXA₄).

Neutrophils convert AA to 5S-HpETE, this is further converted to LTA₄ by two consecutive reactions via 5-LO and FLAP. LTA₄ is further converted by LTA₄H to LTB₄. LTB₄ binds to its high affinity receptor BLT1 and with low affinity to BLT2 receptor. During neutrophil swarming, LTA₄ can also be transferred to other neutrophils where it can be further converted by LTA₄H to LTB₄ in the second neutrophil. Alternately, LTA₄ can be metabolized into LXA₄ and LXB₄ via 12-LO in platelets. LXA₄ acts as the first signal to stop neutrophil swarming.

Additionally, CXCL8, galectin-3, and pentraxin-3 can also modulate LTB₄-driven neutrophil swarming (104). Neutrophil swarming is a highly regulated process to prevent collateral tissue damage and to effectively clear pathogens, which can become impaired in patients with infections, such as cirrhosis and those undergoing solid organ or stem cell transplantation (105, 106). Yersinia pestis, the causative agent of the plague, inhibits LTB₄ biosynthesis in neutrophils to limit their swarming, leading to immunosuppression and death in the host (107, 108). LTB₄ mediates its actions by binding to its high-affinity receptor BLT1. The LTB₄-BLT1 axis controls neutrophil swarming by activating relays of chemotactic signals, long-range calcium waves to neighboring neutrophils, thereby broadening their recruitment range to the area of injury (104, 109, 110). Antagonizing these axes causes defective neutrophil swarming and increased infections (111, 112). Interestingly, cortisone treatment in patients with oropharyngeal candidiasis downregulated BLT1 expression on neutrophils, causing defective swarm formation and increased infection (111). Genetic deletion of LTA₄H or BLT1 in neutrophils substantially impaired neutrophil phagocytic functions to clear Candida albicans and Aspergillus fumigatus (112). Recently, a genetically encoded fluorescent reporter was created in neutrophils that allows the measurement of endogenous and exogenous LTB₄ both in vivo and in vitro, highlighting novel methods to visualize LTB₄-mediated neutrophil swarming and chemotaxis (113).

Neutrophil swarming is also controlled by the metabolic pentose phosphate pathway (PPP) that regulates neutrophil crowding and production of ROS, and provides energy for the swarm (114). Neutrophil swarming also relies on the Arp2/3 complex that generates branched actin networks necessary for maintaining cell integrity (114). Inhibition of Arp2/3 results in apoptosis of the crowding neutrophils at the center of the swarm, which is a crucial component for the resolution of inflammation (114). NADPH-oxidase acts as a negative feedback loop to control neutrophil swarming via a self-extinguish relay mechanism (110). Activation of G protein-coupled receptor kinase 2 (GRK2) is necessary to initiate the GPCRs, mediated by LTB₄ and CXCL2, and is critical for containing bacteria and facilitating phagocytosis (115) (Figure 3). LTB₄ production by neutrophils during the swarm is also essential for monocyte recruitment to the site of injury or infection (116). Additional signals, such as lipid mediators PGD₂ and PGE₂, are also produced during the initial phase of neutrophil swarming (104). Towards the later stages of the swarm, LXA₄ and RvE3 are produced 20-fold higher compared to LTB₄ (104) (Figure 3). SPMs regulate neutrophil transendothelial migration and infiltration by antagonizing BLT1-mediated responses and preventing further LTB₄ formation (117)

Two independent studies demonstrated the protective actions of RvD1 in preventing neutrophil mediated lung injury after lung transplantation (118, 119). Notably, RvD1 was identified in the bronchoalveolar lavage fluid (BALF) of patients after lung transplantation and its levels were positively associated with improved lung function (118). RvD1 reduced neutrophil swarming and their extravasation, thereby preventing early graft rejection of transplanted lungs. Neutrophils from these mice also exhibited a linear trafficking trajectory along the blood flow and had decreased gene expression of TNF-α, hypoxia, TGF-β, and mTORC1 signaling unlike those treated with the vehicle, which displayed multidirectional movement (119). In ALX/FPR2 deficient mice, RvD1-mediated actions were lost, suggesting its role in polarity and directionality of neutrophils is receptor dependent. RvD1 also blocked LTB₄-stimulated expression of adhesion molecules and actin polymerization in neutrophils to further control their diapedeses into the inflammatory site (21). RvD1 also decreased neutrophil accumulation in the ischemic myocardium and reduced fibrosis post myocardial infarction. These protective actions of RvD1 were linked to a positive feedforward biosynthesis of other SPMs in the spleen and the mobilization of reparative monocytes (92). In humans, sleep disturbances downregulate D-series resolvins, potentially delaying inflammation resolution (120). Sleep disruptions may also heighten neutrophil activation and atherosclerosis risk (121, 122).

Excessive levels of LTB₄ are shown in chronic inflammatory diseases like arthritis, cancer, metabolic disorders, and cardiovascular disease (123). It has been proposed the ratio between LTB₄ levels and SPMs in serum may predict disease outcome; for instance, a high ratio is associated with delayed inflammation resolution and atherosclerosis progression (37, 69), airway inflammation (124), and ischemic stroke (53). BLT1 knockout mice show reduced plaque formation, implicating LTB₄ in disease progression. Conversely, transgenic mice lacking ChemR23 and GPR18 receptors exhibit accelerated plaque formation and inflammation, underscoring the role of the resolvin-receptor axis in mitigating atherosclerosis (125–127). The balance between LTB₄ and SPMs could be crucial for regulating neutrophil swarming, facilitating an effective immune response, minimizing tissue damage, and promoting timely inflammation resolution.

Resolvins: New Players in Bone Marrow Dynamics

The BM contains the largest reservoir of neutrophils and is comprised around 50–60%. Neutrophil development begins with a tightly regulated sequence of events from the granulocyte-monocyte progenitors (GMPs) into the myoblasts, promyelocytes, myelocytes, metamyelocytes, band cells (immature neutrophils), and finally with segmented neutrophils (mature neutrophils) (1) (Figure 2). In physiological conditions, mature neutrophils are governed by circadian oscillations within the hematopoietic niche that is orchestrated by the microbiome and clock-related genes, notably Bmal1 and CXCR2 (128, 129). Specifically, Bmal1 and CXCL2 regulate circadian oscillation of neutrophil gene expression and granule content (1, 128). BM neutrophil maturation and mobilization into the peripheral blood is controlled by CXCR2 and CXCR4 (130, 131). Neutrophil retention is signaled via CXCR4 that is expressed on neutrophils and its ligand CXCL12, which is primarily produced by stromal cells in the BM niche (1, 132). The temporal downregulation of CXCL12 by stromal cells leads to the deployment of neutrophils from the BM into the peripheral blood. Disruption of this axis, as seen in acute lung injury, can be modulated by RvD1, which suppress CXCL12 production and CXCR4 expression, thereby regulating the accumulation of alveolar neutrophils in the lungs (133). Moreover, the CXCL12/CXCR4 signaling axis controls neutrophil homing via increasing CXCR4 expression on their surface, which is associated with an “aged phenotype” (CD62L^lowCXCR4^high) (134). These neutrophils are cleared by CD169⁺ resident macrophages via the cholesterol-sensing nuclear liver receptor X pathway (134). Efferocytosis of aged neutrophils in turn promotes the circadian release of hematopoietic progenitor cells into circulation by downregulation of CXCL12 in the stroma. RvD4 increases the clearance of aged neutrophils by CD169⁺ macrophages in vivo during infection. In addition, RvE1 administration in mice with severe aplastic anemia enhances macrophage efferocytosis and platelet production, and restores normal neutrophil numbers, leading to increased host survival (135). For instance, RvD4 triggers the phosphorylation of crucial signaling molecules like STAT3 and ERK1/2, which play pivotal roles in regulating granulopoiesis across various stages of neutrophil maturation in human BM. Notably, the phosphorylation of STAT3 induced by RvD4 appears to occur independently of ALX/FPR2 receptor activation (5). These findings suggest that RvD4 may signal neutrophils in BM via its own receptor, although the identity of this receptor remains elusive. Additionally, RvD4 and its inactive metabolites, namely 17-oxo-RvD4 and 15,16-dihydro-RvD4, were identified in human BM samples (59). Of interest, EPA-derived LXA₄ and LXA₅ were identified in the head kidney gland of trout, the hemopoietic organ equivalent to mammalian BM (136). These studies highlight SPMs to be conserved during evolution.

During the acute inflammatory response, pathogen-associated signals increase BM granulopoiesis and deploy neutrophils to the site of infection to contain, clear, and kill pathogens. Failure to contain infection can lead to systemic bacterial dissemination and evoke emergency granulopoiesis. We recently reported that RvD1 and RvD4 are temporally produced within the BM, where RvD1 exhibited elevated levels during both the initiation and resolution phases of peritonitis, while RvD4 levels consistently remained below baseline infection (5). These changes overlapped with an increase in indicators of emergency granulopoiesis such as MPP2, GMPs, pre-neutrophils, immature and mature neutrophils (5). Using single-cell mass cytometry, RvD4 controls emergency granulopoiesis by regulating GMP, pre-neutrophil, and immature neutrophil differentiation. RvD4 also promoted HSC differentiation toward B and T lymphocytes, controlled granulopoiesis, and attenuated neutrophil deployment, while RvD1 facilitated GMP differentiation into reparative monocytes. Importantly, RvD4 reduced neutrophil accumulation in the site of infection, enhanced bacterial clearance, and accelerated neutrophil apoptosis and efferocytosis (5). RvD4 also blocks mobilization of BM neutrophils and increases BM CXCL12 levels to promote neutrophil retention during infection (5). LTB₄ antagonizes CXCL12 to promote chemotaxis (137). Using a microfluidics chamber, RvD₄ proved to block LTB₄ mediated chemotaxis of human neutrophils. RvD₄ biosynthetic isomers 10E-RvD4, 10E,13E-RvD4, or metabolized 17-oxo-RvD4 did not halt LTB₄ mediated neutrophil chemotaxis and proved to be inactive (59). Also, 10E-RvD4 and 10E,13E-RvD4 both are inactive, and do not induce human neutrophil phagocytosis, when compared to RvD4. The role(s) and functions of other SPMs within the bone marrow and its microenvironment represent an exciting new area of research.

Resolvins and Neutrophil Heterogeneity

Neutrophils, traditionally viewed to be a homogenous population, are now appreciated to exhibit morphological, phenotypical, and functional heterogeneity in a variety of physiological and pathological conditions (1, 138). For example, neutrophils demonstrate variable life fates across different tissue microenvironments, and their states are linked to non-canonical functions such as vascular repair (138). Specifically, neutrophils can be recruited by vascular endothelia growth factor A (VEGF-A) in hypoxic tissues with proangiogenic properties that enable restoration of blood supply (1). In this context, AT-RvD1, the epimer version of RvD1, increases the percentage of CD49d⁺VEGFR1⁺Ly6G⁺ angiogenic neutrophils that have increased expression of ALX/FPR2, and accelerates wound healing and closure in mice (139). The population comprises about 3% of circulating neutrophils in steady-state conditions, and are also identified in hypoxic tissues, liver injury, ischemia, and irradiation. angiogenic neutrophils are reported to upregulate genes involved in in vascular growth and repair (including Apelin, Adamts, or Vegfa) (140). In sepsis patients, neutrophil subsets have been identified based on CD16 surface expression and CD16^bright neutrophils are positively associated with organ failure and exhibit impaired phagocytic ability. Importantly, ex vivo treatment of septic CD16^bright neutrophils with RvD1 partially restores their phagocytic ability for E. coli particles (141) (Table 1). Expression of RvD2 receptors ALX/FPR2 and GPR18 are identified in CD16^bright neutrophils from both healthy and septic patients (141). Sepsis patients also have increased abundance of immature neutrophil subsets with IL1R2⁺, PADI4⁺, MPO⁺ and cycling MK167⁺CYP1B1⁺ neutrophils (142). These neutrophils have been implicated in contributing to increased susceptibility to infections following injury and in promoting metastasis of pancreatic cancer. The involvement of lipid mediators and their corresponding receptors, such as LTB₄ and resolvins, in modulating various neutrophil subtypes in during pathological and physiological conditions remains largely unexplored, but we speculate it will provide important clues in targeting these pathogenic states of neutrophils. Clearly, additional research is essential in further elucidating their role in regulating neutrophil heterogeneity under both physiological and pathological conditions.

Conclusions

Resolvins employ multifaceted effects such as limiting neutrophil infiltration, controlling neutrophil swarming, decreasing pro-inflammatory cytokines, accelerating neutrophil apoptosis, and promoting macrophage phagocytosis and efferocytosis. In doing so, SPMs attenuate neutrophil meditated inflammatory and chronic diseases such as ischemia (26, 27, 59, 118, 119, 143–146), liver failure (147), chronic liver diseases (93, 147–149), sepsis (25, 33–35, 94, 95, 141, 150, 151), cardiovascular disease (37, 67, 69, 72, 78, 92, 126, 152), metabolic syndrome (53, 153, 154), and cancer (155–158). The intricate dynamics between neutrophils and SPMs in the resolution of inflammation begins at their source, in the BM, which highlights the therapeutic potential of targeting these pathways for modulating the full range of immune responses and promoting tissue repair. While SPMs have been extensively studied in the periphery, their function within the BM is an active area of investigation and raise a number of exciting questions. For example, are SPMs the new maestros of myelopoiesis by regulating the bone marrow microenvironment? How are SPM receptors modulated in neutrophil mediated diseases during chronic inflammation? The are particularly relevant questions as dysregulation of SPM signaling pathways and exacerbated production of inflammatory neutrophils may contribute to perpetuation of inflammation and the pathogenesis of chronic inflammatory disorders. A final relevant question is whether all neutrophil subsets equally express SPM receptors, or are there variations that reflect their distinct functional phenotypes? Understanding the differential expression and modulation of SPM receptors, and therefore the possibility of controlling their function, across neutrophil subsets could provide valuable insights into the pathophysiology of inflammatory diseases and inform targeted therapeutic strategies. Taken together, SPMs, biosynthetic pathways, and receptors offer a promising avenue to understand immune physiology and to enhance the activation of resolution mechanisms.

Key points:

1. Resolution of inflammation is partially governed by specialized pro-resolving mediators (SPMs) that are transcellularly biosynthesized via interactions between neutrophils, macrophages, endothelial cells, epithelial cells, and platelets.

2. SPMs control excessive neutrophil bone marrow deployment and infiltration at the site of inflammation, while reducing pro-inflammatory molecules.

3. SPMs enhance neutrophil apoptosis, phagocytosis, and their clearance by macrophages.

4. Leukotriene B₄ (LTB₄) orchestrates neutrophil swarming, amplification, and activation, which is stopped by SPMs.