Pro-Resolving Lipid Mediators in the Respiratory System

Abstract

Lung inflammation, infection and injury can lead to critical illness and death. The current means to pharmacologically treat excessive uncontrolled lung inflammation needs improvement because many treatments are or will become immunosuppressive. The inflammatory response evolved to protect the host from microbes, injury and environmental insults. This response brings phagocytes from the bloodstream to the tissue site to phagocytize and neutralize bacterial invaders and enables airway anti-microbial functions. This physiologic response is ideally self-limited with initiation and resolution phases. Polyunsaturated essential fatty acids (PUFA) are precursors to potent molecules that govern both phases. In the initiation phase, arachidonic acid is converted to prostaglandins and leukotrienes that activate leukocytes to transmigrate from post-capillary venules. The omega-3 fatty acids (e.g. DHA and EPA) are precursors to resolvins, protectins and maresins, which are families of chemically distinct mediators with potent functions in resolution of acute and chronic inflammation in the respiratory system.

Introduction

The respiratory system has the vital function of delivering oxygen to the circulating blood and hence is highly vascularized. The respiratory system from the nose, sinus, and upper and lower airways to the distal lung is protected from invaders by the innate and adaptive immune system. Resident immune cells of the lung and phagocytes delivered by the pulmonary circulation protect and defend the respiratory system from infection, injury and excess inflammation. The essential polyunsaturated fatty acids (PUFAs), arachidonic acid, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are precursors to many chemical signals that instruct cell responses and functions in the respiratory system. For example, arachidonic acid is precursor to eicosanoids including prostaglandins, thromboxanes, leukotrienes and lipoxins that play critical roles in the pulmonary circulation, coagulation and inflammation. Platelet-derived thromboxane and vascular endothelial cell prostacyclin are key lipid mediators in vascular tone and coagulation (1). Leukotrienes such as leukotriene B₄ (LTB₄), the potent chemoattractant, are critical in host defense and bring circulating phagocytes to the site of injury or microbial invasion in the respiratory system. The slow-reacting substance of anaphylaxis (SRS-A), or leukotrienes (LT) LTC₄, LTD₄ and LTE₄, are potent smooth muscle contractors in the respiratory system produced by mast cells, eosinophils, and macrophages, including alveolar macrophages (2). The structures and mechanisms of the enzymes involved in the biosynthesis of the leukotrienes are fully elucidated, as reviewed recently in Haeggström and Newcomer (3). There is increasing evidence from large-scale randomized human clinical trials that the omega-3 essential PUFA EPA and DHA play critical roles in human heart and lung health (4-10). The acute inflammatory response is divided on the basis of cellular infiltrates by pathologists (11) into initiation and resolution phases. The arachidonic acid-derived eicosanoids prostaglandins and leukotrienes help mount leukocyte tissue infiltration, and the omega-3 PUFA DHA and EPA are precursors to resolvins, protectins and maresins, which are predominantly biosynthesized temporally during the resolution phase and function to promote resolution of inflammatory exudates and tissue infiltrates (12-14). These specialized proresolving molecules (SPM) and their biosynthetic pathways were the subject of earlier Annual Reviews. Interested readers are directed to these extensive reviews of the foundational literature (15-17) for detailed accounts. The role of nutrition in resolving inflammation was also the subject of an earlier review by Zhang and Spite (18). The resolvins and SPMs, given their potent anti-inflammatory and proresolving function, open the terrain of resolution biology and pharmacology as new approaches to control excessive inflammation to many investigations, since these novel molecules are now commercially available. Uncontrolled inflammation is today associated with many widely occurring chronic human diseases, heightening the interest in new approaches to controlling the deleterious outcomes of these diseases via exploring agonists of endogenous resolution mechanisms, since the current approaches have some unwanted side effects, including immunosuppression (19).

Authoritative recent reviews on new approaches with resolution pharmacology provide a solid analysis of this new field for readers (see (20, 21) in the Annual Review series). Ji also reviewed initial results for the Annual Review of Pharmacology and Toxicology demonstrating the potent actions of SPM in reducing pain and itch in animal models (22). It has been 10 years since our first review of SPM in the resolution of acute lung inflammation (17). In this Annual Review, we present and review the major steps taken and advances in our appreciation of the function of SPM and novel, more recently uncovered cys-SPM in the control of resolution of inflammation in the respiratory system since our earlier annual review. The therapeutic potential of resolvins in pulmonary diseases (23) and their major structure-function relationships have recently been recognized by others and rigorously reviewed (24), which further underscores our original interest in comprehensively studying the novel molecules produced de novo in the resolution phase of the acute inflammatory response to guide us to new approaches to control excessive and chronic inflammation and collateral tissue damage in the respiratory system relevant in lung injury, sepsis and other life-threatening scenarios that today open new concepts in resolution medicine.

Since our earlier AR review together in 2014 focusing on resolution of inflammation in lung (17) and in our more general resolution of inflammation review (19) highlighting the emergence of the super-family of pro-resolving mediators and fundamental cellular mechanisms in the biosynthesis and functions of pro-resolving mediators, there are many exciting new developments in this period since the 2018 review , in this very rapidly growing field of pro-resolving lipid mediators in general and in the respiratory system. Here, a few of the very impactful discoveries from investigators around the world are briefly highlighted:

• Human in vivo production of the resolvins, protectins and maresins (SPM) is independently confirmed and well documented by others (25, 26) including in human adolescents (27) and in young adults in a population study of 978 subjects at 27 years old (28).

• Resolvin E1, which displays function in the lung and in cardiovascular disease models, is diminished in humans with adiposity (28), elegantly demonstrated using LC-MS-MS targeted metabololipidomics.

• Human SPM tissue amounts are dependent on consumption of the n-3 essential fatty acids EPA and DHA precursors (29) with SPMs dysregulated in SARS-COVID infections in humans (30).

• In human airway, SPM are identified in chronic rhinosinusitis patients (31) and discovered to play an important role in cystic fibrosis (32, 33) as a potential marker of disease (34) and therapeutic potential (35).

• Human Vagus nerve on electrical stimulation ex vivo releases SPMs and reduces prostaglandins (36).

• SPM are regulated by low-dose carbon-monoxide in non-human primates (37).

• Complete stereochemistry and total organic synthesis of the cysteinyl-containing maresins, protectins and resolvins are established (38) and commercially available.

• cys-Maresins counter-regulate the actions of cys-leukotrienes (39) relevant to human asthma (40).

• The resolvin D2 receptor was uncovered (41), and agonist antibody to the resolvin E1 receptor was introduced and demonstrated to be a potent stimulant of endogenous resolution programs as a promising new therapeutic immunoresolvent for chronic inflammatory diseases (42) and are now in clinical development.

• A role for SPMs in the pathogenesis of organ fibrosis and pulmonary fibrosis is recognized (43).

These are just some of the exciting advances in this rapidly growing new field of SPMs in resolution medicine that can impact airway diseases, cardiovascular and many other diseases characterized by excessive inflammation (44).

Resolvin Biosynthesis and Role of Activated Vascular Endothelium

The first resolvin, termed Resolvin E1, was isolated from inflammatory exudates produced in vivo in self-limited acute inflammatory response in the resolution phase (12) and proved to be a potent bioactive molecule produced from EPA. The structure of Resolvin E1 was presented at the International Eicosanoid meeting that year (2000) in Florence, Italy. Resolvin E1 structure was elucidated, and it was shown to stop leukocyte transendothelial migration and leukocyte infiltration in vivo in mice. To explore the biosynthesis with human cells, hypoxic microvascular endothelial cells were coincubated with isolated human neutrophils. The endothelial cells were activated with TNF-α and IL-1β as well as the hypoxic environment that upregulated COX-2 to mimic in vivo conditions during acute inflammation. The activated endothelial cells release omega-3 EPA that is converted by acetylated COX-2 to 18-HEPE, which can be further transformed via transcellular biosynthesis by human neutrophils to Resolvin E1 (12) in this coincubation setting designed to mimic the exudate conditions in which this novel 5,12,18-trihydroxyeicosapentaenoic acid was originally isolated as a bioactive molecule (12). The complete stereochemistry of Resolvin E1 was established by total organic synthesis and matching to the endogenous exudate-derived bioactive molecule, confirming its potent actions and proposed structure (45). Resolvin E2 was identified soon afterwards (46) as the dihydroxy bioactive product of this EPA pathway.

Activated human neutrophils in a hypoxic environment readily convert 18-HEPE to Resolvin E2 in a hypoxia chamber. The reaction mechanism to produce 18R-HEPE and 18S-HEPE with aspirin-acetylated COX-2 was next elucidated (47) and recently confirmed by Cebrián-Prats et al. (48). The biosynthesis of the E-series Resolvins was reviewed in detail recently (49), with focus on the complete stereochemistry and enzymes involved for each step of the biosynthesis. The role of 5-lipoxygenase (5-LOX) and LTA₄ hydrolase in Resolvin E2 was established (46, 50) using isolated and recombinant enzymes. E-series Resolvins are locally biosynthesized, act as autacoids and are locally inactivated. Stable analogs of Resolvin E1 were prepared that demonstrate this principle (49, 51).

Resolvin E4 was uncovered more recently (52); it displays key resolution functions, i.e., limiting neutrophil infiltration and increasing macrophage efferocytosis, but also stimulates the clearance of senescent red blood cells, a physiologic function of splenic macrophages (53). The structure and function of Resolvin E4 was confirmed in two independent total organic syntheses (53, 54). E-series and D-series resolvins require vascular endothelial cells to convert EPA and DHA, respectively, to resolvin precursors to produce these potent molecules via transcellular biosynthesis (55). Both 18-HEPE and 17R-HDHA, in addition to their role as precursor, also possess potent actions of their own (56, 57). The D-series resolvins require transcellular biosynthesis, which also places them in the location where they can effectively regulate leukocyte-endothelial cell interactions and circulate to act on other cell types to regulate detachment (55, 58). Of interest, M2-like macrophages, reparative cells, can produce all of the SPMs on their own because they possess the enzymatic machinery needed to produce and release SPMs (59), including Protectins and Maresins (Figure 1 A&B) (60-62).

Figure 1A. Lipid-derived mediators in programmed resolution of the acute inflammatory response.

Arachidonic acid is the precursor to eicosanoids that have distinct roles as proinflammatory mediators. Prostaglandins and leukotrienes each play specific actions pivotal to the progression of inflammation. Arachidonic acid-derived epoxyeicosatetraenoic acids (EETs) produced via P450 (2, 149) and ω-3 PUFA P450 epoxides may also play roles (150, 151). Cell-cell interactions, exemplified by platelets-leukocytes transcellular biosynthesis of lipid mediators within blood vessels and/or PMN-airway epithelial cell interactions, enhance generation of lipoxins that serve as endogenous anti-inflammatory mediators self-limiting the course of inflammation (13). The essential omega-3 fatty acids eicosapentaenoic acid and docosahexaenoic acid (C20:5 and C22:6) are converted to two novel families of lipid mediators, resolvins and protectins, that play pivotal roles in promoting resolution by regulating leukocyte traffic and functions. Resolvins of the E series are generated from eicosapentaenoic acid (e.g. RvE1), and resolvins of the D series (e.g., resolvin D1) and the protectins, such as neuroprotectin D1, are biosynthesized from DHA. Aspirin-triggering epimers of lipid mediators: Aspirin impacts the formation of lipoxins and resolvins by acetylating. COX-2 (e.g., in human vascular endothelial cells that stereoselectively can generate, in the case of RvE1 biosynthesis, 18R-HPEPE, which is picked up via transcellular cell-cell interactions by leukocytes and converted in a lipoxygenase-like mechanism to RvE1). The complete stereochemistry of RvE1 and at least one of its receptors were established (45). The biosynthesis of RvE1 can also be initiated by P450-like enzymes in microbes (12). Aspirin also impacts the biosynthesis of D-series resolvins. Aspirin catalytically switches COX-2 to a 17R-lipoxygenase-like mechanism that generates 17R-containing series of resolvin D and protectins (e.g., neuroprotectin D1/protectin D1; see text). Please see Serhan et al. (152) and Hamidzadeh et al. (153).

Figure 1B. Resolvins and the Acute inflammatory response in pulmonary disease.

Acute insult by way of injury (surgical intervention, trauma) or microbial invasion. Neutrophils (PMNs) traffic to the site of insult by rolling along the endothelium in the postcapillary venule, eventually adhering and transmigrating out of the vessel into the site of inflammation by swarming along a chemotactic gradient as with leukotriene B₄ (154, 155). Processes such as neutrophil degranulation, neutrophil apoptosis and subsequent macrophage activation increase proinflammatory mediators such as complement components, chemokines, cytokines and cellular debris that in turn continue to promote inflammation and tissue damage. Multiple avenues for resolution medicine to intervene and promote the resolution of inflammation are illustrated;

The cell types shown to produce resolvins and the other SPM, which can impact the respiratory system, are listed in Table 1. Production of prostaglandins, leukotrienes and the proresolving mediators is temporally regulated and follows the trafficking of leukocytes in organs, including the lung (Figure 1 and Figure 2). This lipid mediator class switch is a critical component of the resolution of inflammation with the biosynthesis of specific mediators that are agonists for the resolution of inflammation (63). Both prostaglandin E₂ and prostaglandin D₂ stimulate the expression of the enzymes needed to biosynthesize resolution phase SPM via increasing cAMP in leukocytes (illustrated in Figure 1). Of interest, Dahlke et al demonstrated that FLAP antagonists block conversion of AA by 5-LOX to LTs and LX, but not the conversion of DHA to SPM, which requires the 5-LOX (64). In addition, antagonists of LTA₄ hydrolase decrease conversion to LTB₄ and induce a lipid mediator class switch to increase LTA₄ conversion to lipoxins (65).

Table 1.

SPM-Producing Cell Types of the Lung

Cell Type	SPM	Reference
Alveolar macrophages	RvD1 and LXA4	Townsend et al. (160)
M2-like macrophages	Resolvins, protectins, maresins	Werz et al. (59)
Mast cells	RvD1	Puzzovio et al. (161)
PMN-platelets	MaR1	Abdulnour et al. (121)
PMN-airway epithelial cells	Lipoxins, RvD1	Cox et al. (162) Isopi et al. (32)
Apoptotic PMN	RvDs, MaR1, LX	Dalli and Serhan (68)
Human monocytes Toll-like receptor 7 agonist (COVID-19 patients)	PD1, RvD5	Koltsida et al. (163) Navarini et al. (164)
Eosinophils	LXA4, PDx, RvD2	Miyata et al. (165) Serhan et al. (166)
Microparticles	17-HDHA, 14-HDHA	Dalli and Serhan (68)

DHA, docosahexaenoic acid

LX, lipoxin

Ma, maresin

PD1, protectin D1

Rv, resolvin

SPM, specialized pro-resolving mediators

Figure 2. Cell Surface Receptors for Resolvins, Protectin D1 and MaR1.

The human and mouse specialized proresolving mediator receptors display K_d/EC₅₀ in the nM range in line with their stereoselective actions on human neutrophils, leukocytes, epithelial cells and lymphocytes. Each has been qualified with transgenic and knockout mice. Please see text for details and citations of original contributions.

Mobilization of Omega-3 Fatty Acid Substrates for SPM Production

Increasing evidence from large clinical trials in humans indicates that these essential polyunsaturated fatty acids are important to healthy lung function (7). In experimental animal models, DHA is mobilized from lymph nodes to produce Resolvin D1 and Protectin D1 via the secreted phospholipase A₂ (sPLA₂) group IID enzyme (66), coined the resolving PLA₂. This sPLA₂ also releases precursors 14-DHA and 17-HDHA from human and mouse microparticles (67, 68). In human neural tissues such as microglia, evidence for cPLA₂ to release DHA for NPD1 biosynthesis is available (58, 69).

Vascular leak during inflammation produces edema that can carry DHA and EPA into inflammatory exudates, likely via albumin, to evoke exudate biosynthesis of SPMs to limit further neutrophil recruitment (70). Evidence was also recently obtained for the resolving sPLA acting on triglyceride substrate in M2-like human macrophages to liberate EPA for Resolvin E4 biosynthesis (52). The predominance of these mobilizations of substrate in the respiratory system remains a subject of continued research in our laboratories and is likely a critical target for new approaches to enhance SPM and other resolution pharmacology-based therapies.

In addition to inflammatory exudates, lipid mediator class switching (63) illustrated in Figure 1 occurs within reparative human M2-like macrophages ((71), Table 1). These results, now from several independent laboratories, point to a specific proresolving phospholipase A₂ (58, 66) that can hydrolyze phospholipid esterified EPA and esterified DHA to their free unesterified forms that are next available for conversion to proresolving mediators (Fig. 1). These specific phospholipases can now be targeted for new pro-resolving therapies to control local inflammation in the respiratory system.

Thus, self-limited resolving exudates biosynthesize SPM in a temporally orchestrated manner that coincides with leukocyte traffic to the site of inflammation and their transcellular biosynthesis of key substrates and intermediates (Figure 1).

These include the different types of inflammatory exudates, namely:

Serous exudates:

defined as few in leukocyte numbers, yellowish-colored fluid, containing protein and fibrin-free serum, e.g. “transudative” pleural fluid with pleural fluid/serum total protein < 0.5, lactate dehydrogenase ratio < 0.6, and pleural fluid LDH < (2/3 *upper limit of normal serum LDH) (63).

Purulent exudates:

Infectious, high numbers of leukocytes and cellular debris, creamy white-or green-colored fluid (72).

Hemorrhagic exudates:

can be sterile or infectious; contains leukocytes and red blood cells with red or brown-colored fluid (73).

The presence of large numbers of red blood cells in the exudate indicates vascular damage from infarction, infection, or tumors with excessive leukocyte extravasation contributing to lung tissue damage. Exudates temporally biosynthesize the SPMs to promote tissue repair and the return this injured tissue to a new functional homeostasis.

As the rigorous large-scale human clinical trials with resolvin and SPM biosynthetic precursors yield results indicating improved lung functions (7), the interest in SPM and biosynthetic mechanisms to increase their endogenous production is heightened. Immunoresolvents such as the natural combination medicine Traumeel (Tr14) stimulate the biosynthesis of SPMs in exudates in peritonitis and by human M2-like macrophages activated with Staphylococcus aureus (74). Opportunities to increase wellness and resilience were recently noted by the NIH (75, 76). The therapeutic potential of resolvins in pulmonary diseases was also recently the subject of an independent review (23). The elucidation of the resolvins and other SPM has led to a shift in thinking about treating airway and lung inflammation and infection with molecules that possess the capacity to control of excessive inflammation. The pro-resolving and host-protective actions of the resolvins and SPM (77) are of special interest in the respiratory system (72, 78-80), given that these endogenous mediators can be functionalized to serve as precision therapeutics in airway diseases, for example, the 17R-Resolvin D1 analog 17R-hydroxy-19-para-fluorophenoxy-Resolvin D1 (70, 77, 81) and others including the benzo-resolvin D1 (82, 83).

The SPMs may also have a role in protecting the lung from pulmonary fibrosis (43). This is of urgent special interest because currently there are limited therapeutic options for pulmonary fibrosis as seen in interstitial lung disease, idiopathic pulmonary fibrosis, and sarcoidosis pathogenesis (84, 85) . Administration of SPMs has the potential to prevent transition from inflammation/lung injury to lung fibrosis and respiratory failure (19). The proresolving signaling of the SPM is an advantage in these lung diseases (Table 1).

Human Lung and SPMs

Since the first evidence for leukotrienes in asthma and inflammation appeared in 1989 (86), the human respiratory system has been widely studied and recognized as a substantial source of lipid mediators. Indeed, recent analysis of human lung tissues from human lung grafts in transplant patients, both prereperfusion and postreperfusion, clearly demonstrated, using targeted LC-MS-MS-based methods, that human lung tissue produced substantial amounts of 17-HDHA and D-series resolvins in amounts well within their potent bioactive concentrations on human leukocytes (70, 87). These were found in the low subnanomolar range. While the amounts of resolvins in humans were questioned as enough to evoke their potent pro-resolving actions, Fu et al. independently reported that both human plasma and mouse tissues contain lipoxins and resolvins identified in the picogram range, in line with their potent stereoselective immunoresolving functions (25).

These were also independently confirmed recently by Zhu et al. (88) using targeted LC-MS-MS together with a derivatization method that documented Resolvin D1, Resolvin D2, Resolvin E3 and lipoxins in sera from healthy volunteers. Earlier results clearly demonstrated that resolvin and SPM production in human serum and plasma was dependent on the amounts of EPA and DHA taken by healthy volunteers (89, 90). Thus, if adequate substrates of EPA and DHA are ingested and present, Resolvin D1 is biosynthesized, which can accelerate the resolution of inflammation (Figure 1B) and reduce the impact of acute lung injury (91). Extracellular vesicles are a source of increased SPM substrate availability and biosynthetic enzymes that in inflamed respiratory tissues can locally produce SPM (68, 92).

We next shall consider the new evidence for SPM in human airway and lung disease and specific animal lung disease models.

Chronic rhinosinusitis.

In a recent study with human subjects, Resolvin D2 was elevated in subjects with polyps; both resolvin D1 and resolvin D2 are diminished in smokers compared to non-smokers. Using LC-MS-MS based detection, several SPM correlated with sinonasal mucosal microbiota as well, as with pathogens such as Pseudomonas aeruginosa (31), including LXA₄, LXB₄ and resolvins (RvD1, RvD2, RvD3, RvD5 and RvE1). The presence of protectins (PDX) and MaR1 in nasal polyps and nasal mucosa have also been recently examined. SPM concentrations were significantly higher in the control subjects without chronic inflammation (93).

Cystic fibrosis.

The airway and lung inflammation in cystic fibrosis (CF) are classic examples (Figure 1B) of non-resolving inflammation in upper and lower human airways. Sputum from CF patients analyzed using LC-MS-MS-based methods documents the proresolving mediators resolvin E1 and Lipoxin A₄ among many proinflammatory mediators, e.g. LTB₄, PGE₂ and PGD₂ (94). Prof. Hammock and colleagues point out that patients with detectable resolvin E1 had better lung function than patients with low or undetectable RvE1 in their sputum (94), demonstrating the utility of lipid mediator-metabololipidomic profiling in CF sputum, which may be useful in temporal analysis of a broader range of human pathologic and disease processes (95). Of interest, P. aeruginosa, a key pathogen in CF, can disrupt proresolving lipid mediators (96). Eickmeier et al. demonstrated that, in CF, the D-series Resolvin D1 is a potential marker of lung disease (34). When Resolvin D1 is given as a therapeutic in an animal CF model, Resolvin D1 stimulates resolution of lung inflammation (97) and is a potent broncho-protective SPM (98).

Urbach and colleagues found that in CF patients the airway epithelium plays significant roles in SPM biosynthesis and actions (99) and is sex dependent (35, 100, 101). Both airway inflammation and exercise capacity in CF are improved with resolvin D1 (32, 102, 103).

Macrophages from CF patients exposed to SARS-CoV-2 elaborate a cytokine-chemokine storm that is reduced and resolved with Resolvin D1 and Resolvin D2 (33). Thornton et al. (104) discovered that lipoxin A₄ in biofilm improves antibiotic efficacy with P. aeruginosa, suggesting that the SPMs, which can activate endogenous resolution mechanisms, can help reduce chronic inflammation and infections in the respiratory tract.

Respiratory tract infection and pneumonia.

Several recent publications have provided insights regarding mechanisms for augmentation of airway and lung host defense by SPM. In a mouse model of Escherichia coli pneumonia, lung SPM levels were temporally regulated, and early treatment with exogenous AT-RvD1 (1 h post infection) enhanced clearance of E. coli and P. aeruginosa in vivo, increasing expression of anti-microbial peptides and accelerating lung macrophage phagocytosis of bacteria (14, 105). AT-RvD1 also increased efferocytosis by infiltrating macrophages (CD11b^Hi CD11c^Low) and exudative macrophages (CD11b^Hi CD11c^Hi), and enhanced neutrophil clearance during pneumonia in vivo. Importantly, these anti-bacterial and pro-resolving actions of AT-RvD1 were additive to antibiotic therapy (14).

Lung macrophages are also targeted by the cysteinyl-maresins to augment host defense. Serious respiratory viral infection (e.g., from influenza) can decrease alveolar macrophage numbers and reprogram the cells to promote inflammation. Harnessing endogenous macrophage resolution mechanisms for inflammation with exogenous cysteinyl-maresins increases macrophage resilience after influenza to protect against secondary bacterial pneumonia from Streptococcus pneumoniae (106). Select SPMs serve as endogenous agonists for A20 and single Ig IL-1R-related molecule (SIGIRR) expression to regulate macrophage and airway epithelial cell NF-κB activity as mechanisms for control of lung inflammation and pneumonia resolution (105).

Respiratory syncytial virus (RSV) is a major respiratory pathogen with excess morbidity in early life and older individuals. The morbidity and mortality associated with RSV is secondary to an exacerbated host immune responses and injury to the epithelium that can obstruct the airway and compromise gas exchange. In a mouse model of RSV infection from a human clinical strain (i.e., Line 19), lung PD1 and PCTR1 levels were temporally regulated, and their exogenous administration three days after inoculation enhanced viral clearance and promoted resolution of the host immune response, in part secondary to increased epithelial interferon-lambda and decreased CD4 effector T cell interferon-gamma (107). In addition, MaR1 signaling via its recently identified receptor Lgr6 improves regulatory T cell suppressive function for T cell cytokine production and upregulates host antiviral genes and amphiregulin production to decrease viral burden and pathogen-evoked inflammation, highlighting important roles for SPM-informed regulatory T cells in the restitution of disrupted airway mucosal homeostasis (108). The most serious host response to lung infection is sepsis. Of interest, recent cellular phenotyping from critically ill human patients identified that RvD1 and RvD2 signaling for antiinflammation and resolution are uncoupled from leukocyte activation in early sepsis; findings that point to diminished resolution signaling as a correlate of clinical disease severity (109).

Asthma and allergic lung inflammation.

Asthma is the most common respiratory illness, and immunophenotyping of most patients reveals chronic type 2 inflammation with increased pro-phlogistic mediators, prominent eosinophilia, bronchial epithelial mucous cell metaplasia and airway hyperresponsiveness (110). Asthma control therapy currently emphasizes anti-inflammatory agents, including corticosteroids and type 2 pathway targeted biologics (111); however, these therapies are not curative, failing to fully resolve asthma.

Recent findings have uncovered several intriguing pro-resolving roles for SPM in allergic lung inflammation and asthma. In a DRV2 receptor-dependent manner, resolvin D2 (RvD2) accelerates resolution of mouse lung inflammation evoked by house dust mite sensitization and challenge, and DRV2 receptors are expressed on eosinophils and other leukocytes (112). Eosinophils are heterogeneous with two subsets in mice that are identifiable by CD101 expression with distinct anatomic localization and transcriptional signatures at baseline and during lung inflammation (113). CD101^low Eos are predominantly in a vascular niche and respond to airway allergen challenge by trafficking into the lung interstitium where they acquire an activated phenotype with high expression of CD101 and continued trafficking into the airways where they are detectable in whole lung lavage. RvD2 reduces total Eos numbers and activation by decreasing interleukin 5-dependent lung recruitment of CD101^low Eos and decreasing eosinophil conversion from CD101^low to CD101^high (113).

Cysteinyl leukotrienes (CysLTs) have been assigned important roles in asthma pathophysiology as potent pro-phlogistic mediators; however, inhibition of CysLT1 receptors has not proven consistently effective as a therapeutic strategy. Additional regulatory mechanisms were recently identified to address this gap in knowledge. Cysteinyl-containing lipid mediators derived from DHA, namely the maresin conjugates of tissue regeneration (MCTRs), are produced in human and mouse lung and can block human LTD₄-induced airway contraction and promote resolution in vivo of mouse allergic airway responses (39); endogenous mechanisms for control of asthma phenotypes.

In addition to eosinophil activation, the immunology of the severe asthma airway is notable for decreased NK cell cytotoxicity with increased numbers of NK cell targets, such as granulocytes and effector lymphocytes, which is exacerbated by corticosteroids that further disable NK cell function (114). In contrast to corticosteroids, the SPM LXA₄ preserved NK cell functional responses (114). The LXA₄ receptor ALX/FPR2 can engage both proresolving and proinflammatory ligands for opposing signaling events, enabling these receptors to serve pivotal roles in the fate of lung inflammatory responses. In bronchoalveolar lavage (BAL) fluid, levels of LXA₄ and 15-epi-LXA4 are decreased, and the acute phase reactant serum amyloid A (SAA) is increased in severe asthma relative to non-severe asthma (115). These select ALX receptor ligands define a biochemical endotype for asthma as patients with LXA₄^loSAA^hi levels have characteristics associated with severe asthma, namely increased BAL neutrophils, more asthma symptoms, lower lung function, and increased relative risk for asthma exacerbation, sinusitis, and gastroesophageal reflux disease (115).

Acute respiratory distress syndrome.

Perhaps the most devastating lung disease is the acute respiratory distress syndrome (ARDS). Acute inflammation fills the gas exchanging alveoli, leading to life threatening hypoxemia, so patients most commonly require supplemental oxygen via mechanical ventilation. Of interest, lower plasma levels of SPM are associated with increased duration of ventilatory support and ICU length of stay (116). Both plasma LXA₄ and MaR1 levels are decreased in ARDS (116) and these SPM are protective in experimental acute lung injury in mice (117, 118). These SPM can be produced by circulating leukocyte-platelets aggregates, which are prominent in acute inflammation, augment lung leukocyte infiltration via secondary capture, and are associated with incident human ARDS (119), suggesting that the diminished restraining activities from the lower plasma SPM levels unleash leukocyte activation and transmigration into the lung in ARDS and that exogenous SPM administration could represent a new therapeutic strategy for ARDS.

SPM Therapeutic Potential for Airway Inflammation in Pulmonary Diseases

Given the potent proresolving actions of each SPM demonstrated in many animal disease models, several of the SPMs and their cognate receptors are being considered for new therapeutic approaches in pulmonary diseases (23, 120). This shift in the search for new proresolving therapies from the current immune suppression of pro-inflammatory mediators and pathways is based on the potent stereospecific actions of the SPMs in regulating neutrophil migration for lung protection (87, 121-123), enhancing bacterial and viral clearance (14, 106-108, 123), reducing cytokine and eicosanoid storms (105), stimulating phagocytosis of apoptotic PMNs (efferocytosis), removal of debris, and reducing pain to shorten resolution intervals (R_i) (78).

Following the total organic synthesis and assignment of the complete stereochemistry of each of the first endogenous SPMs originally uncovered to confirm their potent proresolving and anti-inflammatory functions (reviewed in 124), additional total syntheses of specific SPMs have been achieved, for example, refs. (125-129). These enabled further investigations of in vivo SPM functions in animal models and identification of specific proresolving receptors. At this point, five proresolving receptors are identified (illustrated in Figure 2) along with their Kd and concentration range to evoke pro-resolving phagocytosis; for RvD1: ALX/FPR2, human GPR-32 (130-132); for RvE1: ChemR23 (45); RvD2: GPR18 (41); and MaR1: LGR6 (reviewed in 108, 133). Several of these receptors were further validated in receptor knockout mice (134) as for the Resolvin D2 receptor (38, 133) and MaR1 receptor (108).

As part of their intracellular signaling, the SPMs induce heme oxygenase-1 (HO-1) (135), which produces carbon monoxide. Inhaled CO is organ protective in the lung, demonstrated in ischemia-reperfusion lung injury (136). Low-dose inhaled CO (125-250 ppm) reduces PMN-platelet aggregates and reduces PMN infiltration into the lung. Both resolvin D1 and low-dose inhaled CO reduce leukocyte-mediated acute lung injury (122, 136, 137). SPMs restore barrier integrity (122, 138). In baboon pneumonia with S. pneumoniae, prostaglandins and leukotriene B₄ (LTB₄) are increased. Inhaled low-dose CO reduced these proinflammatory eicosanoids and increased SPM, which may shorten the time to resolve pneumonia (37). These results suggest that therapies that increase endogenous SPM may have a beneficial impact on the lung during acute injury or infection. Along these lines, extracts of Hyssopus Cuspidatus Boriss are used to treat asthma in some parts of the world, and these plant extracts have been shown to increase both resolvin and lipoxin production in mice with experimental allergic inflammation using UHPLC-MS-MS for identification and quantitation, suggesting an SPM mechanism of action for this ancient therapy (139). The newly described peptide-containing SPM, namely the cys-SPM (Figure 3) such as MCTR1 (Figure 3), are also lung protective in experimental animal models of lung inflammation (40). Traumeel (Tr14) stimulates SPM biosynthesis, improving the resolution of inflammation in mouse peritonitis (74).

Figure 3. Biosynthesis Pathways for the cys-SPMs and SPMs.

Panel A) The Proposed Biosynthesis of Maresins and cys-Maresins. The complete stereochemistry of each structure shown was established by total organic synthesis and functions confirmed for each potent bioactive molecule of the specialized proresolving mediator superfamily of novel molecules. For examples, see Serhan et al. (61); Dalli et al. (156, 157); and Serhan (62).

Panel B) Biosynthesis proposed for the Protectins and cys-Protectins (PCTRs). The stereochemistry of NPD1/PD1 was established in Serhan et al. (158); Ramon et al. (159); and de la Rosa et al. (81) employing materials prepared by total organic synthesis with each validated using NMR.

Panel C) Proposed Biosynthesis of the cys-Resolvins (RCTRs) and relation to D-Series Resolvin. The stereochemistry of RCTRs 1, 2 and 3 were established; see Ref. (81), and potent actions confirmed with defined synthetic materials prepared by total organic synthesis (81).

Lipid emulsions, which are widely used in critical care medicine as part of total parenteral nutrition, increase the biosynthesis of SPM in human immune cells and mouse tissues (26). In asthmatic patients, 17-HDHA and Resolvin D5 are produced by nasal epithelium, which may be useful to stage the severity of asthma in these patients (140). Indeed, SPM are present in lungs of intubated COVID-19 patients (141) in substantial and sustained amounts.

SPMs are also demonstrable in plasma of human subjects with chronic inflammation (142, 143) and arthritis (29), which increase with EPA and DHA supplementation (89). The identification of SPM in human disease and their increase with supplementation suggest a functional role for SPM in shortening recovery and return to homeostasis (49). To demonstrate the potential of SPM-based resolution therapies, metabolically stable analogs of Resolvin E1 and Resolvin D1 were designed and synthesized; each was demonstrated in vivo in mice to be highly effective (51, 82), giving proof to the new concept that stimulating resolution of inflammation with SPM mimetics and related pro-resolution approaches can be a new line of treatment for airway and lung infections and inflammation.

During studies to map the mechanisms and mediators involved in the resolution of inflammation, we found that the vagus nerve controls resolution of inflammation by stimulating the production of pro-resolving mediators (144) using the vagotomy approach introduced by K. Tracey and colleagues (144, 145). Electrical stimulation of the human vagus ex vivo inhibits the vagus nerve production of prostaglandins and leukotrienes while stimulating the production of pro-resolving mediators, i.e. SPMs (36). Vagus nerve stimulation of SPM production was confirmed by Huang et al. (146), which also demonstrated reduced lung permeability in acute lung injury in mice. Thus, electrical stimulation of the vagus nerve to produce SPM can be a further treatment option in airway inflammation-associated pulmonary diseases. The discovery that dexamethasone can stimulate SPM production in M2-like macrophages and in humans with COVID-19 infections opens many new opportunities to repurpose dexamethasone and related drugs to new SPM-based pro-resolving therapies (147) to bring in resolution pharmacology (78).

Conclusions and Summary

We’ve reviewed herein recent contributions on the Resolvins, Protectins and Maresins focusing on their functions in the respiratory system. Collectively, these endogenous mediators of resolution are referred to as the SPM superfamily of lipid mediators. For this annual review we’ve focused on SPM in the lung/respiratory system with substantial and far-reaching contributions as updates since our earlier annual review on these novel molecules in the physiology of inflammation-resolution lung tissues (17). The SPMs demonstrate proresolving functions independently confirmed by many, and their potent pro-resolution functions and criteria are established as follows a) reduction of pro-inflammatory cytokines and eicosanoid storm, b) enhanced killing, c) clearance of bacteria, d) increased efferocytosis by macrophages, e) removal of cellular debris by phagocytosis, f) reducing the resolution/recovery time interval (R_i), and g) reducing pain. These features and endogenous functions of the SPM are attractive for airway diseases and potentially now provide a wealth of evidence to substantiate a shift from inhibitors – receptor antagonists to exciting new directions for treatment of respiratory disease using activators – receptor agonists of endogenous resolution mechanisms to control excessive inflammation and infections in the respiratory system.

The respiratory tract mucosa is enriched in omega 3 fatty acids EPA and DHA in health, and tissue stores of these SPM substrates decrease in inflammatory disease (148). Local production of the SPMs is lung protective in both vascular and extravascular niches, and their abundance or signaling mechanisms are disrupted in acute and chronic disease. As physiologic counter-regulatory modulators, several experimental systems have provided evidence that the SPMs and cysteinyl-SPMs instruct lung tissue resident cells and macrophages to recapture homeostasis and organ function. We eagerly anticipate the next horizon in resolution biology and pharmacology that will see new therapeutic modalities emerge that leverage the SPM endogenous protective and tissue regenerative mechanisms for resolution medicines to ameliorate the excess morbidity and mortality of acute and chronic diseases of the respiratory tract.