Leukotrienes in Innate Immunity: Still Underappreciated After All These Years?

Abstract

Leukotrienes (LTs) are lipid mediators derived from the 5-lipoxygenase pathway of arachidonate metabolism. Though best known for their role in asthma, they have broad actions that touch on virtually every aspect of mammalian biology. In a Brief Review published in the Journal in 2005, we presented the existing evidence supporting a role for LTs in host defense. In this updated Brief Review, we focus on selected advances since then. We detail new insights into mechanisms and regulation of LT biosynthesis; the protective roles of LTs in the host response to diverse classes of pathogens, with an emphasis on viruses including SARS-CoV2; the phagocyte signal transduction mechanisms by which LTs exert their antimicrobial actions; the capacity for overexuberant LT production to promote tissue damage; and roles of LTs in the non-infectious immune-relevant conditions neuroinflammation and cancer.

Leukotrienes (LTs) are lipid mediators whose synthesis is initiated by the actions of the enzyme 5-lipoxygenase (5-LO) on arachidonic acid (AA). Two major chemical species of LTs exist – leukotriene B₄ (LTB₄) and the cysteinyl LTs (cysLTs) comprised of LTC₄, D₄, and E₄. In a ground-breaking article published in 1979 (1) and subsequently recognized by the Journal as a Pillar of Immunology (2018 commentary in reference (2), cysLTs were demonstrated to be elaborated by mast cells and to account for the long-acting spasmogenic bioactivity first implicated in asthmatic bronchospasm in 1940 and termed “slow-reacting substance” (3). Bengt Samuelsson won the 1982 Nobel Prize for Physiology or Medicine for this and related work. Subsequent research in the LT field was thus understandably dominated by a concerted focus on the rationale for and development of drugs targeting cysLTs (and their predominant G protein-coupled receptor cysLT1) for the treatment of asthma. This effort was rewarded by their introduction into the therapeutic armamentarium in the mid- to late 1990s – the first new class of drugs for the treatment of asthma since corticosteroids were introduced in the 1950s.

As is typical for any field of inquiry, its boundaries gradually expanded as new discoveries elucidated roles for both LTB₄ and cysLTs in diverse aspects of biomedicine. LTB₄ had been identified as a chemotactic factor for polymorphonuclear leukocytes (PMNs) as early as 1980 (4), and the G protein-coupled receptor mediating this response was eventually identified as the Gαi-coupled LTB₄ receptor 1 (BLT1). Indeed, the LTB₄/BLT1 axis began to emerge as an essential mediator of a variety of aspects of phagocyte biology. In early 2005, one of us published a Brief Review in the Journal (5) entitled “Leukotrienes: Underappreciated Mediators of Innate Immune Responses.” In it, we summarized a body of literature which had emerged mainly during the preceding 10–15 years, whose key findings included the following: 1) LTs were generated by phagocytes (predominantly macrophages and PMNs) during encounters with microbes; 2) LTs – particularly LTB₄ more so than cysLTs – promoted antimicrobial effector functions including leukocyte accumulation, phagocytosis, microbial killing, and the generation of other innate immune-active mediators such as cytokines and chemokines; 3) impaired capacity for LT biosynthesis characterized clinically important states of immunosuppression such as HIV infection and malnutrition; and 4) medications with the capacity to alter LT production, or the direct exogenous administration of LTs themselves, had the potential to influence innate immune responses.

In this follow-up Brief Review, we seek to provide a focused update on selected advances in this topic since the original article was published. We examine recent insights into mechanisms of LT biosynthesis, modulation by LTs of innate immune signaling, functional roles for LTs in regulating the inflammasome and PMN movement, and their potential impact on inflammatory diseases including viral infections, specifically Covid-19, as well as non-infectious processes.

Regulation of LT biosynthesis

Overview of established knowledge.

LT formation from endogenous membrane-derived AA proceeds via a multi-enzyme cascade whose early steps include the hydrolysis of AA from membrane phospholipids by cytosolic phospholipase A₂ (cPLA₂) and its oxygenation at the C-5 position by 5-LO in concert with its helper protein 5-LO activating protein (FLAP) to yield LTA₄. This moiety can either be hydrolyzed by LTA₄ hydrolase to form LTB₄ or be conjugated with the tripeptide glutathione by LTC₄ synthase to form LTC₄. LTC4 undergoes stepwise removal of each of these amino acids to form LTD₄ and then LTE₄, respectively. Among these cysLTs, LTD₄ accounts for the bulk of their spasmogenic and edemagenic activities. Extensive research in the quarter century following their discovery delineated a number of key factors regulating LT biosynthesis. For instance, enzymatic activities of both cPLA₂ and 5-LO were shown to require Ca²⁺ and to be enhanced by phosphorylation. Additionally, each of the proteins in this cascade was demonstrated to be transcriptionally upregulated by various proinflammatory mediators. Finally, and quite surprisingly for mediators destined for extracellular secretion, it was the nuclear membrane rather than the plasma membrane at which cPLA₂, 5-LO, FLAP, and LTC₄ synthase were shown to be colocalized in an activated leukocyte (6).

Extracellular vesicles as a novel synthetic site.

A novel form of compartmentalization of biosynthetic enzymes has recently been recognized. Exosomes are nano-sized secreted extracellular vesicles that originate from multivesicular bodies. Multivesicular bodies and secreted exosomes from FMLP-activated PMNs were recently shown (7) to contain LTB₄ and the proteins 5-LO, FLAP, and LTA₄ hydrolase. Such exosomes were shown to mediate secondary PMN movement in a manner dependent on BLT1. This is considered a rapid and efficient means of relaying primary chemotactic signals to amplify PMN recruitment. Although exosome secretion is a conserved function among all leukocytes, it is unknown whether other leukocytes such as macrophages employ this cellular pathway for LT biosynthesis, whether cysLTs can similarly be elaborated in this manner, whether a given leukocyte can employ both exosomal and classical nuclear membrane-based LT biosynthesis, and if so, under what circumstances for each. It is also unclear if the magnitude or duration of PMN chemotaxis differs depending on whether LTB₄ is presented to PMN BLT1 on the surface of exosomes or as a soluble ligand.

Novel forms of modulation.

Additional new modes of modulation of LTB₄ biosynthesis by endogenous molecules, drugs, and environmental conditions have also been identified. First, an endogenous inhibitor of LTA₄ hydrolase activity was recently identified and found to be highly concentrated within the salivary glands of bats (8). This protein inhibited LTB₄ synthesis in vitro and in vivo, and interfered with antiviral activity in bats as well as in a mouse model of influenza A infection, while antiviral activity could be restored by exogenous administration of LTB₄. Second, angiotensin converting enzyme (ACE) inhibitors – which are widely used in the treatment of hypertension, heart failure, and renal disease – were recently reported to inhibit LTB₄ production by PMNs and in parallel to reduce their pro-survival and bactericidal activities (9). This effect was observed in vitro and in vivo in mice, and in PMNs from humans following a one-week treatment interval with drug. Whether this effect translates into clinically relevant immunosuppression of patients on these drugs is unknown. Finally, an acidic environment – which is often observed at sites of inflammation characterized by metabolic reprogramming and lactic acid generation – was recently shown to inhibit PMN chemotaxis towards C5a by a pH-dependent inhibition of LTB₄ generation (10). Its biological implications remain to be determined, but it is possible that this serves to promote retention of PMNs at the acidic locale. In the case of both ACE inhibitors and an acidic environment, neither the mechanistic basis for inhibition of LTB₄ biosynthesis nor their potential relevance in macrophages or other leukocyte populations was determined by investigators.

Participation of LTs in diverse infections

The participation of LTs in host defense against infection, originally reported for Klebsiella pneumonia in the Journal by Bailie et al.(11), was subsequently extended to other pathogens, including other species of bacteria (12, 13), fungi (14–18), protozoan parasites (19–21), and helminths (22, 23). The critical role of the LTB₄/BLT1 axis in these studies was revealed by the use of either BLT1−/− mice or in vivo treatment with BLT1 antagonist (12, 13, 24–26). Research since our original review article (5) has extended these explorations to viral infections, including Covid-19, and selected findings are highlighted below.

Roles in viral infections other than Covid-19.

Protective effects of LTB₄ also extend to viral infection. When mice were treated with LTB₄ i.v, after receiving a lethal dose of murine CMV, the death rate and salivary viral loads decreased compared to the vehicle-control-treated group (27). Administration of i.v LTB₄ similarly protected allogeneic bone marrow recipient mice against murine CMV infection (28). This same group showed that i.v. LTB₄ could protect the lung against influenza infection (29). Complementing the latter finding was our demonstration that BLT1−/− mice are more susceptible to influenza infection despite similar pulmonary viral loads (30). Increased susceptibility to influenza correlated with increased recruitment of monocytes to the lung and higher immunopathology. LTB₄ was found to increase the activation of type 1 IFNα/β production and interstitial macrophage proliferation. Unexpectedly, treatment of mice with LTB₄ during the active infection was able to control tissue damage and increase survival (30).

Roles in Covid-19 infection.

In view of the abilities of LTs to influence both host defense and inflammation programs, it is not surprising that there has been substantial interest in and speculation about their potential role in the Covid-19 pandemic resulting from infection with SARS-CoV 2 (31). An emerging body of evidence supports this possibility. First, elevated levels of LTB₄ and/or cysLTs have been reported in serum (32) and bronchoalveolar lavage fluid (33) of patients with Covid-19. Plasma LTB₄ levels were found to be particularly elevated in infected diabetics requiring intensive care (34). Interestingly, however, elevated lung LT levels were not observed in a murine model of SARS-CoV2 (35). Second, cysLT1 antagonists montelukast or zafirlukast have been shown to inhibit binding of SARS-CoV2 or its spike protein to cells or its ACE2 receptor in vitro or in silico (36, 37), to inhibit virus-induced IL-6 generation by immune cells in vitro (37), to disrupt the viral membrane and reduce infectivity in host cells in vitro (38), and to inhibit platelet activation induced by plasma from Covid-19 patients (39). An open question is the extent to which these actions are related to disruption of cysLT signaling per se, or rather, the chemical actions of the drugs themselves on cellular targets such as viral lipid membranes (38). We are aware of no information regarding the potential actions of LTB₄ or BLT1 antagonists on Covid-19 pathogenesis in vitro, and of either class of LT on coronavirus pathogenesis in vivo. Interestingly, retrospective analyses of patients with Covid-19 infection in both a small single medical center cohort (40) and in the large Department of Veterans Affairs database (41) found that patients who were on montelukast prior to infection had a lower incidence of clinical deterioration or a survival advantage, respectively. Collectively, these data support the advisability of conducting prospective clinical trials of montelukast in patients with Covid-19, and of improving the understanding of the influence of LTs in coronavirus pathogenicity in general.

Cellular mechanisms by which LTs enhance host defense

Host defense is a multi-step process involving the recognition, engulfment, and killing of pathogens by resident and recruited phagocytes (42). Amplification of inflammatory mediator generation can facilitate pathogen clearance but can also inflict tissue damage. Below, we will summarize how LTB₄ influences each of these steps in host defense (see Figure 1, left).

Figure 1. Signaling and innate immune actions of LTB₄ in health and disease.

(left) LTB₄ is generated at homeostatic levels by leukocytes upon encounter with pathogens. Via ligation of BLT1 and reductions in intracellular cAMP, it synergizes with signaling downstream of phagocytic, opsonin, and PRRs to amplify diverse antimicrobial functions that protect the host. These actions include recruitment of PMNs as well as activation of PMNs and resident macrophages to enhance phagocytosis, microbial killing, and generation of pro-inflammatory cytokines via activation of both the inflammasome as well as NFκB-dependent transcription. (right) Excessive and/or prolonged generation of LTB₄ during either infectious or non-infectious inflammation leads to BLT1-dependent activation of the inflammasome as well as signaling via JAK-STAT and NFκB. Collectively, these pathways promote robust cytokine generation and signaling that contribute to exaggerated tissue inflammation and injury. Figure created with BioRender.com

PMN migration and chemotaxis.

LTB₄ is released at the site of infection to mediate PMN recruitment and proinflammatory processes (43). In addition to its intrinsic chemoattractant activity, LTB₄ also enhances PMN chemotaxis towards other chemoattractants such as fMLP, C5a, and heme (44–46). A major advance was the recognition that LTB₄ mediates coordinated intercellular communication and signal relay among extravasated PMNs to facilitate the accumulation of dense clusters in a process termed “PMN swarming” (47, 48). The role of LTB₄/BLT1 as a key player in swarm formation has been identified in both sterile and infectious injuries. In a model of systemic candidiasis, LTB₄-mediated PMN swarming is detrimental to the host, resulting in vessel occlusion and pulmonary hemorrhage (49). The effects of LTB₄/BLT1 in PMN swarming seem to vary depending on the microbe, since Patnode et al have shown that LTB₄ increases the swarming of eosinophils but not PMNs during infections with the nematode Nippostrongylus brasiliensis (23). The basis for these differences in regulation of PMN vs. eosinophil swarming remain to be determined.

Phagocytosis.

Both genetic and pharmacologic blockade of cysLT and LTB₄ synthesis and/or actions significantly reduces ingestion of a variety of opsonized and unopsonized pathogens, including both Gram-positive and -negative bacteria (12, 50–52), fungi (14, 16, 53), and parasites (19, 54). We have shown that LTB₄ and LTD₄ promote FcγR- and dectin 1 (main phagocytic receptor detecting fungal pathogens (55))-dependent phagocytosis in alveolar macrophages (AMs) via BLT1 and cysLT1, respectively (51, 56); however, there are critical differences in the intracellular programs they use to enhance AM functions (50). First, Gαi-coupled BLT1 acts by reducing intracellular cyclic AMP and enhancing activation of Syk and protein kinase C (PKC)-α, whereas cysLT1 is coupled only to Gαq (50) and its effects are independent of Syk and PKC-α but dependent on PKC-δ (51). Second, while cysLT1 is not present in lipid raft microdomains within membranes, we found that FcγRI engagement results in tyrosine phosphorylation of BLT1 by Src family kinases, forming a platform within lipid rafts comprising FcγRI, BLT1, Gαi3, and Src, which drives LTB₄-enhanced signaling and phagocytosis (52).

Microbial killing.

LTB₄-mediated reactive oxygen species production depends on PKC-δ-mediated phosphorylation and resultant membrane translocation of NADPH oxidase complex components p47phox and p40phox in AMs challenged with opsonized K. pneumoniae (57). We also showed that aerosolized LTB₄ increases p47phox expression and membrane translocation during Streptococcus pneumoniae lung infection (58). More recently, we have demonstrated that topical LTB₄ enhances methicillin-resistant Staphylococcus aureus (MRSA) clearance from the skin in a manner dependent on NADPH oxidase activation (13). LTB₄ has likewise been shown to increase reactive nitrogen species accumulation by increasing the expression of inducible nitric oxide synthase (iNOS) in various models of intracellular infection (19, 20, 59). Its increased expression in macrophages depended on the activation of NFκB and STAT1 (60). Whether LTB₄ increases iNOS activity remains to be determined. Finally, LTB₄ also induces the secretion of other antimicrobial molecules. It induces degranulation in human PMNs infected in vitro with L. amazonensis and stimulates release of PMN elastase, myeloperoxidase, defensin 3, Cramp, and LL37 during bacterial and viral infections (43, 61).

Pathogen recognition receptor (PRR) activation and cytokine generation

TLRs are known to detect both pathogen-associated molecular patterns and danger-associated molecular patterns. TLR activation depends on adaptors such as myeloid differentiation factor 88 (MyD88) and TIR-domain-containing adapter-inducing interferon-β (TRIF) (62). MyD88 activation is followed by phosphorylation of downstream components such as IRAKs and TAK-1, leading to NFκB-dependent expression of inflammatory mediators (62). Evidence supports various roles for LTB₄ in TLR activation (60). LTB₄ stimulation upregulates the expression of TLR2 and TLR9 in human PMNs (63, 64). It also can amplify the actions of PRRs in a variety of ways. LTB₄ enhances MyD88 expression and MyD88-dependent activation of NFκB, which intensifies the signaling potential of TLRs and other PRRs (60). The mechanisms involved in LTB₄-enhanced MyD88 expression in macrophages involve BLT1-mediated mRNA degradation of suppressor of cytokine signaling 1 (SOCS1), the major negative regulator of the MyD88 transcription factor STAT1 (60). Finally, it has been shown that LTB₄ amplifies the phosphorylation of IRAK and TAK-1 in human PMNs (63). Altered expression of microRNAs that influence mRNA degradation or translation represents another layer of immune regulation induced by LTs. We found that 5-LO−/− macrophages have lower levels of microRNAs that bind to the SOCS1 3′UTR and thus restrict SOCS1 expression, resulting in increased MyD88 expression(65).

Inflammasome activation

The inflammasome constitutes an intracellular platform required for IL1β maturation and secretion, and recent work has examined the role of inflammasome components in LTB₄ actions. It has been shown that LTB₄-mediated arthritis severity is impaired in mice deficient in the inflammasome components ASC and NLRP3 (66). Also, LTB₄ effects on in vitro leishmanicidal activity depend on NLRP3 activation (67). However, these manuscripts did not investigate the mechanisms underlying the relationship between LTB₄ and inflammasome activation. We have also shown that LTB₄ enhances the assembly of inflammasome components, including NLRP3, AIM2, and NLRC4. LTB₄ mediates the activation of the adaptor ASC in a manner dependent on the activation of Bruton’s tyrosine kinase (68). Finally, LTB₄/BLT1 signaling is indeed required for inflammasome activation in vivo, as evidenced by its necessity for IL-1β-dependent host defense against MRSA skin infection (68).

Excessive LTB₄ production and tissue damage

It is increasingly understood that beyond the elimination of pathogens per se, a crucial component of host defense and survival is the ability to limit the tissue damage caused by the pathogen and/or the ensuing immune response that it engenders – a phenomenon known as disease tolerance (69). Experiences with influenza as well as SARS-CoV2 have revealed that most pulmonary-virus related deaths result from a dysregulated host immune response to the virus (“cytokine storm”) rather than the cytopathic effects of the virus itself (70). Indeed, we and other have shown that disease tolerance plays a crucial role in immunity to a variety of bacterial or viral infections in the lung (30, 71–73). Excessive or prolonged LTB₄ production is a component of overwhelming or chronic inflammation, and this can contribute directly to tissue damage via its abilities to drive PMN recruitment, macrophage activation, and cytokine generation (see Figure 1, right). Furthermore, aberrant LTB₄ production contributes to a variety of chronic inflammatory illnesses that themselves are associated with impaired phagocyte responses and increased susceptibility to infection (74, 75).

LTs in non-infectious inflammatory processes

In addition to their long-recognized actions in leukocyte recruitment and activation, LTs also influence a variety of other processes including vascular permeability, cell proliferation, and diverse aspects of cellular phenotype (76). This broad spectrum of biological actions has resulted in these lipid mediators being investigated, or at least speculated upon, as mediators of virtually every imaginable type of disease process. In some instances, investigators have sought to comprehensively dissect the relative contributions of LTB₄ versus cysLTs, or of specific LT receptors, to the disease process or model in question. In others, treatment with montelukast – a cysLT1 antagonist which is by far the most widely used LT modifier drug – is employed as the lone strategy to implicate LTs. We will touch here on only two of the disease processes that are of interest to the immunology community – neuroinflammation and cancer.

Neuroinflammation is a key component of numerous central nervous system disorders. Increased brain tissue levels of LTs as well as microglial expression of 5-LO have been reported in a mouse model of intracerebral hemorrhage (77) and a rat model of chronic chemical exposure mimicking the inflammation and cognitive impairment observed in Gulf War Illness (78). Neurotoxicity induced by injection of amyloid-β protein into the hippocampi of mice was associated with increased expression of cysLT1 in brain tissue, and administration of a cysLT1 antagonist reduced inflammation, increased cell survival, and improved memory (79). Two recent large retrospective studies examined the association between cysLT1 antagonist use and cognitive function. In one study of Japanese asthmatics over the age of 50 (80), the incidence of developing dementia was significantly lower in those on drug. In another study of patients enrolled in the National Alzheimer’s Disease Research Centers database (81), the use of these agents was associated with a slower rate of cognitive decline. These encouraging data have prompted the initiation of a phase 2A trial of a reformulated version of montelukast designed to facilitate blood-brain barrier penetration in mild-moderate Alzheimer’s disease (NCT03402503).

Chronic inflammation, uncontrolled cell proliferation, and resistance to apoptosis all contribute to the pathogenesis of cancer. Roles for LTs in these cancer-associated processes have been recognized for some time (76). More recently, a link between 5-LO and polycythemia vera has been strongly suggested. Increased 5-LO expression has been reported in hematopoietic stem cells of both patients with this disorder (82) and in a mouse model thereof elicited by their transduction with a mutated version of JAK2 (83). In vitro treatment with the 5-LO inhibitor zileuton abrogated the tumorigenicity of this mutation in patient-derived stem cells, and both its in vivo administration as well as global knockout of 5-LO attenuated disease development in mice. The contributions of LTB₄ versus cysLTs to polycythemia vera were not elucidated in these studies. A recent study linked susceptibility to cancer to impaired innate immunity by showing that BLT1 knockout mice developed increased susceptibility to colon cancer, which was related to alteration of the gut microbiota and which in turn could be overcome by antibiotic treatment (84). Finally, the impact of cysLT1 antagonist treatment on cancer incidence has been investigated in two recent large retrospective insurance claims database studies. In one, treatment was associated with a reduction in overall incidence of cancers, with the magnitude of protection being related to both dose and duration of therapy (85). Interestingly, this protection did not extend to all cancers, and lung cancer was notably unprotected. By contrast, another study exclusively examining lung cancer did show significant protection (86). It is not terribly surprising for retrospective studies in different populations to yield contradictory results, so clarifying the possible effects of LT antagonists on cancer incidence must await prospective studies.

Cross-talk between LTs and other lipid mediators

The family of lipid mediators includes many classes other than LTs, and some of these have actions relevant to innate immunity. Here we will briefly mention two that are derived from AA and one that is derived predominantly from alternative fatty acids. Prostaglandin E₂ (PGE₂) is a cyclooxygenase metabolite of AA which in phagocytes typically signals via increases in cAMP, and as such, it tends to inhibit phagocyte responses that are promoted by LTs and to impair protective host responses to infection (87–91). The fact that AA is a shared substrate for both PGE₂ and LTs means that inhibition or deletion of one oxygenase can result in substrate shunting towards the other, and the resulting biological actions can therefore be attributable to the reduction in one metabolite, the increase of the other, or a combination of both phenomena. In addition to oxygenation reactions carried out by dedicated enzymes such as 5-LO or cyclooxygenases, AA can also be oxygenated by cytochrome P450 epoxygenases into a large array of metabolites that possess broad biological activities. Although the actions of these P450 metabolites in innate immunity remain poorly understood, they have been reported to impair post-viral bacterial defenses as well as TLR signaling (92) but also to potentiate bacterial phagocytosis (93). Furthermore, some of these metabolites have been shown to directly inhibit expression of either 5-LO or BLT1 in endothelial cells (94). The final class of non-LT lipid mediators to be discussed comprise a group of metabolites derived predominantly from n-3 fish oil-derived fatty acids rather than AA itself, and referred to as “specialized pro-resolving mediators” (SPMs)(95). Consisting of protectins, resolvins, and maresins, formation of SPMs is typically catalyzed by 5-LO acting sequentially along with another lipoxygenase. Unlike LTs, SPMs have been implicated in inhibition as well as active resolution of inflammation (95), yet importantly, the dominant phenotype observed when 5-LO is deleted or pharmacologically inhibited is reduction, rather than potentiation, of inflammation (96). Also unexpected for substances with anti-inflammatory properties, SPMs are reported to enhance antimicrobial responses (97). Despite substantial interest in SPMs, significant uncertainty remains about their putative receptors and downstream signaling, and whether they are generated in humans at levels necessary to exert the biological actions that they do when added exogenously (96).

Conclusions

More than forty years after the discovery of LTs, research into their generation and actions continues to extend our understanding of these important substances. Although it is still the case that – relative to protein mediators such as chemokines and cytokines – lipid mediators remain an afterthought in the minds of most investigators studying inflammation and immunity, perhaps the contributions of LTs to innate immunity are not quite as underappreciated today as they were in 2005. Indeed, LTB₄ has emerged as an essential mediator of protective host defense responses towards most classes of pathogens. At the same time, overexuberant or aberrant production of both LTB₄ and cysLTs can also contribute to tissue injury in both infectious and non-infectious settings. Expanding knowledge about these mediators in both homeostatic and pathologic scenarios and translating it into therapeutic opportunities is an important goal for the future, and one that still remains unmet today.