Human Vagus Produces Specialized Pro-Resolving Mediators of Inflammation with Electrical Stimulation Reducing Pro-Inflammatory Eicosanoids

Abstract

Inflammatory-resolution are processes that when uncontrolled impact many organs and diseases. As an active self-limited inflammatory process, resolution involves biosynthesis of specialized pro-resolving mediators (SPM), i.e. lipoxins, resolvins, protectins and maresins. Since vagal stimulation impacts inflammation, we examined human and mouse vagus ex vivo to determine if they produce lipid mediators (LM). Using targeted LM-metabololipidomics, we identified lipoxins, resolvins and protectins, produced by both human and mouse vagus, as well as prostaglandins (PG) and leukotrienes (LT). Human vagus produced SPM (e.g. RvE1, NPD1, MaR1, RvD5, LXA₄) on stimulation that differed from mouse (RvD3, RvD6 and RvE3), demonstrating species-selective SPM. Electrical vagus stimulation (EVS) increased SPM in both human and mouse vagus, as did incubations with E. coli. EVS increased SPM and decreased PGs and leukotrienes (cys-LT). These results provide direct evidence for vagus-SPM and eicosanoids. Moreover, they suggest that this vagus-SPM circuit contributes to a new proresolving vagal reflex.

Introduction

The acute inflammatory response is critical in host defense and, when unresolved, can lead to chronic inflammation associated with many human diseases (1, 2). New therapeutic approaches are needed for diseases where unresolved inflammation contributes to progressive loss of organ function. In recent years, the vagus nerve-based inflammatory reflex uncovered by Tracey and colleagues regulates immune function and inflammation (3). One mechanism of neural-immune control involves activation of macrophage α7-nicotinic acetylcholine receptors that inhibit pro-inflammatory cytokines. This macrophage α7-receptor inhibits NF-κB nuclear translocation and stimulates JAK2/STAT3 pathway to reduce cytokines (4).

Mechanisms controlling the magnitude and duration of inflammatory responses have recently attracted considerable attention (1, 2). Self-limited acute inflammatory responses activate biosynthesis of novel specialized pro-resolving lipid mediators (SPM) that stimulate resolution. SPM function by a) limiting further neutrophil infiltration, b) reducing collateral tissue damage, c) activating macrophages to engulf apoptotic cells and debris as well as d) clearing microbial infections (2). The SPM include lipoxin (LX), resolvin (Rv), protectin (PD) and maresin (MaR) families biosynthesized from essential polyunsaturated fatty acids. Each SPM family member also counter-regulates cytokines, chemokines and pro-inflammatory eicosanoids, e.g. prostaglandin F_2α and leukotrienes, to reduce inflammation and activate IL-10 (2). Resolvins also block macrophage NLRP3 inflammasome reducing IL-1β (5) and reduce pain (6, 7). Recently, new SPM structures containing peptide-conjugates were elucidated that stimulate resolution and activate tissue regeneration (8).

We found that vagotomy delays resolution of inflammation (9). This delay involves shifting LM with reduced resolvins to pro-inflammatory status, demonstrating a novel vagus-resolution circuit (9, 10). During bacterial infection, vagus also controls resolution via biosynthesis of specific SPM that function as immunoresolvents, e.g. protectin conjugate in tissue regeneration (PCTR1) upregulated by acetylcholine via ILC-3 control of macrophage SPM biosynthesis and phenotype (10).

In view of these, we investigated whether vagus can directly produce LM. Here, we report that human vagus produces specific SPM identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabololipidomics that differed from those produced by mouse vagus. E. coli increased LM-SPM, and electrical vagus stimulation (EVS) ex vivo increased SPM and reduced both PGs and LTs.

Materials and Methods

Human and mouse tissues:

Fresh human vagus (de-identified) purchased from Tissue for Research (Ellingham, Bungay, Suffolk, UK) were analyzed under protocol #1999P0001279 approved by Partners Human Research Committee. Each post-mortem, full-length human vagus was thawed on arrival, measured, dissected, and incubated in PBS (with calcium and magnesium) 20 min, 37°C with 5% CO₂, in parallel with direct electrical vagus stimulation (EVS) with 2.5 mA 18V DC, 20 min, in PBS, 37°C (ApeX Type A stimulator, ApeX Electronics LLC, Schenectady, NY), or coincubated with E. coli (10⁹ c.f.u., 3h, 37°C). Deuterium-labeled standards for SPM and eicosanoid extraction recoveries were from Cayman Chemical (Ann Arbor, MI). For abbreviations and stereochemical assignments with full name for each SPM, see (11, 12). Animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Brigham and Women’s Hospital (protocol no. 2016N000145) and complied with institutional and U.S. NIH guidelines. Six- to eight-week-old FVB male mice (Charles River Laboratories, Wilmington, MA) were fed ad libitum Laboratory Rodent Diet 20–5058 (Purina Mills, Great Summit, MO).

Lipid mediator (LM) metabololipidomics:

Cold methanol containing deuterium-labeled (12) internal standards (500pg each/sample) was added to all samples. Following solid-phase extraction, LM-SPM were identified and quantified using LC-MS/MS (13) and for cysteinyl leukotrienes (cys-LT) using published criteria, e.g. 6 ions (12,13). Linear calibration curves were obtained using d₅-LTC₄, d₅-LTD₄, d₂-PCTR3 and others (12) giving r² values of 0.98–0.99.

Statistics:

Results are mean±SEM. Significance was *p<0.05, **p<0.01; one-tailed paired t test using GraphPad^© Prism software(La Jolla, CA).

Results and Discussion

Human vagus produces endogenous SPMs and eicosanoids

To determine if human vagus directly produces LM that could impact inflammation via the neural reflex (3), we assessed LM profiles with fresh human vagus. To this end, using LC-MS/MS-based LM metabololipidomics together with spectral libraries of MS/MS (11–13), we identified in human vagus specific mediators from each major bioactive LM-SPM metabolome. (Fig1A; Table S1). These included resolvins (Rv), protectins (PD), and maresins (MaR) from DHA, E-series Rv from EPA and arachidonic acid-derived lipoxins, LT, thromboxane (TX), and PGs as well as cys-LT (LTC₄, LTD₄). For each, LC-MS/MS results gave at least 6 diagnostic ions for identification (Fig. 1B).

Fig 1: Human vagus produces endogenous SPMs and eicosanoids.

Fresh post-mortem human vagus were each dissected and incubated (20min, 37°C with 5% CO₂), electrically stimulated (2.5mA 18V direct DC, 20min, 37°C), or incubated with 10⁹ c.f.u. of E. coli (3h, 37°C), cold methanol added containing deuterium-labeled internal standards; LM identified and quantified using LC-MS/MS (see Materials and Methods). A) LC-MS/MS chromatographs. B) MS/MS spectra with diagnostic ions for RvD5, RvE1, LXB₄, LXA₄, MaR1, and PD1 are representative of 6 different vagus from 3 human subjects. C) LM-Network visualization of unstimulated vagus using Cytoscape 3.6.1 software and quantitation using LC-MS-MS values. Circle size in pg; Black circle, not detected; Grey square, transient intermediates not monitored. Results are mean values. A-C are representative of 6 different human vagus from 3 subjects.

Human vagus produced several resolvins including RvE1 and specifically RvD3, RvD4 and RvD5 (Fig. 1B). The resolvins of human vagus did not include RvD1, RvD2, RvE2 or RvE3, which are produced by human leukocytes, lymph nodes, spleen (11), and emotional tears (13). These results indicate that, while some tissues produce all of the known D-series resolvins (RvD1-RvD6), human vagus produces those biosynthesized via the 4(5)-epoxy-resolvin intermediate rather than those from 7(8)-epoxy-resolvin intermediate, i.e. RvD1 and RvD2 (cf. 11, 13). D-series Rv control inflammation resolution, infection, and reduce pain (2, 8).

Human vagus also produced both protectin and maresin pathways. This was concluded with identification of neuroprotectin D1 (NPD1/PD1) and its pathway marker (Fig. 1) biosynthesized via double lipoxygenation 10S,17S-diHDHA, known as PDX (14). Also, 17R-NPD1/PD1 was identified in human vagus (Fig. 1A). NPD1/PD1 stimulates resolution and is neuroprotective (15). This 17R epimer of NPD1/PD1 is longer acting and is produced via acetylated COX-2 following aspirin or by p450 that can produce the precursor 17R-hydroxydocosahexaenoic acid (16). Hence, 17R-NPD1/PD1 may have resulted from aspirin use by the organ donors. Alternately, aspirin-triggered resolvins (17R epimer) and lipoxins (15R epimer) are also produced by a new pathway in neural tissues that uses sphingosine kinase 1 to acetylate COX-2 as a mechanism to biosynthesize aspirin-triggered epimers of SPM (17). These longer-acting endogenous epimers of SPM are potent proresolving agonists (2). Human vagus also produced MaR1 and its pathway marker 7S,14S-dihydroxy-DHA (Fig. 1). In addition to MaR1’s potent pro-resolving actions with human leukocytes (2, 14) and platelets (18), MaR1 is neuroprotective and activates recovery from spinal cord injury (19).

In human vagus, SPM from arachidonic acid, i.e. lipoxin A₄ and lipoxin B₄,were also identified (Fig. 1). Along with their ability to activate resolution (2), LXA₄ reduces neuroinflammation and neuropathic pain following hemisection of spinal cord via reducing microglial activation (20), and both LXA₄ and LXB₄ are neuroprotective (21). Thus, their production by human vagus, as well as other SPM documented herein from their physical properties, are of interest as potential mediators from vagal stimulation.

Electrical stimulation of human vagus increased RvD4 and MaR1 with trends for increases in other vagus SPM (Table S1). RvD4 is found in human bone marrow and controls bacterial clearance (22). Vagus express Toll receptors (3), and incubations with live E. coli increased both RvD4 and RvD6. RvD6 was not present in vagus alone or with electrical stimulation (Table S1), as well as increased 15-epi-LXA₄ and MaR1 that may together stimulate clearance of infections. Fig.1 C shows the vagus LM-Network that depicts quantification, biosynthetic relationships between precursors, bioactive LM and pathway marker products of each bioactive metabolome.

Human vagus produces a distinct and unique profile of SPMs and eicosanoids

Because specific SPM were present in human vagus, we investigated LM of mouse vagus. For this, fresh mouse vagus were incubated that demonstrated LM profiles in mice differed from human (Table S1, Fig. S1). Three mouse strains produced the same SPM (Table S3). Mouse vagus produced RvD4, RvE1, RvE3, LXB₄ and 15-epi-LXA₄. Mouse vagus with E. coli increased biosynthesis of only PDX suggesting that this SPM may play a role in vagus control of infection whereas human vagus increased several SPM (e.g. RvD4, NPD1, MaR1, 18-HEPE and 15-epi-LXA₄) that are each potent pro-resolving mediators. Of interest, RvD3 was selectively increased with EVS; vide infra. Multivariate analysis of LM profiles obtained from human or mouse vagus profiles demonstrated a strong association between different species (Fig. S1D); sphere in the 3-dimension score plot represents 95% confidence. Principal component analysis (PCA) confirmed that RvD6, RvE3, and RvD4 were associated with mouse, whereas RvD5, RvE1, MaR1, and NPD1 were with human vagus (Fig. S1D).

Electrical stimulation enhances vagus production of SPMs and reduces eicosanoids

We next investigated whether EVS, ex vivo, also led to LM production. After 20 minutes of electrical stimulation, we found a specific group of SPMs were increased. PCA confirmed that mouse vagus nerve subjected to EVS clustered separately compared to control (Fig. 2A). In multivariant analysis, RvD4, RvE1, RvD3 and PDX (10S,17S-diHDHA) were associated with EVS (Fig. 2B). Also, quantitation of the increase in SPMs gave a statistically significant increases ~ 3X the sum of RvD3, RvD4, and RvE1. Of interest, prostanoids and thromboxane were reduced (Fig. 2C) as were LTC₄, LTD₄, LTE₄ by EVS (Fig. 2D, Table S2). These findings identify LM of human and mouse vagus as well as the first evidence of vagus SPM production. Together, the present findings identify SPMs as vagal products that are known controllers of host response to systemic inflammation (2, 5, 14).

Fig 2. Vagus electrical stimulation of SPM production and reduction of eicosanoids.

Mouse vagus incubated (20min at 37°C with 5 % CO), electrically stimulated (2.5mA 18V direct DC, 20min), or with 10⁹c.f.u. E. coli (3h at 37°C), bioactive metabolomes identified and quantified as in Fig. 1A) PCA 3D score plot; B) Loading 2D plot shows endogenous LM. 3D, 3-dimensional. 2D, 2-dimensional. C) RvD3, RvD4, RvE1, RvE3, and LXB₄ (left) increased, LTB₄, PGD₂, PGE₂, PGF_2α, and TxB₂ (right) diminished. E) LM-SPM-Network pathways visualized with Cytoscape (3.6.1) with mean value changes between unstimulated and stimulated vagus. Up-regulated LM (red), down-regulated (blue). Black circles, not detected; Grey squares, not monitored. A-E are representative from 3 independent animals. Results are mean±SEM, *p<0.05, one-tailed t test.

Vagus from human and mouse also produces PGD₂, PGE₂ and PGF_2α (Figs. 1 and 2) as well as leukotrienes. Leukotriene B₄ (LTB₄,) is a potent chemoattractant, and cys-LT (LTC₄, LTD₄ and LTE₄) are appreciated for their production by mast cells and role as slow-reacting substance of anaphylaxis (SRS-A) in allergic reactions (23). However, cys-LT may also possess physiologic functions in neural and endocrine systems, as in pineal gland control of hormone release (23). Since cys-LT are potent smooth-muscle constrictors and stimulate vascular permability (23), their vagus production is of interest and may contribute to neural reflex pathways that can modulate organ function. Novel SPM, such as PCTR1, regulated by vagal stimulation of ILC3 to control infection (10), along with MCTRs and RCTRs (12), were not present in either mouse or human vagus compared to LTC₄, D₄ and E₄. EVS of mouse vagus increased SPM that included LXB₄, RvE1, RvD3 and RvD4 (Fig. 2A-C). This was accompanied by decreases in both PGs and cysLT (Fig. 2C-E). These findings indicate that vagus stimulation increases pro-resolving mediators that can directly stimulate resolution of inflammation and infections by virtue of their actions on phagocytes and to reduce chemokines, cytokines and pro-inflammatory LM as well as enhance microbial killing and clearance (2). Also, resolvins, i.e. RvE1, reduce pain via SPM receptors on neurons (7).

In PGE synthase-1 (mPGE1) knockout mice, vagus stimulation is abolished, implying that absence of PGE₂ is critical to cholinergic anti-inflammatory pathway (24). In resolution of contained exudates, PGE₂ signals LM class switching increasing SPM (2). Vagus nerve also responds with cytokine-specific neural signals (25) that can contribute to systemic inflammation. Additional regulators of inflammation-resolution that possibly may be vagus controlled include hypoxia-inducible factors (HIFs), purinergic signaling and miRNAs (27–29), which interact with SPMs (2). Vagus-stimulating devices in arthritis patients target the inflammatory reflex reducing TNFα, IL-1β and IL-6 (26).

Our results demonstrate that isolated human vagus produce specific SPM suggesting that EVS may activate resolution of inflammation via SPM and down-regulation of PGs and LT. Excess PG and LTB₄ are known to contribute to chronic inflammation (23). Network mapping in the immune system (Figs. 1 and 2) can highlight species differences in physiologic and pathologic networks (30). The present results demonstrate species differences with human SPM, in that with EVS human vagus produced MaR1 and RvD4 (Fig. 1, Table S1). RvD4 is produced by both human and mouse vagus suggesting pro-resolving functions are intact in both species. Hence, these results document vagus pro-resolving capacity with human and mouse vagus directly producing lipoxins, resolvins and protectins in amounts commensate with their potent pico-nanogram action (2) that can impact multiple organs and immune cells. They also demonstrate that EVS increases SPM and diminishes PG and LT that are known contribute to chronic inflammation and allergic responses (23). Together, these results identify a new vagus pro-resolving reflex that may be targeted via electrical stimulation to improve disease treatments where resolvins and unresolved inflammation are involved, as well as to possibly improve overall health status.