Cutting Edge: Human Vagus Produces Specialized Proresolving Mediators of Inflammation with Electrical Stimulation Reducing Proinflammatory Eicosanoids

Human vagus produces endogenous SPM 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. (Fig. 1A, Supplemental Table I). These included Rv, PD, and MaR from DHA, E-series Rv from EPA, arachidonic acid–derived LX, LT, thromboxane, and PGs, as well as CysLT (LTC₄, LTD₄). For each, LC-MS/MS results gave at least six diagnostic ions for identification (Fig. 1B).

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FIGURE 1.

Human vagus produces endogenous SPM and eicosanoids. Fresh postmortem human vagi were dissected and cold methanol was added containing deuterium-labeled internal standards. LM were identified and quantified using LC-MS/MS (see Materials and Methods). (A) LC-MS/MS chromatographs. (B) MS/MS spectra with diagnostic ions for RvD3, RvD4, RvD5, RvE1, MaR1, and NPD1/PD1 are representative of six different vagi from three 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 picograms. Black circle, not detected; Gray square, transient intermediates not monitored. Results are mean values. (A)–(C) are representative of six different human vagi from three subjects.

Human vagus produced several Rv, including RvE1 and specifically RvD3, RvD4, and RvD5 (Fig. 1B). The Rv 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, although some tissues produce all of the known d-series Rv (RvD1–RvD6), human vagus produces those biosynthesized via the 4(5)-epoxy-Rv intermediate rather than those from 7(8)-epoxy-Rv intermediate (i.e., RvD1 and RvD2) (compare Refs. 11, 13). D-series Rv control inflammation resolution, infection, and pain reduction (2, 8).

Human vagus also produced both PD and MaR pathways. This was concluded through identification of neuroprotectin D1 (NPD1/PD1) and its pathway marker (Fig. 1), biosynthesized via double lipoxygenation, namely 10S,17S-diHDHA (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, which 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 Rv (17R epimer) and LX (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 proresolving 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., LXA₄ and LXB₄) 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 in this article from their physical properties, is 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 (Supplemental Table I). RvD4 is found in human bone marrow and controls bacterial clearance (22). Vagus expresses Toll receptors (3), and incubations with live E. coli increased both RvD4 and RvD6, as well as increased 15-epi-LXA₄ and MaR1, which may together stimulate clearance of infections. RvD6 was not present in vagus alone or with electrical stimulation (Supplemental Table I). Fig. 1C shows the vagus LM network, depicting quantification, biosynthetic relationships between precursors, bioactive LM, and pathway marker products of each bioactive metabolome.

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

Because specific SPM were present in human vagus, we investigated LM of mouse vagus. For this, fresh mouse vagi were incubated, which demonstrated that LM profiles in mice differed from profiles in humans (Supplemental Fig. 1, Supplemental Tables I and II). Three mouse strains produced the same SPM (Supplemental Table III). 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 proresolving mediators. Interestingly, 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 (Supplemental Fig. 1D); the sphere in the three-dimensional score plot represents 95% confidence. Principal component analysis (PCA) confirmed that RvD6, RvE3, and RvD4 were associated with mouse vagus, whereas RvD5, RvE1, MaR1, and NPD1 were associated with human vagus (Supplemental Fig. 1D).

Electrical stimulation enhances vagus production of SPM and reduces eicosanoids

We next investigated whether EVS ex vivo also led to LM production. After 20 min of electrical stimulation, we found a specific group of SPM was increased. PCA confirmed that mouse vagus nerve subjected to EVS clustered separately compared with control (Fig. 2A). In multivariate analysis, RvD4, RvE1, RvD3, and PDX were associated with EVS (Fig. 2B). Also, quantitation of the increase in SPM gave a statistically significant increase of ∼3× the sum of RvD3, RvD4, and RvE1. Interestingly, prostanoids and thromboxane were reduced by EVS (Fig. 2C), as were LTC₄, LTD₄, and LTE₄ (Fig. 2D, Supplemental Table II). These findings identify LM of human and mouse vagus as well as, to our knowledge, the first evidence of vagus SPM production. Together, the present findings identify SPM as vagal products that are known controllers of host response to inflammation and infection (2, 5, 14).

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FIGURE 2.

Vagus electrical stimulation of SPM production and reduction of eicosanoids. Mouse vagus was incubated (20 min at 37°C with 5% CO₂) and electrically stimulated (2.5 mA 18 V direct DC for 20 min). Bioactive metabolomes were identified and quantified as in Fig. 1A. (A) PCA three-dimensional (3D) score plot. (B) Loading two-dimensional (2D) plot shows endogenous LM. (C) RvD3, RvD4, RvE1, RvE3, and LXB₄ (left) increased; LTB₄, PGD₂, PGE₂, PGF_2α, and TxB₂ (right) diminished. (D) CysLT identification (left); LT decreased after EVS (right). (E) LM-SPM network pathways visualized with Cytoscape (3.6.1), with mean value changes between unstimulated and stimulated vagi. Upregulated LM are shown in red, downregulated LM are shown in blue. Black circles, not detected; Gray squares, not monitored. (A) and (B) are representative of three independent animals. Results in (C) and (D) are mean ± SEM. *p < 0.05, one-tailed t test.

Vagus from human and mouse also produces PGD₂, PGE₂, and PGF_2α (Figs. 1, 2) as well as LT. LTB₄ is a potent chemoattractant, and CysLT (LTC₄, LTD₄, and LTE₄) are appreciated for their production by mast cells and role as slow-reacting substance of anaphylaxis in allergic reactions (23). However, CysLT may also possess physiologic functions in neural and endocrine systems, as in pineal gland control of hormone release (23). Because CysLT are potent smooth-muscle constrictors and stimulate vascular permeability (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 maresin conjugates in tissue regeneration and resolvin conjugates in tissue regeneration (12), were not present in either mouse or human vagus, in contrast to LTC₄, LTD₄, and LTE₄. 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 proresolving mediators that can directly stimulate resolution of inflammation and infections by virtue of their actions on phagocytes and reduce chemokines, cytokines, and proinflammatory LM as well as enhance microbial killing and clearance (2). Also, Rv (e.g., 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 the 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, purinergic signaling, and miRNAs (26–28), which interact with SPM (2). Vagus-stimulating devices in arthritis patients target the inflammatory reflex, reducing TNF-α, IL-1β, and IL-6 (29).

Our results demonstrate that isolated human vagi produce specific SPM, suggesting that EVS may activate resolution of inflammation via SPM and downregulation of PGs and LT. Excess PG and LTB₄ are known to contribute to chronic inflammation (23). Network mapping in the immune system (Figs. 1, 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, Supplemental Table I). RvD4 is produced by both human and mouse vagus, suggesting proresolving functions are intact in both species. Hence, these results document vagus proresolving capacity, with human and mouse vagus directly producing LX, Rv, and PD in amounts commensurate with their potent pico- to nanogram actions (2) that can impact multiple organs and immune cells. They also demonstrate that EVS increases SPM and diminishes PGs and LT that are known to contribute to chronic inflammation and allergic responses (23). Together, these results, to our knowledge, identify a new vagus proresolving reflex that may be targeted via electrical stimulation to improve disease treatments in which Rv and unresolved inflammation are involved as well as to possibly improve overall health status.