Cutting Edge: Human Vagus Produces Specialized Proresolving Mediators of with Electrical Stimulation Reducing Proinflammatory This information is current as of September 26, 2021. Charles N. Serhan, Xavier de la Rosa and Charlotte C. Jouvene J Immunol published online 24 October 2018 http://www.jimmunol.org/content/early/2018/10/23/jimmun ol.1800806 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 24, 2018, doi:10.4049/jimmunol.1800806

Cutting Edge: Human Vagus Produces Specialized Proresolving Mediators of Inflammation with Electrical Stimulation Reducing Proinflammatory Eicosanoids Charles N. Serhan, Xavier de la Rosa, and Charlotte C. Jouvene Inflammatory resolution is a process that, when uncon- nuclear translocation and stimulates the JAK2/STAT3 path- trolled, impacts many organs and diseases. As an active, way to reduce (4). self-limited inflammatory process, resolution involves Mechanisms controlling the magnitude and duration of of specialized proresolving mediators inflammatory responses have recently attracted considerable (SPM) (e.g., , [Rv], protectins, and attention (1, 2). Self-limited acute inflammatory responses activate biosynthesis of novel specialized proresolving medi-

). Because vagal stimulation impacts inflam- Downloaded from mation, we examined human and mouse vagus ex vivo ators (SPM) that stimulate resolution. SPM function by 1) to determine if they produce mediators. Using limiting further neutrophil infiltration, 2) reducing collateral targeted lipid mediator metabololipidomics, we identi- tissue damage, and 3) activating to engulf apo- fied lipoxins, Rv, and protectins produced by both hu- ptotic cells and debris as well as 4) clearing microbial infec- man and mouse vagus as well as PGs and . tions (2). The SPM include (LX), (Rv),

Human vagus produced SPM (e.g., RvE1, NPD1/ protectin (PD), and (MaR) families biosynthesized http://www.jimmunol.org/ from essential polyunsaturated fatty acids. Each SPM family PD1, MaR1, RvD5, and LXA4) on stimulation that differed from mouse (RvD3, RvD6, and RvE3), dem- member also counter-regulates cytokines, chemokines, and onstrating species-selective SPM. Electrical vagus stim- proinflammatory eicosanoids (e.g., PGF2a and leukotrienes [LT]) to reduce inflammation and activate IL-10 (2). Rv also ulation increased SPM in both human and mouse b vagus as did incubations with Escherichia coli. Electri- block NLRP3 inflammasome, reducing IL-1 cal vagus stimulation increased SPM and decreased PGs (5), and reduce (6, 7). Recently, new SPM structures containing peptide conjugates were elucidated that stimulate and leukotrienes. These results provide direct evidence resolution and activate tissue regeneration (8). for vagus SPM and eicosanoids. Moreover, they suggest by guest on September 26, 2021 We found that vagotomy delays resolution of inflammation that this vagus SPM circuit contributes to a new prore- (9). This delay involves shifting lipid mediators (LM) with solving vagal reflex. The Journal of Immunology, 2018, reduced Rv to proinflammatory status, demonstrating a novel 201: 000–000. vagus-resolution circuit (9, 10). During bacterial infection, vagus also controls resolution via biosynthesis of specific SPM he acute inflammatory response is critical in host that function as immunoresolvents (e.g., PD conjugate in tissue defense and, when unresolved, can lead to chronic regeneration [PCTR]1) upregulated by acetylcholine via ILC-3 T inflammation associated with many human diseases control of macrophage SPM biosynthesis and phenotype (10). (1, 2). New therapeutic approaches are needed for diseases in In view of these findings, we investigated whether vagus can which unresolved inflammation contributes to progressive loss directly produce LM. In this article, we report that human of organ function. The vagus nerve–based inflammatory reflex vagus produces specific SPM, identified using liquid chro- uncovered by Tracey and colleagues (3) regulates immune matography–tandem mass spectrometry (LC-MS/MS)–based function and inflammation. One mechanism of neural– metabololipidomics, that differed from those produced by immune control involves activation of macrophage a7 nico- mouse vagus. Escherichia coli increased LM-SPM, and elec- tinic acetylcholine receptors that inhibit proinflammatory trical vagus stimulation (EVS) ex vivo increased SPM and cytokines. This macrophage a7 receptor inhibits NF-kB reduced both PGs and LT.

Center for Experimental Therapeutics and , Department of Anesthe- Medical School, Hale Building for Transformative Medicine, Suite 3-016, 60 Fenwood siology, Perioperative and Pain Medicine, Brigham and Women’s Hospital and Harvard Road, Boston, MA 02115. E-mail address: [email protected] Medical School, Boston, MA 02115 The online version of this article contains supplemental material. ORCIDs: 0000-0003-4627-8545 (C.N.S.); 0000-0003-3927-364X (X.d.l.R.); 0000- Abbreviations used in this article: CysLT, cysteinyl LT; DC, direct current; EVS, elec- 0003-3978-8096 (C.C.J.). trical vagus stimulation; LC-MS/MS, liquid chromatography–tandem mass spectrome- Received for publication June 21, 2018. Accepted for publication September 24, 2018. try; LM, lipid mediator; LT, ; LX, lipoxin; MaR, maresin; NPD1/PD1, neuroprotectin D1; PCA, principal component analysis; PCTR, PD conjugate in tissue This work was supported in part by National Institutes of Health Grant R01GM038765 regeneration; PD, protectin; PDX, 10S,17S-diHDHA; Rv, resolvin; SPM, specialized (to C.N.S.). proresolving mediator. Address correspondence and reprint requests to Prof. Charles N. Serhan, Center for Ex- perimental Therapeutics and Reperfusion Injury, Brigham and Women’s Hospital/Harvard Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$37.50

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800806 2 CUTTING EDGE: VAGAL SPM AND EICOSANOIDS

Materials and Methods acid (16). Hence, 17R-NPD1/PD1 may have resulted from Human and mouse tissues use by the organ donors. Alternately, aspirin-triggered Rv (17R epimer) and LX (15R epimer) are also produced by a Fresh human vagi (deidentified) purchased from Tissue for Research (Ellingham, Bungay, Suffolk, U.K.) were analyzed under protocol no. 1999P0001279 approved new pathway in neural tissues that uses sphingosine kinase 1 by the Partners Human Research Committee. Each postmortem, full-length to acetylate COX-2 as a mechanism to biosynthesize aspirin- human vagus was thawed on arrival, measured, dissected, and incubated triggered epimers of SPM (17). These longer-acting endoge- in PBS (with and magnesium) for 20 min at 37˚C with 5% CO2 in parallel with direct EVS with 2.5 mA 18 V direct current (DC) for 20 min in nous epimers of SPM are potent proresolving agonists (2). PBS at 37˚C (ApeX Type A stimulator; ApeX Electronics, Schenectady, NY), Human vagus also produced MaR1 and its pathway marker, or coincubated with E. coli (109 CFU for 3 h at 37˚C). Deuterium-labeled 7S,14S-dihydroxy-DHA (Fig. 1). In addition to MaR1’s po- standards for SPM and extraction recoveries were from Cayman tent proresolving actions with human leukocytes (2, 14) and Chemical (Ann Arbor, MI). For abbreviations and stereochemical assign- ments with the full name for each of the SPM, see (11, 12). Animal exper- (18), MaR1 is neuroprotective and activates recovery imental procedures were approved by the Institutional Animal Care and Use from (19). Committee of Brigham and Women’s Hospital (protocol no. 2016N000145) In human vagus, SPM from (i.e., LXA4 and and complied with institutional and U.S. National Institutes of Health guidelines. Six- to eight-week-old FVB male mice (Charles River Laborato- LXB4) were also identified (Fig. 1). Along with their ability to ries, Wilmington, MA) were fed ad libitum Laboratory Rodent Diet 20-5058 activate resolution (2), LXA4 reduces neuroinflammation and (Purina Mills, Great Summit, MO). neuropathic pain following hemisection of spinal cord via reducing microglial activation (20), and both LXA and LXB LM metabololipidomics 4 4 are neuroprotective (21). Thus, their production by human Downloaded from Cold methanol containing deuterium-labeled (12) internal standards vagus, as well as other SPM documented in this article from (500 pg/sample) were added to all samples. Following solid-phase extraction, LM-SPM were identified and quantified as in Ref. 13 and, for cysteinyl LT their physical properties, is of interest as potential mediators (CysLT), using LC-MS/MS and published criteria (i.e., six ions) (12, 13). from vagal stimulation. Linear calibration curves were obtained using d5-LTC4,d5-LTD4,d2-PCTR3, Electrical stimulation of human vagus increased RvD4 2 and others (12), giving r values of 0.98–0.99. and MaR1, with trends for increases in other vagus SPM

Statistics (Supplemental Table I). RvD4 is found in human bone http://www.jimmunol.org/ marrow and controls bacterial clearance (22). Vagus expresses Results are mean 6 SEM. Significance was calculated using one-tailed paired t test and GraphPad Prism software (La Jolla, CA). The p values were Toll receptors (3), and incubations with live E. coli increased *p , 0.05 and **p , 0.01. both RvD4 and RvD6, as well as increased 15-epi-LXA4 and MaR1, which may together stimulate clearance of infections. Results and Discussion RvD6 was not present in vagus alone or with electrical Human vagus produces endogenous SPM and eicosanoids stimulation (Supplemental Table I). Fig. 1C shows the vagus To determine if human vagus directly produces LM that could LM network, depicting quantification, biosynthetic relation- impact inflammation via the neural reflex (3), we assessed LM ships between precursors, bioactive LM, and pathway marker by guest on September 26, 2021 profiles with fresh human vagus. To this end, using LC-MS/ products of each bioactive metabolome. MS–based LM metabololipidomics together with spectral libraries of MS/MS (11–13), we identified in human vagus Human vagus produces a distinct and unique profile of SPM and eicosanoids specific mediators from each major bioactive LM-SPM metabolome. (Fig. 1A, Supplemental Table I). These in- Because specific SPM were present in human vagus, we in- cluded Rv, PD, and MaR from DHA, E-series Rv from EPA, vestigated LM of mouse vagus. For this, fresh mouse vagi were arachidonic acid–derived LX, LT, , and PGs, as incubated, which demonstrated that LM profiles in mice dif- well as CysLT (LTC4, LTD4). For each, LC-MS/MS results fered from profiles in humans (Supplemental Fig. 1, Supplemental gave at least six diagnostic ions for identification (Fig. 1B). Tables I and II). Three mouse strains produced the same SPM Human vagus produced several Rv, including RvE1 and (Supplemental Table III). Mouse vagus produced RvD4, specifically RvD3, RvD4, and RvD5 (Fig. 1B). The Rv of RvE1, RvE3, LXB4, and 15-epi-LXA4. Mouse vagus with E. human vagus did not include RvD1, RvD2, RvE2, or RvE3, coli increased biosynthesis of only PDX, suggesting that this which are produced by human leukocytes, lymph nodes, SPM may play a role in vagus control of infection, whereas spleen (11), and emotional tears (13). These results indicate human vagus increased several SPM(e.g.,RvD4,NPD1,MaR1, that, although some tissues produce all of the known D-series 18-HEPE, and 15-epi-LXA4) that are each potent proresolving Rv (RvD1–RvD6), human vagus produces those biosynthesized mediators. Interestingly, RvD3 was selectively increased with via the 4(5)-epoxy-Rv intermediate rather than those from 7(8)- EVS(videinfra).MultivariateanalysisofLMprofilesobtained epoxy-Rv intermediate (i.e., RvD1 and RvD2) (compare Refs. from human or mouse vagus profiles demonstrated a strong 11, 13). D-series Rv control inflammation resolution, infection, association between different species (Supplemental Fig. 1D); and pain reduction (2, 8). the sphere in the three-dimensional score plot represents 95% Human vagus also produced both PD and MaR pathways. confidence. Principal component analysis (PCA) confirmed that This was concluded through identification of neuroprotectin RvD6, RvE3, and RvD4 were associated with mouse vagus, D1 (NPD1/PD1) and its pathway marker (Fig. 1), biosynthesized whereas RvD5, RvE1, MaR1, and NPD1 were associated with via double lipoxygenation, namely 10S,17S-diHDHA (PDX) human vagus (Supplemental Fig. 1D). (14). Also, 17R-NPD1/PD1 was identified in human vagus (Fig. 1A). NPD1/PD1 stimulates resolution and is neuroprotective Electrical stimulation enhances vagus production of SPM and (15). This 17R epimer of NPD1/PD1 is longer acting and is reduces eicosanoids produced via acetylated COX-2 following aspirin or by p450, We next investigated whether EVS ex vivo also led to LM which can produce the precursor 17R-hydroxydocosahexaenoic production. After 20 min of electrical stimulation, we found a The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

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. specific group of SPM was increased. PCA confirmed that knowledge, the first evidence of vagus SPM production. To- mouse vagus nerve subjected to EVS clustered separately gether, the present findings identify SPM as vagal products compared with control (Fig. 2A). In multivariate analysis, that are known controllers of host response to inflammation RvD4, RvE1, RvD3, and PDX were associated with EVS and infection (2, 5, 14). (Fig. 2B). Also, quantitation of the increase in SPM gave a Vagus from human and mouse also produces PGD2, PGE2, ∼ 3 statistically significant increase of 3 the sum of RvD3, and PGF2a (Figs. 1, 2) as well as LT. LTB4 is a potent che- RvD4, and RvE1. Interestingly, and thrombox- moattractant, and CysLT (LTC4, LTD4, and LTE4) are ane were reduced by EVS (Fig. 2C), as were LTC4, LTD4, appreciated for their production by mast cells and role as and LTE4 (Fig. 2D, Supplemental Table II). These findings slow-reacting substance of anaphylaxis in allergic reactions identify LM of human and mouse vagus as well as, to our (23). However, CysLT may also possess physiologic functions 4 CUTTING EDGE: VAGAL SPM AND EICOSANOIDS Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

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) B C score plot. ( ) Loading two-dimensional (2D) plot shows endogenous LM. ( ) RvD3, RvD4, RvE1, RvE3, and LXB4 (left) increased; LTB4,PGD2,PGE2,PGF2a, D E and TxB2 (right) diminished. ( ) CysLT identification (left); LT decreased after EVS (right). ( ) 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)aremean6 SEM. *p , 0.05, one-tailed t test. in neural and endocrine systems, as in pineal gland control of both PGs and CysLT (Fig. 2C–E). These findings indicate release (23). Because CysLT are potent smooth- that vagus stimulation increases proresolving mediators that muscle constrictors and stimulate vascular permeability (23), can directly stimulate resolution of inflammation and infec- their vagus production is of interest and may contribute to tions by virtue of their actions on phagocytes and reduce neural reflex pathways that can modulate organ function. chemokines, cytokines, and proinflammatory LM as well as Novel SPM such as PCTR1, regulated by vagal stimulation of enhance microbial killing and clearance (2). Also, Rv (e.g., ILC3 to control infection (10), along with maresin conjugates RvE1) reduce pain via SPM receptors on (7). in tissue regeneration and resolvin conjugates in tissue re- In PGE synthase-1 (mPGE1) knockout mice, vagus stim- generation (12), were not present in either mouse or human ulation is abolished, implying that absence of PGE2 is critical vagus, in contrast to LTC4, LTD4, and LTE4. EVS of mouse to the cholinergic anti-inflammatory pathway (24). In reso- vagus increased SPM that included LXB4, RvE1, RvD3, and lution of contained , PGE2 signals LM class switch- RvD4 (Fig. 2A–C). This was accompanied by decreases in ing, increasing SPM (2). Vagus nerve also responds with The Journal of Immunology 5 -specific neural signals (25) that can contribute to 9. Mirakaj, V., J. Dalli, T. Granja, P. Rosenberger, and C. N. Serhan. 2014. Vagus nerve controls resolution and pro-resolving mediators of inflammation. J. Exp. Med. systemic inflammation. 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