
==== Front
Mol Med
Mol Med
Molecular Medicine
1076-1551
1528-3658
BioMed Central London

39294573
877
10.1186/s10020-024-00877-w
Research Article
Low-dose pro-resolving mediators temporally reset the resolution response to microbial inflammation
http://orcid.org/0000-0003-4627-8545
Serhan Charles N. cserhan@bwh.harvard.edu

http://orcid.org/0000-0003-1963-1585
Chiang Nan
http://orcid.org/0000-0003-0832-9938
Nshimiyimana Robert
https://ror.org/04py2rh25 grid.452687.a 0000 0004 0378 0997 Department of Anesthesiology, Perioperative and Pain Medicine, Center for Experimental Therapeutics and Reperfusion Injury, Mass General Brigham and Harvard Medical School, 60 Fenwood Rd., Hale Building for Transformative Medicine 3-016, Boston, MA 02115 USA
18 9 2024
18 9 2024
2024
30 15310 5 2024
15 7 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by/4.0/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
Background

Specialized pro-resolving mediators (SPMs) promote resolution of inflammation, clear infections and stimulate tissue regeneration. These include resolvins, protectins, and maresins. During self-resolving acute inflammation, SPMs are produced and have key functions activating endogenous resolution response for returning to homeostasis. Herein, we addressed whether infections initiated with ongoing inflammation alter resolution programs, and if low-dose repetitive SPM regimen re-programs the resolution response.

Methods

Inflammation was initiated with zymosan (1 mg/mouse) followed by E. coli (105 CFU/mouse) infections carried out in murine peritonitis, and exudates collected at 4-72 h. Leukocytes were enumerated using light microscopy, percentages of PMN, monocytes and macrophages were determined using flow cytometry, and resolution indices calculated. Lipid mediators and SPM profiles were established using mass spectrometry-based metabololipidomics. Repetitive dosing with a SPM panel consisting of RvD1, RvD2, RvD5, MaR1 and RvE2 (0.1 ng/mouse each, i.p.) was given to mice, followed by zymosan challenge. Leukocyte composition, resolution indices and RNA-sequencing were carried out for the repetitive SPM treatments.

Results

E. coli infections initiated acute inflammation-resolution programs with temporal SPM production in the infectious exudates. Zymosan-induced inflammation prior to E. coli peritonitis shifted exudate resolution indices and delayed E. coli clearance. Lipid mediator metabololipidomics demonstrated that E. coli infection with ongoing zymosan-induced inflammation shifted the time course of exudate SPMs, activating a SPM cluster that included RvD1, RvD5 and MaR1 during the initiation phase of infectious inflammation (0-4 h); RvD5 and MaR1 were present also in the resolution phase (24-48 h). To emulate daily SPM regimens used in humans, a repetitive subthreshold dosing of the SPM panel RvD1, RvD2, RvD5, MaR1 and RvE2 each at 0.1 ng per mouse was administered. This low-dose SPM regimen accelerated exudate PMN clearance following zymosan-induced inflammation, and shortened the resolution interval by > 70%. These low-dose SPMs regulated genes and pathways related to immune response, chemokine clearance and tissue repair, as demonstrated by using RNA-sequencing.

Conclusions

Infections encountered during ongoing inflammation in mice reset the resolution mechanisms of inflammation via SPM clusters. Low-dose SPMs activate innate immune responses and pathways towards the resolution response that can be reprogrammed.

Supplementary Information

The online version contains supplementary material available at 10.1186/s10020-024-00877-w.

Keywords

Macrophage
Neutrophil
Resolvins
Protectins
Maresins
LC–MS–MS
SPMs
http://dx.doi.org/10.13039/100000057 National Institute of General Medical Sciences R35GM139430 Serhan Charles N. issue-copyright-statement© The Feinstein Institute for Medical Research 2024
==== Body
pmcBackground

In the evolution of the innate immune system, the acute inflammatory response initiates the recruitment of leukocytes from circulation to defend and wall off microbial invaders (Majno and Joris 2004). Overall, this is a protective response. The summoning of leukocytes into tissue requires chemotactic signals, as all cells need gradients of chemoattractants to move (Majno and Joris 2004). In the acute inflammatory response, the influx of neutrophils is initiated by chemokines and cytokines. The lion’s share is summoned by leukotriene B4, a well-known pro-inflammatory chemoattractant (Malawista et al. 2008; Hopke et al. 2022; Lammermann et al. 2013). This initial phase also produces prostaglandin E2 that is required to enable neutrophil diapedesis, a pro-inflammatory response, by acting on endothelial cells, stimulating the induction of resolution phase mediators (Serhan 2007). The resolution phase of a normal healthy acute inflammatory response disassembles the defense battleline of neutrophils and returns the tissue to homeostasis by pro-resolving processes such as efferocytosis of apoptotic neutrophils by reparative macrophages (Serhan 2007).

In this resolution phase, omega-3 essential fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are mobilized to produce potent resolvins, protectins and maresins we collectively termed the specialized pro-resolving mediators (SPMs), that each limit further neutrophil recruitment, activate efferocytosis and reduce pain (Serhan 2007; Fredman and Serhan 2024). Since the loss of neutrophils from tissues defined the resolution phase (Majno and Joris 2004; Serhan 2007), earlier we introduced quantitative resolution indices to help pinpoint the precise cellular and molecular actions of each SPM within the resolution phase (Schwab et al. 2007). These indices take into account the numbers of neutrophils in the exudate tissue, the magnitude and duration of the resolution response in vivo. See the definitions of Resolution Indices vide infra in Methods. These are useful in defining resolution agonists to control inflammation rather than inhibitors of the chemical mediators in inflammation (Schwab et al. 2007). These resolution indices are in wide use and proved useful in diverse animal models (Recchiuti et al. 2014; Korner et al. 2018; Bhattacharya et al. 2023). For example, the oral administration of RvD1 impacts peritonitis, shortening the resolution intervals (Recchiuti et al. 2014). Oral omega-3 supplementation increases SPMs and survival in murine sepsis and in peritonitis shortened the resolution interval (Korner et al. 2018).

To further address mechanisms in resolution of inflammation, in our NIH/NIGMS Program Project (P01GM095467 to C.N.S), we prepared synthetic standards for each SPM uncovered and functionally characterized by total organic synthesis to confirm potent pro-resolving functions, and deuterium-labeled SPM standards were also prepared to enable SPM identification and quantification (Hong et al. 2007). Many of these novel molecules are now commercially available, permitting confirmation of their potent pro-resolving functions by others, for example, RvD2 in reducing coronary atherosclerosis (Bardin et al. 2022), diminishing aging (Fitzgerald et al. 2023), in stimulating tissue regeneration (Dort et al. 2021), RvD1 in activating myeloid and muscle stem cells (Markworth et al. 2020), resolvins in reducing lung inflammation (Sekheri et al. 2020), as well as enabling documentation of SPM biosynthesis in humans (Blaauw et al. 2024; Barden et al. 2024; Lau et al. 2023; Hartling et al. 2021) and in animal models (Korner et al. 2019). These were also useful in documenting SPMs in severe SARS-CoV-2 infection in humans (Schwarz et al. 2021; Palmas et al. 2021), giving rise to the notion that dysregulated SPMs and lipid mediators impact the magnitude and duration of the infection (Palmas et al. 2021; Serhan et al. 2022).

In view of these and the potential threat of another global microbial challenge (Huang et al. 2024; Morse et al. 2012), we sought evidence for the production and actions of SPMs during bacterial infection in mice with ongoing inflammation. Here we report that SPM production can reset and upregulate the resolution response of inflammation.

Materials and methods

Murine peritonitis

Animal experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Brigham and Women's Hospital (protocol no. 2016N000145). Mice were anesthetized with isoflurane, and experiments carried out with male C57BL/6 mice (6 to 8 weeks; Jackson Laboratory). Individual mice were used for each timepoint. Mice were sacrificed at designated timepoint using overdose of isoflurane following with cervical dislocation according to approved procedures by IACUC.

E. coli and Zymosan peritonitis: Mice were inoculated with E. coli (serotype O6:K2:H1; 1 × 105 CFU, i.p.) to initiate infectious peritonitis, and exudates collected at indicated time points. Intact leukocytes were identified using trypan blue exclusion, and enumerated using a light microscope. Leukocyte subtypes were assessed using flow cytometry with anti-mouse PerCP/Cy5.5 CD45 (clone 30-F11 Biolegend, CA), anti-mouse PE-CD11b (clone M1/70, eBioscience), anti-mouse APC F4/80 (clone BM8, eBioscience), anti-mouse FITC Ly6C (clone HK1.4, Biolegend) and anti-mouse APCcy7-Ly6G (clone 1A8, Biolegend). Peritoneal exudate populations were determined as PMN (CD45 + CD11b + Ly6G + Ly6C-), monocytes (CD45 + CD11b + Ly6G-Ly6C +) or MΦ (CD45 + CD11b + F4/80 +). In select experiments, Zymosan (1 mg/mL, i.p.) was given two hours before E. coli inoculation. Exudate E. coli titers were determined by plating serial dilutions of exudates onto LB agar plates for 24 h. The E. coli titers were determined by counting E. coli colony forming units (CFU) on the plates.

Zymosan peritonitis with repetitive SPM dosing: For repetitive dosing, mice were given a SPM panel (RvD1, RvD2, RvD5, MaR1 and RvE2, 0.1 ng each/mouse, i.p.) on days 0, 2, 5, 7 and 9. Next, on day 12, the SPM panel was given together with zymosan A (1 mg/mouse, i.p.) to initiate peritonitis. For single dosing of the SPM panel, mice were given vehicle control (0.01% ethanol v/v in 1 mL saline) as control on days 0, 2, 5, 7 and 9. Next, on day 12, the SPM panel was given together with zymosan A (1 mg/mouse, i.p.). Peritoneal exudates were collected at 12, 24 and 48 h, and leukocyte subtypes were assessed using flow cytometry. Aliquots of exudate leukocytes from 12 h time point were used for RNA-seq.

Resolution indices: The resolution of acute inflammation was defined in quantitative terms by resolution indices (Ri) as follows. ψmax: maximal exudate PMN numbers, Tmax: the time point when PMN numbers reach ψmax, R50: 50% of ψmax, T50, the time point when the PMN numbers reached R50, Ri (resolution interval): the time interval from the maximal PMN point ψmax to R50, i.e., T50 – Tmax, calculated as in Schwab et al. (2007).

Lipid mediator metabololipidomics

To monitor the production of lipid mediators in mouse exudates, liquid chromatography and electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS) analysis was carried out. Prior to solid-phase sample extraction, cold methanol (Thermo Fisher Scientific, Waltham, MA) held on ice containing deuterated d4-LTB4, d8-5S-HETE, d4-PGE2, d5-RvD1, d5-RvD2, d5-RvD3, and d5-LXA4 (Cayman Chemical, Ann Arbor, MI) internal standards (500 pg each) were added to each sample. The deuterium-labeled lipid mediators (LM) were initially synthesized to facilitate structural analysis of MS–MS ions, and confirmed the corresponding MS–MS fragmentation mechanisms of these LMs (Hong et al. 2007). These are now commercially available. Here, these deuterated LM were added to each sample to facilitate quantification of recovery from solid-phase extraction. All samples were placed at –80 °C for at least 30 min to allow protein precipitation, followed by solid-phase extraction as detailed earlier in Shay et al. (2021). Next, the extracted samples were subjected to LC–ESI–MS/MS in negative polarity. These experiments were conducted with a QTRAP 6500 + (Sciex, Framingham, MA) equipped with a ExionLC system and a Kinetex® 2.6 μm Polar C18 100 Å, 100 × 3.0 mm column (part no. 00D-4759-Y0, Phenomenex, Torrance, CA) maintained at 50 °C in a column oven. For LC conditions including flow rate, gradient, and mobile phase composition, see Table S1.

Targeted multiple reaction monitoring (MRM) and enhanced product ion (EPI) scan experiments were employed to monitor and quantify lipid mediators of interest. Calibration curves were constructed for each lipid mediator using synthetic and deuterated standard mixtures at, for example, 0.1, 0.5, 1, 5, 10, 25, 50, 125, 250, 500, and 1000 pg ranges, which showed good linearity and gave r2 values of 0.98–0.99. Source and gas parameters were set as follows: collision gas (CAD) = 12, curtain gas (CUR) = 30, ion source gas 1 (GS1, psi) = 85, ion source gas 2 (GS2, psi) = 50, ion spray voltage (IS, V) = -4200, and temperature (TEM, oC) = 500. Each mediator was identified by matching its chromatographic retention time (TR) and ESI–MS/MS to those of synthetic standards. A custom library containing the individual MS/MS spectra of synthetic and authentic standards was utilized to evaluate spectral matching. Spectral library search parameters were set as follows: precursor mass tolerance ± 0.8 Dalton; collision energy ± 5 eV; use polarity, intensity threshold = 0.05; minimal purity = 5.0%; and intensity factor = 100. Of note, the accuracy for data acquisition of the QTRAP 6500 + is ± 0.1 atomic mass units (a.m.u.). The additional digits presented in MS/MS spectra are due to default manufacturer settings. Data were acquired with Analyst 1.7.1 software (Sciex), and the spectral library was constructed in LibraryView™ software version 1.4.0 (Sciex) in conjunction with Sciex OS-Q v1.7.0.36606 and v3.1.5.3945. Analytes in the exudates that gave a signal-to-noise ratio < 5 or MS/MS library fit score < 70% to the synthetic standard were excluded. Partial least squares-discriminant analysis (PLS-DA) and hierarchical clustering heatmaps were carried out using MetaboAnalyst v6.0 (https://www.metaboanalyst.ca/) (Xia et al. 2009).

Authentication of RvD1, RvD2, RvD5, RvE2, and MaR1

Synthetic RvD1, RvD5, and RvE2 were purchased from Cayman Chemical (Ann Arbor, MI); RvD2 and MaR1 were from Vinresol (Budapest, Hungary). Prior to use in the present studies, each of these mediators was authenticated using UV spectrophotometry and LC–ESI–MS/MS to assess physical integrity by evaluating chromatographic retention times and MS/MS spectra. These matched the physical properties of the originally identified chemical mediators, were obtained using a Triple Quad 7500 mass spectrometer (Sciex, Framingham, MA) equipped with the ExionLC system (see Table S1) on a Kinetex® 2.6 μm PS C18 100 Å, 100 × 3.0 mm column (part no. 00D-4780-Y0, Phenomenex, Torrance, CA). Source and gas parameters were set as follows: collision gas = 12, curtain gas = 40, ion source gas 1 (psi) = 45, ion source gas 2 (psi) = 70, ion spray voltage (V) = 2000, and source temperature (°C) = 500. Spectral library parameters were set as stated above, and it should be noted that the accuracy for data resolution on the Sciex triple quad 7500 is also ± 0.1 atomic mass units (a.m.u.). Data were acquired and analyzed using Sciex OS 3.1.5.3945 and are presented as screen captures. UV spectra were recorded on a Cary 3500 Compact Peltier UV–visible Spectrophotometer (Agilent Technologies, Santa Clara, CA).

RNA sequencing and bioinformatics

Mouse exudate leukocytes were collected from peritonitis and submitted for paired-end RNA sequencing using Illumina NovaSeq X (Azenta Life Sciences, Burlington, MA). Sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality using Trimmomatic v.0.36. The trimmed reads were mapped to the Mus musculus GRCm38 ERCC reference genome available on ENSEMBL using the STAR aligner v.2.5.2b. The STAR aligner is a splice aligner that detects splice junctions and incorporates them to help align the entire read sequences. Unique gene hit counts were calculated by using featureCounts from the Subread package v.1.5.2. Only unique reads that fell within exon regions were counted. The unique gene hit counts were then analyzed using DESeq2, a comparison of gene expression between the groups of samples. The Wald test was used to generate p-values and log2 fold changes (Log2FC). Genes with a p-value < 0.05 and absolute Log2FC > 1 or < -1 were called as differentially expressed genes (DEGs). A gene ontology analysis was performed. The GO list was used to cluster the set of genes based on their biological processes and determine their statistical significance. DEGs were clustered by their gene ontology, and the enrichment of gene ontology terms was tested using Fisher exact test (GeneSCF v1.1-p2). An adjusted P-value less than 0.05 is considered statistically significant. The enrichment effect for each pathway was calculated as follows: Ratio 1 = “Significant_genes_count” in a GO category divided by number of significant gene numbers, which was 82 in our dataset; Ratio 2 = “Total_genes_group_count” in the same GO category divided by the total number of genes in the mouse genome, which was 49,671 in our dataset; Enrichment effect = Ratio 1/Ratio 2. Network analyses were also performed using “Atlas of Inflammation Resolution (AIR)” (https://air.bio.informatik.uni-rostock.de) to better understand the non-linear relationship among immune cell types, signaling and regulatory molecules associated with the onset, transition, resolution of acute inflammation and homeostasis (Serhan et al. 2020; Hoch et al. 2022).

Human macrophage and receptor expression

Human peripheral blood mononuclear cells were obtained from the Boston Children's Hospital Blood Bank (Mass General Brigham investigational review board protocol #1999-P-001279). All donors are adults. Peripheral blood mononuclear cells were isolated, and MΦ were differentiated by culturing freshly isolated monocytes in RPMI media supplemented with 10% FBS and recombinant human GM-CSF (10 ng/ml; R&D Systems) for 7 d. Macrophages were then incubated with 10 nM of RvD1, RvD2, MaR1, RvE2 or vehicle control for 24 h. Cells were collected and receptor surface expression monitored by flow cytometry using the following specific antibodies and appropriate isotype controls: GPR18 Antibody [Alexa Fluor® 647] (NBP2-24918AF647, Novus Biologicals) and Rabbit IgG Isotype Control [Alexa Fluor® 647] (NBP2-36463AF647, Novus Biologicals), human ChemR23 PE-conjugated Antibody (FAB362P, R&D systems) and mouse IgG3 PE-conjugated Antibody (IC007P, R&D systems), LGR6 Polyclonal antibody (17658-1-AP, Proteintech) and PE-conjugated F(ab')2-Donkey anti-Rabbit IgG (H + L) Secondary Antibody (12-4739-81, eBioscience).

Statistical analysis

Statistical analyses were performed using 2-tailed Student’s t test for two-group comparisons or one-way ANOVA with multiple group comparisons for three or more independent groups (GraphPad Prism, Version 10.1.0). P values of less than 0.05 were taken as statistically significant.

Results

Bacterial challenge during ongoing inflammation shifts resolution indices

During self-resolving E. coli infectious inflammation, the pro-resolving mediators, i.e., SPMs, are produced and activate endogenous resolution programs (Chiang et al. 2012). Here, we questioned if consecutive challenge with E. coli infection would alter the temporal sequence of SPM production and the resolution programs. To address this, we used a well-established murine peritonitis model (Winyard and Willoughby 2003), relevant to human peritonitis (Moore 1959). Inoculation of E. coli (105 CFU, i.p.) initiated a self-limited innate acute inflammation, characterized by a time-dependent neutrophil infiltration as expected (Fig. 1a and Fig. S1a-b). With these results, we calculated the resolution indices from the present results using equations defined earlier in Schwab et al. (2007). The PMN (CD45 + CD11b + Ly6G + Ly6C-) infiltration into the peritoneum was monitored throughout the time course of the experiments; PMN reached maximum ψmax ~ 1.8 × 106 PMN at 12 h, followed by a rapid decline associated with the resolution response. The resolution interval (Ri) was 12 h (Fig. 1a). The infiltration of monocytes (CD45 + CD11b + Ly6G-Ly6C +) was initially low in numbers at 4 h (Fig, 1b). These monocytes reached maximum at 12 h, and macrophages (MΦ; CD45 + CD11b + F4/80 +) gradually increased between 12 to 48 h (Fig. 1c). In these experiments, exudate E. coli titers were determined and expressed as colony forming units (CFUs). E. coli titers (CFUs) were the highest at 4 h, demonstrating robust infection that was cleared by 12 h (Fig. 1d). These results demonstrated a self-resolving E. coli infection (Fig. 1a–d). These present results are consistent with our earlier findings (Chiang et al. 2012) that enable us to investigate the impact of sequential challenge.Fig. 1 Resolution intervals for E. coli infections shift with ongoing zymosan inflammation. (a-d) E. coli infection. Mice (C57B6, 6-wk old male) were given saline (1 ml, i.p.) two hours prior to inoculation of E. coli (105 CFU, i.p.). Peritoneal exudates were collected by lavaging at indicated time points (Time E. coli). Total leukocytes were enumerated, and leukocyte composition determined using flow cytometry (see representative dot plots in Supplementary Fig. 1). a Exudate PMN time course and resolution indices (ψmax and Ri, see Methods). b exudate monocyte, c macrophage and d E. coli titer time course. e–h E. coli infection with ongoing inflammation. Timeline: Mice were given zymosan (1 mg/ml, i.p.) two hours prior to inoculation of E. coli (105 CFU, i.p.). Peritoneal exudates were collected by lavaging at indicated time points (Time E. coli). e Exudate PMN time course and resolution indices (ψmax and Ri, see Methods). f exudate monocyte, g macrophage and h E. coli titer time course. a–c, e–g Three independent experiments were carried out and 3-4 mice were used for each time point in each experiment. Results are cell numbers/mouse exudate; mean ± SEM; each dot denotes the value obtained from individual mice. (d,h) One dataset from two independent experiments are shown, and in each experiment, E. coli titers of 3-4 mice for each time point were determined. Results are E. coli titers/mouse exudate; mean ± SEM; each dot is the value obtained from one mouse. (a-d) *P < 0.05, **P < 0.01, ***P < 0.001 vs 12 h; ##P < 0.01 vs 4 h. e–g *P < 0.05, **P < 0.01 vs. 0 and 72 h; #P < 0.05 vs 0 h; ‡P < 0.05 vs 4 h using one-way ANOVA with Tukey's post-test

To gain insight into which of the SPM pathways, i.e., resolvins, protectins or maresins, are most critical to activation of the resolution response, we devised a protocol of ongoing zymosan-induced inflammation followed by E. coli infection (Fig. 1e-h). Next, we challenged mice using self-resolving E. coli peritonitis with ongoing sterile yeast-derived zymosan (Fig. 1e), a classic inflammatory stimulus (Winyard and Willoughby 2003). With zymosan challenge prior to E. coli, PMN infiltration was accelerated with a higher magnitude, reaching maximum ψmax 6.3 × 106 PMN (Fig. 1e and Fig. S1 c-d) following E. coli inoculation, compared to ψmax 1.8 × 106 PMN on E. coli challenge alone (Fig. 1a). PMN numbers then declined to half ψmax at 12 h, giving a resolution interval (Ri) of 8 h (Fig. 1e). In these studies, there was a second increase of PMN numbers with *ψmax 5.3 × 106 PMN at 24 h that gradually declined to half *ψmax at ~ 60 h, thus giving a biphasic response with two resolution intervals Ri = 8 h and *Ri = 36 h. In these exudates, monocytes reached maximum at 12 h, macrophages continued to increase till 48 h then declined (Fig. 1f and g). Exudate E. coli titers reached maximum at 24 h then gradually cleared at 48 h (Fig. 1h). These results demonstrated that a self-resolving E. coli peritonitis initiated during ongoing inflammation was reset temporally, thus shifting the exudate leukocyte numbers, E. coli clearance, and resolution intervals (Fig. S2).

Temporal exudate LM-SPM signature profiles

Targeted metabololipidomics profiling was carried out with E. coli exudates from Fig. 1, using mass spectrometry-based lipid mediator metabololipidomics (see Methods), focusing on local-acting lipid mediators including SPMs (Fig. 2 and S3). For identification of exudate lipid mediators, retention times (TR) and prominent MS–MS fragmentation ions present on MS–MS for each eicosanoid and SPM were matched to those obtained with both synthetic and authentic standards, which were in accordance with original published criteria for each molecule (Hong et al. 2007; Serhan and Petasis 2011; Serhan and Chiang 2023; Serhan 2014). Screen captures of the MRM chromatograms for each identified mediator in exudates together with those of validated synthetic standards are shown in Fig. 2. In these experiments, lipid mediators identified in exudates included the arachidonic acid-derived PGE2, LTB4 (both inflammatory (Majno and Joris 2004; Lammermann et al. 2013)) and the resolving mediator LXA4 (Serhan 2007), the eicosapentaenoic acid-derived RvE4 and 18-HEPE, as well as the docosahexaenoic acid-derived RvD1, RvD5, PD1, PDx, MaR1 and MaR2. Each of these pro-resolving mediators is a potent agonist of the resolution of inflammation (Serhan 2007; Fredman and Serhan 2024; Schwab et al. 2007).Fig. 2 Identification of exudate lipid mediators (LM): SPMs and eicosanoids. Exudate SPMs and eicosanoids were identified using LC–MS-MS-based metabololipidomics (see Methods). Screen captures of MRM chromatograms and MS–MS of identified LMs. For each LM, MRM of exudate LM (top panel) and synthetic standards (middle panel) as well as MS–MS of synthetic standard (bottom panel) are shown. See Fig. S3 for the enlarged MS–MS. The retention time (TR) of individual LM matched that obtained with synthetic standard. In MRM chromatograms, shaded blue areas denote the area under the curve used for quantitation. Dark blue data points indicate where the spectra were collected. a AA-derived LMs. PGE2: TR 8.32 min, MRM of m/z 351 > 189, S/N ratio 643; LTB4: TR 12.58 min, MRM of m/z 335 > 195, S/N ratio 402; LXA4: TR 9.21 min; MRM of m/z 351 > 115; S/N ratio 69. b EPA-derived SPMs. RvE4: TR 10.71 min, MRM of m/z 333 > 115, S/N ratio 159; 18-HEPE: TR 14.22 min, MRM of m/z 317 > 259, S/N ratio 730. c DHA-derived Rvs, PDs, and MaRs. RvD1: TR 10.81 min, MRM of m/z 375 > 215, S/N ratio 39; RvD5: TR 12.14 min, MRM of m/z 359 > 199, S/N ratio 332; 17-HDHA: TR 15.64 min, MRM of m/z 343 > 245, S/N ratio 1,610; PD1: TR 12.31 min, MRM of m/z 359 > 153, S/N ratio 127; PDx: TR 12.16 min, MRM of m/z 359 > 153, S/N ratio 409; MaR1: TR 12.48 min, MRM of m/z 359 > 221, S/N ratio 148; MaR2: TR 14.58 min, MRM of m/z 359 > 221, S/N ratio 269

The MS–MS prominent ions used for identification of eicosanoids and SPMs were as follows and shown in Fig. 2 and S3. After extraction of the peritoneal exudates, targeted LC–MS-MS demonstrated the presence of the arachidonic acid inflammation-resolution metabolome. The MS–MS spectra of exudate LXA4 matched those of both synthetic and authentic LXA4 (Fig. 2a) and consisted of a parent ion at m/z 351 = M-H and ions at m/z 315 = M-H-2H2O, 307 = M-H-CO2, 289 = M-H-H2O-CO2, 271 = M-H-2H2O-CO2, 251 = M-H-CHOH-(CH2)4-CH3 + H, 235 = M-H-CHOH-(CH2)3-CO2, 233 = 251-H2O, 217 = 235-H2O, 199 = 235-2H2O, 189 = 251-H2O-CO2, 135 = M-H-115-CHOH-(CH2)4-CH3, 115 = M-H-CHOH-(CH)8-CHOH-(CH2)4-CH3-H. These ions matched those of LXA4 documented in Clish et al. (2000). In the eicosapentaenoic acid metabolome: 18-HEPE is a SPM and precursor to E-series resolvins (RvE), uncovered in resolving inflammatory exudates (reviewed in Serhan and Petasis (2011); Serhan (2014)). Also, 18-HEPE is a potent bioactive SPM that, for example, prevents maladaptive cardiac remodeling (Endo et al. 2014). The MS–MS spectra of exudate 18-HEPE matched those of authentic 18-HEPE (Fig. 2b) that gave a parent ion at m/z 317 = M-H and daughter ions at m/z 299 = M-H-H2O, 273 = M-H-CO2, 259 = M-H-CHOH-CH2-CH3 + H, 255 = M-H-H2O-CO2, 215 = 259-CO2. The MS–MS spectra of exudate RvE4 matched those of authentic RvE4 (Fig. 2b) that gave a parent ion at m/z 333 = M-H and daughter ions at m/z 315 = M-H-H2O, 271 = M-H-H2O-CO2, 253 = M-H-2H2O-CO2, 235 = CHOH-CH2-(CH)2-CH2-CH3 + H, 217 = M-H-CHOH-(CH2)3-CO2, 199 = 217-H2O, 173 = 235-H2O-CO2, 115 = M-H-(CH)4-CH2-(CH)4-CHOH-CH2-(CH)2-CH2-CH3-H, and matched those of synthetic validated RvE4 (cf. (Serhan et al. 2022) and references within).

In the inflammatory exudate docosahexaenoic acid metabolome, the MS–MS spectra of exudate RvD1 matched those of authentic synthetic RvD1 (Fig. 2c), which MS–MS fragmentation consisted of a parent ion at m/z 375 = M-H and daughter ions at m/z 357 = M-H-H2O, 305 = M-H-CH2-(CH)2-CH2-CH3, 277 = M-CHOH-CH2-(CH)2-CH2-CH3 + H, 259 = 277-H2O, 243 = 305-H2O-CO2, 233 = M-H-CHOH-CH2-(CH)2-(CH2)2-CO2, 215 = 233-H2O, 171 = M-H-(CH)8-CHOH-CH2-(CH)2-CH2-CH3-H, 141 = M-H-CHOH-(CH)8-CHOH-CH2-(CH)2-CH2-CH3-H, 135 = 171-2H2O. These ions matched those of synthetic validated RvD1 documented in Hong et al. (2007). The MS–MS spectra of exudate RvD5 also matched those of synthetic RvD5 (Fig. 2c), consisting of a parent ion at m/z 359 = M-H with the daughter ions at m/z 341 = M-H-H2O, 315 = M-H-CO2, 297 = M-H-H2O-CO2, 289 = M-H-CH2-(CH)2-CH2-CH3-H, 279 = M-H-2H2O-CO2, 246 = M-H-CH2-(CH)2-(CH2)2-CO2, 227 = 289-H2O-CO2, 217 = M-H-CHOH-CH2-(CH)2-(CH2)2-CO2, 199 = 217-H2O, 141 = M-H-(CH)4-CH2-(CH)4-CHOH-CH2-(CH)2-CH2-CH3-H. These ions matched those of authentic RvD5 documented in ((Hong et al. 2007; Serhan and Petasis 2011; Serhan and Chiang 2023) and original references within) and Chiang et al., 2012 (Chiang et al. 2012). 17-HDHA is a SPM and pathway marker of both D-series resolvins and protectins (Hong et al. 2007). The MS–MS spectra of exudate 17-HDHA matched those of synthetic 17-HDHA (Fig. 2c) that gave a parent ion at m/z 343 = M-H with daughter ions at m/z 325 = M-H-H2O, 299 = M-H-CO2, 281 = M-H-H2O-CO2, 273 = M-H-CH2-(CH)2-CH2-CH3-H, 255 = 273-H2O, 245 = M-CHOH-CH2-(CH)2-CH2-CH3 + H, 229 = 273-CO2, 227 = 245-H2O, 201 = 245-CO2 (Fig. 2c). These fragmentation ions (Fig. 2c) matched those of authentic 17-HDHA reported earlier in Hong et al. (2007).

In the inflammatory exudates, we also identified protectins (PD) and maresins (MaR). The MS–MS spectra of exudate PD1 matched those of authentic and synthetic PD1 (Fig. 2c), which gave a MS–MS parent ion at m/z 359 = M-H and daughter ions at m/z 341 = M-H-H2O, 315 = M-H-CO2, 297 = M-H-H2O-CO2, 279 = M-H-2H2O-CO2, 261 = M-H-CHOH-CH2-(CH)2-CH2-CH3 + H, 217 = 261-CO2, 177 = M-H-CHOH-CH2-(CH)2-CH2-(CH)2-(CH2)2-CO2, 153 = M-H-CHOH-(CH)6-CHOH-CH2-(CH)2-CH2-CH3 + H (Fig. 2c). These ions matched those of validated synthetic PD1 as documented in Hong et al. (2007); Serhan and Petasis 2011). PDx is a stereoisomer of PD1 and a double dioxygenation product (Hong et al. 2007; Serhan and Petasis 2011). The MS–MS spectra of exudate PDx matched those of synthetic PDx (Fig. 2c), consisting of a parent ion at m/z 359 = M-H and daughter ions at m/z 341 = M-H-H2O, 315 = M-H-CO2, 297 = M-H-H2O-CO2, 290 = M-H-CH2-(CH)2-CH2-CH3, 261 = M-H-CHOH-CH2-(CH)2-CH2-CH3 + H, 181 = M-H-(CH)6-CHOH-CH2-(CH)2-CH2-CH3-H, 177 = M-H-CHOH-CH2-(CH)2-CH2-(CH)2-(CH2)2-CO2, 153 = M-H-CHOH-(CH)6-CHOH-CH2-(CH)2-CH2-CH3 + H, 137 = 181-CO2. In the maresin pathway, the MS–MS spectra of MaR1 obtained from exudates matched those of synthetic authentic MaR1 (Fig. 2c). These consisted of the parent ion at m/z 359 = M-H and daughter ions at m/z 341 = M-H-H2O, 315 = M-H-CO2, 297 = M-H-H2O-CO2, 250 = M-H-CH2-(CH)2-CH2-(CH)2-CH2-CH3, 246 = M-H-CH2-(CH)2-(CH2)2-CO2, 228 = 246-H2O, 221 = M-H-CHOH-CH2-(CH)2-CH2-(CH)2-CH2-CH3 + H, 177 = 221-CO2, 201 = 221-H2O-2H, 123 = 141-H2O. These fragmentation ions matched those of validated synthetic MaR1 as documented in (Lau et al. 2023 and original references within). The MS–MS spectra of MaR2 identified in the inflammatory exudates matched those obtained from synthetic MaR2 (Fig. 2c), which consisted of a parent ion at m/z 359 = M-H and daughter ions at m/z 341 = M-H-H2O, 323 = M-H-2H2O, 313 = M-H-CO2-2H, 297 = M-H-CO2-H2O, 279 = M-H-2H2O-CO2, 249 = M-H-CH2-(CH)2-CH2-(CH)2-CH2-CH3-H, 221 = M-H-CHOH-CH2-(CH)2-CH2-(CH)2-CH2-CH3 + H, 203 = 221-H2O, 191 = M-H-(CHOH)2-CH2-(CH)2-CH2-(CH)2-CH2-CH3 + H, 177 = 221-CO2, 167 = M-H-(CH)6-CH2-(CH)2-(CH2)2-CO2-2H, 159 = 221-H2O-CO2, 147 = 191-CO2. These fragmentation ions matched those of MaR2 ((Serhan and Chiang 2023) and original reports within).

During self-resolving E. coli infection in the DHA metabolome, RvD5 was present at time 0, increased at 4 h followed by a sharp decline (Fig. 3a and Table S2). By comparison, PD1 gradually increased and reached maximum at 48 h, while levels of PDx, the double dioxygenation product (Serhan 2007) were highest at 4 h. MaR1 was also present at time 0, and gradually reduced between 4-24 h, then reached the highest levels in the resolution phase at 48 h, giving a time course akin to that of macrophages (cf. Figure 1c). EPA-derived RvE4 appeared in resolution phase at 48 h, while AA-derived LXA4 remained from 12-48 h. Prostaglandin and leukotriene levels were also determined; both PGE2 and LTB4 reached the highest levels at 4 h (Fig. 3a), preceding maximal PMN infiltration at 12 h (Fig. 1a). Together, these results established the exudate LM temporal profiles in self-resolving E. coli peritonitis. These present results are consistent with the lipid mediator class switching (Serhan 2007; Liu et al. 2019). The lipid mediator class switch defines the temporal appearance and relation of pro-inflammatory lipid mediators such as PGE2 and LTB4 and their time-dependent switch in LM classes to the pro-resolving LMs, e.g. LXA4, resolvins, protectins and maresins (Serhan 2007).Fig. 3 Time course of exudate SPMs and eicosanoids. Exudate SPMs and eicosanoids obtained from mice with a E. coli infections and b E. coli infections with ongoing inflammation were each quantified using LC–MS-MS-based metabololipidomics. See Fig. 2 for screen captures of MRM chromatograms of identified SPMs and eicosanoids. Three independent experiments were carried out. In each experiment, 3–4 mouse exudates collected from the same time point were pooled for lipid mediator extraction and LC–MS-MS. Results are expressed as pg/mouse exudate (2 mL); mean ± SEM, each dot denotes the value obtained from one experiment. *P < 0.05 (RvD5), **P < 0.01 (MaR1) vs. 0 h; #P < 0.05 (PDx) vs. 12, 24 and 48 h; +P < 0.05 (PGE2) vs. 0 h, ‡‡‡P < 0.001 (LTB4) vs. 4, 12, 24 and 48 h using one-way ANOVA with Tukey's post-test

We next questioned whether a sterile stimulus zymosan altered these SPM and eicosanoid profiles during E. coli infection. Upon zymosan challenge, select SPMs are rapidly generated, including RvD1, RvD5, MaR1 and PDx (Fig. 3b); RvD5 and MaR1 reached the highest levels at early inflammation phase, i.e. 0-4 h after E. coli inoculation (TE. coli; see Timeline in Fig. 1), and remained in the exudates in the late resolution phase at 48 h (Fig. 3b and Table S3). In comparison, PD1 and LXA4 reached the maximum at TE. coli 12 h then gradually declined. LTB4 levels quickly reached maximum upon zymosan challenge TE. coli 0 h, while PGE2 levels peaked at TE. coli 4 h (Fig. 3b), coinciding with maximal exudate PMNs (Fig. 1e).

PLS-DA was carried out (Fig. 4 and S4) with the identified SPMs in exudates. In E. coli infections with ongoing inflammation, the score plot (Fig. 4c) showed distinct separation of different time points from TE. coli 0-48 h. The loading plot (Fig. 4d) showed correlations where the measured SPMs contributed to the cluster separation in the score plot. For example, in the early time points following sequential challenge, TE. coli 0 h (red) and 4 h (green) clusters were correlated with select SPMs including RvD5 and MaR1 (Fig. 4c, d). The hierarchical clustering Heatmap (Fig. 4e) further indicated clear separation of two SPM clusters: RvD1, RvD5, MaR1, MaR2 and PDx were associated with early time points 0 and 4 h, denoted Cluster I; PD1, LXA4 and RvE4 are associated with later time points 12, 24 and 48 h, denoted Cluster II (Fig. 4e). The Variable Importance in Projection (VIP) scores obtained by PLS-DA showed highest scores for MaR1 and RvD5 in the SPM cluster I (Fig. 4f).Fig. 4 Exudate SPM profiles and clusters on sequential activation. PLS-DA and heatmaps of a, b E. coli infections and (c-f) E. coli infections with ongoing inflammation. a, c, d Partial Least Squares Discriminant Analysis (PLS-DA) of identified SPMs. a, c The score plot (each dot represents profiles from each time point in each experiment) shows clustering among groups (i.e., time points 0, 2, 12, 24 and 48 h), where closer clusters present higher similarity in the data matrix; d the loading plot demonstrates correlations in which the measured SPMs contribute to the cluster separation in the score plot. b, e The hierarchical clustering heatmaps were generated using normalized data with autoscale features. Euclidean distance was used for distance measure and Ward's method was applied in hierarchical cluster analysis (see Methods). Averages of n = 3 for each SPM in each time point are shown. f Variable Importance in Projection (VIP) score plot of exudate SPMs, depicting the relative levels of each mediator across the 5 experimental groups (0, 4, 12, 24 and 48 h). RvD5 and MaR1 give highest VIP scores > 1. The colored boxes on the right indicate the relative concentrations (red: high, blue: low) of the corresponding SPMs in each group

We also carried out PLS-DA with identified exudate SPMs and eicosanoids including LTB4 and PGE2, which also gave distinct separation of different time points from 0-48 h, as shown in the score plot (Fig. S4). With ANOVA, MaR1, MaR2, RvD5, PDx, 17-HDHA, 18-HEPE, and LTB4 were regulated in a temporal manner from 0-48 h that was statistically significant (P < 0.05; Fig. S4a). Hierarchical Clustering Heatmaps (Fig. S4b) also showed two distinct clusters: (Cluster I) PGE2, LTB4, 18-HEPE, 17-HDHA, RvD1, RvD5, MaR1, MaR2 and PDx associated with early time points 0 and 4 h, and (Cluster II) PD1, LXA4 and RvE4 associated with later time points 12, 24 and 48 h. Together, the metabololipidomics results identified select SPM clusters temporally activated when the host was challenged with E. coli infection during ongoing zymosan-induced inflammation.

Very low dose SPMs program the resolution response in vivo

Since zymosan-induced inflammation with subsequent E. coli infections initiated production of a SPM cluster in vivo within the infectious exudates (Fig. 4), we set out to investigate whether repetitive dosing of these SPMs would impact the resolution response. We selected a SPM panel that included RvD1, RvD5 and MaR1 because they were identified in the SPM Cluster I. We also included RvD2 and RvE2 in this panel because RvD2 exhibits potent actions increasing survival as we found earlier in sepsis models (Spite et al. 2009), and RvE2 limits PMN infiltration in sterile inflammation (Serhan and Chiang 2023). SPMs in this panel accelerate resolution of acute inflammation, shortening resolution intervals. For example, in self-resolving E. coli infection reported in Chiang et al. (2012), RvD1 at 50 ng/mouse shortens Ri by ~ 40%. With zymosan peritonitis, earlier results (Arnardottir et al. 2016) showed that RvD2 and MaR1 at 50 ng/mouse each shortened Ri by ~ 75%. In the present experiments, we first carried out zymosan-initiated murine peritonitis where mice were given an SPM panel consisting of RvD1, RvD2, RvD5, MaR1 and RvE2, i.p. at 0.1, 1 or 10 ng each, compared to vehicle control, to emulate the consequence of daily SPM dosing in humans as in Möller et al. (2023); Ramirez et al. 2019). These SPMs were authenticated prior to experiments (Fig. S5); physical properties of each synthetic SPM were examined and compared to published criteria for authentication (Chiang et al. 2012; Serhan and Petasis 2011; Serhan and Chiang 2023; Spite et al. 2009). This SPM panel at dose of 0.1 ng each reduced exudate PMN ~ 25% at 12 h, albeit did not reach statistical significance (Fig. S6). At 1 or 10 ng each, this SPM panel reduced ~ 45 and ~ 65% exudate PMN, respectively (P < 0.05; Fig. S6).

Next, we carried out consecutive treatments with this SPM panel at subthreshold doses (RvD1, RvD2, RvD5, MaR1 and RvE2, 0.1 ng each) for 5 times from days 0–12. On day 12, the subthreshold SPM panel or vehicle control was given together with zymosan to initiate peritonitis. See the timelines of single (1X) and repetitive (6X) SPM dosings in Fig. S7a. At 12–48 h, peritoneal exudates were collected, and cellular composition determined by flow cytometry (see gating strategy and leukocyte compositions in Fig. 5a and Table S4). At 12 and 24 h, a single dose of subthreshold SPMs did not reduce exudate PMN in a statistically significant manner (Fig. 5b). Repetitive SPMs reduced exudate PMN by ~ 45% (P < 0.01) at 12 h, and ~ 60% (P < 0.05) at 24 h (Fig. 5c). Resolution indices were calculated. At 12 h, ψmax was ~ 9.6 × 106 PMN, that reduced to 50% at ~ 28 h, giving a resolution interval Ri ~ 16 h. With repetitive subthreshold SPMs, Ri markedly reduced 75% to only ~ 4 h. The repetitive SPMs did not significantly alter percentages of exudate macrophages (Table S4). These results indicated that low-dose SPM repetitive dosing regimen accelerated resolution of acute inflammation, as evidenced by shortening the resolution interval (Fig. 5c).Fig. 5 SPM programming in vivo accelerates resolution of inflammation, shortening resolution intervals. Mice were administered with a panel of SPMs (RvD1, RvD2, RvD5, MaR1 and RvE2, 0.1 ng of each SPM in 1 mL saline for each mouse, i.p.) or vehicle control (0.01% ethanol in 1 mL saline) 5 times on Day 0, 2, 5, 7, and 9. On day 12, the panel of SPMs or vehicle was given together with zymosan (1 mg/mouse, i.p.), and exudates collected at indicated time points (see Timelines in Fig. S7a). Total leukocytes were enumerated, and leukocyte composition determined by flow cytometry. a Exudate leukocyte composition identified using flow cytometry. Representative dot plots of exudate samples collected at 12 h with gating strategy to identify PMN, monocytes and macrophages. (Top) Zymosan (Bottom) Zymosan with repetitive subthreshold SPMs. b Zymosan vs zymosan plus one-time (1X) subthreshold SPMs at 12 and 24 h. c Zymosan (red curve) vs. repetitive (6X) subthreshold SPMs (blue curve); time course 12-48 h and resolution indices (ψmax, ψ50 and Ri, see Methods); mean ± SEM, n = 4–6 (12 h) or 3 (24 or 48 h), *P < 0.05, **P < 0.01 using two-tailed Student’s t-test. Each dot denotes cell number obtained from one mouse

RNA-sequencing was carried out with inflammatory exudates collected at 12 h to determine whether select genes and pathways are regulated by the SPM dosing regimen prior to zymosan exposure in vivo. A total of 14,440 transcripts were obtained from inflammatory exudates. There were 57 known transcripts up-regulated (P < 0.05) by repetitive SPM dosing with a fold change (FC) > 2 (log2FC > 1), listed in Fig. S8a. Among them, il-11 (230% increase) is known to have anti-inflammatory properties (Yu et al. 2016); metalloproteinase (mmp)-2 (130% increase) and mmp-3 (180% increase), known to have protective roles in chemokine cleavage (Westermann et al. 2011). The transcripts for SPM receptors (reviewed in Serhan and Chiang (2023)) were present including Fpr2 (a LXA4 and RvD1 receptor), gpr18 (a RvD2 receptor) and Cmklr1 (a RvE1 and RvE2 receptor) (see Table S5). The gpr18 transcript was increased by SPM repetitive dosing by ~ 28% (Log2FC 0.35), albeit they did not reach statistical significance (Table S5). The transcripts for SPM biosynthesis enzymes are also present but did not appear to be regulated in these exudates (Table S5); these include Alox15 (12/15-lipoxygenase), Alox5 (5-lipoxygenase), Ptgs2 (cyclooxygenase) and Lta4h (LTA4 hydrolase) (Haeggström and Newcomer 2023). SPM dosing down-regulated select transcripts; 46 known transcripts were down-regulated (P < 0.05) with log2FC < -0.6 (Fig. S8b).

Gene ontology analysis demonstrated > 300 pathways that were regulated by SPM repetitive dosing (adjusted P values < 0.05). The top 34 pathways related to immune functions are shown in Fig. 6a; some of them are known to have a critical role in the resolution of inflammation and return to homeostasis. Table S6 lists the genes in each significantly regulated GO terms; these include negative regulation of vasoconstriction, cytokine secretion and cell adhesion; positive regulation of innate immune response, cell division, fibroblast and epithelial cell proliferation, and monocyte chemotaxis (Fig. 6a). We next employed Inflammation-resolution network analysis using “Atlas of Inflammation Resolution (AIR)” (https://air.bio.informatik.uni-rostock.de). Inflammation-resolution processes and phenotypes were grouped into 4 phases (inflammation initiation, transition, resolution, and homeostasis), shown in Fig. 6b. SPMs up-regulated select phenotypes in each phase, highlighted in red. Also see Supplementary Figure S9 for SPM-regulated genes in each “phenotype” in the “Atlas of Inflammation Resolution (AIR)”, and a central regulatory network (CRN) representing the molecular interaction associated with the selected phenotype element (e.g., neutrophil, monocyte and fibroblast response). Together, these results using both GO pathway and AIR network analyses indicated that subthreshold SPM dosing in vivo upregulated specific pathways involved in the resolution of inflammation, tissue repair and homeostasis.Fig. 6 SPMs regulate transcriptome and immune pathways towards resolution and homeostasis. a RNA-seq Gene ontology (GO) enrichment analysis of DEGs that are significantly regulated by SPMs, and related to immune functions. X-axis represents enrichment effect (see Methods for the equation) and Y-axis represents different GO terms. The adjusted p-value (Padj-value) are shown on the left for the corresponding GO term when comparing zymosan plus subthreshold SPMs vs. zymosan alone (n = 4), Padj- < 0.05. Also see Supplementary Table S6 for the genes in each GO term. b Inflammation-resolution network analysis using “Atlas of Inflammation Resolution (AIR)” (Serhan et al. 2020; Hoch et al. 2022) (https://air.bio.informatik.uni-rostock.de). Inflammation-resolution processes and phenotypes were grouped into 4 phases (inflammation initiation, transition, resolution, and homeostasis). Repetitive subthreshold SPMs up-regulated select phenotypes in each phase, highlighted in red. See Fig. S9a for a clear view of all processes and phenotypes, genes in each phenotype in “Atlas of Inflammation Resolution”, and Fig. S9b for central regulatory network (CRN) for some of these pathways

To test this principle with human tissue, human monocyte-derived macrophages were incubated with RvD1, RvD2, MaR1 or RvE1 at 10 nM each separately, and surface expression of SPM receptors examined using flow cytometry. These include RvD2 receptor DRV2, MaR1 receptor LGR6 and RvE1 receptor ERV1 (reviewed in Serhan and Chiang (2023)). As shown in Table 1, RvD2 and MaR1 at 24 h each up-regulated (~ 25%, P < 0.01) surface expression of the RvD2 receptor, i.e., DRV2/GPR18. RvD2 also increased surface expression of the MaR1 receptor LGR6 (P < 0.05; Table 1). Under the same conditions, surface expression of ERV1/ChemR23, the receptor for RvE1, was not altered by these SPMs in a statistically significant manner. These findings suggest that the exposure to pro-resolving mediators selectively up-regulates SPM receptors, as a potential feed forward resolution response to accelerate the return to homeostasis.Table 1 Regulation of SPM receptors with human monocyte-derived macrophages

Upregulation of receptor	RvD1	RvD2	MaR1	RvE2	
DRV2/GPR18	RvD2	19.8 ± 7.2% (4)	**26.5 ± 3.4% (6)	**24.8 ± 4.9% (6)	20.5 ± 11.0% (6)	
LGR6	MaR1	2.8 ± 3.5% (5)	*11.2 ± 3.8% (5)	11.2 ± 7.5% (5)	2.2 ± 4.0% (5)	
ERV1/ChemR23	RvE1 & E2	4.4 ± 1.7% (4)	0.0 ± 1.8% (4)	2.9 ± 4.4% (4)	0.0 ± 3.4% (4)	
Human macrophages were incubated with 10 nM of RvD1 RvD2, MaR1, RvE2 or vehicle (0.1% ethanol) for 24 h. Cells were collected, and surface expression of GPR18, LGR6 and ChemR23 was determined using flow cytometry. See Methods for the specific antibodies used for these receptors. Results are percent increase of receptor surface expression above vehicle control; mean ± SEM from 4–6 separate donors, and N numbers are denoted within the brackets. *P < 0.05, **P < 0.01 compared to vehicle control using two-tailed Student’s t-test

Discussion

In the present manuscript, we report that E. coli infections encountered during ongoing inflammation reset the temporal resolution response, and that low-dose SPM repetitive regimen re-programs and up-regulates the endogenous resolution mechanisms. Our challenge protocol (Fig. 1e) enabled us to identify two main SPM clusters that were temporally programmed (Figs. 1, 2, 3 and 4). These proved to be RvD1, RvD5, MaR1, MaR2, PDx in Cluster I, and PD1, LXA4, RvE4 in Cluster II, uncovered using LC–MS–MS-based identification (Shay et al. 2021) with Partial Least Squares Discriminant Analysis (PLS-DA) in Fig. 4.

The production of SPMs such as D-series resolvins and protectins is a highly conserved system in evolution (Hong et al. 2007) supported by the identification and biosynthesis of RvD1, RvD5 and neuroprotectin D1 in brain cells (Hong et al. 2005) and head kidney gland of the rainbow trout (Oncorhynchus mykiss) (Hong et al. 2007). Recently, the head kidney gland cells of the Atlantic Salmon (Salmon salar), which is the hematopoietic organ, were found to biosynthesize nanogram amounts of the D-series resolvins (Araujo et al. 2023). These findings suggest that the conserved SPM molecules are essential to the host defense system, and thus can be important to consider in human resilience (Taylor et al. 2022). Norris et al. (2018) found that supplementation of healthy adults with ω-3 DHA and EPA on LPS challenge gives a plasma SPM cluster that consists of RvE1, RvD1, AT-LXA4, LXB4, 18-HEPE and 17-HDHA. Coagulation of human blood ex vivo initiates temporal production of a SPM cluster that consists of RvD1, RvD5, MaR1, RvE1 and LXB4 (Norris et al. 2017). These pro-resolving lipid mediator clusters from earlier human studies (Norris et al. 2018, 2017) share RvD1, RvE1 and LXB4 pro-resolving mediators. Results in Fig. 4 of the present study identified RvD1, RvD5, MaR1, MaR2 and PDx (Cluster I) production in mice with ongoing inflammation challenged with E. coli. Repetitive low dose (0.1 ng each per mouse) of RvD1, RvD2, RvD5, MaR1 and RvE2 reset the resolution response in vivo.

In humans, SPMs are present in breast milk (Arnardottir et al. 2016), cerebrospinal fluid (Wang et al. 2015), and peripheral blood (Barden et al. 2024; Lau et al. 2023). SPMs increase with omega-3 supplementation (Mas et al. 2012). SPM-containing supplements are increasingly appearing, e.g. SPM-enriched marine oil capsules are commercially available (Möller et al. 2023; Souza et al. 2020) and in daily use, as is the EPA derivative Icosapent Ethyl (Verma et al. 2021) for specific clinical indications. This raises the possibility that daily SPM might down-regulate the endogenous resolution response. On the contrary, here in the present experiments, we found that low-dose SPM in repetitive administration accelerated resolution in this experimental setting. The low-dose SPMs used herein (0.1 ng each) were within the ranges of their quantities present in resolving exudates (Fig. 3 and Table S3). For example, RvD5 range was 64–173 pg and MaR1 214–281 pg per exudate at 0–4 h following sequential challenges, thus in the range of the 100 pg of each SPM per mouse used in these add-back experiments. These low-dose SPMs regulated genes and pathways associated with inflammation and tissue repair (Fig. 6 and S8 and 9). Select SPMs (RvD2 and MaR1) in this panel also significantly regulated SPM receptors present on human monocyte-derived macrophages (Table 1), demonstrating a potential physiologic feed-forward mechanism in resolution.

Along these lines, treatment with a pro-resolving stable mimetic proved to be both safe and effective in oral inflammation (Hasturk et al. 2021). Topical application of the SPM mimetic increased endogenous SPM production documented in vivo in human peripheral blood (Hasturk et al. 2021). Human macrophages are central to resolution and undergo lipid mediator class switch, namely the temporal change in LM classes from the initiation to resolution phase of the acute inflammatory response defined in Serhan (2007) with appropriate stimuli (Liu et al. 2019). EPA and DHA are converted by M2-like macrophages that are a major tissue resource of SPMs (Recchiuti et al. 2014). These SPMs stimulate resolution of inflammation in vivo in experimental animal disease models ((Leroy et al. 2023), reviewed in Serhan and Chiang (2023)). Resolvins also display anti-depressant actions in the brain that are receptor-mediated (Deyama et al. 2018) and evoke brain neurochemical changes in serotonin, dopamine and glutamate (Klein et al. 2014). The nervous system is wired to regulate inflammation via the inflammatory reflex (Diamond and Tracey 2011) that activates resolution mechanisms by stimulating production of resolvins (Mirakaj et al. 2014).

Our present results demonstrate that the resolution response is preemptively activated by both low-dose and repetitive SPM treatment of the innate immune system. Resolvin analogs have been prepared (Murakami et al. 2020) that are highly potent, showing femtomolar activities, which is very promising for therapeutic development (Serhan and Chiang 2023). The activation of SPM receptors stimulates an entire signaling network of anti-inflammation that leads to complete resolution (Suchitha et al. 2024) and activates mesenchymal stem cells (AlZahrani et al. 2022). Our results from RNA-seq with SPMs confirmed and further identified molecules and mechanisms regulated by SPMs. Results in Fig. S8a demonstrated that il11, mmp2/3 and cx3cl1/fractalkine are up-regulated by SPMs (Log2FC > 1, P < 0.05, Fig S8a). IL-11 is protective in ischemia–reperfusion injury (Yu et al. 2016), colitis (Nishina et al. 2023), and accelerates wound healing (Singh et al. 2023). MMP-2 reduces inflammation via cleaving chemokine such as MCP-3 (Westermann et al. 2011). The fractalkine and its receptor CX3CR1 are protective in liver fibrosis (Karlmark et al. 2010). Along these lines, RvD2 in murine model of sepsis increases MMP-2 and MMP-3 in infectious exudates ((Serhan and Chiang 2023) and references within). With human macrophages, select cysteinyl-conjugated SPMs (cys-SPMs), i.e., Maresin conjugate in tissue regeneration 3 (MCTR3) and Resolvin conjugate in tissue regeneration-3 (RCTR3), increase IL-11 in ((Serhan and Chiang 2023) and references within). Of note, in the present experiments, the low-dose SPMs up-regulated Kif21a (Kinesin Family Member 21A; Log2FC 1.17, Fig. S8a) and Lyst (Log2FC 0.45). These genes are also up-regulated by cys-SPMs, MCTR3 and RCTR3 during planaria regeneration uncovered using RNA-seq (Serhan and Chiang 2023 and references within). Lyst (Lysosomal Trafficking Regulator) is a paralog of WDFY3, which is a regulator of macrophage efferocytosis (Shi et al. 2022). The transcription factor spic was down-regulated by SPMs (Fig. S8b); spic (Spi-C Transcription Factor) is up-reregulated in Secondary hemophagocytic lymphohistiocytosis, a hyperinflammatory state in humans (Wang et al. 2019). Thus, these effector functions uncovered in the present experiments (Fig. S8) are in line with the SPM pro-resolving mechanisms (Serhan and Chiang 2023; Serhan 2014). Recently, a resolving neutrophil subset was uncovered that produces RvD1 (Geng et al. 2024), that now joins the cellular and molecular mechanisms in the resolution response (Serhan 2007; Fredman and Serhan 2024; Schwab et al. 2007). Both E. coli and zymosan activate toll-like receptors (TLR)-2 and TLR4, and their intracellular signaling is blocked by resolvins (Croasdell et al. 2016). Trained immunity and tolerance are orchestrated in the innate immune system to a large extent by TLRs (Divangahi et al. 2021). TLR agonism in sequential infection challenges yield hyperinflammation state (Wang et al. 2019). Our present results demonstrated that (A) the TLR sequential agonism can reset the resolution programs, and (B) the resolution response is trained by low dose repetitive SPM treatment.

Conclusions

MaR1 (Jiang et al. 2023), RvD1 (Ferri et al. 2023) and RvD2 (Zhang et al. 2016; Hellmann et al. 2018) have proven to play critical functions controlling inflammation in vivo in animal models since their elucidation originally reported in 2002 and 2009 (reviewed in Serhan and Chiang (2023)) and references within). The potent actions of RvD2 (Spite et al. 2009) are promising in controlling infectious inflammation that occurs in sepsis and tissue injury (Sundarasivarao et al. 2022; Padovani and Yin 2024). These earlier findings and results of the present study provide evidence of positive feed-forward mechanisms in the resolution response evoked by SPM repetitive low-dose therapy that has implications for their long-term use and clinical development. In the present experiments, we used a protocol of infection with underlying inflammation with the goal of identifying which of the superfamily of SPMs are produced and essential to stimulate resolution of infectious inflammation. Importantly, the panel of SPMs that include RvD1, RvD2, RvD5, MaR1 and RvE2 reset the temporal cellular and molecular events in the resolution response. Given in low doses, these SPMs reprogrammed the start sequence of the resolution response code. Together, these findings in mice have direct implications for preventative medicine and resolution pharmacology (Serhan 2014). These may be useful in preparing for the next pandemic (Huang et al. 2024; Morse et al. 2012), particularly in individuals with ongoing inflammation as in arthritis, cardiovascular and neurodegenerative diseases that are co-morbidities in viral pandemics such as COVID-19 by raising resilience, via trained SPM resolution response. These studies warrant further investigation and translation to humans to assess the SPM trained resolution response and nutritional maintenance.

Supplementary Information

Supplementary Material 1.

Abbreviations

AA Arachidonic acid

AIR Atlas of inflammation resolution

CRN Central regulatory network

DEG Differentially expressed genes

DHA Docosahexaenoic acid

EPA Eicosapentaenoic acid

LC–MS/MS Liquid chromatography tandem mass spectrometry

EPI Enhanced product ion

ESI Electrospray ionization

LM Lipid mediators

LT Leukotriene

LX Lipoxin

MΦ Macrophage

MaR Maresin

MMP Metalloproteinase

MRM Multiple reaction monitoring

PD Protectin

PLS-DA Partial least squares-discriminant analysis

PG Prostaglandin

PMN Polymorphonuclear neutrophil

RI Resolution indices

ψmax Maximal exudate PMN numbers

Tmax The time point when PMN numbers reach ψmax

R50 50% Of ψmax

T50 The time point when the PMN numbers reached R50

Ri (resolution interval) The time interval from the maximal PMN point ψmax to R50, i.e., T50−Tmax

Rv Resolvins

SPMs Specialized pro-resolving mediators

TR Retention time

Acknowledgements

The authors thank Mary H. Small for expert assistance in manuscript preparations. The authors wish to acknowledge Dr. Melissa Simard (Research Fellow, Harvard Medical School) for the help with LC-MS-MS analysis, and Holly N Batchelder (Center for Experimental Therapeutics and Reperfusion Injury, Mass General Brigham) for help with authentication of synthetic SPMs.

Author contributions

RN carried out LC-MS-MS analyses and MS-MS interpretation; NC carried out experiments; CNS and NC designed experiments, analyzed results and wrote the manuscript. All authors contributed to writing and figure preparation, read and approved the final manuscript.

Funding

Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R35GM139430 (C.N.S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Availability of data and materials

RNA-seq and LC–MS-MS datasets generated and analyzed in the current study will be available on publication in the repositories: Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) GEO accession no.GSE270972 and BioStudies (https://www.ebi.ac.uk/biostudies/) accession S-BSST1467, respectively.

Declarations

Ethics approval and consent to participate

Human peripheral blood mononuclear cells were obtained from the Boston Children's Hospital Blood Bank under the approved Mass General Brigham investigational review board protocol #1999-P-001279. Animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Brigham and Women's Hospital (protocol no. 2016N000145).

Consent for publication

Not Applicable.

Competing interests

The authors declared that there are no competing interests.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
==== Refs
References

AlZahrani S Shinwari Z Gaafar A Alaiya A Al-Kahtani A Anti-inflammatory effect of specialized proresolving lipid mediators on mesenchymal stem cells: an in vitro study Cells 2022 12 122 10.3390/cells12010122 36611915
AlZahrani S, Shinwari Z, Gaafar A, Alaiya A, Al-Kahtani A. Anti-inflammatory effect of specialized proresolving lipid mediators on mesenchymal stem cells: an in vitro study. Cells. 2022;12:122.36611915
Araujo P Iqbal S Arnø A Espe M Holen E Validation of a liquid-liquid extraction method to study the temporal production of D-Series resolvins by head kidney cells from atlantic salmon (Salmon salar) exposed to docosahexaenoic acid Molecules 2023 28 4728 10.3390/molecules28124728 37375283
Araujo P, Iqbal S, Arnø A, Espe M, Holen E. Validation of a liquid-liquid extraction method to study the temporal production of D-Series resolvins by head kidney cells from atlantic salmon (Salmon salar) exposed to docosahexaenoic acid. Molecules. 2023;28:4728.37375283
Arnardottir H Orr SK Dalli J Serhan CN Human milk proresolving mediators stimulate resolution of acute inflammation Mucosal Immunol 2016 9 757 766 10.1038/mi.2015.99 26462421
Arnardottir H, Orr SK, Dalli J, Serhan CN. Human milk proresolving mediators stimulate resolution of acute inflammation. Mucosal Immunol. 2016;9:757–66.26462421
Barden A Shinde S Beilin LJ Phillips M Adams L Bollmann S Adiposity associates with lower plasma resolvin E1 (Rve1): a population study Int J Obes (lond) 2024 48 725 732 10.1038/s41366-024-01482-x 38347128
Barden A, Shinde S, Beilin LJ, Phillips M, Adams L, Bollmann S, et al. Adiposity associates with lower plasma resolvin E1 (Rve1): a population study. Int J Obes (lond). 2024;48:725–32.38347128
Bardin M Pawelzik SC Lagrange J Mahdi A Arnardottir H Regnault V The resolvin D2 - GPR18 axis is expressed in human coronary atherosclerosis and transduces atheroprotection in apolipoprotein E deficient mice Biochem Pharmacol 2022 201 115075 10.1016/j.bcp.2022.115075 35525326
Bardin M, Pawelzik SC, Lagrange J, Mahdi A, Arnardottir H, Regnault V, et al. The resolvin D2 - GPR18 axis is expressed in human coronary atherosclerosis and transduces atheroprotection in apolipoprotein E deficient mice. Biochem Pharmacol. 2022;201: 115075.35525326
Bhattacharya P Dhawan UK Hussain MT Singh P Bhagat KK Singhal A Efferocytes release extracellular vesicles to resolve inflammation and tissue injury via prosaposin-GPR37 signaling Cell Rep 2023 42 112808 10.1016/j.celrep.2023.112808 37436891
Bhattacharya P, Dhawan UK, Hussain MT, Singh P, Bhagat KK, Singhal A, et al. Efferocytes release extracellular vesicles to resolve inflammation and tissue injury via prosaposin-GPR37 signaling. Cell Rep. 2023;42: 112808.37436891
Blaauw R Calder PC Martindale RG Berger MM Combining proteins with n-3 PUFAs (EPA + DHA) and their inflammation pro-resolution mediators for preservation of skeletal muscle mass Crit Care 2024 28 38 10.1186/s13054-024-04803-8 38302945
Blaauw R, Calder PC, Martindale RG, Berger MM. Combining proteins with n-3 PUFAs (EPA + DHA) and their inflammation pro-resolution mediators for preservation of skeletal muscle mass. Crit Care. 2024;28:38.38302945
Chiang N Fredman G Bäckhed F Oh SF Vickery TW Schmidt BA Infection regulates pro-resolving mediators that lower antibiotic requirements Nature 2012 484 524 528 10.1038/nature11042 22538616
Chiang N, Fredman G, Bäckhed F, Oh SF, Vickery TW, Schmidt BA, et al. Infection regulates pro-resolving mediators that lower antibiotic requirements. Nature. 2012;484:524–8.22538616
Clish CB Levy BD Chiang N Tai H-H Serhan CN Oxidoreductases in lipoxin A4 metabolic inactivation J Biol Chem 2000 275 25372 25380 10.1074/jbc.M002863200 10837478
Clish CB, Levy BD, Chiang N, Tai H-H, Serhan CN. Oxidoreductases in lipoxin A4 metabolic inactivation. J Biol Chem. 2000;275:25372–80.10837478
Croasdell A Sime PJ Phipps RP Resolvin D2 decreases TLR4 expression to mediate resolution in human monocytes FASEB J 2016 30 3181 3193 10.1096/fj.201600375R 27256622
Croasdell A, Sime PJ, Phipps RP. Resolvin D2 decreases TLR4 expression to mediate resolution in human monocytes. FASEB J. 2016;30:3181–93.27256622
Deyama S Shimoda K Suzuki H Ishikawa Y Ishimura K Fukuda H Resolvin E1/E2 ameliorate lipopolysaccharide-induced depression-like behaviors via ChemR23 Psychopharmacology 2018 235 329 336 10.1007/s00213-017-4774-7 29090333
Deyama S, Shimoda K, Suzuki H, Ishikawa Y, Ishimura K, Fukuda H, et al. Resolvin E1/E2 ameliorate lipopolysaccharide-induced depression-like behaviors via ChemR23. Psychopharmacology. 2018;235:329–36.29090333
Diamond B Tracey KJ Mapping the immunological homunculus Proc Natl Acad Sci U S A 2011 108 3461 3462 10.1073/pnas.1100329108 21368214
Diamond B, Tracey KJ. Mapping the immunological homunculus. Proc Natl Acad Sci U S A. 2011;108:3461–2.21368214
Divangahi M Aaby P Khader SA Barreiro LB Bekkering S Chavakis T Trained immunity, tolerance, priming and differentiation: distinct immunological processes Nat Immunol 2021 22 2 6 10.1038/s41590-020-00845-6 33293712
Divangahi M, Aaby P, Khader SA, Barreiro LB, Bekkering S, Chavakis T, et al. Trained immunity, tolerance, priming and differentiation: distinct immunological processes. Nat Immunol. 2021;22:2–6.33293712
Dort J Orfi Z Fabre P Molina T Conte TC Greffard K Resolvin-D2 targets myogenic cells and improves muscle regeneration in Duchenne muscular dystrophy Nat Commun 2021 12 6264 10.1038/s41467-021-26516-0 34716330
Dort J, Orfi Z, Fabre P, Molina T, Conte TC, Greffard K, et al. Resolvin-D2 targets myogenic cells and improves muscle regeneration in Duchenne muscular dystrophy. Nat Commun. 2021;12:6264.34716330
Endo J Sano M Isobe Y Fukuda K Kang JX Arai H 18-HEPE, an n-3 fatty acid metabolite released by macrophages, prevents pressure overload-induced maladaptive cardiac remodeling J Exp Med 2014 211 1673 1687 10.1084/jem.20132011 25049337
Endo J, Sano M, Isobe Y, Fukuda K, Kang JX, Arai H, et al. 18-HEPE, an n-3 fatty acid metabolite released by macrophages, prevents pressure overload-induced maladaptive cardiac remodeling. J Exp Med. 2014;211:1673–87.25049337
Ferri G Serano M Isopi E Mucci M Mattoscio D Pecce R Resolvin D1 improves airway inflammation and exercise capacity in cystic fibrosis lung disease FASEB J 2023 37 e23233 10.1096/fj.202301495R 37823221
Ferri G, Serano M, Isopi E, Mucci M, Mattoscio D, Pecce R, et al. Resolvin D1 improves airway inflammation and exercise capacity in cystic fibrosis lung disease. FASEB J. 2023;37: e23233.37823221
Fitzgerald H Bonin JL Khan S Eid M Sadhu S Rahtes A Resolvin D2-G-Protein Coupled Receptor 18 Enhances Bone Marrow Function and Limits Steatosis and Hepatic Collagen Accumulation in Aging Am J Pathol 2023 193 1953 1968 10.1016/j.ajpath.2023.08.011 37717941
Fitzgerald H, Bonin JL, Khan S, Eid M, Sadhu S, Rahtes A, et al. Resolvin D2-G-Protein Coupled Receptor 18 Enhances Bone Marrow Function and Limits Steatosis and Hepatic Collagen Accumulation in Aging. Am J Pathol. 2023;193:1953–68.37717941
Fredman G Serhan CN Specialized pro-resolving mediators in vascular inflammation and atherosclerotic cardiovascular disease Nat Rev Cardiol 2024 10.1038/s41569-023-00984-x 38216693
Fredman G, Serhan CN. Specialized pro-resolving mediators in vascular inflammation and atherosclerotic cardiovascular disease. Nat Rev Cardiol. 2024. 10.1038/s41569-023-00984-x.38216693
Geng S Zhang Y Lu R Irimia D Li L Resolving neutrophils through genetic deletion of TRAM attenuate atherosclerosis pathogenesis Iscience 2024 27 110097 10.1016/j.isci.2024.110097 38883832
Geng S, Zhang Y, Lu R, Irimia D, Li L. Resolving neutrophils through genetic deletion of TRAM attenuate atherosclerosis pathogenesis. Iscience. 2024;27:110097.38883832
Haeggström JZ Newcomer ME Structures of leukotriene biosynthetic enzymes and development of new therapeutics Annu Rev Pharmacol Toxicol 2023 63 407 428 10.1146/annurev-pharmtox-051921-085014 36130059
Haeggström JZ, Newcomer ME. Structures of leukotriene biosynthetic enzymes and development of new therapeutics. Annu Rev Pharmacol Toxicol. 2023;63:407–28.36130059
Hartling I Cremonesi A Osuna E Lou PH Lucchinetti E Zaugg M Quantitative profiling of inflammatory and pro-resolving lipid mediators in human adolescents and mouse plasma using UHPLC-MS/MS Clin Chem Lab Med 2021 59 1811 1823 10.1515/cclm-2021-0644 34243224
Hartling I, Cremonesi A, Osuna E, Lou PH, Lucchinetti E, Zaugg M, et al. Quantitative profiling of inflammatory and pro-resolving lipid mediators in human adolescents and mouse plasma using UHPLC-MS/MS. Clin Chem Lab Med. 2021;59:1811–23.34243224
Hasturk H Schulte F Martins M Sherzai H Floros C Cugini M Safety and preliminary efficacy of a novel host-modulatory therapy for reducing gingival inflammation Front Immunol 2021 12 704163 10.3389/fimmu.2021.704163 34589083
Hasturk H, Schulte F, Martins M, Sherzai H, Floros C, Cugini M, et al. Safety and preliminary efficacy of a novel host-modulatory therapy for reducing gingival inflammation. Front Immunol. 2021;12: 704163.34589083
Hellmann J Sansbury BE Wong B Li X Singh M Nuutila K Biosynthesis of D-series resolvins in skin provides insights into their role in tissue repair J Invest Dermatol 2018 138 2051 2060 10.1016/j.jid.2018.03.1498 29559341
Hellmann J, Sansbury BE, Wong B, Li X, Singh M, Nuutila K, et al. Biosynthesis of D-series resolvins in skin provides insights into their role in tissue repair. J Invest Dermatol. 2018;138:2051–60.29559341
Hoch M Smita S Cesnulevicius K Lescheid D Schultz M Wolkenhauer O Network- and enrichment-based inference of phenotypes and targets from large-scale disease maps NPJ Syst Biol Appl 2022 8 13 10.1038/s41540-022-00222-z 35473910
Hoch M, Smita S, Cesnulevicius K, Lescheid D, Schultz M, Wolkenhauer O, et al. Network- and enrichment-based inference of phenotypes and targets from large-scale disease maps. NPJ Syst Biol Appl. 2022;8:13.35473910
Hong S Tjonahen E Morgan EL Yu L Serhan CN Rowley AF Rainbow trout (Oncorhynchus mykiss) brain cells biosynthesize novel docosahexaenoic acid-derived resolvins and protectins—mediator lipidomic analysis Prostaglandins Other Lipid Mediat 2005 78 107 116 10.1016/j.prostaglandins.2005.04.004 16303609
Hong S, Tjonahen E, Morgan EL, Yu L, Serhan CN, Rowley AF. Rainbow trout (Oncorhynchus mykiss) brain cells biosynthesize novel docosahexaenoic acid-derived resolvins and protectins—mediator lipidomic analysis. Prostaglandins Other Lipid Mediat. 2005;78:107–16.16303609
Hong S Lu Y Yang R Gotlinger KH Petasis NA Serhan CN Resolvin D1, protectin D1, and related docosahexaenoic acid-derived products: analysis via electrospray/low energy tandem mass spectrometry based on spectra and fragmentation mechanisms J Am Soc Mass Spectrom 2007 18 128 144 10.1016/j.jasms.2006.09.002 17055291
Hong S, Lu Y, Yang R, Gotlinger KH, Petasis NA, Serhan CN. Resolvin D1, protectin D1, and related docosahexaenoic acid-derived products: analysis via electrospray/low energy tandem mass spectrometry based on spectra and fragmentation mechanisms. J Am Soc Mass Spectrom. 2007;18:128–44.17055291
Hopke A Lin T Scherer AK Shay AE Timmer KD Wilson-Mifsud B Transcellular biosynthesis of leukotriene B(4) orchestrates neutrophil swarming to fungi Iscience 2022 25 105226 10.1016/j.isci.2022.105226 36267914
Hopke A, Lin T, Scherer AK, Shay AE, Timmer KD, Wilson-Mifsud B, et al. Transcellular biosynthesis of leukotriene B(4) orchestrates neutrophil swarming to fungi. Iscience. 2022;25:105226.36267914
Huang Z Cheng Z Deng X Yang Y Sun N Hou P Integrated Bioinformatics Exploration and Preliminary Clinical Verification for the Identification of Crucial Biomarkers in Severe Cases of COVID-19 J Inflamm Res 2024 17 1561 1576 10.2147/JIR.S454284 38495341
Huang Z, Cheng Z, Deng X, Yang Y, Sun N, Hou P, et al. Integrated Bioinformatics Exploration and Preliminary Clinical Verification for the Identification of Crucial Biomarkers in Severe Cases of COVID-19. J Inflamm Res. 2024;17:1561–76.38495341
Jiang H Song D Zhou X Chen F Yu Q Ren L Maresin1 ameliorates MSU crystal-induced inflammation by upregulating Prdx5 expression Mol Med 2023 29 158 10.1186/s10020-023-00756-w 37996809
Jiang H, Song D, Zhou X, Chen F, Yu Q, Ren L, et al. Maresin1 ameliorates MSU crystal-induced inflammation by upregulating Prdx5 expression. Mol Med. 2023;29:158.37996809
Karlmark KR Zimmermann HW Roderburg C Gassler N Wasmuth HE Luedde T The fractalkine receptor CX₃CR1 protects against liver fibrosis by controlling differentiation and survival of infiltrating hepatic monocytes Hepatology 2010 52 1769 1782 10.1002/hep.23894 21038415
Karlmark KR, Zimmermann HW, Roderburg C, Gassler N, Wasmuth HE, Luedde T, et al. The fractalkine receptor CX₃CR1 protects against liver fibrosis by controlling differentiation and survival of infiltrating hepatic monocytes. Hepatology. 2010;52:1769–82.21038415
Klein CP Sperotto ND Maciel IS Leite CE Souza AH Campos MM Effects of D-series resolvins on behavioral and neurochemical changes in a fibromyalgia-like model in mice Neuropharmacology 2014 86C 57 66 10.1016/j.neuropharm.2014.05.043
Klein CP, Sperotto ND, Maciel IS, Leite CE, Souza AH, Campos MM. Effects of D-series resolvins on behavioral and neurochemical changes in a fibromyalgia-like model in mice. Neuropharmacology. 2014;86C:57–66.
Korner A Schlegel M Theurer J Frohnmeyer H Adolph M Heijink M Resolution of inflammation and sepsis survival are improved by dietary Omega-3 fatty acids Cell Death Differ 2018 25 421 431 10.1038/cdd.2017.177 29053142
Korner A, Schlegel M, Theurer J, Frohnmeyer H, Adolph M, Heijink M, et al. Resolution of inflammation and sepsis survival are improved by dietary Omega-3 fatty acids. Cell Death Differ. 2018;25:421–31.29053142
Korner A Zhou E Muller C Mohammed Y Herceg S Bracher F Inhibition of Delta24-dehydrocholesterol reductase activates pro-resolving lipid mediator biosynthesis and inflammation resolution Proc Natl Acad Sci U S A 2019 116 20623 20634 10.1073/pnas.1911992116 31548397
Korner A, Zhou E, Muller C, Mohammed Y, Herceg S, Bracher F, et al. Inhibition of Delta24-dehydrocholesterol reductase activates pro-resolving lipid mediator biosynthesis and inflammation resolution. Proc Natl Acad Sci U S A. 2019;116:20623–34.31548397
Lammermann T Afonso PV Angermann BR Wang JM Kastenmuller W Parent CA Neutrophil swarms require LTB4 and integrins at sites of cell death in vivo Nature 2013 498 371 375 10.1038/nature12175 23708969
Lammermann T, Afonso PV, Angermann BR, Wang JM, Kastenmuller W, Parent CA, et al. Neutrophil swarms require LTB4 and integrins at sites of cell death in vivo. Nature. 2013;498:371–5.23708969
Lau ES Roshandelpoor A Zarbafian S Wang D Guseh JS Allen N Eicosanoid and eicosanoid-related inflammatory mediators and exercise intolerance in heart failure with preserved ejection fraction Nat Commun 2023 14 7557 10.1038/s41467-023-43363-3 37985769
Lau ES, Roshandelpoor A, Zarbafian S, Wang D, Guseh JS, Allen N, et al. Eicosanoid and eicosanoid-related inflammatory mediators and exercise intolerance in heart failure with preserved ejection fraction. Nat Commun. 2023;14:7557.37985769
Leroy V Cai J Tu Z McQuiston A Sharma S Emtiazjoo A Resolution of post-lung transplant ischemia-reperfusion injury is modulated via Resolvin D1-FPR2 and Maresin 1-LGR6 signaling J Heart Lung Transplant 2023 42 562 574 10.1016/j.healun.2022.12.013 36628837
Leroy V, Cai J, Tu Z, McQuiston A, Sharma S, Emtiazjoo A, et al. Resolution of post-lung transplant ischemia-reperfusion injury is modulated via Resolvin D1-FPR2 and Maresin 1-LGR6 signaling. J Heart Lung Transplant. 2023;42:562–74.36628837
Liu T Xiang A Peng T Doran AC Tracey KJ Barnes BJ HMGB1-C1q complexes regulate macrophage function by switching between leukotriene and specialized proresolving mediator biosynthesis Proc Natl Acad Sci U S A 2019 116 23254 23263 10.1073/pnas.1907490116 31570601
Liu T, Xiang A, Peng T, Doran AC, Tracey KJ, Barnes BJ, et al. HMGB1-C1q complexes regulate macrophage function by switching between leukotriene and specialized proresolving mediator biosynthesis. Proc Natl Acad Sci U S A. 2019;116:23254–63.31570601
Majno G Joris I Cells, tissues, and disease: principles of general pathology 2004 New York Oxford University Press
Majno G, Joris I. Cells, tissues, and disease: principles of general pathology. New York: Oxford University Press; 2004.
Malawista SE de Boisfleury CA van Damme J Serhan CN Tonic inhibition of chemotaxis in human plasma Proc Natl Acad Sci U S A 2008 105 17949 17954 10.1073/pnas.0802572105 18997012
Malawista SE, de Boisfleury CA, van Damme J, Serhan CN. Tonic inhibition of chemotaxis in human plasma. Proc Natl Acad Sci U S A. 2008;105:17949–54.18997012
Markworth JF Brown LA Lim E Floyd C Larouche J Castor-Macias JA Resolvin D1 supports skeletal myofiber regeneration via actions on myeloid and muscle stem cells JCI Insight 2020 5 e137713 10.1172/jci.insight.137713 32750044
Markworth JF, Brown LA, Lim E, Floyd C, Larouche J, Castor-Macias JA, et al. Resolvin D1 supports skeletal myofiber regeneration via actions on myeloid and muscle stem cells. JCI Insight. 2020;5: e137713.32750044
Mas E Croft KD Zahra P Barden A Mori TA Resolvins D1, D2, and other mediators of self-limited resolution of inflammation in human blood following n-3 fatty acid supplementation Clin Chem 2012 58 1476 1484 10.1373/clinchem.2012.190199 22912397
Mas E, Croft KD, Zahra P, Barden A, Mori TA. Resolvins D1, D2, and other mediators of self-limited resolution of inflammation in human blood following n-3 fatty acid supplementation. Clin Chem. 2012;58:1476–84.22912397
Mirakaj V Dalli J Granja T Rosenberger P Serhan CN Vagus nerve controls resolution and pro-resolving mediators of inflammation J Exp Med 2014 211 1037 1048 10.1084/jem.20132103 24863066
Mirakaj V, Dalli J, Granja T, Rosenberger P, Serhan CN. Vagus nerve controls resolution and pro-resolving mediators of inflammation. J Exp Med. 2014;211:1037–48.24863066
Möller I Rodas G Villalón JM Rodas JA Angulo F Martínez N Randomized, double-blind, placebo-controlled study to evaluate the effect of treatment with an SPMs-enriched oil on chronic pain and inflammation, functionality, and quality of life in patients with symptomatic knee osteoarthritis: GAUDI study J Transl Med 2023 21 423 10.1186/s12967-023-04283-4 37386594
Möller I, Rodas G, Villalón JM, Rodas JA, Angulo F, Martínez N, et al. Randomized, double-blind, placebo-controlled study to evaluate the effect of treatment with an SPMs-enriched oil on chronic pain and inflammation, functionality, and quality of life in patients with symptomatic knee osteoarthritis: GAUDI study. J Transl Med. 2023;21:423.37386594
Moore FD Metabolic care of the surgical patient 1959 Philadelphia and London W.B. Saunders Company
Moore FD. Metabolic care of the surgical patient. Philadelphia and London: W.B. Saunders Company; 1959.
Morse SS Mazet JA Woolhouse M Parrish CR Carroll D Karesh WB Prediction and prevention of the next pandemic zoonosis Lancet 2012 380 1956 1965 10.1016/S0140-6736(12)61684-5 23200504
Morse SS, Mazet JA, Woolhouse M, Parrish CR, Carroll D, Karesh WB, et al. Prediction and prevention of the next pandemic zoonosis. Lancet. 2012;380:1956–65.23200504
Murakami Y Fukuda H Muromoto R Hirashima K Ishimura K Fujiwara K Design and synthesis of benzene congeners of resolvin E2, a proresolving lipid mediator, as its stable equivalents ACS Med Chem Lett 2020 11 479 484 10.1021/acsmedchemlett.9b00596 32292553
Murakami Y, Fukuda H, Muromoto R, Hirashima K, Ishimura K, Fujiwara K, et al. Design and synthesis of benzene congeners of resolvin E2, a proresolving lipid mediator, as its stable equivalents. ACS Med Chem Lett. 2020;11:479–84.32292553
Nishina T Deguchi Y Kawauchi M Xiyu C Yamazaki S Mikami T Interleukin 11 confers resistance to dextran sulfate sodium-induced colitis in mice iScience 2023 26 105934 10.1016/j.isci.2023.105934 36685040
Nishina T, Deguchi Y, Kawauchi M, Xiyu C, Yamazaki S, Mikami T, et al. Interleukin 11 confers resistance to dextran sulfate sodium-induced colitis in mice. iScience. 2023;26:105934.36685040
Norris PC Libreros S Chiang N Serhan CN A cluster of immunoresolvents links coagulation to innate host defense in human blood Sci Signal 2017 10 eaan1471 10.1126/scisignal.aan1471 28765512
Norris PC, Libreros S, Chiang N, Serhan CN. A cluster of immunoresolvents links coagulation to innate host defense in human blood. Sci Signal. 2017;10:eaan1471.28765512
Norris PC Skulas-Ray AC Riley I Richter CK Kris-Etherton PM Jensen GL Identification of specialized pro-resolving mediator clusters from healthy adults after intravenous low-dose endotoxin and omega-3 supplementation: a methodological validation Sci Rep 2018 8 18050 10.1038/s41598-018-36679-4 30575798
Norris PC, Skulas-Ray AC, Riley I, Richter CK, Kris-Etherton PM, Jensen GL, et al. Identification of specialized pro-resolving mediator clusters from healthy adults after intravenous low-dose endotoxin and omega-3 supplementation: a methodological validation. Sci Rep. 2018;8:18050.30575798
Padovani CM Yin K Immunosuppression in sepsis: biomarkers and specialized pro-resolving mediators Biomedicines 2024 12 175 10.3390/biomedicines12010175 38255280
Padovani CM, Yin K. Immunosuppression in sepsis: biomarkers and specialized pro-resolving mediators. Biomedicines. 2024;12:175.38255280
Palmas F Clarke J Colas RA Gomez EA Keogh A Boylan M Dysregulated plasma lipid mediator profiles in critically ill COVID-19 patients PLoS ONE 2021 16 e0256226 10.1371/journal.pone.0256226 34437568
Palmas F, Clarke J, Colas RA, Gomez EA, Keogh A, Boylan M, et al. Dysregulated plasma lipid mediator profiles in critically ill COVID-19 patients. PLoS ONE. 2021;16: e0256226.34437568
Ramirez JL Gasper WJ Khetani SA Zahner GJ Hills NK Mitchell PT Fish oil increases specialized pro-resolving lipid mediators in PAD (The OMEGA-PAD II Trial) J Surg Res 2019 238 164 174 10.1016/j.jss.2019.01.038 30771686
Ramirez JL, Gasper WJ, Khetani SA, Zahner GJ, Hills NK, Mitchell PT, et al. Fish oil increases specialized pro-resolving lipid mediators in PAD (The OMEGA-PAD II Trial). J Surg Res. 2019;238:164–74.30771686
Recchiuti A Codagnone M Pierdomenico AM Rossi C Mari VC Cianci E Immunoresolving actions of oral resolvin D1 include selective regulation of the transcription machinery in resolution-phase mouse macrophages FASEB J 2014 28 3090 3102 10.1096/fj.13-248393 24692596
Recchiuti A, Codagnone M, Pierdomenico AM, Rossi C, Mari VC, Cianci E, et al. Immunoresolving actions of oral resolvin D1 include selective regulation of the transcription machinery in resolution-phase mouse macrophages. FASEB J. 2014;28:3090–102.24692596
Schwab JM Chiang N Arita M Serhan CN Resolvin E1 and protectin D1 activate inflammation-resolution programmes Nature 2007 447 869 874 10.1038/nature05877 17568749
Schwab JM, Chiang N, Arita M, Serhan CN. Resolvin E1 and protectin D1 activate inflammation-resolution programmes. Nature. 2007;447:869–74.17568749
Schwarz B Sharma L Roberts L Peng X Bermejo S Leighton I Cutting Edge: Severe SARS-CoV-2 Infection in Humans Is Defined by a Shift in the Serum Lipidome, Resulting in Dysregulation of Eicosanoid Immune Mediators J Immunol 2021 206 329 334 10.4049/jimmunol.2001025 33277388
Schwarz B, Sharma L, Roberts L, Peng X, Bermejo S, Leighton I, et al. Cutting Edge: Severe SARS-CoV-2 Infection in Humans Is Defined by a Shift in the Serum Lipidome, Resulting in Dysregulation of Eicosanoid Immune Mediators. J Immunol. 2021;206:329–34.33277388
Sekheri M El Kebir D Edner N Filep JG 15-Epi-LXA(4) and 17-epi-RvD1 restore TLR9-mediated impaired neutrophil phagocytosis and accelerate resolution of lung inflammation Proc Natl Acad Sci U S A 2020 117 7971 7980 10.1073/pnas.1920193117 32205444
Sekheri M, El Kebir D, Edner N, Filep JG. 15-Epi-LXA(4) and 17-epi-RvD1 restore TLR9-mediated impaired neutrophil phagocytosis and accelerate resolution of lung inflammation. Proc Natl Acad Sci U S A. 2020;117:7971–80.32205444
Serhan CN Resolution phases of inflammation: novel endogenous anti-inflammatory and pro-resolving lipid mediators and pathways Annu Rev Immunol 2007 25 101 137 10.1146/annurev.immunol.25.022106.141647 17090225
Serhan CN. Resolution phases of inflammation: novel endogenous anti-inflammatory and pro-resolving lipid mediators and pathways. Annu Rev Immunol. 2007;25:101–37.17090225
Serhan CN Pro-resolving lipid mediators are leads for resolution physiology Nature 2014 510 92 101 10.1038/nature13479 24899309
Serhan CN. Pro-resolving lipid mediators are leads for resolution physiology. Nature. 2014;510:92–101.24899309
Serhan CN Petasis NA Resolvins and protectins in inflammation-resolution Chem Rev 2011 111 5922 5943 10.1021/cr100396c 21766791
Serhan CN, Petasis NA. Resolvins and protectins in inflammation-resolution. Chem Rev. 2011;111:5922–43.21766791
Serhan CN Chiang N Resolvins and cysteinyl-containing pro-resolving mediators activate resolution of infectious inflammation and tissue regeneration Prostaglandins Other Lipid Mediat 2023 166 106718 10.1016/j.prostaglandins.2023.106718 36813255
Serhan CN, Chiang N. Resolvins and cysteinyl-containing pro-resolving mediators activate resolution of infectious inflammation and tissue regeneration. Prostaglandins Other Lipid Mediat. 2023;166: 106718.36813255
Serhan CN Gupta SK Perretti M Godson C Brennan E Li Y The Atlas of Inflammation Resolution (AIR) Mol Aspects Med 2020 74 100894 10.1016/j.mam.2020.100894 32893032
Serhan CN, Gupta SK, Perretti M, Godson C, Brennan E, Li Y, et al. The Atlas of Inflammation Resolution (AIR). Mol Aspects Med. 2020;74: 100894.32893032
Serhan CN Libreros S Nshimiyimana R E-series resolvin metabolome, biosynthesis and critical role of stereochemistry of specialized pro-resolving mediators (SPMs) in inflammation-resolution: Preparing SPMs for long COVID-19, human clinical trials, and targeted precision nutrition Semin Immunol 2022 59 101597 10.1016/j.smim.2022.101597 35227568
Serhan CN, Libreros S, Nshimiyimana R. E-series resolvin metabolome, biosynthesis and critical role of stereochemistry of specialized pro-resolving mediators (SPMs) in inflammation-resolution: Preparing SPMs for long COVID-19, human clinical trials, and targeted precision nutrition. Semin Immunol. 2022;59: 101597.35227568
Shay AE Nshimiyimana R Petasis NA Haeggstrom JZ Serhan CN Human leukocytes selectively convert 4S,5S-epoxy-Resolvin to Resolvin D3, Resolvin D4, and a cys-Resolvin isomer Proc Natl Acad Sci USA 2021 118 e2116559118 10.1073/pnas.2116559118 34911767
Shay AE, Nshimiyimana R, Petasis NA, Haeggstrom JZ, Serhan CN. Human leukocytes selectively convert 4S,5S-epoxy-Resolvin to Resolvin D3, Resolvin D4, and a cys-Resolvin isomer. Proc Natl Acad Sci USA. 2021;118: e2116559118.34911767
Shi J Wu X Wang Z Li F Meng Y Moore RM A genome-wide CRISPR screen identifies WDFY3 as a regulator of macrophage efferocytosis Nat Commun 2022 13 7929 10.1038/s41467-022-35604-8 36566259
Shi J, Wu X, Wang Z, Li F, Meng Y, Moore RM, et al. A genome-wide CRISPR screen identifies WDFY3 as a regulator of macrophage efferocytosis. Nat Commun. 2022;13:7929.36566259
Singh A Cho WJ Pulimamidi VK Mittal SK Chauhan SK Interleukin-11 suppresses ocular surface inflammation and accelerates wound healing Invest Ophthalmol vis Sci 2023 64 1 10.1167/iovs.64.14.1 37910094
Singh A, Cho WJ, Pulimamidi VK, Mittal SK, Chauhan SK. Interleukin-11 suppresses ocular surface inflammation and accelerates wound healing. Invest Ophthalmol vis Sci. 2023;64:1.37910094
Souza PR Marques RM Gomez EA Colas RA De Matteis R Zak A Enriched marine oil supplements increase peripheral blood specialized pro-resolving mediators concentrations and reprogram host immune responses: a randomized double-blind placebo-controlled study Circ Res 2020 126 75 90 10.1161/CIRCRESAHA.119.315506 31829100
Souza PR, Marques RM, Gomez EA, Colas RA, De Matteis R, Zak A, et al. Enriched marine oil supplements increase peripheral blood specialized pro-resolving mediators concentrations and reprogram host immune responses: a randomized double-blind placebo-controlled study. Circ Res. 2020;126:75–90.31829100
Spite M Norling LV Summers L Yang R Cooper D Petasis NA Resolvin D2 is a potent regulator of leukocytes and controls microbial sepsis Nature 2009 461 1287 1291 10.1038/nature08541 19865173
Spite M, Norling LV, Summers L, Yang R, Cooper D, Petasis NA, et al. Resolvin D2 is a potent regulator of leukocytes and controls microbial sepsis. Nature. 2009;461:1287–91.19865173
Suchitha GP Devasahayam Arokia Balaya R Prasad TSK Dagamajalu S A signaling network map of Lipoxin (LXA4): an anti-inflammatory molecule Inflamm Res 2024 73 1099 1106 10.1007/s00011-024-01885-6 38668877
Suchitha GP, Devasahayam Arokia Balaya R, Prasad TSK, Dagamajalu S. A signaling network map of Lipoxin (LXA4): an anti-inflammatory molecule. Inflamm Res. 2024;73:1099-1106.38668877
Sundarasivarao PYK Walker JM Rodriguez A Spur BW Yin K Resolvin D2 induces anti-microbial mechanisms in a model of infectious peritonitis and secondary lung infection Front Immunol 2022 13 1011944 10.3389/fimmu.2022.1011944 36532055
Sundarasivarao PYK, Walker JM, Rodriguez A, Spur BW, Yin K. Resolvin D2 induces anti-microbial mechanisms in a model of infectious peritonitis and secondary lung infection. Front Immunol. 2022;13:1011944.36532055
Taylor HA Finkel T Gao Y Ballinger SW Campo R Chen R Scientific opportunities in resilience research for cardiovascular health and wellness. Report from a National Heart, Lung, and Blood Institute workshop FASEB J 2022 36 e22639 10.1096/fj.202201407R 36322029
Taylor HA, Finkel T, Gao Y, Ballinger SW, Campo R, Chen R, et al. Scientific opportunities in resilience research for cardiovascular health and wellness. Report from a National Heart, Lung, and Blood Institute workshop. FASEB J. 2022;36:e22639.36322029
Verma S Bhatt DL Steg PG Miller M Brinton EA Jacobson TA Icosapent ethyl reduces ischemic events in patients with a history of previous coronary artery bypass grafting: REDUCE-IT CABG Circulation 2021 144 1845 1855 10.1161/CIRCULATIONAHA.121.056290 34710343
Verma S, Bhatt DL, Steg PG, Miller M, Brinton EA, Jacobson TA, et al. Icosapent ethyl reduces ischemic events in patients with a history of previous coronary artery bypass grafting: REDUCE-IT CABG. Circulation. 2021;144:1845–55.34710343
Wang A Pope SD Weinstein JS Yu S Zhang C Booth CJ Specific sequences of infectious challenge lead to secondary hemophagocytic lymphohistiocytosis-like disease in mice Proc Natl Acad Sci U S A 2019 116 2200 2209 10.1073/pnas.1820704116 30674681
Wang A, Pope SD, Weinstein JS, Yu S, Zhang C, Booth CJ, et al. Specific sequences of infectious challenge lead to secondary hemophagocytic lymphohistiocytosis-like disease in mice. Proc Natl Acad Sci U S A. 2019;116:2200–9.30674681
Wang X, Zhu M, Hjorth E, Cortés-Toro V, Eyjolfsdottir H, Graff C, et al. Resolution of inflammation is altered in Alzheimer's disease. Alzheimers Dement. 2015;11:40–50.e41–42.
Westermann D Savvatis K Lindner D Zietsch C Becher PM Hammer E Reduced degradation of the chemokine MCP-3 by matrix metalloproteinase-2 exacerbates myocardial inflammation in experimental viral cardiomyopathy Circulation 2011 124 2082 2093 10.1161/CIRCULATIONAHA.111.035964 21986287
Westermann D, Savvatis K, Lindner D, Zietsch C, Becher PM, Hammer E, et al. Reduced degradation of the chemokine MCP-3 by matrix metalloproteinase-2 exacerbates myocardial inflammation in experimental viral cardiomyopathy. Circulation. 2011;124:2082–93.21986287
Winyard PG, Willoughby DA. Inflammation protocols. In: Methods Mol. Biol. Walker JM (ed.) Humana, Totowa, NJ. 2003.
Xia J Psychogios N Young N Wishart DS MetaboAnalyst: a web server for metabolomic data analysis and interpretation Nucleic Acids Res 2009 37 W652 660 10.1093/nar/gkp356 19429898
Xia J, Psychogios N, Young N, Wishart DS. MetaboAnalyst: a web server for metabolomic data analysis and interpretation. Nucleic Acids Res. 2009;37:W652-660.19429898
Yu J Feng Z Tan L Pu L Kong L Interleukin-11 protects mouse liver from warm ischemia/reperfusion (WI/Rp) injury Clin Res Hepatol Gastroenterol 2016 40 562 570 10.1016/j.clinre.2015.11.009 27016892
Yu J, Feng Z, Tan L, Pu L, Kong L. Interleukin-11 protects mouse liver from warm ischemia/reperfusion (WI/Rp) injury. Clin Res Hepatol Gastroenterol. 2016;40:562–70.27016892
Zhang MJ Sansbury BE Hellmann J Baker JF Guo L Parmer CM Resolvin D2 enhances postischemic revascularization while resolving inflammation Circulation 2016 134 666 680 10.1161/CIRCULATIONAHA.116.021894 27507404
Zhang MJ, Sansbury BE, Hellmann J, Baker JF, Guo L, Parmer CM, et al. Resolvin D2 enhances postischemic revascularization while resolving inflammation. Circulation. 2016;134:666–80.27507404
