The Natural Combination Medicine Traumeel (Tr14) Improves Resolution of Inflammation by Promoting the Biosynthesis of Specialized Pro-Resolving Mediators

The resolution of inflammation is an integral part of the acute inflammatory response and eventually leads to the return to homeostasis. It is supported by specialized pro-resolving mediators (SPMs) that act as immunoresolvents via specific G-protein-coupled receptors. In contrast to classical non-steroidal anti-inflammatory drugs (NSAIDs) that suppress the formation of pro-inflammatory lipid mediators such as prostaglandins, novel pharmacotherapeutic concepts propose to foster the biosynthesis of beneficial SPMs. Here, we demonstrate that the natural combination medicine Traumeel (Tr14) improves resolution of inflammation by promoting SPM formation. Tr14 enhanced the biosynthesis of 12-/15-lipoxygenase (LOX) products and of SPMs in zymosan-induced mouse peritonitis as well as in human monocyte-derived macrophages challenged with Staphylococcus aureus. Importantly, in the peritonitis model, Tr14 supported the recruitment of innate leukocytes and the efferocytotic capacity of macrophages, and positively influenced the inflammation resolution index. Taken together, we suggest that based on these properties Tr14 may possess therapeutic potential as an enhancer for the resolution of inflammatory processes.


Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) are amongst the most popular medicines and are widely used as analgesics and for reducing inflammation [1]. Despite their efficacy in alleviating pain and inflammatory reactions, NSAIDs exert adverse gastrointestinal, cardiovascular, and renal effects [2], and they are essentially inefficient at promoting resolution and tissue repair [3]. Acute inflammation is usually self-limited and terminated in a temporal manner governing the return to tissue homeostasis [4,5]. The resolution of inflammation is part of the inflammatory process that actively terminates inflammation and leads to tissue repair and regeneration [6]. It is characterized by normalization of chemokine gradients and by the clearance of apoptotic neutrophils by macrophages [7][8][9][10]. In the early stages of acute inflammation, pro-inflammatory lipid mediators (LMs) are generated by resident cells in injured tissue which elicit recruitment of polymorphonuclear leukocytes (PMNs) that further enhance pro-inflammatory mediator production [6,[11][12][13]. The proinflammatory LMs are comprised of leukotrienes (LTs) and prostaglandins (PGs) produced

Tr14 Affects Lipid Mediator Pathways and Promotes Resolution of Inflammation in Murine Peritonitis
To study the effects of Tr14 on the inflammation-resolving mechanism in vivo, we employed the well-characterized acute inflammation model of zymosan-induced peritonitis in mice. Two different experimental settings were applied (see Section 4 and Figure S1 for more details): (1) Tr14 was given intraperitoneally (i.p.) at low (1.5 mL/kg) or high dose (3 mL/kg) once daily for six days prior to zymosan (i.p.) injection and peritoneal exudates were collected after 4, 8 and 24 h ( Figure 1A); (2) Tr14 was given i.p. at low (1.5 mL/kg) or high dose (3 mL/kg) 4 and 8 h post-zymosan (i.p.) injection and peritoneal exudates were collected after 4,8,24,192 and 360 h (Figure 2A).
In the experimental setting (1), where Tr14 was given before zymosan, the maximum cell infiltration was reached at 4 h post-zymosan (i.p.) injection. Tr14 at the high dose significantly increased the total cell number in the peritoneum after 4 h ( Figure 1B). The infiltrate was predominantly composed of PMNs that were significantly decreased in numbers by the low dose of Tr14 at 8 h ( Figure 1C, Figure S2A). Additionally, Tr14 significantly enhanced the recruitment of monocytes/macrophages after 4 and 24 h and significantly increased eosinophil recruitment after 4 h ( Figure S2A,B). Tr14 shortened the resolution interval (R i -interval between T max and T 50 ) with the high dose by 1.3 h and with the low dose by  Figure 1C). Tr14 did not alter the release of IL-6, TNF-α, monocyte chemoattractant protein-1 (MCP-1), eotaxin-1, KC (IL-8) or IL-10 after 4 h ( Figures 1D and S2C) and did not affect the ratio of anti-inflammatory IL-10 versus the sum of the pro-inflammatory cytokines/chemokines IL-6, TNF-α, MCP-1, eotaxin-1, and KC ( Figure 1E). We then investigated the effect of Tr14 on the expression of 13 genes related to LM-biosynthetic enzymes or LM receptors, and of other 17 inflammation-related genes (mainly cytokines) in the peritoneal lavage. After 4 h, Tr14 at high and low dose slightly increased the mRNA levels of 15-LOX ( Figure 1F), IL-2, IL-4, and IL-15 ( Figure S2D), which are involved in antiinflammatory signaling. Furthermore, Tr14 decreased mRNA levels of pro-inflammatory cytokines such as IL-5 and IL-17A ( Figure S2D). Next, we assessed the levels of LM in the peritoneal exudates using UPLC-MS-MS [25]. In the absence of Tr14, the pro-inflammatory COX-derived PGE 2 and TXB 2 along with 5-LOX-derived LTB 4 were mainly produced 4 to 8 h post-zymosan and their levels were diminished after 24 h (Figures 1G and S2E). In contrast, the levels of monohydroxylated SPM precursors (i.e., 14-HDHA and 17-HDHA) and of various SPMs (i.e., PD1, MaR1, RvD2, RvD5 and LXA 4 ) were continuously formed from 8 h up to 24 h ( Figure 1G and Figure S2E). Pre-treatment with Tr14 prior zymosan did not markedly affect the overall production of LMs, although a tendency of decreased COX products (PGE 2 and TXB 2 ) was observed after 24 h ( Figures 1G and S2E). However, Tr14 significantly increased the ratio of 12/15-LOX-derived products (14-HDHA and 17-HDHA) and SPMs (PD1, MaR1, RvD2, RvD5 and LXA 4 ) versus COX-derived (PGE 2 and TXB 2 ) LMs implying that Tr14 promotes a switch from pro-inflammatory towards pro-resolving LMs ( Figure 1H).
When Tr14 was administered after zymosan injection (experimental setting (2), Figure 2A), no significant change in cell counts was observed after 24 h versus vehicle group ( Figures 2B and S3A). However, for the post-resolution phase (192 h up to 360 h after zymosan), Tr14 at low dose significantly increased numbers of resident macrophages and lymphocytes ( Figure S3A). In comparison to vehicle control, the cell-type composition remained unaltered after 24 h of Tr14 treatment ( Figure S3B) but slightly changed to more lymphocytes and resident macrophages after low-dose Tr14 treatment after 360 h ( Figure S3B). Note that Tr14 at both doses did not affect the resolution indices ( Figure 2C). Released cytokines in the exudates peaked 4 h post-zymosan administration and were barely present at other timepoints. Tr14 weakly influenced the release of cytokines ( Figure  S3C) with a tendency towards elevated amounts of anti-inflammatory IL-10 and chemokine KC (IL-8) ( Figure 2D) and towards an increase of the ratio of anti-inflammatory IL-10 versus the sum of the pro-inflammatory cytokines/chemokines IL-6, TNF-α, MCP-1, eotaxin-1, and KC after 8 h post-zymosan administration ( Figure 2E). In the absence of Tr14, SPMs and especially 12/15-LOX products (14-HDHA and 17-HDHA) as well as the 5-LOX product LTB 4 were increasingly produced up to 360 h ( Figures 2F and S3D). Strikingly, Tr14, especially at the low dose, significantly increased the levels of SPMs such as RvD2, RvD5 and LXA 4 after 24 h as well as for the post-resolution time (360 h) against vehicle ( Figures 2F and S3D). In comparison to experimental setting (1), application of Tr14 after zymosan injection did not significantly alter the ratio of 12/15-LOX-derived products (14-HDHA and 17-HDHA) and SPMs (PD1, MaR1, RvD2, RvD5 and LXA 4 ) versus COX-derived (PGE 2 and TXB 2 ) LMs ( Figure 2G).

Tr14 Enhances Macrophage Efferocytotic Actions In Vivo
Efferocytosis of apoptotic cells and cellular debris by recruited macrophages is an essential step in the resolution process that is strongly promoted by SPMs [6]. We investigated the effect of Tr14 administered i.p. once daily for six days prior to zymosan injection (experimental setting (1)) on macrophage efferocytotic functions in the murine peritonitis model ( Figure S1). First, we assessed the total numbers of non-apoptotic and apoptotic PMNs after 4, 8 and 24 h in the exudates. Tr14 did not significantly change the numbers of apoptotic and non-apoptotic PMNs ( Figure 3A). Next, we assessed the number of efferocytotic macrophages. Interestingly, Tr14 significantly enhanced the number of efferocytotic macrophages after 4 h ( Figure 3B). The ratio of efferocytotic macrophages versus non-apoptotic PMNs was higher in Tr14-pre-treated mice after 4 h, which suggests acceleration of inflammation resolution ( Figure 3C). Moreover, mice pretreated with Tr14 revealed a tendency towards enhanced capacity for PMN uptake by each efferocytotic macrophage versus cells from vehicle-treated animals ( Figure 3D). Together, our data show that Tr14 enhances the formation of SPM and, arguably as a consequence, promotes the efferocytosis of apoptotic PMNs by macrophages in vivo during peritonitis in mice.

Tr14 Increases 15-Lox-1-Mediated Lipid Mediator Biosynthesis in Human M2 Macrophages
To investigate the effect of Tr14 on the biosynthesis of 5-LOX-, COX-, and 12/15-LOX-derived LM biosynthesis in vitro in human innate immune cells, we preincubated MDM that had been polarized towards M1 and M2 phenotypes for 48 h, with Tr14 (0.1 or 10%) for 15 min and exposed them to S. aureus (MOI = 1:50) as stimulus to elicit LM formation. Previous studies showed that S. aureus elicits the formation of a broad spectrum of LM in such human MDM, where the M1 phenotype mainly generates pro-inflammatory PGs and LTs, and M2 are a major source for SPMs and 15-LOX-derived precursors [27]. First, we excluded that Tr14 possesses cytotoxic effects on MDMs over an incubation time of 48 h ( Figure S4A). Tr14 did not significantly impact the LM biosynthesis in short-term incubations (up to 3 h, Figure 4A,B), different from classical NSAIDs ibuprofen and diclofenac, the 5-LOX inhibitor zileuton, and the specific 15-LOX-1 inhibitor BLX-3887 [28].  in pg/mL exudate as single values and mean + SEM, *p < 0.05; **p < 0.

Tr14 Enhances Macrophage Efferocytotic Actions In Vivo
Efferocytosis of apoptotic cells and cellular debris by recruited macrophages is an essential step in the resolution process that is strongly promoted by SPMs [6]. We investigated the effect of Tr14 administered i.p. once daily for six days prior to zymosan injection (experimental setting (1)) on macrophage efferocytotic functions in the murine peritonitis model ( Figure S1). First, we assessed the total numbers of non-apoptotic and apoptotic PMNs after 4, 8 and 24 h in the exudates. Tr14 did not significantly change the numbers of apoptotic and non-apoptotic PMNs ( Figure 3A). Next, we assessed the number of efferocytotic macrophages. Interestingly, Tr14 significantly enhanced the number of efferocytotic macrophages after 4 h ( Figure 3B). The ratio of efferocytotic macrophages versus non-apoptotic PMNs was higher in Tr14-pre-treated mice after 4 h, which suggests acceleration of inflammation resolution ( Figure 3C). Moreover, mice pretreated with Tr14 revealed a tendency towards enhanced capacity for PMN uptake by each efferocytotic macrophage versus cells from vehicle-treated animals ( Figure 3D). Together, our data show that Tr14 enhances the formation of SPM and, arguably as a consequence, promotes the efferocytosis of apoptotic PMNs by macrophages in vivo during peritonitis in mice.   In contrast, when naïve MDMs were pre-treated with Tr14 for 15 min, then polarized towards M1-or M2-like phenotypes for 48 h, and subsequently challenged by S. aureus for LM formation, an enhanced formation of 15-LOX-1-mediated LM, especially SPMs, was observed in M2-MDM ( Figure 4C,D and Figure S4B). As a result, Tr14 diminished the proportion of produced LTB 4 and PGE 2 versus SPMs ( Figure 4E) suggesting that Tr14 shifts LM formation in M2-MDM towards a more pro-resolution profile during polarization of macrophages.

Discussion
NSAIDs are used for pain and inflammation management with decades of documented success. However, they are also well-known for their gastric, cardiovascular, renal, hepatic, and hematologic side effects [29]. It is suggested that, among many reasons, LM biosynthesis shunting phenomena and impairment of SPM formation are accounting for these side effects [28]. However, if SPM formation and thus resolution signaling is impaired, pro-inflammatory signals may accumulate, leading to excessive inflammation and, subsequently, to disease [17,18]. A frequently used approach to reduce the severity and number of side effects of NSAIDs is the use of respective prodrugs of the active substances that especially limit gastric toxicity [30]. Alternative strategies to NSAIDs could be smart manipulation of the overall LM networks to stimulate signaling in resolution pathways. In this respect, SPMs as novel immunoresolvents have been investigated in numerous experimental studies and showed promising therapeutic potential for treatment of inflammatory diseases [6,7,18,31].
The objective of this study was to investigate the potential pro-resolution properties of Tr14. We show that Tr14, which has been studied in various experimental models and clinical trials for its effect on inflammatory conditions [19][20][21][22][23][24], has a favorable impact on the inflammation resolution processes in mice along with a significant ability to elevate SPM levels. We found that in a mouse model of self-resolving inflammation triggered by zymosan in vivo, Tr14 did not consistently inhibit pro-inflammatory actions, such as recruitment of PMNs, release of cytokines or production of LTs and PGs, which contrasts with classical NSAIDs [1]. In this respect, Tr14 should not elicit the typical side effects of NSAIDs that essentially act as COX inhibitors. Our data demonstrate that Tr14 rather promotes the resolution process of inflammation, reflected by shortened resolution intervals by pre-treatment with Tr14 and elevated numbers of efferocytotic macrophages important for clearance of apoptotic PMNs and cellular debris produced during the onset of inflammation [32]. These macrophage actions are typically stimulated by SPMs such as MaR1 [33], and our data show that Tr14 shifts the LM profiles towards SPMs in the mouse peritonitis model in vivo as well as in human macrophages on the cellular level. Taken together, Tr14 positively influenced several pro-resolution properties, that is, it shortened the resolution interval, enhanced macrophage efferocytosis and increased SPM levels.
Our findings reveal that Tr14 directly targets cells of the innate immune system supported by the stimulatory effect on SPM formation in human M2-MDM. We recently showed that, in contrast to M1-MDM, the M2 phenotype produces substantial amounts of SPMs upon exposure to pathogenic bacteria [27] that release exotoxins such as HLA to elicit 15-LOX-1 activation and concomitant SPM formation involving ADAM10 [34]. The capacity to produce SPMs and their 15-LOX-derived precursors by M2-MDM was increased when Tr14 was present during macrophage polarization. Note that short-term pre-treatment of polarized M2-MDM with Tr14 failed to increase 15-LOX-derived products. This would exclude direct stimulatory effects of Tr14 on the LM-biosynthetic machinery and signaling such as Ca 2+ mobilization, 15-LOX translocation or supply of fatty acid substrates (i.e., AA, EPA or DHA) [34]. Possibly, Tr14 increases 15-LOX-1 or 15-LOX-2 protein levels, but priming of the M2-MDMs for more sustained Ca 2+ mobilization is also reasonable. Along these lines, we previously reported that besides the expression of 15-LOXs the activation on 15-LOX-1 depends on a slow and sustained Ca 2+ -influx into M2-MDMs [27].
Our data showing that Tr14 increased SPM production, support previous clinical reports about Tr14 s analgesic action. Indeed, SPMs were shown to reduce pain [35] and in the management of osteoarthritis of the knee, Tr14 co-administered with Zeel (Ze14), another natural combination medicine, reduced moderate-to-severe pain in the affected knee [36]. Since Tr14 enhanced efferocytotic actions of macrophages in mice, increased SPM levels and promoted inflammation resolution (shortening the resolution interval), we suggest that Tr14 may diminish pain in arthritic diseases by promoting the clearance of damage-associated molecular patterns (DAMPs) that are major causes for activated nociceptive neurons in the osteoarthritic joints [37]. This is in line with the reported effect of topical Traumeel application that successfully reduced mild-to-moderate post-injury pain and improved ankle mobility as effectively as 1% diclofenac gel [38].

Interventions
Traumeel (Tr14) was obtained free of charge from Heel GmbH (Baden-Baden, Germany). Tr14 was prepared in accordance with GMP standards and supplied in glass ampoules prepared for injection. The active ingredients of Tr14 are listed in Table S1. Each 1.1 mL ampoule of the vehicle control contained 0.9% sodium chloride for injection.

Animals, Animal Care and the Ethical Statement
Male C57BL/6J mice (7-12 weeks of age) with a weight range from 21 to 26 g, obtained from Charles River (Écully, France), were housed in a controlled environment (21 ± 2 • C) and provided with standard rodent chow and water. Animals were allowed to acclimate for seven days prior to experiments and were subjected to 12 h light/12 h dark schedule. Experiments were conducted during the light phase. The experimental procedures were approved ethically by the Comité d'Ethique en Expérimentation Animale (CEEA) 122 with the protocol number of 22923-201911251226765v3.

Study Design
The in vivo experiment was a randomized, three-arm parallel, vehicle-controlled exploratory study. Three intervention conditions were defined for each experimental setting: vehicle, Tr14 "high-dose", and Tr14 "low-dose". Two different experimental settings were applied: (1) preventive pre-treatment injection, when the intervention was administered intraperitoneally (i.p.) before induction of inflammatory response and peritoneal exudates were collected after 4, 8 and 24 h; (2) curative post-induction injection, when the intervention was administered i.p. after induction of inflammatory response and peritoneal exudates were collected after 4,8,24,192 and 360 h ( Figure S1). The experimental baseline was expressed as time point 0 h and resulted from the samples of a separate group of 6 naïve untreated animals.
For each time point and each intervention condition, one group of 8 mice was used. Each animal was considered as an experimental unit ( Figure S1). Mice were assigned for the experiments according to their body weights.
For the in vitro studies peripheral blood mononuclear cells (PBMC) isolated from three healthy volunteers were used and differentiated to macrophages. These monocyte-derived macrophages (MDM) were subjected to polarization towards M1 or M2 phenotypes and incubated with Staphylococcus aureus for induction of LM formation. Three intervention conditions were defined: Tr14 (low concentration = 0.1%) or Tr14 (high concentration = 10%) and vehicle with three independent biological replications (different donors). Experiments were performed in singlet technical measurements with cells from n = 3 different donors at different days, according to experiences and results from previous studies [27,34].
To explore whether and how Tr14 affects LM biosynthesis in M1-and M2-MDMs two experimental settings were applied: (1) polarized M1-and M2-MDMs were treated with the test item after polarization to investigate an effect on LM-producing enzyme activation; (2) MDMs were pre-treated with the test item prior to polarization to investigate the impact on LM-producing enzymes production. The assessed parameters were cell viability (MTT assay), and LM profiling (metabololipidomics).

Zymosan-Induced Murine Peritonitis and Peritoneal Lavage Collection
Zymosan (0.1 mg/mouse; InvivoGen, Toulouse, France) was injected into the peritoneum of mice for all tested groups, except for the baseline group (time point 0 h). Tr14 at 3 mL/kg (high dose), diluted with 0.9% NaCl to a concentration of 1.5 mL/kg (low dose), and 0.9% NaCl as vehicle were administered i.p. once daily for 6 days before zymosan injection (pre-treatment) for experimental setting (1) and i.p. 4 h and again 8 h after zymosan injection (post-induction) for experimental setting (2). At 4,8,24,192 or 360 h after zymosan injection, mice were euthanized. The inflammatory exudate was obtained by washing the peritoneal cavity with 2 mL PBS. Cells in the exudates were immediately counted using Scepter 2.0 cell counter (Merck Millipore, Burlington, MA, USA) and labeled for subpopulation identification and quantification by Macs quant analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany).

Measurement of Lipid Mediators and Cytokines in Peritoneal Lavage
The extraction protocol and analysis of LM in peritoneal lavages was performed as previously described [25] and adapted according to the Ambiotis SAS (Toulouse, France) standard operating procedure. Dedicated samples of peritoneal lavages were extracted using oasis HLB 96 wells solid phase extraction (Waters Corporation, Milford, MA, USA). The LC-MS/MS analysis was performed on a 1290 Infinity UHPLC system coupled to a 6490 triple quadrupole MS (Agilent Technologies, Santa Clara, CA, USA), equipped with electrospray ionization source, and performed in negative ion mode. Reverse-phase UHPLC was performed with a Kinetex Biphenyl column (2.1 mm × 50 mm × 1.7 µm; Phenomenex Inc., Torrance, CA, USA) maintained at 50 • C. For monitoring and quantification of LM, analyses were run in multiple reaction monitoring (MRM) detection mode.

Measurement of Gene Expression in the Peritoneal Lavage
mRNA was extracted from the peritoneal lavages using the TurboCapture 96 mRNA kit (Qiagen, Hilden, Germany). The reverse transcription was then carried out and the specific preparation steps for the chip (96 × 96) were conducted according to Fluidigm protocol version PN100-1201B1. In brief, a pre-amplification step was performed in the presence of the primers used on the chip using the Fluidigm Preamp Master Mix (Fluidigm, San Francisco, CA, USA). This pre-amplification was performed on a Veriti 96-well Fast Thermal Cycler (Applied Biosystems, Foster City, CA, USA) at 95 • C for 10 min followed by 14 cycles at 95 • C (15 s) and 60 • C (4 min). Each pre-amplified cDNA sample was diluted 1:5 and then deposited in a 96-well plate according to a pre-defined plate pan with a mix containing Taqman Gene expression Master Mix (Applied Biosystems), EvaGreen (Interchim, Montluçon, France) and Dye Sample Loading Reagent (Fluidigm). In parallel, on another 96-well plate, each pair of primer was prepared at a concentration of 5 µM according to a pre-defined plate plan. Then, each plate was deposited on both sides of the chip. The mixture of each well of each plate was made by the IFC controller within the chip and then the chip was placed in the BioMark (Fluidigm) to perform the RT-PCR. The data was extracted using Fluidigm Real-Time PCR analysis software (Fluidigm). The expression level of the mRNAs was normalized using the following reference genes: YWHAZ, OAZ1, HPRT and β-actin. Data were analyzed using the ∆∆Ct method where the Ct corresponds to the number of cycles necessary to generate a fluorescent signal above the pre-defined threshold and with the cut-off criteria of Ct > 23. The results are shown as fold-increase against the mean of the vehicle group at each time point.

Isolation of Human Monocytes and Preparation of Monocyte-Derived Macrophages
Leukocyte concentrates from freshly withdrawn peripheral blood of healthy adult human donors were provided by the Institute of Transfusion Medicine (University Hospital Jena, Germany). The experimental protocol was approved by the ethical committee of the University Hospital Jena. All methods were performed in accordance with the relevant guidelines and regulations. PBMC were isolated using dextran sedimentation and Ficoll-Histopaque 1077-1 (Sigma-Aldrich, Taufkirchen, Germany) centrifugation. PBMC were seeded in PBS containing 1 mM Ca 2+ and 0.5 mM Mg 2+ in cell culture flasks (Greiner Bio-one, Frickenhausen, Germany) for 1.5 h at 37 • C and 5% CO 2 for adherence of monocytes. For differentiation and polarization of monocytes towards M1-and M2-MDM, published criteria [27] were used. Thus, M1-MDM were generated by incubating monocytes with 20 ng/mL GM-CSF (Peprotech, Hamburg, Germany) for 6 days in RPMI 1640 supplemented with 10% fetal calf serum, 2 mmol/L L-glutamine (Biochrom/Merck, Berlin, Germany), and penicillin-streptomycin (Biochrom/Merck), followed by 100 ng/mL LPS (Sigma-Aldrich) and 20 ng/mL IFN-γ (Peprotech) treatment for another 48 h. M2-MDM were obtained after differentiation of monocytes with 20 ng/mL M-CSF (Peprotech) for 6 days, and then with 20 ng/mL IL-4 (Peprotech) for additional 48 h of polarization.

Quantification and Statistical Analysis
Results are expressed as mean ± standard error of the mean (SEM) of n observations, where n represents the number of experiments with separate donors, performed on different days, as indicated or mentioned otherwise. For the in vivo studies, n represents the number of animals. Analyses of data were conducted using GraphPad Prism 8 software (San Diego, CA, USA). Two-tailed t test was used for the comparison of two groups. For multiple comparisons, one-way and two-way analysis of variance (ANOVA) with Bonferroni, Dunnett s and Tukey's post hoc tests were applied as indicated. To identify outliers, ROUT test (Q = 0.1%) was performed and further outliers were excluded. The criterion for statistical significance is p < 0.05.

Conclusions
We demonstrated that Tr14 enhances the biosynthesis of 12-/15-LOX products and SPMs in zymosan-induced mouse peritonitis as well as in human MDM challenged with bacterial exotoxins. In the mouse peritonitis model, Tr14 also positively influenced the inflammation resolution index and supported efferocytosis. Our data reveal Tr14 as an immunoresolvent agent with diverse actions that promote resolution of inflammation, which may benefit patients with non-resolving inflammation due to defects in the resolution response.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/ph14111123/s1, Figure S1: Study plan for investigation of the effects of Tr14 on the resolution of inflammation in zymosan-induced mouse peritonitis, Figure S2: Self-resolving inflammation was initiated by injection of zymosan (0.1 mg/mouse, i.p.) into mice. Before zymosan injection, Tr14 (3 mL/kg or 1.5 mL/kg) or vehicle (veh., 0.9% NaCl) were administered i.p. once daily for six days.