The N-Formyl Peptide Receptor 2 (FPR2) Agonist MR-39 Exhibits Anti-Inflammatory Activity in LPS-Stimulated Organotypic Hippocampal Cultures

Accumulating evidence indicates a pivotal role for chronic inflammatory processes in the pathogenesis of neurodegenerative and psychiatric disorders. G protein-coupled formyl peptide receptor 2 (FPR2) mediates pro-inflammatory or anti-/pro-resolving effects upon stimulation with biased agonists. We aimed to evaluate the effects of a new FPR2 ureidopropanamide agonist, compound MR-39, on neuroinflammatory processes in organotypic hippocampal cultures (OHCs) derived from control (WT) and knockout FPR2−/− mice (KO) exposed to bacterial endotoxin (lipopolysaccharide; LPS). Higher LPS-induced cytokine expression and basal release were observed in KO FPR2 cultures than in WT cultures, suggesting that a lack of FPR2 enhances the OHCs response to inflammatory stimuli. Pretreatment with MR-39 abolished some of the LPS-induced changes in the expression of genes related to the M1/M2 phenotypes (including Il-1β, Il-6, Arg1, Il-4, Cd74, Fizz and Cx3cr1) and TNF-α, IL-1β and IL-4 release in tissue derived from WT but not KO mice. Receptor specificity was confirmed by adding the FPR2 antagonist WRW4, which abolished the abovementioned effects of MR-39. Further biochemical data showed an increase in the phospho-p65/total p65 ratio after LPS stimulation in hippocampal tissues from both WT and KO mice, and MR-39 only reversed this effect on WT OHCs. LPS also increased TRAF6 levels, which are critical for the TLR4-mediated NF-κB pro-inflammatory responses. MR-39 attenuated the LPS-evoked increase in the levels of the NLRP3 and caspase-1 proteins in WT but not KO hippocampal cultures. Since NLRP3 may be involved in the pyroptosis, a lytic type of programmed cell death in which the main role is played by Gasdermin D (GSDMD), we examined the effects of LPS and/or MR-39 on the GSDMD protein level. LPS only increased GSDMD production in the WT tissues, and this effect was ameliorated by MR-39. Collectively, this study indicates that the new FPR2 agonist efficiently abrogates LPS-induced neuroinflammation in an ex vivo model, as evidenced by a decrease in pro-inflammatory cytokine expression and release as well as the downregulation of NLRP3 inflammasome-related pathways.


Introduction
Neuroinflammation is a complex multicellular process that plays an important role in the onset and progression of several neurodegenerative and psychiatric disorders [1,2].
we evaluated the effects of MR-39 on cell death, nitric oxide release, and the levels of pro-and anti-inflammatory cytokines under both basal and LPS-stimulated conditions. The receptor specificity of the effect of MR-39 was verified using the FPR2 antagonist WRW4. We analyzed the intracellular pathways related to the inflammatory response, including MyD88/TRAF6/NF-кB and inflammasome 3 (NLRP3) signaling, to investigate the mechanisms underlying the effect of MR-39 on organotypic hippocampal cultures obtained from both WT and FPR2−/− mice. Recent data suggested that NLRP3 may be involved in the pyroptosis, a lytic type of programmed cell death in which the main role is played by Gasdermin D (GSDMD). Thus, we also examined the effects of LPS and/or MR-39 on the GSDMD protein level.

Animals
FPR2-deficient mice (knockout; KO; FPR2−/−) were obtained from Dr. Lars-Ove Brandenburg of the Department of Anatomy and Cell Biology, RWTH Aachen University, Aachen, Germany. Briefly, FPR2−/− KO mice on a C57BL/6 background were generated as described previously [22,24]. The wild type (WT) mice were back-crossed onto the C57BL/6J background for at least five generations. All mice were maintained under standard conditions (a room temperature of 23 • C, 12/12 h light/dark cycle, and lights on at 06:00 a.m.), with food and water available ad libitum. Females were mated with syngeneic male KO or WT mice. All experimental protocols were performed in accordance with guidelines from the Committee for Laboratory Animal Welfare and Ethics of the Maj Institute of Pharmacology, Polish Academy of Sciences, Krakow, Poland.

OHC Treatment
On the 7th day, OHCs obtained from WT and KO mice were pretreated with the FPR2 antagonist WRW4 (10 µM) for 30 min. Afterward, MR-39 (1 µM) was added for 1 h, and then OHCs were stimulated for 24 h by adding LPS to the medium (final concentration in the well: 1 µg/mL). Control (unstimulated) OHCs were treated with vehicle (phosphatebuffered saline (PBS)).

Determination of Lactate Dehydrogenase (LDH) Activity
Twenty-four hours after culture stimulation, the level of lactate dehydrogenase released to the culture medium was measured as previously described [35] using a colorimetric method according to the supplier's protocol (Cytotoxicity Detection Kit, Roche Diagnostic, Mannheim, Germany). The data were normalized to the activity of LDH released from control slices (100%; vehicle-treated WT OHCs) and are reported as a percentage of the control ± SEM (standard error of the mean).

Nitric Oxide (NO) Release Assay
The Griess test was used for the nitric oxide (NO) secretion in the culture medium. According to the protocol, 24 h after stimulation, 50 µL of supernatant were mixed with an equal volume of Griess reagent (Griess A-0.1% N-1-naphthylethylenediamine dihydrochloride and Griess B-1% sulfanilamide in 5% phosphoric acid; Sigma-Aldrich, St. Louis, MO, USA) in a 96-well plate. The absorbance was measured at 540 nm using an Infinite ® 200 PRO Detector (TECAN, Switzerland). The data were normalized to the NO released from vehicle-treated cells (100%; vehicle-treated WT OHCs) and reported as a percentage of the control ± SEM.

RNA Extraction and cDNA Preparation
Hippocampal slices were lysed by adding 200 µL of TRI Reagent (Sigma-Aldrich, St. Louis, MO, USA) 24 h after LPS (1 µg/mL) treatment and stored at −20 • C until isolation. Total RNA was extracted using TRIzol ® Reagent according to the User Guide (Thermo Fisher Scientific, Waltham, MA, USA) based on the Chomczyński (1993) method [37]. After isolation, the RNA concentration was assessed using a NanoDrop spectrophotometer (ND/1000 UV/Vis, Thermo Fisher NanoDrop, Waltham, MA, USA) and the synthesis of cDNA was carried out via reverse transcription using an NG dART RT Kit (EURx, Gdansk, Poland) according to the manufacturer's instructions.

Statistical Analysis
Statistical analyses were performed using Statistica 10.0 software (Statsoft, Tulsa, OK, USA). All biochemical experiments were carried out under the same conditions for all samples, regardless of the type of treatment. The results presented in this study were derived from three independent KO FPR2−/− or WT OHCs, and the "n" for each culture was 2-5. All data were obtained in independent experiments and are presented as the means ± SEM (standard errors of the means). The results of the death processes (LDH) and NO release are presented as the mean percentages ± SEM of the control (vehicle-treated WT OHCs). The data obtained from the ELISA study are presented as the means ± SEM, and those for RT-PCR are presented as the average fold changes ± SEM. All groups were compared using a factorial analysis of variance (ANOVA) to determine the effects of the factors, followed, when appropriate, by Duncan's post hoc test. In the case of the qRT-PCR analysis, factorial ANOVA was performed for those groups in which we obtained a value for measured factors. When appropriate, a two-way ANOVA was used. When the level of the tested factor was undetectable, a statistical analysis was not performed. A p-value < 0.05 was considered statistically significant. GraphPad Prism 5 was used for graphs preparation.

Dynamics of OHCs Obtained from the Offspring of WT and KO Mice
In the first set of experiments, the dynamics of organotypic cultures were assessed based on the lactate dehydrogenase (LDH) release test. The LDH assay takes advantage of the release of the enzyme lactate dehydrogenase into the culture medium upon cell damage (disruption of the cell membrane). From the 7th day in vitro (DIV) of cultivation, the amount of LDH released into the medium remained at a low and constant level (until the 13th DIV) in both the WT and KO cultures ( Figure 1). Based on this result, indicating a culture stabilization process, further experiments were carried out on the 7th day after culture stabilization.
tors, followed, when appropriate, by Duncan's post hoc test. In the case of the qRTanalysis, factorial ANOVA was performed for those groups in which we obtained a v for measured factors. When appropriate, a two-way ANOVA was used. When the l of the tested factor was undetectable, a statistical analysis was not performed. A p-v < 0.05 was considered statistically significant. GraphPad Prism 5 was used for gra preparation.

Dynamics of OHCs Obtained from the Offspring of WT and KO Mice
In the first set of experiments, the dynamics of organotypic cultures were asse based on the lactate dehydrogenase (LDH) release test. The LDH assay takes advan of the release of the enzyme lactate dehydrogenase into the culture medium upon damage (disruption of the cell membrane). From the 7th day in vitro (DIV) of cultiva the amount of LDH released into the medium remained at a low and constant level (u the 13th DIV) in both the WT and KO cultures ( Figure 1). Based on this result, indica a culture stabilization process, further experiments were carried out on the 7th day culture stabilization.    We used an assay based on the Griess reaction to assess the effect of MR-39 on NO secretion from OHCs. OHCs were stimulated for 24 h, after which NO release was measured. As shown in Figure 2b, we did not observe an effect of either LPS (1 µg/mL) or MR-39 pretreatment on NO release in OHCs obtained from WT or KO mice. Control cultures were treated with the appropriate vehicle. NO release was measured using the Griess reaction. The data are presented as the mean percentages ± SEM of the control (vehicle-treated WT OHCs) from three independent experiments. The results were statistically evaluated using a two-way analysis of variance (ANOVA) with the Duncan post hoc test to assess the differences between the treatment groups. Significant differences are indicated by * p < 0.05. LDH-lactate dehydrogenase; NO-nitric oxide. +-with LPS or MR treatment; --without LPS or MR treatment.

The Effects of LPS and/or MR-39 on the mRNA Expression of Pro-Inflammatory and Anti-Inflammatory Factors in OHCs Obtained from the Offspring of WT and KO Mice
In the central nervous system, microglia are responsible for maintaining the physiological condition, while the shift from the M1-to M2-like profile is important in RoI. Generally, the M1-like phenotype is characterized by pro-inflammatory properties, while the M2-like phenotype is characterized by anti-inflammatory actions, which promotes tissue remodeling and repair. We decided to explore the effects of MR-39 on the pro-and antiinflammatory phenotypes in OHCs from WT and KO mice under basal conditions and after LPS stimulation.
Additionally, the OHCs obtained from KO mice exhibited an altered microglial phenotype, since we noted abnormalities in the expression of genes related to both M1 and M2 phenotypes. The expression of the following factors was not detected: Il-1β, Tnf-α, Il- Control cultures were treated with the appropriate vehicle. NO release was measured using the Griess reaction. The data are presented as the mean percentages ± SEM of the control (vehicle-treated WT OHCs) from three independent experiments. The results were statistically evaluated using a two-way analysis of variance (ANOVA) with the Duncan post hoc test to assess the differences between the treatment groups. Significant differences are indicated by * p < 0.05. LDH-lactate dehydrogenase; NO-nitric oxide. +-with LPS or MR treatment; --without LPS or MR treatment.
We used an assay based on the Griess reaction to assess the effect of MR-39 on NO secretion from OHCs. OHCs were stimulated for 24 h, after which NO release was measured. As shown in Figure 2b, we did not observe an effect of either LPS (1 µg/mL) or MR-39 pretreatment on NO release in OHCs obtained from WT or KO mice.

The Effects of LPS and/or MR-39 on the mRNA Expression of Pro-Inflammatory and Anti-Inflammatory Factors in OHCs Obtained from the Offspring of WT and KO Mice
In the central nervous system, microglia are responsible for maintaining the physiological condition, while the shift from the M1-to M2-like profile is important in RoI. Generally, the M1-like phenotype is characterized by pro-inflammatory properties, while the M2-like phenotype is characterized by anti-inflammatory actions, which promotes tissue remodeling and repair. We decided to explore the effects of MR-39 on the pro-and anti-inflammatory phenotypes in OHCs from WT and KO mice under basal conditions and after LPS stimulation.
Additionally, the OHCs obtained from KO mice exhibited an altered microglial phenotype, since we noted abnormalities in the expression of genes related to both M1 and M2 phenotypes. The expression of the following factors was not detected: Il-1β, Tnf-α, Il-6, Il-23a, Il-10, IL-13, Fizz and Il-27. However, in the case of M1 phenotype-related factors, stimulation with LPS (1 µg/mL) increased the expression of Cd40. We assessed the anti-inflammatory and pro-resolving effects of the FPR2 agonist MR-39 on the production of the pro-inflammatory factors IL-1β, TNF-α and IL-6 and the anti-inflammatory factors IL-4, IL-10 and TGF-β in LPS-stimulated OHCs. Furthermore, to ensure that the observed effect was linked with interactions between ligand and FPR2, we pretreated cultures with the FPR2 antagonist WRW4.
Stimulation with LPS (1 µg/mL) significantly increased IL-1β levels in the medium of the WT (p < 0.0001) and KO (p < 0.0001) cultures, as shown in Figure 3a Moreover, in cultures obtained from KO mice, the level of TNF-α was higher than that in WT hippocampal cultures under basal conditions (Figure 3b; p < 0.0001). LPS (1 µg/mL) induced a significant upregulation of TNF-α production in the WT cultures (p < 0.02663) (Figure 3b). Importantly, a decrease in TNF-α secretion was observed after MR-39 (1 µM) treatment (p = 0.025743) only in WT hippocampal cultures, but was blocked by WRW4 (Figure 3b; p = 0.024152). These effects of MR-39 and WRW4 were not observed in the KO cultures. We did not observe statistically significant changes in IL-6 levels between hippocampal cultures obtained from WT and KO mice (under basal conditions). LPS (1 µg/mL) induced a significant increase in the production of this cytokine only in the KO cultures (Figure 3c; p < 0.0001). Therefore, the LPS (1 µg/mL)-induced stimulation of IL-6 release was significantly higher in the KO cultures than in the WT cultures (Figure 3c; p < 0.0001).
Next, we measured the effects of LPS and/or MR-39 on anti-inflammatory cytokine levels. Experiments conducted on WT and KO OHCs demonstrated that under basal conditions, no changes were detected in the levels of IL-4 and IL-10 between the examined cultures (Figure 4a,b). LPS (1 µg/mL) treatment increased the levels of the IL-4 and IL-10 proteins only in WT cultures (p < 0.0001). In the case of IL-4, MR-39 suppressed the effect of LPS stimulation, while WRW inhibited MR-39 actions (Figure 4a,b; p < 0.0001; p < 0.0001). This effect was not observed on the KO cultures. Additionally, we did not observe effects of MR-39 on IL-10 levels. Meanwhile, the significant decrease in TGF-β release in KO hippocampal cultures evoked by LPS (1 µg/mL) (Figure 4c; p < 0.0001) was not modulated by the MR-39 treatment.

The Effects of LPS and/or MR-39 on TLR4-Related Pathways in OHCs Obtained from the Offspring of WT and KO Mice
One of the most important targets of LPS is TLR4, whose activation causes the upregulation of downstream proteins, such as the MyD88 adapter protein and transcription factors, including NF-κB, consequently leading to the synthesis of inflammatory genes. Additionally, TRAF6 is critical for both MyD88-dependent and MyD88-independent downstream signaling pathways mediated by NF-κB pro-inflammatory responses. Thus, we examined the effects of LPS and/or MR-39 on MyD88 and TRAF6 protein levels.
None of the administered treatments affected the level of the MyD88 protein in the hippocampal cultures obtained from both WT and KO mice (Figure 5a). After LPS (1 µg/mL) stimulation, the level of TRAF6 was increased in cultures obtained from both WT (p = 0.018564) and KO (p = 0.04718) hippocampi (Figure 5b), but the MR-39 treatment was not able to effectively reverse these effects.
We did not observe any changes in the phospho-p65/total p65 ratio between WT and KO cultures under basal conditions. However, an increase in the phospho-p65/total p65 ratio was observed after LPS (1 µg/mL) stimulation in the WT (p = 0.015451) and KO cultures (Figure 5c; p < 0.0001). Moreover, the effect of LPS on the phospho-p65/total p65 ratio observed in the KO cultures was significantly stronger than that observed in the WT OHCs (p = 0.002216). Importantly, MR-39 only decreased the phospho-p65/total p65 ratio in the WT cultures (Figure 5c; p < 0.0001), while WRW4 blocked this effect (Figure 5c; p = 0.009803).

The Effects of LPS and/or MR-39 on the Levels of Proteins Involved in the NLRP3 Inflammasome Signaling Pathway in OHCs Obtained from the Offspring of WT and KO Mice
The NLRP3 inflammasome is a molecular platform within which inactive forms of the pro-inflammatory cytokines IL-1β and IL-18 are transformed into active forms. These cytokines are released from the cell to the extracellular space with the participation of NLRP3 to regulate the immune response in the central nervous system. Therefore, we estimated the effects of LPS and/or MR-39 on the protein levels of components of the NLRP3 inflammasome, such as NLRP3, caspase-1 and ASC.
No changes in the levels of examined proteins were observed under basal conditions in either of the examined groups (WT and KO). Stimulation with LPS (1 µg/mL) led to increased NLRP3 and Caspase-1 levels in both WT (p = 0.01030 and p = 0.002245, respectively) and KO (p = 0.04801 and p = 0.00353, respectively) hippocampal cultures (Figure 6a,b). Interestingly, MR-39 treatment effectively diminished the levels of the NLRP3 (p = 0.01402) and caspase-1 (p = 0.001881) proteins but only in WT hippocampal cultures (Figure 6a-c). Pretreatment with the antagonist WRW4 suppressed this effect on Caspase-1 (Figure 6b; p = 0.003693). On the other hand, none of the treatments affected the level of the ASC protein in the hippocampal cultures from the examined groups (Figure 6c). The results were statistically evaluated using a factorial analysis of variance (ANOVA) with Duncan's post hoc test to assess the differences between the treatment groups. Significant differences are indicated by * p < 0.05. ASC-apoptosisassociated speck-like protein containing a caspase recruitment domain; NLRP3-Nod-like receptor pyrin-containing 3 subunit; GSDMD-gasdermin D. +-with LPS, MR or WRW treatment; --without LPS, MR or WRW treatment.

Discussion
The present study provided several lines of evidence that the new FPR2 agonist MR-39 exerts anti-inflammatory and cell-protective effects on an ex vivo model of neuroinflammation based on hippocampal organotypic cultures exposed to bacterial endotoxin.
Regarding the effects of the new FPR2 agonist MR-39, it inhibited LDH release and attenuated the OHC inflammatory status by affecting pro-and anti-inflammatory gene expression, as well as IL-1β, TNF-α and IL-4 release. Moreover, our biochemical data indicated that inhibition of the NLRP3 inflammasome pathways appears to play a key role in the anti-inflammatory mechanism of MR-39 action. The FPR2 specificity of MR-39 was supported by the ability of the antagonist, WRW4, to abrogate its actions, as well as by the absence of favorable MR-39 activity in hippocampal cultures derived from knockout mice.
In the present study, we employed organotypic hippocampal cultures (OHCs) derived from 6-to 7-day-old offspring of both WT and FPR2 −/− KO mice. OHCs are innovative and reliable ex vivo models because they retain the neuronal-glia architecture and connectivity and permit studies of the effects of tested compounds on various cell types The results are presented as the means ± SEM. The data were obtained from three independent experiments. The results were statistically evaluated using a factorial analysis of variance (ANOVA) with Duncan's post hoc test to assess the differences between the treatment groups. Significant differences are indicated by * p < 0.05. ASC-apoptosisassociated speck-like protein containing a caspase recruitment domain; NLRP3-Nod-like receptor pyrin-containing 3 subunit; GSDMD-gasdermin D. +-with LPS, MR or WRW treatment; --without LPS, MR or WRW treatment.
Caspase-1 can also cleaves gasdermin D (GSDMD), which forms pores in the host cell membrane through which pro-inflammatory cytokines (IL-1β and IL-18) are released. On the other hand, GSDMD may induces the activation of the NLRP3 inflammasome and the associated proteolytic cleavage of pro-IL-1β and pro-IL-18. Thus, we also examined the effects of LPS and/or MR-39 on the GSDMD protein level.
In cultures obtained from KO mice, the level of GSDMD was higher than that in WT hippocampal cultures under basal conditions (Figure 6d; p = 0.046578). LPS (1 µg/mL) induced a significant upregulation of GSDMD production only in the WT (p = 0.048635) cultures. Importantly, after MR-39 (1 µM) pretreatment, the GSDMD level was decreased (p = 0.024790) in WT cultures, while this effect of MR-39 was blocked by WRW4 (Figure 6d; p < 0.0001). These effects were not observed on the KO cultures.

Discussion
The present study provided several lines of evidence that the new FPR2 agonist MR-39 exerts anti-inflammatory and cell-protective effects on an ex vivo model of neuroinflammation based on hippocampal organotypic cultures exposed to bacterial endotoxin.
Regarding the effects of the new FPR2 agonist MR-39, it inhibited LDH release and attenuated the OHC inflammatory status by affecting pro-and anti-inflammatory gene expression, as well as IL-1β, TNF-α and IL-4 release. Moreover, our biochemical data indicated that inhibition of the NLRP3 inflammasome pathways appears to play a key role in the anti-inflammatory mechanism of MR-39 action. The FPR2 specificity of MR-39 was supported by the ability of the antagonist, WRW4, to abrogate its actions, as well as by the absence of favorable MR-39 activity in hippocampal cultures derived from knockout mice.
In the present study, we employed organotypic hippocampal cultures (OHCs) derived from 6-to 7-day-old offspring of both WT and FPR2−/− KO mice. OHCs are innovative and reliable ex vivo models because they retain the neuronal-glia architecture and connectivity and permit studies of the effects of tested compounds on various cell types [39]. Furthermore, these ex vivo models enable pharmacological manipulations to investigate the mechanisms of inflammatory processes in the brain. FPR2 is expressed on microglia [40], astrocytes and hippocampal neurons [41]. OHC represents a particularly useful model for analyzing the role of new FPR2 ligands in the inflammatory response evoked by bacterial endotoxin.
In our research, we did not observe any changes in the dynamics/stabilization of OHCs obtained from WT and KO FPR2 mice or in lactate dehydrogenase (LDH) release evoked by LPS stimulation. As expected, MR-39 attenuated the LPS-induced increase of LDH levels in WT but not KO cultures. LDH is a stable cytoplasmic enzyme that is suddenly released into the cell culture medium because of cell membrane damages, and thus, it may be considered a key feature of cells undergoing apoptosis, necrosis and other forms of cell death [42]. Therefore, the inhibitory effect of MR-39 on LPS-induced LDH release in WT OHCs can be regarded as neuroprotective.
The endotoxin of gram-negative bacteria (lipopolysaccharide, LPS) is one of the most potent bacterial inducers of cytokine release, including the pro-inflammatory cytokines TNF-α, IL-1β and IL-6 [43], and gene expression of various other pro-inflammatory markers and factors. Consistent with these data, LPS increased the expression of Cd40 in both WT and KO cultures. Moreover, as expected, in WT cultures, we observed upregulated expression of the TNF-α, IL-1β, IL-6 and IL-23a mRNAs. Concurrently, MR-39 abolished the stimulatory effect of LPS administration on IL-1β, and IL-6 expression but only in WT cultures. Furthermore, by exploring the effect of MR-39 on cytokine levels under basal conditions, we showed the upregulation of TNF-α synthesis in OHCs from KO FPR2 mice compared with WT cultures. Simultaneously, LPS treatment induced a more potent increase in the secretion of TNF-α from KO FPR2 hippocampal cultures. Similarly, IL-1β and IL-6 release were increased in KO FPR2 cultures after LPS treatment, potentially suggesting that OHCs obtained from KO mouse offspring have an altered ability to respond to inflammatory stimuli due to the lack of FPR2, which balances the response towards the resolution of inflammation [44]. This hypothesis appears to confirm the lack of the upregulation of various anti-inflammatory mediators, including Ym-1, Cx3cr1, Il-10 and Il-13, as well as the release of the anti-inflammatory cytokines IL-10 and TGF-β in response to endotoxin stimulation observed in FPR2 KO hippocampal cultures. IL-10 exerts antiinflammatory effects at least in part by regulating IL-1β production. Moreover, LPS specifically activates IL-10, triggering the induction of IL-10 secretion, which efficiently prevented pro-IL-1β expression. Thus, the equipoise between IL-10 induction and the amount of pro-IL1β potentially determines the final level of IL-1β [45]. IL-10 may also exert an anti-inflammatory effect through the fractalkine receptor (CX3CR1), which is mainly expressed in microglial cells [46,47]. Consistent with the results reported by Cunha et al. [48], in the present study, we observed that LPS diminishes Cx3cr1 expression, but only in WT cultures. Thus, the upregulation of Cx3cr1 mRNA expression by MR-39 in the presence of FPR2 might reinforce the regulatory effect of IL-10 on IL-1β production. Therefore, our study suggests that the lack of FPR2 affects the balance between pro-and anti-inflammatory responses in hippocampal cultures, shifting the profile of expressed factors and cytokines towards pro-inflammatory mediators.
Intriguingly, MR-39 only inhibited the pro-inflammatory response induced by LPS in the WT hippocampal cultures, as evidenced by increased TNF-α and IL-1β production, while it was blocked by WRW4 pretreatment. This observation indicates that the favorable effect of MR-39 was mediated by its interaction with FPR2, probably predominantly on microglial cells, which are the main source of IL-1β and TNF-α. Accordingly, we did not detect an effect of MR-39 on the increased level of IL-6 in KO FPR2 cultures.
In addition to the favorable effect of MR-39 on the pro-inflammatory cytokine profile, the data from the present study showed that the absence of FPR2 limited the effect of MR-39 on IL-4 release. In the brain, IL-4 is mainly produced by astroglial cells and induces both pro-and anti-inflammatory responses, depending on the treatment and timing paradigm. In some studies, IL-4 treatment reduced NO production and iNOS protein synthesis, as well as the secretion of TNF-α upon LPS stimulation [49]; thus, IL-4 elicits neuroprotective phenotypes in astrocytes [50]. In the current study, the upregulation of Il-4 mRNA expression evoked by MR-39 pretreatment correlated at least in part with the upregulation of Arg-1 and Fizz expression because Il-4-dependent M2 polarization of microglia is widely postulated [51]. Moreover, neurons from IL-4−/− mice were less effective than WT neurons at attenuating an inflammatory response, indicating that the absence of IL-4 increases the vulnerability to neuroinflammation. On the other hand, an IL-1β pretreatment of primary mouse astrocytes increased IL-6 production when cells were subsequently treated with IL-4 [52]. Moreover, long-term exposure to IL-4 induces MHCII expression in microglial cells [53]. Hence, the potential biological functions of IL-4 are complex and depend on the environment through mechanisms that are probably mediated by different processes in different tissues or conditions. Thus, the effect of IL-4 on OHCs in which both neuronal and glial cells are present should be considered complex, while the observed effect of MR-39 is ambiguous and requires further research.
The primary downstream signaling pathways affected by LPS include the MyD66/ TRAF6/NF-кB pathway, whose activation leads to the synthesis of TNF-α, IL-1β and other inflammation-related factors [54]. Accordingly, in the next part of our study, we assayed the levels of proteins involved in the MYD88/TRAF6/NF-кB pathway in OHCs from both WT and KO FPR2 mice to provide more insights into the potential mechanism underlying the anti-inflammatory effect of MR-39. MR-39 had no effect on the MyD88 level and only tended to diminish the LPS-evoked increase in TRAF6 levels in WT hippocampal cultures. Nevertheless, the LPS treatment exerted a more pronounced effect on increasing the phospho-p65/p65 subunit ratio in KO FPR2 cultures compared with that in WT OHCs. We also found that the MR-39 treatment diminished the stimulatory effect of LPS on the NF-кB level only in WT cultures, and the WRW4 pretreatment blocked this effect of MR-39. Hence, we postulated that the anti-inflammatory activity of MR-39 toward TNF-α and/or IL-1β release was at least in part linked to the inhibition of the TRAF6/NF-кB pathway, while the presence of FPR2 was pivotal for this effect.
Growing pieces of evidence demonstrate that IL-1β is biologically inactive and must be cleaved and transformed into the bioactive form through the enzymatic activity of caspase-1. The proteolytic cleavage of procaspase-1 into active caspase-1 and maturation of IL-1β from its precursor form are triggered by NLRP3 inflammasome activation [55][56][57]. Activation of the NLRP3 inflammasome is strictly regulated. Indeed, its stimulation involves priming induced by the Toll-like receptor (TLR) and nuclear factor (NF-κB) [58]. Interestingly, activation of NF-κB evoked by lipopolysaccharide leads to the regulation of NLRP3 transcription by binding to NF-κB binding sites in the NLRP3 promoter [59,60]. Consequently, once primed, the following activation of the NLRP3 inflammasome, indicated as 'the second signal,' leads to the oligomerization of NLRP3 and the later assembly of NLRP3, ASC and procaspase-1 into a platform [61,62].
In the present study, LPS increased the levels of NLRP3 and caspase-1. We showed for the first time that MR-39 suppressed the increase in caspase-1 levels and that this effect was abolished by the WRW4 pretreatment, although only in WT cultures. This result suggests a possible association between FPR2 and the potential inhibitory effects of agonists of this receptor on NLRP3 pathways. In fact, annexin 1 (AnxA1), an agonist of FPR2, is required for IL-1β release in response to NLRP3 activators and is involved in NLRP3 inflammasome priming and assembly. Moreover, the absence of AnxA1 reduces the production and release of IL-1β in response to NLRP3 stimulation [63,64]. On the other hand, further studies are needed to determine whether this AnxA1 action depends on FPR2.
Notably, within the canonical pathway, the NLRP3 inflammasome serves as a platform for activating the proteolytic enzyme caspase-1, but this enzyme also processes gasdermin D (GSDMD) into a 30 kD fragment capable of oligomerizing and inserting in the plasma membrane, thus forming pores [65][66][67][68]. More advanced stages of GSDMD-mediated loss of membrane integrity result in pyroptosis. Nonetheless, membrane lysis and complete loss of its integrity are not an obstacle to the release of both pro-inflammatory cytokines-IL1β and IL-18-because of the presence of GSDMS pores. [69,70]. On the other hand, GSDMD may induce the activation of the NLRP3 inflammasome and the associated proteolytic cleavage of pro-IL-1β and pro-IL-18; thus, the GSDMD-NLRP3 interaction is complex and reciprocal.
Here, we documented increased GSDMD levels under basal conditions in KO FPR2 hippocampal cultures, which were affected neither by additional LPS stimulation nor by MR-39 treatment. However, LPS stimulation of WT hippocampal cultures increased GSDMD levels, which were reduced by the administration of MR-39, while WRW4 blocked these effects. This observation is in line with recent data pointing that LPS may directly targeting GSDMD [71]. We are fully aware that our present data have some methodological limitations (including the lack of evaluation of the caspase-1 activity and/or GSDMD cleavage); nonetheless, they shed more light on the MR-39 potential in the modulation of the NLRP3-related pathways in the inflammatory response.

Conclusions
Taken together, the results of the present study showed that the absence of FPR2 in hippocampal cultures leads to an aberrant inflammatory response to LPS stimulation and that the new FPR2 agonist MR-39 is a powerful inhibitor of some neuroinflammatory events. Moreover, our findings provide more insights into pathways inhibiting the NLRP3 inflammasome as a new molecular platform engaged in the protective effects of MR-39 on restraining inflammation in the brain. In our opinion, FPR2 offers a broad perspective for the development of promising new pro-resolving mediators, which deserve to be studied in further preclinical studies as candidates for the treatment of some brain disorders.