An increased autophagic flux contributes to the anti-inflammatory potential of urolithin a in macrophages

: Background: An extract of Phyllanthus muellerianus and its constituent geraniin have been reported to exert anti-inflammatory activity in vivo . However, orally consumed geraniin, an ellagitannin, shows low bioavailability and undergoes metabolization to urolithins by gut microbiota. This study aimed at comparing geraniin and urolithin A with respect to inhibition of M1 (LPS) polarization of murine J774.1 macrophages and shedding more light on possible underlying mechanisms. Methods: Photometric, fluorimetric as well as luminescence-based assays monitored production of reactive oxygen species (ROS) and nitric oxide (NO), cell viability or reporter gene expression. Western blot analyses and confocal microscopy showed abundance and localization of target proteins, respectively. Results: Urolithin A is a stronger inhibitor of M1 (LPS) macrophage polarization (production of NO, ROS and pro-inflammatory proteins) than geraniin. Urolithin A leads to an elevated autophagic flux in macrophages. Inhibition of autophagy in M1 (LPS) macrophages overcomes the suppressed nuclear translocation of p65 (NF-kB; nuclear factor kB), the reduced expression of pro-inflammatory genes as well as the diminished NO production brought about by urolithin A. The increased autophagic flux is furthermore associated with impaired Akt/mTOR (mammalian target of rapamycin) signaling in urolithin A-treated macrophages. Conclusions and general significance : Intestinal metabolization may boost the potential health benefit of widely consumed dietary ellagitannins, as suggested by side by side comparison of geraniin and urolithin A in M1(LPS) macrophages. Increased activity of the autophagic cellular recycling machinery aids the anti-inflammatory bioactivity of urolithin A.

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Introduction
Phyllanthus muellerianus belongs to the family Euphorbiaceae, and leaves, bark and fruits are used in Traditional African Medicine for the treatment of dysentry, wounds and inflammatory disorders, among others [1]. Our recent study confirmed anti-inflammatory activity of an orally administered aqueous Phyllanthus leaf extract in rats and assigned part of its activity to its constituent geraniin [2].
Geraniin is an ellagitannin made up of a galloyl, a hexahydrodiphenoyl and a dehydrohexahydrodiphenoyl group esterified to a glucose molecule, most prominently present in different plant parts (leaf, root, seed, fruit) of the genera Euphorbiaceae, Geraniaceae and Phyllanthaceae [3]. Ellagitannins are generally very poorly absorbed from the gastrointestinal tract, possibly due to their bulky structure which does not facilitate simple diffusion. Therefore, metabolization of geraniin by intestinal microflora into ellagic acid, gallic acid, urolithin A, B and C and other related compounds is crucial for absorption [4,5]. Several bioactivities were assigned to geraniin, including improved wound healing [6], antiviral [7][8][9], growth inhibitory [10,11], hepato-, neuro-and cytoprotective [12][13][14][15][16] as well as anti-inflammatory [17] properties. However, given the low bioavailability and metabolization of geraniin it is questionable whether mere in vitro data may become relevant in an in vivo setting or whether the active principle beyond the observation made upon oral administration of geraniin is mainly the parent ellagitannin. In this line, it is of note that the reported bioactivities of urolithins partly overlap with those of geraniin, such as growth inhibition and

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4 produce pro-inflammatory cytokines and mediators (interleukin (IL)-12, IL-6, IL-1, tumor necrosis factor (TNF)-, reactive oxygen species (ROS) and reactive nitrogen species produced by inducible NO synthase (iNOS) or eicosanoids). In contrast, M2 polarization occurs upon exposure to cytokines connected with Th2 responses (such as IL-4 or IL-13). These cells are considered anti-inflammatory, produce IL-10 and highly express arginase 1 as well as scavenger receptors [24][25][26]. Despite being a vital defense reaction in the acute setting, ongoing chronic inflammation and production of cytokines and other pro-inflammatory factors can lead to tissue damage and is associated with chronic diseases, such as inflammatory bowel disease, the metabolic syndrome or declining cognitive function [27,28].
Therefore, compounds able to prevent or alleviate M1 polarization or boost the M1 M2 shift may be promising candidates for countering those disorders and understanding their mode of action may even open up new therapeutic and intervention strategies.
Prompted by the results from our previous in vivo study with geraniin and from observations that ellagitannin-rich diets appear beneficial for patients with e.g. intestinal inflammation [29,30], we aimed in this study at comparing geraniin and the selected metabolite urolithin A side-by-side in their impact on M1 macrophage polarization and revealing the potential underlying anti-inflammatory mechanism.
A C C E P T E D M A N U S C R I P T to determine nitrite as surrogate readout for NO release. Absorbance of the pink azo-dye was assessed at 550 nm. To the cells (still covered with 100 µL medium), 100 µL of the ready-to-go Cell TiterGlo solution (Promega, Austria) were added, the plate was incubated in the dark for one hour and then luminescence was recorded using a multiplate reader (Tecan, Austria).

Determination of intracellular reactive oxygen species (ROS)
A C C E P T E D M A N U S C R I P T 6 Cells were treated as desired and then exposed to the ROS sensitive dye H2DCF-DA (20 µM) for 30 minutes. Cells were washed and mean green fluorescence of 20 000 cells /sample was analyzed by flow cytometry (FL1 channel) with a FACSCalibur™ (BD Biosciences, Austria) instrument. Obtained values were corrected for autofluorescene of control cells without dye.

Western blot analysis
After the desired cell treatment extraction of total cell lysates, SDS-polyacrylamide electrophoresis and immunoblot analysis were performed essentially as described previously [32]. To separate nuclear from cytosolic proteins, cells were first washed with cold PBS and then exposed to buffer 1 (10 mM Successful separation of cytosolic and nuclear fractions was routinely validated by immunoblotting of tubulin (cytosolic marker) and lamin (nuclear marker), respectively.

Reporter gene assays
Peroxisome proliferator activated receptor (PPAR), liver X receptors (LXR)  and retinoid X receptor (RXR) reporter gene assays utilized HEK293 cells that were transiently transfected with the expression plasmid and response element of the respective nuclear receptor as well as a plasmid coding for enhanced green fluorescent protein (EGFP) as control. The nuclear factor erythroid 2 related factor 2 (Nrf2)-dependent reporter gene assay was done in CHO cells stably expressing an ARE-dependent luciferase reporter gene and a control EGFP expression plasmid. The procedure was essentially the one described in previous studies [31, 33, 34].

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Confocal laser scanning microscopy
Cells were grown on coverslips and treated as indicated. The coverslips were then washed with PBS and cells were fixed with 3.7% formaldehyde in PBS followed by permeabilization with 0.5 % Triton® X-100 in PBS. After two washing steps with PBS, the coverslips were incubated with blocking solution (2% BSA in PBS) for 30 minutes, then with anti-p65 antibody (1:50 in PBS/BSA) for two hours, washed and then exposed to FITC-coupled anti-rabbit antibody (1:500 in PBS/BSA) for another hour. After washing, the coverslips were mounted on a glass slide using ProLong antifade-mounting medium (New England Biolabs, Germany) and kept at 4°C overnight. Confocal laser scanning microscopy was performed using a TCS SP5 II system (Leica, Germany). Images were recorded with a 63x oil immersion objective using the manufacturer's LAS AF imaging software. All compared samples within one experiment were detected with constant gain, zoom and exposure time.

Statistics
Experiments were performed at least three times. Error bars in the pictures represent the standard deviation (SD). Statistical significance was determined by using Student's t test (for two groups) or ANOVA (>2 groups) followed by Dunnett´s or Bonferroni's post test in the GraphPad Prism software.
P-values <0.05 were considered as significant and are designated with * in the figures.

Urolithin A is more potent in impeding M1(LPS) macrophage polarization than geraniin
In a first step, we compared the influence of geraniin and urolithin A ( A direct side by side comparison revealed that the metabolite urolithin A hinders the M1(LPS) polarization and is hereby more potent and versatile than the parent ellagitannin geraniin, suggesting that microbial metabolization boosts the anti-inflammatory potential.

Urolithin A and geraniin have no influence on peroxisome proliferator activated receptor (PPAR), liver X receptors (LXR), retinoid X receptor  (RXR) or nuclear factor erythroid 2 related factor 2 (Nrf2)-mediated gene transactivation
The anti-inflammatory properties of Urolithin A and geraniin have been mainly assigned to an impaired nuclear factor  B (NF-B) signaling in previous studies [21,36]. However, other anti-inflammatory targets have not been investigated to a great extent. We therefore examined potential agonism of geraniin and urolithin A on nuclear receptors with anti-inflammatory properties. Those studies

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9 included PPAR, LXR and , which counter pro-inflammatory gene expression and seem to determine macrophage polarization also by controlling lipid metabolism [37][38][39] as well as RXR, the heterodimerization partner of the permissive PPAR and LXR. However, neither geraniin nor urolithin A was able to markedly activate luciferase expression in the respective reporter gene assay, whereas the used positive controls (pioglitazone for PPAR, GW 3965 for the LXRs and retinoic acid for RXR) elicited significant activation. (Figures 2 a-d). At 50 µM, urolithin A was able to slightly (approx.

Urolithin A leads to an elevated autophagic flux in macrophages
A recent study reported life span prolongation in the nematode C. elegans by urolithin A-mediated induction of autophagy [42]. The authors also investigated induction of autophagy in myo-and hepatocytes, but not in macrophages. To close this gap and because autophagy and In the presence of bafilomycin urolithin A significantly increased the LC3II level compared to DMSO,

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10 indicating an increased autophagic flux in naïve and LPS-stimulated macrophages (Figure 3a) [46]. As expected, the mTOR inhibitor rapamycin also led to an increased autophagic flux. Moreover, the proautophagic effect of urolithin A occurred in a concentration dependent manner ( Figure 3b). Thus, urolithin A is able to enhance the autophagic flux in macrophages.

Urolithin A interferes with the Akt/mTOR signaling pathway
Autophagy is a highly regulated and finely tuned complex process that integrates cues from a plethora of signaling molecules. However, mTOR and AMP-activated kinase (AMPK) are considered as main control hubs of autophagy [47][48][49][50]. Time course experiments revealed that LPS-treatment leads to an increase in the Akt/mTOR/p70 S6K signaling axis after approximately 1 hour in macrophages that lasts up to 8 hours (suppl Figure. 1). An increased phosphorylation and activation of AKT is transduced to an increased phosphorylation of tuberin (TSC2) at threonine 1462 which in turn leads to activation of the mTOR complex via the small G-protein Rheb. Activated mTOR finally phosphorylates its substrates including p70S6K at Ser 389 [51]. In order to examine the influence of urolithin A on the mTOR signaling pathway, macrophages were treated with 20 and 40 µM urolithin A in the absence or presence of LPS for 2 and 6 hours. Subsequent western blot analysis of total cell lysates revealed that urolithin A was able to suppress LPS-induced AKT, TSC2 and as well as basal and LPS-induced p70S6K phosphorylation (Figures 4 a-c). In contrast, levels of phosphorylated (Ser79) acetyl-CoA carboxylase (ACC) as readout for AMPK activity did not obviously change between control and urolithin A -treated cells (Suppl. Figure   2). Thus, urolithin A is able to interfere with the mTOR signaling pathway in macrophages which is likely to account for the observed increase in autophagic flux.

The increased autophagic flux contributes to the prevention of M1(LPS) macrophage polarization by urolithin A
In a next step we examined whether the increased autophagic flux is linked with the anti-inflammatory activity of urolithin A. The most primordial function of autophagy is adaptation to nutrient deprivation.
However, the autophagy machinery has also been found to entangle with immunity and inflammation

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11 [43][44][45]. Monitoring LPS-triggered NO production uncovered inhibition by urolithin A (in line with we went on to investigate the relationship between increased autophagy and inhibited nuclear accumulation of p65 (NF-B) in urolithin A-treated macrophages. One hour after LPS stimulation appeared as optimal time point to study nuclear p65 in the used murine macrophages (Suppl. figure   3). Employing confocal laser scanning microscopy ( Figure 6b) and western blot analysis of nuclear extracts (Figure 6c) confirmed that urolithin A impedes nuclear accumulation of p65. Of note, cotreatment with the autophagy inhibitor bafilomycin overcame the blunted nuclear translocation of p65 by urolithin A. Bafilomycin alone had no influence on nuclear p65 levels. These data imply that

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12 urolithin A interferes with NF-B (p65) function at a step between release from the IB complex and transport into the nucleus in an autophagy-dependent manner.

Discussion
The main findings of this study are that (i) microbial metabolization of geraniin to urolithin A increases the anti-inflammatory potential, (ii) urolithin A triggers an increased autophagic flux in macrophages which contributes to reduced nuclear abundance of p65 (NF-B) and M1(LPS) polarization and that (iii) urolithin A exerts a negative influence on the AKT/TSC/mTOR signaling axis.
The concept that the intestinal microbiome is a critical player in metabolization and exploitation of ingested food and drugs is commonly accepted. For instance, metabolization of dietary fiber (precursor) into short chain fatty acids (active principle), such as butyric acid acting mainly as epigenetic modulator, is thought to bring about the benefits of a fiber-rich diet [52,53]. Secondary Geraniin and urolithin A did not show a marked agonism on the nuclear receptors PPAR, RXR and LXR or on activation of the stress responsive transcription factor Nrf2. Their polar nature may prevent binding to the nuclear receptors that usually habour lipopilic ligands, such fatty acids or sterols. Accordingly, in endothelial cells urolithin A led to increased PPARexpression rather than acted as ligand [64]. The putative positive impact on PPARsignaling due to increased abundance could not be confirmed in macrophages, though, as we did not see increased PPAR levels nor target gene expression upon urolithin A exposure (data not shown). Although reported for geraniin in previous studies [35,65], we did not observe any activation of Nrf2 sigalling with urolithin A or geraniin. The

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15 reasons for the divergent results on geraniin may be based on the different readouts for Nrf2 activation (Nrf2 accumulation/ heme oxygenase 1 expression [35] versus consensus sequence-based reporter gene assays (ARE-LUC (here)), different experimental setups (pre-stressed cells [35] versus naïve cells (here)) or an actual metabolite of geraniin being the active principle for Nrf2 activation in the in vivo setting [65].
Looking at the two major prominent control hubs in autophagy, we showed unaltered AMPK activity but impaired mTOR signaling in urolithin-treated cells. Phosphorylation of p70 S6K, indicative for an active mTOR signaling, was strongly and concentration-dependently decreased by urolithin A treatment in naïve and LPS-stimulated macrophages. Therefore, induction of autophagy is likely to occur via an inhibited mTOR axis in urolithin-exposed M1(LPS) macrophages. Notably, mTOR inhibition occurred as anti-inflammatory strategy in several recent publications [e.g. 66,67], but was also found to aggravate (adipose tissue) inflammation in other studies [68,69]. More studies are needed to completely decipher the context-and tissue specific action of mTOR in inflammation, to understand the possible distinct responses to pharmacological mTOR inhibition and absolute mTOR deficiency as well as the detailed mode of action underlying the augmented autophagic flux upon urolithin A.
Urolithin A also increased the life span of the nematode C.elegans via increased autophagy [42], and was reported to inhibit proliferation of various cancer cell lines, to exert vaso-and cardioprotective effects or improve lipid profiles [18,19,58,64,70,71]. To what extent autophagy induction plays a role for these effects has not been investigated so far although there is good chance that autophagy is

Acknowledgments
The authors thank Drs. Simone Latkolik and Angela Ladurner, University of Vienna, for her initial help with reporter gene assays in HEK cells. This work was supported by the FWF (P29392 to EHH), the Herzfelder Jubiläumsstiftung (to EHH) and a Willmar Schwabe research fellowship from the Society of Medicinal Plant and Natural Product Research (to YDB).