The gut microbiota metabolite urolithin A inhibits NF-κB activation in LPS stimulated BMDMs

Inflammation is a natural defense process of the innate immune system, associated with the release of proinflammatory cytokines such as interleukin-1β, interleukin-6, interleukin-12 and TNFα; and enzymes including iNOS through the activation and nuclear translocation of NF-κB p65 due to the phosphorylation of IκBα. Regulation of intracellular Ca2+ is considered a promising strategy for the prevention of reactive oxygen species (ROS) production and accumulation of DNA double strand breaks (DSBs) that occurs in inflammatory-associated-diseases. Among the metabolites of ellagitannins that are produced in the gut microbiome, urolithin A (UA) has received an increasing attention as a novel candidate with anti-inflammatory and anti-oxidant effects. Here, we investigated the effect of UA on the suppression of pro-inflammatory molecules and NF-κB activation by targeting TLR4 signalling pathway. We also identified the influence of UA on Ca2+ entry, ROS production and DSBs availability in murine bone-marrow-derived macrophages challenged with lipopolysaccharides (LPS). We found that UA inhibits IκBα phosphorylation and supresses MAPK and PI3K activation. In addition, UA was able to reduce calcium entry, ROS production and DSBs availability. In conclusion, we suggest that urolithin A is a promising therapeutic agent for treating inflammatory diseases through suppression of NF-κB and preserving DNA through maintaining intracellular calcium and ROS homeostasis.


Supplementary Figure Legends
Supplementary Figure 1 Effect of urolithin A or DMSO on superoxide production in murine BMDMs Murine BMDMs were treated either by UA (25 µM or 50 µM) or DMSO for 48h. Both DMSO and UA did not record any observable changes. Nuclei were counterstained with DAPI (blue). Scale bar represents 50 μm. Murine BMDMs treated with DMSO were used as negative control. Abbreviations: DMSO, dimethyl sulfoxide; UA, urolithin A.

Supplementary Figure 2 Influence of urolithin A or DMSO on DSBs in murine BMDMs after 48h
Murine BMDMs were treated either by UA (25 µM or 50 µM) or DMSO for 48h. Both UA and DMSO did not register any significant effect on DSBs after 48h. Nuclei were counterstained with DAPI (blue). Scale bar represents 50 μm. BMDMs treated with DMSO were used as negative control. Abbreviations: DMSO, dimethyl sulfoxide; UA, urolithin A.

Supplementary Figure 4 Sensitivity of TLR4 expressions to urolithin A (UA) in LPS-stimulated murine BMDMs
Murine BMDMs were stimulated by 1µg/ml of LPS in the presence or absence of UA (25 µM or 50 µM), then harvested and subjected to western blot after 72h. (a) Bar graph represents relative band intensities of TLR4 normalized to GAPDH. The unstimulated and untreated BMDMs were used as control. BMDMs treated with DMSO were used as negative control. (b) Representative image of TLR4 protein expression assessed by western blot analysis. Data are shown as means ± SEM from four independent experiments. One way ANOVA was used and * (p < 0.05), ** (p < 0.001), and *** (p < 0.001) indicate statistically significant differences compared to untreated control. + (p < 0.05) indicate statistically significant differences compared to LPS. Abbreviations: DMSO, dimethyl sulfoxide; LPS, lipopolysaccharides; UA, urolithin A; TLR, Toll like receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Supplementary Figure 6 FACS phenotyping of BMDMs
Naive bone marrow cells were isolated and subsequently cultured for one week in DMEM complete media (M0) and then analyzed for macrophage markers. (a) FACS histogram displaying the harvested BMDMs (M0) which were defined as F4/80 high , CD11b high and MHCII high . (b) Arithmetic means ± SEM (n = 5) of percentage of the positive gated populations of M0.

Supplementary Figure 7 FACS phenotyping of BMDMs after 72h of stimulation
Naive bone marrow cells were isolated and subsequently cultured for one week in DMEM complete media and then stimulated with 1µg/ml LPS in the presence or absence of UA (25 µM or 50 µM). (a) FACS histogram displaying the expression of macrophage marker after 72h of desired treatment using CD11b, F4/80 and MHCII. Arithmetic means ± SEM (n = 5) of percentage of the positive gated populations of stimulated macrophages which were defined as CD11b high (b), F4/80 high (c) and MHCII high (d). * (p<0.05) and ** (P<0.001) indicates statistically significant difference compared to control. Abbreviations: DMSO, dimethyl sulfoxide; UA, urolithin A; RAD, radiation.

Supplementary Figure 8 Influence of urolithin A on the viability of LPS-stimulated murine BMDMs after 72h
Naive bone marrow cells were isolated and subsequently cultured for one week in DMEM complete media and then stimulated with 1µg/ml LPS in the presence or absence of UA (25 µM or 50 µM). (a) Dot plots of mature BMDMs which were treated as indicated. After 72h the cell viability was measured by Annexin-V /PI compared to untreated control cells. (b) Arithmetic means ± SEM (n = 5) of BMDMs which were treated with different concentrations of UA for 72h. (c) Values show the percentage of live BMDMs Annexin-V -/PIafter indicated treatments. Arithmetic means ± SEM (n = 5). *** (P<0.001) and **** (P<0.0001) indicate statistically significant differences compared to control. Abbreviations: DMSO, dimethyl sulfoxide; UA, urolithin A; LPS, lipopolysaccharides.

Supplementary western blots (whole bands)
In this project only the first seven bands were used and the other bands belong to another project.
 Prosieve ladder 300 kda (Lonza) was used (see image below).  Usually the membrane was cut at 70 kda and the upper part was used for TLR4, mTOR, phospho-mTOR and the lower part for the other depicted proteins.  The membrane was first incubated with antibodies against the phospho-protein, then stripped and controlled, blocked and incubated with the antibodies against the total protein.  Either phospho-or total proteins were controlled to their corresponding GAPDH.