Gut bacterial metabolite Urolithin A (UA) mitigates Ca2+ entry in T cells by regulating miR-10a-5p

The gut microbiota influences several biological functions including immune response. Inflammatory bowel disease is favourably influenced by consumption of several dietary natural plant products such as pomegranate, walnuts and berries containing polyphenolic compounds such as ellagitannins and ellagic acid. The gut microbiota metabolises ellagic acid leading to formation of bioactive urolithins A, B, C and D. Urolithin A (UA) is the most active and effective gut metabolite and acts as a potent anti-inflammatory and anti-oxidant agent. However, how gut metabolite UA affects the function of immune cells remained incompletely understood. T cell proliferation is stimulated by store operated Ca2+ entry (SOCE) resulting from stimulation of Orai1 by STIM1/STIM2. We show here that treatment of murine CD4+ T cells with UA (10 µM, 3 days) significantly blunted SOCE in CD4+ T cells, an effect paralleled by significant downregulation of Orai1 and STIM1/2 transcript levels and protein abundance. UA treatment further increased miR-10a-5p abundance in CD4+ T cells in a dose dependent fashion. Overexpression of miR-10a-5p significantly decreased STIM1/2 and Orai1 mRNA and protein levels as well as SOCE in CD4+ T cells. UA further decreased CD4+ T cell proliferation. Thus, bacterial metabolite UA up-regulates miR-10a-5p thus interfering with Orai1/STIM1/STIM2 expression, store operated Ca2+ entry and proliferation of murine CD4+ T cells.


miR-10a-5p overexpression and inhibition inversely influence SOCE in CD4 + T cells 167
To examine, whether the downregulation of Orai1 and STIM1/2 expression following miR-10a-168 5p overexpression was paralleled by a similar decrease of SOCE, both control mimic and miR-10a-5p 169 mimic transfected CD4 + T cells were activated for 3 days in the presence of plate-bound anti-CD3 and 170 anti-CD28 (1:2 ratio). Ca 2+ entry was measured at day 3 after transfection of miR-10a-5p 171 overexpression using miRNAs mimic. The activated cells were again loaded with Fura-2 for 30 minutes 172 in standard HEPES and washed once with standard HEPES. [Ca 2+ ]i was measured first in standard 173 HEPES, which was subsequently replaced by Ca 2+ -free HEPES. In a next step the intracellular Ca 2+ 174 stores were depleted by addition of thapsigargin (1 µM) in the nominal absence of extracellular Ca 2+ . 175 The subsequent re-addition of extracellular Ca 2+ was followed by a sharp increase of [Ca 2+ ]i. Both, 176 slope and peak of the [Ca 2+ ]i increase were significantly lower in miR-10a-5p mimic transfected than 177 in control mimic transfected cells (Fig. 5A&B). Thus, our data suggest indeed that overexpression of 178 miR-10a-5p contributes to the downregulation of Orai1 and STIM1/2 expression following UA 179 treatment. Conversely, inhibition of miR-10a-5p augments significantly both slope and peak of the 180 [Ca 2+ ]i increase following Ca 2+ re-addition (Fig. 5C&D). Our gain-of-function and loss-of-function data 181 suggested that indeed miR-10a-5p is a powerful regulator of SOCE. 182

Effect of UA on cell proliferation 184
As stimulation of SOCE is involved in the signalling triggering T-cell proliferation (25), cell 185 proliferation was quantified using the dye CFSE. As illustrated in Fig. 6, cell proliferation was 186 significantly decreased in the presence of 10 µM UA. 187

DISCUSSION 189
The polyphenolic compounds are known to control inflammation, angiogenesis, drug and 190 radiation resistance (1). These findings have been further corroborated by the observations that 191 Ellagitannin-rich food have beneficial effects on inflammatory bowel disease and other inflammatory 192 diseases (2). However, the bioavailability of these compounds (ellagitannins and ellagic acid) is limited 193 and the compounds must be metabolized by the gut microbiota to produce bioactive molecules that can 194 be easily absorbed (7). Ellagic acids (by the help of gut bacteria) are converted into different Urolithins 195 (A-D) (7). Gut metabolites such as Urolithins can directly activate adaptive immune T cells (8-11). In 196 this report, we reveal that UA can suppress store operated Ca 2+ entry by modulating Orai/STIM 197 regulating miRNAs, thus affecting Ca 2+ sensitive cellular functions including CD4 + T cell proliferation. 198 High levels of intracellular Ca 2+ are necessary to maintain numerous functions of T cells such 199 as the interaction between a T-cell and antigen-presenting cell (APC) that leads to formation of the 200 specialized contact surface known as the immunological synapse and activation of different 201 transcription factors (12,18,22,33,34). Several hours of oscillating Ca 2+ influx are required to complete 202 the T-cell activation program, which involves expression of a large number of activation-associated 203 genes (25). Our observations describe for the first time that bacterial metabolite product UA is a 204 negative regulator of store operated Ca 2+ entry into murine CD4 + T cells, an effect paralleled by 205 downregulation of Orai1/STIM1/2 expression. Thus, the present observations also uncover a 206 completely novel mechanism accounting for the effect of UA on SOCE, i.e. the upregulation of miR-207 10a-5p, which in turn downregulates Orai1 and STIM1/2 transcript and protein levels as well as SOCE. 208 Thus, UA changes the post-transcriptional machinery of the key players undertaking SOCE in CD4 + T 209 cells, i.e. Orai1 and STIM1/2. 210 Emerging evidence indicates that UA is involved in the regulation of inflammatory pathways, 211 cell cycle and cell death (1,35,36). Some studies further suggested that UA is also involved in 212 controlling the growth of cancer cells, and could thus be used as anti-cancer agent (1,35). It is tempting 213 to speculate that UA interferes similarly with tumor cell proliferation by downregulating SOCE. 214 Recently, we have demonstrated that miRNAs processing protein dicer is involved in the 215 regulation of SOCE in CD4 + T cells (30). Thus, identifying the role of individual miRNAs which could 216 be involved in the regulation of Ca 2+ pathways open a new avenue to therapeutic intervention. Using 217 bioinformatics tools, we found that miR-10-5p could regulate Orai1 proteins thus modifying STIM1/2 218 expression. Previous studies have reported that miR-10a-5p is involved in the development and function 219 of regulatory T cells (Tregs) (37). Our results reveal that miR-10a-5p was upregulated after treatment 220 with UA in T cells. Thus, miR-10a-5p appears to be involved in the regulation of Ca 2+ entry and thus 221 Ca 2+ sensitive cellular functions such as gene expression, proliferation, cell motility and cytokine 222 expression. However, a role of other miRNAs or further signalling pathways cannot be excluded. 223 In conclusion, the present observations reveal a completely novel role of gut bacterial 224 metabolite UA in the regulation of Ca 2+ entry into CD4 + T cells leading to suppression of activation of 225 CD4 + T cell activation. UA upregulates the expression of miR-10a-5p which in turn decreases SOCE 226 by downregulating Orai1 and STIM1/2 expression. Thus, our results suggest that upregulation of miR-227 10a-5p by UA restrains store operated Ca 2+ entry in murine CD4 + T cells and UA could be used a natural 228 immune-suppressant during various inflammatory disorders including inflammatory bowel disease. 229

MATERIALS AND METHODS 231
Mice 232 Naïve CD4 + T Cells were isolated from C57BL/6 mice (male and female) between 8 -16 weeks 233 of age. All the animals were kept in standard housing conditions with 12 ± dark/light cycle and fed on 234 Fluorescence measurements were performed using an inverted light incidence fluorescence phase-249 contrast microscope (Axiovert 100, Zeiss, Germany). Cells were excited alternatively at λ = 340 or 380 250 nm and the light deflected by a dichroic mirror into either the objective (Fluar 40×/1.30 oil, Zeiss, 251 Germany) or a camera (Proxitronic, Germany). Emitted fluorescence intensity recorded at λ = 505 nm 252 and data were acquired by using specialized computer software (Metafluor, Universal Imaging, USA) 253

310
Immunoblotting 311 CD4 + T cells were activated in presence of anti-CD3 (1µg/ml)/anti-CD28 (2µg/ml) and treated 312 with 10 µM UA. After 72 hours of activation and treatment, CD4 + T cells were washed once with PBS, 313 counted and equal amounts cells were taken for cell lysis using H 2 O and 2×Lammelli's Buffer. Proteins 314 were denatured at 95 0 C for 5-10 minutes and stored at -20 0 C. Sample proteins were loaded on 8% or 315 10% gel depending on protein size and run at 80 V until crossing of stacking gel then voltage was 316 increased to 120 V during the separation phase and total gel run for 90-100 minutes. Proteins were 317 electro-transferred onto PVDF membranes. Membranes were probed with the indicated primary  Data were analysed by ImageJ software (https://imagej.nih.gov/ij/). 324

CFSE staining 326
The proliferation of CD4 + T cells was detected by CellTrace TM CFSE Cell Proliferation Kit 327 (#C34554, eBioscience, USA). Briefly, cells were washed with PBS (#D8537, Sigma, Germany) once, 328 stained with CellTrace TM CFSE (1:1000 dilution) and re-suspended gently, incubated at 37 0 C for 15 329 minutes in the dark, then washed with R-10 medium twice and activated as described in CD4 + T cell isolation and culture in Materials and Methods above. After 72 hours, cells were collected to perform 331 the flow cytometry. Data were analysed by Flowjo software (FLOWJO LLC, USA). 332 333

Statistics 334
Data are provided as means ± SEM, n represents the number of independent experiments. All 335 data were tested for significance using unpaired Student's t-test or ANOVA. Data were analysed by 336 Excel 2010 or GraphPad Prism Software, USA. P value ≤0.05 was considered statistically significant. 337 338