Butyrate mediates anti-inflammatory effects of Faecalibacterium prausnitzii in intestinal epithelial cells through Dact3

Butyrate mediates anti-inflammatory effects of Faecalibacterium prausnitzii in intestinal epithelial cells through Dact3 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Butyrate mediates anti-inflammatory effects of Faecalibacterium prausnitzii in intestinal epithelial cells through Dact3 Marion Lenoir, Rebeca Martin, Edgar Torres-Maravilla, Sead Chadi, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-28864/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Oct, 2020 Read the published version in Gut Microbes → Version 1 posted You are reading this latest preprint version Abstract Background The commensal bacterium Faecalibacterium prausnitzii plays a key role in inflammatory bowel disease (IBD) pathogenesis and serves as a general health biomarker in humans. However, the host molecular mechanisms that underlie its anti-inflammatory effects remain unknown. Methods A transcriptomic approach on human intestinal epithelial cells (HT-29) that were stimulated with TNF-α and exposed to F. prausnitzii culture supernatant (SN) was used. Modulation of the most upregulated gene after F. prausnitzii SN contact was validated both in vitro and in vivo . Results F. prausnitzii SN upregulates the expression of Dact3 , a gene linked to the Wnt/JNK pathway. Interestingly, when we silenced Dact3 expression, the effect of F. prausnitzii SN was lost. Butyrate was identified as the F. prausnitzii effector responsible for Dact3 modulation. Dact3 upregulation was also validated in vivo in both healthy and inflamed mice treated with either F. prausnitzii SN or the live bacteria, respectively. Finally, we demonstrated by colon transcriptomics that gut microbiota directly influences Dact3 expression. Conclusions Our results provide new clues about the host molecular mechanisms involved in the anti-inflammatory effects of the beneficial commensal bacterium F. prausnitzii . *Contributed equally to this work General Microbiology Commensal bacteria Faecalibacterium prausnitzii inflammatory bowel disease transcriptomic analysis signaling pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Inflammatory bowel disease (IBD) is a group of disorders characterized by chronic inflammation in the gastrointestinal tract [ 1 , 2 ]. One potential cause (and/or consequence) of IBD is the disruption of the intestinal ecosystem equilibrium. For example, gut microbiota analysis of Crohn’s disease (a type of IBD) patients revealed markedly lower diversity of Firmicutes (in particular of the Clostridium leptum group) compared to healthy individuals [ 3 ]. These assemblages are also relatively poor in Faecalibacterium prausnitzii , a major member of the C. leptum group and one of the most abundant intestinal bacteria in healthy adults [ 4 , 5 ]. Because a potential approach to prevent and treat IBD is the oral administration of probiotic and commensal bacteria [ 6 ], F. prausnitzii may represent a relevant target for the development of diagnostic, prognostic, or therapeutic tools. In different pre-clinical models of IBD, F. prausnitzii efficiently improves intestinal inflammation [ 7 , 8 ] and gut barrier function [ 9 ]. Indeed, through secreted metabolites this bacterium able to block NF-κB activation and IL-8 production, which both contribute to inflammation [ 10 ]. In addition, this species produces high quantities of butyrate [ 4 ], a short-chain fatty acid (SCFA) which is important in gut physiology [ 11 ] [ 12 ]. F. prausnitzii also produces several bioactive molecules that affect inflammation and gut barrier function: shikimic and salicylic acids [ 12 ] and a microbial anti-inflammatory molecule (MAM; [ 13 ]). Despite many advances in the study of F. prausnitzii ’s anti-inflammatory effects within the host [ 4 ], we still do not understand the exact molecular responses of the host, which could represent targets for new therapies. To identify the host receptor and signaling pathways involved in the beneficial effects of this anti-inflammatory bacterium, we performed DNA chip-based transcriptomic analyses in human intestinal epithelial cells (IECs; ie. , HT-29 cells) that were stimulated with the proinflammatory cytokine TNF-α and exposed to F. prausnitzii culture supernatant (SN). These analyses led us to focus on Dact3 , a member of the Dishevelled Binding Antagonist Of Beta Catenin (DACT) gene family which negatively regulates the Wnt/JNK signaling pathway [ 14 ]. We present here evidence from both in vitro and in vivo experiments which provide the first clues about the important role of Dact3 in the host molecular mechanisms involved in the anti-inflammatory effects of the beneficial commensal bacterium F. prausnitzii . Methods Bacterial strains Faecalibacterium prausnitzii strain A2-165 (DSM N°17677, DSMZ collection, Braunschweig, Germany) was grown in LYBHI medium (BHI, Difco, Detroit, USA) supplemented with 0.5% yeast extract, 1 mg/ml cellobiose (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland), 1 mg/ml maltose and 0.5 mg/ml cysteine (Sigma-Aldrich), at 37 °C in an anaerobic chamber. F. prausnitzii supernatant (SN) was recovered by centrifugation and filtered through 0.45-µm-pore-size filters (VWR, Haasrode, Belgium) and stored at -80 °C. Cell lines and co-incubations The human colon carcinoma cell line HT-29 (ATCC HTB-38) was grown in Dulbecco’s Modified Eagle’s minimal essential medium with 4.5 g/L glucose (DMEM) (Sigma-Aldrich), supplemented with 10% (w/v) heat-inactivated fetal calf serum (FCS) (GibcoBRL, Eragny, France), 4 mM L-glutamine, and penicillin G/streptomycin (5000 IU/mL, 5000 µg/mL) (Sigma-Aldrich). Cultures were incubated in 25-cm 2 tissue culture flasks (Nunc, Roskilde, Denmark) at 37ºC in a 10% (v/v) CO 2 atmosphere until confluence. For co-culture experiments, HT-29 cells were seeded in 24-well culture plates (Nunc) in DMEM supplemented with 10% heat-inactivated FCS-1% glutamine at 37 °C in a 10% CO 2 -air atmosphere. Culture medium was changed every day. Experiments began on day 7 after seeding, when cells were at confluence (approx. 1.83 × 10 6 cells/well). On day 6, 24 h before co-culture with F. prausnitzii SN, the culture medium was changed to one with 5% heat-inactivated FCS and 1% glutamine. The day of the co-culture, either SN or LYBHI medium was added at a concentration of 10% (v/v) in a total volume of 500 µl. Cells were stimulated simultaneously with recombinant human TNF-α (5 ng/ml; Peprotech, NJ, USA) for 6 h at 37 °C in 10% CO 2 . All samples were analyzed in triplicate. ELISA Supernatants of mock non-stimulated and TNF-α-stimulated HT-29 cells used for transcriptomic analysis were first validated for IL-8 modulation by ELISA. Supernatants for ELISA and cells for RNA extractions and transcriptomic analysis were collected at the same time and from the same culture plates. IL-8 concentrations were determined by ELISA (Biolegend, San Diego, CA), according to the manufacturer’s instructions. Results were reported as the mean values of duplicate ELISA wells. RNA isolation After co-incubation with either LYBHI or F. prausnitzii SN, HT-29 cells were collected and RNA-purified for DNA transcriptomic hybridization. Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen, USA) and purified by on-column digestion of DNA with DNase I as recommended by the manufacturer to eliminate residual genomic DNA. RNA concentration was determined by Nanodrop quantification (Thermo Fisher Scientific Inc., France). RNA quality was checked on an Agilent 2100 Expert Bioanalyzer (Agilent Technologies, France). Only RNAs with a RIN > 8 were used for transcriptomic and qRT-PCR experiments. Microarray hybridization A reference design with complete dye-swap including two biological replicates was used to compare HT-29 cells among different treatments, for a total of 6 Agilent 4 × 44K Whole Human Genome Microarrays (Agilent Technologies, France). Scheme design is available in Fig. S3 . For labeling, 100 ng of total RNA was reverse-transcribed and stained with Cy3 or Cy5 using the two-color Low Input Quick Amp Gene Expression Labeling Kit (Agilent Technologies, France) according to the manufacturer's instructions. The CyDye-labeled cRNAs were then purified using the RNeasy Mini Kit (Qiagen, France). cRNA quantity was determined by Nanodrop and quality was checked on the Agilent 2100 Expert Bioanalyzer. Yield and specific activity were determined per manufacturer's instructions. We mixed 825 ng of Cy3-labeled cRNA from one treatment with the same amount of Cy5-labeled cRNA from another treatment. cRNAs were hybridized to the Agilent 4 × 44K Whole Human Genome Microarray (Agilent Technologies, France) at 65 °C for 17 h in a rotating incubator. After hybridization, slides were washed and then scanned using an Agilent G2565CA scanner (Agilent Technologies, France). Raw data were extracted using Feature Extraction software version 10.5.1.1 (Agilent Technologies, France). Microarray analysis R 3.0.2 software and the LIMMA package [ 15 ] were used to analyze microarray data (within-array normalization by loess method followed by between-array normalization by quantile method) and to generate lists of differentially expressed genes. Microarray data were deposited in the NCBI-GEO database (accession number GSE72048). To create gene lists, we filtered by expression levels (|(FC)|>1.5 as a cut-off) as well as by adjusted p- values, using a 0.05 threshold with the Benjamini and Hochberg false discovery rate [ 16 ] as multiple testing correction. The complete data set is available through the Gene Expression Omnibus GSE72048. Selected gene lists (log ratio and p- value data) were loaded into Ingenuity Pathway Analysis (IPA) and Multiarray Experiment Viewer [ 17 ] to analyze pathways and generate data displays. Quantitative real time RT-PCR (qRT-PCR) Three µg of DNase I-treated total RNAs were reverse-transcribed using Oligo(dT) primers and 1 µl of SuperScript II reverse transcriptase (Invitrogen, France). The resulting cDNAs were quantified by Nanodrop (Thermo Fisher Scientific Inc., France) and diluted to a working concentration of 100 ng/µl. Reactions were performed in a final volume of 25 µl with 500 ng cDNA, 10 pM primers, and SYBR Green PCR Master Mix (Applied Biosystems, USA), using a Mastercycler Realplex (Eppendorf, France). Primers are listed in Table S1 . The genes B2M (for human analysis) and HMBS (for mice analysis) were used as internal references [ 18 ] and the 2 −ΔΔCT method [ 19 ] was used to calculate the FC in gene expression. Dact3 siRNA in HT-29 cells HT-29 cells were cultured as described above. siGENOME® Human Dact3 siRNA-SMARTpool®, Dact3 siRNA D-015690-01, D-015690-02, D-015690-03, D-015690-17, and control siRNA (Non-Targeting siRNA D001136-01-05 and Cyclophilin B D-001210-02-05) siRNAs were transfected into HT-29 cells using Dharmafect 1 Transfection Reagent (Dharmacon, USA) following manufacturer's instructions with some modifications. A total of 1 × 10 5 cells were plated in 12-well plates and transfected using 30 nmol siRNA and 2 µL of Dharmafect 1 Transfection Reagent per well in DMEM containing 5% FCS and 1% L-Glutamine. After 24 h, the medium was changed. After another 24 h, control medium, F. prausnitzii SN, or LYBHI medium was added at a concentration of 10% (v/v) in a total volume of 1 ml. Cells were simultaneously stimulated with recombinant human TNF-α (5 ng/ml; Peprotech, NJ, USA) at 37 °C in 10% CO 2 . After 6 h of co-culture, supernatants were tested for IL-8 production by ELISA and RNA was isolated from cells for Dact3 expression analysis, as described above. All samples were geretaed and analyzed in triplicate. Dact3 overexpression in HT-29 cells HT-29 cells were cultured as above. The S-adenosylhomocystein hydrolase inhibitor 3-Deazaneplanocin A (DZNep) and Trichostatin A (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) were used as described in [ 20 ] with some modifications. Cells were plated at 0.5 × 10 5 cells/well; 24 h later, 12.5 M DZNep was added, and 48 hours after that, 0.5 µM TSA was added for the final 24 h of culture. After treatment, cells were stimulated with TNF-α (5 ng/ml; Peprotech, NJ, USA) and F. prausnitzii SN or LYBHI (added at 10% v/v). After 6 h of co-incubation, supernatants were tested by ELISA for IL-8 production and RNA was isolated from cells for Dact3 expression analysis. All samples were performed in triplicate. Dact3 -microbiota modulation in vivo Microarray data comparing germ-free and conventionalized mice were described and published previously ([ 21 ], GEO accession number: GSE63299). Conventionalization of germ-free C3H/HeN mice was performed with fresh stools from C3H/HeN donor mice. Dact3-F. prausnitzii SN modulation in vivo C57BL/6 mice (males, 6–8 weeks of age; Janvier, Le Genest Saint Isle, France) were maintained at the animal care facilities of the National Institute of Agricultural Research (IERP, INRA, Jouy-en-Josas, France) under specific pathogen-free conditions. Mice were housed under standard conditions for a minimum of 1 week before experimentation. All experiments were performed in accordance with European Community rules and approved by the animal care committee COMETHEA (Comité d’Ethique en Expérimentation Animale du Centre INRA de Jouy-en-Josas et AgroParisTech, Jouy-en-Josas, France). All assays were carried out under agreement N°3445-2016010615159974. The protocol for Dact3 modulation and quantification in vivo is illustrated in Fig. S1A . Three groups of mice ( n = 8) were intragastrically administered 200 µl of F. prausnitzii SN and sacrificed 3 h (T3), 6 h (T6) or 9 h (T9) after gavage. Control (T0) mice were not treated. Colitis was induced as described in [ 4 ], by intrarectal injection of 200 mg/kg of DNBS solution (ICN, Biomedical Inc.) in 30% ethanol. Fourteen days following the first injection, a 200-µl solution of either 1 × 10 9 CFU of F. prausnitzii or PBS was administered intragastrically for 10 days. Colitis was reactivated 21 days after the first DNBS injection with a second injection of 100 mg/kg of DNBS solution. Mice were sacrificed 3 days after the second DNBS injection ( Fig. S1A ) by cervical dislocation and different parameters of inflammation were recorded as previously described [ 8 ] Colon samples (one centimeter from distal colon) were frozen in 500 µl of RNAlater solution (Ambion, France) in liquid nitrogen and stored at -80 °C until use. Total RNA was extracted from individual samples with the RNeasy mini kit (Qiagen) according to the manufacturer’s instructions, using homogenization with Tissue Lyser (Qiagen) and purification by on-column digestion of DNA with DNase I. Total RNA was determined by Nanodrop quantification. Dact3 expression was analyzed by qRT-PCR. Statistical Analysis Statistical analysis, with the exception of transcriptomic analysis, was completed using GraphPad (GraphPad Software, La Jolla, CA, USA). A p- value of less than 0.05 was considered significant. Significant differences in the relative expression values of the target genes were tested with REST software using pairwise fixed reallocation randomization [ 22 ]. Results Transcriptomic analysis reveals Dact3 as a target of F. prausnitzii We used TNF-α to stimulate inflammation in HT-29 cells. A specific trait of this stimulation is upregulation of IL-8 [ 15 , 16 , 23 – 25 ], which we then measured as a readout for cell inflammatory status. To extend previous research on F. prausnitzii SN in TNF-α-stimulated HT-29 cells [ 12 ], we performed a SN dose-effect experiment and determined the stability ( ie. , putative degradation) of both IL-8 and TNF-α in the presence of the SN. F. prausnitzii SN had a significant dose-response effect (5–30%) while the bacterial culture medium LYBHI did not ( Fig. S2A ). Of note, F. prausnitzii SN did not directly degrade either IL-8 ( Fig. S2B ) or TNF-α ( Fig. S2C ). To elucidate the host molecular mechanisms involved ( eg. , the modulatory effects on IL-8 production), we performed a transcriptomic analysis of TNF-α-stimulated HT-29 cells exposed to F. prausnitzii SN (microarray hybridization schema is presented in Fig. S3 ). Before evaluating the effects of the SN, we first determined the regulatory changes due to TNF-α stimulation alone. Compared to control HT-29 cells, 227 genes were upregulated and 60 genes were downregulated in TNF-α-stimulated cells (adj. p 1.5). The genes with the largest changes in expression are listed in Table S2 . As expected, IL-8 was among the most upregulated genes. According to Ingenuity Pathway Analysis (IPA), upstream regulators such as TNF-α, IFN-γ, and NF-κB were also activated, confirming the inflamed status of the HT-29 cells. Next, we evaluated the effect of the LYBHI medium used to culture F. prausnitzii. Here, only 84 genes were differentially regulated (Fig. 1 A), and LYBHI did not seem to reduce cellular inflammation. Finally, we introduced F. prausnitzii SN. There were extensive changes in gene regulation: 913 genes were upregulated and 585 were downregulated after TNF-α stimulation and SN treatment (Fig. 1 A). Interestingly, these genes are involved in several inflammatory pathways such as NF-κβ, p38 and other ERK/MAPK pathways. Among these, 25% of the genes that had been activated by TNF-α exposure (in our first comparison) were inactivated by treatment with F. prausnitzii SN (70 out of 287). To explore this complex gene regulation network, we submitted this dataset to IPA, which highlighted the SN-affected pathways. This analysis confirmed the ability of F. prausnitzii SN to regulate ERK/MAPK signaling pathway as the most important pathway related to inflammation modulated specifically by F. prausnitzii SN (Fig. 1 C). Notably, 7% of all genes regulated by F. prausnitzii SN were linked to MAPK pathways, including JNK. The 10 genes with the largest fold-change in regulation are shown in Fig. 1 B and Table S3 , IL-8 was found to be downregulated, while the most upregulated gene was Dact3 (FC = 17.2). Dact3 belongs to the Dact gene family, whose members interact with the Dsh protein to inhibit Dsh-induced activation of the JNK pathway [ 26 , 27 ]. The Dact3/JNK pathway is presented in Fig. 2 A. Finally, in order to validate our transcriptomic data, the differential expression of up- and downregulated genes, including Dact3 , was validated by RT-qPCR (only Dact3 data shown). B2M was used as reference for data normalization as described in [ 28 ]. Compared to LYBHI, F. prausnitzii SN led to significant Dact3 upregulation in TNF-α-stimulated HT-29 cells (Fig. 2 B), confirming transcriptomic observations. Role of Dact3 in TNF-α-stimulated HT-29 cells As Dact3 was the most upregulated gene in the transcriptomics analysis, we thus decided to focus our investigations on this gene. To investigate the effect of Dact3 on IL-8 production in TNF-α-stimulated HT-29 cells, we overexpressed or knocked down Dact3 in IECs. First, we tried to overexpress Dact3 using transient transfection in HT-29 cells; unfortunately, we were unable to efficiently transfect these cells (data not shown). This could be due to the strong upregulation of Dact3 that led to massive apoptosis of the cells, as was previously reported for colorectal cancer cells [ 20 ]. We then used the siRNA technology to knock down Dact3 mRNA in TNF-α-inflamed HT-29 cells. As shown in Fig. 3 A, RT-qPCR analysis confirmed that: i) both the non-targeting (NT) siRNA and LYBHI had no effect on Dact3 expression (FC = 1); ii) F. prausnitzii SN strongly upregulated Dact3 (FC = 30) in presence of NT siRNA; and iii) Dact3 siRNA significantly reduced the abundance of Dact3 mRNA induced by F. prausnitzii SN. Besides, treatment with Dact3 siRNA in TNF-α-stimulated HT-29 cells tends to increase IL-8 production (Fig. 3 B) suggesting an important role of Dact3 activation in intestinal homeostasis. Moreover, the inhibitory effect of F. prausnitzii SN on IL-8 production was partially lost in Dact3 -silencing condition siRNA. Next, we evaluated the effect of factors modulating Dact3 expression on IL-8 production. As histone modification has been reported to modulate Dact3 expression [ 20 ], we used a mix of TSA (a histone deacetylase inhibitor) and DZNep (a histone methylation inhibitor) drugs as previously described [ 20 ]. These drugs led to a strong upregulation of Dact3 similar to that obtained with F. prausnitzii SN (Fig. 3 C). Furthermore, after drug treatment and TNF-α stimulation, Dact3 upregulation strongly inhibited IL-8 production in HT-29 cells, to an even stronger extent than was observed with F. prausnitzii SN (Fig. 3 D). Characterization of the CCCA KO HEK293 cell line Identification of the F. prausnitzii-effectors responsible for Dact3 modulation To identify the bacterial effector(s) responsible for Dact3 modulation, we tested some molecules implicated in the anti-inflammatory effects of F. prausnitzii : the MAM protein [ 29 ] and salicylic acid [ 12 ]. In addition, as F. prausnitzii abundantly produces butyrate, which reduces IL-8 production in TNF-α-stimulated HT-29 cells [ 12 ], we also tested its effect on Dact3 modulation (only results for butyrate are shown). Treatment with 10% of a solution of 10 mM of butyrate (the concentration present in the F. prausnitzii SN [ 30 ]) led to strong upregulation of Dact3 (Fig. 4 A) and a decrease in IL-8 production (Fig. 4 B), whereas no Dact3 modulation was observed with either salicylic acid or MAM. Altogether, these results reveal that butyrate is one of the F. prausnitzii effectors responsible for Dact3 modulation. In order to validate this hypothesis, we tested other gut bacterial strains ( eg. Roseburia intestinalis , Akkermansia muciniphila and Bacteroides thetaiotaomicron ), known to produce or not butyrate ( Table S4 ), for their capacities to upregulate Dact3 (Fig. 4 C) and downregulate IL-8 (Fig. 4 D). We also tested a probiotic lactic acid bacterium (LAB) with anti-inflammatory capacities, L. casei BL23, that does not produce butyrate. Strikingly, the SN from the only another butyrate-producing bacterium ( R. intestinalis ) highly upregulated Dact3 expression and thus downregulated IL-8 production. In contrast, the three other bacterial strains that do not produce butyrate ( A. muciniphila, B. thetaiotaomicron and L. casei BL23) did not have any effect on neither Dact3 nor IL-8. In vivo validation of Dact3 modulation Dact3 modulation was first investigated in healthy mice orally administered with F. prausnitzii SN and sacrificed at different time points ( e.g. 0, 3, 6 and 9 hours after administration) to evaluate its effects in normal physiological conditions ( Fig. S1A ). F. prausnitzii SN led to a significant increase in Dact3 mRNA in colonic samples 9 h after its administration ( Fig. S1B ). Other groups of mice were orally administered with either F. prausnitzii SN, R. intestinalis SN or butyrate (1 mM) and euthanized 9 h later. As shown in Fig. 5 A, butyrate was detected in colonic samples of mice treated with either F. prausnitzii SN or R. intestinalis SN but not with butyrate itself, suggesting that soluble butyrate is rapidly absorbed in the colon. Strikingly, all treatments, including butyrate, results in Dact3 upregulation in samples from proximal colon (Fig. 5 B). We then investigated in vivo Dact3 modulation in an inflammatory context, using a murine model in which chronic moderate inflammation was induced by intrarectal injection of dinitrobenzene sulfonic acid (DNBS). Animals were given daily oral gavages of live F. prausnitzii bacteria (Fig. 6 A), as previously described [ 8 ]. As expected, F. prausnitzii administration led to significant reductions in markers of colitis with improvements in weight loss and a decrease in macroscopic scores (Fig. 6 B), MPO activity (Fig. 6 C) and a reduction of the pro-inflammatory cytokines IFN-γ (Fig. 6 D), IL-6 (Fig. 6 E), IL-17A (Fig. 6 F) and the chemokine MCP-1 (Fig. 6 G). Moreover, Dact3 expression was upregulated in the colon of F. prausnitzii -treated mice compared to the PBS-treated mice (Fig. 6 H), confirming that Dact3 is induced by F. prausnitzii in an inflammatory context too. Finally, to assess the effect of the gut microbiota on Dact3 modulation in in vivo physiological conditions, we investigated a dataset that we previously published comparing the colonic transcriptome of germ-free and conventionalized (Conv) mice [ 21 ]. We found that Dact3 expression was significantly reduced in germ-free compared to Conv mice (Fig. 7 ). These observations confirm that gut microbiota has a major impact on Dact3 expression. Discussion F. prausnitzii is a commensal bacterium well-known for its immuno-modulatory properties and more specifically for its anti-inflammatory effects both in vitro [ 7 , 12 , 13 ] and in vivo [ 4 , 7 , 9 , 12 , 13 , 31 ]. Here, we demonstrated that F. prausnitzii SN is able to block IL-8 production in TNF-α-activated HT-29, but not through the proteolytic degradation of either TNF-α or IL-8. Because of this, and because F. prausnitzii is extremely oxygen sensitive (EOS) and cannot be easily cultured with human cells [ 32 ], we decided to further study the immunomodulatory effects of its SN. F. prausnitzii SN modulated the expression of a massive number of genes, in fact, more than the TNF-α treatment itself ( Table S2 ). Among the inflammation-related pathways regulated by F. prausnitzii SN, we found enrichment in genes related to MAPKs. In particular, Dact3 , a gene implicated in Wnt/JNK regulation, was among the top upregulated genes by F. prausnitzii SN in non-stimulated as well as in TNF-α-stimulated cells. The Dact gene family was initially reported in studies of embryonic development [ 14 , 33 ]. Although Dact3 is involved in postnatal development (adult Dact3 −/− mice show a mild reduction in body weight), the fact that Dact3 −/− mice are viable means that this gene is not essential for mouse embryogenesis, postnatal survival, and reproduction [ 34 ]. Dact3 has a postnatal role as a regulator in the Wnt/β-catenin signaling pathway [ 20 ] and has been associated with several types of cancer: colorectal [ 20 ], breast [ 35 ], ovarian [ 36 ], lung [ 37 ], papillary thyroid [ 38 ], and renal fibrosis [ 34 ]. In colorectal cancer cell lines, Dact3 transcription is epigenetically downregulated by a bivalent histone modification [ 20 ]. In particular, drugs that target both histone methylation (DZNep) and deacetylation (TSA) strongly induce Dact3 expression [ 20 ], a result that we confirmed here. Moreover, we also observed that these drugs reduced IL-8 production by TNF-α-stimulated HT-29 cells in a way that was similar to the effect of F. prausnitzii SN. These results show that Dact3 has an important function in the IL-8 pathway and that IL-8 inhibition by F. prausnitzii SN is, at least partly, mediated via Dact3 upregulation. Dact3 could thus be an epigenetic regulator of inflammation and play a key role in intestinal homeostasis. To further decipher this, we evaluated the effect of Dact3 silencing in HT-29 using siRNA. Silencing of Dact3 in IECs prevented F. prausnitzii SN from blocking IL-8 production. Dact3 histone modulation could provide clues about the bacterial effectors responsible for this regulation. In this study, only butyrate was able to both upregulate Dact3 expression and to block IL-8 production by TNF-α-stimulated HT-29 cells. Indeed, butyrate-induced reduction in IL-8 production was similar to that observed with the F. prausnitzii SN, but Dact3 upregulation was higher in the presence of butyrate than of SN. This is probably because Dact3 is also modulated by histone deacetylases [ 20 ] which are known to be inhibited by butyrate [ 39 ]. In this context, our results confirm those obtained by Fung et al. [ 40 ], who found that Dact3 appeared to be differentially modulated in HT-29 cells treated with butyrate. However, these authors did not further explore Dact3 in their study. Altogether, our results provide evidence that F. prausnitzii SN regulates Dact3 /IL-8 production and suggest that butyrate produced by F. prausnitzii is the main actor in this regulation. As butyrate is known to have pleiotropic effects in the intestinal cell life cycle and numerous beneficial effects for human health (e.g., anti-inflammatory and anti-tumorigenic properties) [ 11 ], it is plausible that butyrate production is a means by which F. prausnitzii affects its host physiological functions and homeostasis to maintain health. However, further studies are necessary to confirm this hypothesis. In particular, genetic manipulation of F. prausnitzii to inactivate the gene encoding the butyril-CoA synthase involved in butyrate metabolism would be helpful to answer to this question. Indeed, O’Cuiv et al. [ 41 ] have isolated F. prausnitzii transconjugants using metaparental mating and this strategy opens promising perspectives to manipulate F. prausnitzii. Finally, in vitro observations were validated in vivo in two different models: first, in healthy mice that were orally administered F. prausnitzii SN one time ( Fig. S3 ) and second, in inflamed mice that were given live F. prausnitzii orally for 10 days (Fig. 7 ). In both in vivo models, either F. prausnitzii or F. prausnitzii SN positively regulated Dact3 , confirming the key effect of F. prausnitzii on this gene. Conclusions In conclusion, we propose in this study a new role for Dact3 as a master regulator of intestinal homeostasis, particularly in inflamed cells. Although it is expressed in IECs at a low level, Dact3 seems to be essential for intestinal homeostasis, as its downregulation or loss leads to a global increase in inflammation. We hypothesize that Dact3 upregulation inhibits the AP-1 transcription factor, which in turn leads to a global downregulation of genes encoding for pro-inflammatory cytokines such as IL-2, IL-6, and IL-8 [ 27 ]. Additionally, the ability of F. prausnitzii SN to modulate other genes involved in cancer pathways (as revealed in the transcriptomic analysis) represents a novel potential beneficial effect of this commensal anti-inflammatory bacterium, which is currently being investigated by our laboratory. In conclusion, our study provides the first clues on one of the host molecular targets involved in the anti-inflammatory effects of F. prausnitzii in IECs. Moreover, these results point out Dact3 as a potential master regulator of inflammation in IECs. As there is increasing interest in exploring new alternatives for IBD treatment, this research suggests at least three potential opportunities: i) use of F. prausnitzii itself, ii) use of drugs to modulate Dact3 expression (such as histone deacetylases) and iii) heterologous delivery of Dact3 (either as a cDNA or a protein) using food-grade live vectors [ 42 , 43 ]. For these, further studies on Dact3 -knockout mice will be necessary to understand the physiological functions of Dact3 . Declarations Availability of data and materials The complete data set is available through the Gene Expression Omnibus GSE72048. Ethics approval and consent to participate All experiments were performed in accordance with European Community rules and approved by the animal care committee COMETHEA (Comité d’Ethique en Expérimentation Animale du Centre INRA de Jouy-en-Josas et AgroParisTech, Jouy-en-Josas, France) under agreement N°3445-2016010615159974. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Funding This study was a part of FPARIS collaborative project selected and supported by the Vitagora Competitive Cluster and funded by the French Fond Unique Interministériel, n°F1010012D, the Fonds Européen de Développement Régional (Bourgogne: 34606), the Burgundy Region, the Conseil Général 21, and the Grand Dijon. This work was also supported by Merck Médication Familiale (Dijon, France) and Biovitis (Saint Etienne de Chomeil, France). RM received a salary from these grants and ML received a PhD grant from ABIES. Authors’ contributions Author contributions: L.G.B.H. and F.C. designed research; M.L., R.M., E.T.M., S.C. and F.C. performed research; H.S. and P.G.D contributed analytic tools; M.L., R.M., H.S., F.C., and L.G.B.H., analyzed data; and M.L., R.M., H.S., P.L., F.C., and L.G.B.H wrote the paper. All read and approved the final manuscript. Acknowledgements The authors would like to thank Marco Moroldo, Jérôme Lecardonnel, and Déborah Jardet from the GABI/CRB GABI platform, and Valentin Loux and all IERP personnel for their help. We also thank Dr. Hélène Bierne for her precious advices. References Khor B, Gardet A, Xavier RJ. Genetics and pathogenesis of inflammatory bowel disease. Nature. 2011;474(7351):307–17. Abraham C, Cho JH. Inflammatory bowel disease. N Engl J Med. 2009;361(21):2066–78. Sokol H, Seksik P, Furet JP, Firmesse O, Nion-Larmurier I, Beaugerie L, Cosnes J, Corthier G, Marteau P, Dore J. Low counts of Faecalibacterium prausnitzii in colitis microbiota. Inflamm Bowel Dis. 2009;15(8):1183–9. Miquel S, Martin R, Bridonneau C, Robert V, Sokol H, Bermudez-Humaran LG, Thomas M, Langella P. Ecology and metabolism of the beneficial intestinal commensal bacterium Faecalibacterium prausnitzii. Gut Microbes. 2014;5(2):146–51. 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Xi SC, Yang MC, Tao YG, Xu H, Shan JG, Inchauste S, Zhang M, Mercedes L, Hong JA, Rao M, et al: Cigarette Smoke Induces C/EBP-beta-Mediated Activation of miR-31 in Normal Human Respiratory Epithelia and Lung Cancer Cells . PLoS One 2010, 5(10). Neta G, Brenner AV, Sturgis EM, Pfeiffer RM, Hutchinson AA, Aschebrook-Kilfoy B, Yeager M, Xu L, Wheeler W, Abend M, et al. Common genetic variants related to genomic integrity and risk of papillary thyroid cancer. Carcinogenesis. 2011;32(8):1231–7. Leonel AJ, Alvarez-Leite JI. Butyrate: implications for intestinal function. Curr Opin Clin Nutr. 2012;15(5):474–9. Fung KYC, Kerr C, Henderson S, Ilka P, Shaw J, Buckley MJ, Lockett T, Head R, Cosgrove L. Mechanisms Associated With Acquisition Of Resistance To Butyrate-Induced Apoptosis In Colorectal Cancer Cells Using Gene Expression Analysis. Journal of Proteomics Genomics Research. 2014;1(4):16–30. Cuiv PO, Smith WJ, Pottenger S, Burman S, Shanahan ER, Morrison M. 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Supplementary Files FigS2MSDact3Microbiome.pptx FigS1MSDact3Microbiome.pptx TablesSupplMSDact3Microbiome.pptx FigS3MSDact3Microbiome.pptx Cite Share Download PDF Status: Published Journal Publication published 15 Oct, 2020 Read the published version in Gut Microbes → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-28864","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":575503,"identity":"e44741ea-415a-40fb-99c6-675574cd59f3","order_by":1,"name":"Marion Lenoir","email":"","orcid":"","institution":"INRAE","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Marion","middleName":"","lastName":"Lenoir","suffix":""},{"id":575504,"identity":"b01212c1-ab12-4490-bade-78ac548e8d8d","order_by":2,"name":"Rebeca Martin","email":"","orcid":"","institution":"INRAE","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rebeca","middleName":"","lastName":"Martin","suffix":""},{"id":575505,"identity":"be9980af-3a08-4626-9af8-2d6b111e8862","order_by":3,"name":"Edgar Torres-Maravilla","email":"","orcid":"","institution":"INRAE","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Edgar","middleName":"","lastName":"Torres-Maravilla","suffix":""},{"id":575506,"identity":"bd957b35-ba14-4492-9d5c-2a5b62f4b244","order_by":4,"name":"Sead Chadi","email":"","orcid":"","institution":"INRAE","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sead","middleName":"","lastName":"Chadi","suffix":""},{"id":575507,"identity":"56787c6a-70c7-47f6-9ce1-23eabbb6b265","order_by":5,"name":"Pamela González-Dávila","email":"","orcid":"","institution":"INRAE","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Pamela","middleName":"","lastName":"González-Dávila","suffix":""},{"id":575508,"identity":"d712858e-6aa5-4687-9e8d-a9c7b247bd28","order_by":6,"name":"Harry Sokol","email":"","orcid":"","institution":"INRAE","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Harry","middleName":"","lastName":"Sokol","suffix":""},{"id":575509,"identity":"ff13d9f4-72b1-4c0d-982f-1fe4774432d7","order_by":7,"name":"Philippe Langella","email":"","orcid":"","institution":"INRAE","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Philippe","middleName":"","lastName":"Langella","suffix":""},{"id":575510,"identity":"0ae130ca-7c89-451b-a243-3ef3f72c8a3a","order_by":8,"name":"Florian Chain","email":"","orcid":"","institution":"INRAE","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Florian","middleName":"","lastName":"Chain","suffix":""},{"id":575511,"identity":"31999bc1-c7f6-4b7b-83aa-cb163c98b4c0","order_by":9,"name":"Luis G. Bermudez Humaran","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIiWNgGAWjYBACPgbGBhACAQMGhgoJiHAFHi1sqFrOQLWcwasFCOBaGNsYiNAikdz8gXGHTb7ujOSND3/Os8gzbz98gOHgHnxaEtskGM+kWW67kVZsILlNoljmTFoCw4Fn+LUA3XPYwOxGjpmE4TaJxBkMOQbMHw7g1QJ0WNt/kBbzH4lzgFr4339gOIBfS4MEY9sBsC0MBxuAWiRyGPBr4XnYJpF4JtnA7MyzYsmGYyAtzwwO4NPCz57++MPHHXYGZseTN378UVMHdFjywwf4tIBBAroAIQ2jYBSMglEwCggAALQxVLZesDMIAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-7525-3131","institution":"INRAE","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Luis","middleName":"G. Bermudez","lastName":"Humaran","suffix":""}],"badges":[],"createdAt":"2020-05-14 12:10:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-28864/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-28864/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1080/19490976.2020.1826748","type":"published","date":"2020-10-15T18:50:45+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":1107642,"identity":"4893756e-de78-4373-a32b-c48f3d55077c","added_by":"auto","created_at":"2020-05-15 16:45:57","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":346922,"visible":true,"origin":"","legend":"Gene and cellular pathways modulation by F. prausnitzii SN. (A) Comparison of the genes modulated by LYBHI+TNF-vs TNF- and SN+TNF- vs TNF-. The genes specifically modulated by SN and linked to inflammatory pathways have been represented using IPA canonical pathway display: y-axis displays the -log of the p-value which is calculated by Fisher's exact test right-tailed. The orange points interconnected by a thin line represent the ratio. This ratio is calculated as follows: # of genes in a given pathway that meet the cutoff criteria, divided by the total # of genes that make up that pathway and that are in the reference gene set. (B) Histogram of the ten most up- and downregulated genes in the comparison LYBHI+TNF-vs SN+TNF- (C) IPA canonical pathway display of the genes modulated in the comparison LYBHI+TNF-vs SN+TNF- y-axis displays the -log of p-value which is calculated by Fisher's exact test right-tailed. The orange and blue colored bars indicate predicted pathway activation, or predicted inhibition, respectively (z-score). Only genes with z-score are represented. The orange points interconnected by a thin line represent the ratio","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-28864/v1/Figure1.jpg"},{"id":1107644,"identity":"108e0c66-15e8-463e-adb7-99fb79454494","added_by":"auto","created_at":"2020-05-15 16:45:57","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":84054,"visible":true,"origin":"","legend":"F. prausnitzii SN modulates Dact3 expression in vitro. (D) Schematic representation of Dact3 pathway. (E) Validation of Dact3 expression by F. prausnitzii SN in TNF--stimulated HT-29 cells by RT-qPCR. Results are expressed as the fold change (FC) of Dact3 expression relative to the B2M housekeeping gene.","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-28864/v1/Figure2.jpg"},{"id":1107646,"identity":"0ebafe89-db40-4002-b603-6d551553d57e","added_by":"auto","created_at":"2020-05-15 16:45:57","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":271859,"visible":true,"origin":"","legend":"Modulation of Dact3 expression in TNF-α-stimulated HT-29 cells under different conditions. (A) Dact3 expression and (B) IL-8 production by HT-29 cells transfected with either Dact3 or non-targeting (NT) siRNA and treated with both F. prausnitzii SN and TNF-α. Dact3 expression was analyzed by RT-qPCR and IL-8 production by ELISA. Results are expressed as the FC of Dact3 expression relative to LYBHI with NT siRNA and as IL-8 % of DMEM under NT siRNA conditions. (C) Dact3 expression and (D) IL-8 production by TNF-α-stimulated HT-29 cells co-incubated with 0.5 µM of TSA or 12.5 µM of DZNep, or both. Results are expressed as the FC of Dact3 expression relative to LYBHI and as IL-8 % of DMEM.","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-28864/v1/Figure3.jpg"},{"id":1107648,"identity":"4c2d1f4d-0ada-416b-ba2b-728bcecb4321","added_by":"auto","created_at":"2020-05-15 16:45:58","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":152988,"visible":true,"origin":"","legend":"Effect of butyrate on Dact3 expression. (A) Dact3 expression and (B) IL-8 production by HT-29 cells co-incubated with 1 mM of butyrate. Results are expressed as the FC of Dact3 expression relative to LYBHI and as IL-8 % of DMEM. All experiments were performed in triplicate. Non-parametric Kruskal-Wallis and Dunn’s post hoc test *p\u003c0.05; **p\u003c0.01; ***p\u003c0.001. (C) Dact3 expression and (D) IL-8 production in TNF-α-stimulated HT-29 cells and treated with the SN of different bacterial strains producing or not butyrate. LYBHI and MRS were used as negative control of the SN from the different bacterial strains. Results are expressed as the FC of Dact3 expression relative to LYBHI and as the concentration of IL-8 % of DMEM. All experiments were performed in triplicate. Non-parametric Kruskal-Wallis and Dunn’s post hoc test *p\u003c0.05.","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-28864/v1/Figure4.jpg"},{"id":1107649,"identity":"cd3e46a6-4034-4fee-b4eb-6b315aac1a73","added_by":"auto","created_at":"2020-05-15 16:45:58","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":68274,"visible":true,"origin":"","legend":"Modulation of Dact3 expression in vivo by F. prausnitzii SN. Animals were intragastrically administered with either butyrate (1 mM) F. prausnitzii SN or R. intestinalis SN and sacrificed 9 after. (A) Quantification of butyrate in samples from proximal colon of treated mice. (B) FC of Dact3 expression in colonic samples relative to actin housekeeping gene.","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-28864/v1/Figure5.jpg"},{"id":1107650,"identity":"9b3910e2-a376-4554-8ebe-27b9928417df","added_by":"auto","created_at":"2020-05-15 16:45:58","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":159455,"visible":true,"origin":"","legend":"Modulation of Dact3 expression in vivo by F. prausnitzii (A) Experimental protocol used for the analysis of the in vivo effects of F. prausnitzii in a mouse model of chronic inflammation (described in [8]: (B) Macroscopic scores; (C) MPO activity; (D-G) colonic pro-inflammatory cytokine and chemokine concentrations and (H) FC of Dact3 expression in colonic samples relative to HMBS housekeeping gene.","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-28864/v1/Figure6.jpg"},{"id":1107651,"identity":"8e2408c8-381f-4339-88d3-dc596eeb73fb","added_by":"auto","created_at":"2020-05-15 16:45:58","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":44818,"visible":true,"origin":"","legend":"Dact3 modulation by endogenous microbiota. Total RNA was extracted from colon tissues of both germ-free and conventional (Conv) mice and analyzed by microarrays to determine modulation of Dact3.","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-28864/v1/Figure7.jpg"},{"id":13503821,"identity":"293559f3-5612-46e0-879a-79fba32493dc","added_by":"auto","created_at":"2021-09-16 23:20:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1204639,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-28864/v1/c2983f8a-f417-49d1-9b45-f51e423c3ead.pdf"},{"id":1107647,"identity":"cb899338-1c2a-41fd-b3f3-a9101fb9a3f5","added_by":"auto","created_at":"2020-05-15 16:45:58","extension":"pptx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":131777,"visible":true,"origin":"","legend":"","description":"","filename":"FigS2MSDact3Microbiome.pptx","url":"https://assets-eu.researchsquare.com/files/rs-28864/v1/FigS2MSDact3Microbiome.pptx"},{"id":1107641,"identity":"f7bf83e3-d9af-48bc-98b1-210846960e3d","added_by":"auto","created_at":"2020-05-15 16:45:57","extension":"pptx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":59467,"visible":true,"origin":"","legend":"","description":"","filename":"FigS1MSDact3Microbiome.pptx","url":"https://assets-eu.researchsquare.com/files/rs-28864/v1/FigS1MSDact3Microbiome.pptx"},{"id":1107645,"identity":"8852af30-ab4a-4e49-b2cf-fd6a55d53b7f","added_by":"auto","created_at":"2020-05-15 16:45:57","extension":"pptx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":62132,"visible":true,"origin":"","legend":"","description":"","filename":"TablesSupplMSDact3Microbiome.pptx","url":"https://assets-eu.researchsquare.com/files/rs-28864/v1/TablesSupplMSDact3Microbiome.pptx"},{"id":1107643,"identity":"1435ba81-891b-48db-905c-40d23c822a37","added_by":"auto","created_at":"2020-05-15 16:45:57","extension":"pptx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":36750,"visible":true,"origin":"","legend":"","description":"","filename":"FigS3MSDact3Microbiome.pptx","url":"https://assets-eu.researchsquare.com/files/rs-28864/v1/FigS3MSDact3Microbiome.pptx"}],"financialInterests":"","formattedTitle":"Butyrate mediates anti-inflammatory effects of Faecalibacterium prausnitzii in intestinal epithelial cells through Dact3","fulltext":[{"header":"Background","content":" \u003cp\u003eInflammatory bowel disease (IBD) is a group of disorders characterized by chronic inflammation in the gastrointestinal tract [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. One potential cause (and/or consequence) of IBD is the disruption of the intestinal ecosystem equilibrium. For example, gut microbiota analysis of Crohn\u0026rsquo;s disease (a type of IBD) patients revealed markedly lower diversity of Firmicutes (in particular of the \u003cem\u003eClostridium leptum\u003c/em\u003e group) compared to healthy individuals [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. These assemblages are also relatively poor in \u003cem\u003eFaecalibacterium prausnitzii\u003c/em\u003e, a major member of the \u003cem\u003eC. leptum\u003c/em\u003e group and one of the most abundant intestinal bacteria in healthy adults [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Because a potential approach to prevent and treat IBD is the oral administration of probiotic and commensal bacteria [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], \u003cem\u003eF. prausnitzii\u003c/em\u003e may represent a relevant target for the development of diagnostic, prognostic, or therapeutic tools.\u003c/p\u003e \u003cp\u003eIn different pre-clinical models of IBD, \u003cem\u003eF. prausnitzii\u003c/em\u003e efficiently improves intestinal inflammation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and gut barrier function [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Indeed, through secreted metabolites this bacterium able to block NF-κB activation and IL-8 production, which both contribute to inflammation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In addition, this species produces high quantities of butyrate [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], a short-chain fatty acid (SCFA) which is important in gut physiology [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. \u003cem\u003eF. prausnitzii\u003c/em\u003e also produces several bioactive molecules that affect inflammation and gut barrier function: shikimic and salicylic acids [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and a microbial anti-inflammatory molecule (MAM; [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]). Despite many advances in the study of \u003cem\u003eF. prausnitzii\u003c/em\u003e\u0026rsquo;s anti-inflammatory effects within the host [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], we still do not understand the exact molecular responses of the host, which could represent targets for new therapies. To identify the host receptor and signaling pathways involved in the beneficial effects of this anti-inflammatory bacterium, we performed DNA chip-based transcriptomic analyses in human intestinal epithelial cells (IECs; \u003cem\u003eie.\u003c/em\u003e, HT-29 cells) that were stimulated with the proinflammatory cytokine TNF-α and exposed to \u003cem\u003eF. prausnitzii\u003c/em\u003e culture supernatant (SN). These analyses led us to focus on \u003cem\u003eDact3\u003c/em\u003e, a member of the Dishevelled Binding Antagonist Of Beta Catenin (DACT) gene family which negatively regulates the Wnt/JNK signaling pathway [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. We present here evidence from both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments which provide the first clues about the important role of \u003cem\u003eDact3\u003c/em\u003e in the host molecular mechanisms involved in the anti-inflammatory effects of the beneficial commensal bacterium \u003cem\u003eF. prausnitzii\u003c/em\u003e.\u003c/p\u003e "},{"header":"Methods","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBacterial strains\u003c/h2\u003e \u003cp\u003e \u003cem\u003eFaecalibacterium prausnitzii\u003c/em\u003e strain A2-165 (DSM N\u0026deg;17677, DSMZ collection, Braunschweig, Germany) was grown in LYBHI medium (BHI, Difco, Detroit, USA) supplemented with 0.5% yeast extract, 1\u0026nbsp;mg/ml cellobiose (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland), 1\u0026nbsp;mg/ml maltose and 0.5\u0026nbsp;mg/ml cysteine (Sigma-Aldrich), at 37\u0026nbsp;\u0026deg;C in an anaerobic chamber. \u003cem\u003eF. prausnitzii\u003c/em\u003e supernatant (SN) was recovered by centrifugation and filtered through 0.45-\u0026micro;m-pore-size filters (VWR, Haasrode, Belgium) and stored at -80\u0026nbsp;\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell lines and co-incubations\u003c/h2\u003e \u003cp\u003eThe human colon carcinoma cell line HT-29 (ATCC HTB-38) was grown in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s minimal essential medium with 4.5\u0026nbsp;g/L glucose (DMEM) (Sigma-Aldrich), supplemented with 10% (w/v) heat-inactivated fetal calf serum (FCS) (GibcoBRL, Eragny, France), 4\u0026nbsp;mM L-glutamine, and penicillin G/streptomycin (5000\u0026nbsp;IU/mL, 5000\u0026nbsp;\u0026micro;g/mL) (Sigma-Aldrich). Cultures were incubated in 25-cm\u003csup\u003e2\u003c/sup\u003e tissue culture flasks (Nunc, Roskilde, Denmark) at 37\u0026ordm;C in a 10% (v/v) CO\u003csub\u003e2\u003c/sub\u003e atmosphere until confluence.\u003c/p\u003e \u003cp\u003eFor co-culture experiments, HT-29 cells were seeded in 24-well culture plates (Nunc) in DMEM supplemented with 10% heat-inactivated FCS-1% glutamine at 37\u0026nbsp;\u0026deg;C in a 10% CO\u003csub\u003e2\u003c/sub\u003e-air atmosphere. Culture medium was changed every day. Experiments began on day 7 after seeding, when cells were at confluence (approx. 1.83\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e6\u003c/sup\u003e cells/well). On day 6, 24\u0026nbsp;h before co-culture with \u003cem\u003eF. prausnitzii\u003c/em\u003e SN, the culture medium was changed to one with 5% heat-inactivated FCS and 1% glutamine. The day of the co-culture, either SN or LYBHI medium was added at a concentration of 10% (v/v) in a total volume of 500\u0026nbsp;\u0026micro;l. Cells were stimulated simultaneously with recombinant human TNF-α (5\u0026nbsp;ng/ml; Peprotech, NJ, USA) for 6\u0026nbsp;h at 37\u0026nbsp;\u0026deg;C in 10% CO\u003csub\u003e2\u003c/sub\u003e. All samples were analyzed in triplicate.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003eELISA\u003c/h2\u003e \u003cp\u003eSupernatants of mock non-stimulated and TNF-α-stimulated HT-29 cells used for transcriptomic analysis were first validated for IL-8 modulation by ELISA. Supernatants for ELISA and cells for RNA extractions and transcriptomic analysis were collected at the same time and from the same culture plates. IL-8 concentrations were determined by ELISA (Biolegend, San Diego, CA), according to the manufacturer\u0026rsquo;s instructions. Results were reported as the mean values of duplicate ELISA wells.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eRNA isolation\u003c/h2\u003e \u003cp\u003eAfter co-incubation with either LYBHI or \u003cem\u003eF. prausnitzii\u003c/em\u003e SN, HT-29 cells were collected and RNA-purified for DNA transcriptomic hybridization. Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen, USA) and purified by on-column digestion of DNA with DNase I as recommended by the manufacturer to eliminate residual genomic DNA. RNA concentration was determined by Nanodrop quantification (Thermo Fisher Scientific Inc., France). RNA quality was checked on an Agilent 2100 Expert Bioanalyzer (Agilent Technologies, France). Only RNAs with a RIN\u0026thinsp;\u0026gt;\u0026thinsp;8 were used for transcriptomic and qRT-PCR experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMicroarray hybridization\u003c/h2\u003e \u003cp\u003eA reference design with complete dye-swap including two biological replicates was used to compare HT-29 cells among different treatments, for a total of 6 Agilent 4\u0026thinsp;\u0026times;\u0026thinsp;44K Whole Human Genome Microarrays (Agilent Technologies, France). Scheme design is available in \u003cb\u003eFig. S3\u003c/b\u003e. For labeling, 100\u0026nbsp;ng of total RNA was reverse-transcribed and stained with Cy3 or Cy5 using the two-color Low Input Quick Amp Gene Expression Labeling Kit (Agilent Technologies, France) according to the manufacturer's instructions. The CyDye-labeled cRNAs were then purified using the RNeasy Mini Kit (Qiagen, France). cRNA quantity was determined by Nanodrop and quality was checked on the Agilent 2100 Expert Bioanalyzer. Yield and specific activity were determined per manufacturer's instructions. We mixed 825\u0026nbsp;ng of Cy3-labeled cRNA from one treatment with the same amount of Cy5-labeled cRNA from another treatment. cRNAs were hybridized to the Agilent 4\u0026thinsp;\u0026times;\u0026thinsp;44K Whole Human Genome Microarray (Agilent Technologies, France) at 65\u0026nbsp;\u0026deg;C for 17\u0026nbsp;h in a rotating incubator. After hybridization, slides were washed and then scanned using an Agilent G2565CA scanner (Agilent Technologies, France). Raw data were extracted using Feature Extraction software version 10.5.1.1 (Agilent Technologies, France).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMicroarray analysis\u003c/h2\u003e \u003cp\u003eR 3.0.2 software and the LIMMA package [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] were used to analyze microarray data (within-array normalization by loess method followed by between-array normalization by quantile method) and to generate lists of differentially expressed genes. Microarray data were deposited in the NCBI-GEO database (accession number GSE72048). To create gene lists, we filtered by expression levels (|(FC)|\u0026gt;1.5 as a cut-off) as well as by adjusted \u003cem\u003ep-\u003c/em\u003evalues, using a 0.05 threshold with the Benjamini and Hochberg false discovery rate [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] as multiple testing correction. The complete data set is available through the Gene Expression Omnibus GSE72048. Selected gene lists (log ratio and \u003cem\u003ep-\u003c/em\u003evalue data) were loaded into Ingenuity Pathway Analysis (IPA) and Multiarray Experiment Viewer [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] to analyze pathways and generate data displays.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative real time RT-PCR (qRT-PCR)\u003c/h2\u003e \u003cp\u003eThree \u0026micro;g of DNase I-treated total RNAs were reverse-transcribed using Oligo(dT) primers and 1\u0026nbsp;\u0026micro;l of SuperScript II reverse transcriptase (Invitrogen, France). The resulting cDNAs were quantified by Nanodrop (Thermo Fisher Scientific Inc., France) and diluted to a working concentration of 100\u0026nbsp;ng/\u0026micro;l. Reactions were performed in a final volume of 25\u0026nbsp;\u0026micro;l with 500\u0026nbsp;ng cDNA, 10 pM primers, and SYBR Green PCR Master Mix (Applied Biosystems, USA), using a Mastercycler Realplex (Eppendorf, France). Primers are listed in \u003cb\u003eTable S1\u003c/b\u003e. The genes B2M (for human analysis) and HMBS (for mice analysis) were used as internal references [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] was used to calculate the FC in gene expression.\u003c/p\u003e \u003ch2\u003eDact3 siRNA in HT-29 cells\u003c/h2\u003e \u003cp\u003eHT-29 cells were cultured as described above. siGENOME\u0026reg; Human Dact3 siRNA-SMARTpool\u0026reg;, Dact3 siRNA D-015690-01, D-015690-02, D-015690-03, D-015690-17, and control siRNA (Non-Targeting siRNA D001136-01-05 and Cyclophilin B D-001210-02-05) siRNAs were transfected into HT-29 cells using Dharmafect 1 Transfection Reagent (Dharmacon, USA) following manufacturer's instructions with some modifications. A total of 1\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e5\u003c/sup\u003e cells were plated in 12-well plates and transfected using 30\u0026nbsp;nmol siRNA and 2\u0026nbsp;\u0026micro;L of Dharmafect 1 Transfection Reagent per well in DMEM containing 5% FCS and 1% L-Glutamine. After 24\u0026nbsp;h, the medium was changed. After another 24\u0026nbsp;h, control medium, \u003cem\u003eF. prausnitzii\u003c/em\u003e SN, or LYBHI medium was added at a concentration of 10% (v/v) in a total volume of 1\u0026nbsp;ml. Cells were simultaneously stimulated with recombinant human TNF-α (5\u0026nbsp;ng/ml; Peprotech, NJ, USA) at 37\u0026nbsp;\u0026deg;C in 10% CO\u003csub\u003e2\u003c/sub\u003e. After 6\u0026nbsp;h of co-culture, supernatants were tested for IL-8 production by ELISA and RNA was isolated from cells for \u003cem\u003eDact3\u003c/em\u003e expression analysis, as described above. All samples were geretaed and analyzed in triplicate.\u003c/p\u003e \u003ch2\u003e Dact3 overexpression in HT-29 cells \u003c/h2\u003e \u003cp\u003eHT-29 cells were cultured as above. The S-adenosylhomocystein hydrolase inhibitor 3-Deazaneplanocin A (DZNep) and Trichostatin A (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) were used as described in [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] with some modifications. Cells were plated at 0.5\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e5\u003c/sup\u003e cells/well; 24\u0026nbsp;h later, 12.5\u0026nbsp;M DZNep was added, and 48 hours after that, 0.5\u0026nbsp;\u0026micro;M TSA was added for the final 24\u0026nbsp;h of culture. After treatment, cells were stimulated with TNF-α (5\u0026nbsp;ng/ml; Peprotech, NJ, USA) and \u003cem\u003eF. prausnitzii\u003c/em\u003e SN or LYBHI (added at 10% v/v). After 6\u0026nbsp;h of co-incubation, supernatants were tested by ELISA for IL-8 production and RNA was isolated from cells for \u003cem\u003eDact3\u003c/em\u003e expression analysis. All samples were performed in triplicate.\u003c/p\u003e \u003ch2\u003eDact3 -microbiota modulation in vivo\u003c/h2\u003e \u003cp\u003eMicroarray data comparing germ-free and conventionalized mice were described and published previously ([\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], GEO accession number: GSE63299). Conventionalization of germ-free C3H/HeN mice was performed with fresh stools from C3H/HeN donor mice.\u003c/p\u003e \u003ch2\u003eDact3-F. prausnitzii SN modulation in vivo\u003c/h2\u003e \u003cp\u003eC57BL/6 mice (males, 6\u0026ndash;8 weeks of age; Janvier, Le Genest Saint Isle, France) were maintained at the animal care facilities of the National Institute of Agricultural Research (IERP, INRA, Jouy-en-Josas, France) under specific pathogen-free conditions. Mice were housed under standard conditions for a minimum of 1 week before experimentation. All experiments were performed in accordance with European Community rules and approved by the animal care committee COMETHEA (Comit\u0026eacute; d\u0026rsquo;Ethique en Exp\u0026eacute;rimentation Animale du Centre INRA de Jouy-en-Josas et AgroParisTech, Jouy-en-Josas, France). All assays were carried out under agreement N\u0026deg;3445-2016010615159974.\u003c/p\u003e \u003cp\u003eThe protocol for \u003cem\u003eDact3\u003c/em\u003e modulation and quantification \u003cem\u003ein vivo\u003c/em\u003e is illustrated in \u003cb\u003eFig. S1A\u003c/b\u003e. Three groups of mice (\u003cem\u003en\u0026thinsp;=\u003c/em\u003e\u0026thinsp;8) were intragastrically administered 200\u0026nbsp;\u0026micro;l of \u003cem\u003eF. prausnitzii\u003c/em\u003e SN and sacrificed 3\u0026nbsp;h (T3), 6\u0026nbsp;h (T6) or 9\u0026nbsp;h (T9) after gavage. Control (T0) mice were not treated. Colitis was induced as described in [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], by intrarectal injection of 200\u0026nbsp;mg/kg of DNBS solution (ICN, Biomedical Inc.) in 30% ethanol. Fourteen days following the first injection, a 200-\u0026micro;l solution of either 1\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e9\u003c/sup\u003e CFU of \u003cem\u003eF. prausnitzii\u003c/em\u003e or PBS was administered intragastrically for 10 days. Colitis was reactivated 21 days after the first DNBS injection with a second injection of 100\u0026nbsp;mg/kg of DNBS solution. Mice were sacrificed 3 days after the second DNBS injection (\u003cb\u003eFig. S1A\u003c/b\u003e) by cervical dislocation and different parameters of inflammation were recorded as previously described [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eColon samples (one centimeter from distal colon) were frozen in 500\u0026nbsp;\u0026micro;l of RNAlater solution (Ambion, France) in liquid nitrogen and stored at -80\u0026nbsp;\u0026deg;C until use. Total RNA was extracted from individual samples with the RNeasy mini kit (Qiagen) according to the manufacturer\u0026rsquo;s instructions, using homogenization with Tissue Lyser (Qiagen) and purification by on-column digestion of DNA with DNase I. Total RNA was determined by Nanodrop quantification. \u003cem\u003eDact3\u003c/em\u003e expression was analyzed by qRT-PCR.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis, with the exception of transcriptomic analysis, was completed using GraphPad (GraphPad Software, La Jolla, CA, USA). A \u003cem\u003ep-\u003c/em\u003evalue of less than 0.05 was considered significant. Significant differences in the relative expression values of the target genes were tested with REST software using pairwise fixed reallocation randomization [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e "},{"header":"Results","content":" \u003ch2\u003eTranscriptomic analysis reveals Dact3 as a target of F. prausnitzii\u003c/h2\u003e \u003cp\u003eWe used TNF-α to stimulate inflammation in HT-29 cells. A specific trait of this stimulation is upregulation of IL-8 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], which we then measured as a readout for cell inflammatory status. To extend previous research on \u003cem\u003eF. prausnitzii\u003c/em\u003e SN in TNF-α-stimulated HT-29 cells [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], we performed a SN dose-effect experiment and determined the stability (\u003cem\u003eie.\u003c/em\u003e, putative degradation) of both IL-8 and TNF-α in the presence of the SN. \u003cem\u003eF. prausnitzii\u003c/em\u003e SN had a significant dose-response effect (5\u0026ndash;30%) while the bacterial culture medium LYBHI did not (\u003cb\u003eFig. S2A\u003c/b\u003e). Of note, \u003cem\u003eF. prausnitzii\u003c/em\u003e SN did not directly degrade either IL-8 (\u003cb\u003eFig. S2B\u003c/b\u003e) or TNF-α (\u003cb\u003eFig. S2C\u003c/b\u003e). To elucidate the host molecular mechanisms involved (\u003cem\u003eeg.\u003c/em\u003e, the modulatory effects on IL-8 production), we performed a transcriptomic analysis of TNF-α-stimulated HT-29 cells exposed to \u003cem\u003eF. prausnitzii\u003c/em\u003e SN (microarray hybridization schema is presented in \u003cb\u003eFig. S3\u003c/b\u003e). Before evaluating the effects of the SN, we first determined the regulatory changes due to TNF-α stimulation alone. Compared to control HT-29 cells, 227 genes were upregulated and 60 genes were downregulated in TNF-α-stimulated cells (adj. \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05, |fold change (FC)|\u0026gt;1.5). The genes with the largest changes in expression are listed in \u003cb\u003eTable S2\u003c/b\u003e. As expected, IL-8 was among the most upregulated genes. According to Ingenuity Pathway Analysis (IPA), upstream regulators such as TNF-α, IFN-γ, and NF-κB were also activated, confirming the inflamed status of the HT-29 cells. Next, we evaluated the effect of the LYBHI medium used to culture \u003cem\u003eF. prausnitzii.\u003c/em\u003e Here, only 84 genes were differentially regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), and LYBHI did not seem to reduce cellular inflammation. Finally, we introduced \u003cem\u003eF. prausnitzii\u003c/em\u003e SN. There were extensive changes in gene regulation: 913 genes were upregulated and 585 were downregulated after TNF-α stimulation and SN treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Interestingly, these genes are involved in several inflammatory pathways such as NF-κβ, p38 and other ERK/MAPK pathways. Among these, 25% of the genes that had been activated by TNF-α exposure (in our first comparison) were inactivated by treatment with \u003cem\u003eF. prausnitzii\u003c/em\u003e SN (70 out of 287). To explore this complex gene regulation network, we submitted this dataset to IPA, which highlighted the SN-affected pathways. This analysis confirmed the ability of \u003cem\u003eF. prausnitzii\u003c/em\u003e SN to regulate ERK/MAPK signaling pathway as the most important pathway related to inflammation modulated specifically by \u003cem\u003eF. prausnitzii\u003c/em\u003e SN (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Notably, 7% of all genes regulated by \u003cem\u003eF. prausnitzii\u003c/em\u003e SN were linked to MAPK pathways, including JNK. The 10 genes with the largest fold-change in regulation are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cb\u003eTable S3\u003c/b\u003e, IL-8 was found to be downregulated, while the most upregulated gene was \u003cem\u003eDact3\u003c/em\u003e (FC\u0026thinsp;=\u0026thinsp;17.2). \u003cem\u003eDact3\u003c/em\u003e belongs to the \u003cem\u003eDact\u003c/em\u003e gene family, whose members interact with the Dsh protein to inhibit Dsh-induced activation of the JNK pathway [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The Dact3/JNK pathway is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. Finally, in order to validate our transcriptomic data, the differential expression of up- and downregulated genes, including \u003cem\u003eDact3\u003c/em\u003e, was validated by RT-qPCR (only \u003cem\u003eDact3\u003c/em\u003e data shown). B2M was used as reference for data normalization as described in [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Compared to LYBHI, \u003cem\u003eF. prausnitzii\u003c/em\u003e SN led to significant \u003cem\u003eDact3\u003c/em\u003e upregulation in TNF-α-stimulated HT-29 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), confirming transcriptomic observations.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003ch2\u003eRole of Dact3 in TNF-α-stimulated HT-29 cells\u003c/h2\u003e \u003cp\u003eAs \u003cem\u003eDact3\u003c/em\u003e was the most upregulated gene in the transcriptomics analysis, we thus decided to focus our investigations on this gene. To investigate the effect of \u003cem\u003eDact3\u003c/em\u003e on IL-8 production in TNF-α-stimulated HT-29 cells, we overexpressed or knocked down \u003cem\u003eDact3\u003c/em\u003e in IECs. First, we tried to overexpress \u003cem\u003eDact3\u003c/em\u003e using transient transfection in HT-29 cells; unfortunately, we were unable to efficiently transfect these cells (data not shown). This could be due to the strong upregulation of \u003cem\u003eDact3\u003c/em\u003e that led to massive apoptosis of the cells, as was previously reported for colorectal cancer cells [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. We then used the siRNA technology to knock down \u003cem\u003eDact3\u003c/em\u003e mRNA in TNF-α-inflamed HT-29 cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, RT-qPCR analysis confirmed that: i) both the non-targeting (NT) siRNA and LYBHI had no effect on \u003cem\u003eDact3\u003c/em\u003e expression (FC\u0026thinsp;=\u0026thinsp;1); ii) \u003cem\u003eF. prausnitzii\u003c/em\u003e SN strongly upregulated \u003cem\u003eDact3\u003c/em\u003e (FC\u0026thinsp;=\u0026thinsp;30) in presence of NT siRNA; and iii) \u003cem\u003eDact3\u003c/em\u003e siRNA significantly reduced the abundance of \u003cem\u003eDact3\u003c/em\u003e mRNA induced by \u003cem\u003eF. prausnitzii\u003c/em\u003e SN. Besides, treatment with \u003cem\u003eDact3\u003c/em\u003e siRNA in TNF-α-stimulated HT-29 cells tends to increase IL-8 production (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) suggesting an important role of \u003cem\u003eDact3\u003c/em\u003e activation in intestinal homeostasis. Moreover, the inhibitory effect of \u003cem\u003eF. prausnitzii\u003c/em\u003e SN on IL-8 production was partially lost in \u003cem\u003eDact3\u003c/em\u003e-silencing condition siRNA. Next, we evaluated the effect of factors modulating \u003cem\u003eDact3\u003c/em\u003e expression on IL-8 production. As histone modification has been reported to modulate \u003cem\u003eDact3\u003c/em\u003e expression [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], we used a mix of TSA (a histone deacetylase inhibitor) and DZNep (a histone methylation inhibitor) drugs as previously described [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. These drugs led to a strong upregulation of \u003cem\u003eDact3\u003c/em\u003e similar to that obtained with \u003cem\u003eF. prausnitzii\u003c/em\u003e SN (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Furthermore, after drug treatment and TNF-α stimulation, \u003cem\u003eDact3\u003c/em\u003e upregulation strongly inhibited IL-8 production in HT-29 cells, to an even stronger extent than was observed with \u003cem\u003eF. prausnitzii\u003c/em\u003e SN (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCharacterization of the CCCA KO HEK293 cell\u003c/p\u003e \u003cp\u003eline\u003c/p\u003e \u003ch2\u003eIdentification of the F. prausnitzii-effectors responsible for Dact3 modulation\u003c/h2\u003e \u003cp\u003eTo identify the bacterial effector(s) responsible for \u003cem\u003eDact3\u003c/em\u003e modulation, we tested some molecules implicated in the anti-inflammatory effects of \u003cem\u003eF. prausnitzii\u003c/em\u003e: the MAM protein [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and salicylic acid [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In addition, as \u003cem\u003eF. prausnitzii\u003c/em\u003e abundantly produces butyrate, which reduces IL-8 production in TNF-α-stimulated HT-29 cells [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], we also tested its effect on \u003cem\u003eDact3\u003c/em\u003e modulation (only results for butyrate are shown). Treatment with 10% of a solution of 10\u0026nbsp;mM of butyrate (the concentration present in the \u003cem\u003eF. prausnitzii\u003c/em\u003e SN [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]) led to strong upregulation of \u003cem\u003eDact3\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) and a decrease in IL-8 production (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), whereas no \u003cem\u003eDact3\u003c/em\u003e modulation was observed with either salicylic acid or MAM. Altogether, these results reveal that butyrate is one of the \u003cem\u003eF. prausnitzii\u003c/em\u003e effectors responsible for \u003cem\u003eDact3\u003c/em\u003e modulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn order to validate this hypothesis, we tested other gut bacterial strains (\u003cem\u003eeg. Roseburia intestinalis\u003c/em\u003e, \u003cem\u003eAkkermansia muciniphila\u003c/em\u003e and \u003cem\u003eBacteroides thetaiotaomicron\u003c/em\u003e), known to produce or not butyrate (\u003cb\u003eTable S4\u003c/b\u003e), for their capacities to upregulate \u003cem\u003eDact3\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) and downregulate IL-8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). We also tested a probiotic lactic acid bacterium (LAB) with anti-inflammatory capacities, \u003cem\u003eL. casei\u003c/em\u003e BL23, that does not produce butyrate. Strikingly, the SN from the only another butyrate-producing bacterium (\u003cem\u003eR. intestinalis\u003c/em\u003e) highly upregulated \u003cem\u003eDact3\u003c/em\u003e expression and thus downregulated IL-8 production. In contrast, the three other bacterial strains that do not produce butyrate (\u003cem\u003eA. muciniphila, B. thetaiotaomicron\u003c/em\u003e and \u003cem\u003eL. casei\u003c/em\u003e BL23) did not have any effect on neither \u003cem\u003eDact3\u003c/em\u003e nor IL-8.\u003c/p\u003e \u003ch2\u003e In vivo validation of Dact3 modulation\u003c/h2\u003e \u003cp\u003e \u003cem\u003eDact3\u003c/em\u003e modulation was first investigated in healthy mice orally administered with \u003cem\u003eF. prausnitzii\u003c/em\u003e SN and sacrificed at different time points (\u003cem\u003ee.g.\u003c/em\u003e 0, 3, 6 and 9 hours after administration) to evaluate its effects in normal physiological conditions (\u003cb\u003eFig. S1A\u003c/b\u003e). \u003cem\u003eF. prausnitzii\u003c/em\u003e SN led to a significant increase in \u003cem\u003eDact3\u003c/em\u003e mRNA in colonic samples 9\u0026nbsp;h after its administration (\u003cb\u003eFig. S1B\u003c/b\u003e). Other groups of mice were orally administered with either \u003cem\u003eF. prausnitzii\u003c/em\u003e SN, \u003cem\u003eR. intestinalis\u003c/em\u003e SN or butyrate (1\u0026nbsp;mM) and euthanized 9\u0026nbsp;h later. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, butyrate was detected in colonic samples of mice treated with either \u003cem\u003eF. prausnitzii\u003c/em\u003e SN or \u003cem\u003eR. intestinalis\u003c/em\u003e SN but not with butyrate itself, suggesting that soluble butyrate is rapidly absorbed in the colon. Strikingly, all treatments, including butyrate, results in \u003cem\u003eDact3\u003c/em\u003e upregulation in samples from proximal colon (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then investigated \u003cem\u003ein vivo Dact3\u003c/em\u003e modulation in an inflammatory context, using a murine model in which chronic moderate inflammation was induced by intrarectal injection of dinitrobenzene sulfonic acid (DNBS). Animals were given daily oral gavages of live \u003cem\u003eF. prausnitzii\u003c/em\u003e bacteria (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), as previously described [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. As expected, \u003cem\u003eF. prausnitzii\u003c/em\u003e administration led to significant reductions in markers of colitis with improvements in weight loss and a decrease in macroscopic scores (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), MPO activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC) and a reduction of the pro-inflammatory cytokines IFN-γ (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), IL-6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE), IL-17A (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF) and the chemokine MCP-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). Moreover, \u003cem\u003eDact3\u003c/em\u003e expression was upregulated in the colon of \u003cem\u003eF. prausnitzii\u003c/em\u003e-treated mice compared to the PBS-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH), confirming that \u003cem\u003eDact3\u003c/em\u003e is induced by \u003cem\u003eF. prausnitzii\u003c/em\u003e in an inflammatory context too.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFinally, to assess the effect of the gut microbiota on \u003cem\u003eDact3\u003c/em\u003e modulation in \u003cem\u003ein vivo\u003c/em\u003e physiological conditions, we investigated a dataset that we previously published comparing the colonic transcriptome of germ-free and conventionalized (Conv) mice [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. We found that \u003cem\u003eDact3\u003c/em\u003e expression was significantly reduced in germ-free compared to Conv mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These observations confirm that gut microbiota has a major impact on \u003cem\u003eDact3\u003c/em\u003e expression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e "},{"header":"Discussion","content":" \u003cp\u003e \u003cem\u003eF. prausnitzii\u003c/em\u003e is a commensal bacterium well-known for its immuno-modulatory properties and more specifically for its anti-inflammatory effects both \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Here, we demonstrated that \u003cem\u003eF. prausnitzii\u003c/em\u003e SN is able to block IL-8 production in TNF-α-activated HT-29, but not through the proteolytic degradation of either TNF-α or IL-8. Because of this, and because \u003cem\u003eF. prausnitzii\u003c/em\u003e is extremely oxygen sensitive (EOS) and cannot be easily cultured with human cells [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], we decided to further study the immunomodulatory effects of its SN. \u003cem\u003eF. prausnitzii\u003c/em\u003e SN modulated the expression of a massive number of genes, in fact, more than the TNF-α treatment itself (\u003cb\u003eTable S2\u003c/b\u003e). Among the inflammation-related pathways regulated by \u003cem\u003eF. prausnitzii\u003c/em\u003e SN, we found enrichment in genes related to MAPKs. In particular, \u003cem\u003eDact3\u003c/em\u003e, a gene implicated in Wnt/JNK regulation, was among the top upregulated genes by \u003cem\u003eF. prausnitzii\u003c/em\u003e SN in non-stimulated as well as in TNF-α-stimulated cells. The \u003cem\u003eDact\u003c/em\u003e gene family was initially reported in studies of embryonic development [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Although \u003cem\u003eDact3\u003c/em\u003e is involved in postnatal development (adult \u003cem\u003eDact3\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice show a mild reduction in body weight), the fact that \u003cem\u003eDact3\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice are viable means that this gene is not essential for mouse embryogenesis, postnatal survival, and reproduction [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. \u003cem\u003eDact3\u003c/em\u003e has a postnatal role as a regulator in the Wnt/β-catenin signaling pathway [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and has been associated with several types of cancer: colorectal [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], breast [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], ovarian [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], lung [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], papillary thyroid [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], and renal fibrosis [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In colorectal cancer cell lines, \u003cem\u003eDact3\u003c/em\u003e transcription is epigenetically downregulated by a bivalent histone modification [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In particular, drugs that target both histone methylation (DZNep) and deacetylation (TSA) strongly induce \u003cem\u003eDact3\u003c/em\u003e expression [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], a result that we confirmed here. Moreover, we also observed that these drugs reduced IL-8 production by TNF-α-stimulated HT-29 cells in a way that was similar to the effect of \u003cem\u003eF. prausnitzii\u003c/em\u003e SN. These results show that \u003cem\u003eDact3\u003c/em\u003e has an important function in the IL-8 pathway and that IL-8 inhibition by \u003cem\u003eF. prausnitzii\u003c/em\u003e SN is, at least partly, mediated via \u003cem\u003eDact3\u003c/em\u003e upregulation. \u003cem\u003eDact3\u003c/em\u003e could thus be an epigenetic regulator of inflammation and play a key role in intestinal homeostasis. To further decipher this, we evaluated the effect of \u003cem\u003eDact3\u003c/em\u003e silencing in HT-29 using siRNA. Silencing of \u003cem\u003eDact3\u003c/em\u003e in IECs prevented \u003cem\u003eF. prausnitzii\u003c/em\u003e SN from blocking IL-8 production.\u003c/p\u003e \u003cp\u003e \u003cem\u003eDact3\u003c/em\u003e histone modulation could provide clues about the bacterial effectors responsible for this regulation. In this study, only butyrate was able to both upregulate \u003cem\u003eDact3\u003c/em\u003e expression and to block IL-8 production by TNF-α-stimulated HT-29 cells. Indeed, butyrate-induced reduction in IL-8 production was similar to that observed with the \u003cem\u003eF. prausnitzii\u003c/em\u003e SN, but \u003cem\u003eDact3\u003c/em\u003e upregulation was higher in the presence of butyrate than of SN. This is probably because \u003cem\u003eDact3\u003c/em\u003e is also modulated by histone deacetylases [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] which are known to be inhibited by butyrate [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In this context, our results confirm those obtained by Fung \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], who found that \u003cem\u003eDact3\u003c/em\u003e appeared to be differentially modulated in HT-29 cells treated with butyrate. However, these authors did not further explore \u003cem\u003eDact3\u003c/em\u003e in their study.\u003c/p\u003e \u003cp\u003eAltogether, our results provide evidence that \u003cem\u003eF. prausnitzii\u003c/em\u003e SN regulates \u003cem\u003eDact3\u003c/em\u003e/IL-8 production and suggest that butyrate produced by \u003cem\u003eF. prausnitzii\u003c/em\u003e is the main actor in this regulation. As butyrate is known to have pleiotropic effects in the intestinal cell life cycle and numerous beneficial effects for human health (e.g., anti-inflammatory and anti-tumorigenic properties) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], it is plausible that butyrate production is a means by which \u003cem\u003eF. prausnitzii\u003c/em\u003e affects its host physiological functions and homeostasis to maintain health. However, further studies are necessary to confirm this hypothesis. In particular, genetic manipulation of \u003cem\u003eF. prausnitzii\u003c/em\u003e to inactivate the gene encoding the butyril-CoA synthase involved in butyrate metabolism would be helpful to answer to this question. Indeed, O\u0026rsquo;Cuiv \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] have isolated \u003cem\u003eF. prausnitzii\u003c/em\u003e transconjugants using metaparental mating and this strategy opens promising perspectives to manipulate \u003cem\u003eF. prausnitzii.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eFinally, \u003cem\u003ein vitro\u003c/em\u003e observations were validated \u003cem\u003ein vivo\u003c/em\u003e in two different models: first, in healthy mice that were orally administered \u003cem\u003eF. prausnitzii\u003c/em\u003e SN one time (\u003cb\u003eFig. S3\u003c/b\u003e) and second, in inflamed mice that were given live \u003cem\u003eF. prausnitzii\u003c/em\u003e orally for 10 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). In both \u003cem\u003ein vivo\u003c/em\u003e models, either \u003cem\u003eF. prausnitzii\u003c/em\u003e or \u003cem\u003eF. prausnitzii\u003c/em\u003e SN positively regulated \u003cem\u003eDact3\u003c/em\u003e, confirming the key effect of \u003cem\u003eF. prausnitzii\u003c/em\u003e on this gene.\u003c/p\u003e "},{"header":"Conclusions","content":" \u003cp\u003eIn conclusion, we propose in this study a new role for \u003cem\u003eDact3\u003c/em\u003e as a master regulator of intestinal homeostasis, particularly in inflamed cells. Although it is expressed in IECs at a low level, \u003cem\u003eDact3\u003c/em\u003e seems to be essential for intestinal homeostasis, as its downregulation or loss leads to a global increase in inflammation. We hypothesize that \u003cem\u003eDact3\u003c/em\u003e upregulation inhibits the AP-1 transcription factor, which in turn leads to a global downregulation of genes encoding for pro-inflammatory cytokines such as IL-2, IL-6, and IL-8 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Additionally, the ability of \u003cem\u003eF. prausnitzii\u003c/em\u003e SN to modulate other genes involved in cancer pathways (as revealed in the transcriptomic analysis) represents a novel potential beneficial effect of this commensal anti-inflammatory bacterium, which is currently being investigated by our laboratory.\u003c/p\u003e \u003cp\u003eIn conclusion, our study provides the first clues on one of the host molecular targets involved in the anti-inflammatory effects of \u003cem\u003eF. prausnitzii\u003c/em\u003e in IECs. Moreover, these results point out \u003cem\u003eDact3\u003c/em\u003e as a potential master regulator of inflammation in IECs. As there is increasing interest in exploring new alternatives for IBD treatment, this research suggests at least three potential opportunities: i) use of \u003cem\u003eF. prausnitzii\u003c/em\u003e itself, ii) use of drugs to modulate \u003cem\u003eDact3\u003c/em\u003e expression (such as histone deacetylases) and iii) heterologous delivery of \u003cem\u003eDact3\u003c/em\u003e (either as a cDNA or a protein) using food-grade live vectors [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. For these, further studies on \u003cem\u003eDact3\u003c/em\u003e-knockout mice will be necessary to understand the physiological functions of \u003cem\u003eDact3\u003c/em\u003e.\u003c/p\u003e "},{"header":"Declarations","content":" \u003cp\u003e \u003ch2\u003eAvailability of data and materials\u003c/h2\u003e \u003cp\u003eThe complete data set is available through the Gene Expression Omnibus GSE72048.\u003c/p\u003e \u003c/p\u003e \u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e \u003cp\u003eAll experiments were performed in accordance with European Community rules and approved by the animal care committee COMETHEA (Comit\u0026eacute; d\u0026rsquo;Ethique en Exp\u0026eacute;rimentation Animale du Centre INRA de Jouy-en-Josas et AgroParisTech, Jouy-en-Josas, France) under agreement N\u0026deg;3445-2016010615159974.\u003c/p\u003e \u003ch2\u003eConsent for publication\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was a part of FPARIS collaborative project selected and supported by the Vitagora Competitive Cluster and funded by the French Fond Unique Interminist\u0026eacute;riel, n\u0026deg;F1010012D, the Fonds Europ\u0026eacute;en de D\u0026eacute;veloppement R\u0026eacute;gional (Bourgogne: 34606), the Burgundy Region, the Conseil G\u0026eacute;n\u0026eacute;ral 21, and the Grand Dijon. This work was also supported by Merck M\u0026eacute;dication Familiale (Dijon, France) and Biovitis (Saint Etienne de Chomeil, France). RM received a salary from these grants and ML received a PhD grant from ABIES.\u003c/p\u003e \u003ch2\u003eAuthors\u0026rsquo; contributions\u003c/h2\u003e \u003cp\u003eAuthor contributions: L.G.B.H. and F.C. designed research; M.L., R.M., E.T.M., S.C. and F.C. performed research; H.S. and P.G.D contributed analytic tools; M.L., R.M., H.S., F.C., and L.G.B.H., analyzed data; and M.L., R.M., H.S., P.L., F.C., and L.G.B.H wrote the paper. All read and approved the final manuscript.\u003c/p\u003e \u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe authors would like to thank Marco Moroldo, J\u0026eacute;r\u0026ocirc;me Lecardonnel, and D\u0026eacute;borah Jardet from the GABI/CRB GABI platform, and Valentin Loux and all IERP personnel for their help. 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EMBO Mol Med. 2011;3(9):559\u0026ndash;72.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eBenbouziane B, Ribelles P, Aubry C, Martin R, Kharrat P, Riazi A, Langella P, Bermudez-Humaran LG. Development of a Stress-Inducible Controlled Expression (SICE) system in Lactococcus lactis for the production and delivery of therapeutic molecules at mucosal surfaces. J Biotechnol. 2013;168(2):120\u0026ndash;9.\u003c/span\u003e \u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Commensal bacteria, Faecalibacterium prausnitzii, inflammatory bowel disease, transcriptomic analysis, signaling pathway","lastPublishedDoi":"10.21203/rs.3.rs-28864/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-28864/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground\u003c/p\u003e\u003cp\u003eThe commensal bacterium \u003cem\u003eFaecalibacterium prausnitzii\u003c/em\u003e plays a key role in inflammatory bowel disease (IBD) pathogenesis and serves as a general health biomarker in humans. However, the host molecular mechanisms that underlie its anti-inflammatory effects remain unknown.\u003c/p\u003e\u003cp\u003eMethods\u003c/p\u003e\u003cp\u003eA transcriptomic approach on human intestinal epithelial cells (HT-29) that were stimulated with TNF-α and exposed to \u003cem\u003eF. prausnitzii\u003c/em\u003e culture supernatant (SN) was used. Modulation of the most upregulated gene after \u003cem\u003eF. prausnitzii\u003c/em\u003e SN contact was validated both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eResults\u003c/p\u003e\u003cp\u003e\u003cem\u003eF. prausnitzii\u003c/em\u003e SN upregulates the expression of \u003cem\u003eDact3\u003c/em\u003e, a gene linked to the Wnt/JNK pathway. Interestingly, when we silenced \u003cem\u003eDact3\u003c/em\u003e expression, the effect of \u003cem\u003eF. prausnitzii\u003c/em\u003e SN was lost. Butyrate was identified as the \u003cem\u003eF. prausnitzii\u003c/em\u003e effector responsible for \u003cem\u003eDact3\u003c/em\u003e modulation. \u003cem\u003eDact3\u003c/em\u003e upregulation was also validated \u003cem\u003ein vivo\u003c/em\u003e in both healthy and inflamed mice treated with either \u003cem\u003eF. prausnitzii\u003c/em\u003e SN or the live bacteria, respectively. Finally, we demonstrated by colon transcriptomics that gut microbiota directly influences \u003cem\u003eDact3\u003c/em\u003e expression.\u003c/p\u003e\u003cp\u003eConclusions\u003c/p\u003e\u003cp\u003eOur results provide new clues about the host molecular mechanisms involved in the anti-inflammatory effects of the beneficial commensal bacterium \u003cem\u003eF. prausnitzii\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e*Contributed equally to this work\u003c/p\u003e","manuscriptTitle":"Butyrate mediates anti-inflammatory effects of Faecalibacterium prausnitzii in intestinal epithelial cells through Dact3","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-05-15 16:45:56","doi":"10.21203/rs.3.rs-28864/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a75bd16a-aadf-43d5-99c1-9bc5dcc579ca","owner":[],"postedDate":"May 15th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":100872,"name":"General Microbiology"}],"tags":[],"updatedAt":"2021-07-22T18:50:45+00:00","versionOfRecord":{"articleIdentity":"rs-28864","link":"https://doi.org/10.1080/19490976.2020.1826748","journal":{"identity":"gut-microbes","isVorOnly":true,"title":"Gut Microbes"},"publishedOn":"2020-10-15 18:50:45","publishedOnDateReadable":"October 15th, 2020"},"versionCreatedAt":"2020-05-15 16:45:56","video":"","vorDoi":"10.1080/19490976.2020.1826748","vorDoiUrl":"https://doi.org/10.1080/19490976.2020.1826748","workflowStages":[]},"version":"v1","identity":"rs-28864","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-28864","identity":"rs-28864","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}