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Commensal bacteria are crucial for the development and maintenance of a healthy immune system therefore contributing to the global well-being of their host. A wide variety of metabolites produced by commensal bacteria are influencing host health but the characterization of the multiple molecular mechanisms involved in host-microbiota interactions is still only partially unraveled. The intestinal epithelial cells (IECs) take a central part in the host-microbiota dialogue by inducing the first microbial-derived immune signals. Amongst the numerous effector molecules modulating the immune responses produced by IECs, indoleamine 2,3-dioxygenase-1 (IDO-1) is essential for gut homeostasis. IDO-1 expression is dependent on the microbiota and despites its central role, how the commensal bacteria impacts its expression is still unclear. Therefore, we investigated the impact of individual cultivable commensal bacteria on IDO-1 transcriptional expression and found that the short chain fatty acid (SCFA) butyrate was the main metabolite controlling IDO-1 expression in human primary IECs and IEC cell-lines. This butyrate-driven effect was independent of the G-protein coupled receptors GPR41, GPR43, and GPR109a and of the transcription factors SP1, AP1, and PPARγ for which binding sites were reported in the IDO-1 promoter. We demonstrated for the first time that butyrate represses IDO-1 expression by two distinct mechanisms. Firstly, butyrate decreases STAT1 expression leading to the inhibition of the IFNγ-dependent and phosphoSTAT1-driven transcription of IDO-1 . In addition, we described a second mechanism by which butyrate impairs IDO-1 transcription in a STAT1-independent manner that could be attributed to its histone deacetylase (HDAC) inhibitor property. In conclusion, our results showed that IDO-1 expression is down-regulated by butyrate via a dual mechanism: the reduction of STAT1 level and the HDAC inhibitor property of SCFAs.

The intestinal microbiota contributes to the global wellbeing of their host by their fundamental role in the induction and maintenance of a healthy immune system. Commensal bacteria shape the mucosal immune system by influencing the proportion and the activation state of anti-inflammatory regulatory T cells (Treg) by metabolites that are still only partially unravelled. Microbiota members such as Clostridiales provide a transforming growth factor β (TGFβ)-rich environment that promotes the accumulation of Treg cells in the gut. The intestinal epithelial cells (IECs) take a central part in this process, as they are a major source of TGFβ1 upon bacterial colonisation. In this study, we investigated which gut commensal bacteria were able to regulate the TGFB1 human promoter in IECs using supernatants from cultured bacteria. We reported that Firmicutes and Fusobacteria supernatants were the most potent TGFB1 modulators in HT-29 cells. Furthermore, we demonstrated that butyrate was the main metabolite in bacterial supernatants accounting for TGFβ1 increase. This butyrate-driven effect was independent of the G-protein coupled receptors GPR41, GPR43 and GPR109a, the transporter MCT1 as well as the transcription factors NF-κB and AP-1 present on TGFB1 promoter. Interestingly, HDAC inhibitors were inducing a similar TGFB1 increase suggesting that butyrate acted through its HDAC inhibitor properties. Finally, our results showed that SP1 was the main transcription factor mediating the HDAC inhibitor effect of butyrate on TGFB1 expression. This is, to our knowledge, the first characterisation of the mechanisms underlying TGFB1 regulation in IEC by commensal bacteria derived butyrate.

The intestinal microbiota has many beneficial roles for its host. However, the precise mechanisms developed by the microbiota to influence the host intestinal cell responses are only partially known. The complexity of the ecosystem and our inability to culture most of these micro-organisms have led to the development of molecular approaches such as functional metagenomics, i.e. the heterologous expression of a metagenome in order to identify functions. This elegant strategy coupled to high throughput screening allowed to identify novel enzymes from different ecosystems where culture methods have not yet been adapted to isolate the candidate microorganisms. We have proposed to use this functional metagenomic approach in order to model the microbiota's interaction with the host by combining this heterologous expression with intestinal reporter cell lines. The addition of the cellular component to this functional metagenomic approach introduced a second important source of variability resulting in a novel challenge for high throughput screening. First attempts of high throughput screening with various reporter cell-lines showed a high distribution of the response and consequent difficulties to reproduce the response, impairing an easy and clear identification of confirmed hits. In this study, we developed a robust and reproducible methodology to combine these two biological systems for high throughput application. We optimized experimental setups and completed them by appropriate statistical analysis tools allowing the use this innovative approach in a high throughput manner and on a broad range of reporter assays. We herewith present a methodology allowing a high throughput screening combining two biological systems. Therefore ideal conditions for homogeneity, sensitivity and reproducibility of both metagenomic clones as well as reporter cell lines have been identified and validated. We believe that this innovative method will allow the identification of new bioactive microbial molecules and, subsequently, will promote understanding of host-microbiota interactions.