Als and methods section. (A ) Analysis of Disease activity index (DAI) in MyD88+/+ (A) and Rebaudioside A cost MyD882/2 mice (C). (B ) Analysis of mucosal damage score in MyD88+/+ (B) and MyD882/2 mice (D). (E ) H E staining of representative paraffin-embedded sections from distal colons after administration of vehicle (control mice), TNBS or TNBS plus 6-ECDCA in MyD88+/+ (panels E ) and MyD882/2 (H ) mice. Data are mean 6 SE of 6 animals.*P,0.05 versus wild type naive mice. #P,0.05 versus wild type mice 
administered TNBS. doi:10.1371/journal.pone.0054472.gindicating that the FXR signaling pathways lies downstream to TLR9 and MyD88 and is conserved in mice lacking the expression of these genes. Previous studies have shown that CpG rescues wild type mice from murine colitis, highlighting a role for TLR9 generated signals in repressing intestinal inflammation [20] and activation of TLR9 is instrumental to the immune-regulatory activity of 1326631 probiotics in rodent models of colitis [21]. However, since in vivo CpG administration failed to rescue FXR2/2 from colitis induced by TNBS, it appears that FXR is a non-dispensable component of the immune-modulatory activity of TLR9. Another important finding of this study was the demonstration that regulation of FXR by TLR9 is mediated by activation of IRF7. IRF7 is a member of the interferon regulatory family of transcription factors involved in the transcriptional activation of virus-inducible cellular genes, including the type I interferon genes [22]. IRF7 is essential for the MedChemExpress Biotin N-hydroxysuccinimide ester induction of IFN-a/b genes via the virus-activated, MyD88-independent and the TLR-activated MyD88-dependent pathway [22]. Viral induction of IFN-a/b genes is severely impaired in IRF2/2 fibroblasts and IRF2/2 mice are more vulnerable than Myd882/2 mice to viral infection, and this correlates with a decrease in serum IFN levels, indicating theimportance of the IRF7-dependent induction of systemic IFN responses for innate antiviral immunity [28]. Furthermore, robust induction of IFN production by activation of the TLR9 subfamily is entirely dependent on IRF7 [22]. IRF7-RE have been detected in the promoter of several CpG responsive genes [29]. In the present study we report the detection of a IRF7-RE in the promoter of FXR. This IRF7-RE was conserved across species and its functionality was examined by a variety of molecular approaches. Results from ChIP, EMSA and transactivation assays have shown that not only IRF7 binds to the FXR promoter in response to TLR9 stimulation with CpG, but that this interaction results in a IRF7 mediated transcription of the FXR gene. Furthermore, 
by EMSA we have demonstrated that IRF7 binds to a specific IRF7 consensus and that mutation of this consensus results in the abrogation of the binding. Present findings might have a therapeutic relevance because probiotics that are increasingly used for treating IBDs and other intestinal disorders are positive regulator of FXR expression [30]. Since FXR functions as a negative regulator of inflammatory responses, present data uncover a striking mechanism through which nuclear receptors function as a gatekeeper signaling in regulating intestinal immune response to microbiota.FXR Is a Novel TLR-9 Target GeneFigure 5. An intact FXR signalling is required to preserve TLR9 action. TNBS colitis was induced in FXR+/+ and FXR2/2 mice. Mice were administered CpG as described in materials and methods. (A ) Analysis of Disease activity index (DAI) in FXR+/+ (A) and FXR2/2 mice (C). (B.Als and methods section. (A ) Analysis of Disease activity index (DAI) in MyD88+/+ (A) and MyD882/2 mice (C). (B ) Analysis of mucosal damage score in MyD88+/+ (B) and MyD882/2 mice (D). (E ) H E staining of representative paraffin-embedded sections from distal colons after administration of vehicle (control mice), TNBS or TNBS plus 6-ECDCA in MyD88+/+ (panels E ) and MyD882/2 (H ) mice. Data are mean 6 SE of 6 animals.*P,0.05 versus wild type naive mice. #P,0.05 versus wild type mice administered TNBS. doi:10.1371/journal.pone.0054472.gindicating that the FXR signaling pathways lies downstream to TLR9 and MyD88 and is conserved in mice lacking the expression of these genes. Previous studies have shown that CpG rescues wild type mice from murine colitis, highlighting a role for TLR9 generated signals in repressing intestinal inflammation [20] and activation of TLR9 is instrumental to the immune-regulatory activity of 1326631 probiotics in rodent models of colitis [21]. However, since in vivo CpG administration failed to rescue FXR2/2 from colitis induced by TNBS, it appears that FXR is a non-dispensable component of the immune-modulatory activity of TLR9. Another important finding of this study was the demonstration that regulation of FXR by TLR9 is mediated by activation of IRF7. IRF7 is a member of the interferon regulatory family of transcription factors involved in the transcriptional activation of virus-inducible cellular genes, including the type I interferon genes [22]. IRF7 is essential for the induction of IFN-a/b genes via the virus-activated, MyD88-independent and the TLR-activated MyD88-dependent pathway [22]. Viral induction of IFN-a/b genes is severely impaired in IRF2/2 fibroblasts and IRF2/2 mice are more vulnerable than Myd882/2 mice to viral infection, and this correlates with a decrease in serum IFN levels, indicating theimportance of the IRF7-dependent induction of systemic IFN responses for innate antiviral immunity [28]. Furthermore, robust induction of IFN production by activation of the TLR9 subfamily is entirely dependent on IRF7 [22]. IRF7-RE have been detected in the promoter of several CpG responsive genes [29]. In the present study we report the detection of a IRF7-RE in the promoter of FXR. This IRF7-RE was conserved across species and its functionality was examined by a variety of molecular approaches. Results from ChIP, EMSA and transactivation assays have shown that not only IRF7 binds to the FXR promoter in response to TLR9 stimulation with CpG, but that this interaction results in a IRF7 mediated transcription of the FXR gene. Furthermore, by EMSA we have demonstrated that IRF7 binds to a specific IRF7 consensus and that mutation of this consensus results in the abrogation of the binding. Present findings might have a therapeutic relevance because probiotics that are increasingly used for treating IBDs and other intestinal disorders are positive regulator of FXR expression [30]. Since FXR functions as a negative regulator of inflammatory responses, present data uncover a striking mechanism through which nuclear receptors function as a gatekeeper signaling in regulating intestinal immune response to microbiota.FXR Is a Novel TLR-9 Target GeneFigure 5. An intact FXR signalling is required to preserve TLR9 action. TNBS colitis was induced in FXR+/+ and FXR2/2 mice. Mice were administered CpG as described in materials and methods. (A ) Analysis of Disease activity index (DAI) in FXR+/+ (A) and FXR2/2 mice (C). (B.