Claudia Stanzel

Doxycycline, metronidazole and isotretinoin: Do they modify microRNA/mRNA expression profiles and function in murine T-cells?

Inflammatory bowel disease (IBD) may develop due to an inflammatory response to commensal gut microbiota triggered by environmental factors in a genetically susceptible host. Isotretinoin (acne therapy) has been inconsistently associated with IBD onset and flares but prior treatment with antibiotics, also associated with IBD development, complicates the confirmation of this association. Here we studied in mice whether doxycycline, metronidazole or isotretinoin induce epigenetic modifications, and consequently change T-cell mRNA expression and/or function directly after treatment and after a 4 week recovery period. Isotretinoin induced IL-10 signaling in Tregs and naive T-cells directly after treatment and reduced effector T-cell proliferation alone and in co-culture with Tregs. Metronidazole activated processes associated with anti-inflammatory pathways in both T-cell subsets directly after the treatment period whereas doxycycline induced an immediate pro-inflammatory expression profile that resolved after the recovery period. Long-term changes indicated an inhibition of proliferation by doxycycline and induction of beneficial immune and metabolic pathways by metronidazole. Persistent alterations in microRNA and mRNA expression profiles after the recovery period indicate that all three medications may induce long-term epigenetic modifications in both T-cell subsets. Yet, our data do not support the induction of a long-term pro-inflammatory phenotype in murine Tregs and naive T-cells.

Effects of oral antibiotics and isotretinoin on the murine gut microbiota

Inflammatory bowel disease (IBD) may develop due to an immunogenic response to commensal gut microbiota triggered by environmental factors in the genetically susceptible host. Isotretinoin, applied in the treatment of severe acne, has been variably associated with IBD, but prior treatment with antibiotics, also associated with IBD development, confounds confirmation of this association. This study investigated the effects of doxycycline, metronidazole (frequently used in the treatment of acne and IBD, respectively) and isotretinoin on murine gut (faecal) microbiota after 2 weeks of treatment and after a 4-week recovery period. Faecal microbiota composition was assessed by 16S rRNA gene sequencing on the GS-FLX 454 platform with primers directed against the variable regions V1-V2. Doxycycline had a modest effect on bacterial richness and evenness, but had pronounced persistent and significant effects on the abundance of certain operational taxonomic units compared with the control group. In contrast, metronidazole induced a pronounced reduction in diversity after treatment, but these effects did not persist after the recovery period. This study demonstrates differential effects of antibiotics on the gut microbiota with doxycycline, unlike metronidazole, mediating long-term changes in the murine gut microbiota. Isotretinoin had no significant effect on the faecal microbiota.

Gp96 deficiency affects TLR4 functionality and impairs ERK and p38 phosphorylation

Gp96 is an endoplasmic reticulum chaperone for multiple protein substrates. Its lack in intestinal macrophages of Crohn’s disease (CD) patients is correlated with loss of tolerance against the host gut flora. Gp96 has been stablished to be an essential chaperone for Toll-like receptors (TLRs). We studied the impact of gp96-knockdown on TLR-function in macrophages. TLR2 and TLR4 expression was only decreased but not abolished when gp96 was knocked-down in cell lines, whereas in a monocyte/macrophage specific knock-out mouse model (LysMCre) TLR4 was abolished, while TLR2 was still present. Lipopolysaccharide (LPS)-induced NF-κB activation was still observed in the absence of gp96, and gp96-deficient macrophages were able to up-regulate surface TLR4 upon LPS treatment, suggesting that there is another chaperone involved in the folding of TLR4 upon stress responses. Moreover, LPS-dependent pro-inflammatory cytokines were still expressed, although to a lesser extent in the absence of gp96, which reinforces the fact that gp96 is involved in regulating signaling cascades downstream of TLR4 are impaired upon loss of gp96. In addition, we have also found a reduced phosphorylation of ERK and p38 kinases and an impaired response upon CSF1R activation in gp96 deficient macrophages. Our findings indicate that the loss of gp96 not only impairs TLR4 signaling, but is also associated with a diminished phosphorylation of ERK and mitogen-activated stress kinases resulting in an impaired signalling through several receptors, including CSF1R.

P081 ER stress leads to epithelial to mesenchymal transition (EMT) in intestinal epithelial cells in a JNK- and Src-dependent manner

broken down to enable IEC to invade. Here, we investigated the impact of pathogenassociated molecular patters (PAMPs) such as muramyldipeptide (MDP) on the induction of EMT in a three-dimensional multicellular model of HT-29 IEC and matrix destruction in human fistula tissue. Methods: Multicellular three-dimensional HT-29 IEC constructs were exposed to MDP (100 ng/ml) for seven days and observed microscopically over a timeline of seven days as well as on a molecular level. Human perianal fistula tissue was assessed for matrix metalloproteinases (MMP) using immunohistochemical procedures. Results: After exposure to MDP for seven days, the spheroids lost the well-defined globular shape and the epithelial cells separated indicating the onset of EMT. This was not seen in untreated control spheroids. In accordance with these microscopic findings, MDP treated spheroids up-regulated mRNA levels of EMT-related genes, including SNAIL1 (p < 0.05), TGF-β (p < 0.05) and IL-13 (p < 0.05) after treatment for seven days. In contrast to MDP, interleukin (IL)-22 did not induce EMT in spheroids. Furthermore, we found that MMP-3 was highly expressed around fistula tracts in human CD fistula tissue while only few stained cells were found in the surrounding inflamed tissue. Conclusions: Here, we demonstrate that PAMPs might contribute to the onset of EMT and therefore to the development of CD fistulae. The excessive expression of MMP-3 around the fistula tracts might contribute to the invasive growth of the EMT cells into deeper tissue layers. In summary, our data suggest that bacterial pathogens likely impact the pathogenesis of CD-associated fistulae.