@phdthesis{Ganesh2013, author = {Ganesh, Bhanu Priya}, title = {Host-microbe interactions in the inflamed gut}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-69558}, school = {Universit{\"a}t Potsdam}, year = {2013}, abstract = {Initiation and perpetuation of inflammatory bowel diseases (IBD) may result from an exaggerated mucosal immune response to the luminal microbiota in a susceptible host. We proposed that this may be caused either 1) by an abnormal microbial composition or 2) by weakening of the protective mucus layer due to excessive mucus degradation, which may lead to an easy access of luminal antigens to the host mucosa triggering inflammation. We tested whether the probiotic Enterococcus faecium NCIMB 10415 (NCIMB) is capable of reducing chronic gut inflammation by changing the existing gut microbiota composition and aimed to identify mechanisms that are involved in possible beneficial effects of the probiotic. To identify health-promoting mechanisms of the strain, we used interleukin (IL)-10 deficient mice that spontaneously develop gut inflammation and fed these mice a diet containing NCIMB (106 cells g-1) for 3, 8 and 24 weeks, respectively. Control mice were fed an identically composed diet but without the probiotic strain. No clear-cut differences between the animals were observed in pro-inflammatory cytokine gene expression and in intestinal microbiota composition after probiotic supplementation. However, we observed a low abundance of the mucin-degrading bacterium Akkermansia muciniphila in the mice that were fed NCIMB for 8 weeks. These low cell numbers were associated with significantly lower interferon gamma (IFN-γ) and IFN-γ-inducible protein (IP-10) mRNA levels as compared to the NCIMB-treated mice that were killed after 3 and 24 weeks of intervention. In conclusion, NCIMB was not capable of reducing gut inflammation in the IL-10-/- mouse model. To further identify the exact role of A. muciniphila and uncover a possible interaction between this bacterium, NCIMB and the host in relation to inflammation, we performed in vitro studies using HT-29 colon cancer cells. The HT-29 cells were treated with bacterial conditioned media obtained by growing either A. muciniphila (AM-CM) or NCIMB (NCIMB-CM) or both together (COMB-CM) in Dulbecco's Modified Eagle Medium (DMEM) for 2 h at 37 °C followed by bacterial cell removal. HT-29 cells treated with COMB-CM displayed reduced cell viability after 18 h (p<0.01) and no viable cells were detected after 24 h of treatment, in contrast to the other groups or heated COMB-CM. Detection of activated caspase-3 in COMB-CM treated groups indicated that death of the HT-29 cells was brought about by apoptosis. It was concluded that either NCIMB or A. muciniphila produce a soluble and heat-sensitive factor during their concomitant presence that influences cell viability in an in vitro system. We currently hypothesize that this factor is a protein, which has not yet been identified. Based on the potential effect of A. muciniphila on inflammation (in vivo) and cell-viability (in vitro) in the presence of NCIMB, we investigated how the presence of A. muciniphila affects the severity of an intestinal Salmonella enterica Typhimurium (STm)-induced gut inflammation using gnotobiotic C3H mice with a background microbiota of eight bacterial species (SIHUMI, referred to as simplified human intestinal microbiota). Presence of A. muciniphila in STm-infected SIHUMI (SIHUMI-AS) mice caused significantly increased histopathology scores and elevated mRNA levels of IFN-γ, IP-10, tumor necrosis factor alpha (TNF-α), IL-12, IL-17 and IL-6 in cecal and colonic tissue. The number of mucin filled goblet cells was 2- to 3- fold lower in cecal tissue of SIHUMI-AS mice compared to SIHUMI mice associated with STm (SIHUMI-S) or A. muciniphila (SIHUMI-A) or SIHUMI mice. Reduced goblet cell numbers significantly correlated with increased IFN-γ (r2 = -0.86, ***P<0.001) in all infected mice. In addition, loss of cecal mucin sulphation was observed in SIHUMI-AS mice. Concomitant presence of A. muciniphila and STm resulted in a drastic change in microbiota composition of the SIHUMI consortium. The proportion of Bacteroides thetaiotaomicron in SIHUMI, SIHUMI-A and SIHUMI-S mice made up to 80-90\% but was completely taken over by STm in SIHUMI-AS mice contributing 94\% to total bacteria. These results suggest that A. muciniphila exacerbates STm-induced intestinal inflammation by its ability to disturb host mucus homeostasis. In conclusion, abnormal microbiota composition together with excessive mucus degradation contributes to severe intestinal inflammation in a susceptible host.}, language = {en} } @article{MaaresKeilKozaetal.2018, author = {Maares, Maria and Keil, Claudia and Koza, Jenny and Straubing, Sophia and Schwerdtle, Tanja and Haase, Hajo}, title = {In Vitro Studies on Zinc Binding and Buffering by Intestinal Mucins}, series = {International Journal of Molecular Sciences}, volume = {19}, journal = {International Journal of Molecular Sciences}, number = {9}, issn = {1422-0067}, doi = {10.3390/ijms19092662}, pages = {20}, year = {2018}, abstract = {The investigation of luminal factors influencing zinc availability and accessibility in the intestine is of great interest when analyzing parameters regulating intestinal zinc resorption. Of note, intestinal mucins were suggested to play a beneficial role in the luminal availability of zinc. Their exact zinc binding properties, however, remain unknown and the impact of these glycoproteins on human intestinal zinc resorption has not been investigated in detail. Thus, the aim of this study is to elucidate the impact of intestinal mucins on luminal uptake of zinc into enterocytes and its transfer into the blood. In the present study, in vitro zinc binding properties of mucins were analyzed using commercially available porcine mucins and secreted mucins of the goblet cell line HT-29-MTX. The molecular zinc binding capacity and average zinc binding affinity of these glycoproteins demonstrates that mucins contain multiple zinc-binding sites with biologically relevant affinity within one mucin molecule. Zinc uptake into the enterocyte cell line Caco-2 was impaired by zinc-depleted mucins. Yet this does not represent their form in the intestinal lumen in vivo under zinc adequate conditions. In fact, zinc-uptake studies into enterocytes in the presence of mucins with differing degree of zinc saturation revealed zinc buffering by these glycoproteins, indicating that mucin-bound zinc is still available for the cells. Finally, the impact of mucins on zinc resorption using three-dimensional cultures was studied comparing the zinc transfer of a Caco-2/HT-29-MTX co-culture and conventional Caco-2 monoculture. Here, the mucin secreting co-cultures yielded higher fractional zinc resorption and elevated zinc transport rates, suggesting that intestinal mucins facilitate the zinc uptake into enterocytes and act as a zinc delivery system for the intestinal epithelium.}, language = {en} } @misc{MaaresKeilKozaetal.2018, author = {Maares, Maria and Keil, Claudia and Koza, Jenny and Straubing, Sophia and Schwerdtle, Tanja and Haase, Hajo}, title = {In Vitro Studies on Zinc Binding and Buffering by Intestinal Mucins}, series = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {1079}, issn = {1866-8372}, doi = {10.25932/publishup-46907}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-469078}, pages = {22}, year = {2018}, abstract = {The investigation of luminal factors influencing zinc availability and accessibility in the intestine is of great interest when analyzing parameters regulating intestinal zinc resorption. Of note, intestinal mucins were suggested to play a beneficial role in the luminal availability of zinc. Their exact zinc binding properties, however, remain unknown and the impact of these glycoproteins on human intestinal zinc resorption has not been investigated in detail. Thus, the aim of this study is to elucidate the impact of intestinal mucins on luminal uptake of zinc into enterocytes and its transfer into the blood. In the present study, in vitro zinc binding properties of mucins were analyzed using commercially available porcine mucins and secreted mucins of the goblet cell line HT-29-MTX. The molecular zinc binding capacity and average zinc binding affinity of these glycoproteins demonstrates that mucins contain multiple zinc-binding sites with biologically relevant affinity within one mucin molecule. Zinc uptake into the enterocyte cell line Caco-2 was impaired by zinc-depleted mucins. Yet this does not represent their form in the intestinal lumen in vivo under zinc adequate conditions. In fact, zinc-uptake studies into enterocytes in the presence of mucins with differing degree of zinc saturation revealed zinc buffering by these glycoproteins, indicating that mucin-bound zinc is still available for the cells. Finally, the impact of mucins on zinc resorption using three-dimensional cultures was studied comparing the zinc transfer of a Caco-2/HT-29-MTX co-culture and conventional Caco-2 monoculture. Here, the mucin secreting co-cultures yielded higher fractional zinc resorption and elevated zinc transport rates, suggesting that intestinal mucins facilitate the zinc uptake into enterocytes and act as a zinc delivery system for the intestinal epithelium.}, language = {en} }