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Patnode Lab
Background
Background
Dietary plant fibers can't be digested by host
Plants in our diet expose us to complex plant glycans (dietary fiber)
Dietary fiber is not digested by the host and are fermented into short chain fatty acids by gut microbes
Direct effects of dietary fiber on the immune system is understudied
Supplementing mouse diet with fibers result in anti-fiber antibodies in blood
Immune mechanisms to degrade plant polysaccharides are not well characterized
Inability to degrade them may cause immune cell dysfunction
Nitric oxide can be used by immune cells to degrade polysaccharides
Macrophages = innate immune cells that phagocytose and degrade pathogens, cellular debris, and foreign materials in our tissues
Nitric oxide (NO) = reactive molecule with free radical properties that can break down other molecules through oxidation
NO is used to non-selectively degrade large molecules including bacterial polysaccharides
But what about dietary fibers?
How macrophages interact with dietary fibers and the role of NO is not fully characterized
Inability to degrade dietary polysaccharides and their accumulation in tissues could lead to inflammation and worsen Inflammatory Bowel Disease symptoms
Determining whether xylan induces NO production in Macrophages
Determining whether xylan induces NO production in Macrophages
NO Quantification - Griess Assay
NO Quantification - Griess Assay
NO stimulating treatment (xylan) is incubated with macrophages for 46 hrs
Supernatant is collected and Griess reagents are added and incubated for 30 min
In presence of NO, Griess reagent causes a color change
Absorbance at 560 nm is measured
Xylan, but not Xylose, induces NO production by RAW 264.7 Macrophages
Xylan, but not Xylose, induces NO production by RAW 264.7 Macrophages
RAW 264.7 macrophages were treated with LPS and xylan for 46 hrs
LPS is known to induce NO production in macrophages and is used as a positive control
Xylose, the monosaccharide of xylan, was also tested for NO production
NO in the supernatants was quantified by the Griess assay
Determining Whether NO is Required for the Degradation of Xylan by Macrophages
Determining Whether NO is Required for the Degradation of Xylan by Macrophages
Xylan Detection in Macropahge Lysate - ELISA
Xylan Detection in Macropahge Lysate - ELISA
ELISA by Fatima Rizvi
RAW 264.7 macrophages were seeded overnight at 1.5 x10^5 cells/ml
The wells without macrophages were seeded with complete medium
The next day, the cells were incubated with biotin-xylan for 1 hr
After 1 hr, the wells were washed to remove xylan not bound or internalized by the macrophages
The cells were lysed and xylan was detected in lysates using ELISA with an anti-xylan antibody
Conclusions
Conclusions
Xylan, but not xylose, induces NO production in RAW 264.7 macrophages, indicating that the polysaccharide is required for this induction
NO production in response to xylan may be a potential mechanism macrophages use to degrade xylan
Whether or not xylan stimulates NO by macrophages upon binding or internalization remains to be shown
Future Directions
Future Directions
Detect and measure xylan in lysate of wildtype and iNOS deficient murine bone marrow derived macrophages at different time points to determine if degradation is occurring and whether NO is required
Test whether other dietary fibers also induce NO and whether they are degraded by macrophages
Acknowledgements
Acknowledgements
Faculty Mentor:
Michael Patnode
Patnode Lab Members:
Fatima Rizvi
Giovanni Vega
Bo Huey Chiang
Christian Montiel
Katie Miller
Meghan Graham
Myrka Carmona
Sanridhi Semwal
Natalie Ceculli
Thank you to the Koret Foundation for financially supporting this project.
References
References
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