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Kayla Costan, Karminna Ezpeleta, Traci Pressley, Megan ShuminskiRole of Toll-like Receptors in Spontaneous Commensal-Dependent ColitisSeth Rakoff-Nahoum, 2006 1.     What is IBD?Irritable Bowel Disease. This is a classification of a group of conditions that cause inflammation in the bowels. Conditions such as Colitis, Crohn’s Disease, and Irritable Bowel Syndrome with Constipation/ Diarrhea (IBS-C/D) all fall under this classification. 2.     What is CD?CD stands for Crohn’s Disease. This disease is apart of the IBD group. It is characterized for causing chronic inflammatory along the GI tract.3.     What is IL-10?  How might a deficiency result in chronic inflammation?IL-10 is interleukin 10. This is a type of cytokine, which regulates immune responses in particular cells.  As stated in the article, colitis occurs in IL-10 deficient mice when the mouse has commensal microbial colonization. Immune responses triggered by commensal microbiota, such as inflammation, can be inhibited by IL-10. So, the deficiency of IL-10 would eliminate the normal preventative mechanisms that IL-10 would usually activate in the immune systems of mice, thus causing chronic inflammation. 4.     What is IL-2?  How might a deficiency result in chronic inflammation?IL-2 is interleukin 2. This interleukin is a cytokine associated with immune responses that prevent colitis. This study examines the effects of removing IL-2 on immune responses that cause colitis. The removal of IL-2 will remove it’s preventative effects thus allowing chronic inflammation to occur. 5.     What is the BIG question addressed in the paper?Are toll-like receptors related to the development of spontaneous commensal microflora dependent colitis? 6.     What was the biological question addressed in Figure 1?  What methods were used to answer this question?  What does the data show?To explore the role that TLR-MyD88 plays in commensal-dependent colitis in both single mutant IL-10 and IL-2 deficient mice. The figures show the gross pathological changes between both single mutant IL-10 and IL-2 deficient mice, with compound mutant of both IL-10 MyD88 and IL-2 MyD88 double deficient mice. These were compared with a wild-type. The intestinal tract, lymph nodes, spleen and colon were used to show the gross pathology changes caused by colitis. Colitis can be qualitatively characterized by swollen lymph nodes, an enlarged spleen and a shortened inflamed colon. The data showed the macroscopic manifestation of colitis of both single mutant IL-10 and IL-2 mice and IL2 MyD88 double deficient mice. The gross pathological change in IL-2 deficient mice without TLR- MyD88 developed colitis , where mice with both an IL-10 and TLR-MyD88 double deficiency did not develop the disease. Showing that IL-2 is indepent of the MyD88 signaling pathway, whereas IL-10 is dependent on this pathway. 7.     Complete the same questions as in number 6 for 3 additional figures presented in the paper.Figure 2:How is epithelial hyperplasia within the colon, an indication of colitis, dependent upon signaling through the TLR MyD88 pathway?(2A) Histopathological evaluation showed that the absence of MyD88 eradicated all suggestion of colitis in IL-10, but not IL-2 mice. This was done quantitatively by scoring the histopathology of the colon based on it’s epithelial hyperplasia and mononuclear and polymorphonuclear infiltrate. (2B) Representative photomicrographs, at both x40 and x100 magnification, qualitatively showed traces of colitis in IL-10 deficient, IL-2 deficient, and IL-2 MyD88 double deficient mice. These mice developed leukocytic infiltrate, epithelial hyperplasia, and abnormal colon features. Figure 4: Does the presence of MyD88 drive Th1 activation? In colitic mice, Th1 polarization increases with the activation of T cells. To determine if MyD88 has an effect on the T cell activation of IL-2 and IL-10 deficient mice, researchers measured the numbers of IFN-gamma and TNF in intestinal cells, which indicates Th1 polarization. Th1 polarization was significantly increased in both IL-2 and IL-10 deficient mice. The same method was utilized to IL-10 MyD88 and IL-2 MyD88 double deficient mice. Results showed that IL-10 MyD88 double deficient mice had no apparent Th1 polarization, thus no colitis. Th1 polarization in IL-2 MyD88 double deficient mice was still apparent. These results show that MyD88 is significant in colitis of IL-10 deficient mice, but not IL-2 deficient mice.Figure 6: Is MyD88 independent colitis, due to IL-2 deficiency, affected by other TLR signaling pathways? This figure examines the effects of MyD88-independent TLR signaling pathways on the IL-2 deficient mice, which present with MyD88 independent colitis. MyD88-independent signaling pathways use a common signaling adaptor, TRIF. In the figure, levels of mRNA for 3 proteins known for their inflammatory effects were examined. In triply-deficient mice, mice deficient of IL-2, MyD88, and TRIF, an increase in all three proteins was observed. This suggests that they are produced completely independent of the TLR signaling pathways. Further research is needed to determine if commensal-dependent colitis in IL-2 deficient mice is caused by non-TLR signaling immune responses or commensal non-self-antigens, but it is clear that commensal-dependent colitis in IL-2 deficient mice is not caused by TLR signaling pathways.  8.     What is the main conclusion of the paper?  Which pieces of data are the most important for supporting their conclusions?The development of colitis in IL-10 deficient mice is dependent on a TLR signaling pathway, while the development in IL-2 deficient mice is not. IL-10 regulates the response from the TLR receptors’ detection of commensal microflora, occuring due to Myd88; but this regulatory response from TLR receptors is not impacted by IL-2 deficient mice. To conclude that the TLR pathway in IL-10 deficient mice is significant in colitis generation, researchers observed double deficient mice, whom of which were deficient in either IL-2 or IL-10, as well as a crucial adapter protein in TLR signaling, MyD88. Observations upon intestinal organs of a series of mice, as seen in Figure 1, confirmed that the MyD88 signaling pathway has an effect on IL-10 deficient mice, as the double deficiency of IL-10 and MyD88 did not bring about colitic pathological symptoms. Similar conclusions could be made from Figures 2 through 5, through observing the colon tissue, T Cell activation and several other immune responses in mice.In contrast to IL-10 deficient mice, TLR signaling is not a contributing factor to colitis in IL-2 deficient mice. This is confirmed in the experiment correlating to Figure 6, displaying chronic intestinal inflammation on IL-2 deficient mice even in the absence of all TLR signaling. IL-2 deficiency leads to an organ specific autoimmunity that independently occurs from the commensal microflora.