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Mo1837 Inhibition of microRNA-29a in Human Bone Marrow Mesenchymal Stem Cells Increases Their Immunomodulatory Function
Mo1838
Viral Products Elicit Different Phenotypes From Small Intestine and Colonic Crypt Cultures Julie M. Davies, Rebeca Santaolalla, Richard J. von Furstenberg, Susan J. Henning, Maria T. Abreu
Effect of Chang Medium on Proliferation, Differentiation Capacity and Immunomodulatory Function of Human Tonsil-Derived Mesenchymal Stem Cells: Feasibility of Therapeutic Application to Inflammatory Bowel Disease Yeonsil Yu, Sung-Ae Jung, Seong-Eun Kim, Chang Mo Moon, Yang-Hee Joo, Ha Yeong Kim, Yoon Shin Park, Sangmee Ahn Jo, Inho Jo
The small intestine and colon differ greatly in both function and exposure to microbial products. Specific studies are needed to assess the response of microbial signaling in the small intestine versus the colon. Double-stranded RNAs (ds-RNA) are primarily viral products, but can also be derived from bacteria. Acute enteric viruses usually impact the small intestine, but viruses have also been investigated in inflammatory bowel disease and in post-infective irritable bowel syndrome and viruses are frequently found in colon cancer tissue. As such, understanding differences in the response of epithelial cells from the small intestine and colon to exposure to viral products will be important for dissecting the impact of infection at the different anatomical locations. Using newly developed culture techniques for growing primary enteroids and colonoids, we sought to compare the responses of enteroids versus colonoids to stimulation with the synthetic ds-RNA polyinosinic-polycytidylic acid (poly I:C). Murine crypts from jejunum or colon were plated in Matrigel for two days prior to the addition of poly I:C for the duration of culture. Stimulation of enteroids with poly I:C significantly altered the surface area but decreased the number of surviving enteroids compared to unstimulated. In colonoids, stimulation with poly I:C resulted in a significant decrease in bud count, but did not impact the area or survival of the organoids. Gene expression measured by the probe-based Nanostring technology in enteroids following poly I:C stimulation showed significantly decreased expression of stem cell markers including: Sox9, Lgr5, Dclk1, Tert and Lrig1. Additionally, differentiation markers Sis and Muc2 were significantly decreased. In contrast, fewer genes were significantly impacted by poly I:C stimulation in colonoids. Stem cell markers were not altered by poly I:C stimulation in colonoids. As expected, in both enteroids and colonoids inflammatory markers were significantly increased in response to poly I:C stimulation. Additionally, pro-apoptotic genes in both enteroids and colonoids were decreased following poly I:C stimulation. Comparing the response of enteroids and colonoids to poly I:C stimulation we found differences in the magnitude or direction of change in several classes of genes. These distinctions may derive from baseline differences in expression levels. Enteroids had significantly increased expression of stem cell markers at baseline compared to colonoids, while colonoids had increased levels of inflammatory markers and toll-like receptors. Together, these data indicate that important functional differences exist between enteroids and colonoids at baseline and after viral product stimulation providing evidence which may help explain the different anatomical responses to viral infection throughout the intestinal tract.
Backgrounds/Aims: Mesenchymal stem cells (MSC) have multipotent differentiation potentials and immunomodulation function, which largely turn on MSC expansion culture conditions. Here, we investigated the effect of hormone-supplemented Chang medium on MSC properties, and compared them with Dulbecco's modified Eagle's medium containing low glucose (DMEM-LG), which has been the most widely used for MSC culture. Methods: Tonsil-derived MSC (T-MSC) were used as a model of MSC. MSC properties included the proliferation, MSC-specific surface markers, embryonic stem cells genes such as Nanog, Oct4A and Sox-2, differentiation potentials and immunomodulatory function. Results: The use of Chang medium increased T-MSC proliferation greater than DMEM-LG. Cell morphology and MSC-specific markers were clearer in T-MSC cultured in Chang medium. Although the expression of CD146, a recently-identified MSC marker, decreased dramatically in DMEMLG with increasing passages, Chang medium significantly attenuated this decrease. Both adipogenic and chondrogenic differentiation potential were decreased less in Chang medium than in DMEM-LG, as evidenced by Oil Red O and Alcian blue staining, respectively. These findings were also supported by increased expressions of PPARγ and aggrecan, respectively. Compared with DMEM-LG, Chang medium increased Alizarin Red S staining and the expression of TAZ and CCN1, suggesting an increased osteogenic differentiation potential. However, the level of Nanog expression was decreased in T-MSC cultured with Chang medium than DMEM-LG. More interestingly and unexpectedly, T-MSC cultured in Chang medium didn't display the immunomodulatory capacity in in vitro cell and in vivo inflammatory bowel disease (IBD) model. Conclusion: These results suggest that the proper culture conditions of MSC including T-MSC should be defined before application to cell-based therapy. Mo1839 A Leaky Colonic Vascular Barrier Caused by Clostridium difficile Toxins A and B Contributes to the Pathogenesis of C. difficile Infection Jun Huang, Ciaran P. Kelly, Xiaotong Yang, Hua Xu, Kelsey S. Shields, Jeffrey D. Goldsmith, Joshua Hansen, Ishan Patel, Meijin Huang, Seppo Yla-Herttuala, Alan C. Moss, Jianping Wang, Xinhua Chen Background & Aims: Clostridium difficile infection (CDI) is mediated by two major exotoxins, toxin A (TcdA) and toxin B (TcdB). Vascular endothelial growth factor (VEGF) is known to contribute to Inflammatory Bowel Disease (IBD) pathogenesis in both clinical and experimental models, but its role in acute colitis, such as in CDI, is unknown. We aimed to examine VEGF production in response to C. difficile toxins and to evaluate its role in CDI pathogenesis in vitro, in vivo, and ex vivo. Methods: Human colonocytes were exposed to TcdA or TcdB in vitro. Mouse models of CDI and IBD-associated CDI were used in vivo. Colonic vascular permeability was assessed using Evans Blue extravasation. A kinase inhibitor of vascular endothelial growth factor receptor 2 (VEGFR-2) and an anti-mouse VEGF-A antibody were examined in the CDI mouse model. Human colonic mucosal biopsies were exposed to TcdA or TcdB using ex vivo culture to assess VEGF-A production. Results: VEGF-A production and colonic and cecal vascular permeability were increased in CDI mice. TcdA and TcdB induced VEGF-A production in human colonocytes; this was attenuated by inhibitors of HIF1α/2α and MAPK pathways. In vivo inhibition of VEGFR-2 kinase and use of an antibody to neutralize VEGF-A each significantly reduced gut vascular permeability and increased survival in CDI mice. In human colonic mucosa ex vivo stimulation with TcdA or TcdB induced VEGF-A production. In a mouse model that mimics IBD associated CDI, VEGF-2 kinase inhibition also attenuated disease severity. Conclusions: C. difficile toxins induced elevated levels of VEGF-A in vitro, in vivo, and ex vivio. We conclude that VEGF-A mediates increased colonic and cecal vascular permeability, which contributes to CDI pathogenesis. Therefore, TcdA and TcdB not only directly cause a breach in the epithelial barrier by disruption of epithelial tight junction, but also indirectly induce VEGFA production leading to a more permeable vascular barrier in the gut. These findings indicate a critical role for VEGF-A in CDI pathogenesis and may enable new therapeutic approaches for severe, refractory CDI or IBD-associated CDI.
Mo1837 Inhibition of microRNA-29a in Human Bone Marrow Mesenchymal Stem Cells Increases Their Immunomodulatory Function Angelos Oikonomopoulos, Christos Polytarchou, Precious Lacey, Tamera A. Tomakili, Maria Hatziapostolou, Georgios Koukos, Daniel W. Hommes Background: Mesenchymal stem cells (MSC) offer new options for therapy of refractory IBD due to their potent immunomodulatory function that can be increased by pre-stimulation (priming) with inflammatory cytokines. MSC have been proven beneficial in numerous immunological conditions, including graft versus host disease, ulcerative colitis and Crohn's disease. Nevertheless, the exact regulatory mechanisms of MSC immunomodulatory function remain largely unknown. MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression and are involved in the pathophysiology of numerous disorders, IBD included. The role of miRNAs on the immunomodulatory function of MSC remains unidentified. Our hypothesis is that miRNAs regulate the immunomodulatory function of bone marrow MSC (BMMSC) and that manipulation of miRNA levels might potentiate the salutary properties of BMMSC. Methods: Human donor derived BMMSC (n=3) were primed with IFN-γ (50ng/mL) for a period of 24 hours. MiRNA expression profiling (800 miRNAs) was performed in resting and primed BMMSC via Nanostring nCounter assays (n=3). Cytokine levels were determined in resting and primed BMMSC via multiplex immunoassays (40 cytokines) (n=3). BMMSC immunomodulatory function were accessed via co-culture assays (n=3) of resting and primed BMMSC with human CFSE-loaded CD3/CD28-stimulated peripheral blood mononuclear cells (PBMC). Gene expression was measured by quantitative RT-PCR. Transfection of BMMSC with miRNA-29a oligonucleotides (inhibitors or mimics) or with scramble was performed by lipofectamine assays. Results: MiRNA-29a was found to be the most robustly expressed miRNA in rested (non-primed) BMMSC and it was the most highly up-regulated following IFN-γ priming (1.7-fold). In independent samples of IFN-γ primed BMMSC, miRNA-29a was found to be 2-fold up-regulated by conventional real-time PCR. Co-culture assays between resting and primed BMMSC with human PBMC revealed that inhibition of miRNA-29a in IFN-γ primed BMMSC augments their immunosuppressive properties in comparison to scramble transfected counterparts. Cytokine profiling revealed that IFN-γ induces the expression of numerous immunomodulators including IDO1, CSF1, CCL5, TNFSF10, CCL2, CXCL16, CCL7, CCL13, CX3CL1, CXCL9, and CCL8. Inhibition of miRNA-29a in primed BMMSC further upregulated the expression of IDO1, but down-regulated the expression of other cytokines such as CSF1, CCL5, CCL2 and TNFSF10. Conclusion: Thus, miRNA-29a is capable of modulating the immune functions of BMMSC by regulating the expression of inflammatory cytokines. Overall, our data strongly suggest that miRNA-29a is a novel regulator of the immunomodulatory function of human BMMSC. Elucidating the molecular networks governing the immunomodulatory function of MSC will advance the designing of stem cell trials for IBD and other inflammatory conditions.
Mo1840 Fecal Microbiota Transplantation for Recurrent and/or Refractory Clostridium difficile Infection: A Large Retrospective Study of Failure Rates, Predictors of Failure and Outcomes Chetan Mittal, Ajin John, Benjamin R. Hart, Nichole Miller, Alireza Meighani, Mayur Ramesh Background/Objective Clostridium difficile infection (CDI) is a significant health care burden, accruing $1.3-$3.4 billion yearly in healthcare costs and is associated with increased morbidity and mortality. Traditional treatment regimens using Vancomycin and Metronidazole for recurrent CDI have a 30-50% success rate compared to 80-90% success rate with fecal microbiota transplantation (FMT). Although, numerous studies document the success rate of FMT, there is paucity of data on failure rates, relapse rates, and mortality after FMT. There is no study, to our knowledge, that identifies predictors of FMT failure in patients with recurrent CDI. The main aim of this study is to identify failure rates and predictors of failure of FMT in patients with recurrent and/or refractory CDI. In addition, we aim to identify relapse rates and mortality after FMT. Methods A large retrospective cohort study was performed including all patients who underwent FMT from December 2012 through May 2014. Data was obtained from procedure notes and extensive chart review. Patient factors (demographics, comorbidities, immune-suppression, transplant history, antibiotics used, hospitalization and surgeries), disease factors (number of episodes of CDI, treatments and severity), and transplant factors (route and number of FMT) were examined. Primary
S-723
AGA Abstracts
AGA Abstracts
Mo1836
Viral Products Elicit Different Phenotypes From Small Intestine and Colonic Crypt Cultures Julie M. Davies, Rebeca Santaolalla, Richard J. von Furstenberg, Susan J. Henning, Maria T. Abreu
Effect of Chang Medium on Proliferation, Differentiation Capacity and Immunomodulatory Function of Human Tonsil-Derived Mesenchymal Stem Cells: Feasibility of Therapeutic Application to Inflammatory Bowel Disease Yeonsil Yu, Sung-Ae Jung, Seong-Eun Kim, Chang Mo Moon, Yang-Hee Joo, Ha Yeong Kim, Yoon Shin Park, Sangmee Ahn Jo, Inho Jo
The small intestine and colon differ greatly in both function and exposure to microbial products. Specific studies are needed to assess the response of microbial signaling in the small intestine versus the colon. Double-stranded RNAs (ds-RNA) are primarily viral products, but can also be derived from bacteria. Acute enteric viruses usually impact the small intestine, but viruses have also been investigated in inflammatory bowel disease and in post-infective irritable bowel syndrome and viruses are frequently found in colon cancer tissue. As such, understanding differences in the response of epithelial cells from the small intestine and colon to exposure to viral products will be important for dissecting the impact of infection at the different anatomical locations. Using newly developed culture techniques for growing primary enteroids and colonoids, we sought to compare the responses of enteroids versus colonoids to stimulation with the synthetic ds-RNA polyinosinic-polycytidylic acid (poly I:C). Murine crypts from jejunum or colon were plated in Matrigel for two days prior to the addition of poly I:C for the duration of culture. Stimulation of enteroids with poly I:C significantly altered the surface area but decreased the number of surviving enteroids compared to unstimulated. In colonoids, stimulation with poly I:C resulted in a significant decrease in bud count, but did not impact the area or survival of the organoids. Gene expression measured by the probe-based Nanostring technology in enteroids following poly I:C stimulation showed significantly decreased expression of stem cell markers including: Sox9, Lgr5, Dclk1, Tert and Lrig1. Additionally, differentiation markers Sis and Muc2 were significantly decreased. In contrast, fewer genes were significantly impacted by poly I:C stimulation in colonoids. Stem cell markers were not altered by poly I:C stimulation in colonoids. As expected, in both enteroids and colonoids inflammatory markers were significantly increased in response to poly I:C stimulation. Additionally, pro-apoptotic genes in both enteroids and colonoids were decreased following poly I:C stimulation. Comparing the response of enteroids and colonoids to poly I:C stimulation we found differences in the magnitude or direction of change in several classes of genes. These distinctions may derive from baseline differences in expression levels. Enteroids had significantly increased expression of stem cell markers at baseline compared to colonoids, while colonoids had increased levels of inflammatory markers and toll-like receptors. Together, these data indicate that important functional differences exist between enteroids and colonoids at baseline and after viral product stimulation providing evidence which may help explain the different anatomical responses to viral infection throughout the intestinal tract.
Backgrounds/Aims: Mesenchymal stem cells (MSC) have multipotent differentiation potentials and immunomodulation function, which largely turn on MSC expansion culture conditions. Here, we investigated the effect of hormone-supplemented Chang medium on MSC properties, and compared them with Dulbecco's modified Eagle's medium containing low glucose (DMEM-LG), which has been the most widely used for MSC culture. Methods: Tonsil-derived MSC (T-MSC) were used as a model of MSC. MSC properties included the proliferation, MSC-specific surface markers, embryonic stem cells genes such as Nanog, Oct4A and Sox-2, differentiation potentials and immunomodulatory function. Results: The use of Chang medium increased T-MSC proliferation greater than DMEM-LG. Cell morphology and MSC-specific markers were clearer in T-MSC cultured in Chang medium. Although the expression of CD146, a recently-identified MSC marker, decreased dramatically in DMEMLG with increasing passages, Chang medium significantly attenuated this decrease. Both adipogenic and chondrogenic differentiation potential were decreased less in Chang medium than in DMEM-LG, as evidenced by Oil Red O and Alcian blue staining, respectively. These findings were also supported by increased expressions of PPARγ and aggrecan, respectively. Compared with DMEM-LG, Chang medium increased Alizarin Red S staining and the expression of TAZ and CCN1, suggesting an increased osteogenic differentiation potential. However, the level of Nanog expression was decreased in T-MSC cultured with Chang medium than DMEM-LG. More interestingly and unexpectedly, T-MSC cultured in Chang medium didn't display the immunomodulatory capacity in in vitro cell and in vivo inflammatory bowel disease (IBD) model. Conclusion: These results suggest that the proper culture conditions of MSC including T-MSC should be defined before application to cell-based therapy. Mo1839 A Leaky Colonic Vascular Barrier Caused by Clostridium difficile Toxins A and B Contributes to the Pathogenesis of C. difficile Infection Jun Huang, Ciaran P. Kelly, Xiaotong Yang, Hua Xu, Kelsey S. Shields, Jeffrey D. Goldsmith, Joshua Hansen, Ishan Patel, Meijin Huang, Seppo Yla-Herttuala, Alan C. Moss, Jianping Wang, Xinhua Chen Background & Aims: Clostridium difficile infection (CDI) is mediated by two major exotoxins, toxin A (TcdA) and toxin B (TcdB). Vascular endothelial growth factor (VEGF) is known to contribute to Inflammatory Bowel Disease (IBD) pathogenesis in both clinical and experimental models, but its role in acute colitis, such as in CDI, is unknown. We aimed to examine VEGF production in response to C. difficile toxins and to evaluate its role in CDI pathogenesis in vitro, in vivo, and ex vivo. Methods: Human colonocytes were exposed to TcdA or TcdB in vitro. Mouse models of CDI and IBD-associated CDI were used in vivo. Colonic vascular permeability was assessed using Evans Blue extravasation. A kinase inhibitor of vascular endothelial growth factor receptor 2 (VEGFR-2) and an anti-mouse VEGF-A antibody were examined in the CDI mouse model. Human colonic mucosal biopsies were exposed to TcdA or TcdB using ex vivo culture to assess VEGF-A production. Results: VEGF-A production and colonic and cecal vascular permeability were increased in CDI mice. TcdA and TcdB induced VEGF-A production in human colonocytes; this was attenuated by inhibitors of HIF1α/2α and MAPK pathways. In vivo inhibition of VEGFR-2 kinase and use of an antibody to neutralize VEGF-A each significantly reduced gut vascular permeability and increased survival in CDI mice. In human colonic mucosa ex vivo stimulation with TcdA or TcdB induced VEGF-A production. In a mouse model that mimics IBD associated CDI, VEGF-2 kinase inhibition also attenuated disease severity. Conclusions: C. difficile toxins induced elevated levels of VEGF-A in vitro, in vivo, and ex vivio. We conclude that VEGF-A mediates increased colonic and cecal vascular permeability, which contributes to CDI pathogenesis. Therefore, TcdA and TcdB not only directly cause a breach in the epithelial barrier by disruption of epithelial tight junction, but also indirectly induce VEGFA production leading to a more permeable vascular barrier in the gut. These findings indicate a critical role for VEGF-A in CDI pathogenesis and may enable new therapeutic approaches for severe, refractory CDI or IBD-associated CDI.
Mo1837 Inhibition of microRNA-29a in Human Bone Marrow Mesenchymal Stem Cells Increases Their Immunomodulatory Function Angelos Oikonomopoulos, Christos Polytarchou, Precious Lacey, Tamera A. Tomakili, Maria Hatziapostolou, Georgios Koukos, Daniel W. Hommes Background: Mesenchymal stem cells (MSC) offer new options for therapy of refractory IBD due to their potent immunomodulatory function that can be increased by pre-stimulation (priming) with inflammatory cytokines. MSC have been proven beneficial in numerous immunological conditions, including graft versus host disease, ulcerative colitis and Crohn's disease. Nevertheless, the exact regulatory mechanisms of MSC immunomodulatory function remain largely unknown. MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression and are involved in the pathophysiology of numerous disorders, IBD included. The role of miRNAs on the immunomodulatory function of MSC remains unidentified. Our hypothesis is that miRNAs regulate the immunomodulatory function of bone marrow MSC (BMMSC) and that manipulation of miRNA levels might potentiate the salutary properties of BMMSC. Methods: Human donor derived BMMSC (n=3) were primed with IFN-γ (50ng/mL) for a period of 24 hours. MiRNA expression profiling (800 miRNAs) was performed in resting and primed BMMSC via Nanostring nCounter assays (n=3). Cytokine levels were determined in resting and primed BMMSC via multiplex immunoassays (40 cytokines) (n=3). BMMSC immunomodulatory function were accessed via co-culture assays (n=3) of resting and primed BMMSC with human CFSE-loaded CD3/CD28-stimulated peripheral blood mononuclear cells (PBMC). Gene expression was measured by quantitative RT-PCR. Transfection of BMMSC with miRNA-29a oligonucleotides (inhibitors or mimics) or with scramble was performed by lipofectamine assays. Results: MiRNA-29a was found to be the most robustly expressed miRNA in rested (non-primed) BMMSC and it was the most highly up-regulated following IFN-γ priming (1.7-fold). In independent samples of IFN-γ primed BMMSC, miRNA-29a was found to be 2-fold up-regulated by conventional real-time PCR. Co-culture assays between resting and primed BMMSC with human PBMC revealed that inhibition of miRNA-29a in IFN-γ primed BMMSC augments their immunosuppressive properties in comparison to scramble transfected counterparts. Cytokine profiling revealed that IFN-γ induces the expression of numerous immunomodulators including IDO1, CSF1, CCL5, TNFSF10, CCL2, CXCL16, CCL7, CCL13, CX3CL1, CXCL9, and CCL8. Inhibition of miRNA-29a in primed BMMSC further upregulated the expression of IDO1, but down-regulated the expression of other cytokines such as CSF1, CCL5, CCL2 and TNFSF10. Conclusion: Thus, miRNA-29a is capable of modulating the immune functions of BMMSC by regulating the expression of inflammatory cytokines. Overall, our data strongly suggest that miRNA-29a is a novel regulator of the immunomodulatory function of human BMMSC. Elucidating the molecular networks governing the immunomodulatory function of MSC will advance the designing of stem cell trials for IBD and other inflammatory conditions.
Mo1840 Fecal Microbiota Transplantation for Recurrent and/or Refractory Clostridium difficile Infection: A Large Retrospective Study of Failure Rates, Predictors of Failure and Outcomes Chetan Mittal, Ajin John, Benjamin R. Hart, Nichole Miller, Alireza Meighani, Mayur Ramesh Background/Objective Clostridium difficile infection (CDI) is a significant health care burden, accruing $1.3-$3.4 billion yearly in healthcare costs and is associated with increased morbidity and mortality. Traditional treatment regimens using Vancomycin and Metronidazole for recurrent CDI have a 30-50% success rate compared to 80-90% success rate with fecal microbiota transplantation (FMT). Although, numerous studies document the success rate of FMT, there is paucity of data on failure rates, relapse rates, and mortality after FMT. There is no study, to our knowledge, that identifies predictors of FMT failure in patients with recurrent CDI. The main aim of this study is to identify failure rates and predictors of failure of FMT in patients with recurrent and/or refractory CDI. In addition, we aim to identify relapse rates and mortality after FMT. Methods A large retrospective cohort study was performed including all patients who underwent FMT from December 2012 through May 2014. Data was obtained from procedure notes and extensive chart review. Patient factors (demographics, comorbidities, immune-suppression, transplant history, antibiotics used, hospitalization and surgeries), disease factors (number of episodes of CDI, treatments and severity), and transplant factors (route and number of FMT) were examined. Primary
S-723
AGA Abstracts
AGA Abstracts
Mo1836