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Tu1945 Induction of Autophagy Upon DC-T Cell Contact is Mediated by LKB1-AMPK-mTOR Signalling
Tu1945
and 1,25(OH)2D3, n=10) and Control group (received peanut oil, n=10). The disease activity index (DAI), the change of body weight, colon length and damage store of the mice were observed. The spleen length, weight, spleen index and the mononuclear cells of the spleen were measured. The expressions of protein and mRNA of TH 1 and TH17 cytokines in the colon mucosa and the spleen were measured by Immunohistochemisty and Real time-PCR, respectively. Results: The results revealed that the colons and the spleen from DSS group showed more severe ulceration and the infiltration of inflammatory cells than those in the control group and the ulceration and the infiltration of inflammatory cells significantly reduced after 1,25(OH)2D3 treatment compared with that in the model group (P<0.05). The number of dendritic cells and macrophages in spleen was obviously larger in DSS group than that in the control group by FACS (P<0.05), and the number was smaller in 1,25(OH)2D3 group compared to DSS group (P<0.05).The protein and mRNA levels of Interferon (IFN)-γ and interleukin (IL)-17 significantly increased in the model group compared with the control group and down-regulated in 1,25(OH)2D3 group (P<0.05). Conclusions: Our results reveal that 1,25(OH)2D3 is an important modulator in the development of chronic inflammation by decreasing TH1 and TH17 effector functions. The central role of 1,25(OH)2D3 represents an attractive, novel therapeutic drug for the treatment of IBD.
AGA Abstracts
Induction of Autophagy Upon DC-T Cell Contact is Mediated by LKB1-AMPKmTOR Signalling Manon Wildenberg, Anne Christine Vos, Marjolijn Duijvestein, Auke P. Verhaar, Daniel W. Hommes, Gijs R. van den Brink Background We have previously shown that upon formation of immunogenic dendritic cell (DC) - T cell contacts, autophagy is induced. This process then functions in a negative feedback loop by destabilization of the interaction. In a subset of Crohn's disease patients, this mechanism is impaired, leading to hyperstable interactions and increased immunogenicity. The correction of autophagy induction after DC-T cell contact in risk allele carriers may form a therapeutic possibilty resulting in normal interaction stability and decreased T cell activation. We therefore sought to determine the signaling events linking DC- T cell interactions with the induction of autophagy. Methods Interactions were induced between human monocyte derived DC and allogeneic T cells. Activation of signalling pathways was determined by Western Blot analysis. Inihibition of signalling mediators was achieved through siRNA and pharmacological inhibitors. Results Upon immunogenic DC-T cell interactions, phosphorylation of AMPK was increased. AMPK is an inhibitor of mTOR signaling, and indeed mTOR pathway activity was markedly reduced, as shown by decreased phosphorylation of mTOR itself as well as its downstream target S6. It has been shown previously that inhibition of mTOR is a strong inducer of autophagy. Conversely, HMGB1 and tissue transglutaminase 2, which are also known to influence autophagic activity, did not appear to be involved in autophagy induction under these circumstances. Interestingly, AMPK activation was mediated through activation of LKB1, a molecule which has been shown to be involved in mediating the polarity of intestinal epithelial cells. We show that LKB1 is recruited to the site of DCT cell contact, and that inhibition of LKB1 signalling results in impaired induction of autophagy after cell-cell contact. Consequently, cell contacts formed by LKB1 deficient DC were hyperstable and resulted in increased immunogenicity, similar to the effects of autophagy deficiency. Conclusion This data shows that upon DC-T cell interaction, autophagy is induced as a regulatory mechanism and this induction is mediated through the LKB1-AMPK- mTOR signaling pathway. In subsets of CD patients this regulatory mechanism is disturbed, leading to hyperstable interactions and excessive immunity. Boosting the mTOR pathway may be an interesting therapeutic option in CD patients carrying risk alleles in autophagyrelated genes.
Tu1948 KIR2DL3- and 3DL1-Mediated NK Cell Education Confers Crohn's Disease Susceptibility Lin Lin, Chao Ma, Bo Wei, Ian McHardy, Stephan R. Targan, Dermot P. McGovern, Jonathan Braun Background: Innate lymphocytes have recently emerged as protective and pathogenic participants in chronic inflammatory states. KIR2DL2/3, an inhibitory killer immunoglobulinlike receptor (KIR) expressed by innate lymphocytes, has been genetically associated with Crohn's disease (CD) and other chronic inflammatory diseases in the presence of its cognate ligand HLA-C1. However, the nature and mechanism of its contribution to IBD disease risk and activity is unknown. Natural killer (NK) cells, the main KIR-expressing innate lymphocyte population, enter two paths of differentiation, dependent on whether they undergo “education” via inhibitory KIR ligation with cognate MHC ligands. We therefore tested the hypothesis that NK education is required for their inducing role for IBD. Methods: The frequencies of HLA-C1 and HLA-Bw4, the respective ligands for KIR2DL3 and KIR3DL1, respectively, were compared in CD and healthy control cohorts bearing the simplified AA KIR haplotype by Chi square test. An NK co-culture assay was performed to test their effect on TCRmediated CD4+ T cell activation and interaction mechanisms. NK culture media were profiled for cytokine and chemokine production using microfluidic multiplex immunoassay, and biostatistical analysis was performed using R package. NK subsets expressing KIR2DL3, 3DL1, and 2DL1 were analyzed for cytokine production by flow cytometry and single cell barcode chip (SCBC). Results: The HLA C1C1 haplotype significantly (p<0.0001) predisposes Bw6/6 individuals to CD. NK education conferred their capacity to augment CD4+ T cells proliferation. That is, only strong KIR isoforms (KIR2DL3 and KIR3DL1) in the genetic context of their cognate MHC ligands permitted NK augmentation of CD4+ T cell proliferation. This was mirrored by the same genetic associations with disease risk. Blocking of NK costimulatory molecules, and transwell separation of NK from CD4 T cells, did not influence augmentation, indicating the interaction is contact-independent. NK cells from C1C1 individuals have significantly (p<0.05) increased production for 11 out of 19 soluble mediators, including IFNγ, IL-6, TNFα, and TNFβ. Principal component analysis and hierarchical clustering readily distinguished C1C1 and C2 individual NKs based on their secretion profile. Within each individual, educated KIR2DL3+ NKs are the most potent cytokines producers; cross comparison of different individual KIR2DL3+ NKs showed preferential cytokine production by the educated NK cells. Conclusions: Strong NK education mediated by KIR2DL3 or 3DL1 signaling license them to produce larger amount of proinflammatory cytokines and chemokines, and to reduce the threshold for CD4+ T cell activation. These findings for the first time uncover a biologic basis for KIR-associated susceptibility to Crohn's Disease and other chronic inflammatory syndromes.
Tu1946 Osteopontin Prevents Onset of Immune-Mediated Colitis by Inducing Tolerogenic Dendritic Cells Hiroshi Nakase, Minoru Matsuura, Tsutomu Chiba INTRODUCTION: Osteopontin (OPN), a matricelluar glycoprotein that exhibits a broad range of functional activities in physiological and pathological processes, is up-regulated in epithelial cells, mesenchymal cells, and inflammatory cells during inflammation and wound healing. OPN is expressed in activated T cells, dendritic cells (DC) and macrophages within 24hr of viral or bacterial infection and may account for resistance to microbial infection. On the contrary, an over-expression of OPN has been reported to be correlated with the severity of Th1 cell-mediated disease pathogenesis in both mouse model and several types of human autoimmune disease. However, there has been no report analyzing the role of OPN in immune-mediated colitis models, most resembling human IBD. AIMS: To clarify the role and mechanism of OPN in the development of immune-mediated spontaneous colitis model. METHODS (1) We evaluated the expression of OPN in colonic mucosa of both IL-10 KO mice and TCRαKO mice by immnohistochemistry. (2) We generated OPN×IL10 double KO (DKO) mice and OPN×TCRαDKO mice to compare the grade of colonic inflammation of OPN IL-10DKO mice to IL-10 KO mice at 4, 8, 12 and 16 weeks by investigating histological examination and the gene expression of pro-inflammatory cytokines in colonic mucosa. Also, we compared the grade of colonic inflammation of OPN TCRαDKO mice to TCRα KO mice in a similar fashion. (3) To evaluate the relationship between OPN and tolerogenic dendritic cells (DCs) induced by phagocytosis of apoptotic T cells, we cocultured immature bone marrow-derived DCs with anti-CD3/CD28-activated CD3 cells for 24hrs RESULTS: (1) Immunohistochemical examination with anti-OPN antibody revealed that OPN was mainly expressed in infiltrating (DC, macrophage, and CD4 T cells) of colitis of both IL-10 KO mice and TCRαKO mice, suggesting that OPN is involved in the pathophysiology of both Th1 and Th2 immune-mediated colitis. (2) Histological examination revealed that colonic inflammation of OPN IL-10DKO mice and OPN TCRαDKO mice developed at earlier age compared to that of IL-10KO mice and TCRαKO mice, respectively. Gene expressions of pro-inflammatory cytokines in colonic mucosa were significantly upregulated in OPN IL-10DKO mice and OPN TCRαDKO mice at 4weeks compared to those in IL-10KO mice and TCRαKO mice, respectively. (3) Confocal microscopy showed that no significant difference of DC phagocytotic activity was observed between OPNKO and WT. However, the level of TGF-β in the supernatant of DCs lacking OPN cocultured with apoptotic T cells was significantly lower than that of wild type DC. CONCLUSION: Our data demonstrated that OPN contributes to prevent onset of immune-mediated colitis by inducing tolerogenic dendritic cells.
Tu1949 Abrogation of TGFβ Signaling in Dendritic Cells Leads to Autoimmunity Through Regulatory T Cell Dependent and Independent Mechanisms Rajalakshmy Ramalingam, Robert D. Thurston, Claire B. Larmonier, Fayez K. Ghishan, Pawel R. Kiela Background: TGFβ is a pleiotropic cytokine that has profound immunoregulatory properties. It exerts its regulatory function on different cell types including lymphocytes, NK cells, dendritic cells (DCs), macrophages and granulocytes. We have recently shown that mice with targeted (Cre-lox) deletion of TGFβRII receptor dendritic cells (DC-TGFβRII KO) develop systemic autoimmunity. Since the levels of expression of MHCII and co-stimulatory molecules were not altered in DCs from DC-TGFβRII KO mice, the mechanism underlying the development of autoimmune disease was not clear. We hypothesized that In Vivo, TGFβ signaling controls DC functions other than antigen presentation. Results: GMCSF/IL-4 differentiated bone marrow derived DCs (BMDCs) from DC-TGFβRII KO mice had a more pro-inflammatory phenotype. Unstimulated DCs had significantly higher basal expression of TNFα compared to those generated from control mice. LPS stimulation increased IL-6 and IL-12 expression to levels significantly higher in the DCs from KO mice compared to those from control mice. When adoptively transferred with wild-type naïve T-cells to Rag1-/mice, DCs from DC-TGFβRII KO mice exacerbated the severity of colitis as evidenced by the increased colonic expression of pro-inflammatory cytokines. Microarray analysis revealed increased expression of TNFα and ~10 IFNγ responsive genes in splenic CD11c+ DCs from DC-TGFβRII KO mice whereas in MLN CD103+ and CD103- DCs from KO mice, IFNγ itself was significantly upregulated. Consistently, IFNγ was also over-expressed in Flt3L derived BMDCs. We utilized blocking anti-IFNγ antibody to test whether elevated expression of IFNγ inhibited DC mediated antigen-specific (Ova) regulatory OT-II T cell (Treg) conversion. Neutralization of IFNγ restored Treg differentiation to levels similar to that observed with control DCs. In Vivo, we observed a significant increase in the frequency and numbers of Foxp3+ T cells in both the spleen and MLNs of DC-TGFβRII KO mice but further analysis
Tu1947 1,25-Dihydroxyvitamin D3 Regulates the Development of Chronic Colitis by Modulating Both T Helper (Th)1 and TH17 Activation Lei Liu, Hui Li, Hong Zhang, Hui Wu, Guochao Niu, Chenyang Li, Xiaolan Zhang, David Q. Shih Background: T helper (TH) 1 and TH17 cytokines have been reported to be involved in the genesis and maintenance of inflammatory bowel disease (IBD). 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the active form of vitamin D, has emerged recently as a direct regulator of immune system function. Aim: To investigate the possible immunomodulatory effects of 1,25(OH)2D3 on chronic colitis. Methods: Mice were grouped as follows: DSS group (received 2% DSS drinking water only, n=10), 1,25(OH)2D3 group (received 2% DSS drinking water
AGA Abstracts
S-884
and 1,25(OH)2D3, n=10) and Control group (received peanut oil, n=10). The disease activity index (DAI), the change of body weight, colon length and damage store of the mice were observed. The spleen length, weight, spleen index and the mononuclear cells of the spleen were measured. The expressions of protein and mRNA of TH 1 and TH17 cytokines in the colon mucosa and the spleen were measured by Immunohistochemisty and Real time-PCR, respectively. Results: The results revealed that the colons and the spleen from DSS group showed more severe ulceration and the infiltration of inflammatory cells than those in the control group and the ulceration and the infiltration of inflammatory cells significantly reduced after 1,25(OH)2D3 treatment compared with that in the model group (P<0.05). The number of dendritic cells and macrophages in spleen was obviously larger in DSS group than that in the control group by FACS (P<0.05), and the number was smaller in 1,25(OH)2D3 group compared to DSS group (P<0.05).The protein and mRNA levels of Interferon (IFN)-γ and interleukin (IL)-17 significantly increased in the model group compared with the control group and down-regulated in 1,25(OH)2D3 group (P<0.05). Conclusions: Our results reveal that 1,25(OH)2D3 is an important modulator in the development of chronic inflammation by decreasing TH1 and TH17 effector functions. The central role of 1,25(OH)2D3 represents an attractive, novel therapeutic drug for the treatment of IBD.
AGA Abstracts
Induction of Autophagy Upon DC-T Cell Contact is Mediated by LKB1-AMPKmTOR Signalling Manon Wildenberg, Anne Christine Vos, Marjolijn Duijvestein, Auke P. Verhaar, Daniel W. Hommes, Gijs R. van den Brink Background We have previously shown that upon formation of immunogenic dendritic cell (DC) - T cell contacts, autophagy is induced. This process then functions in a negative feedback loop by destabilization of the interaction. In a subset of Crohn's disease patients, this mechanism is impaired, leading to hyperstable interactions and increased immunogenicity. The correction of autophagy induction after DC-T cell contact in risk allele carriers may form a therapeutic possibilty resulting in normal interaction stability and decreased T cell activation. We therefore sought to determine the signaling events linking DC- T cell interactions with the induction of autophagy. Methods Interactions were induced between human monocyte derived DC and allogeneic T cells. Activation of signalling pathways was determined by Western Blot analysis. Inihibition of signalling mediators was achieved through siRNA and pharmacological inhibitors. Results Upon immunogenic DC-T cell interactions, phosphorylation of AMPK was increased. AMPK is an inhibitor of mTOR signaling, and indeed mTOR pathway activity was markedly reduced, as shown by decreased phosphorylation of mTOR itself as well as its downstream target S6. It has been shown previously that inhibition of mTOR is a strong inducer of autophagy. Conversely, HMGB1 and tissue transglutaminase 2, which are also known to influence autophagic activity, did not appear to be involved in autophagy induction under these circumstances. Interestingly, AMPK activation was mediated through activation of LKB1, a molecule which has been shown to be involved in mediating the polarity of intestinal epithelial cells. We show that LKB1 is recruited to the site of DCT cell contact, and that inhibition of LKB1 signalling results in impaired induction of autophagy after cell-cell contact. Consequently, cell contacts formed by LKB1 deficient DC were hyperstable and resulted in increased immunogenicity, similar to the effects of autophagy deficiency. Conclusion This data shows that upon DC-T cell interaction, autophagy is induced as a regulatory mechanism and this induction is mediated through the LKB1-AMPK- mTOR signaling pathway. In subsets of CD patients this regulatory mechanism is disturbed, leading to hyperstable interactions and excessive immunity. Boosting the mTOR pathway may be an interesting therapeutic option in CD patients carrying risk alleles in autophagyrelated genes.
Tu1948 KIR2DL3- and 3DL1-Mediated NK Cell Education Confers Crohn's Disease Susceptibility Lin Lin, Chao Ma, Bo Wei, Ian McHardy, Stephan R. Targan, Dermot P. McGovern, Jonathan Braun Background: Innate lymphocytes have recently emerged as protective and pathogenic participants in chronic inflammatory states. KIR2DL2/3, an inhibitory killer immunoglobulinlike receptor (KIR) expressed by innate lymphocytes, has been genetically associated with Crohn's disease (CD) and other chronic inflammatory diseases in the presence of its cognate ligand HLA-C1. However, the nature and mechanism of its contribution to IBD disease risk and activity is unknown. Natural killer (NK) cells, the main KIR-expressing innate lymphocyte population, enter two paths of differentiation, dependent on whether they undergo “education” via inhibitory KIR ligation with cognate MHC ligands. We therefore tested the hypothesis that NK education is required for their inducing role for IBD. Methods: The frequencies of HLA-C1 and HLA-Bw4, the respective ligands for KIR2DL3 and KIR3DL1, respectively, were compared in CD and healthy control cohorts bearing the simplified AA KIR haplotype by Chi square test. An NK co-culture assay was performed to test their effect on TCRmediated CD4+ T cell activation and interaction mechanisms. NK culture media were profiled for cytokine and chemokine production using microfluidic multiplex immunoassay, and biostatistical analysis was performed using R package. NK subsets expressing KIR2DL3, 3DL1, and 2DL1 were analyzed for cytokine production by flow cytometry and single cell barcode chip (SCBC). Results: The HLA C1C1 haplotype significantly (p<0.0001) predisposes Bw6/6 individuals to CD. NK education conferred their capacity to augment CD4+ T cells proliferation. That is, only strong KIR isoforms (KIR2DL3 and KIR3DL1) in the genetic context of their cognate MHC ligands permitted NK augmentation of CD4+ T cell proliferation. This was mirrored by the same genetic associations with disease risk. Blocking of NK costimulatory molecules, and transwell separation of NK from CD4 T cells, did not influence augmentation, indicating the interaction is contact-independent. NK cells from C1C1 individuals have significantly (p<0.05) increased production for 11 out of 19 soluble mediators, including IFNγ, IL-6, TNFα, and TNFβ. Principal component analysis and hierarchical clustering readily distinguished C1C1 and C2 individual NKs based on their secretion profile. Within each individual, educated KIR2DL3+ NKs are the most potent cytokines producers; cross comparison of different individual KIR2DL3+ NKs showed preferential cytokine production by the educated NK cells. Conclusions: Strong NK education mediated by KIR2DL3 or 3DL1 signaling license them to produce larger amount of proinflammatory cytokines and chemokines, and to reduce the threshold for CD4+ T cell activation. These findings for the first time uncover a biologic basis for KIR-associated susceptibility to Crohn's Disease and other chronic inflammatory syndromes.
Tu1946 Osteopontin Prevents Onset of Immune-Mediated Colitis by Inducing Tolerogenic Dendritic Cells Hiroshi Nakase, Minoru Matsuura, Tsutomu Chiba INTRODUCTION: Osteopontin (OPN), a matricelluar glycoprotein that exhibits a broad range of functional activities in physiological and pathological processes, is up-regulated in epithelial cells, mesenchymal cells, and inflammatory cells during inflammation and wound healing. OPN is expressed in activated T cells, dendritic cells (DC) and macrophages within 24hr of viral or bacterial infection and may account for resistance to microbial infection. On the contrary, an over-expression of OPN has been reported to be correlated with the severity of Th1 cell-mediated disease pathogenesis in both mouse model and several types of human autoimmune disease. However, there has been no report analyzing the role of OPN in immune-mediated colitis models, most resembling human IBD. AIMS: To clarify the role and mechanism of OPN in the development of immune-mediated spontaneous colitis model. METHODS (1) We evaluated the expression of OPN in colonic mucosa of both IL-10 KO mice and TCRαKO mice by immnohistochemistry. (2) We generated OPN×IL10 double KO (DKO) mice and OPN×TCRαDKO mice to compare the grade of colonic inflammation of OPN IL-10DKO mice to IL-10 KO mice at 4, 8, 12 and 16 weeks by investigating histological examination and the gene expression of pro-inflammatory cytokines in colonic mucosa. Also, we compared the grade of colonic inflammation of OPN TCRαDKO mice to TCRα KO mice in a similar fashion. (3) To evaluate the relationship between OPN and tolerogenic dendritic cells (DCs) induced by phagocytosis of apoptotic T cells, we cocultured immature bone marrow-derived DCs with anti-CD3/CD28-activated CD3 cells for 24hrs RESULTS: (1) Immunohistochemical examination with anti-OPN antibody revealed that OPN was mainly expressed in infiltrating (DC, macrophage, and CD4 T cells) of colitis of both IL-10 KO mice and TCRαKO mice, suggesting that OPN is involved in the pathophysiology of both Th1 and Th2 immune-mediated colitis. (2) Histological examination revealed that colonic inflammation of OPN IL-10DKO mice and OPN TCRαDKO mice developed at earlier age compared to that of IL-10KO mice and TCRαKO mice, respectively. Gene expressions of pro-inflammatory cytokines in colonic mucosa were significantly upregulated in OPN IL-10DKO mice and OPN TCRαDKO mice at 4weeks compared to those in IL-10KO mice and TCRαKO mice, respectively. (3) Confocal microscopy showed that no significant difference of DC phagocytotic activity was observed between OPNKO and WT. However, the level of TGF-β in the supernatant of DCs lacking OPN cocultured with apoptotic T cells was significantly lower than that of wild type DC. CONCLUSION: Our data demonstrated that OPN contributes to prevent onset of immune-mediated colitis by inducing tolerogenic dendritic cells.
Tu1949 Abrogation of TGFβ Signaling in Dendritic Cells Leads to Autoimmunity Through Regulatory T Cell Dependent and Independent Mechanisms Rajalakshmy Ramalingam, Robert D. Thurston, Claire B. Larmonier, Fayez K. Ghishan, Pawel R. Kiela Background: TGFβ is a pleiotropic cytokine that has profound immunoregulatory properties. It exerts its regulatory function on different cell types including lymphocytes, NK cells, dendritic cells (DCs), macrophages and granulocytes. We have recently shown that mice with targeted (Cre-lox) deletion of TGFβRII receptor dendritic cells (DC-TGFβRII KO) develop systemic autoimmunity. Since the levels of expression of MHCII and co-stimulatory molecules were not altered in DCs from DC-TGFβRII KO mice, the mechanism underlying the development of autoimmune disease was not clear. We hypothesized that In Vivo, TGFβ signaling controls DC functions other than antigen presentation. Results: GMCSF/IL-4 differentiated bone marrow derived DCs (BMDCs) from DC-TGFβRII KO mice had a more pro-inflammatory phenotype. Unstimulated DCs had significantly higher basal expression of TNFα compared to those generated from control mice. LPS stimulation increased IL-6 and IL-12 expression to levels significantly higher in the DCs from KO mice compared to those from control mice. When adoptively transferred with wild-type naïve T-cells to Rag1-/mice, DCs from DC-TGFβRII KO mice exacerbated the severity of colitis as evidenced by the increased colonic expression of pro-inflammatory cytokines. Microarray analysis revealed increased expression of TNFα and ~10 IFNγ responsive genes in splenic CD11c+ DCs from DC-TGFβRII KO mice whereas in MLN CD103+ and CD103- DCs from KO mice, IFNγ itself was significantly upregulated. Consistently, IFNγ was also over-expressed in Flt3L derived BMDCs. We utilized blocking anti-IFNγ antibody to test whether elevated expression of IFNγ inhibited DC mediated antigen-specific (Ova) regulatory OT-II T cell (Treg) conversion. Neutralization of IFNγ restored Treg differentiation to levels similar to that observed with control DCs. In Vivo, we observed a significant increase in the frequency and numbers of Foxp3+ T cells in both the spleen and MLNs of DC-TGFβRII KO mice but further analysis
Tu1947 1,25-Dihydroxyvitamin D3 Regulates the Development of Chronic Colitis by Modulating Both T Helper (Th)1 and TH17 Activation Lei Liu, Hui Li, Hong Zhang, Hui Wu, Guochao Niu, Chenyang Li, Xiaolan Zhang, David Q. Shih Background: T helper (TH) 1 and TH17 cytokines have been reported to be involved in the genesis and maintenance of inflammatory bowel disease (IBD). 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the active form of vitamin D, has emerged recently as a direct regulator of immune system function. Aim: To investigate the possible immunomodulatory effects of 1,25(OH)2D3 on chronic colitis. Methods: Mice were grouped as follows: DSS group (received 2% DSS drinking water only, n=10), 1,25(OH)2D3 group (received 2% DSS drinking water
AGA Abstracts
S-884