- Email: [email protected]
the Fatty Acid Imbalance of Cystic Fibrosis Impairs Glucose-stimulated Insulin Secretion From Pancreatic Islets
treated once a day by s. c. injection of a dose range of 0.1 -3 mg/kg. Results: We obtained a yield of >400 mg/L of hBD2 on a 10 L scale. HBD2 showed antimicrobial activity in radial diffusion assays while we did not find any cytotoxic effect against human PBMCs and murine fibroblasts. Treatment of LPS-activated human PBMCs with hBD2 decreased the release of pro-inflammatory TNFα, IL-1β and IL-23 while upregulating anti-inflammatory IL-10 and IL-24. In all colitis models tested s. c. application of hBD2 led to a remarkable improvement of disease at an optimal dose of 0.1 mg/kg (DSS and TNBS) and 1 mg/kg (transfer) on par with anti-TNFα (DSS), prednisolone (TNBS) and dexamethasone (transfer), respectively. In the DSS model hBD2 ameliorated loss of body weight (P<0.05), improved the disease activity index (P<0.001) as well as the histological score (P<0.001), significantly. In the TNBS model the histological score was significantly improved by hBD2 treatment (P<0.001) while in the T-cell-transfer model hBD2 again improved the disease activity index. Summary: We were able to produce and purify hBD2 in relevant amounts. Since hBD2 showed no cytotoxicity, strong anti-inflammatory properties and protected mice from colitis our data provided evidence that hBD2 could be used as potential therapeutic agent in the treatment of inflammatory bowel diseases.
THE FATTY ACID IMBALANCE OF CYSTIC FIBROSIS IMPAIRS GLUCOSESTIMULATED INSULIN SECRETION FROM PANCREATIC ISLETS Jamal Kriem, Andrew Norris Background: Cystic fibrosis (CF) is accompanied by a prominent imbalance of serum and tissue fatty acids. This imbalance includes low levels of the essential fatty acids linoleic and docosahexaenoic acid. Furthermore, the levels of mead acid, a normally scarce fatty acid, are elevated. Insulin secretion is impaired in CF due to unclear mechanisms. Because fatty acids alter beta-cell function, we hypothesized that the fatty acid imbalance found in CF reduces glucose stimulated insulin secretion. Methods: Mouse islets were cultured in media containing a 200 micromolar fatty acid mixture buffered in 1% albumin. Two different fatty acid mixtures were employed, representing a control or CF-skewed balance. The CF-skewed fatty acid mixture contained less linoleic and docosahexaenoic acids and increased mead acid, reflective of serum fatty acid imbalance in serum of CF patients. Insulin secretion was measured in the basal state in 1.67 mM glucose and in the stimulated state in 16.7 mM glucose. Results: The fatty acid mixtures contained linoleic, docosahexaenoic, mead, palmitic, oleic, and arachidonic acids at 27, 3, 0, 35, 28, and 7% (control) or 20, 1, 1, 38, 33, and 7% (CF). Glucose stimulated insulin secretion was measured after 24 hours culture in either of the two fatty acid mixtures and referenced to the insulin secretion observed islets without added fatty acids. Islets cultured in the CF-skewed fatty acid mixture exhibited significantly less glucose stimulated insulin secretion as compared to the control fatty acid mixture (CF: 57.9 +/- 8.3% of reference; control 89.2 +/- 12.5% of reference; N=15, p<0.001 by Wilcoxon Signed Rank Test). Conclusion: The imbalanced fatty acid profile that commonly occurs in cystic fibrosis reduces glucose stimulated insulin secretion from islets. Thus the imbalance of fatty acids in CF may contribute to the etiology of cystic fibrosis related diabetes.
Su1836 CURCUMA LONGA-DERIVED NANOPARTICLES REDUCE COLITIS AND PROMOTE INTESTINAL WOUND REPAIR BY INACTIVATING THE NF-ΚB PATHWAY Mingzhen Zhang, Didier Merlin Background: The inflammatory bowel diseases (IBD), which include Crohn's disease and Ulcerative colitis, affect over a million people in North America. Current therapies (antiinflammatory drugs, immune-suppressants, antibiotics, and drugs for symptomatic relief) are only modestly effective and often cause unacceptable adverse events, particularly with long-term use. Thus, we need novel therapeutics. The use of nanoparticles derived from natural sources may represent a new and more effective treatment for IBD. Additionally, strategies are also needed to elucidate the molecular mechanisms underlying any beneficial effects. Method: As a proof of concept, we herein isolated natural nanoparticles from turmeric (Curcuma Longa, which contains anti-inflammatory and anti-oxidant compounds and has long been used as a traditional herbal medicine), tested their efficacy for targeting the colon, and assessed their intrinsic anti-inflammatory and anti-oxidant potentials using the wellestablished model of dextran sulfate sodium (DSS) induced-colitis in mice. Results: We characterized a specific population of nanoparticles derived from turmeric (TDNPs 2) using a sucrose gradient centrifugation procedure. TDNPs 2 had an average size of around 178 nm and exhibited a negative zeta potential (-21.7 mV), as assessed using dynamic light scattering technology. TDNPs 2 contained high levels of lipids, a few proteins, and a large amount of curcumin (10.84±0.14 µg/mg), which is a main bioactive constituent of turmeric. Our experiments showed that TDNPs 2 could be taken up by the intestinal epithelial cells (IEC) and macrophages of the colon, and was nontoxic in our in vitro and in vivo systems. In mouse models of colitis, TDNPs 2 could reduce colitis symptoms and enhance intestinal repair compared with controls. Oral administration of TDNPs 2 increased the expression of the anti-oxidation gene, heme oxygenase 1 (HO-1), and reduced the expressions of proinflammatory cytokines (IL-6, IL-1β, and TNF-α), suggesting that TDNPs 2 have the potential to promote a healing factor while attenuating damaging factors. Experiments performed in NF-kB-RE-luc mice indicated that TDNPs 2-mediated inactivation of the NF-kB pathway might partially contribute to this protection against colitis. Conclusion: TDNPs 2, which are derived from edible turmeric, represent a novel, natural delivery mechanism for IBD therapeutics; our findings also expand current concepts of drug delivery systems (DDS) and lay the foundation for an effective and nontoxic targeted drug delivery approach for the treatment of IBD.
Su1834 TOPICAL DELIVERY OF IL-10 USING A THERMOSENSITIVE DRUG DELIVERY PLATFORM TO TREAT MURINE COLITIS Sidhartha R. Sinha, Linh Nguyen, Anika N. Ullah, Aida Habtezion Background: Systemic therapies for inflammatory bowel disease such as biologics are effective, but carry increased risks of infections and malignancies. Topical therapy is effective at treating colitis while reducing systemic exposure, but can be challenging to retain. We have developed a thermosensitive delivery platform (TDP) to administer therapeutics that is liquid at room temperature, ensuring proximal delivery; at body temperature, it rapidly becomes a mucoadhesive gel, allowing for easier retention. We have shown that TDP can effectively deliver established topical therapies and now explore its use in delivering protein-based therapeutics. IL-10 holds promise as a treatment for colitis, but systemic testing has been limited by factors including the inability to reach appropriate tissue concentrations, while avoiding systemic toxicities. Here we explore TDP to deliver IL-10 (IL-10/TDP) topically to treat colitis in a murine model. Aim: To determine the ability of TDP to delivery IL-10 and the therapeutic effect of IL-10/TDP in a murine colitis model. Materials and methods: To test whether bioactive IL-10 can be released from TDP, we first measured the IL-10 concentration eluted over time in diffusate from IL-10/TDP formulation. We then tested the ability of the eluted IL-10 supernatant to suppress the pro-inflammatory response of stimulated splenocyte derived T cells. We utilized the dextran sodium sulfate (DSS) murine model of colitis to test the efficacy of rectally delivered IL-10/TDP compared to that of liquid IL-10 enema (IL-10/vehicle) and vehicle enema controls. Body weight, colon length, and histology were used to assess response to treatment. Results: IL-10 released from TDP retains bioactivity and suppressed TNF-a expression in cultured spenocytes. Results from our colitis studies show IL-10/TDP to be superior to liquid IL-10 enema (IL-10/Water) and vehicle controls in treating colitis, as demonstrated by weight recovery, colon length, and histopathology. We also tested the effect of topical IL-10 on pro-inflammatory response by assessing the intracellular cytokine profiles of CD4+ effector/memory T cells of the mesenteric lymph nodes. Intracellular levels of TNFa, IL-17, and IFNg indicated a trend in reduction of proinflammatory cytokines in mice treated with IL-10/TDP compared to IL-10/vehicle and vehicle control. Conclusion: These data collectively support strong therapeutic potential for IL-10/TDP. Being a mucoadhesive gel that is easier to retain, TDP would reduce side effects/risks that can accumulate over time with systemic therapies. In addition, by overcoming retention and urgency issues with existing enema therapy, TDP can improve quality of life for people using topical therapy and potentially reduce dosing frequency.
Su1837 REGULATION OF IL-12P40 BY HIF CONTROLS TH1/TH17 RESPONSES TO PREVENT MUCOSAL INFLAMMATION Ellen Marks, Crystal Naudin, Marjorie M. Walker, Martin Veysey, Paul Foster, Nicholas J. Talley, Graham L. Radford-Smith, Simon Keely Background Intestinal inflammatory lesions are inherently hypoxic, due to both the increased metabolic demands created by cellular infiltration and proliferation, and the reduced oxygen supply with vascular damage. In mucosal tissues, hypoxia stabilises the transcription factor; hypoxia-inducible factor (HIF)-1α, leading to a co-ordinated induction of endogenously protective pathways. HIF-stabilising prolyl-hydroxylase inhibitors (PHDi) protect against colitis in murine models, promoting mucosal healing and offer potential as a novel therapy for inflammatory bowel disease (IBD). Aberrant CD4+ T cell responses have been ascribed a central role in IBD-associated pathology, with differentiation of naïve T cells into T helper (Th) subsets, such as Th1, Th2, and Th17 largely dictated by the production of polarising cytokines. In IBD patients, two important IL-12 family cytokines, IL-12p70 and IL-23, are key to the development of pathogenic Th cell responses. IL-12 and IL-23 are heterodimers with a shared subunit, IL-12p40 which pairs with IL-12p35 or IL-23p19, respectively. In animal models of colitis treated with PHDi, we observed increased mRNA expression of IL12B the gene that codes for IL-12p40 without concurrent changes to IL-12p35 or IL23p19 gene expression. Hypothesis and Aims We hypothesised that HIF-regulation of IL12p40 signalling mediates immune responses during the hypoxia of mucosal inflammation. We aimed to define the role of HIF-mediated IL-12p40 regulation in intestinal inflammation. Methods: IL12B promoter analysis was performed to identify and examine hypoxia-responsive elements. Intestinal lamina propria lymphocytes (LPL) were characterised in wildtype and IL-12p40-/- murine models of colitis (DSS or C. rodentium) treated with vehicle or PHDi (AKB-4924 5 mg/Kg). Immunoblot analysis of PHDi-treated murine and human LPL supernatants was performed to characterise IL-12 secretion. Results: We observed selective induction of IL-12p40 following PHDi-treatment, concurrent with suppression of Th1 responses in DSS colitis and Th17 responses in C. rodentium infection. In the absence of IL-12p40, PHDi-treatment was ineffective, while depletion of IL-23p19 did not influence PHDi efficacy. Analysis of the IL12B promoter identified canonical HIF-binding sites and HIF stabilisation in LPLs resulted in production of IL-12p40 homodimer which was protective
Su1835 RECOMBINANT PRODUCTION OF HUMAN BETA-DEFENSIN 2 (HBD2) AS AN IMMUNE-MODULATOR: IMPROVEMENT OF EXPERIMENTAL COLITIS Daniela Mailänder-Sánchez, Soeren Kjaerulf, Karoline Sidelmann Brinch, Birgitte Andersen, Eduard F. Stange, Nisar P. Malek, Peter Nordkild, Jan Wehkamp Introduction and Aims: Antimicrobial peptides (AMPs) are small endogenous peptide antibiotics which are produced by all multicellular organisms. In recent years, the significance of antimicrobial peptides for the maintenance of epithelial barriers has been recognized and various diseases have been associated with compromised antimicrobial barrier function. Of note, colonic Crohns disease has been related to an attenuated induction of human beta defensin 2 (hBD2) in the colon. In addition to their antimicrobial activity, defensins also have important immune-modulatory functions. Here, we screened hBD2 for potency and toxicity and produced this peptide using a microbial expression system. Furthermore, we tested its preclinical efficacy using different in vivo models of colitis. Methods: We established a cellular expression-system, Saccharomyces cerevisiae, to produce sufficient amounts of hBD2 for in vivo screening and determined its antimicrobial activity in vitro. Next, we tested hBD2 for cytotoxicity in human PBMCs and in murine fibroblasts and assessed its anti-inflammatory properties in human PBMCs stimulated with LPS. Finally, we tested hBD2 in different mouse models of colitis using subcutaneous (s.c.) application. We used DSS (dextran sulfate sodium, n=10), TNBS (Trinitrobenzenesulfonic acid, n=15) and T-cell transfer from wild-type into SCID mice to induce colitis (n=11). To test the protective effect of hBD2, animals were
S-567
AGA Abstracts
AGA Abstracts
Su1833
THE FATTY ACID IMBALANCE OF CYSTIC FIBROSIS IMPAIRS GLUCOSESTIMULATED INSULIN SECRETION FROM PANCREATIC ISLETS Jamal Kriem, Andrew Norris Background: Cystic fibrosis (CF) is accompanied by a prominent imbalance of serum and tissue fatty acids. This imbalance includes low levels of the essential fatty acids linoleic and docosahexaenoic acid. Furthermore, the levels of mead acid, a normally scarce fatty acid, are elevated. Insulin secretion is impaired in CF due to unclear mechanisms. Because fatty acids alter beta-cell function, we hypothesized that the fatty acid imbalance found in CF reduces glucose stimulated insulin secretion. Methods: Mouse islets were cultured in media containing a 200 micromolar fatty acid mixture buffered in 1% albumin. Two different fatty acid mixtures were employed, representing a control or CF-skewed balance. The CF-skewed fatty acid mixture contained less linoleic and docosahexaenoic acids and increased mead acid, reflective of serum fatty acid imbalance in serum of CF patients. Insulin secretion was measured in the basal state in 1.67 mM glucose and in the stimulated state in 16.7 mM glucose. Results: The fatty acid mixtures contained linoleic, docosahexaenoic, mead, palmitic, oleic, and arachidonic acids at 27, 3, 0, 35, 28, and 7% (control) or 20, 1, 1, 38, 33, and 7% (CF). Glucose stimulated insulin secretion was measured after 24 hours culture in either of the two fatty acid mixtures and referenced to the insulin secretion observed islets without added fatty acids. Islets cultured in the CF-skewed fatty acid mixture exhibited significantly less glucose stimulated insulin secretion as compared to the control fatty acid mixture (CF: 57.9 +/- 8.3% of reference; control 89.2 +/- 12.5% of reference; N=15, p<0.001 by Wilcoxon Signed Rank Test). Conclusion: The imbalanced fatty acid profile that commonly occurs in cystic fibrosis reduces glucose stimulated insulin secretion from islets. Thus the imbalance of fatty acids in CF may contribute to the etiology of cystic fibrosis related diabetes.
Su1836 CURCUMA LONGA-DERIVED NANOPARTICLES REDUCE COLITIS AND PROMOTE INTESTINAL WOUND REPAIR BY INACTIVATING THE NF-ΚB PATHWAY Mingzhen Zhang, Didier Merlin Background: The inflammatory bowel diseases (IBD), which include Crohn's disease and Ulcerative colitis, affect over a million people in North America. Current therapies (antiinflammatory drugs, immune-suppressants, antibiotics, and drugs for symptomatic relief) are only modestly effective and often cause unacceptable adverse events, particularly with long-term use. Thus, we need novel therapeutics. The use of nanoparticles derived from natural sources may represent a new and more effective treatment for IBD. Additionally, strategies are also needed to elucidate the molecular mechanisms underlying any beneficial effects. Method: As a proof of concept, we herein isolated natural nanoparticles from turmeric (Curcuma Longa, which contains anti-inflammatory and anti-oxidant compounds and has long been used as a traditional herbal medicine), tested their efficacy for targeting the colon, and assessed their intrinsic anti-inflammatory and anti-oxidant potentials using the wellestablished model of dextran sulfate sodium (DSS) induced-colitis in mice. Results: We characterized a specific population of nanoparticles derived from turmeric (TDNPs 2) using a sucrose gradient centrifugation procedure. TDNPs 2 had an average size of around 178 nm and exhibited a negative zeta potential (-21.7 mV), as assessed using dynamic light scattering technology. TDNPs 2 contained high levels of lipids, a few proteins, and a large amount of curcumin (10.84±0.14 µg/mg), which is a main bioactive constituent of turmeric. Our experiments showed that TDNPs 2 could be taken up by the intestinal epithelial cells (IEC) and macrophages of the colon, and was nontoxic in our in vitro and in vivo systems. In mouse models of colitis, TDNPs 2 could reduce colitis symptoms and enhance intestinal repair compared with controls. Oral administration of TDNPs 2 increased the expression of the anti-oxidation gene, heme oxygenase 1 (HO-1), and reduced the expressions of proinflammatory cytokines (IL-6, IL-1β, and TNF-α), suggesting that TDNPs 2 have the potential to promote a healing factor while attenuating damaging factors. Experiments performed in NF-kB-RE-luc mice indicated that TDNPs 2-mediated inactivation of the NF-kB pathway might partially contribute to this protection against colitis. Conclusion: TDNPs 2, which are derived from edible turmeric, represent a novel, natural delivery mechanism for IBD therapeutics; our findings also expand current concepts of drug delivery systems (DDS) and lay the foundation for an effective and nontoxic targeted drug delivery approach for the treatment of IBD.
Su1834 TOPICAL DELIVERY OF IL-10 USING A THERMOSENSITIVE DRUG DELIVERY PLATFORM TO TREAT MURINE COLITIS Sidhartha R. Sinha, Linh Nguyen, Anika N. Ullah, Aida Habtezion Background: Systemic therapies for inflammatory bowel disease such as biologics are effective, but carry increased risks of infections and malignancies. Topical therapy is effective at treating colitis while reducing systemic exposure, but can be challenging to retain. We have developed a thermosensitive delivery platform (TDP) to administer therapeutics that is liquid at room temperature, ensuring proximal delivery; at body temperature, it rapidly becomes a mucoadhesive gel, allowing for easier retention. We have shown that TDP can effectively deliver established topical therapies and now explore its use in delivering protein-based therapeutics. IL-10 holds promise as a treatment for colitis, but systemic testing has been limited by factors including the inability to reach appropriate tissue concentrations, while avoiding systemic toxicities. Here we explore TDP to deliver IL-10 (IL-10/TDP) topically to treat colitis in a murine model. Aim: To determine the ability of TDP to delivery IL-10 and the therapeutic effect of IL-10/TDP in a murine colitis model. Materials and methods: To test whether bioactive IL-10 can be released from TDP, we first measured the IL-10 concentration eluted over time in diffusate from IL-10/TDP formulation. We then tested the ability of the eluted IL-10 supernatant to suppress the pro-inflammatory response of stimulated splenocyte derived T cells. We utilized the dextran sodium sulfate (DSS) murine model of colitis to test the efficacy of rectally delivered IL-10/TDP compared to that of liquid IL-10 enema (IL-10/vehicle) and vehicle enema controls. Body weight, colon length, and histology were used to assess response to treatment. Results: IL-10 released from TDP retains bioactivity and suppressed TNF-a expression in cultured spenocytes. Results from our colitis studies show IL-10/TDP to be superior to liquid IL-10 enema (IL-10/Water) and vehicle controls in treating colitis, as demonstrated by weight recovery, colon length, and histopathology. We also tested the effect of topical IL-10 on pro-inflammatory response by assessing the intracellular cytokine profiles of CD4+ effector/memory T cells of the mesenteric lymph nodes. Intracellular levels of TNFa, IL-17, and IFNg indicated a trend in reduction of proinflammatory cytokines in mice treated with IL-10/TDP compared to IL-10/vehicle and vehicle control. Conclusion: These data collectively support strong therapeutic potential for IL-10/TDP. Being a mucoadhesive gel that is easier to retain, TDP would reduce side effects/risks that can accumulate over time with systemic therapies. In addition, by overcoming retention and urgency issues with existing enema therapy, TDP can improve quality of life for people using topical therapy and potentially reduce dosing frequency.
Su1837 REGULATION OF IL-12P40 BY HIF CONTROLS TH1/TH17 RESPONSES TO PREVENT MUCOSAL INFLAMMATION Ellen Marks, Crystal Naudin, Marjorie M. Walker, Martin Veysey, Paul Foster, Nicholas J. Talley, Graham L. Radford-Smith, Simon Keely Background Intestinal inflammatory lesions are inherently hypoxic, due to both the increased metabolic demands created by cellular infiltration and proliferation, and the reduced oxygen supply with vascular damage. In mucosal tissues, hypoxia stabilises the transcription factor; hypoxia-inducible factor (HIF)-1α, leading to a co-ordinated induction of endogenously protective pathways. HIF-stabilising prolyl-hydroxylase inhibitors (PHDi) protect against colitis in murine models, promoting mucosal healing and offer potential as a novel therapy for inflammatory bowel disease (IBD). Aberrant CD4+ T cell responses have been ascribed a central role in IBD-associated pathology, with differentiation of naïve T cells into T helper (Th) subsets, such as Th1, Th2, and Th17 largely dictated by the production of polarising cytokines. In IBD patients, two important IL-12 family cytokines, IL-12p70 and IL-23, are key to the development of pathogenic Th cell responses. IL-12 and IL-23 are heterodimers with a shared subunit, IL-12p40 which pairs with IL-12p35 or IL-23p19, respectively. In animal models of colitis treated with PHDi, we observed increased mRNA expression of IL12B the gene that codes for IL-12p40 without concurrent changes to IL-12p35 or IL23p19 gene expression. Hypothesis and Aims We hypothesised that HIF-regulation of IL12p40 signalling mediates immune responses during the hypoxia of mucosal inflammation. We aimed to define the role of HIF-mediated IL-12p40 regulation in intestinal inflammation. Methods: IL12B promoter analysis was performed to identify and examine hypoxia-responsive elements. Intestinal lamina propria lymphocytes (LPL) were characterised in wildtype and IL-12p40-/- murine models of colitis (DSS or C. rodentium) treated with vehicle or PHDi (AKB-4924 5 mg/Kg). Immunoblot analysis of PHDi-treated murine and human LPL supernatants was performed to characterise IL-12 secretion. Results: We observed selective induction of IL-12p40 following PHDi-treatment, concurrent with suppression of Th1 responses in DSS colitis and Th17 responses in C. rodentium infection. In the absence of IL-12p40, PHDi-treatment was ineffective, while depletion of IL-23p19 did not influence PHDi efficacy. Analysis of the IL12B promoter identified canonical HIF-binding sites and HIF stabilisation in LPLs resulted in production of IL-12p40 homodimer which was protective
Su1835 RECOMBINANT PRODUCTION OF HUMAN BETA-DEFENSIN 2 (HBD2) AS AN IMMUNE-MODULATOR: IMPROVEMENT OF EXPERIMENTAL COLITIS Daniela Mailänder-Sánchez, Soeren Kjaerulf, Karoline Sidelmann Brinch, Birgitte Andersen, Eduard F. Stange, Nisar P. Malek, Peter Nordkild, Jan Wehkamp Introduction and Aims: Antimicrobial peptides (AMPs) are small endogenous peptide antibiotics which are produced by all multicellular organisms. In recent years, the significance of antimicrobial peptides for the maintenance of epithelial barriers has been recognized and various diseases have been associated with compromised antimicrobial barrier function. Of note, colonic Crohns disease has been related to an attenuated induction of human beta defensin 2 (hBD2) in the colon. In addition to their antimicrobial activity, defensins also have important immune-modulatory functions. Here, we screened hBD2 for potency and toxicity and produced this peptide using a microbial expression system. Furthermore, we tested its preclinical efficacy using different in vivo models of colitis. Methods: We established a cellular expression-system, Saccharomyces cerevisiae, to produce sufficient amounts of hBD2 for in vivo screening and determined its antimicrobial activity in vitro. Next, we tested hBD2 for cytotoxicity in human PBMCs and in murine fibroblasts and assessed its anti-inflammatory properties in human PBMCs stimulated with LPS. Finally, we tested hBD2 in different mouse models of colitis using subcutaneous (s.c.) application. We used DSS (dextran sulfate sodium, n=10), TNBS (Trinitrobenzenesulfonic acid, n=15) and T-cell transfer from wild-type into SCID mice to induce colitis (n=11). To test the protective effect of hBD2, animals were
S-567
AGA Abstracts
AGA Abstracts
Su1833